pharmacognostic evaluation and pharmacological …
TRANSCRIPT
PHARMACOGNOSTIC EVALUATION AND
PHARMACOLOGICAL EXPLORATION OF ALNUS
NITIDA (SPACH) ENDL.
Ph.D THESIS
BY
NAGINA
DEPARTMENT OF BOTANY
UNIVERSITY OF PESHAWAR
(2020)
PHARMACOGNOSTIC EVALUATION AND
PHARMACOLOGICAL EXPLORATION OF ALNUS
NITIDA (SPACH) ENDL.
BY
NAGINA
A dissertation submitted to the Department of Botany, University
of Peshawar, Peshawar, Pakistan in partial fulfillment for the
award of degree of
DOCTOR OF PHILOSOPHY
IN
BOTANY
DEPARTMENT OF BOTANY
UNIVERSITY OF PESHAWAR
2020
AUTHER’S DECLARATION
I Nagina hereby stated that my Ph.D. thesis “Pharmacognostic evaluation and
pharmacological exploration of Alnus nitida (Spach) Endl.” is my own work and has
not been submitted previously by me for taking degree from this University (University
of Peshawar) or anywhere in the country/world. At any time if my statement is found to
be incorrect even after my graduation the university has the right to withdraw my Ph.D.
degree.
Nagina
Date:22 /07/2020
DEDICATION
Dedicated to my loving father and mother
Field: Botany Field of specialization: Pharmacognosy
Courses studied
(Theory + Lab. work) Teachers names
1. Fresh water algae Dr. Nadeem Ahmad
2. Pharmacognosy Dr. Muhammad Ibrar
3. Vegetation ecology Dr. Lal Badshah
4. Limnology Dr. Barkat Ullah
5. Soil algae Dr. Nadeem Ahmad
LIST OF CONTENTS
S. No. Contents Page
No. Acknowledgement i List of abbreviations iii List of figures v
List of tables vii
Abstract viii
CHAPTER-1 INTRODUCTION 1-18
1.1 Pharmacognosy 1
1.2 Medicinal plants 2
1.3 Ethnobotany 3
1.4 Pharmacognostic evaluation 4
1.4.1 Standardization 5
1.4.2 Identification and authentication 5
1.4.3.1 Macroscopic evaluation 6
1.4.3.2 Microscopic evaluation 6
1.4.3.2a Histology 7
1.4.3.2b Powder drug study 7
1.4.4. Phytochemical analysis 7
1.4.5 Physicochemical analysis 8
1.4.5a Fluorescence study 8
1.4.5b Extractive values 8
1.4.5c Ash analysis 8
1.4.5d Nutritional analysis 9
1.4.5e Elemental analysis 9
1.5 Pharmacology 10
1.5.1 Analgesic activities 11
1.5.2 Anti-inflammatory activities 11
1.5.3 Antipyretic activities 11
1.5.4 Cytotoxic activities 12
1.5.5 Antiviral activities 12
1.5.6 Aflatoxin degradation activity 13
1.5.7 Phytotoxic activities 14
1.5.8 Antioxidant activities 14
1.6 The family Description 14
1.6.1 Taxa of Betulaceae 16
1.6.2 Ethnobotanical uses 16
1.6.3 Phytochemicals of the Genus Alnus 16
1.7.1 Plant Description 17
1.7.2 Taxonomic position 17
1.7.3 Ethnobotanical uses 18
Objectives of the study 20
CHAPTER-2 REVIEW OF LITERATURE
21-36
2.1 Review of literature for A. nitida 21
2.2 Review of literature for other Alnus species 22
2.3 Ethnobotany 23
2.3. Review of literature for pharmacognostic evaluation and
pharmacological exploration of other plants 23
2.3.1 Ethnobotany 23
2.3.2 Pharmacognostic study 25
2.3.3. Extractive values 26
2.3.4 Ash values 26
2.3.5 Fluorescence study 27
2.3.6 Phytochemical screening 27
2.3.7 Elemental analysis 28
2.3.8 Nutritional analysis 30 Pharmacological activities
2.3.9 Analgesic activities 31
2.3.10 Anti-inflammatory activities 32
2.3.11 Antipyretic activities 33
2.3.12 Cytotoxic activities 33
2.3.13 Antiviral activities 34
2.3.14 Aflatoxin degradation activities 35
2.3.15 Phytotoxic activities 35
2.3.16 Antioxidant activities 36
CHAPTER-3 MATERIALS AND METHODS 38-75
3.1 Plant Morphology 38
3.2 Ethnobotany 38
3.3 Pharmacognosy 38
3.3.1 Macroscopy 39
3.3.2 Microscopy 40
3.3.2.1 Micromorphology 40
3.3.2.1a Stomatal Number and stomatal index 40
3.3.2.1b Vein islets and vein termination number 41
3.3.2.1c Palisade ratio 41
3.3.2.2 Scanning electron microscopy 42
3.4 Physicochemical characteristics of powder drugs 42
3.4.1 Powder drug study 42
3.4.2 Ash analysis 43
3.4.2. a Determination of total ash 43
3.4.2.b Determination of acid insoluble ash 44
3.4.2.c Determination of water soluble ash 44
3.4.3 Fluorescence study 45
3.4.4 Determination of extractive values 46
3.4.5 Elemental analysis 46
3.4.6 Nutritional analysis 48
3.4.6.a Determination of ash 48
3.4.6.b Determination of the moisture content 48
3.4.6.c Determination of crude proteins 49
3.4.6.d Determination of fat (ether extract) 50
3.4.6.e Determination of crude fiber 51
3.4.6.f Carbohydrates contents 52
Phytochemistry
3.5 Extraction with organic solvent 53
3.6 Qualitative tests for phytochemical screening 53
3.6.1 Carbohydrates detection tests 53
3.6.2 Detection of proteins & amino acids 54
3.6.3 Alkaloid detection 54
3.6.4 Detection of phytosterols and triterpenoids 54
3.6.5 Detection of phenols 55
3.6.6 Detection of flavonoids 55
3.6.7 Detection of tannins 55
3.6.8 Detection of anthocyanins 55
3.6.9 Detection of saponin 56
3.6.10 Detection of steroidal glycosides 56
3.6.11 Detection of fixed oils 56
3.6.12 Detection of volatile oil 56
3.7 Quantitative analysis of phytochemicals 56
3.7.1 Determination of total phenols 57
3.7.2 Determination of total flavonoids 58
3.7.3 Determination of sterols 59
3.8 Pharmacological activities 59
3.8.1 Analgesic activity 60
3.8.2 Anti-inflammatory activity 61
3.8.3 Antipyretic activity 62
3.8.4 In vitro cytotoxic activity 64
3.8.5 Antiviral activity 67
3.8.6 Aflatoxin degradation activity 70
3.8.7 Phytotoxic activity 73
3.8.8 Antioxidant activity 74
CHAPTER-4 RESULTS AND DISCUSSION 76
4.1 Morphology of A. nitida 76
4.2 Ethnobotany 76
4.2.1 Ethnobotany of A. nitida 77
4.3 Pharmacognosy 79
4.3.1 Macroscopy 79
4.3.2 Microscopy 87
4.3.2.1 Micromorphology of A. nitida 87
4.4 Physicochemical characteristics of crude drug 94
4.4.1 Powder drug study 95
4.4.2 Ash analysis of the powdered plant parts 101
4.4.3 Fluorescence study 102
4.4.4 Determination of extractive values 106
4.4.5 Elemental analysis 108
4.4.6 Nutritional analysis 112
4.5 Phytochemical screening 114
4.6 Pharmacological activities 118
4.6.1 Analgesic activity 118
4.6.2 Anti-inflammatory activity 122
4.6.3 Antipyretic activity 129
4.6.4 In vitro cytotoxic activity 133
4.6.5 Antiviral activity 141
4.6.6 Aflatoxin degradation activity 146
4.6.7 Phytotoxic activity 149
4.6.8 Antioxidant activity 150
Conclusions 154
Recommendations 157
References 159-
211
Appendices 212-
220
i
ACKNOWLEDGEMENT
The entire commendations of magnificence, greatness, compassion and endowment are
only for immortal Allah. Who provided me the nerve, strength and vision to complete this
research work despite all hurdles that i encountered during this intact course of work. I also
pay my respect to Hazrat Muhammad (P.B.U.H.), the last messenger of ALLAH and his
faithful companions, who are forever a true source of guidance for humanity.
I feel utmost pride to extend my cordial thanks to my Research Supervisor Prof. Dr.
Muhammad Ibrar for keen interest, cooperation, persistent valuable suggestions, and
constructive criticism which were the real sources of inspiration for me during this research
work.
I also express my cordial thanks to Prof. Dr. Ghulam Dastagir, Chairman,
Department of Botany, University of Peshawar. Prof. Dr. Siraj-Ud-Din, Dr. Nadeem
Ahmad, Dr. Zahir Muhammad, Dr. Lal Badshah, Dr. Tanvir Burni, Dr. Fazal Hadi, Dr.
Rehman Ullah, Dr. Sami-Ullah, Sir Ghulam Jelani and all the administrative staff of
Department of Botany University of Peshawar, who have been helpful for me during my
work.
I am, thankful to Mr. Ghulam Jilani, curator at department of Botany,
University of Peshawar for identification of plant.
I feel great pleasure and honor to express sincere appreciation to Dr. Ahmad
Naveed and Dr. Mirza Ali khan, Director General Veterinary Research Institute Peshawar,
for providing Lab. facilities, help and moral support.
I also offer my special thanks to Dr. Baitullah, Dr. Sibghat-Ullah, senior
research officers; Mr. Imran Ullah, Mr. Rahmat-Ullah and Mr. Abdur- Rahman, lab
assistants including the whole staff at FMD Research center for helping me a lot during
lab. work and providing me assistance from time to time.
My sincere thanks go to Director General PCSIR Laboratories Complex, for
allowing me to carry out my research activities at their esteemed institute.
I am thankful to Mr. Farid-Ullah Khan, Director P&D, PCSIR Laboratories
Complex for his help and support.
I am also thankful to Dr. Farah Gul, Senior Scientist, Mr. Muhammad Anwar,
laboratory assistant and whole staff at Pharmacology section, PCSIR Laboratories
Complex, Peshawar for assisting me in my research work.
ii
I am also thankful to Dr. Shafqat-Ullah and Dr. Arshad Hussain Research
officers at mycotoxin Lab. PCSIR, Laboratories complex Peshawar for their valuable
guidance and assistance in lab work.
I offer my sincere thanks to the Chairman, Department of Chemistry,
Agricultural University Peshawar, for providing me laboratory facilities to carry out my
research activities there.
Last but not the least i am very much thankful to my unforgettable,
loving and kind parents, brothers and sisters, for their endless cooperation and prayers for
my success.
NAGINA
iii
LIST OF ABBREVIATIONS
Abbreviation Full name
˚C Centigrade
µm Micrometer
AFB1 Aflatoxin B1
An.st Anomocytic stomata
ATCC American type culture collection
B Bark of Alnus nitida
BHK21 Baby hamster kidney 21 fibroblast cell line
Ca Calcium
CPEs Cytopathic effects
Cu Copper
CuSO4 Copper sulphate
DMSO Dimethyl sulfoxide
Ep Epidermal cells number (per sq. mm).
Fe Iron
FMDRC Foot and mouth disease research center
FMDV Foot and mouth disease virus
GAE Gallic acid equivalent
GMEM Glasgow Minimum Essential Medium
H2SO4 Sulphuric acid
HCl Hydrochloric acid
HClO4 Perchloric acid
HNO3 Nitric acid
Is stomatal index
K Potassium
K2SO4 Potassium sulphate
L Leaf of A. nitida
LM Light microscope
M Molar
±SEM plus/minus standard error mean
Mg Magnesium
mg Milligram
ml Milliliter
mm Millimeter
mm2 Millimeter square
Mn Manganese
MNTC Maximum nontoxic concentration
MTT 3-(4, 5-Dimethylthiazol-2-yl)-2,5
Diphenyltetrazoliumbromide
Na Sodium
NADPH Nicotinamide adenine dinucleotide phosphate
NaOH Sodium hydroxide
Nm Nanometer
OD Optical density
iv
PBS Phosphate buffered saline
PC Pistillate cone of A. nitida
PCSIR Pakistan council of scientific and industrial research
PGT Peltate glandular trichome
QE Quercetin equivalent
SC Staminate catkin of A. nitida
SEM Scanning electron microscopy
Sn Stomatal number (per sq. mm).
TCID50 Tissue culture infective dose 50
TLC Thin layer chromatography
UV Ultraviolet
VRI Veterinary research institute
Zn Zinc
v
LIST OF FIGURES
Fig. No. Title Page
No.
Fig.1.1 Alnus nitida (Spach) Endl. in its natural habitat 19
Fig. 4.1. Images of A. nitida bark upper and lower surface, bark fracture and
seeds.
84
Fig. 4.2. Images of A. nitida twig and upper and lower surface of leaf 85
Fig. 4.3. Images of A. nitida fresh and dry staminate catkin (SC) and pistillate
(PC)
86
Fig. 4.4a Light microscope image of A. nitida leaf abaxial epidermis with
anomocytic stomata.
90
Fig. 4.4b Light microscope image of A. nitida leaf abaxial epidermis with
anomocytic stomata, peltate glandular trichome and trichome base.
90
Fig.4.4c Light microscope image of A. nitida leaf adaxial epidermal cells. 90
Fig.4.4d Light microscope image of A. nitida leaf adaxial epidermis with non
glandular (NGT) and peltate glandular trichome (PGT).
90
Fig.4.4e Light microscope image of leaf adaxial epidermis with non glandular
trichome (NGT) along vein.
90
Fig.4.4f Light microscope image of A. nitida adaxial leaf surface epidermal
cells.
90
Fig.4.5a Scanning electron microscopy image of A. nitida abaxial epidermis
with Stomata, Vein and Peltate glandular trichome.
91
Fig.4.5b. Scanning electron microscopy image of adaxial leaf surface with
trichome
91
Fig.4.6. Scanning electron microscopy images of A. nitida leaf stomata, guard
cell, cuticular striation, giant stomata and normal sized stomata.
92
Fig.4.7. Scanning electron microscopy image of staminate catkin powder. 93
Fig. 4.8. Arrangement of veins in lamina of A. nitida leaf. 94
Fig. 4.9 a. Scanning electron microscopy image of A. nitida leaf powder. 95
Fig. 4.9b-c Scanning electron microscopy images of trichomes in A. nitida leaf
powder.
96
Fig. 4.10. Microscopic fragments in leaf powder of A. nitida 97
Fig. 4.11. Microscopic fragments in bark powder of A. nitida 98
Fig. 4.12. Microscopic fragments in staminate catkin powder of A. nitida 99
Fig. 4.13 Scanning electron microscopy image of A. nitida staminate catkin
powder.
99
Fig.4.14. Microscopic fragments in pistillate cone powder of A. nitida. 100
Fig. 4.15a Effect of A. nitida extracts on number of acetic acid induced
writhings in mice
120
Fig.4.15b Effect of A. nitida extracts on acetic acid induced % pain reduction 110
Fig.4.16a Anti-inflammatory effects of A. nitida extracts on carrageenan
induced paw edema in mice after one hour
123
vi
Figs.4.16b Anti-inflammatory effects of A. nitida extracts on carrageenan
induced paw edema in mice after two hours
123
Figs.4.16c Anti-inflammatory effects of A. nitida extracts on carrageenan
induced paw edema in mice after three hours
123
Figs.4.16d Anti-inflammatory effects of A. nitida extracts on carrageenan
induced paw edema in mice after four hours.
124
Figs.4.16e Anti-inflammatory effects of A. nitida extracts on carrageenan
induced paw edema in mice after five hours.
124
Figs.4.17a-d Comparison between % inhibitions of different doses from the same
extract of A. nitida with respect to time.
125
Figs. 4.18a-
e
Antipyretic effect of A. nitida extracts on brewer’s yeast induced
pyrexia in mice after 1, 2, 3, 4 and 5h.
132
Figs. 4.19a-
d.
BHK 21 cell culture 134
Figs.4.20a-
c.
Cytopathic effects (CPEs) of A. nitida on BHK 21 cell line 135
Fig. 4.21. BHK 21 cells culture with CPEs at 31.25 µg/ml concentration of A.
nitida extracts.
136
Fig. 4.22. BHK 21 cells culture with CPEs at 1000 µg/ml concentration of the
A. nitida extracts.
136
Fig.4.23. Differences in cell viability, caused by A. nitida extracts 138
Figs.4.24a-d In vitro cytotoxicity and IC50 values of A. nitida extracts against
BHK21 cells
139
Figs.4.25. TCID50 determination of FMDV 142
Figs.4.26a-j CPEs of FMDV on BHK 21 cell line in antiviral activity of A. nitida
extracts.
144
Fig.4.27. Antiviral activity of A. nitida against FMD virus 145
Fig.4.28a-d Percent protection and EC50 values of A. nitida extracts against
FMDV.
145
Fig. 4.29. Aflatoxin B1 degradation by A. nitida extracts 147
Fig. 4.30. Antioxidant potential of aqueous ethanolic extracts of A. nitida bark,
leaf, staminate catkin and pistillate cone with seeds
152
vii
LIST OF TABLES
Table. No. Title Page No.
Tab. 3.1 Instrumental conditions for detection elements 48
Tab. 3.2 E-Medium composition 74
Table 4.1 Ethnobotanical uses of A. nitida 78
Tab. 4.2 Macroscopic features of A. nitida stem bark. 80
Tab. 4.3 Macroscopic features of A. nitida leaf. 81
Tab. 4.4 Macroscopic features of staminate catkin of A. nitida 82
Tab. 4.5 Macroscopic features of pistillate cone of A. nitida 82
Tab. 4.6 Leaf surface features of A. nitida. 89
Tab. 4.7 Stomatal features of A. nitida leaf. 89
Tab. 4.8 Leaf constant values of A. nitida 94
Tab. 4.9 Ash contents of different parts of A. nitida 102
Tab. 4.10 Fluorescence analysis of stem bark and leaf powder of A. nitida 103
Tab. 4.11 Fluorescence analysis of staminate catkin and pistillate cone
powder of A. nitida 104
Tab. 4.12. Fluorescence analysis of seed powder and extracts of A. nitida 105
Tab. 4.13 Percent extractive values of stem bark, leaf, staminate catkin and
pistillate cone of A. nitida with different solvents 107
Tab. 4.14 Elemental analysis of A. nitida 112
Tab. 4.15 Proximate analysis of A. nitida stem bark, leaf, staminate catkin
and pistillate cone. 114
Tab. 4.16 Preliminary phytochemical screening of A. nitida stem
bark, leaf, staminate catkin and pistillate cone 115
Tab.4.17 Quantitative chemical analysis of the stem bark, leaf,
staminate catkin and pistillate cone of A. nitida 116
Tab. 4.18 Antinociceptive effect of different ethanolic extracts of A. nitida 119
Tab. 4.19 Effect of A. nitida extracts on carrageenan induced paw edema
in mice 126
Tab. 4.20 Antipyretic effect of the bark, leaf, staminate catkin and cone
extracts of A. nitida 131
Tab. 4.21 In vitro cytotoxic activity of A. nitida extracts 133
Tab. 4.22 Cytotoxicity of the A. nitida extracts against BHK21 cell line 137
Tab. 4.23 Antiviral activity of A. nitida extracts against FMD virus 142
Tab. 4.24 Samples for aflatoxin B1degradation 146
Tab. 4.25 Degradation of aflatoxin B1 by ethanolic extracts of A. nitida 147
Tab. 4.26 Phytotoxic activity of A. nitida extracts 149
Tab. 4.27 Percent antioxidant (DPPH Scavenging) activity of A. nitida 151
viii
PHARMACOGNOSTIC EVALUATION AND PHARMACOLOGICAL
EXPLORATION OF ALNUS NITIDA (SPACH) ENDL.
ABSTRACT
Present study included ethnobotanical, pharmacognostic, physicochemical and
pharmacological activities of the bark, leaf, staminate catkin and pistillate cone of Alnus
nitida.
Ethnobotanical study was conducted by visiting some areas in district Swat to
know uses of A. nitida by the local people. Ethnobotanical uses of the plant were almost
the same in all visited areas which was also confirmed from the ethnobotanical literature
review. A. nitida was more frequently used for wood, for agricultural tools making, as
source of dye and as soil binder. In traditional medicines its leaf and bark are used as
treatment for pain and swellings.
Pharmacognostic study determined the color, odor, size, shape, fracture etc. of
the samples. Mean length of cells on abaxial and adaxial surfaces were 28.4 ± 3.1µm and
28 ± 2.8µm; while mean width was 14±1.8µm and 13±1.7µm respectively. Cuticle was
present on both surfaces. Anomocytic stomata were present on abaxial surface. Normal
(up to 20μm) and giant stomata (up to 36μm) of variable length and width were found on
leaf. Non glandular trichome with mean length and width of 253±12.4 µm and 12 ±1.3µm
respectively; and orange colored peltate glandular trichome having rounded head with
mean diameter of 75.5±6.6 µm were also present on both surfaces. Both tetraporate and
pentaporate pollens were found on staminate catkin surface, with dominance of
pentaporate pollen. Mean vein islet number was 10±0.7 per mm2. Vein termination
number was 6.4±0.74 per mm2 and mean stomatal number was 140.4±4.86. Mean palisade
ratio and stomatal index were 5.7±0.3 and 7.6±0.247 respectively. Powder drug study of
the samples showed fragments of various types of cells. Ash content was analyzed,
fluorescence properties were found, elemental and nutritional contents were determined.
All samples showed higher quantities of Fe, K, Mg and Ca. Crude fiber contents were
much higher in bark and pistillate cone samples. 7% proteins were present in staminate
catkin while pistillate cone and leaf contained 6% protein. Carbohydrate contents were
high in staminate catkin (48%) and leaf (46%).
Qualitative and quantitative phytochemical studies showed the presence of phenols,
ix
flavonoids, tannins, sterols, triterpenoids, saponins, steroidal glycosides, fixed oil and fats
in the studied samples. Phenols and flavonoids were the most abundantly found
phytochemicals.
Highest analgesic activity was shown by the leaf (77.87±1.01%), followed by
bark (76.00±1.09) and pistillate cone (52.98±1.01) at dose of 200 mg/kg. Bark extract
showed highly significant anti-inflammatory activity (81.9±2.3%) after 4hrs, leaf extract
caused 81± 3.6 % reduction in carrageenan induced paw edema after 3 hrs. Cones extract
showed 42±1.89% anti-inflammatory potential after 4 hrs at dose of 200 mg/kg.
Highly significant (p<0.01) antipyretic activity was noted for leaf (66%) and
bark (63.8%) extracts at dose of 300 mg/kg after 4hrs while antipyretic activity observed
for the pistillate cone and staminate catkin extracts were 41% and 22% respectively at
dose of 300 mg/kg after 4 hrs.
Significantly higher cytotoxicity was shown by the pistillate cone extract with
IC50 value of 93.05 µg/ml followed by leaf, staminate catkin and bark extracts with IC50
values of 118.2, 119.4 and 152 µg/ml respectively.
Antiviral bioassay showed that bark extract was more effective against FMD
virus (EC50 value of 3.8μg/ml), followed by staminate catkin (EC50 of 3.87 μg/ml),
pistillate cone (EC50 of 4.8 μg/ml) and leaf (EC50 of 5 μg/ml).
The bark and pistillate cone extracts significantly reduced aflatoxin B1
fluorescence compared to standard aflatoxin having same concentrations and kept under
same conditions showing the aflatoxin B1 degrading potential of bark and pistillate cone
(65 and 50 % respectively) at 1000 μg/ml.
The cone, seed and bark extracts showed highest phytotoxic potential of 83%,
83.2% and 80% respectively against Lemna minor plant at 1000 μg/ml.
The tested samples of A. nitida exhibited highly significant free radical
scavenging potential with highest antioxidant potential of leaf (IC50 of 42.56 µg/ml),
followed by bark (IC50 of 49.47), pistillate cone (IC50 of 52.94 µg /ml) and staminate
catkin (IC50 of 56.59µg /ml) respectively.
The present work has revealed the highly significant potentials of the bark, leaf,
staminate catkin and pistillate cone (Pistillate catkin with seeds) ethanolic extracts (70%)
against multiple ailments. This plant can be a rich source of bioactive constituents and can
provide valuable commercial products on further exploration.
1
CHAPTER-1
1.1 Pharmacognosy
INTRODUCTION
Pharmacognosy is the knowledge of drug materials (Evans, 2002). Its history is very
old. The ancient Roman, Egyptian, Greek, Chinese and Indians have enormously
contributed to its development. Pharmacognosy deals with the structural, physical,
biological and chemical aspects of crude drug in a systematic and scientific way in addition
to their history, cultivation method and collection techniques for commerce (Gokhale et
al., 2008). It helps in evolution of new medicines and has played a vital role to find,
characterize, standardize and manufacture phytomedicines/plant material based on their
biochemical, microscopic and macroscopic features (Kaplan, 2001; Kinghorn, 2002;
Gokhale et al., 2008). Pharmacognostic study usually consists of botanical, physical,
organoleptic, chemical as well as pharmacological parameters to investigate the unique
aspects of crude drugs in three steps i.e., correct identification of the therapeutic materials,
isolation of bioactive constituents and then testing for bioactivities. In addition to crude
drugs pharmacognosy deals with therapeutic products (vitamins, pesticides, allergens,
enzymes, and antibiotics etc.) and excipients (emulsifiers, coloring, suspending and
flavoring agents, bulking agents, disintegrants, sweeteners, solidifiers, adhesives and
diluents etc.). The study of teratogenic, poisonous and hallucinogenic plants as well as
condiments, spices and beverages are also part of Pharmacognosy (Alamgir, 2017a).
Pharmacognosy is an interdisciplinary science because; it is closely linked to botany
and phytochemistry on one hand, while on the other hand it is naturally related to the other
biological branches for instance, tissue culture, pharmacology, microbiology, analytical
chemistry and biotechnology etc (Rangari, 2002; Balunasa & Kinghorn, 2005). Nowadays,
pharmacognostic studies comprises of products obtained from plant, fungus, marine
species and nutritional supplements along with treatments from plants (Cardellina, 2002).
Pharmacognosists frequently encounter the common practices of adulteration and
substitution in trade. Generally, adulteration is means of degradation of an object and
includes conditions like admixture, spoilage, inferiority, deterioration and
2
substitution. Adulteration adversely affects the drug industry. Substitution generally means
to put an object in place of another one, while pharmacognostical substitution is to use or
sell a completely different object instead of the requested/ required object (Selvam, 2010).
Pharmacognosy also includes identification and isolation of active constituents and
screening for biological assays (Sarker, 2012).
1.2 Medicinal Plants
Plants have been utilized by man for medicinal purposes since the dawn of human
civilization. Almost 80% population worldwide utilizes plants for medicinal purposes
because of ease in access, in addition to its fewer harmful effects than synthetic drugs (Ibrar
et al., 2007). According to an estimate, Pakistan has 400-600 species of medicinal plants
(Shinwari & Qaisar, 2011). Almost 70% medicinally important plants and animals in
Himalayan ranges are wild species while about 70-80% of total population use local plants
for cure (Pie & Manadhar, 1987; Shaheen et al., 2011; Shaheen & Shinwari, 2012). Various
systems of medications i.e. Unani, Chinese, homeopathy and Ayurveda, throughout the
world use medicinal plants to treat ailments. Secondary metabolites of medicinally
important plants such as phenolics, flavonoids, alkaloids and terpenoids etc., provide
therapeutically active constituents used in modern medicine. The global trade of medicinal
plants is expanding and by 2050 it is likely to reach about US$ 5 trillion (Alamgir, 2017b).
The poor people around the world trust and widely use medicinal plants to alleviate
numerous ailments. Some of the plants are used to treat specific diseases while others as
cure for multiple ailments (Shinwari & Qaiser, 2011). In both developed and
underdeveloped countries demand for medicinal plants is gradually growing to overcome
the high cost and severe side effects of modern medicines.
Pakistan has great diversity of medicinal plants. This high diversity demands further
research to explore their medicinal value and utilize it to treat various novel and old
diseases. It would be more economical to explore species of medicinal plants that are
abundant in wild and also easier to collect. The conservation and cultivation of medicinally
important plants can be made a thriving industry in Pakistan (Noman et al., 2013). The
current research work was carried out on the underutilized plant species Alnus
3
nitida (Spach) Endl. commonly known as Himalayan Alder, found abundantly in
northern areas of Pakistan. The species got little attention as medicinal plant by the local
community and researchers. Therefore, it was studied in detail to explore its medicinal
and economic values besides its more frequent use for wood and dye.
The main points which were taken into consideration to select A. nitida for the current
research work included the ethnobotanical uses of the plant and its Genus (mentioned
in section 1.7.3; 1.6.2 ), easier access, faster and easier growth from its seeds, its stable
population trend( mentioned in section 1.7.1) reports of the presence of
diarylheptanoids ( polyphenolic compounds which have shown remarkable
pharmacological properties in many Alnus species) including two new diarylheptanoids
from A. nitida (mentioned in section 2.1; 2.2), the importance of other species of the
Genus Alnus, showing a variety of pharmacological properties (mentioned in section 2.2
) and lesser laboratory work done on A. nitida as compared to other Alnus species
(mentioned in section 2.1).
1.3. Ethnobotany
Humans are using plants for different purposes since the beginning of their life on
earth (Venkataswamy et al., 2010; Lulekal et al., 2013). The treatment of different diseases
with traditional medicines is greatly increasing (Khan & Shinwari, 2016). The rural people
of Pakistan more rely on local herbal drugs than modern medicines. Approximately 80%
people of the developing countries still use herbal medications (Tareen et al., 2016).
In search of new medicines, the pharmacological and biological properties of
medicinal plant extracts are studied. It provides evidence for its traditional use to cure
diseases. Thus, information on traditional utilization of curative plants by the local people
has a vital role in development of novel and more efficient medicines (Kumar et al., 2015a).
Pakistan is rich in biodiversity of flora and has a unique position in the world due to variable
climatic and soil conditions (Tareen et al., 2016). Literature survey shows that the use of
plant based drugs is very common in the culture of Pakistan. A number of studies have
documented the traditional knowledge of plants used to treat several diseases in different
parts of the country including diabetes, scorpion bites, skin and respiratory tract disorders
4
(Abbasi et al., 2010; Kayani et al., 2014; Butt et al., 2015; Khan et al., 2015; Shah et al.,
2015a; Yaseen et al., 2015a; Khan & Shinwari, 2016; Tareen et al., 2016). About 6000
wild species of plants have been used as medicines while, many plants are under
exploration for their medicinal value (Shinwari, 1996; Ishtiaq et al., 2007). Almost 80% of
endemic flowering plants are located in the northern and western hilly ranges of Pakistan
(Shinwari et al., 2017). Indigenous people rely on wild plants for food timber, vegetables,
medicines, wood and fruits. The phytochemical screening and pharmacological exploration
of several reported indigenous medicinal plants are still needed (Shinwari et al., 2017).
The history of traditional uses of plants is very old. People have acquired the traditional
knowledge of medicinal plants by trial and error method. Today, the pharmaceutical
technology has significantly reduced the folk phytotherapy. But, the old knowledge of
phytotherapy is still alive. Hundreds of plant species were in use either as whole plant part
or in the form of extract. Synthetic medicines have replaced the traditional medicinal plants
in developed countries. But, plants are still used as source of raw material in drug
development research. Several modern medicines are derived from plants. Since 1990,
people have again shown more interest in plant based remedies. Industries are taking more
interest to explore areas of the world where traditional medicinal plants were mainly used
to cure different diseases. In developing countries medicinal plants are extensively used by
practitioners in system of traditional medicines as well as home remedies due to limited
services of public health care. Besides, the industrialized countries are showing interest in
traditional as well as contemporary and alternative medicine (Khan et al., 2012).
1.4. Pharmacognostic evaluation
Pharmacognostic evaluation of crude drugs includes morphology, anatomy,
different parameters of leaf, extractive values (yield to solvent), ash analysis, fluorescence
study and powder drug studies. Such studies aid in proper identification and quality
assessment of medicinal plants by detecting adulterants in powder drug and in whole plants
(Evans, 2002).
A number of synthetic drugs have shown harmful effects on humans as well as on
environment. Therefore, interest in plant based medicines is increased, which are
considered safe. Natural drugs are easily available, economical with less or no side effects.
5
But, they can be easily adulterated. As the availability of more efficient natural drugs
decreases with its increasing demand, they are adulterated with low grade material, another
plant or substance to decrease the cost. The quality and amount of bioactive component(s)
determine the efficacy of plants against various diseases. Medicinal plants are often
misused due to incorrect identification. Most commonly, two or more completely different
species are given the same vernacular name which leads to incorrect identification.
Pharmacognostic studies helps to solve these problems. Thus, pharmacognostic
specifications are vital for drugs. Pharmacognostic study mainly deals with standardization
and authentication of crude drugs by morphological, phytochemical and physicochemical
evaluation. It has helped in identification and authentication of many traditionally used
medicinal plants. In contrast to taxonomy, pharmacognostic evaluation includes
parameters for detection of adulterants in dry powder form as well. The dried powdered
plants lose morphological identity and are easily disposed to adulteration. Therefore,
powdered drug study is essential. Pharmacognostic study confirms distinctiveness of
plants, sets parameters to standardize a crude drug and evade adulterations. It ensures
reproducible quality, safety and efficacy of medicines derived from plants (Chanda, 2014).
1.4.1. Standardization
Standardization means to confirm the identity, quality and purity of drugs. The
basic requirement to making sure the safe utilization of medicines is to establish standards
for safety with quality (Bhat et al., 2012). Different techniques and methodologies such as
pharmacognostic and phytochemical studies are employed for identification and
standardization. Accuracy in characterization and guarantee of drug quality is vital to
ensure reproducible efficacy (Akbar et al., 2014). Pharmacognostic evaluation includes the
following parameters.
1.4.2. Identification and authentication
The primitive people have identified medicinal plants by trial and error method.
Medicinal value of these plants is determined by the quality and quantity of its bioactive
6
constituents. Some exclusively different plant species have the same local names, making
the accurate identification of the desired plant species more difficult. Pharmacognostic
specification of medicinal plants helps to solve these problems (Chanda, 2014).
The exact taxonomic identification of plant species is the first and a vital step in
standardizing herbal drugs whether in dried state, powder or fresh (Springfield et al., 2005).
Plants are correctly identified before starting research work on it. Identified plants are
collected and compared with herbarium sheet in addition to its comparison with published
description of the plant (Tylor et al., 2001).
1.4.3.1. Macroscopic evaluation
Plant parts are authenticated by observing macroscopic traits such as color, odor and
texture (herbaceous, woody or semi woody); shape, size and morphology of leaf (such as
margins of leaf as serrate, entire, lobed, dentate, pinnatified or undulate); floral
morphology, type of inflorescence (corymb, panicle, spike, cyme, head, raceme etc.);
number and shape of stamens, carpels and seeds. These traits are used to distinguish the
desired plant part or species from closely related species that can occur as its adulterants or
substitutes (Smillie & Khan, 2010).
1.4.3.2 Microscopic evaluation
Microscopic techniques such as light or electron microscopy help to examine traits
such as presence or absence of trichomes (hairs), morphology of seed and pollen grains,
vascular traces, oil glands and cell types (Smillie and Khan, 2010). Microscopic
evaluation includes observation of crude drug tissues in cross sectional and powdered
form (William, 2000). Evaluation through microscope is very important in plants
identification. Because by information of specific tissue features in a drug, the small
fragments of powder/crude drugs and adulterants such as molds, insects and fungi are
easily detected.
7
1.4.3.2a. Histology
Histology deals with study of internal structure of plants. It facilitates accurate
identification of plant species. The microscopic evaluation, aid in correct
identification of plant taxa by revealing presence and arrangement of internal
structures viz epidermis, stomatal type, thickening of cell wall, collenchyma,
sclerenchyma, vascular bundles, crystals of calcium oxalate and other specific
structures present in plant sample (Upreti et al., 2013). Such studies also show
similarity of a plant with other taxa of the family.
Other procedures employed to evolve drugs include quantitative microscopy,
linear measurement and leaf constants determination. Starch grain size, trichome, fiber
length and width etc. are included in linear measurement. While leaf constants i.e.
palisade ratio, stomatal number, vein islet number, stomatal index and vein
termination number are extensively used for the microscopic assessment of drugs, in
the form of leaf (Jarald & Jarald, 2007).
1.4.3.2b. Powder drug study
The dried powdered plants lose morphological identity and are easily disposed
to adulteration. Therefore, microscopic study of powdered drug is essential. Powdered
drug study confirms identity of powdered plants by setting parameters for
standardization such as, presence and type or absence of calcium oxalate crystals,
grains of starch, fibers, xylem vessels and trichomes etc (Shrikumar et al., 2006).
1.4.4. Phytochemical analysis
Phytochemicals are chemicals, mainly secondary metabolites produced in plants
that may have significant biological activity but are not recognized as fundamental
nutrients in plants (Tiwari et al., 2011). The vital phytochemicals of plants are
alkaloids, anthraquinones, saponins, phenols, flavonoids, phytosterols and tannins etc.
Phytochemicals may provide new economical and safe sources for manufacturing
various commercial products and medicines (Wu et al., 2013). Various biochemical
quantitative and qualitative tests are employed for screening of phytochemicals. These
tests are significant in evaluation of drugs and detection of adulterants (Grover &
8
Patni, 2013). The curative properties of medicinal plants are determined by its
phytochemicals. Therefore, phytochemical screening of medicinal plant is a
preliminary step towards discovery of novel plant based drugs (Waweru et al., 2017a).
1.4.5. Physicochemical analysis
Physiochemical features of powdered drug assist in estimating the quantity of
adulteration i.e. presence of adventitious and earthy particles etc. in drug.
Physicochemical analysis of drug includes:
a. Fluorescence study
The emission of light by powder drug or extract, which has absorbed
light/electromagnetic radiation, is called fluorescence. Substances show fluorescence
because of its specific chemical composition. Similar powders or extract may appear
dissimilar under light with distinct wave lengths. Some of the extract components
exhibit fluorescence in daylight in visible range whereas others in ultraviolet range of
light. Sometimes substances are made fluorescent by treating their decomposition
products or derivatives with different reagents. By this method many drugs (in crude
form) are evaluated (qualitatively) and standardized. Therefore, fluorescence property
of a drug can be employed as indicator for its identification (Wallis, 1985; Reddy &
Chaturvedi, 2010).
b. Extractive values
Extraction means separating therapeutically active fractions from tissues of
medicinal plants /animals employing standard methods and selective solvents. In
extraction process solvents are used to solubilize compounds having similar polarity
by diffusing gradually into plant/drug material. In evaluation of crude drug extractive
values with various solvents are vital parameters because they show nature of the
chemical compounds that a crude drug contains. Extractive values are essential to
evaluate crude drugs. Extractive values obtained by using dissimilar solvents assure
different kinds of exhausted materials and adulterants (Tatiya et al., 2012).
c. Ash analysis
Ash analysis is a significant tool used to detect adulterants in crude drug. For
9
detection purpose, various ash values i.e. total ash, water soluble ash and acid insoluble ash
are employed. Total ash value is valuable in detecting siliceous contaminants, lime, powder
chalk and other earthy materials. Excessive earthy materials are identified by acid insoluble
ash, which mainly contain silica. On the other hand, water exhausted materials are detected
by water soluble ash (Chanda, 2014).
d. Nutritional analysis
Plants are regarded as fundamental source of nutrition for humans and animals
because; plants contain nutrients essential for development and growth. Carbohydrates,
vitamins, fats, protein, water, oil and minerals including trace elements from plants fulfill
caloric as well as metabolic needs of human body. In developing countries people mostly
use plant proteins in their diets, particularly the high-quality proteins from seeds. There is
always need for searching low priced and better-quality plant protein (Kabir et al., 2015).
Majority of countries in the world including Pakistan have malnutrition problems and
proteins deficiency particularly is very common in food and feed. Furthermore, the rapidly
increasing population growth and climatic changes would result in increased demand of
plant based food in future. Therefore, search for alternate nutritious food is essential to
fulfill the growing demand for food. In remote areas of Pakistan several plants are also
employed as food supplements. These plants are also easily accessible substitute of the
common plant based expensive food. But, the medicinal and nutritionally valuable plants
are often overexploited. Therefore, continuous research is needed to explore the proximate
composition of wild medicinal plants (Rehman & Adnan, 2018).
e. Elemental analysis
Elemental analysis of herbal medicines is important to know their nutritive value
and effects on biological activity of human body. Usually plants contain larger amounts of
essential elements such as K, P, Ca, Fe, Na, Zn, Mn and Mg. However, the concentration
of these essential elements, other trace elements (Cu, Co, Ni, Cr, V and Se) as well as
amounts of some elements (CO, Pb, Al, Sb, Ni, Ba, Cr, Cd, Hg, Sn and As) that may
negatively affect the human body must be confirmed (Pohl et al., 2016). WHO (1998), has
also recommended the checking of medicinal plants for various contaminations including
10
heavy metals. Taking excessive doses or prolong use of medicinal plants can cause health
problems, resulting from increasing contents of heavy metals in body (WHO, 1992; Sharma
et al., 2009). Recent researches have shown that ingestion of essential elements in high
quantity can be lethal. On the other hand, very low concentrations of non-essential trace
elements are toxic for human beings. Therefore, elemental analysis of medicinally useful
plants is very important.
1.5. Pharmacology
Pharmacology is the science of utilization of drugs for prevention, diagnosis and
cure of ailments. It is the blending of Greek words pharmakon (drug) and logia/ logy (study
of). In a much broader sense pharmacology is concerned with interaction of living
body/cells with chemical drugs, including all aspects of information on drugs (Tripathi,
2013). Bioassay, also known as biological standardization is an experiment conducted to
find out the effects of chemicals on physiology of living organisms. Bioassays play key
role in novel drug development and are also important for checking pollutants in
environment. Effective doses of drugs are also determined by bioassays. Some biological
assays were also used in present work to evaluate bioactivities of A. nitida.
The ethanolic extracts obtained from different parts of research plant were
evaluated for their effectiveness by the following activities.
➢ Analgesic activity
➢ Anti-inflammatory activity
➢ Antipyretic activity
➢ Cytotoxic activity
➢ Antiviral activity
➢ Aflatoxin degradation activity
➢ Antioxidant activity
➢ Phytotoxic activity
11
1.5.1. Analgesic activity
Pain is a sensory and disturbing experience coupled with tissue damages in body.
Its nature is protective, but it leads to discomfort and many side effects (Raquibul et al.,
2010). Analgesics are medicines which reduce/alleviate the sensation of pain (Matthew et
al., 2013). The traditionally used analgesics (non-steroidal anti-inflammatory drugs and
opiates) are derived from plants. But, many synthetic analgesics developed with similar
mechanism of action have shown severe side effects such as vomiting, gastrointestinal
bleeding, respiratory discomfort and ulceration (Laurence et al 1997; Mate et al., 2008).
Development of new synthetic analgesics is very costly and may also have severe side
effects. Conversely, many medicines derived from plants are being used for years with no
serious side effects (Kumar et al., 2010). Therefore, search for new bioactive analgesics
from natural resources, particularly from plants is essential.
1.5.2 Anti-inflammatory Activity
Inflammation is a defensive reaction of injured body tissues. Inflammation is
usually caused by injury in living tissues due to microbial infection, defective response of
immune system or physical factors. The basic function of inflammation is localization and
removal of harmful agent(s) as well as the elimination of remains of injured tissue (Barnes,
2009; Garrett et al., 2010; Ahmed, 2011). It can result in acute and chronic diseases if not
controlled. Anti-inflammatory constituents of medicinal plants extract reduce
inflammation by inhibiting inflammatory mediators. In modern medicines research is
focusing on anti-inflammatory activities of plants. Because, the knowledge on traditional
use of plants for inflammation, is yet waiting to be further explored (Oguntibeju et al.,
2018).
1.5.3. Antipyretic activity
Fever (pyrexia) is the raise in body temperature from normal range as result of
physiological stress (increase in thyroid secretion, infections caused by microbes,
leukemia, too much exercise, CNS injury etc.). Fever results in quicker progression of
diseases because of dehydration, increase in catabolic activities of tissue and in case of
12
some chronic infections including HIV, persistent illness. Antipyretic drugs decrease
higher body temperature by inhibiting expression of cyclooxygenase-2 (COX-2) which
further inhibits synthesis of prostaglandin. These antipyretic synthetic drugs cause highly
selective inhibition of COX-2. However, these show toxicities to further organs like brain
cortex, cardiac muscles, glomeruli and liver cells. While, COX-2 inhibitors derived from
natural products (particularly plants) show less side effects and are lower in selectivity
(Sultana et al., 2015a). Plants have been utilized as natural source for antipyretic agents
since ancient times. So, modern research is based on search for herbal medicines with
antipyretic properties, reduced side effects, lower toxicity and as an alternate of synthetic
drug (Ahmad et al., 2017).
1.5.4 Cytotoxic activity
In vitro assays are validated and accepted as substitute to tests on whole animals.
Therefore, in vitro screening of the plant extract for its cytotoxic potential is an appropriate
strategy to explore plants with antineoplastic properties. Use of continuous cell lines for
evaluation of in vitro cytotoxicity is the most usually employed approach (Bhanushali, et
al., 2010). In vitro Cytotoxic studies aid to select non-cytotoxic concentrations of plant
extracts for bioactivities. Also, the extracts with cytotoxic potency can be analyzed for
cytotoxic constituents and further investigated on appropriate cell lines for anticancer
potential (Swiatek et al., 2013). Baby Hamster Kidney-21 fibroblast cell line (BHK 21) has
been used for evaluating cytotoxicity of compounds, drugs and plant extracts (Mekawey et
al., 2009; Ankita & Chauhan, 2012; Zhou et al., 2012; Bisht et al., 2014), through
microscopic study of changes in cell morphology, appoptosis and antiproliferative effects;
and determining percentage viability by measurement of metabolic activity.
1.5.5. Antiviral activity
The increasing viral infections all over the world are demanding exploration of
natural, cost effective and more efficient antiviral drugs. Many plant extracts are reported
for their antiviral activity (Chattopadhyay & Naik, 2007; Naithani et al., 2008). Besides,
studies have revealed antiviral potential of certain plants extracts against strains of viruses,
13
resistant to common antiviral drugs (Ser-kedjieva, 2003; Tolo et al., 2006). This has
challenged the practices made in current drug discovery processes and has enforced the
exploration of antiviral medicinal plants and their antiviral constituents. Foot and mouth
disease (FMD) is an infectious viral disease of livestock (cloven hoofed). It is responsible
for annual loss of about 10 billion USD approximately, in more than 100 countries of the
world (Knight-Jones & Rushton, 2013). In Pakistan, data for total loss is not available but,
the six months’ survey of only twelve FMD infected villages has reported losses of about
0.32 million USD (Gorsi et al., 2011). Vaccines for FMD consist of inactivated FMDV
(foot and mouth disease virus) and require a week to immunize an animal against this
disease (Deshpande & Chaphalkar, 2013). Combination of antiviral agents and vaccines
are suggested as more effective approach to cure the infected animals. Many researchers
have tested antiviral potency of plant extract against FMD (Deshpande & Chaphalkar,
2013; Boseila, 2011; Boseila &Hatab, 2011). But, at present there are no approved drugs
to completely prevent or cure FMDV (Vagnozzi et al., 2007).
Many constituents of plants such as flavonoids, alkaloids, coumarins, phenols,
saponins and terpenoids have shown antiviral effects on different viruses and have been
suggested as substitutes to conventionally used antiviral drugs. Herbal teas and volatile
essential oil of some common herbs and spices have also shown significant antiviral effects
(Jassim & Naji, 2003; Liu & Du, 2012). Plants are regarded as potential sources of antiviral
drugs because, secondary metabolites of plants are supposed comparatively safer. In
addition, the phytochemicals also have new and multiple target sites (Vlietinck & Vanden-
Berghe, 1991; Willium, 2001; Raskin et al., 2002, Jassium & Naji, 2003).
1.5.6 Aflatoxin degradation activity
Aflatoxin B1 is the mycotoxin produced in food products by toxigenic strain of
molds, Aspergillus flavus. Aflatoxin B1 has mutagenic, carcinogenic teratogenic and
thermostability potential as well as accumulation capacity in human body (WHO, 2006;
Wu, 2006; Wagacha & Muthomi, 2008). Synthetic preservatives are reported to have more
negative effects on food products (Tolouee et al., 2010; Prakash et al., 2011a). Conversely,
many plants derived products have become significant as harmless food preservatives in
agricultural industries and are identified as substitute of synthetic preservatives because of
14
their biologically active components. Extracts of many plants are reported as anti-
mycotoxin due to phytochemical contents (Ebana & Madunagu, 1993; Mahoney &
Molyneux, 2004; Radulovic et al., 2006; Palumbo et al., 2007; Samapundo et al., 2007).
Aflatoxin B1 is resistant to degradation by high temperature treatment (Raters & Matissek,
2008). Other physical or chemical agents used for its detoxification are harmful, high-
priced, and also affect the quality of food products (Womack et al., 2014). Several plant
extracts have revealed aflatoxin B1 degrading potential (Hajare et al., 2005; Sandosskumar
et al., 2007; Velazhahan et al., 2010; Hassan et al., 2012; Fapohunda et al., 2014).
Medicinal plants can be a more effective, safer, economical, and better-quality substitute
of other agents used to degrade aflatoxin. Majority of the investigators recommend high
quantity of phenolic compounds in plant extracts accountable for their anti-aflatoxigenicity
(Selvi et al., 2003; Prakash et al., 2010, 2011b; Garcia et al., 2011).
1.5.7. Phytotoxic activity
Improper control of weeds causes enormous wastage of important food crops in
Pakistan. The level of damage caused by weeds is generally more than crop diseases and
pests however; its effects are unnoticed/ ignored. Crop yield is affected by weeds because
these compete with plants for accessible resources. Therefore, planning is necessary to
control weed growth. The technique of Lemna minor bioassay is used to discover weed’s
natural inhibitors. L. minor, an aquatic monocot plant has a filamentous root and an oval
central frond with two attached daughter fronds. Vegetative reproduction in L. minor takes
place through buds produced from fronds and pouches present on sides of the main frond
(Atta-ur-Rehman et al., 2001). Being sessile, plants produce some toxic chemicals to
protect themselves from insects, fungal and bacterial attacks and compete with other plants
for food, light and habitat. The secondary metabolites/chemicals produced by plants for
defense can also act as herbicides to other plants (Hussain et al., 2010 b). Nearly every
plant and its roots, flowers, seeds, stems, buds, bark and leaves have phytotoxic chemicals.
These may be released under appropriate conditions and affect growth of the adjacent plants
(Weston, 1996).
1.5.8. Antioxidant activity
Natural antioxidants are abundantly found in plants. These antioxidants
15
(specifically the carotenoids and polyphenols) have shown significant bioactivities such as
anti-inflammatory, anticancer, antiaging, anti-diabetes, anti-obesity and antihypertensive
(Zhang et al., 2015). Thus, exploration of potent antioxidants from plants is vital to promote
their use in medicines and foods (Xu et al., 2017). The excessive production of free radicals
(such as hydroxyl, nitric oxide and superoxide radicals) due to smoking, alcohol, radiation
and other toxic environmental pollutants disturb the natural balance of oxidation and anti-
oxidation in human body (Li et al.,2015; Wang et al., 2016; Zhou et al., 2016). Free radicals
oxidize biological molecules and leads to cancer, coronary heart diseases, dementia as well
as aging. Consumption of exogenous antioxidants would prevent these oxidative chain
reactions in the body cells (Baiano & Del -Nobile, 2015). But, the more commonly used
synthetic antioxidants are expected to cause carcinogenesis and are harmful to liver as well
(Qi et al., 2005). Therefore, more efficient natural antioxidants must be developed and used
to defend human beings against free radical attack and various chronic diseases.
1.6. The family description
The family Betulaceae include shrubs and trees of six genera i.e. Alnus (Alder),
Carpinus (Hornbeam), Ostrya (Hop Hornbeam), Betula (Birch), Ostryopsis, and Corylus
(Hazel). The number of total species as well as infraspecific taxa is taxonomically uncertain
(Shaw et al., 2014a). The largest genus of family Betulaceae is Betula with 40- 50 species
(Ashburner & McAllister, 2013); also, total 62 species of Betula are reported by world
check list for selected plants families (Govaerts, 2014). Ostryopsis having 3 reported
species is the smallest genus of Betulaceae. Genus Alnus, is closely related to Betula, both
having the number of chromosomes as multiple of 14. On the other hand, number of
chromosomes in Carpinus, Ostryopsis and Ostrya are the multiples of 8, while in Genus
Corylus 11. The latter mentioned four genera were earlier placed in family Corylaceae due
to their distinct features. Now Taxonomists are agreed to place them in family Betulaceae
with Alnus and Betula (Shaw et al., 2014a)
16
1.6.1. Taxa of Betulaceae
Taxa of family Betulaceae have distribution on a big part of northern hemisphere,
Canada to China on west side; while in East side on Siberia and Japan. Many species spread
in south of equator, reported in northern areas of Southern America as well as in Central
America. These taxa have large distribution naturally and across their range occur
commonly e.g. Corylus avellana and Betula pendula. But, few taxa have very low number
of population and are confined to small areas such as Carpinus putoensis with a single
known tree (Shaw et al., 2014a). The Genus Alnus of family Betulaceae comprises of
about 40 species, having wide distribution in Europe, Asia, North America and Africa (Ren
et al., 2017).
1.6.2. Ethnobotanical uses
Several species of the genus Betula and Alnus contains bioactive compounds such
as betulin and lupeol which have been useful in combating cancers. Researchers are now
searching its efficacy against herpes virus, replication of HIV-1 virus and hepatitis (Sati et
al., 2011a). Aerial part of Alnus jorullensis Kunth subsp. jorulensis, is reported to inhibit
tumor sarcoma, Adenocarcinoma of the duodenum as well as Lymphoid leukemia (Abbott
et al., 1966). In traditional system of medicines Alnus japonica Steudel is used as treatment
for alcoholism, hemorrhage, fever and diarrhea. Bark of stems is antioxidant, antiviral (used
against influenza virus) and hepatoprotective (Kuete et al., 2013).
Many species of this genus have been utilized as cure for rheumatism, cancer
(Hammond et al., 1998), inflammation of nail, dental abscesses (Loi et al., 2004), many
diseases of skin like prurigo and eczema (Choi et al., 2011), hemorrhoids, chronic herpes,
healing of wounds (Neves et al., 2009) and as antiperspirant (Loi et al., 2004).
1.6.3. Phytochemicals of the Genus Alnus
Phytochemical evaluation of Alnus species have shown the presence of flavonoids,
phenolic compounds, tannins, terpenoids and steroids in addition to diarylheptanoids which
are the major substances amongst all. Lupeol, Betulinic acid, betulin, oregonin and ursolic
acid were also reported from Alnus species (Sati et al., 2011).
17
1.7.1 Plant description
Name of the Taxon: Alnus nitida (Spach) Endl. (Shaw et al., 2014b)
Synonym: Clethropsis nitida Spach.
English name: Alder
Common name: Himalayan Alder
Local names
In Pakistan: Geiray (Pushto), Sharol (Urdu)
In India: Indian Alder, Kosh, Kunish (Chauhan et al., 2014)
Flowering period: September-November (Chauhan et al., 2014).
Occurrence:
A. nitida (Spach) Endl. is found in temperate Himalayas usually occurring at lower
elevations (Ranges between 1,000 m & 3,000 m above sea level). A. nitida is common
along water course in its range. Its present population trend is stable. It is found as pure
stands in its range areas. It is native to Pakistan (Swat, Dir, Hazara and Azad Kashmir),
Afghanistan, India and Nepal (Shaw et al., 2014b).
Threats
It is assessed in IUCN red list of threatened species as least concern; because
currently it has no major known threat to impact its survival. Degradation, overgrazing,
encroachment and over exploitation are the threats known to Himalayan forests areas.
Himalayan trees are threatened by the locals’ collection of tree as fuel wood and timber.
1.7.2. Taxonomic position
Kingdom: Plantae
Phylum Tracheophyta
Division: Magnoliophyta
18
Class: Magnoliopsida
Order: Fagales
Family: Betulaceae
Genus: Alnus
Species: nitida (Spach) Endl.
1.7.3. Ethnobotanical uses
Bark of A. nitida cure swelling and body pain. Bark is employed for tanning as well
as dyeing (Shaw et al., 2014b). Leaves are used to cure sour feet and to relieve body pain
(Ilyas et al., 2013). Catkins are expectorant, sedative, and diuretic (Hazrat et al., 2011).
Fresh leaves used to cure diabetes (Yaseen et al., 2015b). A. nitida cure scorpions bite
(Nasim et al., 2013). This plant is also used as soil binder (Ilyas et al., 2013). Wood is used
for fencing, construction, roofing, utensils and in making of furniture and agricultural tools
(Ahmad et al., 2009; Hazrat et al., 2011).
19
Fig.1.1. Alnus nitida (Spach) Endl. in its natural habitat
20
OBJECTIVES OF THE STUDY
The current research work on A. nitida (Spach) Endl. was conducted with the
following objectives.
• To study the morphology of research plant parts of A. nitida for accurate
identification.
• To determine leaf epidermal features such as palisade ratio, stomatal number, types
of stomata, stomatal index, and vein termination number and vein islet number.
• To Physicochemicaly analyze the powder drug including ash analysis, fluorescence
properties and extractive values to authenticate the plant samples and find out
solvent with maximum extractive values of the samples.
• To evaluate the phytochemicals /secondary metabolites of the research plant parts
in the solvent used for extraction qualitatively as well as quantitatively.
• To document different fragments, present in powder drug samples.
• To evaluate the aflatoxin B1 degradation potential of the Alnus nitida plant
samples for exploration of its potential to be used as food preservatives against
aflatoxin B1 contamination.
• To evaluate the A. nitida extracts for anti-inflammatory, analgesic, antipyretic, anti-
oxidant, cytotoxic and antiviral activities.
• To assess the A. nitida extracts for phytotoxic potential.
• To document the nutritional and elemental contents of the A. nitida samples.
21
CHAPTER-2
REVIEW OF LITERATURE
2.1. Review of literature for Alnus nitida
Alnus nitida (Spach) Endl. belongs to family Betulaceae. Its wood is used in
construction, roofing, fencing, making furniture and utensils (Ahmad et al., 2009).
Siddique et al. (2010) isolated two new diarylheptanoids (nitidone A and nitidone
B) from A. nitida.
Barkatullah & Ibrar (2011) reported the A. nitida tree as soil binder and its flower
and wood for having medicinal uses. Its wood is also used to make agricultural tools while,
catkins are sedative, diuretic, expectorant and are also used in cosmetics (Hazrat et al.,
2011).
Bano et al. (2013) reported utilization of A. nitida as fodder, fuel and medicine in
Azad Kashmir. Its leaf decoction is applied to cure pain and sour feet. Ilyas et al., (2013)
also reported it as valuable soil binder and fuel wood. It has been useful against scorpion
bite (Nasim et al, 2013).
Bark concoction of A. nitida is used to cure swelling and body pain (Shaw et al.,
2014).
Fresh leaves of A. nitida are placed in water for a night and half cup of this water
is taken before breakfast as cure for diabetes (Yaseen et al., 2015b).
Sajid et al. (2016) reported Catechin, Gallic acid and Rutin compounds as well as
hepatoprotective potential in bark methanolic extract of A. nitida.
Sajid et al. (2017) revealed analgesic, as well as anti-inflammatory action for A.
nitida bark methanolic extract (95%).
Sajid et al., (2019) studied lung cancer inhibitory potential of the A. nitida bark
and leaf methanolic extract against A-549 and H460 cells. They reported that these
22
extracts significantly inhibited the cells survival, stopped cell cycle at G1 phase and
depressed the anti-apoptotic proteins expression.
2.2. Review of literature for other Alnus species
Above 40 species are included in the genus Alnus, which are mostly found in
Europe, Africa, North America and Asia (Ren et al., 2017). Alnus species are usually used
in traditional medicines (Sati et al., 2011). Following is the literature review for some other
Alnus species.
Many triterpenoids from Alnus species have shown inhibition of tumor and HIV-1
viral enzyme while some of these compounds exhibited hepatoprotective activity (Sheth et
al., 1973; Yu et al., 2007; Lee et al., 2011).
Anti-inflammatory effects are reported for phenolic glycosides and
diarylheptanoids from various Alnus species (Kim, 2005; Aguilar, 2011; Lai, 2012).
Tung et al., (2010a) explored the anti-influenza effects for the bark of Alnus
japonica. Alnus hirsuta Turcz. is used to cure fever, burns, diarrhea, hemorrhages etc (Park
et al., 2010). Diarylheptanoids from A. hirsuta and A. Japonica are reported for significant
hepatoprotective activity (Park et al., 2010; Tang et al., 2010b). Stevic et al., (2010)
reported the cone, leaf and bark extracts from A. viridis and A. incana for significantly
higher scavenging of DPPH free radical and strong cytotoxicity on HeLa cells line.
Ludwiczuk et al., (2011) investigated Galangin isolated from A. sieboldiana and
reported its inhibitory potential for gene expression of TNF-α (tumor necrosis factor-α) in
A549 cells.
A. Japonica Steudel. has been reported for anti-inflammatory, anticancer,
antioxidant, and hepatoprotective activity (Lim et al., 2011).
Choi et al., (2012a) revealed ethanolic extract of A. pendula bark for highly
significant antibacterial potential against MRSA (methicillin resistant Staphylococcus
aureus).
23
Several degenerative diseases (aging, heart problems, cancer etc.), are connected
with the presence of free radicals. Antioxidants reduce the negative effects of ROS
(reactive oxygen species) (Poljsak et al., 2013).
Hirsutenone isolated from A. pendula, A. japonica and A. hirsuta has been reported
for anticancer properties (Choi et al., 2012b; Leon-Gonzalez et al., 2014). Diarylheptanoids
from A. glutinosa can protect normal cells from toxicity of chemotherapeutic drugs without
lessening their therapeutic effects (Novakovic et al., 2013; Dinic et al., 2014).
Diarylheptanoids are considered as the most important bioactive constituents of
Alnus species with antioxidant and anticancer potency (Hu &Wang, 2011; Novakovic et
al., 2014).
Alnus glutinosa (L.) Gaertn. has shown significantly higher chemo-protective,
antioxidant as well as antimicrobial properties (Dahija et al., 2014; Dinic et al., 2015;
Abedini et al., 2016).
Ren et al., (2017) have summarized 273 chemical compounds isolated from Alnus
species including diarylheptanoids, terpenoids, polyphenols, steroids and flavonoids etc.
2.3. Review of literature for pharmacognostic evaluation and pharmacological
exploration of other plants including some Pakistani as well as international
plants.
2.3.1. Ethnobotany
Plants have been an important source of medicines, inherited in the health care
system all over the world. In Pakistan also plants are used as traditional medicines by large
part of population (Khan et al., 2012). Numerous researchers have reported traditional uses
of local plants in different areas, some of which are the following.
Noman et al. (2013) documented medicinal plants of Omara, Gawadar, Pakistan,
used as drugs, spices, fodder, and food. 45% were utilized as medicines, 26% were having
many uses and 29% were used as fodder.
24
Ahmad et al. (2014) enlisted 50 species of plants from Chail valley. The
documented plants were from 48 genera belonging to 35 families. 12% were trees, 58% of
plants were herbs, 2% were climbers and 28% were shrubs. Parts of the plant used included
seed, rhizome, fruit, bark, leaf and stem. The maximum numbers of cured ailments were
urinary, digestive and skin problems followed by diarrhea, asthma, jaundice, dysentery and
angina.
Ajaib et al. (2014) collected plants from Tehsil Kharian, District Gujrat. They
identified 50 plants from 32 families, used as drugs, fodder, fuel and shelter. Plants from
Asteracae and Poaceae were more abundantly found.
Silva et al. (2015) conducted ethnobotanical survey and enlisted plants from
community of Sobradinho, Luís Correia, Piaui in Brazil. Total 57 species from 33 families
were enlisted including native (56%) and exotic (44%) plants. The most abundantly used
plant part was leaf while the most frequently found species were Morinda citrifolia and
Cymbopogon citratus. Large numbers of species were used to cure inflammation, fever and
pain.
Uddin et al. (2016) studied plants of Ikrampur village at Mardan. 68 species of
plants representing 63 genera from 34 families were reported. These plants were utilized
for different purposes such as fuel, ornamental, furniture, shelter, fodder and as vegetable.
Mostly found plant family was Asteraceae and Poaceae with 6 species. The collected data
was helpful to use and control weeds in the study area.
Shinwari et al. (2017) enlisted traditional therapeutic plants and their uses in
Kohistan and Shangla areas of Northern Pakistan. Local people provided information about
61 plants from 49 genera of 34 families. The dominant family was Lamiaceae with 6
species from 6 genera. Leaves were the most utilized part. Many plants were used
effectively for many diseases.
Khan et al. (2018) documented local use of medicinal plants in the valley of Talash,
Lower Dir, Pakistan. The elder people and herbalist were interviewed. 50 plants of 46
genera from 33 families were identified. Maximum number of plants belonged to
Lamiaceae. 68% of the treatments comprised of herbs. Most used part was the leaf (41%).
25
All of the documented plants were suggested for further study/screening to verify the
reported effective usage by the local people against various ailments.
2.3.2. Pharmacognostic study
Pharmacognostic study provides scientific facts on morphological, microscopical
and physicochemical features of crude drug to determine its quality and purity (Sindhu,
2010).
Sultana et al. (2011) used various pharmacognostic techniques to authenticate the
herbal drug Azadirachta indica A. Juss (Neem). Quality and standardization was ensured
by studying morphological and organoleptic features as well as SEM (scanning electron
microscopy) study of pollen grains anatomy.
Alam & Saqib (2015) studied macroscopic, microscopic parameters of Gaultheria
trichophylla for its pharmacognostic standardization. Determination of extractive and ash
values, analysis of fluorescence and phytochemical constituents helped to identify and
standardize G. trichophylla. Valuable diagnostic features of its leaf cross section and
powder were also observed under light and scanning electron microscope.
Patill et al. (2016) compared anatomical features of Boerhaavia diffusa L. and its
adulterants Sesuvium portulacastrum L. and Trianthema portulacastrum L. Anisocytic
stomata was present in Boerhaavia diffusa while its adulterant plants showed the presence
of only paracytic stomata. Many starch grains were observed in the ground parenchyma
tissues between cambium and xylem parenchymatous tissues of B. diffusa which were
absent in T. portulacastrum. Also, the B. diffusa root was having semicircular patches in
phloem cells surrounding the xylem while, in T. portulacastrum only thin strips were
found.
Sarkar et al. (2017) determined the total ash, water soluble ash as well as acid
insoluble ash, extractive values in water and alcohol as well as fluorescence properties and
phytochemical constituents of the leaf powder of Bauhinia purpurea and Centipeda
minima. Alkaloids, flavonoids, phenolic compounds, tannins, glycosides, carbohydrates
and fats were found in both plants leaves.
26
Nilam et al (2018) evaluated Ipomoea pes-caprae by using microscopic,
physicochemical and phytochemical parameters to establish pharmacognostic standards
for accurate identification and authentication of its leaf and stem. The microscopic
observation showed paracytic type of stomata, large number of larger palisade tissue cells
as well as open collateral and conjoint vascular bundles. Powder drug study confirmed the
presence of unicellular trichomes, vessels of xylem with border pits and paracytic type of
stomata etc. Ash analysis, extractive values and phytochemical studies were also
conducted. Analysis of leaf and stem confirmed the presence of tannins, phenols, alkaloids,
flavonoids and steroids.
These studies provided important distinguishing features for identification and
authentication of the evaluated plants.
2.3.3. Extractive Values
Extractive values with different solvents help in identification of drug adulterants.
There are many references available for determination of extractive values to authenticate
drugs which include Mary et al.,2019 (Capparis erythrocarpos Isert, leaf , root and stem),
Ramadurga et al., 2019 (Careya arborea root), Mehta et al., 2018 (Swertia chirayita, S.
purpurascens, S. cordata, S. alata, and S. angustifolia), Pal et al. 2018 (Benincasa hispida
root), Abdullahi et al., 2018 (Microtrichia perotitii leaf), Sundar and Habibur, 2018 (Leaf,
fruit and stem bark of Gardenia latifolia Aiton), Chaudhari and Griase, 2015 (bark of
Sesbania sesban (L) Merr. and Sanjeeva et al., 2014 (whole plant of Ipomoea quamoclit
Linn).
2.3.4. Ash values
Ash values (total ash, water soluble and acid insoluble ash) are employed to find
adulteration in herbal crude drugs (Jarald & Jarald, 2007). Many researchers have
determined ash values for different plants to standardize the crude drugs, some references
are Melissa parviflora (Bhat et al., 2012), Sesbania sesban, Sesbania rostrata, and
Sesbania exaltata (Kadam et al., 2013), Zaleya govindia (Shailendra et al., 2014),
Eucalyptus globulus, Acacia nilotica, Butea monosperma and Bombax malbaricum
(Kumbhar & Godghate, 2015), Blepharis sindica (Priyadarshi et al., 2016), Mallotus
27
rhamnifolius (Loganathan et al., 2017). Phania matricarioides (Gutierrez et al., 2018) and
Costus spicatus (Azhagumadhavan et al., 2019).
2.3.5. Fluorescence study
Herbal drugs treated with different solvents show characteristic fluorescence under
UV and visible light. Fluorescence is due to different chemical compounds present in
plants. The difference in color observed under ultraviolet and visible light is a useful
parameter for authentication and standardization of these drugs (Kasthuri & Ramesh,
2018). Gayathri & Kiruba (2015), Ruba & Mohan (2016), Mandal et al. (2017), Ishtiaq et
al. (2018), Ranjith et al. (2018) and Jothi et al. (2019), have also standardized various
medicinal plants through fluorescence analysis.
2.3.6. Phytochemical screening
Plants are screened for secondary metabolites (flavonoids, alkaloids, tannins,
steroids, saponins, and phenols etc.), which have shown significant therapeutic potential.
Phytochemical evaluation of plants is significant for identification and isolation of new
bioactive compounds against various diseases. It includes both qualitative and quantitative
tests (Tripathi & Mishra, 2015). Some of the studies conducted on phytochemical analysis
are given below.
Madhu et al., (2016) carried out quantitative phytochemical studies on ten
medicinal plants. They reported variation in concentration of phytochemicals extracted in
different solvents. Highest alkaloid concentration was found in Petroleum Ether (PE)
extract of Foeniculum vulgare (stem) and Levisticum officinale (leaf). Flavonoids content
was higher in water (AQ) and PE extract of Sapindus saponaria, Garcinia indica and
Dracaena loureiri, moderate amounts of phenols were present in PE and AQ extracts of
Sapindus saponaria and Jatropha curcas.While the PE extract of Sapindus saponaria
(pericarp of fruit) was reported for high steroid contents.
Ahmad et al. (2016a) investigated the rhizome and aerial parts of Meconopsis
aculeata for various phytochemical constituents. They reported flavonoids, phlobatannins,
alkaloids and terpenoids in the studied parts.
28
Ajuru et al. (2017) identified and quantified bioactive constituents present in
aqueous leaf extract of Piper nigrum L., Phyllanthus amarus Schum and Thonn, Senna
occidentalis L., Gongronema latifolium Benth. and Euphorbia heterophylla Linn.
Qualitative study revealed the presence of glycoside tannin, alkaloid and sugar in all plants
while quantitative analysis showed variation of tannin, phenol, flavonoid, alkaloid and
saponin contents among the studied plants.
Loganathan et al. (2017) conducted phytochemical screening of Mallotus
rhamnifolius leaf and found glycosides, quinones, saponins, flavonoids, carbohydrates,
amino acids, terpenoids, alkaloids, tannins, phenolic compounds, proteins and phytosterols
in its extract.
Rao & Kumar (2017) tested the ethyl acetate extract of Hygrophyla auriculata for
the presence of different phytochemicals. They found large content of Alkaloids and
moderate amounts of terpenoids, saponins, cardio glycosides, flavonoids and steroids in
H. auriculata while, quinones and tannins were absent.
Kumar et al. (2018) reported that flavonoids, sugar and phenols contents in dialyzed
protein extracts of Coleus aromaticus were lower than that found in its chloroform and
ethyl alcoholic extracts.
Arulmozhi et al. (2018) reported cardiac glycosides, tannins and Steroids in ethyl
acetate extract of Capparis zeylanica.
Behera et al. (2019) screened twenty medicinal plants for different phytochemicals
and revealed that Gymnema sylvestre and Andrographis paniculata contain maximum
classes of plant secondary metabolites.
2.3.7. Elemental Analysis
Raju et al. (2016) evaluated the leaves of Cassia fistula and Sphaeranthus indicus
for the presence of inorganic elements. Concentrations of Na, Al, Zn, Ca, K, Mg, Sc, Fe,
V, Br, La and Mn were found. They also reported that the contents of various elements
were present in different proportions. The quantities of elements detected were dependent
29
on the location, climate as well as composition of soil.
Anal & Chase (2016) determined the concentration of trace elements Ca, Cd, V,
Mn, Cu, Mo, Mg, Zn, Cr and Fe in Elsholtzia blanda Bentham, Potentilla fulgens Wallich
ex Hooker, Lycopodium cernuum Linnaeus, Swertia macrosperma C.B. Clarke, and
Valeriana jatamansi Jones, Cynoglossum furcatum Wallich, Thalictrum foliolosum DC.
All of the analysed elements were found in the studied samples. Highest concentration of
Ca and Mg was detected in both the leaf and root. Among trace elements Fe was detected
in highest contents while, Cd contents were present in limit permitted by WHO and FAO.
Teerthe & Kerur (2017) carried out elemental analysis of Punica granatum and
Vitex negundo. They reported highest Ca contents in the studied plants while, Cu, Zn, Mg,
Cr, Al, Mn and K contents were different among these plants. On the other hand, Cd, Si,
Mo, Ti and V were detected in trace amounts.
Bola et al. (2017) investigated trace elements (Se, Mn, Zn, Mg and Cu) in
Cymbopogon citratus, Azadirachta indica and Angelica keiskei. Concentration range was
found between 7.7 to 8.9 mg/kg (Zn), 1.2-1.4 mg/Kg (Cu), 167-190 mg/kg (Mg), 6.5-6.7
(Se) and 1.6-1.8 (Mn) mg/kg.
Sunitha et al. (2018) analysed the leaf of Abroma augusta and reported various
elements in its ethanolic extract, including Cu (copper), Fe (iron), Mg (magnesium), Ni
(nickel), Ca (calcium), Mn (manganese), Pb (lead), Zn (zinc) and Na (sodium).
Dilawar et al. (2018) quantified trace elements in the bark, flower, leaf, stem and
fruit of Moringa oleifera found in D.I. Khan, Laki Marwat and Bannu areas. Analysis was
made through AAS (Atomic absorption spectrophotometer). All studied parts of Moringa
oleifera collected from Laki Marwat were reported with comparatively higher contents of
Cu, Se, Fe and Zn than the other two areas.
Derkach & Khomenko (2018) analyzed the contents of Mn, Cu, Zn and Fe, Pb, Cr,
Cd and Co in Hyperichi herba (St John’s wort), Chamomile flowers and Urtica folia
(nettle) from Ukraine by AAS (atomic absorption spectrophotometer). There was gradual
increase in Mn and Zn. While, decrease was noted in Fe contents of Urtica folia, followed
by Chamomile flowers and Hyperichi herba respectively. Cd concentration was high in
30
Hyperichi herba while Pb concentration was higher in Urtica folia.
Anjum et al. (2019) evaluated elemental contents of Hertia intermedia (Bioss) O.
Ktze, Achillea wilhelmsii C. Koch, Sophora mollis (Royle) Baker, Seriphidium quettense
(Podlech.) Ling, Nepeta praetervisa Rech. F. Peganum harmala Linn and Perovskia
atriplicifolia Benth. C contents were highest in all studied plants, followed by H and K. On
the other hand, moderate quantity of Fe, Cl and Na was detected in these plants.
Saifullah et al. (2019) investigated various elements in Convolvulus leiocalycinus
and Haloxylon griffithii. Highest concentration of H was detected in H. griffithii followed
by K, Na, Fe, Ni, Mn and Cu. In C. leiocalycinus concentration of K was highest followed
by Na, Fe, Cu, Mn and Ni. Whereas, trace amounts of Co, Pb and Cd were found in both
plants.
2.3.8. Nutritional Analysis
Shukla et al. (2016) assessed nutritional contents of Reinwardtia indica leaf. The
carbohydrates, ash, fiber, moisture, protein and fat contents reported were 11%, 2.4%,
12.6%, 29% and11.4% respectively. The nutritional value estimated for 100 g leaves was
403.05 Kcal.
Achi et al. (2017) analysed leaves of Ficus capensis for its nutritional value.
Percent nutritional contents detected were 1.83 (lipids), 104.53 (moisture), 6.31 (protein),
73.77 (carbohydrates), 4.77 (fiber) and 6.65% (ash). Similarly, large contents of Vitamin
A (6.06 ± 0.004%) and moderate level of vitamin B, and E were also found.
Ahongshangbam & Devi (2017) estimated nutritional value of Anethum graveolens
L., Polygonum posumbu Buch.Ham. ex. D. Don, Eryngium foetidum L., Allium hookeri
Thwaites, Zanthoxylum acanthopodium DC., Allium ramosum L., Houttuynia cordata
Thunb., Lepidium sativum L., and Citrus hystrix DC. Range of crude fat contents detected
were 3.182±0.070 to 1.433±0.019% and contents of crude protein were ranged from
34.993±0.303 to 15.035±0.075%. Maximum contents of crude fiber (7.290±0.115%) were
noted for Z. acanthopodium DC.
31
2.3.9. Analgesic activities
Shojaii et al. (2015) investigated the analgesic potential of the Astragalus hamosus
pods extract by using hot plate and acetic acid induced writhing tests. Hydro alcoholic
extract of A. hamosus pods showed significant antinociceptive potential in both models.
Highest activity was noted at doses of 1000 and 700 mg/Kg.Whereas, the ethylacetate
and hexane extracts revealed analgesic potency comparable to morphine in hot plate
method.
Singh et al. (2016) explored analgesic activity of Murraya koenigii Linn. leaf, using
male wistar rats. Significant and dose dependent increase in paw licking latency (hot plate
test) and reduction in writhing numbers (acetic acid induced response) was observed for
aqueous extract.
Waweru et al. (2017b) investigated analgesic effects of Tradescantia fluminensis
leaf ethanolic extract. Models of mice paw licking and acetic acid induced writhings were
used which showed significant analgesic effects of the leaf extract. Kopaei et al. (2017)
studied analgesic effects of Linum usitatissimum L. and reported its significantly higher
analgesic activity at doses of 500 and 200 mg/kg. Hijazi et al. (2017) explored analgesic
activity of Papaver libanoticum ethanolic extract and observed significant analgesic effects
in both tail flick and hot plate method. Kumari et al. (2017b) evaluated leaf extract of
Quisqualis indica Linn. for analgesic effects on wistar rats. The hydroalcoholic extract of
Q. indica Linn showed significant analgesic effect at 200 and 100 mg/kg. Fahmy et al.
(2017) investigated analgesic effects of Terminalia Muelleri Benth. in mice by hot plate
and acetic acid induced writhing models. Significant reduction in number of writhings
(63%) was noted at dose of 400 mg/kg.
Zihad et al. (2018) evaluated Chrysopogon aciculatus for pain reducing effects.
Ethanolic extract of the whole plant significantly reduced the number of acetic acid induced
writhings in mice at doses of 750 and 500 mg/kg. Similarly, analgesic effects were also
observed in hot plate model.
Khanum et al. (2019) revealed pain relieving potential of Wedelia chinensis. They
reported the stem and leaf ethanolic extract of W. chinensis for significantly inhibiting
acetic acid induced writhings in mice.
32
2.3.10. Anti-inflammatory activities
Lucarini et al. (2015) reported anti-inflammatory properties for Gochnatia pulchra
hydroethanolic extract. Mice and Rats were employed in carrageenan induced paw edema
and pleurisy inflammation models. Significant inhibition in inflammation was noted at 100,
250 and 500 mg/kg.
Shaikh et al. (2016) explored anti-inflammatory properties of Terminalia chebula,
Cissus quadrangularis, Terminalia bellarica and Plumbago zeylanica. Significant
inhibitory potential of COX-2 observed was 74.81 % and 73.34 % for the extracts of T.
chebula and T. bellarica respectively.
Manouze et al. (2017) studied the anti-inflammatory effects of the Anacyclus
pyrethrum. Methanolic and aqueous extract of its root tested in ear and paw edema showed
significant anti-inflammatory effects at doses of 250 and 500 mg/kg.
Kosala et al. (2018) reported the anti-inflammatory potential of Coptosapelta
flavescens Korth. The root methanolic extract significantly inhibited the carrageenan
induced edema at doses of 600 and 1200 mg/kg.
Amri et al. (2018) assessed the anti-inflammatory potency of Pistacia atlantica
leaves and observed significant reduction in carrageenan induced paw edema, at doses of
100 and 250 mg/kg after third and sixth hour.
Umeti et al. (2019) explored significant anti-inflammatory potential of C. adansonii
leaf (ethyl acetate fraction). Mah et al. (2019) verified the traditional use of Malaysian
Calophyllum species as anti-inflammatory agents.
33
2.3.11. Antipyretic activities
Alam et al. (2016) compared the antipyretic activity of five medicinal plants. The
ethanolic extract of these plants showed significant reduction in pyrexia at doses of 250
mg/kg (Cymbopogon jwarancusa), 750 mg/kg (Echinops echinatus), 500mg/kg (Fagonia
cretica), 500 and 750 mg/kg (both Panicum turgidum and Alhagi maurorum) in rabbits.
Ashfaq et al. (2016) investigated the anti-inflammatory potential of Acacia jacquemontii
Benth. The methanolic extract of A. jacquemontii root bark showed highly significant
reduction in pyrexia induced by brewer’s yeast, at doses of 100 and 50 mg/kg.
Gaichu et al. (2017) explored the significant antipyretic effects of Ximenia
americana (dichloromethane and methanolic extract) in male Wistar rats at doses of 100
and 150 mg/kg. Hajjaj et al. (2017) evaluated antipyretic effects of Pistacia atlantica,
Matricaria chamomilla L. (MC) and Ormenis mixta L. They reported significant reduction
in fever by aqueous extracts of all these plants in yeast induced pyrexia at dose of 400
mg/kg.
Sultan et al. (2018) determined antipyretic activity of Trachyspermum ammi Linn.
The seed hydromethanolic extract of T. ammi significantly reduced yeast induced Pyrexia
in rabits at doses of 500 and 250 mg/kg.
Pokala et al. (2019) studied and compared the antipyretic effects of Andrographis
paniculata and Vitex negundo. The aqueous leaf extract of both plants were reported for
significantly higher antipyretic activity in rabbits at doses of 800 and 400 mg/kg. While,
V. negundo exhibited faster antipyretic action compared to A. paniculata.
2.3.12. Cytotoxic activities
Dos-Reis et al. (2015) explored low cytotoxicity of the hexane extract of Talinum
paniculatum leaf on BHK21 cell line by MTT assay. Rezk et al. (2015) investigated
cytotoxic effects of Rhododendron species on intestinal mucosa epithelial cells and
epidermal keratinocytes. At doses of 500 μg/mL extracts of all Rhododendron species
except R. hippophaeoides showed negative effects on both types of cells.
34
Akhtar et al. (2016) determined cytotoxic potential of Terminalia citrine in baby
hamster kidney (BHK21) cell line. Both aqueous and ethanolic extracts exhibited 50% cell
viability at doses of 545 and 260 µg/ml respectively. Artun et al. (2016) evaluated
cytotoxicity of some endemic plant species from Anatolia. IC50 values of 293 mg/ml, 265
μg/ml, 2 μg/ml and 427 μg/ml were observed for Cotinus coggygria Scop., Rosa
damascena Miller, Colchicum sanguicolle K.M. Perss and Centaurea antiochia Boiss. on
HeLa cells line. On Vero cells the IC50 values were > 1000 mg/ml (Rosa damascene,
Cotinus coggygria),>1000 μg/ml (Centaurea antiochia) and 454 mg/ml (Colchicum
sanguicolle).
Selvakumar & Sarkar (2017) revealed the toxic effects of poly herbal
formulations on kidney epithelial cells of monkey through MTT assay. Sharif et al. (2017)
assessed cytotoxic effects of Kalanchoe laciniata extract on BHK 21 cell line by MTT
assay. Both, the hydro-methanolic and n-hexane extracts showed cytotoxicity with IC50
values of 638.5 and 321.9µg/ml.
Gotep et al. (2018) evaluated cytotoxic effects of Euphorbia hirta in albino rats and
BHK-21 cells. CPE were increased by rising concentration of the extract from 25 to 200
μg/ml. In Albino rats, E. hirta extract showed toxic effects on kidney and liver.
Zhao et al. (2019) reported that extract of Euphorbiaceae exhibited inhibitory
effects on viability of Lewis lung adenocarcinoma cell in MTT assay.
2.3.13. Antiviral activities
Deshpande and Chaphalkar (2013) determined the antiviral effects of Withania
somnifera (root and leaf), Ficus bengalensis Vad (stems), Azadirachta indica (bark and
leaf), Ocimum sanctum (leaf) and Acacia catechu (bark) and Curcuma longa. The root and
leaf extract of Withania somnifera, leaf of Ocimum sanctum as well as Curcuma loga
(turmeric extract) showed antiviral effects against FMDV in BHK21 cell line.
Daoud & Soliman (2015) documented the antiviral effects of Spirulina platensis on
FMDV (foot and mouth disease virus) and assessed its replication in baby mice and BHK21
cell line. At 50 µg/ml dose of S. platensis extract, titer of FMDV type A, SAT2 and O,
35
showed reduction of 28, 31 and 35.7% respectively.
Younus et al. (2016) investigated the antiviral potential of Morus alba, Azadirachta
indica and Moringa oleifera against FMDV by MTT colorimetric assay. Significant
antiviral activity was observed for Azadirachta indica followed by Moringa oleifera.
However, Morus alba exhibited no antiviral effects.
Shakiba et al. (2018) evaluated antiviral activity of Alhagi maurorum against
FMDV (foot and mouth disease virus) by MTT colorimetric assay. Aqueous acetic acid
and hydro-alcoholic extracts of A. maurorum showed significant anti FMDV effects and
were suggested for further investigation to develop anti FMDV drug. Saher et al. (2018)
studied anti FMDV (foot and mouth disease virus) potential of Calotropis procera extract
by MTT assay. Maximum anti FMDV activity was noted for the leaf methanolic extract.
Besides, the aqueous extracts from root and flowers were also effective against FMDV.
2.3.14. Aflatoxin degradation activities
Velazhahan et al. (2010) evaluated aqueous extract of Trachyspermum ammi (L.)
seed for its aflatoxin degradation/detoxification activity. Highest degradation of AFG1
(Aflatoxin G1) noted was 65%. Significantly higher degradation of 61%, 54%, 46% were
observed for aflatoxin B1, aflatoxin B2 and aflatoxin G2 respectively.
Vijayanandraj et al. (2014) reported the aflatoxin B1 degradation potential for
aqueous leaf extract of Adhatoda vasica Nees. Significantly higher degradation (≥98%)
was observed at 37°C after 24 hours‟ incubation period.
Iram et al. (2016) compared the aflatoxin B1 and B2 degradation activity of Cassia
fistula and Ocimum basilicum. Extract of O. basilicum was highly effective with 90 and
89% degradation of Aflatoxin B1 and B2 respectively.
Velazhahan (2017) suggested plant products as a harmless substitute to be used for
degradation of aflatoxin.
2.3.15. Phytotoxic activities
Shah et al. (2015b) assessed the bark extract of Cornus macrophylla for its
36
phytotoxic effects on Lamna minor plant. Highly significant inhibition of growth was
revealed at 1000 μg/ml. Barkatullah et al. (2015a) reported phytotoxicity of Callicarpa
macrophylla leaf extract against Lemna minor plant and observed FI50 Values of 464.55.
Saadullah et al. (2016) observed the growth inhibitory effect of Conocarpus
lancifolius by using Lemna minor assay.
Alam & Saqib (2017) reported toxic effects of Zanthoxylum armatum on Lemna
minor plant. Z. armatum fruit extract revealed significant herbicidal effects with 90%
growth inhibition at concentration of 1000 μg/ml.
Ayaz et al. (2018a) investigated phytotoxic potential of Chrysophthalmum
dichotomum on Lemna minor plant. 100% inhibition in Lemna minor growth was observed
with chloroform and n-hexane extract concentration of 1000 μg/ml. Ayaz et al. (2018b)
investigated phytotoxic effects of Chrysophthalmum gueneri on Lemna minor. Phytotoxic
activity of 47 and 53 % were reported for the n-butanol and methanolic (80%) fraction of
C. gueneri extract.
Baloch et al. (2019) evaluated phytoxicity of Heliotropium dasycarpum methanolic
extract and reported significant growth inhibition of 100% against Lemna minor L., and
Eichhornia crassipes (Mart.) Solms-laub. While, 75% growth inhibition was found against
Convolvulus arvensis L. and Elymus repens (L.) Gould. Jan et al. (2019) reported moderate
growth inhibitory effects of Tagetes minuta L. on Lemna minor.
2.3.16. Antioxidant activities
Sadeghi et al. (2015) evaluated antioxidant effects of Boerhavia elegans L. by ferric
reducing antioxidant power (FRAP) and 2, 2-diphenyl-1 picryl hydrazyl (DPPH) methods.
Highly significant antioxidant potential was observed for methanolic extract followed by
the aqueous, ethyl acetate and chloroform extracts respectively in all of the studied plant
parts (root, leaf and stem).
Attanayake et al. (2016) found different IC50 ranges for the ferric reducing power
(1.1-26 μM), DPPH free radical scavenging (19.5-245.7 μg/mL) and nitric oxide inhibition
(103-485 μg/ml) for different medicinal plants from Siri Lanka. Jayathilake et al. (2016)
37
explored significant antioxidant potential of Phyllanthus emblica and Coscinium
fenestratum aqueous extracts. They also reported phenols and flavonoids as dominant
antioxidants in the studied extracts.
Neffati et al. (2017) revealed highly significant antioxidant potential of Lavandula
multifidi and Rhus tripartitum with IC50 values of 5.1 and 5.16 μg /ml respectively.
Behera et al. (2018) evaluated Aerva lanata Linn flowers extract for
antioxidant property. Higher antioxidant potency was reported for its methanolic extract
followed by ethyl acetate and chloroform. Whereas, the water extract showed lower
antioxidant activity.
Faitanin et al. (2018) studied antioxidant activity of the ethanolic extract of
Myrciaria strigipes and revealed its significantly high scavenging activity of DPPH free
radical with EC50 value of 61.79 ± 2.97.
Ondua et al. (2019) explored the nitric oxide inhibition and free radical scavenging
potential of Typha capensis. The n-Hexane extact of T. capensis revealed 86% inhibition
of nitric oxide at concentration of 50µg/ml, while its acetone fraction showed significantly
higher DPPH free radical scavenging property with IC50 value of 7.11µg/ml.
38
CHAPTER-3
3.1. Plant morphology
MATERIALS AND METHODS
Morphology of Alnus nitida was studied in its natural habitat following Wallis
(2005) and Evans (2002).
3.2. Ethnobotany
To know the traditional use of Alnus nitida, different areas of district Swat were
visited. Information on its ethnobotanical usage was gathered from the elderly inhabitants
of the visited area. Literature and research papers on ethnobotanical studies of different
areas were also studied. Plant local name, habitat, flowering season, traditional uses of its
different parts, distribution, native countries, synonym, taxonomic position, elevation,
threats, and population trend were studied. Information gathered was then reported with
references.
3.3. Pharmacognosy
Plant Specimens were first identified by Prof. Dr. Muhammad Ibrar and Sir.
Ghulam Jelani, Department of Botany University of Peshawar (UOP) Pakistan. Plant
specimen was then properly pressed, dried and mounted on herbarium sheet and assigned
voucher number (Bot.20151-PUP). It was also identified through Flora of Pakistan and
kept at department of Botany, UOP, Peshawar, Pakistan for future reference.
Parts of the research plant were collected at appropriate times. Macroscopic
features were studied at collection spot. Leaves (L) and Bark (B) were collected in August,
Staminate catkin (SC) and Pistillate Cone (PC) were collected in November. The collected
plant parts were cleaned, washed and then shade dried. These were then powdered in
electric grinder and sealed in bottles, to protect it from moisture as well as deterioration.
Powdered drugs of the plant parts were then used for microscopic studies, microchemical
tests, elemental and nutritional analysis. Fresh and dried samples of leaf were stored and
later used for leaf surface (including epidermal and stomatal) studies.
39
3.3.1. Macroscopy
Macroscopic features of the plant parts were studied on the spot by methodology of
Wallis (2005) and Evans (2002).
The following characteristics were studied for leaf.
i) Size
ii) Duration
iii) Color
iv) Taste
v) Odor
vi) Phyllotaxis
vii) Insertion
viii) Leaf base
ix) Petiole/stipule
x) Lamina
a. Composition
b. Apex
c. Venation
d. Surface
e. Incision
f. Fracture of dry leaf
g. Texture
Each of the following features was studied for Bark, staminate catkin and pistillate cone.
i) Color
ii) Shape
iii) Taste
iv) Thickness
v) Outer surface
vi) Inner surface
vii) Fracture
viii) Fracture surface
ix) Odor
x) Dimension
xi) Texture
40
3.3.2. Microscopy
3.3.2.1. Micromorphology
Most of the structures of the crude drug are destroyed in powder form including
tissues and their arrangement. Therefore, for evaluation of the plant, microscopic features
of the leaf surface were examined under Labomed microscope, fitted with camera using
methodology of Chaffey (2001). Studied features include; epidermal cells, presence and
kinds of trichomes and stomata on leaf surfaces, vein arrangement, stomatal number and
stomatal index, vein islets and vein termination number and palisade cell ratio. These
features were studied by making permanent slides of fresh leaf, following Akbar et al
(2014).
Both (upper and lower) surfaces of dry leaf were also observed under scanning
electron microscope (SEM) for further confirmation and more clear view of the leaf surface
features. For SEM study, method of Alam et al. (2015) was followed.
a) Stomatal Number and Stomatal index
Stomatal number is the average number of stomata observed in one mm2 (square
millimeter) of a leaf epidermis on both upper and lower surfaces. Whereas, stomatal index
is the percentage of stomata to the total number of epidermal cells (Evans, 2002). These
are the most distinctive features used to identify, standardize and characterize the leaf crude
drug.
Procedure
Epidermis was peeled off from both surfaces of a fresh and clean leaf by forceps.
The peels were mounted in diluted glycerin. Observations under microscope were recorded
for the number of epidermal cells and stomata per square millimeter with 100x
magnification.
Types of stomata, presence and types of trichomes were also noted. Stomatal index
was calculated by the following formula (Evans, 2002).
41
IS= Sn/Sn+Ep ×100
Is= stomatal index
Sn= Stomatal number (per sq. mm).
Ep= Epidermal cells number (per sq. mm).
b) Vein islets and vein termination number
Vein islet is the tiny part of leaf photosynthetic tissue, enclosed by eventual
divisions of the leaf veins. The number of those islets in one square mm area of leaf is
called vein islet number (Evans, 2002). The final free ending of the veinlet is called vein
termination and their number in one square millimeter area of leaf is termed as vein
termination number. The range of these values is constant for a species. These values can
be used as an important tool to identify leaf drug.
Procedure
Many small pieces of lamina from base, margins, midway from margins to midrib
and tip of the leaf were taken. For clarity of specimen, the pieces were boiled in
concentrated solution of chloral hydrate in a test tube and placed on water bath. 1mm area
was fixed using stage micrometer and 4mm objective lens of the microscope. Then, stage
micrometer was removed, and the leaf piece mounted in a drop of dilute glycerin on clean
glass slide was focused. Total vein islets and vein termination number observed under
microscope (at power 5x) were counted and noted. For accuracy of data, average results
were calculated from ten different areas (mm2) of leaf (Evans, 2002, Akbar et al., 2014).
c) Palisade ratio
The average number of palisade cells present under the upper epidermal cells of a
leaf is called palisade ratio (Evans, 2002). The ranges of palisade ratios are constant for
species and do not vary with geographical changes. That why, it is used as an analytical
feature to identify, characterize and standardize specific species of plants (Shruthi et al.,
2010).
42
Procedure
Small parts of leaf lamina from region between midrib and margins were cleared
by boiling in concentrated solution of chloral hydrate in test tube (Shruthi et al., 2010).
Leaf pieces were then mounted in a drop of dilute glycerin on glass slide and observed
under light microscope. The microscope was focused as such that the upper epidermal cells
and palisade cells lying under them could be examined at the same time by slight
adjustment. At first, a group of four epidermal cells and subsequently the palisade cells
lying below them were focused by slightly rotating the fine adjustment. Palisade cells
within the epidermal cells, in addition to those which were covered more than half by the
epidermal cells were counted. The palisade ratio was found by dividing this resultant
number by 4 (Evans, 2002; Akbar et al., 2014).
3.3.2.2. Scanning electron microscopy
For Scanning electron microscopy (SEM) samples were attached to Aluminium
stubs. They were coated with gold (30-40nm), dried with CO2 and then examined under
scanning electron microscope, JEOL, JSM-5910 (JEOL, Tokyo, Japan; SEM interface
version 5.05) at 10 and 5KV, following Alam & Saqib (2015); and Aline et al (2013).
3.4. Physicochemical characteristics of powder drugs
3.4.1 Powder drug study
Powdered bark, leaf, staminate catkin and pistillate cone of Alnus nitida were
studied for their odor, taste and color (physical characteristics). For microscopic
examination a pinch of fine powder of each sample taken on a clean glass slide was treated
with water and then with solutions of chloral hydrate and iodine separately. It was
examined under microscope at objective lenses of 10x and 45x. Different structures were
observed with Labomed microscope fitted with camera and photographs were taken. Some
of the microscopic structures were later sketched (Wallis 1985).
43
3.4.2. Ash analysis
The ash analysis of powdered bark, leaf, staminate catkin and pistillate cone of A.
nitida was carried out following Wallis (1985). It included total ash, acid insoluble and
water soluble ash.
Principle
The term ash is used for the inorganic residue left behind ignition of organic
material at very high temperature. Ash varies in composition from original plant material
due to interaction among chemical constituents or volatilization (Jarald & Jarald, 2007).
Ash analysis help to detect exhausted drug material and sandy or earthy adulterants in drugs
(Wallis, 1985).
a. Determination of total ash
Equipment and glassware
Silica crucibles, muffle furnace, dessicators, analytical balance, tongs and burner.
Procedure
2g of powdered sample was taken in an oven dried, clean and flat bottomed silica
crucible (w1). It was heated on bunsen burner to burn the powdered plant sample and make
it free from smoke. The sample was then transferred to muffle furnace. Temperature of
furnace was gradually increased up to 550°C, which was maintained for several hours till
carbon was completely burnt and it turned the sample into greyish or white colored ash.
The furnace was turned off and allowed to cool down up to 100°C. The crucible along with
ash was placed in desiccators for cooling and then weighed (w2). Ash values were
determined by using the following formula (Wallis, 1985; A. O. A. C., 2000). Weight of
crucible (empty) = w1
Weight of crucible (empty) + Ash = w2
Total ash (mg/g) = (w2- w1)
Weight of the sample(g)
44
b. Determination of acid insoluble ash
Samples with variable quantity of ca-oxalate crystals or which may have clay,
lime or sand adulterants are analyzed for acid insoluble ash (Wallis, 1985).
Procedure
25 ml of 10% HCl was poured into a crucible containing total ash of a sample
(obtained as mentioned in 3.4.2.a. above). It was covered with watch glass and gently
boiled on a burner for 5 minutes, 5 ml of hot distilled water utilized to rinse the watch glass
was added to crucible. This liquid was filtered by ash less filter paper (Whatman No. 41).
The ash less filter paper with left over insoluble residue was shifted to its own crucible. It
was dried on a hot plate and then placed in furnace to ignite it till constant weight (w3) at
500 °C. After ignition the crucible was cooled for 30 minutes in desiccator and weighed.
The value of acid insoluble ash in mg per g of air dried sample was calculated as follows
(Wallis, 1985; A. O. A. C., 2000).
Weight of the crucible + ash= w2
Weight of crucible + residue left on ash less filter paper= w3
Acid insoluble ash (mg/g) = w2 – w3
c. Determination of water soluble ash
Water insoluble ash is useful to detect water exhausted materials in samples (Jarald &
Jarald, 2007). Total ash (w2) of the sample (obtained by the above mentioned procedure,
3.4.2.a.) was taken in a pre-weighed crucible (w1). 25 ml of distilled water was added in
it. The crucible was then covered with watch glass and gently boiled on burner for 5
minutes. 5 ml of hot distilled water was used to rinse the watch glass and this water is again
collected in the crucible. The ash less filter paper was used to collect insoluble matter. Filter
paper with insoluble filtrate was then placed in its respective crucible. It was dehydrated
on hot plate and ignited in furnace for 15 minutes at 500 °C. The crucible was placed in
desiccator for about 30 minutes to lower its temperature and then weighed (w3), from
which weight of water insoluble matter was calculated. The weight
45
of water soluble ash was calculated by subtracting the weight of insoluble matter from
total ash weight (Wallis, 1985; AOAC., 2000).
Wt (weight) of the empty crucible= w1
Total ash = w2
Wt of the empty crucible + insoluble ash = w3
Wt of the insoluble ash = w3 - w1 = x mg/g
Water soluble ash = w2 – x = y mg/g
3.4.3 Florescence study
Certain drugs emit different lights under ultraviolet radiation and ordinary visible
light, when treated with specific chemical reagents. This property of materials, called
florescence, is employed to identify and evaluate whole or powdered drugs (Jarald &
Jarald, 2007).
Materials required:
i) Equipment: UV Lamp with different wavelength (254nm, 366nm).
ii) Glassware: Glass slides.
iii) Reagents: NH3 solution, Ethanol, Iodine Solution, 50% HCL, Ethyl acetate, Acetic
acid, 50% H2SO4, Acetone, Butanol.
Procedure
The bark, leaf, staminate catkin, pistillate cone and seed of Alnus nitida and their
extracts as whole, as well as their powder were taken on slide, treated with different
reagents (NH3 solution, Ethanol, Iodine Solution, 50% HCL, Ethyl acetate, Acetic acid,
50% H2SO4, Acetone and Butanol) and observed under both the short and long
wavelengths of UV light and visible day light (Evans, 2002, Akbar et al., 2014).
46
3.4.4. Determination of extractive values
Extractive values show the nature of chemical constituents in crude drugs. These
values help in detection of adulterants or contaminants. Therefore, these are considered
more useful for evaluation of crude drugs. Polarity of solvent is more important for
extraction of chemicals from crude drugs. For instance, alcohols extract tannins and resins
while, fats and oils are extracted in ether (Kokate, 1994). The bark, leaf, staminate catkin
and pistillate cone powder of A. nitida were extracted with ethanol, n-hexane, ethyl-acetate,
methanol and water.
Material required:
Analytical balance, rotary evaporator, funnel, beakers, filter paper, air tight bottle
and different solvents (methanol, n-hexane, water, ethanol and ethyl acetate).
Procedure:
Extractive values were found by dissolving 10 g of each powdered drug in 200 ml
of different organic solvents in airtight bottles for a week with infrequent shaking. Each of
these extracts was filtered. All filtrates were dried in rotary evaporator to a semi solid
residue. The percent extractive value of each sample with different solvent was calculated
as follows (Wallis, 1985; Ansari, 2006).
Extract weight
Percent extractive value= Sample weight
× 100
3.4.5. Elemental analysis
Elemental content of the powdered samples of the bark, leaf, staminate catkin and
pistillate cone were analyzed with atomic absorption spectrophotometer (AAS 700,
Perkin Elmer, USA).
47
Analyzed elements:
Copper (Cu), Iron (Fe), Zinc (Zn), Manganese (Mn), Magnesium (Mg), Sodium
(Na), Calcium (Ca) and Potassium (K).
Materials required:
Reagents:
Nitric acid (HNO3) and Perchloric acid (HClO4) were Merck (Germany) made. Fe,
Zn, Na and Cu were Aldrich made while Mn, K, Ca and Mg were Sigma made. All the
glassware was properly cleaned with distilled water prior to use.
Preparation of sample:
For elemental analysis, samples were prepared by wet digestion technique of Hseu
(2004). 1 g powder of each sample was placed in a conical flask. 10 ml of concentrated
HNO3 (67%) was added and kept at room temperature for 24 hours (overnight). Then 4 ml
of perchloric acid (67%) was added. Flasks were kept on hot plate after 30 minutes. The
contents of flasks were allowed, to evaporate until volume of digested material reached to
approximately 1 ml of clear solution. After cooling, double distilled water was added to
make the final volume of this solution up to 100 ml. The solution was filtered through
Whatman No. 42 filter paper. These filtrates were stored in sealed bottles and then used for
elemental analysis. Each element in all samples was analyzed in triplicate and stock
solutions were properly diluted to prepare calibration standards for elements (Saeed et al.,
2010).
Statistical analysis
Data for elemental content was statistically analyzed using mean of three replicates
and standard deviation (Saeed et al., 2010).
48
Table 3.1. Instrumental conditions for detection of elements.
Elements Wavelength
(nm) Flame type Slit
width
(nm)
Cathode
lamp
current
(mA)
Acetylene
flow
(L/min)
Air oxide
flow (L/min)
Cu 324.8 Air/Acetylene 0.7H 15 2.0 17
Fe 248.3 Air/Acetylene 0.2H 30 2.3 17
Zn 213.9 Air/Acetylene 0.7H 15 2.0 17
Mn 279.5 Air/Acetylene 0.2H 20 2.0 17
Mg 285.2 Air/Acetylene 0.7H 6 2.0 17
Na 589 Air/Acetylene 0.2H 8 2.0 17
Ca 422.7 Air/Acetylene 0.7H 10 2.0 17
K 766.5 Air/Acetylene 0.7H 12 2.0 17
3.4.6. Nutritional (Proximate) Analysis
Plants provide nutrients including carbohydrates, protein and fats, essential for
development and growth of humans and animals (Nisar et al., 2009). The following
parameters were quantified in the bark, leaf, staminate catkin and pistillate cone.
3.4.6. a. Determination of ash
The methodology used for ash determination is mentioned in section 3.4.2.
3.4.6. b. Determination of moisture content
Equipment and glassware:
Electric balance, desiccators, electric oven and petri plate.
Procedure
2 g powder of each sample was taken in a clean and pre-weighted (w1) petri plate,
roofed partially with lid, and was kept at 105°C in electric oven for 5-6 hours to acquire
stable weight and then shifted to desiccators for cooling. After 30 minutes the petri plate
was again weighted (w2). Moisture percentage was calculated as follows (AOAC, 2000).
49
% Moisture =
X
× 100
Sample wt
X = Sample Wt (after heating) = w2 - w1
w2 = Wt of empty Petri plate+ sample wt (after heating)
w1 =Wt of the empty Petri plate
3.4.6.c. Determination of crude proteins
Protein contents (% N x 6.25) in plant samples were measured by using Macro
Kjeldahl distillation method (AOAC, 2003; Gul & Safdar, 2009).
Reagents:
Standard solution of HCl (0.1 N), Conc. H2SO4, Boric Acid (4%), NaOH (40 %)
and digestion mixture containing CuSO4 and K2SO4.
Mixed indicator (Methyl red and bromocresol green indicator):
It is prepared by dissolving methyl red (0.016 g) and bromocresol green (0.03 g)
in alcohol (100 ml).
Apparatus
Digestion and distillation apparatus, Kjeldahl flasks and burettes etc.
Principle:
Powdered samples of the plant were heated for digestion in concentrated H2SO4
and digestion mixture. NaOH was added to make the mixture alkaline. Ammonium
sulphate produced in the mixture liberated ammonia which was passed into the 4% solution
of boric acid and then titrated against 0.1 N HCl. Percent Nitrogen (% N) was multiplied
with 6.25 to calculate the % Protein content.
Procedure:
1 g dry powder of each sample was placed in digestion flask. 15 ml of conc. H2SO4
and 8 g of digestion mixture (CuSO4 and K2SO4 having 8:1 respectively) were mixed with
it and heated for digestion. After 3-6 hours digestion was completed, the
50
cleared (blue green color) mixture was allowed to cool, shifted to volumetric flask and
distilled water was added to increase its volume up to 100 ml. 10 ml of the digest was
transferred to the distillation tube and then 10 ml of NaOH (0.5 N) was also added in it.
This resulted in liberation of ammonia which was collected and converted into NH4OH in
a flask containing 20 ml boric acid solution (4%) with 2-3 drops of mixed indicator. The
formation of NH4OH results in appearance of yellowish color. At the completion of
distillation, the flask was taken out for titration. 0.1 N HCl was taken in burette and the
flask content was titrated against it, till pink color appeared. End point reading was noted.
Similar method was used for the blank. Percent nitrogen and % crude protein were
calculated as follows (AOAC., 2003; Gul & Safdar, 2009).
N % = S - B × N × D × 0.014 × 100 Wt of sample × volume of digest taken for distillation
S= titration reading of sample
B= titration reading of blank
N= normality of HCl
D= dilution factor
0.014=milliequivalent Wt of Nitrogen.
% Protein = % N x 6.25 (Correction factor)
3.4.6.d. Determination of fat (ether extract)
Equipment and glassware:
Soxhlet extraction apparatus, Extraction thimbles (Whatman), Water bath and
heating mantle.
Chemical
Petroleum ether
51
Procedure:
Crude fat was extracted with Soxhlet apparatus (Zarnowski & Suzuki, 2004). 2 g
powder of each sample was put in extraction thimble made up of cellulose. Absorbent
cotton wool was plugged in thimble. Thimble was then kept in extraction tube. 250 ml,
cleaned, dried and pre weighted (W1) flask was filled up to one third, with petroleum ether.
The flask was joined with the thimble containing extraction tube. Soxhlet apparatus was
allowed to run, continuing extraction for 6 hours. Siphoning occurred at condensation rate
of 2-3 drops per second, after every 6-10 minutes. Thimble was separated from extractor.
Flask was allowed to heat on water bath and solvent from extract was evaporated. The flask
was then dried at 105°C for one hour, cooled and re- weighted (W2). Percentage of the
crude fat in each sample was calculated with the following formula (AOAC., 2000).
% Crude fat (Ether extract) = Y × 100
Wt of the sample
Whereas,
Y = Weight of the fats = W2 - W1
W1 =Weight of the empty flask
W2 = Weight of the empty flask + fat
3.4.6.e. Determination of crude fiber
Equipment and glassware:
Muffle furnace, Crude fiber extraction apparatus (Fiber Tec System M. Tecator),
Oven, Electric balance, Suction pump, Gooch crucible, Beaker, Funnel, Cotton cloth or
Filter paper.
Reagents:
Sulphuric Acid (0.255N), Ethyl Alcohol (95%), Asbestos, Petroleum Ether, Sodium
Hydroxide (0.313 N).
52
Procedure:
3 g powder of each sample was placed for drying in oven. 2 g powder from dried
sample was extracted with petroleum ether. Residue of the sample was placed in digestion
flask .0.5 g of asbestos was also added to the flask. Furthermore, 200 ml of hot Sulphuric
acid (0.255 N) was poured in this flask, it was linked to condenser and allowed to boil for
half an hour. This content was then filtered in fluted funnel through linen cloth. The
insoluble matter left on cloth was washed with boiling water to remove acid and again
washed to digestion flask with wash bottle containing Sodium hydroxide (0.313 N). NaOH
solution in flask was raised up to 200 ml. Flask was attached to reflux condenser and the
mixture was boiled for half an hour. Gooch crucible made with mat of asbestos was used
to filter the heated material, which was cleaned with boiled water and Ethanol (15 ml)
respectively. It was transferred to crucible and dried in oven at 100°C till constant weight,
placed in desiccator for cooling and weighed (w1). The crucible with residue was placed
in muffle furnace to ignite (at 550° C) till white, shifted to desiccator for cooling and then
weighed (w2). The amount of crude fiber is calculated from loss in weight as follows
(AOAC, 2000).
Percent crude fiber = w2 - w1 × 100
Wt of the sample
Whereas,
w2 – w1 = Crude fiber
3.4.6. f. Carbohydrates contents
Carbohydrate content in each sample was computed by subtraction of the total
weights of ash, crude fat, moisture, crude proteins and crude fibers from hundred (Merrill
& Watt, 1973).
Percent carbohydrates = 100 – (ash + fat+ moisture + proteins+ fibers)
53
Phytochemistry
3.5. Extraction with organic solvent
1 Kg powder of each sample (Bark, leaf, staminate catkin and pistillate cone) was
separately soaked for about two weeks, in 4 liters of Ethyl alcohol. Each of the soaked
sample was occasionally shaked. Extracts were twice collected from each of the sample.
The steps of filtration and then concentration in rotary evaporator were repeated for each
extract. Combined extracts of each sample were collected in vials, used for phytochemical
analysis and pharmacological studies (Miliauskas et al., 2004).
3.6. Qualitative tests for phytochemical screening
The following preliminary, phytochemical screening tests for the bark, leaf,
staminate catkin and pistillate cone of A. nitida were carried out to investigate the chemical
composition of these plant parts.
3.6.1. Carbohydrates detection.
a. Benedict’s test
Each extract solution (1 ml) and 2-3 drops of Benedict’s reagent were mixed,
placed on water bath for boiling. Appearance of reddish brown precipitate confirm
presence of sugar (Evans, 2002).
Benedict’s reagent
100 and 173 grams of sodium carbonate and sodium citrate respectively were
completely dissolved in 800 ml of distilled water by boiling. An aqueous solution (100 ml)
of 173 grams of copper sulphate was mixed with it.
b. Fehling’s test
Extract solution of each sample was mixed with the same volume of Fehling’s
solutions A and B, and then boiled. Appearance of brick red color precipitates (cuprous
oxide) confirmed the presence of sugar (Evans, 2002).
54
Fehling’s solutions
I. For Fehling’s solution A, Copper sulphate (34.66 g) was dissolved in distilled water
and volume of solution was increased up to 500 ml with distilled water.
II. Fehling’s solution B was prepared by dissolving potassium sodium tartarate (173
g) and sodium hydroxide (50 g) in distilled water and then making its volume up to
500 ml with distilled water (Evans, 2002).
3.6.2. Detection of Proteins & amino acids
Ninhydrin test
Few drops of Ninhydrin solution (0.2%) were mixed with 2ml solution of the
sample extract. Indication of violet colour on boiling show the presence of proteins (Kumar
& Kiladi, 2009).
3.6.3. Alkaloid detection
Hager’s test
2 ml extract solution with 2-3 drops of Hager’s reagent was taken in test tube.
Yellow colored precipitates in solution detect Alkaloids (Tiwari et al, 2011).
3.6.4. Detection of phytosterols and triterpenoids
a. Libermann - Burchard test
Extract solutions taken in test tube were treated with acetic anhydride (2-3
drops). After boiling and cooling, the addition of concentrated sulphuric acid from sides
resulted in a brown ring at joining of the two layers. Change in colour of upper layer into
green indicates sterols while if it turns into deep red color then triterpenoids are present
(Harborne, 1998; Tiwari et al., 2011).
b. Salkowski’s test
Solution of each sample extract in chloroform was taken in a test tube with conc.
Sulphuric acid (3-4 drops), properly shaken and allowed to stand. Appearance of yellow
55
or red color confirms triterpenes or sterols respectively in the sample (Harborne, 1998;
Tiwari et al., 2011).
3.6.5. Detection of phenol
Ferric chloride test
Extract solution (2 ml) was added in a test tube with equal volume of ferric chloride
solution. Test for phenols is positive if dark bluish green color appears (Tiwari et al., 2011).
3.6.6. Detection of flavonoids
Alkali reagent test
Extract solution of each sample and sodium hydroxide were added in test tube.
Appearance of yellow to red precipitates confirms flavonoids in samples (Kokate, 1994).
3.6.7. Tannins
a. Ferric chloride test
Extract solution (2ml) was added with ferric chloride (2ml) solution.
Turning of solution into blue green color indicate tannins in sample (Kokate, 1994).
b. Alkali reagent test
Solution of sample extract with sodium hydroxide (2-3 drops) was taken in
test tube. Appearance of yellowish to reddish precipitates indicates tannins in
sample (Kokate, 1994).
3.6.8. Detection of anthocyanins
Hydrochloric acid test
Solution of extract was treated with 2 N HCl (2 ml), if reddish pink color appears
and turn blue violet on treatment with ammonia then anthocyanins are present (Harborne,
1998).
56
3.6.9. Detection of saponin
Froth test
Diluted solution of extract was vigorously shaken. Formation of froth which
persisted for some time indicates saponins in sample (Tiwari et al., 2011).
3.6.10. Detection of steroidal glycosides
Killaer kilani test
Glacial acetic acid (1 ml), ferric chloride (5%) drop and concentrated sulphuric acid
(1ml) were added in a test tube containing extract solution (2 ml). Reddish brown
coloration at the joining of two layers and blue green coloration in top layer indicate
presence of steroidal glycosides (Harborne, 1998).
3.6.11. Detection of fixed oils
Spot test
Extract of the sample was folded and pressed in filter paper. Oily stain on filter
paper confirm that the sample contain fixed oil (Gomathi, 2010).
3.6.12. Detection of volatile oil
Sample extract when pressed between folds of filter paper left no permanent stain,
indicate presence of volatile oil in sample (Kumar & Kiladi, 2009).
3.7. Quantitative analysis of phytochemicals
Phytochemical studies have revealed a number of phytochemicals in Alnus species.
The contents of phenols, flavonoids and sterols were quantified in present work. Phenols
and flavonoids are the most abundantly found bioactive phytoconstituents in Alnus species
and Alnus nitida is traditionally used to cure inflammation (Ren et al., 2017). Many plants
with anti-inflammatory activity are reported in preclinical and clinical investigation for
having steroid compounds having structural similarity with the anti-inflammatory agent
Glucocorticoids (Patel& Savjani. 2015). Moreover, Steroids have
57
also been isolated from the bark of A. nepalensis, A. glutinosa and A. acuminata; from the
aerial parts A. rugose and pollen of A. glutinosa (Ren et al., 2017) Therefore, the total sterol
contents were also found in A. nitida samples.
Quantitative analysis for phenols, flavonoids, and sterols were carried out in the
bark, leaf, staminate catkin and pistillate cone extract of A. nitida as follows.
3.7.1. Determination of total phenols
Required material
Chemicals and reagents:
Sodium carbonate, Folin Ciocalteu’s phenol reagent and Standard Gallic acid
obtained from PCSIR Laboratory complex Peshawar, Peshawar, Pakistan.
Instrument:
UV Visible Spectrophotometer.
Procedure:
Contents of phenols in extract samples of A. nitida were determined by Folin
Ciocalteu assay using spectrophotometer (Tambe & Bhambar, 2014). 1ml of each extract
(mg/ml) solution and 9 ml distilled water were added in 25ml volumetric flasks
(separately). Folin Ciocalteu phenol reagent (1ml) was added, shaken and kept for 5
minutes. Then, 10 ml of sodium carbonate (7%) solution was also added in it and the
volume was increased to 25 ml by adding more distilled water. Standard Gallic acid
solutions of different concentrations (20 to 100 μg/ml) were prepared by the same method.
All of these prepared mixtures were kept for 90 minutes to incubate at room temperature
(25±2 °C). Absorbances were then measured by UV visible spectrophotometer at 700nm
against reagent blank. Total phenols in each sample were calculated as GAE mg/g (Gallic
acid equivalent milligram per gram) of sample extract using the following equation.
Total Phenols GAE mg/g of extract = C1 × V1 × D M1
58
C1 = Gallic acid conc. (mg/ml) measured from the calibration curve.
V1 = Extract solution volume (ml) used.
D = Dilution made, M1 = Extract weight in g.
3.7.2. Determination of total flavonoids
Materials required
Sodium nitrite, Sodium hydroxide, Aluminium chloride and Standard Quercetin
obtained from PCSIR Laboratory complex Peshawar, Peshawar, Pakistan.
Procedure
Aluminium chloride colorimetric assay was used for determination of total
flavonoid content in extract samples (Kostic et al., 2013; Biju et al., 2014). In 10 ml
volumetric flasks, mixture of 1ml extract solution (mg/ml), distilled water (4 ml) and 300
µl of NaNO3 (5%) was added and flasks were kept for 5 minutes. Subsequently, 300 µl of
Aluminium Chloride solution (10%) was added. After 5 minutes, 1 Molar solution of
NaOH (2ml) was also added and total volume was made up to 10 ml with distilled water.
Standard solutions Quercetin (20 to120 µg/ml) were prepared by the above mentioned
procedure. Absorbances of all these reaction mixtures were measured against reagent blank
at 510 nm wavelength with UV/Visible spectrophotometer. Flavonoid contents were
determined in triplicate for each sample. Total flavonoids calculated from calibration curve
were measured as QE mg/g (Quercetin equivalent milligram per gram) of sample extract
by using the following equation.
Total Flavonoids (QE mg/g) of extract = Cq × Ve × De
Me
Cq = Conc. of Quercetin (mg/ml) calculated from the calibration curve.
Ve = Volume of each extract solution in ml.
De = Dilution of the extract.
59
Me = Extract Wt. in gram
3.7.3. Determination of sterols
Required materials:
Electric balance, filter paper, flasks, water bath, separatory funnel, petroleum
ether, and ethanolic potassium hydroxide solution (10%).
Procedure:
Sample solution was prepared by dissolving 2 g of extract in 75 ml of distilled
water. Addition of 25 ml of potassium hydroxide (10%) solution in it changed chlorophylls
into chlorophyllins (a water soluble salt). Three times extraction of this mixture was carried
out in a separatory funnel with petroleum ether (75 ml). A pre weighted (w1) flask was
used to separate ether fraction. The flask was kept on a hot water bath to concentrate and
dry the ether fraction and then placed in desiccator for cooling. Weight (w2) of the flask
was noted again. Contents of sterol were calculated in all samples by the following formula
(Huang et al., 2010).
Sterols (mg/g) =
SW
Wt of the sample
Percent sterols = SW × 100 Wt of the sample
Whereas,
SW = Sterols Wt (weight) = w2- w1
w1 = Wt of flask
w2= Wt of flask residue
3.8. Pharmacological activities
The therapeutic potential of A. nitida (Spach) Endl. (bark, leaf, staminate catkin
and pistillate cone) was evaluated by pharmacological activities.
60
• All the bioassays carried out in the present work were permitted by the ethical
committee of the University of Peshawar, Pakistan.
• Experimental animals BALB/c mice (either sex), provided by NIH (National
Institute of Health), Islamabad, were supplied standard settings of light (12 hours light
/dark cycles) and temperature (25±2°C) in laboratory, standard foodstuff and water.
One day before experimentation the food supply was stopped but water was still
provided to animals.
3.8.1 Analgesic activity
Material required.
Equipment’s, glassware and chemicals:
Electric balance, Beakers, Test tubes, Stopwatch, 70% Ethanolic extract of the plant
samples. Acetic acid solution (1%). Aspirin was employed as +ve control and sterile
normal saline as -ve control. Normal saline was also employed in preparation of all extract
solutions.
Procedure
Animals were separated in XIV groups (6 animals in each group). Group I was
administered normal saline which served as -ve control. Group II injected with aspirin
(10mg/kg) was +ve control. Group III-XIV was injected with doses 50, 100 and 200mg/kg
i.p. of the bark, leaf, staminate catkin and pistillate cone extracts. After 30 minutes each
animal was injected with 1% acetic acid. Five minutes later, the numbers of writhes
(abdominal constrictions) were counted. Reduction in pain was computed as follows
(Muhammad et al., 2012).
% Reduction in pain=Wc-Wt / Wc ×100
Wc = No. of writhings in –ve control group.
Wt = No. of writhings in tested groups.
61
Statistical analysis
All experimental results were statistically analyzed using one-way ANOVA with
Turkey’s multiple comparison test. Differences at p<0.05 were considered significant.
Graph pad prism software (version 6.01) was employed for statistical analysis.
3.8.2. Anti-inflammatory activity
Material required.
Equipment and glass ware
Electric balance, beakers, test tube, graduated cylinder. Plethysmometer (LE 7500
Plan Lab S.L, Italy).
Chemicals
Ethanolic extract of plant samples, Diclofenac sodium (Suzhou Ausun Chemical
Co, Lit.,China), Carrageenan (Sigma Lambda, USA) as +ve control. Sterile solution of
normal saline as -ve control and solutions of all extract in normal saline.
Carrageenan-induced paw edema test
The carrageenan induced hind paw edema assay (Winter, 1962), was used to
investigate anti-inflammatory activity of the A. nitida extracts.
Mice (25-30g) were used for the bioassay. Normal paw volume (NPV) of all mice
was measured. Fourteen groups of mice (each with 6 mice) were made. Group I (negative
control) was treated with 0.2ml of normal saline. Group II (positive control) was injected
with 10mg/kg of diclofenac sodium, Group III-XIV with Alnus nitida bark, leaf, staminate
catkin and pistillate cone extracts (50,100 and 200 mg/kg of each extract). 30 minutes later,
carrageenan (0.05ml; 1%) was injected subcutaneously in right hind paw of mice into
subplantar tissue. Plethysmometer (LE 7500 Plan Lab S.L, Italy) was used to measure paw
edema immediately after carrageenan injection and then after each hour till 5th hour (from
1st to 5th hour respectively). Percent inhibition of edema and edema volume of paw was
calculated as follows (Afsar et al, 2015).
62
% Inhibition of edema = Edc- Edt / Edc ×100. Ed= PVA-PVI
Whereas,
Edc = Edema volume of -ve control, Edt = Edema volume of test.
Ed = Edema volume, PVA = Paw volume following treatment with carrageenan
and PVI = Paw volume before treatment with carrageenan.
Statistical analysis
All the results were expressed as mean±SEM (n=6). Data was statistically analyzed
by using ANOVA and Dennett’s post hoc test was carried out for multiple comparison.
Results of anti-inflammatory activity were considered significant at
*p<0.05 and highly significant at **p<0.01. Data was statistically analyzed by Graph pad
prism (version 6.01).
3.8.3. Antipyretic activity
Ethanolic extract of A. nitida bark (B), leaf (L), staminate catkin (SC) and pistillate
cone (PC) were evaluated for antipyretic potential by following Barkatullah et al. 2013).
Material required.
Electric balance, ethanolic extract of plant samples, brewer’s yeast, thermometer,
beakers test tube, graduated cylinder, normal saline solution (0.9%) as -ve control,
paracetamol as +ve control. All extract solutions and different doses were also prepared in
normal saline.
Procedure
25-30 g 0f BALB/c mice (either sex) adapted to standard laboratory conditions for
two weeks, were separated into fourteen groups (6 mice in each). Mice were deprived of
foodstuff overnight before experiment, but, were having free access to drinking water
(Taesotikul et al., 2003). Digital thermometer was employed to note normal body
temperature (T) of all animals. Aqueous suspension of the brewer’s yeast (20%) in saline
63
solution (0.9%) was injected in animals subcutaneously at dose of 10 ml/kg, to make them
hyperthermic (Patra et al., 2009). 18 hours after injection of brewer’s yeast, the rectal
temperature of animals was again recorded. Animals that showed greater than 1.2°C
increase in rectal temperature were arranged in XVI groups (each group with six animals).
Groups (Gp) of animals were subcutaneously injected with specific extract doses.
Gp-i Treated with saline water (-ve control Gp)
Gp-ii Treated with paracetamol (150 mg/kg) (+ve control Gp)
Gp-iii Treated with B (bark) 100 mg/kg
Gp-iv Treated with B 200 mg/kg
Gp-v Treated with B 300 mg/kg
Gp-vi Treated with L(leaf) 100 mg/kg
Gp-vii Treated with L 200 mg/kg
Gp-viii Treated with L 300 mg/kg
Gp-ix Treated with SC (staminate catkin) 100 mg/kg
Gp-x Treated with SC 200 mg/kg
Gp-xi Treated with SC 300 mg/kg
Gp-xii Treated with PC (pistillate cone) 100 mg/kg
Gp-xiii Treated with PC 200 mg/kg
Gp-xiv Treated with PC 300 mg/kg
Rectal temperature of each animal was recorded after 1,2,3,4, and 5 hours of the dose
administration.
% Reduction in temperature was calculated as follows.
% Reduction in temperature = (Ty- tn) ×100 (Ty-T)
T= normal temperature
Ty= Temperature after injection of yeast
64
tn = rectal temperature of each group after 1, 2, 3, 4 and 5 hours (Taesotikul et al. 2003).
Statistical analysis
Results were expressed as mean ±SE. ANOVA (one way) with Turkey’s multiple
comparison test was used for evaluation of results with *p<0.05 considered significant
(Barkatullah et al., 2013).
3.8.4. In vitro cytotoxic activity (Mosmann et al., 1983)
In vitro cytotoxicity of A. nitida sample extracts was evaluated on BHK21 cell lines
by microscopic observation of cytopathic effects (CPEs) and using MTT (3-(4, 5-
dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay (Annex c, ISO,
109993-5: 2009). Lab. facilities for research work, BHK21 cells and medium were
provided by FMD (foot and mouth diseases) research center of VRI (Veterinary research
institute) Peshawar.
Principle of MTT assay
The dehydrogenases and reducing agents of only metabolically active cells (viable
cells) reduce the yellow MTT, turning it into a violet blue formazan product which is
insoluble in water. Culture medium is removed, formazan deposits are solubilized in
DMSO and colorimetrically assessed (Mosmann, 1983). As MTT reduction occur only in
metabolically active cells. Therefore, formazan product increases with increased number
of viable cells and decrease in formazan production indicates cytotoxicity of the tested
samples.
65
Materials and methods
Equipments / instruments:
Inverted microscope, Electric balance, Vortex mixer, Biosafety cabinet (II),
Incubator with 5% CO2, Refrigerator (4°C). ELISA microplate reader, Pipettor for glass
pipette, single and multichannel micropipettes.
Glass/ plastic ware:
Syringe filters (0.2-0.45µm pore size), Micropipettes, 96 Well plate, blue and
yellow tips, beakers, cell culture flasks (angled neck), waste container, glass pipettes, petri
dishes, sealing tape, plastic racks, tissue culture tubes, Eppendorf tubes.
Chemicals and reagent
GMEM (Glasgow Minimum Essential Medium), Nystatin, Streptomycin,
Penicillin, Amphotericin, Sodium bicarbonate, Phosphate buffer saline (PBS), Trypsin,
MTT (3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide), DMSO, Ethanol,
Fetal bovine serum.
Cell culture
BHK21 (Baby hamster kidney 21 fibroblast) cell line of ATCC (American type
culture collection), obtained from FMDRC (foot and mouth diseases research center) of
VRI (Veterinary research institute), Peshawar were cultured in GMEM (Glasgow
Minimum Essential Medium), supplemented with nystatin, streptomycin, penicillin,
amphotericin, and sodium bicarbonate, adjusted PH was 7.2 – 7.4.
MTT solution
Fresh MTT solution was prepared in PBS (Phosphate buffered saline), stored in
dark at 4°C, filtered before use through 0.22µm syringe filter and used within 2 weeks.
66
Preparation of Extract solutions
Plant extract stock solution (30mg/ml) was made in 1% DMSO (Dimethyl
sulfoxide) and filtered in biosafety cabinet through 0.2µm membrane filter and collected
in sterile eppendorf tubes. Diluted working solution (1000μg/ml) from the stock solution
and its further 2-fold serial dilutions (1000 μg/ml to 31.25 μg/ml) were made using GMEM
medium.
Procedure
Cytotoxicity of the extract at each concentration was evaluated by following Bisht
et al., (2014) and Mosmann (1983). BHK21 cells with fresh GMEM media, were cultured
(2×105 cells per well) in 96 well culture plate. All plates were kept for incubation at 37°C
in incubator (with 5% CO2) for 24 hours. Plates were observed under inverted microscope
for cells growth (confluency). On minimum 80 % confluency in wells, exhausted media
was removed with multi-channel pipette. Then 200 µl of each extract dilutions (1000, 500,
250,125, 62.5 and 31.2 µg/ml), prepared on the spot with fresh GMEM medium (with 3-
5% serum for maintenance / stationary phase cells in 80 to 90% confluent wells; 10% serum
for proliferating cells in 50% confluent wells) were added in each well, labelled, and
incubated with 5% of CO2 incubator for 2 to 3 days at 37°C.
1) Microscopic observations of cytopathic effects (CPEs) of extracts on cells.
Cytopathic effects were observed under inverted microscope, for 2 to 3 days after
incubation and scored as; 76 to 100% CPE = 4, 51 to 75% CPE= 3, 26–50% CPE = 2, 0–
25%= 1, and 0% CPE = 0 (Serkedjieva & Ivancheva, 1999; Zhao, 2014).
2) MTT colorimetric assay.
10µl of MTT (5mg/ml solution in PBS) was mixed in each well and plates were
kept for 4 hours in incubator with 5% CO2 at 37 °C. Then 100 µl of DMSO was mixed
properly in each well to dissolve crystals of formazan. Absorbances of the wells were
67
recorded at 570 nm (with 630 nm as reference wavelength) using ELISA plate reader
(multiwell microplate reader, Thermo Scientific USA). Control sets containing only
medium and medium with cells were run under the same condition as blank and negative
control for cytotoxicity. The smaller the number of Formazan, the less intense is the dye
color, which results in lower OD (Optical density) values. That is, a large number of cells
were metabolically inactive (dead), which showed the cytotoxicity of sample extracts
when compared to control. % Cell viability and % cell inhibition (cytotoxicity) were
calculated by using the given formula.
% Cell viability = Mean sample absorbance - mean blank absorbance × 100 Mean control absorbance - mean blank absorbance
% Cell inhibition= 100-% Cell viability
Statistical analysis
Each sample concentration in triplicate was analyzed resulting in mean ± SD values
of % cell inhibition, which was plotted against log concentration as dose response curve
and IC50 values were computed by Graph pad prism (version 6.01).
3.8.5. Antiviral Activity
Antiviral potential of B (bark), L(leaf), SC (staminate catkin) and PC (pistillate
cone) extracts of A. nitida were evaluated against FMD (foot and mouth disease) virus.
Materials
Virus used:
Asia -1, FMD (Foot and mouth disease) virus strain was obtained from viral seed
bank maintained at Foot and mouth disease research center (FMDRC), Veterinary research
institute (VRI) Peshawar, Pakistan. Other materials needed were same as mentioned above
in section 3.8.4.
68
MNTC (maximum nontoxic concentration)
To find antiviral potential of A. nitida plant samples on FMD virus, the maximum
non-toxic concentrations (MNTC) of the extracts were determined, which are not toxic to
BHK 21 cell line so that virus can be cultured in it. Based on the in vitro cytotoxicity result
of sample extract, 5 serial dilutions (2-fold) were prepared for each extract (15.6, 7.8,3.9,
1.95 and 1 µg/ml).
For determination of MNTC same procedure was used as mentioned above in
section 8.3.5. Cells were observed under inverted microscope, doses with no cytopathic
effects were used for antiviral assay.
Preparation of viral dilutions
0.9 ml GMEM media (with 5% serum) was added to ten sterile eppendorf tubes.
0.1ml of the virus was added to 1st tube, properly mixed. Then 0.1ml of the 1st tube was
shifted to the 2nd tube and mixed properly. From 2nd tube 0.1 ml was transferred to third
and so on up to the 10th tube. So, ten viral dilutions 10-1 to10-10 made by 10- fold serial
dilutions were used for determination of TCID50 (dose of virus that will produce
cytopathic effects in 50% of the seeded cells).
TCID50 (Tissue culture infective dose 50) calculation
Infectious titer of the FMD virus was determined, that can cause cytopathic effects
(CPE) in 50% cells in cell culture for 2 to 3 days while cells remain viable. Tissue culture
induced infective dose is calculated to quantify infectious virus in a solution.
Procedure
100 µl BHK21 cells (1×105 cells per ml) in GMEM medium (with 10 % serum)
were seeded in 96 well plates, incubated at 37°C in CO2 (5%) incubator for 24 hours. Then,
observed under inverted microscope, plates with 50% confluency were selected. Old media
was removed and100µl from each dilution of FMD virus (in GMEM with 5% serum) were
added in 5 wells (i.e. 5 replicates for each dilution of virus). Virus dilution was not added
to control wells (5 replicates). Plate was covered with lid, kept in
69
incubator with 5% CO2 at 37°C and observed for cytopathic effect for next 48 hrs. Wells
with cytopathic effect (indicated virus infecting cells) were marked. Number of wells
showing CPE (cytopathic effects) for each virus dilution was noted and TCID50 was
calculated as follows (Nadgir et al, 2013; Reed & Muench, 1938).
TCID50/ml = 10(pd-A)
B
Where,
Pd=proportional distance, A = log dilution for CPE greater than 50%, and B = volume
(ml) of virus added (100µl).
Pd = (% of wells with CPE above 50% - 50%)
(% of wells with CPE above 50%) - (% of wells with less than 50% CPE).
CPE noted include swelling (indicating virus replication), detachment of cells from
flask wall, rounding, floating of cells in culture medium, apoptosis and lysis.
Antiviral assay
Antiviral potential of the non-cytotoxic concentration of the plant extracts were
tested against the FMDV (Serotype, Asia 1) by observation of cytopathic effects inhibition
in BHK21 cell line under inverted microscope (Chungsamarnyart et al., 2007) and MTT
assay (Serkedjieva & Ivancheva, 1999; Gupta et al., 2010).
MTT Assay
Procedure
i. BHK 21 cells (1×105) in GMEM (with 10% serum), were seeded in each well
of a 96 wells microtiter plate and incubated for 24 hours at 37°C with 5% CO2
in incubator.
70
ii. After 24 hours, cells growth and confluency were observed under inverted
microscope. When plates were more than 80% confluent, they were kept in
biosafety cabinet for further treatment.
iii. Then 10 TCID50 dose of the virus was mixed with the non cytotoxic
concentrations of each extract, incubated for 1 hour at 37 °C (with 5% CO2).
iv. Exhausted media was taken out from the BHK21 cells cultured in 96 wells plate,
mixture of extract and virus (step iii above) was added in each well (3 replicates
each concentration) in GMEM maintenance medium (with 5% serum). Then,
Kept in incubator at 37°C for 48-72 hours.
v. Growth medium without extract and virus suspension were added to cell
cultures as controls for the cell and virus respectively. Cells were observed for
cytopathic effects under inverted microscope every 24 hours.
vi. After third day 10 μl of MTT (5mg/ml) was added and incubated for 24 hours
at 37 °C in 5% CO2. Then medium was removed, 100μl of DMSO was mixed,
tapped for shaking, incubated for 20 minutes and then kept in microplate reader
to determine optical density at 570nm.
Percentage protection was determined as follows:
(ODS)V - (ODC)V / (ODC)M - (ODC)V ×100%
Where, (ODS)V = Absorbence of virus infected cells with sample extract
(ODC)V = Absorbence of virus infected cells without sample extract
(ODC)M = Absorbence of cells without infection of virus.
3.8.6 Aflatoxin degradation Activity
AFB1 content was qualitatively identified by visually comparing intensity of
fluorescence of the samples with AFB1 standard spots using standard method of
Association of Official Analytical Chemist (AOAC, 2000).
71
Materials required.
Electric balance, test tubes, graduated cylinders, beakers, micro-pipettes, micro
syringes, TLC plate, Sample concentrator, Autospotter, Developing tank, Viewing
chamber with long UV lamp (365 nm).
Chemicals
Ethanol, Chloroform, Aflatoxin standard solution, Benzene: Acetonitrile (98:2)
spot sols AF (AOAC), Developing solvent Chloroform: Xylene: Acetone (60:30:10),
sulphuric acid (50%) solution.
Preparation of extract solution and dilutions
Stock solutions (1000 ppm) of the, bark, leaf, staminate catkin and pistillate cone
were prepared by dissolving 10mg of the extract in ethanol (10 ml). From which 500 µl
and 100 µl were taken and diluted by adding in 500µl and 900 µl of ethanol making their
final concentration 500 ppm and 100 ppm respectively.
Spiking of extract dilutions
The concentration of Aflatoxin B1 Stock solution was 2.06 µg/ml. 10 µl of this
standard stock solution was added in 900 µl of the bark, leaf, staminate catkin and pistillate
cone extracts (having 100 ppm, 500ppm and 1000 ppm concentrations) and total volume
was made up to 1ml by adding the same extract solutions with micro syringe. So, final
aflatoxin concentration in each extract solution was 20.6 ppb (20.6 ng/ml).
Preparation of standard dilutions
5 dilutions of aflatoxin standard (18.54ppb, 14.42ppb, 12.36ppb, 10.3ppb and
7.21ppb) were made by adding 9, 7, 6, 5 and 3.5 µl of standard solution with final volume
of 1ml.
Sample preparation for TLC (Thin layer chromatography):
These extract solutions in test tubes were then kept for 48hrs incubation. After 48
hrs 1 ml chloroform was added in each of these test tubes and kept in sample concentrator
72
at 60°C under N2 stream to evaporate chloroform fraction. After drying of test tube samples
200µl of Benzene: Acetonitrile (98:2) spot sols AF (AOAC) were added in it and then kept
on vortex mixer to dissolve the residue in solution. 20 µl of these sample and standard
solutions were spotted on TLC plate through autospotter.
Spotting and Development of TLC
Small and uniform spots of samples and standard solutions were directly applied
on the TLC plate one after another, at equal distance so, that a justified comparison can be
made between samples and standards by passing them through same developing conditions
on plate. For visual estimation, 5 spots of aflatoxin standard with different concentrations
were used, so that the sample spot aflatoxin concentration fall within the range of increasing
aflatoxin concentrations of standard spots. Developing solvent, Chloroform: Xylene:
Acetone (60:30:10) was poured in developing tank. Solvent level was kept below the
spotting line. Spotted TLC plate was kept in developing tank. The plate was then observed
under long wave UV light (365 nm wave length) in viewing chamber.
Interpretation and calculation
Aflatoxin B1 (AFB1) is detected under UV light by its characteristic (blue)
fluorescence because it has strong UV absorbing ability. This energy is then emitted as
fluorescent light and help in aflatoxin detection in samples. The spots of aflatoxin from
extracts and standards were matched visually by (blue) color and intensity to detect and
quantify aflatoxin concentration (FAO, 1990). Aflatoxin B1 was also identified by spraying
sulphuric acid (50%) solution on developed TLC plate (AOAC, 2000). Percent AFB1
degradation was calculated by the following formula:
% AFB1 Degradation = (CAFB1 - SAFB1 ) ×100 CAFB1
SAFB1= Concentration of AFB1 recovered in extract treated samples
73
CAFB1= Concentration of AFB1 in control (i.e. AFB1 standard A having same concentration
as spiked in sample containing extracts).
3.8.7 Phytotoxic Activity
The phytotoxic potential of aqueous ethanolic extract (70%) of the A. nitida bark,
leaf, staminate catkin and pistillate cone were evaluated on Lemna minor plant, following
Atta-Ur-Rahman et al, (2001).
Materials required
Lemna minor plant, flasks, distilled water, micropipettes, glass vials, plant samples
extracts, filter paper, oven, beakers, laminar flow hood, brush, E- medium and Atrazine
etc.
Media preparation
Mineral nutrients required in different proportions in preparation of the E- medium
were weighed, added in distilled water and dissolved volume of the E- medium was made
up to 1000 ml. KOH was added to adjust pH between 5.5-6 (Table.3.2).
Procedure
From stock solution (1g/100ml) of each sample 100, 1000 and 2000 µl of the
extract was transferred to cleaned, dried flasks (3 replicates of each), solvent was
evaporated overnight in laminar flow under sterile conditions. 20 ml of E- medium was
transferred in each of the flasks, extract present in its bottom was again dissolved in it so
that the final concentration of extracts in flasks became 50, 500 and 1000µg/ml. Negative
and positive controls were also prepared by addition of E medium and atrazine (standard
drug) respectively to other flasks (3 replicates for each). Ten plants of lemna minor having
2-3 fronds were transferred to each of these flasks, kept under conditions of about 12 hours
day light and daily observed. On the third and seventh day fronds number was recorded. %
growth inhibition was determined compared to negative control as follows.
74
% Inhibition in growth = 100 – ( FS ) × 100 FC
FS = No. of fronds in sample
FC = No. of fronds in –ve control
Statistical analysis. The percent phytotoxicity of each sample was measured from
Mean±SEM of three replicates. One way ANOVA was used to find significance (p<0.05)
with Graph pad prism version 6.01.
Table. 3.2. E- medium composition
S.No. Mineral nutrient Concentration (mg/L)
1 KH2PO4 (Potassium dihydrogen phosphate) 680
2 FeCl2.4H2O (Ferric chloride) 5.40
3 KNO3 (Potassium nitrate) 1515
4 ZnSO4.5H2O (Zinc sulfate) 0.22
5 Ca(NO2)2.4H2O (Calcium nitrate) 1.180
6 CuSO4.5H2O (Copper sulfate) 0.22
7 MgSO4.7H2O (Magnesium sulfate) 492
8 EDTA (Ethylene diamino tetra acetic acid) 11.20
9 H3BO3 (Boric acid) 2.86
10 Na2MO4.2H2O Sodium molybdate 0.12
11 MnCl2.4H2O (Manganous chloride) 3.62
3.8.8 Antioxidant Activity
In present work the DPPH free radical scavenging potential of the bark, leaf,
staminate catkin and pistillate cone of A. nitida extracts was measured to evaluate the
antioxidant potential of these plant parts, following Kato et al., 1988.
75
Required materials
Spectrophotometer, Micropipettes, Electric balance, Incubator, Ethanol, Methanol,
Test tubes, DPPH radical and Plant extracts etc.
Procedure
Stock solution of each plant sample was prepared by dissolving 1 g of the extract
in 100 ml of methanol. Then diluted solutions of different concentration (20 µg/ml,
40µg/ml,60µg/ml,80µg/ml,100 µg/ml and 120µg/ml) were prepared from stock solution of
each sample. 1ml from each of these solutions was added in a separate test tube. 2ml of
freshly prepared DPPH solution (0.003%) was also added in the test tubes. Control was
prepared by the addition of 2 ml DPPH solution with 0.9 ml of methanol and 0.1 ml of 50%
ethanol. All of these mixtures were kept for 30 minutes in dark and then absorbance was
recorded by Optima UV Visible Spectrophotometer at 517nm. Blank was prepared by
adding 2.9 ml of methanol with 0.1 ml of the plant extract for neutralization of the extract
color. Antioxidants present in extracts changes the purple color of DPPH to yellow by
reducing it. Antioxidant potential was determined as follows.
% Scavenging activity of DPPH = AC-AS × 100 AC
AC =absorbance of control
AS= absorbance of sample
Statistical Analysis
% Scavenging activities of all samples were calculated using Mean ± SE (3
replicates each). ANOVA (One way) with Tukey’s multiple comparison test was used to
find significance at p<0.05. A curve of percent scavenging was plotted against
concentration and IC50 values were calculated using graph pad prism version 6.01.
76
CHAPTER -04
RESULTS AND DISCUSSION
4.1. Morphology of Alnus nitida
Alnus nitida (Spach) Endl. is a deciduous, large tree, 24- 30 m tall. The shoots are
pubescent when young. But, it becomes glabrescent with age. Leaves size ranges from 5-
15 cm × 3-9 cm, which are ovate to elliptic ovate, pubescent to pilose, villous at veins angle
on under side, acuminate or acute, sub-serrate to serrate, leaf base cuneate to rounded.
Petiole is glabrous to pubescent. Catkins (staminate flowers) are up to 19 cm in length, with
5-6.5 mm long peduncle, bract 1.2 mm long, ovate, smaller and suborbiculate bracteoles.
Taples are spathulate to oblong-obovate 1mm in lengh, with minutely toothed apex and
margin. Anthers1mm in length. Its filaments are scarcely forked and smaller than taples.
Pistillate flowers are woody, erect cones, 3-3.5cm × 1.2 cm; suborbiculate bracteoles with
broadly ovate bract. 2, linear styles. 5-6 mm long fruiting scale with 5 lobes and obliquely
truncate apex. Nut is 2.5 to 4mm in length, fringed by thin and almost leathery wings.
These, descriptions were similar to those described in Flora of Pakistan.
4.2 Ethnobotany
In biological studies, ethnobotany is one of the valuable steps. Such studies preserve
the knowledge of traditional uses of medicinal plants. These also provide reports on plants
with new traditional uses that can cure many ailments. Medicinal plants with higher
ethnobotanical index values are classified as most important for herbal medicines and
clinical practices. Plant species reported for novel treatments can be preserved for reference
in future (Ahmad et al., 2018). The use of medicinal plants has been an old practice, with
transfer of traditional knowledge from one generation to the next. The traditional uses of
herbal medicines by ethnic and rural communities are documented through conduction of
ethno-pharmacological surveys (Vitalini et al., 2013). Reports of these surveys provide
significant information for taxonomists, new drug researchers, ecologists and wild life
managers (Ibrar et al., 2007). Ethnobotanical information for the present research plant
(Alnus nitida) was collected from inhabitants of different areas, where this plant grows
wildly.
77
4.2.1. Ethnobotany of A. nitida
Vernacular Name: Geiray, Sharol.
Part used: Bark, Leaf, Staminate catkin, Pistillate cone, Wood.
Ethnobotanical uses.
In present study, usage of A. nitida in different areas of district Swat (Kabal,
Charbagh, Ahingaro dherai, Shamozai, Ningolai), was the same as reported by earlier
researchers such as fencing, agricultural tools, fuel, roofing, relieving pain, inflammation,
as dye and in construction. Literature survey revealed that in Pakistan, especially in
Khyber-Pakhtunkhwa, similar ethnobotanical uses were reported by many authors from
different areas. Poultice of A. nitida leaves is used to alleviate body pain while leaves
decoction is applied to treat sour feet. This plant is also valuable as soil binder and as fuel
wood (Ilyas et al., 2013). Wood is used in construction, furniture and utensils making,
roofing and fencing (Ahmad et al., 2009). Catkins of A. nitida are sedative and reported as
diuretic and expectorant; catkins are used in cosmetics, while wood is used to make
agricultural tools (Hazrat et al., 2011). For the treatment of diabetes, fresh leaves of A.
nitida are placed in water for a night and half cup of this water is taken before breakfast
(Yaseen et al., 2015b). Flower and wood of A. nitida have medicinal properties and tree is
also used as soil binder (Barkatullah & Ibrar, 2011). A. nitida (Spach) Endl, has been
helpful against scorpion bite (Nasim et al, 2013).
Majority of people depends on wild plants for their livelihood. Over exploitation of
these plants and grazing can disrupt the natural balance of these plants. The ethnobotanical
uses of diverse medicinal plants in Pakistan require further studies to explore, investigate
and verify their efficacy to treat various ailments. Besides, knowledge of ethnobotany will
be helpful to provide new sources of materials from wild; that will provide novel and more
efficient goods for different industries and new sources of income for the local people. The
results of the present work (as will be discussed in the forthcoming pages) on A. nitida
signify the value and importance of this valuable indigenous plant.
78
Table. 4.1. Ethnobotanical uses of A. nitida from literature survey and visited areas
S.
No.
Study
Area
Fencing/
hedging
Fuel Fodder/
grazing
Agricultural
tools
Medicinal
value
Soil
binder
Dye Roofing
1. Kabal + + - + Pain, sour
feet
+ + +
2. Charbagh + + - + - + - +
3. Ahingaro
dherai
+ + - + - + - +
4. Shamozai + + - + - + - +
5 Ningolai + + - + - + - +
6 Literature
survey of A.
nitida
ethnobotany
+ + - + Sedative,
diuretic,
expectorant.
antidiabetic,
cure
Scorpion
bite, Pain
and sour feet
+ + +
79
4.3 Pharmacognosy
Pharmacognosy is the science of medicines derived from natural resources. It
includes the biological, chemical as well as physical features of drugs. It also deals with
the history of drugs, their collection, cultivation as well as commercial use (Gokhale et al.,
2008). Pharmacognosy is concerned with crude drugs derived from plants, animals,
minerals and metals. About 90% crude drugs are of plant origin (Joy et al., 1998).
Pharmacognostic study is very significant for accurate identification of crude drugs in
which standardization of medicinally important plants is more emphasized. Despite,
modern techniques, pharmacognostic procedures are still considered reliable to identify
and standardize a drug (Najafi & Deokule, 2010). In present study bark, leaf, staminate
catkins and pistillate cone (pistillate catkin with seeds) were evaluated for pharmacognostic
features. These include macroscopic and microscopic study as well as physicochemical
studies on powder of the research plant parts.
4.3.1. Macroscopy
Macroscopic features are the main pharmacognostic parameters to correctly
identify a crude drug. The bark (Fig.4.1a, 4.1b, 4.1c), leaf (Fig. 4.2), staminate catkin (Fig.
4.3a, 4.3b) and pistillate cone (Fig. 4.3c, 4.3d) of A. nitida were macroscopically described
to set standard parameters.
Bark of A. nitida was irregular, curved shaped, grayish brown in color with
astringent taste, strong aromatic odor and up to 0.8cm in thickness. Outer surface was
grayish brown, slightly ridged with rough texture; inner side was smooth and yellowish.
Its fracture was short with fibrous, uneven surface and rough, woody texture (Table. 4.2)
80
Table 4.2. Macroscopic features of A. nitida stem bark.
S. No. Feature Observation
1 Shape Irregular, curved.
2 Colour Grayish brown
3 Taste Astringent
4 Thickness Up to 0.8 cm
5 Outer surface Grayesh brown in colour, rough texture,
slightly ridged.
6 Inner surface Yellowish in colour, smooth.
7 Fracture Short
8 Fracture surface Uneven and fibrous
9 Odor Strong aromatic
10 Texture Rough, woody
Leaf of A. nitida has a conspicuous midrib (broad at base and gradually narrows
towards apex), with 10-12 pairs of secondaries. The thickness of secondaries was almost
half to that of the midrib. Tertiaries arising from secondaries form meshes. Leaf often sticky
from resin. Leaf size range was 5-15 cm in length and 3-9cm in width, leaves were
deciduous, upper surface was dark green in color while lower surface was light green, taste
was pleasant, odor was strong and aromatic, leaves arrangement was alternate, ramel, leaf
base was cuneate to rounded with glabrous to pubescent petiole. Leaf lamina was simple
ovate to elliptic ovate with acuminate or acute apex, reticulate and unicostate venation,
surface glabrous but slightly pubescent to pilose near midrib on lower side. Margins of the
leaf were serrate to sub serrate. Dry leaf showed short and smooth fracture and soft texture
(Table 4.3).
81
Table 4.3. Macroscopic features of A. nitida leaf.
S.NO Characteristic Observation
1 Size Length= 5-15cm, Width= 3-9cm
2 Duration Deciduous.
3 Color Upper surface dark green, Lower surface light green.
4 Taste Pleasant
5 Odour Strong aromatic
6 Phyllotaxis Alternate
7 Insertion Ramel i.e. inserted on branches.
8 Leaf base Cuneate to rounded
9 Petiole Glabrous to pubescent
10 Lamina Composition Simple ovate and simple elliptic ovate
Apex Acute or acuminate
Venation Reticulate and unicostate
Surface Glabrous, slightly pubescent to pilose on lower
surface near midrib.
Incision Serrate, sub serrate
Fracture of dry
leaf Short and smooth
Texture Thin, Soft, Herbaceous
Staminate catkins (SC) were narrowly cylindrical in shape, up to 19 cm in length
and 0.5cm in thickness. It has astringent taste, with light pleasant odor and soft texture with
uneven surface, fresh SC are yellowish green which becomes dark brown in dried form
(Table. 4.4).
82
Table 4.4. Macroscopic features of staminate catkin of A. nitida.
S.No. Feature Observation
1 Shape Narrowly cylindrical
2 Taste Astringent
3 Thickness Up to 0.5 cm in diameter
4 Color yellowish green (fresh), dark brown (dry form)
5 Fracture Short
6 Fracture surface Uneven
7 Odor Light pleasant
8 Dimension Up to 19 cm in length and up to 0.5cm in width.
9 Texture Soft
Macroscopic features observed for Pistillate catkin (PC) are presented in Table
4.5. PC of A. nitida was cylindrical cone shaped, with slightly bitter taste and strong
aromatic odor. Its thickness was up to 1.5 cm in diameter and 3-3.5 cm in length. Mature
cones were of brown color with brown seeds; its fracture was short with rough fibrous
uneven surface and texture was hard.
Table 4.5. Macroscopic features of pistillate cone of A. nitida.
S. No. Feature Observation
1 Shape Cylindrical cone
2 Taste Slightly bitter
3 Thickness Up to 1.5 cm in diameter
4 Color Mature cones brown and woody with brown
seeds. (soft, green and closed when young)
5 Fracture Short
6 Fracture surface Uneven and fibrous, rough.
7 Odor Strong aromatic
8 Length 3 to 3.5 cm in length
9 Texture Woody, hard
83
The significance of macroscopic studies of medicinal plants is evident from several
studies, conducted to evaluate macroscopic features of medicinal plants including members
of the genus Alnus and family Betulaceae. Basic et al. (2014) studied A. glutinosa (L.)
Gaertn (black Alder), A. incana (L.) Moench (grey Alder) and their hybrid species called
A. ×Pubescens for the morphometric characters including variability in length (4.8 – 7.4,
4.7 – 7.6 and 4.5 – 7.3 respectively) and width (3.9 – 6.3, 3.0 – 5.57 and
1.0 – 5.9 respectively) of the leaf blade. Our results for A. nitida shows higher range in
length and width of leaf as reported for these species. Mohlenbrock (2009) described the
bark, leaf, staminate catkin and pistillate cone of A. serrulata for almost similar features.
The color of bark, leaf shape and apex; and range in length of staminate catkins and
pistillate cone were different from our present result for A. nitida. However, leaf of A. nitida
showed similarity to the that of A. serrulata leaf in having pubescence at lower surface on
veins (midrib). Alam & Saqib (2015) also used the same macroscopical characteristics to
evaluate the leaf, stem and fruit of Gaultheria trichophylla and determined its
standardization parameters according to the guidelines of WHO. Eom et al. (2011) also
studied Alnus incana subsp. tchangbokii and Alnus incana subsp. hirsuta for almost similar
macroscopic features to compare and differentiate them. The species Alnus incana subsp.
hirsuta and Alnus incana subsp. tchangbokii are reported for cuneate and rounded leaf base
respectively which is common to our observations for the leaf of A. nitida. Sharma &
Kumar (2016) used macroscopic features such as colour, length, arrangement and surface
features for phramcognostic evaluation and standardization of the leaf, stem and flower of
Justicia adhatoda. Mathur et al. (2010) have also used macroscopic features of Bauhinia
Purpurea for its pharmacognostic standardization. Goyal et al. (2011) and Ahmad & Urooj
(2011) studied macroscopic features of bark of Careya arborea and Ficus racemosa
respectively for pharmacognostic standardization. These studies support our course of
work. Our present study will set pharmacognostic parameters for accurate identification of
the bark, leaf, staminate catkin and pistillate cone of A. nitida and will be of great help to
future researchers in the fields of pharmacognosy, pharmacology and phytochemistry.
84
a.
b.
c. d.
Fig. 4.1. a. A. nitida. Bark upper surface; b, Bark lower surface; c, Bark fracture; d, seeds.
85
a.
b.
c
.
Fig. 4.2. a-twig; b-upper leaf surface; c-lower leaf surface of A. nitida.
86
a. b.
c.
d.
Fig. 4.3. a, Fresh staminate catkin (SC) ; b, Dry staminate catkin (SC)
c, Fresh Pistillate cone (i.e. pistillate catkins with seeds, PC); d, Dry pistillate
cones (PC) of A. nitida.
87
4.3.2. Microscopy
Microscopic studies of the leaf surface features (epidermal cells, trichomes, types of
stomata, stomatal number, stomatal index, vein islet number, vein termination number,
palisade ratio) and pollen study of staminate catkin as well as powder drug of leaf, bark,
staminate catkin and pistillate cone were carried out to set pharmacognostic standards for
their accurate identification.
4.3.2.1. Micromorphology of A. nitida
Microscopic studies revealed that epidermis of A. nitida leaf is composed of
irregular shaped epidermal cells with slightly convex or flat walls (Figs. 4.4a-4.4f.). Cells
of both abaxial and adaxial leaf surfaces have variable length (10-40µm and 12-40µm
respectively) and width (5-17µm and 8-17µm respectively) with mean length of 28.4 ±
3.1µm and 28 ± 2.8µm; and mean width 14±1.8µm and 13±1.7µm respectively (Table,
4.6).
Both surfaces are covered with cuticle. Abaxial surface of A. nitida is stomatiferous
with large number of stomata irregularly scattered, having no proper orientation to each
other (Fig.4.5). The stomata of A. nitida are of anomocytic type with striated cuticle.
Cuticular striations are perpendicular around the stomata (Figs.4.6a - 4.6c). Stomata
showed variability in size with small up to 20µm and giant stomata up to 36µm in length
when opened (Figs.4.6a - 4.6b). The normal and giant stomata showed variability in length
(14-20µm and 24-27µm respectively) and width (3-10µm and 7.5-11 µm respectively) of
closed guard cells with mean length 16±1.18 and 26.1±0.85 and mean width of 5±1.04 and
9±0.88 respectively. Variability range of the open pore of normal and giant stomata length
was 12-14.28µm and 14-22µm with mean pore length of 12.8±0.38 and 17±1.9
respectively. Width range for open pore of normal and giant stomata was 2- 2.28µm and
4-6µm with mean pore width of 2.5±0.26 and 4 ±0.5 respectively (Table.4.7).
Non-glandular trichomes were present on both the upper and lower epidermis along
the veins (Fig. 4.4d-4.4e) with length range of 200-300µm and mean length of 253±12.4
µm. The width range of non-glandular unicellular trichomes was 10-16µm with average
width of 12 ±1.3µm (Table.4.6).
88
4-5 celled bases (16-30 µm) of almost rounded glandular peltate trichome with
mean diameter of 21±3.1 (Table.4.6) were found on both adaxial and abaxial surfaces
(Fig4.4b .4.5b…). Peltate glandular trichome with rounded secretory head (60-90µm in
diameter) and mean diameter of 75.5±6.6 µm (Table.4.6), having characteristic orange to
brownish colour were also present on both surfaces, on or near veins (Fig..4.4d).
The present SEM (scanning electron microscopy) study of pollens showed the pore
number range of A. nitida pollens as 4-5, while the dominant pore number was 5 (Fig.4.7)
and arci showed thickenings between pores. Leopold et al. (2012) reported similar results
for pollens of A. nitida. Pollen morphology provides informative patterns for taxonomic
identification which can be used in clarification of evolutionary and biogeographic history
of the genus as well. This information also helps to interpret the record of fossil pollens of
Alnus. The pollen grains of Alnus have unique morphology within Betulaceae as well as
among other angiosperms (Leopold et al. 2012) and pore number is specifically regarded
very significant diagnostic feature (Erdtman, 1943).
Our results for the size range of epidermal cells, hypostomatic leaf, presence of
cuticular striation perpendicular to stomata on abaxial leaf surface and presence of non-
glandular and peltate glandular trichome on both surfaces, are similar to the reports and
descriptions of Worobiec & Szynkiewicz (2007) for the leaf fossil remains of A. gaudinii
(Heer) Knobloch et Kvacek, which was also reported as most similar to A. nitida (Spach)
Endl. by Knobloch &Kvacek (1976) as well as by Mai & Walther (1988).
The giant stomata in A. nitida leaf is at least 20% larger in size (>24µm) than normal
sized stomata (up to 20 µm), solitary, having larger distance from smaller stomata and have
noticeable striae around (Fig.4.6a) or lateral (Fig.4.6c) to the guard cells, which fulfill the
criteria for identification of giant stomata described by Carr & Carr (1990). Boldt & Rank
(2010) have reported presence of giant stomata which is minimum 20% larger than normal
stomata, in some dicotyledonous plants including Alnus maximowiczii Callier ex C. K.
Schneid and Alnus glutinosa (L.) Gaertn. Ascensao et al. (1999) have also reported orange
to brownish coloured, peltate glandular trichome with round head on the surface of
Plectranthus ornatus.
The heads of peltate glandular trichome appear wrinkled (Fig.4.5a.) or smooth (Fig.4.4d),
89
which represent close attachment of the secretory upper cell walls with cuticle, highlighting
outlines of cells. Smooth surface is due to formation of a large subcuticular space by the
cuticle detachment along the outer part of cell wall. The trichome head appear almost
rounded due to trapping of secretion in this space and the diameter of the trichome is about
70 µm (±10) at secretory stage (Ascensao et al. 1999), which is also very close to our
findings. All of these studied features might be used as diagnostic features for leaf
evaluation and in turn help in identification of A. nitida.
Table. 4.6. Leaf surface features of A. nitida
Cuticle Adaxial epidermal
cell
Abaxial epidermal
cell
Non Glandular
trichome
Glandular
trichome
bases
Peltate
glandular
Trichome head
Covering
both
adaxial and
abaxial leaf
surface
Length Width Length Width Length Width of Base
Diameter Diameter
10-
40µm
(28.4±3)
5-17µm
(14±1.8)
12-40µm
(28 ±2.8)
8-17µm
(13±1.7)
200-
300µm
(253±12)
10-16µm
(12 ±1.3)
16-
30µm
(21±3.1)
60-90µm
(75.5±6.6)
Table. 4.7. Stomatal features of A. nitida leaf.
Normal Stomata Giant stomata
Size of closed Guard cells Size of stoma
(open pore)
Size of closed guard cells Size of stoma
(open pore)
Length Width Length Width Length Width length width
14-20µm
(16±1.18)
3-10µm
(5±1.04)
12-
14.28µm
(12.8±0.38)
2-2.28µm
(2.5±0.26)
24-27µm
(26.1±0.85)
7.5-11 µm
(9±0.88) 14-
22µm
(17±1.9)
4-6µm
(4 ±0.5)
90
4.4a 4.4b.
Fig. 4.4a and 4.4b, LM (light microscopic) images of A. nitida leaf abaxial epidermis.
An.st, anomocytic stomata; GT, glandular trichome; Bt, trichome base
4.4c. 4.4d.
Fig. 4.4c, LM images. Closer view of A. nitida leaf adaxial epidermal cells. 4.4d,
adaxial epidermis with glandular trichome (GT).
4.4e. 4.4f.
Fig. 4.4e. LM image of A. nitida leaf adaxial epidermis with non glandular trichome
(NGT) along vein. 4.4f adaxial leaf surface epidermal cells.
91
Fig.4.5a. Scanning electron microscopy image of A. nitida abaxial epidermis. St-Stomata; V-Vein; PGT- Peltate glandular trichome.
Fig. 4.5b. Scanning electron microscopy image of adaxial leaf surface with trichome.
92
a. b.
c. d. e.
Fig.4.6. Scanning electron microscopy images of A. nitida leaf. (a) Stomata, GC- Guard cell; CS-Cuticular
striation. (b) Abaxial epidermis, GSt-Giant stomata, CS-Cuticular striae, NS-Normal stomata (c). GSt-Giant
stomata, CS-Cuticular striae, NS-Normal stomata(d)Stomata, OS- Open stoma. (e) Stomata, GC-Guard cell, Ct-
cuticle, s-stoma.
93
Fig.4.7. Scanning electron microscopy image of staminate catkin powder. P=Pollen; Pr=Pore
a). Leaf surface values
The vein islet and vein termination number for the leaf of A. nitida (Spach) Endl.
were in range of 8-12 (10±0.7) and 5-9 (6.4±0.74) per mm2 respectively. The vein islets
were of polygonal, squaresh or elongated shape and veinlets free endings were simple or
2-3 branched (Fig.4.8). Palisade ratio values were ranging from 5 to 6.75 (5.7±0.32)
palisade cells beneath each upper epidermal cell. Stomata was observed only on abaxial
surface (Figs.4.4b; 4.5a) with stomatal number 130 to 158 (140.4±4.86) per mm2 and
stomatal index of 7 to 8.4 (7.6±0.247) per mm2 (Table 4.8).
Plant taxonomists give prime importance to the epidermal features of leaf to find
the phylogenetic and taxonomic relationship of closely related species (Taia, 2005).
Palaeobotanist have also used stomatal features for reformation of palaeoclimates
(McBlwain & Chaloner 1995). Our results for the shape of vein islets (areoles) and veinlets
branching agree with Worobiec and Szynkiewicz (2007) who reported these features for
the genus Alnus. Pool et al. (1996) have investigated Alnus glutinosa (L.) Gaertn for
stomatal density and stomatal index. Leaves of many other plants, including Malva
parviflora L. (Akbar et al. 2014) and Microtrichia perotitii DC. (Abdullahi et al. 2018)
were studied for vein islet number, vein termination number, palisade ratio, stomatal index
and stomatal number to set peculiar identities for standardization. The variability in values
of these plants suggests that these features can provide significant referential information
for authentication and identification of the leaf crude drugs.
94
Table. 4.8. Leaf constant values of A. nitida.
S.NO. Parameter Range Average
Palisade ratio 5 to 6.75 5.7±0.32
1
2 Vein islets number 8 to 12 10±0.7
3 Vein termination number 5 to 9 6.4±0.74
4 Stomatal number 130 to 158 140.4±4.86
5 Stomatal Index 7 to 8.4 7.6±0.247
Fig. 4.8. Arrangement of veins in lamina of A. nitida leaf.
V.Tr, vein termination; V. Isl, vein islet.
4.4. Physicochemical characteristics of crude drug
The following physicochemical characteristics were carried out for the powder of leaf,
bark, staminate catkin and pistillate cone of A. nitida (Spach) Endl.
V.Isl
V.Tr
95
4.4.1. Powder drug study
a). Leaf powder
Powder of the A. nitida leaf is light green in appearance with pleasant taste and
strong aromatic odor just like that of henna (leaf powder of Lawsonia alba Lam.). Leaf
powder showed glandular and non glandular trichome, fragments of abaxial epidermis with
anomocytic stomata, crystals of calcium oxalate, Uniseriate, long and twisted aduncate type
trichome, anomocytic stomata and simple non glandular trichomes also found in SEM of
the leaf powder (Figs. 4.9a; 4.9b; 4.9c.)
Leaf powder also showed the presence of epidermal cells with palisade cells
attached. Parenchymatous cells patches, Parenchyma cells attached to vessels, grains of
starch, vein fibers attached with parenchymatous cells. Crystals of Ca oxalate under
inverted light microscope (Fig.4.10).
Fig. 4.9 a. Scanning electron microscopy image of powdered A. nitida leaf. St-stomata; NGT-non
glandular trichome; G-glandular trichome. Ct- cuticle; B- trichome (gland) base. Cr- crystal ca-
oxalate.
96
Fig. 4.9b. Scanning electron microscopy image of A. nitida leaf powder. AT- Aduncate non glandular trichome; ST- Anomocytic stomata on abaxial epidermis.
Fig.4.9c. Scanning electron microscopy image of A. nitida leaf powder. NGT- Non glandular trichome.
97
Fig.4.10. Leaf powder of Alnus nitida. a. Upper epidermal cells with attached palisade
cells; b. Parenchymatous cells; c. Parenchyma cells with vessels; d. Starch grains; e. Parenchyma cells attached with vein fibers; f. Ca-oxalate crystals.
Stem bark powder
Stem bark powder is light orange in color, with astringent taste and aromatic odor.
Microscopic study of the powdered bark revealed the presence of phloem fiber bundle, ca-
oxalate crystals, sieve elements, thick walled cork cells, starch granules aggregates of ca-
oxalate crystals, cells of collenchyma, medullary rays attached parenchymatous cells
(Fig.4.11)
98
Fig. 4.11. Bark powder of A. nitida. a. Ca-oxalate crystals; b. Sieve
elements; c. Collenchyma cells with ca oxalate crystals;d. starch grain;
e. fibers; f. Phloem parenchyma with attached medullary rays; g.Cork cells.
99
Staminate catkin powder:
Staminate catkin powder was yellowish green in color with pleasant odor and
astringent taste. Microscopic study of powdered staminate catkin showed fragments of
parenchymatous cells, starch granules, calcium oxalate crystals, fibers (Fig. 4.12) SEM of
the powder confirmed the presence of tetraporate and pentaporate pollens (Fig. 4.7).
Fig. 4.12. Staminate catkin powder of A. nitida .a. Thick walled
parenchyma cells; b. Fibers; c. Calcium oxalate crystals; d. Starch grains.
Fig. 4.13. Scanning electron microscopy image of A. nitida staminate catkin powder. F=Fiber; P=Pollen grain
100
Pistillate cone powder:
Pistillate cone powder was light brown in color, with a slight bitter taste and
strong aromatic odor. Microscopic study of cone powder revealed the presence of fibers,
Sclerenchyma cells, Pitted vessels, patches of parenchyma cells, Ca-oxalate crystals and
starch granules (Fig.4.14).
Fig.4.14. Pistillate cone powder of A. nitida. a. Fibers; b. Sclerenchyma cells; c. Pitted vessels; Ca oxalate crystals; e. Strach grains; f. Parenchyma cells.
The present powder study of bark, leaf, staminate catkin and pistillate cone of A.
nitida, showed the presence of different types of oxalate crystals, fibers, vessels, cork cells,
simple and aduncate type of non glandular trichome, peltate glandular trichome, cork cells,
parenchymatous cells with anomocytic stomata, cuticle covering of epidermal cells and
starch granules.
101
These specific microscopic features in powder of different plant parts provide
important tool for accurate identity, authenticity and standardization of plant based crude
drugs. Similar studies were conducted by other researchers for pharmacognostic evaluation
of different crude drugs such as; Abdullahi et al. (2018) included powder drug study in
pharmacognostic evaluation of Microtrichia perotitii DC. with the objective of establishing
pharmacognostic parameter for addition to pharmacopoeias that can be used for
authenticity and standardization of the leaf drug. Similarly, Jadhav et al. (2018) studied
leaf powder of Lagerstroemia lanceolata Wall to standardize it for specimen identification,
quality and purity.
4.4.2 Ash analysis of the powdered plant parts
The total ash, water soluble and acid insoluble ash values for the bark(B), leaf(L),
staminate catkin (SC) and pistillate cone (PC) of A. nitida were determined in the present
study (Table. 4.9). Highest value of the total ash was calculated for PC (100 mg/g) followed
by L (90 mg/g), SC (87.5 mg/g) and B (70 mg/g). Water soluble ash values were highest
for L (50 mg/g) and PC (50 mg/g) followed by B (20mg/g) and SC (10mg/g).While, the
highest acid insoluble values were noted for SC (70 mg/g), followed by B (45 mg/g), PC
(45 mg/g) and L (30 mg/g).
Ash analysis helps in detection of adulterants or silica and inorganic earthy
materials in drugs. Water soluble ash values reveal the presence of water exhausted
material in powder drug samples, while acid insoluble ash values detect earthy materials
like clay, sand etc. and crystals of calcium oxalate (Wallis, 1985; Rangari, 2002; Jarald &
Jarald, 2007). The present study is valuable for evaluation of the B, L, SC, and PC samples
of A. nitida. Several researchers have carried out similar ash analysis of medicinal plants
for standardization of herbal crude drugs including Bisht et al., 2011; Kumar et al., 2011;
Sarkar, 2017; Nilam et al., 2018; Mehta et al., 2018).
102
Table. 4.9. Ash contents of different parts of A. nitida (mg/g of powder).
S.NO. Powder Total ash
(mg/g)
Water soluble
ash mg/g)
Acid insoluble
ash (mg/g)
1 B 70 20 45
2 L 90 50 30
3 SC 87.5 10 70
4 PC 100 50 45
4.4.3 Fluorescence study
Plants contain different chemical constituents; therefore, they show different
fluorescence when observed under UV (ultra violet) and normal visible light. The
characteristic fluorescence of drugs under UV light is used as valuable tool to authenticate
and standardize crude drugs (Wallis, 1985; Reddy & Chaturvedi, 2010).
In present study fluorescence analysis was carried out in visible light as well as
under UV long (365 nm) and short wave lengths (254 nm). Powdered samples were also
analyzed after treatment with different reagents. Results for powdered samples of the B
and L are presented in Table 4.10; for powders of SC and PC samples in Table 4.11; and
seed powder and extracts of all samples in Table 4.12. The characteristic fluorescence
shown by these samples will help in authentication of the studied plant samples powder as
well as their extracts.
A number of studies have been conducted by other researchers to authenticate the
crude herbal drugs using fluorescence study such as Shrivastava & Leelavathi (2010)
studied leaves of Catunaregum spinose. Kumar et al. (2011) observed leaves of Crocus
sativus and Sarkar et al. (2017) carried out fluorescence study for the leaf powder of
Bauhinia purpurea and Centipeda minim. Fluorescence analysis of the plant samples and
extracts is valuable, fast and easy method to detect adulterants in herbal drug samples.
This method is not only very useful for qualitative evaluation, but to a certain
extent, can also help in quantitative evaluation of powder drugs (Wallis, 2005).
103
Table.4.10. Fluorescence analysis of stem bark (B) and leaf (L) powder of A.
nitida with different reagents.
S.NO. Reagents Visible light UV365 UV254
1 B powder as such Light orange Brown Light brown
2 B Powder + NH3 Orange red Red Orange
3 B Powder + Iodine Red Dark red Orange red
4 B Powder + HCL Whitish brown, Cream Yellowish
Brown
5 B Powder + H2SO4 Dark brown Blackish brown Blackish brown
6 B Powder + Acetic acid Light brown,
Cream
Light pink Yellowish
Green
7 B Powder + Acetone Light brown Light brown Yellow
8 B Powder + Ethanol As powder
Color
As powder
color
As powder
Color
9 B Powder + Butanol As powder
Color
Pink Light orange
10 B Powder + Ethyl
acetate
As powder
Color
Brown Light brown
11. L Powder as such Green Brown Yellow
12 L Powder + NH3 Brown Black Brown
13 L Powder + Iodine Brown Light brown
14 L Powder + HCL Light green Dark blue Yellowish
Green
15 L Powder + H2SO4 Dark brown black Brownish
Green
16 L Powder + Acetic acid Yellowish
Green
Greenish blue Yellow green
17 L Powder + Acetone Dark green Dark blue Dark green
18 L Powder + Ethanol Light green Dark brown Yellowish
Green
19 L Powder + Butanol Light green Orange brown Dark green
20 L Powder + Ethyl
acetate
Light green Blackish
brown
Light green
104
Table.4.11. Fluorescence analysis of staminate catkin (SC) and pistillate cone
(PC) powder of A. nitida with different reagents.
S.NO. Reagents Visible light UV365 UV254
1 SC Powder as such Yellowish
Green
Brown Light yellow
2 SC Powder + NH3 Orange Reddish brown Brown
3 SC Powder + Iodine Yellowish
Green
Dark brown Yellowish
Brown
4 SC Powder + HCL Light brown Blackish
brown
Light green
5 SC Powder + H2SO4 Reddish orange Red Orange brown
6 SC Powder + Acetic acid Light brown Yellow brown Yellowish brown
7 SC Powder + Acetone Yellowish
Green
dark brown Yellow
8 SC Powder + Ethanol Yellowish
Brown
Black Yellow brown
9 SC Powder + Butanol Yellow brown Dark blue Yellow green
10 SC Powder + Ethyl
acetate
Yellow green Dark brown Yellowish
Brown
11 PC Powder as such Brown Light brown Light yellow
12 PC Powder + NH3 Red Dark red Orange red
13 PC Powder + Iodine Brown Black Orange brown
14 PC Powder + HCL Light brown Purple Olive green
15 PC Powder + H2SO4 Orange red Dark red Reddish brown
16 PC Powder + Acetic
acid
Brown Brown Light yellow
17 PC Powder + Acetone Yellow brown Light purple Light green
18 PC Powder + Ethanol Yellow brown Dark brown Yellowish brown
19 PC Powder + Butanol Light brown Purple Yellow green
20 PC Powder + Ethyl
acetate
Yellow brown Pink Yellow
105
Table 4.12. Fluorescence analysis of seed powder (S) and extracts of stem bark (B),
leaf (L), staminate catkin (SC) and pistillate cone (PC) of A. nitida with different
reagents.
S.NO. Reagents Visible light UV365 UV254
1 S Powder as such Dark brown Brown Yellow brown
2 S Powder + NH3 Orange brown Red Orange brown
3 S Powder + Iodine Orange brown Purple Brown
4 S Powder + HCL Orange brown Purple Light brown
5 S Powder + H2SO4 Black red Black Orange brown
6 S Powder + Acetic acid yellow brown Green Yellowish brown
7 S Powder + Acetone Yellow brown Dark green Yellow brown
8 S Powder + Ethanol Yellowish
Brown
Green Light brown
9 S Powder + Butanol Yellow brown Dark green Yellow green
10 S C Powder + Ethyl
acetate
Yellow brown Purple Orange
11 B Extract Dark brown Black Yellowish brown
12 L Extract Black Dark green Light green
13 SC Extract Yellow green Black Olive green
14 PC Extract Olive brown Olive green Yellow green
15 S Extract Brown Black Light green
106
4.4.4. Determination of extractive values
Extractive values of the 10g powder of bark (B), leaf (L), staminate catkin (SC) and
pistillate cone (PC) of A. nitida with different solvents (ethanol, hexane, ethyl acetate,
methanol and water) were determined. Results are shown in Table. 4.13. Maximum
extractive values of 20.5%, 19.8%, 19.7% and 19.5% for sample B, SC, L and PC
respectively were obtained with 90 % Ethanol solvent followed by 15.5% (B), 15.7% (L),
17.1% (PC) and 13.5% (SC) extractive values in Methanol; 11.7%(L), 10.7%(SC),
10.3%PC and 8.5%(B) in solvent Ethyl acetate; 8.7%(PC), 4.4%(SC), 4.2%(L) and 2.4%
(B) in solvent water. Minimum extractive values of 2.3% (SC), 1.8%
(B) 1.6%(L) and 1.5% (PC) were obtained with Hexane solvent.
Extractive values of drugs in different solvents are a useful tool in evaluation of
drugs. It helps to detect adulterants and exhausted materials in crude drugs and assist in
selection of appropriate solvents to get the desired and maximum extractives. Solvents of
different polarity were selected to find extractive values in present work. Based on the
maximum extractive values for each sample 70% ethanol was used for extract preparation
from powdered B, L, SC and PC samples for subsequent research work. Several workers
have determined extractive values for many medicinal plants, suggesting it as valuable tool
to select a suitable solvent for extraction and to find out adulteration in crude drugs as well
(Hussain et al.,2011; Kumar et al., 2011; Nilam et al., 2018; Mehta et al., 2018).
107
Table. 4.13. Percent extractive values of stem bark, leaf, staminate
catkin and pistillate cone of A. nitida with different solvents.
S.NO. Sample Solvent % Extracts
1 B (Bark) Ethanol 20.5
Hexane 1.8
Ethyl acetate 8.5
Methanol 15.5
Water 2.4
2 L(Leaf) Ethanol 19.7
Hexane 1.6
Ethyl acetate 11.7
Methanol 15.7
Water 4.2
3 SC (Staminate catkin) Ethanol 19.8
Hexane 2.3
Ethyl acetate 10.7
Methanol 13.5
Water 4.4
4 PC (Pistillate cone) Ethanol 19.5
Hexane 1.5
Ethyl acetate 10.3
Methanol 17.1
Water 8.7
108
4.4.5. Elemental analysis
Elemental analysis of the B (bark), L(leaf), SC (staminate catkin) and PC (pistillate
cone) of A. nitida were carried out for Cu, Fe, Zn, Mn, Mg, Na, Ca and K, by using atomic
absorption spectrophotometer. Results for these elements are displayed in Table.4.14.
Following were the elements detected in all samples.
Cu (Copper)
Cu contents (ppm) in B, L, SC and PC are presented in Table 4.14. Highest Cu
contents were found in B (40.8±0.003) followed by SC (23.10±.001), L (23.2±0.007) and
PC (21.5±0.005) respectively. Cu is an important nutrient. Many proteins in human body
depends on copper (Huang & Failla, 2000). Cu has a vital role in neurotransmitter
metabolism, oxidation reduction reactions, formation of myelin sheath and connective
tissues as well as energy production (Turnlund, 2006; Harris, 1997; Amina et al., 2003).
Deficiency of Cu affects transport of iron in tissues of body, leading to iron deficiency in
cells. It causes hypochromic microcytic anemia, a condition similar to that triggered by
deficiency of iron (Arredondo & Nunez, 2005).
Permissible limit of the copper content in a plant is 10 ppm set by WHO/FAO.
(Markert, 1994) and RDA (recommended dietary allowance) of Cu for humans is 340– 900
μg /day (Saeed et al., 2010) While, in Singapore and China permissible limit of Cu for
medicinal plants is 150 ppm and 20 ppm respectively. The higher Cu content of the plant
samples suggests their usefulness to cure hypochromic microcytic anemia and other
disorders caused by Cu deficiency (Barkatullah, et al. 2015b). Also, increased copper levels
can result in discoloration of skin, nausea and dermatitis (Maobe et al., 2012). The
present results showed that Cu is present in non-toxic and permissible limits in all of the
studied plant parts.
Zinc (Zn)
Zn content (ppm) was higher in L (38.4±0.001), followed by PC (35.7±0.002), B
(33.8±0.004) and SC (31.9±0.004). Zinc is an essential micro mineral nutrient. As cofactor
of several enzymes, Zn performs significant functions in living cells. In human body above
300 proteins and enzymes depend on zinc. Zn has important role in increasing glucose
109
metabolism, functioning and strengthening of bones, release of hormones, apoptosis
signaling of cells and healing of wounds (Saeed et al., 2010). Zinc deficiency can affect
metabolism of glucose, loss of body weight, vomiting, abdominal pain and diarrhea (Ibrar
et al, 2003; Lokhande et al. 2010; Anonymous, 2001). Recommended daily intake range
of Zn for adult humans is 15 to 25 mg (Prasad, 1982). While, in plants the recommended
limit of zinc set by WHO is 50 ppm (Saeed et al., 2010; Shah et al., 2013). Due to high
concentrations of its zinc contents, the present investigated plant parts may cure skin
diseases, bleeding and may heal wounds (Lokhande et al. 2010; Saeed et al.; 2010; Zafar
et al., 2010).
Manganese (Mn)
Mn contents were higher in L (75.6±0.008) followed by SC (26.3±0.0038), B
(21.4±0.004) and PC (20.5±0.003) (Table.4.14). All of these detected concentrations were
under permissible range for plants (200 ppm). Mn is an essential trace element and Co-
factor for several enzymes. It is needed for normal growth and glucose metabolism. Mn
intoxication leads to Parkinsonism (Ibrar et al., 2003; Wang et al., 2008), while its
inadequate supply cause glycaemia (Donsbach & Ayne, 1982). High Mn concentration in
A. nitida suggests that it can be a better source of Mn for nutritional and curative purposes
(Barkatullah, 2015b).
Iron (Fe)
In present study highest iron contents (ppm) were present in L (438.3±0.0502)
followed by SC (176.5±0.008), PC (139.4±0.009), and B (114.5±0.032). Iron contents in
SC, PC and B were under the permissible limit of Fe in plants (36-241 ppm), except for L
which is very rich in iron.
Iron is an important and abundant trace element in human body (Arredondo &
Nunez, 2005). It is an essential constituent of hemoglobin molecule, responsible for
exchange of oxygen and carbon dioxide between body tissues and lungs. Its deficiency
leads to anemia and affect normal functioning of brain (Sigel, 1978; Beard, 2001).
Deficiency of Fe also causes myocardial infarction, gastrointestinal infection and bleeding
of nose (Hunt, 1994). RDA (recommended daily allowance) range of iron for adult human
110
is 8 to 10 mg/day (Anonymous, 2004). The sufficient Fe content in A. nitida may serve as
a good source of iron.
Sodium (Na)
Sodium contents were higher in L (117.3±0.000) followed by SC (100.0±0.002), B
(99.8±0.003) and PC (97.9±0.004). No, internationally recommended limit for Na content
is available for plants. Its daily recommended intake is 1 to 3.8 mg/day (Anonymous, 2001).
Sodium, a macronutrient is needed for many metabolic processes in human body. Sodium
(cation) regulates irritability of muscles, nerve impulses conduction, membrane potential
and osmotic pressure (Hays; 1985; Murray, 2000; Lokhande, et al., 2010). Table salt is the
most common dietary source of sodium. It is important for excitation and transmission of
nerve impulse (Saeed et al., 2010; Underwood, 1977) and distribution of fluid inside and
outside of the cells (Morris et al., 2008). Its deficiency results in hypotension, dehydration,
muscle cramps, change in mood and fatigue etc. (Harper et al., 1997). In present study
sufficient concentration of sodium in all samples shows that these may be used in disorders
related to deficiency of sodium (Barkatullah, 2015b).
Potassium (K)
Potassium contents of A. nitida were highest in PC (3298±0.005) followed by SC
(3286±0.006), L (2761 ±0.133) and B (787.3±0.0609). The minimum K intake value is
3500 mg/day (Baysal, 2002) and no, international limit has been reported for K contents in
plants. Potassium, a macronutrient for plants and animals has significant role in
metabolism. Potassium has a vital role in many biological processes including conduction
of nerve impulse, balance of acid and bases, osmotic pressure regulation and muscles
movement. Potassium is the main cation of intracellular fluid. Its deficiency in body leads
to paralysis, cardiac arrhythmias, intolerance of carbohydrates, weakness of muscles etc.
(Hays & Swenson, 1985; Martin, 1985; Murray, 2000; Streeten & Williams, 1952;
Wadhwa, 2015). The present results suggest A. nitida as rich source of K. Other workers
have also reported large quantity of K in plants (Jan et al. 2011; Ravi et al. 2011;
Barkatullah et al., 2015b).
Calcium (Ca)
111
In present study highest Ca contents were detected in SC (314.3±0.007) followed
by B (278.7±0.005), L (186.9±0.002) and PC (110.9±0.001) (Table, 4.14).
The Ca recommended intake for a body’s ordinary biochemical activities is 1500
mg/day. Ca is needed for bones and teeth composition, nerve impulses transmission and
permeability of membrane (Murray, 2000; Indrayan, 2005; Wadhwa, 2015). Ca help in
coagulation of blood by converting prothrombin to thrombin. Ca increases vitamin D
absorption, activates enzymes like ATPase succinic dehydrogenase and lipase. Excessive
absorption of Ca may lead to Ca toxicity which results in cardiac failure (Soetan, 2010).
Deficiency of Ca cause rickets in children and osteomalacia in adults and may also result
in porous weak bones (Murray, 2000; Saiki, 1990).
Magnesium (Mg)
Mg contents were highest in SC (357.3±0.003) followed by PC (328.8±0.033), L
(303.3±0.001) and B (182.2±0.003). Predicted Mg intake value per day is 400 ppm. Mg is
required for many enzyme systems, bones and teeth (Murray, 2000), Osmotic pressure
maintenance in plasma and extracellular fluid. Magnesium deficiency causes vasodilation
with hyperemia and erythema, chronic vomiting and diarrhea. Long term deficiency of Mg
results in cardiac arrhythmia and neuromuscular hyperirritability (Soetan, 2010; Chatterjee
& Shinde, 1995). The overall results of elemental analysis suggested that A. nitida has
balanced and useful contents of the studied biologically important elements; furthermore,
no element was present in toxic concentration.
112
Table. 4.14. Elemental analysis of A. nitida.
Metal B(ppm) L(ppm) SC (ppm) PC (ppm)
Cu 40.8±0.003 23.2±0.007 23.10±.001 21.5±0.005
Fe 114.5±0.032 438.3±0.0502 176.5±0.008 139.4±0.009
Zn 33.8±0.004 38.4±0.001 31.9±0.004 35.7±0.002
Mn 21.4±0.004 75.6±0.008 26.3±0.0038 20.5±0.003
Mg 182.2±0.003 303.3±0.001 357.3±0.003 328.8±0.033
Na 99.8±0.003 117.3±0.000 100.0±0.002 97.9±0.004
Ca 278.7±0.005 186.9±0.002 314.3±0.007 110.9±0.001
K 787.3±0.060 2761.±0.133 3286±0.006 3298±0.005
4.4.6. Nutritional Analysis
Nutritional value of plants is determined by proximate and nutrient analysis
(Pandey et al., 2006). In present study the B, L, SC and PC samples of A. nitida were
evaluated for their nutritional values. Results obtained are presented in (Table. 4.15).
Carbohydrate value was higher in SC (48.45%) followed by L (46.025%), PC
(20.025%) and B (3.575%). Carbohydrates are considered as the main energy source for
all organisms. Besides nutrition, they have structural role in the body as well. Higher
contents of carbohydrates suggest the plants stability as feed (Abighor et al; 1997). Many
workers have reported high contents of carbohydrates in different parts of plants (Bukhsh
et al. 2007; Hussain et al., 2011b; Barkatullah et al., 2015b).
Protein contents were 7% in SC, 6.125% in L, 6.125% in PC and 0.875% in B.
Flowers (SC and PC) and leaf have more protein content than stem bark. Kabir et al., (2015)
have also reported high protein contents in flowers and leaf than stem. Similarly,
Gonz´alez-Hern´Andez et al., (2000) has reported 15% crude protein in leaf of Alnus rubra.
In developing countries people use plants as source of proteins in their diets (Kabir et al.,
2015). Protein are necessary for the formation of hormones, controlling growth and repair
(Mau et al.,1999). Shanker (1989), have reported 17 to 22% protein in A. nitida
113
leaf from different localities. Protein contents in plants may vary with ecological
conditions and seasonal variations (Kabir et al., 2015).
New sources of economical and good quality proteins are needed to fulfill its
increasing demands by rapidly growing population in Pakistan (Nisar et al., 2009). For this
purpose, many workers have carried out protein analysis of medicinal plants (Anwar &
Rashid 2007; Hussain et al. 2010a; Barkatullah, 2015b). Kabir et al., (2015) has reported
8 to 26% proteins in various plants. In present study results indicated that A. nitida is good
in protein content but not a rich source of proteins.
Fats contents in present study were 5% in PC, 5% in L, 3% in SC and 2% in B.
Excessive consumption of fats leads to atherosclerosis, aging and cancer. Plant materials
with 1 to 2 % fats are considered as significant energy source for human beings (Antia et
al., 2006). In present study Fat contents in PC and L samples are high. 4 to 8% fat contents
were also detected in plants by Iniaghe et al. (2009) and Zain-Ullah et al. (2013).
Crude fibers
Present results showed highest crude fiber content of 73% in Bark followed by
48.5% in PC, 20.3% in SC and 22% in L. Non starchy materials have high contents of
fibers (Agostoni et al., 1995) which are useful for curing obesity, diabetes, cancer and
gastrointestinal disorders (Saldanha, 1995). Gonzalez-Hernandez et al., (2000) reported 12
to 40% fibers in leaf of Alnus rubra. Tuncturk et al., (2015) reported 44.60 ± 1.650,
48.98 ± 2.090, and 43.14 ± 0.890 %, fibers in Scorzonera cana, Scorzonera suberosa and
Scorzonera tomentosa respectively. Many other workers have also carried out similar tests
(Naseem et al., 2006; Hussain et al., 2011b). Our present study revealed that bark and cone
of A. nitida are very rich sources of fibers. Shanker (1989) have reported range of 765-
2000 µm for Fibre length of A. nitida.
The ash contents in present study were10 % in PC, 9% in L, 8.7% in SC and 7% in
B. High ash content shows large amounts of minerals in plant sample (Antia et al.,
114
2006). Ash analysis have also been carried out on many other plants (Iniaghe et al. 2009;
Akindahunsi & Salawu, 2005).
Moisture contents were higher in B (13.55%), followed by SC (12.55%), L
(11.85%) and PC (10.35%). Shanker et al., (1989) have reported 8 to 13% wood moisture
for A. nitida samples from various loacalities indicating moisture dependence on ecological
factors. Many workers have reported moisture contents for other plants (Kochhar et al.,
2006; Hameed et al., 2008 and Hussain et al., 2011b).
Table. 4.15. Proximate analysis of A. nitida stem bark (B), leaf (L) and staminate
catkin (SC) and pistillate cone (PC).
Sample % Moisture % Ash %Proteins % Fats % Carbohydrates % Crude Fiber
B 13.55 7 0.875 2 3.575 73
L 11.85 9 6.125 5 46.025 22
SC 12.55 8.7 7 3 48.45 20.3
PC 10.35 10 6.125 5 20.025 48.5
4.5. Phytochemical screening
i. Qualitative screening
Plants are source of pharmacologically important constituents. Preliminary
phytochemical screening help to identify and isolate bioactive constituents from a plant
(Ming et al., 2005; Sugumaran & Vetrichelvan, 2008).
Alkaloids are group of secondary metabolites with low molecular weight nitrogen
containing compounds, having strong biological activities such as anticancer (vincristine
and vinblastine from Catharanthus roseus) and analgesic (morphine from Papaver
somniferum) (Croteau, 2000). Most of the alkaloid act against infections of microbes and
attack of herbivore (Pagare, 2015). Saponins cure diseases and provide protection against
pathogens (Fluck, 1973; Sodipo et al., 1991). Tannins and phenolics are useful in many
biological activities (Asquith & Butler, 1986; Havsteen, 2002).
115
In present study qualitative as well as quantitative phytochemical screening of A.
nitida bark, leaf, staminate catkin and pistillate cone were carried out. Results for
qualitative screening are presented in Table. 4.16. Carbohydrates, proteins, saponins,
tannins, phenol, steroidal glycosides, fixed oil and fats were detected in all samples.
Volatile oil was found only in SC and PC. Anthocyanins were absent in all samples.
Triterpenes were detected in bark and leaf only. Sterols were present in bark, staminate
catkin and pistillate cone. Bikovens et al. (2013) reported triterpenoids, tannins, sterols and
fats in bark and cone of Alnus incana. Several other workers have carried out
phytochemical screening of different plants, reporting a number of phytochemicals in them
(Patra et al. 2009; Shrivastava & Leelavathi, 2010; Kumar et al. 2011).
Table. 4.16. Preliminary phytochemical screening of stem bark, leaf, staminate catkin
and pistillate cone of A. nitida.
S.NO. Constituents Test name B L SC PC
1 Carbohydrates Fehling’s test + + + +
Benedict’s test + + + +
2 Protein Ninhydrin test + + + +
3 Alkaloid Hager’s test + + + +
4 Saponins Frothing test + + + +
5 Flavonoids Alkali test + + + +
6 Phytosterols
Salkowski’s test + - + +
Liebermann-Burchard Test
- - + +
7 Triterpenoids Salkowski’s test - + - -
Liebermann-Burchard Test
+ + - -
8 Tannins Ferric chloride test
+ + + +
Alkali test + + + +
8
Phenol
Ferric chloride test
+ + + +
9 Anthocyanins HCl test _ _ _ _
10 Volatile oil Spot test _ _ + +
11 Steroidal Glycosides
Keller kiliani test + + + +
12 Fixed oil and fats
Spot test + + + +
116
Studies on the genus Alnus have revealed the presence of about 273 chemical
constituents so far, including flavonoids, diarylheptanoids, steroids, polyphenols and
terpenoids etc. Alnus species have shown notable anticancer, antioxidant, antiviral,
anti- inflammatory and hepatoprotective activities (Ren et al., 2017). In the present
work preliminary phytochemical screening revealed the presence of different classes
of chemical constituents in bark, leaf, staminate catkin and pistillate cone. The
presence of those metabolites in various parts speaks for the marked physiological and
pharmacological activities as worked out in the present study.
ii. Quantitative evaluation
Quantitative evaluation of A. nitida was carried out for Flavonoids, Phenols and
Sterols. Results of quantitative analysis are presented in Table. 4.17. In present study total
flavonoids (QE mg/g of extract) were 103.6 in B, 105 in L, 90 in PC and 70 in SC. Total
phenolic contents (GAE mg/g of extract) found were 472 in B, 409 in L, 332 in SC and
637 in PC. Total sterol contents (mg/g) were 80 in B, 40 in L, 200 in SC and 140 in PC.
Table.4. 17.Quantitative chemical analysis of stem bark (B), leaf
(L), staminate catkin (SC) and pistillate cone (PC) of A. nitida.
S.NO. Extract Flavonoids
QE (mg/g of
extract) (Mean±SD)
Phenols GAE
(mg/g of
extract) (Mean±SD)
Sterols (mg/g)
(Mean±SD)
1 B 103.6±0.5 472±2.5 80±2.5
2 L 105.0±1.5 409±1.5 40±3.0
3 SC 70.3±1.5 332±1.5 200±4.5
4 PC 90.0±2.0 637±0.5 140±1.5
Several similar studies on quantitative chemical analysis have been carried out by
other workers on different plants including Alnus species.
Contents of total phenols in leaf extract of A. viridis, A. glutinosa and A. incana, were
reported as 397.00±15.64, 338.00±9.38 and 555.00±15.04 mgCAT/g respectively. While,
their bark extract have shown 780.00±12.75, 333.00±15.91 and 410.00±13.05 mgCAT/g
of total phenols. Similarly, total flavonoid contents of 11.85±0.22, 15.05±1.74 and
30.01±2.001mgRUT/g were reported in leaf of A. glutinosa, A. incana and A. viridis
respectively. While, total flavonoids in their bark were 10.26±0.37, 11.40±0.20 and ±0.05
117
mgRUT/g respectively (Dahija et al., 2014). On the other hand, Stevic et al. (2010) have
reported total phenolic contents of 316.2±6.9 and 238.6±6.6 mg GAE/g in methanolic
extracts of A. incana and A. viridis respectively. Choi et al., (2018) found the total phenolic
content of 436.26±3.30 mg GAE/g in A. firma while, its total flavonoid content was
73.82±0.54 mg QE/g. Similarly, Acero and Muñoz-Mingarro (2012) have calculated the
total flavonoid contents of 34.55±0.19 mg QE/g in methanolic extract of A. glutinosa.
Flavonoids and phenols are the extensively found secondary metabolites in plants
and have shown significant antioxidant activity (Wang et al. 2008). A number of phenolic
compounds have shown anticancer, antiviral, antioxidant, anti-inflammatory and
antibacterial activities (Cassidy et al., 2000; Tapiero et al., 2002). Flavonoids are usually
found in pollen, leaf and flowering tissues (Larson, 1998) and are reported as very effective
scavengers of many oxidizing molecules (Bravo, 1998).
The present study showed that A. nitida is a rich source of various phytochemicals
(Table. 4.16), especially phenolic contents were higher in all studied samples (Table. 4.17).
A. nitida may contain bioactive constituents against many ailments. The total phenols
(631.5±1.7 Gallic acid equivalents mg/g of extract) contents reported by Sajjid et al. (2016)
for bark methanolic extract of A. nitida collected in March were higher than our present
result. The reason may be seasonal variation of phytochemical contents in plants because;
we have collected the bark in autumn. Other reason may be 70% ethanolic extract as solvent
used for extraction in present study instead of absolute methanol. Ahmad et al. (2017) have
also reported seasonal decrease in total phenolic content from spring to autumn in Pistacia
atlantica ssp. leaf and stated that the harvest time and growing region have impact on
contents of its secondary metabolites.
Steroids from plants (corticosteroids, digitoxin and digoxin, steroidal glycosides)
have shown many agrochemical and pharmacological activities including cardiotonic,
antitumor, antibacterial, cytotoxic etc (Patel & Savjani. 2015). Sterols have also been
reported from many Alnus species (Ren et al., 2017). The bioactivities observed for A.
ntitda may be due to the presence of bioactive sterol compound(s) which needs further
detailed investigation of its compounds.
118
4.6. Parmacological activities
Bioassays are pharmacological tools, used to screen out extracts of plants for their
therapeutic potential in different diseases (Srirama et al., 2007). The following bioassays
(pharmacological activities) were used to explore the therapeutic potential of A. nitida.
4.6.1. Analgesic activity
The effect of ethanolic extracts of the bark, leaf, staminate catkin and pistillate cone
of A. nitida (Spach) Endl. at doses of 50, 100 and 200 mg/kg on the acetic induced
abdominal constrictions in mice is presented in Table.4.18.
Leaf extract at dose of 200 mg/kg showed highest and highly significant
(**p<0.001) reduction in abdominal constriction and showed 77.87±1.01 % reduction in
pain, followed by % pain reduction of 76.00±1.09 at 200 mg/kg by bark extract. These
values were even higher than Aspirin (73.10±0.93%), followed by 59.44±1.70 and
56.67±0.92 % reduction in pain by leaf and bark extract respectively at 100 mg/kg. pistillate
cone extract showed 52.98±1.01 % pain reduction at dose of 200 mg/kg. Bark and leaf
extract showed 37.31±0.92 and 36.84±1.66% reduction in pain respectively at 50 mg/kg
while, % pain reduction by pistillate cone extract at 100 and 50mg/kg were 42.84±1.70%,
and 35.01±1.18% respectively. The % reduction in pain by staminate catkin extract at doses
50, 100 and 200mg/kg were 15.19±2.83%, 27.17±1.17% and 33.62±1.59% respectively
(Fig. 4.15b). All treatments at all doses showed highly significant reduction in pain
compared to saline (-ve control) (Fig.4.15b). The order of % reduction in pain was
Leaf>Bark> Pistillate cone> staminate catkin.
119
Table.4.18. Antinociceptive effect of different doses of ethanolic extract of Bark(B),
Leaf (L), Staminate catkin (SC) and Pistillate cone (PC) of A. nitida (Spach) Endl. on acetic acid induced writhing in mice.
S. No. Treatments Dose
(mg/kg)
No. of writhings
(mean±SEM)
% Inhibition
(mean±SEM)
1 Saline
(-ve control)
10
(ml/kg)
36.17±0.32
2 Aspirin
(+ve control)
10 9.667±0.33** 73.10±0.93
3 B 50 22.67±0.33** 37.31±0.92
4 100 15.67±0.33** 56.67±0.92
5 200 8.50±0.44** 76.00±1.09
6 L 50 23.00±0.63** 36.84±1.66
7 100 14.67±0.61** 59.44±1.70
8 200 8.00±0.36** 77.87±1.01
9 SC 50 30.67±28.04** 15.19±2.83
10 100 26.33±0.42** 27.17±1.17
11 200 24.00±0.58** 33.62±1.59
12 PC 50 23.50±0.45** 35.01±1.18
13 100 20.67±0.61** 42.84±1.70
14 200 17.00±0.36** 52.98±1.01
Note. Values presented in table are mean±SEM (n=6), showing highly significant (**p<0.001) reduction
in number of writhings compared to saline (-ve control).
120
Fig. 4.15a. Effect of A. nitida extracts on number of acetic acid induced writhings in mice.
Bars represent number of writhings (mean ± SEM) (n=6). One-way ANOVA withTukey’s
multiple comparison test was used to find significant difference between groups. Asterisk
represent highly significant reduction in number of writhings vs Saline (-ve control) at
**P<0.001. Bars with different superscript number represent significant difference at p<0.05.
B =Bark =Leaf, SC=Staminate catkin; PC=Pistillate cone (Pistillate catkin with seeds).
Fig. 4. 15 b. Effect of A. nitida extracts reduction of acetic acid induced pain in mice.
Bars represent % reduction in pain (mean ± SEM) (n=6). One-way ANOVA with Tukey’s
multiple comparison test was used to find significant difference between groups. Asterisks
represent highly significant difference at **p<0.001 vs Saline (-ve control). Bars with
different superscript letters have significant difference at P<0.05. B=Bark, L=Leaf, SC=
Staminate catkin. PC=Pistillate cone (Pistillate catkin with seeds).
121
All types of pain start from inflammations (Omoigui, 2007) during which many
proinflammatory mediators are released such as cyclooxygenase-2 (COX-2), interferon
(INF-γ), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF) and
interleukin 6 (IL-6), IL-12 (Chiu, 2012; Moncada, 1991).
Researchers throughout the world use primarily acetic acid induced writhings test
to evaluate antinociceptive potential of natural compounds (Okokon & Nwafor, 2010;
Ahmed et al., 2011a). Acetic acid trigger release of endogenous detrimental mediators like
histamine, bradykinin substance P and serotonin (Mazid et al., 2010; Dellai, 2012) which
results in pain. The pain produced by acetic acid injection in mice was symbolized by
abdominal muscle contraction, accompanied by forelimbs extension and elongation of the
body collectively called “writhingsˮ. Peripheral nociceptive fibers are sensitive to both
NSAIDs and narcotic analgesic drugs (Khan et al., 2011; Khan et al., 2009; Muhammad et
al., 2012). Inhibition of COX enzyme is necessary for reduction in writhings (Kumar et al.,
2015b). Researchers have also shown that any agent that induces reduction in writhings
will cause analgesic effect preferably by peripheral pain inhibitory mechanism through
inhibition of prostaglandins synthesis (Ferdous et al., 2008). In Alnus species frequently
found phenolic compounds are recognized as inhibitors of prostaglandins synthesizing
enzymes (Mohammad et al., 2015).
In our present study the bark, leaf, staminate catkin and pistillate cone extracts
showed highly significant reduction in pain. It is suggested that these extracts may contain
pharmacologically active constituents which can interfere with or inhibit the release or
action of pain inducing mediators. Our results are similar to Sajjid et al., (2017) who
reported significantly higher antinociceptive effect of the A. nitida bark chloroform extract
as compared to aspirin in acetic acid induced pain model, Similar, results were reported for
other species of the genus Alnus that explored the presence of active constituents in bark,
leaf, pistillate cone and seeds, which inhibited the release, synthesis or action of
inflammatory mediators (O‟Rourke et al., 2005; Kuo, et al., 2008; Choi et al., 2011; Sati
et al., 2011; Sajjid et al.,2017). Sajjid et al., (2017) have also
122
reported analgesic activity for bark methanolic extract of A. nitida. Our results justified the
folkloric use of the plant leaves for analgesic effect and also explored the pain relieving
potency of the staminate catkin and pistillate cone extracts.
4.6.2. Anti-inflammatory activity
Anti-inflammatory effects of the crude ethanolic extracts of bark, leaf, staminate
catkin and pistillate cone of A. nitida (Spach) Endl. at doses of 50,100 and 200 mg/kg, with
respect to time and to -ve control are presented in Figs. 5.16a- 5.16e. All extracts showed
significant (*p<0.05) and dose dependent percent inhibition against carrageenan induced
paw edema in mice from 1st hour and was maintained till 4th hour. The only exceptions
were bark (B) and staminate catkin (SC) at doses of 50 mg/kg which showed non-
significant (p>0.05) percent inhibitions after 1st hour of carrageenan administration.
Ethanolic extract of bark (B) at dose of 200 mg/kg showed highest and highly significant
(**p<0.01) anti-inflammatory activity of 81.9±2.3% after 4 hrs followed by 77.88±4.4%
after 5 hrs, 77.44±2% after 3 hrs and 65.3±1.6% after 2 hrs. Leaf (L) showed highest and
highly significant (**p<0.01) anti-inflammatory activities of 61.6±3.8%, 81± 3.6 %,
79±3% and 76.9±1.86% after 2, 3,4 and 5 hrs respectively, at a dose of 200 mg/ kg. The %
inhibition by leaf (L) extract at 50 and 100mg/kg after 2, 3,4 and 5 hrs were 45±1.3%,
56.7.4±2.7%, 57.6±2.9%, 51±5.79% and 49.6±2.67%, 70±3%,
68±3% and 66±1.475% respectively. While, the percent inhibitions by staminate
catkin(SC), observed at doses of 50, 100 and 200 mg/kg were 17 ±2.768%, 18.9±2.4% and
18±1.5% respectively after 4 hrs. Pistillate cone extract (PC) displayed higher anti-
inflammatory activity of 42±1.89% at 200mg/kg dose after 4 hrs while at 50 and 100 mg/kg
doses its highest percent inhibitions were 18.9 ±4% after 3 hrs and 23±3.078% after 4 hrs
respectively .The percent anti- inflammatory effect of the remaining doses at their
respective time were significantly lower compared to +ve standard (Diclofenac) and the
test doses mentioned here (Fig.4.16a- 4.16e) Order of percent anti-inflammatory activity
was Bark >Leaf> Pistillate cone> Staminate catkin.
123
% I
nh
ib
itio
n o
f e
de
ma
(M
ea
n
SE
M)
Saline 1
0(m
l/kg)
Dic
lofe
nac 1
0(m
g/k
g)
B50
B100
B200
L50
L100
L200
SC
50
SC
100
SC
200
PC
50
PC
100
PC
200
0
1 0
2 0
3 0
A n t iin f la m m a t o r y a c t iv i t y
a f te r o n e h o u r* *
****
**
**
**
n s
**
*
** **
n s
n s
E x t r a c t c o n c e n t r a t io n ( m g /k g )
Fig. 4.16a
Fig.4.16b
A n t iin f la m m a to r y a c t iv i t y
a f t e r t h r e e h o u r s
% I
nh
ib
itio
n o
f e
de
ma
(M
ea
n
SE
M)
Saline 1
0(m
l/kg)
Dic
lofe
nac 1
0(m
g/k
g)
B50
B100
B200
L50
L100
L200
SC
50
SC
100
SC
200
PC
50
PC
100
PC
200
0
2 0
4 0
6 0
8 0
1 0 0
**
**
**
**
**
**
**
* * * ****
**
E x t r a c t c o n c e n t r a t io n ( m g /k g )
Fig.4.16c
124
Comparative % inhibition of same extract at doses of 50, 100, 200 mg/kg with
respect to time is displayed in Figs.4.17a – 4.17d.
100
80
60
40
20
0
% Inhibition of edema
by bark extract
1 2 3 4 5
Time (hr)
Diclofenac
B50
B100
B200
100
80
60
40
20
0
% inhibition of edema by
leaf extract
1 2 3 4 5
Time(hr)
Diclofenac
L50
L100
L200
Fig.4.17a. Fig.4.17b
100
80
60
40
20
0
% Inhibition of edema by
staminate catkins extract
1 2 3 4 5
Time (hr)
Diclofenac
SC50
SC100
SC200
100
80
60
40
20
0
% Inhibition of edema by
pistillate cone extract
1 2 3 4 5
Time (hr)
Diclofenac
PC50
PC100
PC200
Fig.4.17c. Fig.4.17d.
Figs.4.17a-4.17d. Comparison between % inhibitions of different doses from the same extract of A. nitida with
respect to time. B50= bark extract 50 mg/kg; B100=bark extract 100 mg/kg; B200= bark extract 200mg/kg; L50= leaf extract 50
mg/kg; L100=Leaf extract 100 mg/kg;L200= leaf extract 200 mg/kg; SC50= Staminate catkin extract 50mg/kg;
SC100=Staminate catkin extract 100 mg/kg; SC200= Staminate catkin extract 200 mg/kg; PC50= Pistillate cone extract 50 mg/kg; PC100= Pistillate cone extract 100 mg/kg; PC200=Pistillate cone extract 200 mg/kg.
The overall results showing changes in paw volume after carrageenan injection and
percent inhibition of edema as compared to -ve control and with respect to time period are
shown in Table.4.19.
% In
hib
itio
n
% In
hib
itio
n
% In
hib
itio
n
% In
hib
itio
n
125
Table. 4.19. Effect of A. nitida extracts on carrageenan induced paw edema in mice.
Treatment Dose NPV Increase in paw volume(mean±SEM) after carrageenan injection
(Percent inhibition in edema±SEM)
1hr 2hr 3hr 4hr 5hr
Saline
(-ve control)
10ml/kg 0.095±0.002 0.219±0.002 00.2196±0.02 0.206±0.003 0.206±0.001 0.208±0.002
B 50mg/kg 0.095±0.002 0.208±0.001 0.180±0.003 0.156±0.004 0.158±0.003 0.16±0.003
(8.6±1.7ns) (32± 3.42**) (44± 4.87**) (43±3.00**) (42.48±4.4**)
100mg/kg 0.098±0.001 0.205±0.003 0.166±0.004 0.141±0.006 0.138±0.003 0.143±0.002
(13.9±1.6**) (45.3±3.2**) (61±6.00**) (63.9±3.27**) (60±3.02**)
200mg/kg 0.096±0.002 0.201±0.001 0.14±0.002 0.121±0.003 0.116±0.002 0.121±0.003
(15±1.8**) (65.3±1.6**) (77.44±2.03**) (81.9±2.33**) (77.88±4.4**)
L 50mg/kg 0.098±0.001 0.20±0.002 0.166±0.002 0.146±0.002 0.145±0.002 0.153±0.002
(18±2.48**) (45±1.33**) (56.4±2.758**) (57.6±2.99**) (51±5.796**)
100mg/kg 0.095±0.002 0.201±0.004 0.158±0.002 0.128±0.003 0.13±0.002 0.133±0.002
(14.5±3.9**) (49.6±2.67**) (70±3.01**) (68±3.086**) (66±1.475**)
200mg/kg 0.095±0.002 0.20±0.003 0.143±0.003 0.116±0.002 0.118±0.001 0.121±0.001
(15±2.75*) (61.6±3.8**) (81± 3.63**) (79±3.017**) (76.9±1.866**)
Sc 50mg/kg 0.096 ±0.002 0.208 ±0.001 0.206±0.002 0.19±0.002 0.188±0.003 0.19±0.003
(9.6±2.479ns
) (12±2.92*) (15±1.897*) (17±2.768**) (16.8±3.728*)
100mg/kg 0.095±0.002 0.205±0.002 0.203±0.003 0.188±0.003 0.185±0.002 0.188±0.004
ns
(12±2.08) (13.6±3.21*) (16±3.003*) (18.9±2.4**) (17.6±3.728*)
200mg/kg 0.095±0.002 0.201±0.003 0.2± 0.003 0.186 ±0.004 0.186±0.002 0.191±0.003
(14.5±3.39*) (16±3.5**) (18±3.616*) (18±1.5**) (15±3.729ns
)
PC 50mg/kg 0.096±0.002 0.205±0.002 0.203±0.002 0.186±0.003 0.188±0.003 0.191±0.003
ns
(12±1.34) (14.4±2.66**) (18.9±4.028**) (17±3.616**) (15.9±4.42*)
100mg/kg 0.095±0.002 0.196±0.003 0.195±0.002 0.181±0.003 0.18±0.002 0.185±0.002
(18.5±3.8**) (20±2.06**) (22.5±3.799**) (23±3.078**) (20±2.285**)
200mg/kg 0.096±0.002 0.196±0.002 0.191±0.006 0.161±0.003 0.16±0.002 0.166±0.002
(19±2.08**) (24±1.789**) (41±3.078**) (42±1.899**) (38±3.232**)
Diclofenac
(+ve control)
10mg/kg 0.096±0.003 0.191±0.001 0.141±0.003 0.118±0.004 0.115±0.005 0.126±0.004
(23±4.99**) (64±4.00**) (82±5.427**) (83±5.426**) (73.45±5.11**)
Note: Values shown in table represent mean ± SEM (n = 6). One-way ANOVA followed by Dunnett’s multiple comparison test used to find
significance versus saline (-ve control) group. Asterisks represent significance level at *p < 0.05 and **p < 0.01. Percent inhibition in
edema±SEM is shown in parenthesis.
NPV= Normal paw volume, B=Bark extract, L=Leaf extract, SC=Staminate catkin extract, PC= Pistillate cone extract (Pistillate catkin with seeds).
126
Inflammation has its implications in almost all diseases of man and animals.
Therefore, it has been given more emphasis by the global scientific research. Use of non-
steroidal anti-inflammatory drugs (NSAIDs) can result in adverse effects like gastric
lesions, while opiates induce tolerance and dependence; therefore, these drugs have not
been suitable in all inflammatory conditions (Phanse et al., 2012).
Mechanisms suggested to elucidate the anti-inflammatory effects of
phytoconstituents include modulation of the activities of cells associated with
inflammation (neutrophils, mast cells, lymphocytes and macrophages), proinflammatory
enzymes (Cycloxygenase, Lipoxygenase, Phospholipase, Nitric oxide producing enzymes
and Nitric oxide synthase), other proinflammatory molecules production, expression of
proinflammatory gene as well as their antioxidant and free radical scavenging activities.
Most studied phytoconstituents with anti-inflammatory effects include terpenes, alkaloids
and polyphenols. Many inflammatory diseases are associated with activation of NF-κB
(nuclear transcription factor-kappa B), while a number of studies emphasize the importance
of phytochemicals to inhibit its activation pathway (Bellik et al., 2013).
Phytochemicals are revealed to modulate many points of inflammatory processes
(Kim et al., 2009), which acts as key points for disconnecting the amplification of
inflammatory processes and decrease the risk of succeeding diseases as well. Phenolic
glycosides and diarylheptanoids from several Alnus species (A. nepalensis, A. hirsuta, A.
acuminata, A. formosana, A. japonica, A. firma) and bark extract of A. nitida have shown
significant anti-inflammatory activity (Jin, 2007; Lee, 2010; Aguilar, 2011; Lai, 2012;
Sajid, 2017; Kim, 2005; Saxena, 2016).
Carrageenan induced hind paw edema assay has been extensively used to evaluate
antiinflammatory effects of novel drugs (Afsar et al., 2015). It is a biphasic model. The
early phase of edema (0-1hr) is mostly due to the release of mediators; histamine, 5-
HT(Serotonin) and bradykinin. In the later phase of edema development, cyclooxygenase
enzyme (COX) plays significant role of arachidonic acid conversion into prostaglandins
(Gilani & Janbaz, 1993; Blokhina et al., 2003; Briben et al., 2012).
127
For validation of anti-inflammatory effects of A. nitida extracts, diclofenac sodium
was used as standard drug (+ve control) in present study. The percent inhibition of A. nitida
bark and leaf extract with respect to time, at 200 mg/kg were comparable to diclofenac (10
mg/kg). Furthermore, highest inhibition was achieved at later phase of edema and at 4th hr,
showing similar inhibitory pattern as the positive standard diclofenac, which show anti-
inflammatory effect by inhibiting prostaglandin synthesis through inhibition of enzymes
cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-
2) (Gan, 2010). These results are also similar to Afsar et al (2015), who reported significant
inhibitory effect (p<0.001) of Acacia hydaspica methanolic extract in later phase of edema,
showing diclofenac like activity, which was further investigated and PGE2 (prostaglandin-
E2) inhibition by the extract was proved.
The antiinflammatory effect shown by the A. nitida extracts may be due to the
presence of active constituents that inhibited the action, release or synthesis of
inflammatory mediators. Our results are similar to literature reports for extracts of other
species of the genus Alnus, having Anti-inflammatory effects which shows the presence of
active constituents against inflammation. Leaf and bark extracts of A. japonica are reported
to inhibit COX-2 while leaf extract of Alnus hirsuta and bark extract of A. firma have shown
significant reduction in NO (nitric oxide) production (Choi et al., 2011; Sati et al., 2011).
Leaf extract of Alnus viridis has revealed potential to inhibit NF-κB. Oregonin, a
diarylheptanoid derivative was suggested as responsible for this effect, because it is
reported for antiinflammmatory activities and mostly found in species of the genus Alnus
(Kuo, et al., 2008). Our results are similar to Sajjid et al. (2017), who suggested that
inhibitory effects of Alnus nitida bark chloroform extract both in initial and later phase of
edema may be the result of its inhibitory effect on release of inflammatory mediators and
also proposed the inhibition of neutrophils infiltration by the Alnus nitida extract and its
fraction as cause of its anti-inflammatory (anti edematic) potential. Similarly, ellagitannins
isolated from A. glutinosa cones were reported to have anti-inflammatory activity (Sati et
al., 2011). While, seeds of A. glutinosa are reported to contain hirsutanonol, oregonin and
genkwanin. (O‟Rourke et al., 2005) which have shown anti-inflammatory potential by
inhibiting inflammatory mediators production
128
through inhibition of; NF-κB (nuclear factor kappa B), iNOS (inducible nitric oxide
synthase) gene transcription by suppressing activator protein-1 (AP-1) as well as
transcriptional activity of NFκB and proinflammatory mediators TNF-α, IL-6, IL-1β at the
transcriptional and translational levels respectively (Kim et al., 2005; Lee et al., 2005; Gao,
et al., 2014). As PC (pistillate cone) extract contained pistillate catkins along with seeds,
these reports probably explain the anti-inflammatory effect showed by PC (pistillate
catkins with seeds) extract may be due to the presence of potent biochemical constituent(s).
The non-significant inhibitory effect of staminate catkins (SC) at higher dose
(200mg/kg, after 5 hrs) and its significantly higher effect at lower dose may be due to
tolerance phenomenon at higher dose. Similar results were shown by Kumar and Kashyap
(2015) for methanolic extract of Fraxinus micrantha.
During an earlier preliminary phytochemical screening of A. nitida, alkaloids,
saponins, triterpenoids, flavonoid, phenols and tannins were detected in these extracts.
Therefore, the observed effects on inflammation can be attributed to the presence of these
classes of compounds. It is concluded from the present study that the use of A. nitida in
traditional treatment of inflammation is justified on scientific grounds especially that of the
bark and leaf.
4.6.3. Antipyretic activity
The antipyretic activity of A. nitida crude extracts as compared to –ve control
(Saline) is shown in Table. 4.20 and Fig.4.18a-e. Leaf extract (L) showed highest and
highly significant (*p<0.01) % antipyretic activity of 66% after 4 hours followed by 65%,
64.47%, 55% and 35.8% after 3,5,2 and 1 hours respectively at a dose of 300 mg/kg.
Percent antipyretic activity of leaf extract (L) at a dose of 200 mg/kg was 49.06%, 48.59%,
47.66%, 35.5% and 21.49% after 4,3,5,2 and 1 hours respectively, while at 100 mg/kg of
leaf extract (L) highly significant (**p<0.01) antipyretic activity of 39.6%, 38.4%, 36.5%
and significant (*p<0.05) antipyretic activity of 24.39% was noted after 3,4,5 and 2 hours
respectively. Bark ethanolic extract showed highly significant (**p<0.01) % antipyretic
activity of 63.8% after 4 hours followed by 62.7%, 61.6%, 55%
129
and 38% after 3,5,2 and 1 hours respectively at dose of 300 mg/kg. % antipyretic activity
of the bark extract at 200 mg/kg was 47%, 45%, 44%, 32.35% and 21% after 4,3,5,2 and 1
hours respectively, while at 100 mg/kg of bark extract highly significant antipyretic activity
of 38%, 37.8% and 32% was observed at 4,3 and 5 hours respectively. Pistillate
cone extract showed antipyretic activity of 41%,40%,38.7% and 27.45% after 4,3,5 and 2
hours respectively at dose of 300mg/kg, which was significantly higher than –ve control
(saline). Staminate catkin extract showed significantly higher antipyretic activity of 22%
and 21% after 4 and 3 hours respectively at dose of 300 mg/kg.
Probably this is the 1st report on antipyretic effects of A. nitida. Paracetamol was
used as positive standard to justify the antipyretic potential of A. nitida. The % antipyretic
activity of bark and leaf extracts at dose of 300 mg/kg after 3,4 and 5 hours were
comparable to that of paracetamol (150 mg/kg) after 2 hours. In present study A. nitida
extracts have shown antipyretic activity in dose dependent manner. We also found tannins,
saponins, phenols, flavonoids and steroids in these plant samples (Table.4.16). Flavonoids,
steroids and tannins are the principle inhibitors of cyclooxygenase or lipoxygenase and
prostaglandins synthetase which help to prevent pyrexia (Raj- Narayana et al., 2001). The
presence of these phytochemicals in A. nitida plant samples may be the cause of the
observed antipyretic activity. Some endogenous substances such as prostaglandins are
responsible for increase in body temperature. All types of drugs used to cure pyrexia are
able to prevent prostaglandins formation (Alam et al., 2016). The fever induced by
Brewer’s yeast, called as pathogenic fever results from production of prostaglandins.
Researchers have a general agreement that antipyretics works by interrupting
synthesis of prostaglandin through inhibition of COX enzyme (Aronoff & Neilson, 2001;
Van-Arman et al., 1985). The possible mechanism of the A. nitida extracts to inhibit
antipyretic activity could be the inhibition of prostaglandins production.
Many plants including Alnus species are reported to inhibit pyrexia. The extracts of
A. japonica leaf and bark have shown inhibition of COX-2 (Choi et al., 2011). Antipyretic
activity has also been reported for many other plant extracts against pyrexia
130
induced by brewer’s yeast (Ahmed, et al., 2016b; Sultana et al., 2015b; Barkatullah,
2013).
Table.4.20. Antipyretic activity of the bark, leaf, staminate catkin and pistillate cone of
A. nitida.
Treatment Dose
(mg/kg)
Initial rectal temperature
(C°)
Rectal temperature (C°) after injection of extract (tn)
T(Normal) Ty
(After 24hrs)
t1 (1 hr) t2 (2hr) t3 (3hr) t4(4hr) t5(5hr)
Saline 10ml 36.7±0.1 38.9±0.04 38.8±0.05 38.86±0.03 38.85±0.04 38.8±0.03 38.80±0.03
Paracetamol 150 36.94±0.03 38.5 ±0.14 37.76±0.08
(47%)
37.5±0.05
(64%)
37.4±0.07
(70%)
37.38±0.07
(72%)
37.42±0.07
(69%)
B100 100 37.0±0.02 38.4±0.09 38.25±0.07
(10.7%)
38.16±0.06
(17%)
37.87±0.03
(37.8%)
37.86±0.02
(38%)
37.95±0.08
(32%)
B200 200 36.9±0.04 38.6±0.06 38.25±0.03
(21%)
38.05±0.03
32.35%
37.83±0.05
45%
37.8±0.02
47%
37.85±0.02
44%
B300 300 36.9±0.08 38.7±0.057 38.05±0.04
38%
37.7±0.03
55%
37.57±0.03
62.7%
37.55±0.03
63.8%
37.59±0.02
(61.6%)
L100 100 36.96±0.08 38.6±0.12 38.4±0.11
12.2%
38.2±0.11
24.39%
37.95±0.04
39.6%
37.97±0.16
38.4%
38.0±0.014
36.5%
L200 200 36.7±0.06 38.84±0.03 38.38±0.08
21.49%
38.08±0.03
35.5%
37.8±0.03
48.59%
37.79±0.03
49.06%
37.82±0.04
47.66%
L300 300 37.0±0.05 38.56±0.13 38.0±0.04
35.8%
37.7±0.06
55%
37.54±0.07
65%
37.53±0.07
66%
37.55±0.07
64.47%
SC100 100 36.6±0.05 38.4±0.12 38.3±0.13
5.5%
38.2±0.13
11%
38.15±0.13
14%
38.12±0.14
15.5%
38.17±0.14
13%
SC200 200 36.87±0.06 38.3±0.09 38.17±0.1
9.09%
38.1±0.14
13.9%
38.04±0.15
14%
38.0±0.15
21%
38.07±0.14
16%
SC300 300 36.58±0.09 38.4±0.12 38.18±0.07
12.08%
38.08±0.06
17.58%
38.01±0.06
21.4%
38.0±0.04
22%
38.05±0.03
19%
PC100 100 36.86±0.08 38.48±0.14 38.26±0.15
13.58%
38.2±0.16
17.28%
38.13±0.06
21.6%
38.1±0.06
23.45%
38.15±0.06
20.37%
PC200 200 36.76±0.08 38.6±0.09 38.3±0.08
16.3%
38.2±0.06
22%
38.13±0.06
25.54%
38.09±0.05
28%
38.15±0.04
24.45%
PC300 300 36.66±0.07 38.7±±0.08 38.3±0.04
19.6%
38.14±0.02
27.45%
37.88±0.03
40%
37.86±0.02
41%
37.91±0.03
38.7%
Table. 4.20 Shows mean values ± SEM, (n = 5). B=Bark, L=Leaf extract, SC=Staminate catkin extract,
PC=Pistillate cones extract (Pistillate catkin with seeds). tn = Rectal temperature after 1-5 hours.
T= Normal temperature, Ty= Temperature after yeast injection. Values in parenthesis show % antipyretic
activity at given time.
131
A n t ip y r e t ic a c t iv i t y
a f te r 1 h r
% P
yr
ex
ia i
nh
ibit
ion
(Me
an
SE
M)
Sa li
ne 1
0 (ml/
kg )
Pa r a c e ta
mo l 1
0 (mg /k
g )
B1 0 0
B2 0 0
B3 0 0
L1 0 0
L2 0 0
L3 0 0
SC
1 0 0
SC
2 0 0
SC
3 0 0
PC
1 0 0
PC
2 0 0
PC
3 0 0
0
2 0
4 0
6 0
8 0
**
n s
**
**
n s
**
**
n s n s
n s
n s
n s *
E x t r a c t c o n c e n t r a t io n ( m g /k g )
Fig.4.18a
A n t ip y r e t ic a c t iv i t y
a f t e r 2 h r s
% p
yr
ex
ia i
nh
ibit
ion
(Me
an
SE
M)
Sa li
ne 1
0 (ml/
kg )
Pa r a c e ta
mo l
1 0 (mg /k
g )
B1 0 0
B2 0 0
B3 0 0
L1 0 0
L2 0 0
L3 0 0
SC
1 0 0
SC
2 0 0
SC
3 0 0
PC
1 0 0
PC
2 0 0
PC
3 0 0
0
2 0
4 0
6 0
8 0
**
n s
**
**
*
**
**
n s
n sn s n s
n s
**
E x t r a c t c o n c e n t r a t io n ( m g /k g )
Fig.4.18b
A n t ip y r e t ic a c t iv it y
a f t e r 3 h r s
% P
yrex
ia in
hib
itio
n
(Mea
n S
EM
)
S a line 1
0 (ml/k
g )
P a r a c e ta mo l 1
0 (mg /k
g )
B1 0 0
B2 0 0
B3 0 0
L1 0 0
L2 0 0
L3 0 0
S C1 0 0
S C2 0 0
S C3 0 0
P C1 0 0
P C2 0 0
P C3 0 0
0
2 0
4 0
6 0
8 0
n s*
**
**
**
**
**
**
**
****
**
**
E x t r a c t c o n c e n t r a t io n ( m g /k g )
Fig.4.18c.
A n t ip y r e t ic a c t iv it y
a f t e r 4 h r s
% P
yr
ex
ia i
nh
ibit
ion
(Me
an
SE
M)
Sa li
ne 1
0 (ml/
kg )
Pa r a c e ta
mo l
1 0 (mg /k
g )
B1 0 0
B2 0 0
B3 0 0
L1 0 0
L2 0 0
L3 0 0
SC
1 0 0
SC
2 0 0
SC
3 0 0
PC
1 0 0
PC
2 0 0
PC
3 0 0
0
2 0
4 0
6 0
8 0
n s
**
**
**
**
**
**
**
****
****
**
E x t r a c t c o n c e n t r a t io n ( m g /k g )
Fig.4.18d
A n t ip y r e t ic a c t iv i t y
a f t e r 5 h r s
% P
yr
ex
ia i
nh
ibit
ion
(Mea
n
SE
M)
S a line 1
0 (ml/k
g )
Pa r a c e ta
mo l 1
0 (mg /k
g )
B1 0 0
B2 0 0
B3 0 0
L1 0 0
L2 0 0
L3 0 0
S C1 0 0
S C2 0 0
S C3 0 0
PC
1 0 0
PC
2 0 0
PC
3 0 0
0
2 0
4 0
6 0
8 0
**
**
**
**
**
**
**
n s
n s n s ***
**
E x t r a c t c o n c e n t r a t io n ( m g /k g )
Fig.4.18e
Figs. 4.18a-4.18e. Antipyretic activity of A. nitida. Bars represent percent antipyretic activity mean values ± SEM (n=5).
One-way ANOVA followed by Dunnett’s multiple comparison test was used to find significance at**p<0.01 and
*p<0.05 compared to –ve control (normal saline). ns= non significant, B=Bark, L=Leaf extract, SC=Staminate catkin
extract, PC= Pistillate cone extract
133
4.6.4. In vitro cytotoxic activity
In vitro cytotoxicity of A. nitida extracts was tested by microscopic observation of
cell morphology at different concentrations of each extract as shown in Table.4.21.
Cytopathic effects (CPE) observed (under inverted microscope) include rounding,
detaching from flask surface resulting in free floating of cells, cells clumping. Some
apoptosis like CPEs were also observed after incubation. Cell culture and cytopathic effects
shown by the extracts are shown in Figs 4.19a-d and Figs 4.20a-c. Cytopathic effects were
increased gradually with increase in extract concentration. Highest CPEs were shown by
bark and catkin extract at 1000 µg/ml. Minimum CPEs were observed for each extract at
31.25 µg/ml in which bark extract showed the lowest CPEs (Table.4.21)
Table. 4.21. In vitro cytotoxic activity of A. nitida extracts.
Microscopic observations. (Cytopathic effects scoring).
Concentration (µg/ml) B L SC PC
31.25 1 1 1 1
62.5 1 1 1 2
125 2 2 2 2
250 2 2 2 3
500 3 3 3 3
1000 3 3 3 3
Extract control 0
Note: CPE scores; 4= 75 to 100%, 3 = 50 to 75%, 2= 25–50%, 1 = 0– 25% and 0 = 0%. B, Bark; L, Leaf;
SC, Staminate catkin; PC, Pistillate cone.
134
Fig.4.19a.BHK 21 Cells after trypsinization Fig.4.19b.BHK 21 cells after splitting and
Subculture
Fig.4.19c. Cells attachment with flask surface Fig.4.19d. Rapidly Dividing Cells
Fig. 4.19a-d. BHK 21 cell culture.
135
Fig.4.20a. Apoptosis like CPEs
Fig.4.20b. Cells detachment from
surface and rounding
Fig. 4.20c. Clumping of cells
Figs. 4.20a- 4.20c. Cytopathic effects (CPEs) of A. nitida on BHK 21 Cell line
136
Control with only GMEM
B1 L1 SC1 PC1
Fig. 4.21. BHK 21 cells culture with 31.25 µg/ml concentration of the bark (B1), leaf (L1), staminate catkin (SC1) and
pistillate cone (PC1) extracts after 48 hours.
B2
L2
SC2
PC2
Fig. 4.22. BHK 21 cells culture with CPEs at 1000 µg/ml concentration of the bark (B2), leaf (L2), staminate catkin (SC2)
and pistillate cone (PC2) extracts showing cytotoxicity of A. nitida extracts (After 48 hours).
137
MTT Assay is a colorimetric method based on tetrazolium salt, used to test in vitro
cell viability. It helps to indicate cytotoxicity, anti-proliferative and cell activation potency
of drugs, environmental pollutants and toxic compounds (Lupu & Popescu, 2013). In
viable cells oxido-reductases present in cytoplasm, mitochondria and cell membrane
converts MTT into measureable formazan (Berridge et al., 2005). In MTT assay highest
and highly significant (p<0.01) % cell viability of 87.7±3.22, 86±5.24, 85.1±2.55 and
82.2±1.0 was observed for SC (catkin), B (bark), L (leaf) and PC (Pistillate cone) extracts
respectively at 31.25µg/ml. At concentrations of 62.5,125,250,500 and 1000 µg/ml the %
cell viability for the bark (B) and staminate catkin (SC) extracts were 83±1.67, 74±2.55,
63±4.41, 47±2.55 and 37±3.33%; and 77. ±4.02, 68.3±4.51, 61.3±1.67, 48.8±3.47 and
37.2±3.25% respectively. While at these concentrations of the leaf (L) and pistillate cone
(PC) extracts the % cell viability noted were 77±4.02, 68.3±4.51, 61.3±1.67, 48.8±3.47
and 37.2±3.25; and 72.4±3.3, 64.4±2.5,
49.4±3.5, 43.3±2.5 and 32.6±3.1% respectively (Table.4.22). The IC50 values for the B, L,
SC and PC extracts were 152, 118.2, 119.4 and 93.05 µg/ml respectively (Fig.4.24a-4.24d).
Highly significant (p<0.01) differences in cell viability caused by A. nitida extracts
compared to control is shown in Fig. 4.23.
Table. 4.22. Cytotoxicity of the bark, leaf, staminate catkin and pistillate cone extracts of A. nitida against BHK 21cells
Concentration
(μg/ml)
% Cell viability (Mean ± SD)
B (Bark) Leaf (L) Catkin (SC) PC (Cone)
31.25 86±5.24 85.1±2.55 87.7±3.22 82.2±1.0
62.5 83±1.67 77.±4.02 79.1±3.84 72.4±3.3
125 74±2.55 68.3±4.51 70.8±3.82 64.4±2.5
250 63±4.41 61.3±1.67 62.2±3.47 49.4±3.5
500 47±2.55 48.8±3.47 47.2±4.19 43.3±2.5
1000 37±3.33 37.2±3.25 40.0±2.89 32.6±3.1
138
Fig.4.23. Differences in cell viability, caused by A. nitida extracts. Bars represent % cell viability mean ±SD of three
replicates. Data was analysed by One-way ANOVA with Dunnett’s post hoc comparison test by Graph pad prism
6.01 version. All plotted data was significantly different from control with P<0.01.
139
Cytotoxicity of bioactive agents is determined to select their non cytotoxic
concentration for biological activities. Such studies are also helpful to explore the cytotoxic
agents that can help to combat cancer cells. The Baby Hamster Kidney-21 fibroblast cell line
(BHK 21) has been used extensively to evaluate cytotoxic effects of compounds, extracts
and drugs etc. in anticancer drug discovery research (Mekawey et al., 2009; Ankita &
Chauhan, 2012; Zhou et al., 2012; Bisht et al., 2014). The efficacy of cytotoxic agents is
determined from the mode of cells death (Fisher, 1994). Researches are more focused on
compounds from plants that can influence apoptosis and in mechanism of their action (Jin-
Mu et al. 2003). Cells go through specific biochemical and morphological features during
apoptosis such as aggregation of chromatin, condensation of nucleus and cytoplasm and
membrane bounded vesicles formation (Kerr et al., 1972). In present study all extracts of A.
nitida have shown dose dependent cytotoxicity. The bark and pistillate cone extracts have
shown apoptosis and significant (p<0.05) cytotoxicity at 1000µg/ml. Phytochemical studies
I n v i t r o c y t o t o x ic i t y o f
A . n it id a b a r k (B )
L o g 1 0 C o n c .
% C
ell
in
hib
itio
n
0 1 2 3
0
2 5
5 0
7 5
1 0 0L o g I C 5 0 = 2 .1 8 2
I C 5 0 = 1 5 2 g /m l
R2= 0 .9 5 5 1
I n v i t r o c y t o t o x ic i t y o f
A . n it id a le a f (L )
L o g 1 0 C o n c .
% C
ell
in
hib
itio
n
0 1 2 3
0
2 5
5 0
7 5
1 0 0
L o g I C 5 0 = 2 .0 7 2
I C 5 0 = 1 1 8 .2 g /m l
R2= 0 .9 6 4 0
Fig. 4.24a Fig. 4.24b
I n v i t r o c y t o t o x ic i t y o f
A . n it id a s t a m in a te c a t k in (S C )
L o g 1 0 C o n c .
% C
ell
in
hib
itio
n
0 1 2 3
0
2 5
5 0
7 5
1 0 0
L o g I C 5 0 = 2 .0 7 7
I C 5 0 = 1 1 9 .4 g /m l
R2= 0 .9 6 3 6
I n v i t r o c y t o t o x ic i t y o f
A . n it id a p is t i l l a t e c o n e ( P C )
L o g 1 0 C o n c .
% C
ell
in
hib
itio
n
0 1 2 3
0
2 5
5 0
7 5
1 0 0L o g I C 5 0 = 1 .9 6 9
I C 5 0 = 9 3 .0 5 g /m l
R2= 0 .9 7 7 6
Fig. 4.24c. Fig. 4.24d.
Figs.4.24a- 4.24d. IC50 values of A. nitida extracts against BHK21 cells.
140
of the B, L, SC and PC extracts have revealed the presence of various secondary metabolites
such as flavonoids, phenols, tannins and sterols as mentioned in Table.4.16.
The phytochemicals present in aqueous ethanolic extract of B, L, SC and PC may
be responsible for the observed cytotoxic effects. Many workers have reported cytotoxicity
of different plant extracts and their compounds including Alnus species, against various
cell lines.
The present study revealed highest(p<0.01) cytotoxicity by the A. nitida pistillate
cone extract on BHK21 Cell line , with IC50 value of 93.05 µg/ml which is higher than the
IC50 values reported by Stevic et al (2010) for the cone extracts of A. viridis
(IC50=39.9µg/ml) and A. incana (IC50=47.4 µg/ml) on Hela cell line. Similarly, in our
present study the IC50 values noted for the cytotoxicity of L (118.2µg/ml), B (152 µg/ml)
and PC (119.4 µg/ml) were higher than the IC50 values of Alnus species reported for the
leaf (Stevic et al., 2010) and bark extracts (Sajid et al., 2019) against cancer cell lines; and
staminate catkin extract against VERO (African green monkey kidney) and HEK293
(human embryonic kidney cells lines in MTT assay (Swiatek et al., 2013). Lee et al., 1992
have also reported significant cytotoxic effects for A. hirsuta, and A. japonica extracts
against cancer cell lines.
Differences in IC50 values of extracts are also dependent on the type of cells. As
bark extract of A. nitida is reported for having lower IC50 values against cancer cell lines
(sajid, et al., 2019). It shows that the IC50 values of A. nitida extracts can be different on
different cell lines. Thus, we can expect a better result with lowest IC50 values of these
sample extracts on variety of cancer cell lines. All these studies are in agreement with our
present findings and reveals the remarkable potential of Alnus species as anticancer agents.
The present work on A. nitida will aid in selection of aqueous ethanolic extract
concentration of the studied plant parts for evaluation of their biological potential. The
present study, has revealed the cytotoxic potential of the studied extracts. The cytotoxic
value of the studied extracts can be depicted from the CPEs produced in present assay
(Figs.4.20a- 4.20c).
141
4.6.5. Antiviral Activity
Antiviral potential of A. nitida was determined by observation of cytopathic effects
under microscope as well as by MTT assay. Based on result of cytotoxicity of the
A. nitida extracts against BHK 21 cell lines, MNTC of 15.6,7. 8 ,3.9, 1.95 and 1 µg/ml of
each extract was used in antiviral activity against FMDV. TCID50 (105.75 /ml) for the
FMDV was determined (Fig.4.25). 10 TCID50 dose of virus was used for this experiment.
CPE caused by FMDV on BHK21 cells include rounding (Fig.4.26d), detachment of cells
from flask wall and floating (Fig. 4.26e), clumping (Fig.4.26 f) swelling indicating virus
replication (Fig.4.26g) and lysis (Fig.4.26 h) of cells. Negative control, showed no CPE. It
was a healthy monolayer of cells (Fig. 4.26i), not detached from flasks.
MTT assay showed highest and significant (p<0.05) cell protection of 57.6±2.8%
for the bark (B) extract at concentration of 15.6µg/ml, followed by 45±1.3%, 38±1.3%,
15±3% and 10±3.9% at concentrations of 7.8, 3.9, 1.95 and 1µg/ml. Leaf (L) extract
showed highest and significant (p<0.05) cell protection of 51.7±0.8% at 15.6µg/ml,
followed 49.7±2.5%, 36±1.45%, 32±1.9% and 29±1.5% protection of cells at
concentrations of 7.8, 3.9, 1.95 and 1µg/ml (Table. 4.23). Similarly, the staminate catkin
(SC) and pistillate cone extracts showed cell protection of 54.4±4.12, 45.9±2.78, 33.9±0.9,
14.35±4.1, 9.38±3.3 and 51.84±1.16, 42.45±4.02, 33±2.3, 23.8±3.25, 20.59±2.4 % at
concentrations of 15.6, 7.8 ,3.9, 1.95 and 1 µg/ml respectively (Table.4.23). ANOVA (one
way) with Dunnett’s post hoc multiple comparison test showed all samples were
significantly different from both controls with p<0.05(Fig.4.27). Highest antiviral activity
was shown by B extract with EC50 of 3.8μg/ml, followed by the SC, PC and L with EC50
values of 3.87, 4.8 and 5 μg/ml respectively (Fig. 4.28a-4.28e).
142
Fig. 4.25. TCID50 determination of FMDV
Table. 4.23. Antiviral activity of A. nitida extracts against FMD virus.
Concentration
(μg/ml) % Cell Viability (Mean ± SD)
B (Bark) Leaf (L) Staminate
catkin (SC)
Pistillate cone
(PC)
1 9.6±2.94 28.97±1.51 9.38±3.3 20.59±2.40
1.95 15±2.99 32.46±1.9 14.35±4.1 23.8±3.25
3.9 38.4±1.3 36±1.45 33.9±0.9 33.06±2.30
7.8 45.94±1.34 49.76±2.54 45.9±2.78 42.45±4.02
15.6 57.6±2.82 51.7±0.87 54.4 ± 4.12 51.84±1.16
Control V 0
Control M 100±0.02
FMD is a transmissible infection caused by a Picnovirus, FMDV (Ayers et al.,
2001). It is endemic to Africa and Asia including Pakistan. It causes enormous harms
to live stock (Davies, 2002; Klein et al., 2008). Several studies have been conducted
on plant extracts and different compounds from plants to find their antiviral effects on
FMDV (Dibarka, 2011). Myrtle oil was reported for antiviral effects on FMDV (Najafi
et al., 2011). Similarly, antiviral activity is reported for many Alnus species.
Flavonoids and triterpenoids from Alnus firma leaf methanolic extract have inhibited
143
essential enzymes as well as replication of HIV-1(Yu et al., 2007). Similarly, betulinic
aldehyde isolated from A. japonica is reported for significantly higher antiviral
potential with EC50 value of 12.5 µg/ml against influenza virus (Tung et al., 2010a).
In present study the B (bark), L (leaf), SC (staminate catkin) and PC (pistillate catkin
with seeds) extracts of A. nitida have shown dose dependent antiviral effects against
FMDV.
144
a. Trypsinized cells b. Monolayer formation c. Appearance of viral
CPEs
d.50% Cells rounding
e. Cells complete
rounding and detachment
f. Cells clumping g. Cells swelling h. Cell lysis
i. Healthy dividing
cells
j. Formazon crystals
Figs. 4.26a-4.26j. Antiviral activity of A. nitida extracts (CPEs of FMDV on BHK21cell line.
145
C o n c e n t r a t io n s ( µ g /m l)
% P
ro
te
ctio
n
Con
trol V
Con
trol M
B(1
)
B(1
.95)
B(3
.9)
B(7
.8)
B(1
5.6
)
L(1
)
L(1
.95)
L(3
.9)
L(7
.8)
L(1
5.6
)
SC
(1)
SC
(1.9
5)
SC
(3.9
)
SC
(7.8
)
SC
(15.6
)
PC
(1)
PC
(1.9
5)
PC
(3.9
)
PC
(7.8
)
PC
(15.6
)
0
2 5
5 0
7 5
1 0 0
Fig.4.27. Antiviral activity of A. nitida against FMD virus. Data presented in graph are
mean±SD of three replicates. Data was analyzed using ANOVA (one way) with Dunnett’s
comparison test and p<0.05 was considered significant. All samples were significantly
different (p<0.05) from both control V and control M. Control V= -ve control, containing
virus with no extract. Control M=+ve Control with only growth media.
A n t iv ir a l a c t iv it y o f
A . n it id a b a r k (B )
L o g ( C o n c e n t r a t io n )
% P
ro
te
ctio
n
0 .0 0 .5 1 .0 1 .5
0
2 5
5 0
7 5
1 0 0
L o g E C 5 0 = 0 .5 8 1 1
E C 5 0 = 3 .8 1 2
R2
= 0 .9 6 4 7
g /m l
A n t iv ir a l a c t iv it y o f
A . n it id a le a f (L )
L o g ( C o n c e n t r a t io n )
% P
ro
te
ctio
n
0 .0 0 .5 1 .0 1 .5
0
2 5
5 0
7 5
1 0 0
L o g E C 5 0 = 0 .6 9 8
E C 5 0 = 5 .0 g /m l
R2= 0 .9 6 8 8
Fig.4.28a Fig.4.28b
A n t iv ir a l a c t iv it y o f A . n it id a
S ta m in a te c a tk in ( S C )
L o g ( C o n c e n t r a t io n )
% P
ro
tec
tio
n
0 .0 0 .5 1 .0 1 .5
0
2 5
5 0
7 5
1 0 0
L o g E C 5 0 = 0 .5 8 8 0
E C 5 0 = 3 .8 7 2
R2= 0 .9 8 7 1
A n t iv ir a l a c t iv it y o f A . n it id a
p is t i l la t e c o n e ( P C )
L o g ( C o n c e n t r a t io n )
% P
ro
te
ctio
n
0 .0 0 .5 1 .0 1 .5
0
2 5
5 0
7 5
1 0 0
L o g E C 5 0 = 0 .6 8 4 0
E C 5 0 = 4 .8 3 0
R2= 0 .9 6 4 2
Fig.4.28c. Fig.4.28d. Figs. 4.28a-4.28d. Percent protection and EC50 determination of A. nitida extracts against FMDV.
146
4.6.6. Aflatoxin degradation activity
In present study the aflatoxin degrading potential of B (bark), L (leaf), SC
(staminate catkin) and PC (Pistillate catkin with seeds) extracts of A. nitida was
investigated. The extracts (un-spiked) were found free of aflatoxin (Table. 4.24), when
spiked with aflatoxin standard (2.06 µg/ml), were incubated for 48 hrs. The bark extract
showed degradation of 40 and 65% while the pistillate cone extract showed degradation of
30 and 50% at concentrations of 500 and 1000ppm respectively; however, the leaf and
staminate catkin extracts showed no reduction in aflatoxin compared to its standard control
(Table.4.25).
WHO (1979) has reported 17 types of aflatoxins; found in animals, soil, plants,
peanuts, rice, walnuts and soybean etc (Begum & Samajpati, 2000). Aflatoxin B1,
produced by Aspergillus flavus and Aspergillus parasiticus is highly toxic to many species.
It has blue fluorescence under UV light (FAO, 1990). Food and feed contaminated with
lower levels of AFB1 can cause serious health problems, when taken in large quantities
(Wild et al., 1992). Williams et al. (2004) reported AFB1 (Aflatoxin B1), as the most
powerful hepatocarcinogen identified to man. 4 to 30 ppb of AFB1 are acceptable range
for different countries in human food (Williams et al., 2004).
Table.4.24. Samples for Aflatoxin B1 (AFB1) degradation activity
Control Aflatoxin B1 (ppb)
Unspiked bark extract 0.00
Unspiked leaf extract 0.00
Unspiked staminate catkins extract 0.00
Unspiked pistillate cones Extract 0.00
Standard A 20.6 ppb
Standard B 18.5 ppb
Standard C 14.42ppb
Standard D 12.36 ppb
Standard E 10.3ppb
Standard F 7.21 ppb
147
Table. 4.25. Degradation of AFB1 by ethanolic extracts of A. nitida.
Sample
(Spiked extract)
Extract
Concentration
(μg/ml)
AFB1
Recovered
(ng/ml)
% AFB1
Degradation
B(bark) 100 18.5 10%
500 12.36 40%
1000 7.21 65%
L(leaf) 100 20.6 0%
500 20.6 0%
1000 20.6 0%
SC (staminate catkin) 100 20.6 0%
500 20.6 0%
1000 20.6 0%
PC (pistillate cone) 100 18.5 10%
500 14.42 30%
1000 10.3 50%
Control (AFB1 Standard A) 0 20.6 0%
Fig.4.29. Aflatoxin B1 degradation by different concentrations of bark (B), leaf (L), staminate
catkin (SC) and pistillate cone extract of A. nitida. Bars represent mean±SEM (n=3).
148
Various methods have been tried to detoxify the food and feed contaminated with
mycotoxin (Blunt, 2006). Several compounds including, oxidizing agents, gases, acids,
bases and bisulphites can react and change aflatoxin to less harmful form (Dollear et al.,
1968; Mann et al., 1970; Mendez-Albores, 2007). Many plants extracts and compounds
such as phenol-propanoids, terpenoids and alkaloids are also reported for inhibitory effects
on biosynthesis of aflatoxin (Holmes et al., 2008).
Aflatoxins structure consists of cyclopentene ring with furan moiety. The toxicity
and carcinogenic effects of AFB1 are mainly due to double bond in its terminal furan ring
(Wang et al., 2011). Studies on the degraded products of AFB1 has revealed decrease in
both florescence and toxicity of aflatoxins on cleavage of its lactone ring (Lee et al., 1981).
Thus, the degraded product with reduced fluorescence may also have no or less toxicity.
This will require its further exploration.
In present work the B (bark) and PC (pistillate catkin with seeds) extracts have
shown degradation of AFB1. Our results are in agreement with Aviala et al., (2016) who
has reported AFB1 degradation by the leaf extract of Cymbopogon citratus and Irum et al
(2016) who reported highly significant degradation of AFB1 (90.4%) by the leaf extract of
Ocimum basilicum. Similarly, Al-Saidy et al (2014) reported degradation of aflatoxin B1
by extracts of Thymus vulgaris, Cinnamomum zylanicum and Syzygium aromaticum. The
leaf extracts of Adhatoda vasica have also shown 95% degradation of AFB1 after 24 hours
incubation (Vijayanandraj et al., 2014). These plant extracts were reported for having
important phytochemicals which are also found in the extracts of A. nitida (Table.5.5.). A.
nitida B and PC spiked extracts degraded aflatoxin B1, detected by its significantly reduced
blue fluorescence compared to control standard. The active constituents of A. nitida B and
PC extracts may have interacted with active groups in AFB1 and disrupted its active ring,
that resulted in AFB1 destruction or conversion into other compounds. Further exploration
of the active constituent(s) of the bark and cone extracts of A. nitida may provide natural,
safe /less harmful, cost effective, easily available, efficient and ecofriendly agents for
degradation of AFB1.
149
4.6.7. Phytotoxic Activity
Weed control is also a problem in Pakistan. Weeds cause great losses to crops
through utilization of the available resources for crop growth. Synthetic herbicides have
shown harmful effect on human body and polluted our soil and water (Barkatullah et al.,
2011). Plant based herbicides are needed to eliminate weeds and protect living organisms
and environment from pollution. Plant produce some metabolites (phytochemicals) which
protect them from hostile organisms. These metabolites cause death, chlorosis or wilting
of antagonistic plants (Lungu et al., 2011). Plants extracts with selective phytotoxic
potential can be used for development of novel herbicides (Parvez et al., 2014).
Lemna minor bioassay is a simple, cost effective and easy method that can help to
explore phytotoxic constituents of plants (Hussain et al., 2010b; Ayatollahi et al., 2010).
In present study Lemna minor bioassay was used to check phytotoxicity of A. nitida
(Spach) Endl. Results of phytotoxic assay are presented in Table.4.26.
Table.4.26. Phytotoxic activity of A. nitida plant extract in Lemna minor assay.
Sample Concentration
(µg/ml)
No. of
fronds
survived
% Growth
inhibition
-ve Control (Medium
only)
0.0 30 0
B(bark) 50 22±1.50 27±2.9
500 18±1.53 40±3.0
1000 6±1.00 80±2.9
L(leaf) 50 27±1.00 10±1.9
500 24±1.50 20±3.0
1000 18±1.00 40±2.9
SC (staminate catkin) 50 00±0.00 00±0.0
500 24±1.53 20±2.9
1000 19±2.52 37±3.0
PC (pistillate cone) 50 25±1.53 17±4.8
500 19±2.08 37±2.9
1000 5±2.52 83±4.0
150
The extracts showed dose dependent phytotoxicity. The highest % growth
inhibition was shown by the pistillate cone extract (83±4.0%), followed by bark (80±2.9%),
leaf (40±2.9%) and staminate catkin extracts (37±3.0%) at 1000 µg/ml as compared to
control. From these results it is concluded that A. nitida pistillate cone, bark, leaf and
staminate catkin extracts can be a great source of phytotoxic constituents and further
studies may result in development of new, natural and useful herbicides (weedicides).
Other workers have also investigated and reported phytotoxic potential of different plant
extracts in Lemna minor assay such as, Khurm et al (2016), have reported phytotoxic
potential for Heliotropium strigosum. Barkatullah et al (2015a), have reported phytotoxic
activity for extract of Callicarpa macrophylla and phytotoxic effects are also reported at
1000µg/ml for extracts of Zizyphus jujube by Ahmad et al, (2011b). These findings also
support the results of present work of plants extracts toxicity against Lemna minor.
4.6.8. Antioxidant Activity
Free radicals oxidize biological molecules which lead to cancer, early aging and
heart diseases (Siriwardhana et al., 2003). Antioxidants reduce the detrimental effects of
free radicals. DPPH free radical scavenging assay is a simple and fast method for
antioxidants evaluation through spectrophotometer (Huang et al., 2005).The free radical
nature of DPPH is neutralized by antioxidants through hydrogen / electron transfer (Leong
& Shui, 2002). Natural antioxidants are comparatively safer than synthetic ones used in
food processing, medicines and cosmetics. A number of medicinal plants have shown
antioxidant activity (Krishnaiah et al., 2011; Sati et al., 2011). DPPH (2, 2- diphenyl-1-
picrylhydrazyl) is reduced by antioxidants present in plant extracts, making its solution
colorless.
In present research the 70% Ethanolic extracts of A. nitida (bark, leaf, staminate
catkin and pistillate cone) were evaluated for free radical scavenging ability. The
scavenging potential of the extracts was revealed by reduction in colour of DPPH. Results
of DPPH free radical scavenging are displayed in Table.4.27.
151
All extracts showed dose dependent increase in DPPH scavenging activity. Highest
and significant (p<0.05) % scavenging ability for the bark (95.8±0.77), pistillate cone
(95.6±0.9) and leaf (94±2.3) extracts was recorded at 120 µg/ml of the extracts. Staminate
catkin extract showed highest (93±1.2) and significant (p<0.05) % scavenging of DPPH at
100 µg/ml . Significantly higher (p<0.05) DPPH free radical scavenging by each of A.
nitida extract compared to control as well as significant (p<0.05) differences in their
antioxidant activity is shown in Fig.4.30. The IC50 values were calculated from the curve,
by plotting % scavenging of DPPH against log conc. of the samples in Graph pad prism
version 6.01. IC50 values of B, L, SC and PC were 49.47, 42.56, 56.59 and 52.94µg /ml
respectively.
Table. 4.27. % Antioxidant (DPPH Scavenging) activity of A. nitida
Concentration
(µg/ml)
% DPPH Scavenging (Mean±SEM)
B(Bark) L(Leaf) SC (Staminate
catkin)
PC (Pistillate
cone)
20 38±0.588 40±1.176 28±0.89 36±1.2
40 55.6±0.89 66.6±0.89 47±1.17 54±0.56
60 77.6±1.48 78.6±0.9 61±1.2 69±1.2
80 86±1.17 89±0.59 78±0.59 86±1.2
100 95±0.89 92.6±1.48 93±1.2 94±1.5
120 95.8±0.77 94±2.3 91.6±0.9 95.6±0.9
Control
(without extract)
0±0.00
152
Fig.4.30. Antioxidant activity of A. nitida. Bar represent % antioxidant activity mean±SEM (three
replicates) of the plant samples at given concentration. One-way ANOVA with Turkey’s multiple
comparison test was used to find significant difference at p<0.05. Bars with same superscript have no
significant difference (i.e.>0.05). All bars connected by a line with superscript “ j ˮ have non significant
difference (i.e. p >0.05). Two bars connected by a line with superscript f also have no significant difference
(i.e. p >0.05). B= Bark; L=Leaf; SC=Catkin; PC= (Pistillate catkin with seeds)
Plant extracts as well as isolated compounds (such as hirsutenone and oregonin)
from other species of the genus Alnus are also reported for significant antioxidant potential
(Ren et al., 2017). The cone, leaf and bark extracts of A. viridis and A. incana have shown
highly significant DPPH free radical scavenging activity with the range of IC50 values
from 3.3 to 18.9 μg/ml (Stevic et al., 2010). Similarly, free radical scavenging activity is
reported for the bark extracts of A. glutinosa and A. nitida (Altınyay, et al., 2016; Sajid et
al., 2016).
The extensively found plant secondary metabolites with antioxidant potential are
phenolic and flavonoid compounds (Wang et al. 2008). Flavonoids, usually present in
pollens, leaves and flowering tissue (Larson, 1998) are significant antioxidants and very
effectual scavengers of nearly all kinds of oxidizing molecules including a variety of free
radicals and singlet oxygen (Bravo, 1998). Stevic et al. (2010) suggested that antioxidant
potential of A. viridis and A. incana might be due to their triterpenoids and diarylheptanoids
contents. The ethanolic (70%) extracts of A. nitida used in present study have also shown
the presence of phenolic, flavonoids, tannins and triterpenoids etc. as mentioned earlier in
Table. 4.16. The highly significant antioxidant activity of these extracts may be attributed
153
to the presence of these bioactive secondary metabolites present in them. Results of present
work showed that A. nitida bark, leaf, catkin and pistillate cone (pistillate catkin with seeds)
are very rich sources of antioxidants and can be exploited for useful antioxidant drug
preparation on commercial scale.
154
CONCLUSIONS
• Alnus nitida is a deciduous tree, abundantly found in Pakistan in its natural habitat.
It is most commonly used for wood and as soil binder. It is frequently growing
species with no threat of extinction. It is usually found at elevation of 1000- 3000m
above sea level and locally named as Geiray. Traditionally its bark and leaf has
been used to treat inflammation and pain.
• Macroscopic studies determined the morphological features including ranges in
size, color, odour fracture, texture etc. of the plant sample as distinguishing
characters for identification.
• Microscopic study of leaf showed the presence of giant and normal, anomocytic
stomata with striated cuticle, found abundantly on abaxial epidermis.
• Leaf surface values, vein termination and vein islet number, number and size of
stomata, presence of trichomes including peltate glandular trichome and aduncate
type of trichome, 4-5 celled bases of peltate glandular trichome and the pollens
with 4-5 numbers of pores are some distinguishing pharmacognostic characters of
A. nitida leaf.
• All samples showed characteristic fluorescence under UV light, showed specific
extractive values based on the nature of compounds present, specific ash values,
nutritional and elemental composition.
• Phytochemical screening showed the presence of phenols, flavonoids, tannins,
sterols, saponins, and triterpenoids. Quantitatively phenols were most abundantly
found class of compounds in B, L and PC. Sterols were dominant in staminate
catkins.
• All samples have shown highly significant analgesic, anti-inflammatory and
antipyretic activities, validating its traditional therapeutic use in pain and
inflammation.
155
• Cytotoxic activity of the sample showed, remarkable CPEs, showing the possible
potency of A. nitida extracts to combat cancer cells.
• Samples, of A. nitida have also shown anti FMDV activity, necessitating further
study to isolate the bioactive constituent(s) in these extracts causing the observed
effects.
• The bark and leaf sample of A. nitida have significantly reduced the blue
fluorescence of AFB1 (under UV light) showing its potential to degrade Aflatoxin
B1.
• A. nitida samples have shown significant phytotoxicity against Lemna minor plant
and have significant DPPH scavenging activity which can be attributed to its higher
phenolic and flavonoid contents. It can be depicted that constituent of A. nitida can
provide protection against oxidative damage of free radicals.
Following are the novel and most important conclusions drawn from present
research work.
• From the micromorphological investigation it is confirmed that A. nitida leaf has
anomocytic type of giant stomata on the abaxial surface at least 20% larger in size than
its normal sized stomata, solitary, having larger distance from smaller stomata and
have noticeable cuticular striae around or lateral to the guard cells that can be used as
diagnostic features for its identification.
• Powder drug of the plant has some specific fragments and fluorescence behaviours
and ash values which provide important tool for accurate identity, authenticity and
standardization of plant based crude drugs.
• The present results for the size range of epidermal cells, hypostomatic leaf, presence
of more conspicuous cuticular striations perpendicular to stomata on abaxial leaf
surface and presence of non- glandular and peltate glandular trichome on both surfaces
of leaf can be used as tools for identification of the A. nitida leaf. In addition, these
156
features also confirm similarity of A. nitida to the leaf fossil remains of A. gaudinii
(Heer) Knobloch et Kvacek, depicting its close phylogenetic and taxonomic
relationship with this Alnus species.
• Aqueous ethanolic extract of A. nitida contains various phytochemicals including
carbohydrates, protein, alkaloid, saponins, flavonoids, phytosterols, triterpenoids,
tannins, phenol, anthocyanins, volatile oil, steroidal glycosides, fixed oil and fats.
• A. nitida contains the useful elements Fe, Zn, Mn, Mg, Cu, Na, Ca and K. In addition,
carbohydrates contents are adequate in staminate catkin and leaf while bark and cone
were rich in crude fibers.
• Results of the present studies confirm the remarkable potential of aqueous ethanolic
extract of A. nitida as more economical and accessible source for analgesic, anti-
inflammatory, antipyretic, anti foot and mouth disease virus, antiaflatoxin, cytotoxic,
phytotoxic and remarkable antioxidant agents.
157
RECOMMENDATIONS
In light of current findings, the following recommendations are provided to assist
in future research work.
• The present study revealed valuable information about the native, easily grown A.
nitida (Spach)Endl. with stable population trend that can provide valuable, cost
effective and easily accessible ingredients for medicinal or industrial uses. However,
large scale trials are recommended for isolation of its active constituents and their
mechanism of action on both animals and cell lines for the explored bioactivities to
ensure its safety along with efficacy.
• The present study showed highly significant results of A. nitida against pain,
inflammation and fever. These results validated the ethnobotanical usage of the plant
against pain and inflammation and predicts the presence of more efficient bioactive
compounds responsible for the revealed results that interrupted the related mechanism
responsible for these distresses. However, isolation of responsible compounds and its
safety for health must be ensured through further investigation at molecular level.
• The remarkable antioxidant and aflatoxin B1 degradation properties depicts the
preservative potency of these extracts. But novel investigations should be focused on
the effects of these extracts on food safety and quality as well as the safety of degraded
products of AFB1 for both human and animal consumption.
• The morphological features, leaf constant values, fluorescence behaviors and
physiochemical properties documented in present study must be referred to set
standards for correct identification, quality assessment and detection of any adulterant,
prior to exploitation of this plant for medicinal or other commercial purposes.
158
• A. nitida is a source of important phytochemicals that can be employed in food,
cosmetics and pharmaceutical industries; but the heavy metal contents must be
checked before utilizing parts of this plant for its useful purposes.
159
REFERENCES
Abbasi, A.M., M. Khan, M. Ahmad, M. Zafar, S. Jahan and S. Sultana. 2010.
Ethnopharmacological application of medicinal plants to cure skin diseases and in
folk cosmetics among the tribal communities of North-West Frontier Province,
Pakistan. J. Ethnopharmacol.,128(2): 322-335.
Abbott, B.J., J. Leiter, J.L. Hartwell, M.E. Caldwell, J.L. Beal, R.E. Perdue Jr. and S.A.
Schepartz. 1966. Screening data from the Cancer Chemotherapy National Service
Center Screening Laboratories XXXIV. Plant extracts. Cancer Res., 26:761–928.
Abdullahi M. N., N. Ilyas, I. Hajara, and Y.M. Kabir.2018. Pharmacognostic evaluation of
the leaf of Microtrichia perotitii DC. (Asteraceae). J. Pharmacognosy
Phytother.,10(4):76-84. DOI: 10.5897/JPP2018.0490
Abedini, A., S. Chollet, A. Angelis, N. Borie, J.M. Nuzillard, A.L. Skaltsounis, R.
Reynaud, S.C. Gangloff, J.H. Renault and J. Hubert. 2016. Bioactivity-guided
identification of antimicrobial metabolites in Alnus glutinosa bark and optimization
of oregonin purification by centrifugal partition chromatography. J. Chromatogr.
B.,1029:121–127.
Abighor, R. A., E. Okpe, M. E. Bafor, P. O. Udia and A. Osagie. 1997. The
physicochemical properties of the seed and seed oil of Jatropha curcas. L. Riv. Ital.
Grass., 74: 465-466.
Acero, N., and D. Muñoz-Mingarro. 2012. Effect on tumor necrosis factor-α production
and antioxidant ability of black alder, as factors related to its anti-inflammatory
properties. J Med Food ,15: 542-548.
Achi, N.K., C. Onyeabo, C. A. Ekeleme-Egedigwe and J. C. Onyeanula. 2017.
Phytochemical, Proximate Analysis, Vitamin and Mineral Composition of Aqueous
Extract of Ficus capensis leaves in South Eastern Nigeria. J. Appl. Pharm. Sci., 7
(03):117-122.
160
Afsar, T., M. R. Khan, S. Razak, S. Ullah and B. Mirza. 2015. Antipyretic, anti-
inflammatory and analgesic activity of Acacia hydaspica R. Parker and its
phytochemical analysis. BMC Complem. Altern. Med., 15:136.
http://dx.doi.org/10.1186/s12906-015-0658-8
Agostoni, C., S. Trojan, R. Bellu, E. Riva and M. Giovannini. 1995. Neurodevelopmental
quotient of healthy term infants at 4 months and feeding practise: the role of
longchain polyunsaturated fatty acids. Pediatr. Res., 38: 262-266
Aguilar, M.I., R. Rovelo, J.G. Verjan, O. Illescas, A.E. Baeza, M.D.L. Fuente, I. Avila and
A. Navarrete. 2011. Anti-inflammatory activities, triterpenoids, and
diarylheptanoids of Alnus acuminate ssp. arguta. Pharm. Biol., 49: 1052–1057.
Ahmad, B., I. Khan, S. Bashir, S. Azam and F. Hussain.2011b. Screening of Zizyphus
jujuba for antibacterial, phytotoxic and haemagglutination activities. Afr. J.
Biotech.,10(13): 2514-2519.
Ahmad, H., S.M. Khan, S. Ghafoor and N. Ali. 2009. Ethnobotanical Study of Upper Siran,
J Herbs Spices Med Plants.,15:1,86 – 97. DOI: 10.1080/10496470902787519.
Ahmad, I.,H. Khan, A.H. Gilani, M.A. Kamal.2017. Potential of Plant Alkaloids as
Antipyretic Drugs of Future. Current Drug Metabolism., 18(2):138-144.
Ahmad, M., M. Zafar, N. Shahzadi, G. Yaseen,T. M. Murphey, S. Sultana. 2018.
Ethnobotanical importance of medicinal plants traded in Herbal markets of
Rawalpindi- Pakistan.J Herb Med., 11:78-89.
Ahmad, M., S. Sultana, S. F. Hadi, T. B. Hadda, S. Rashid, M. Zafar, M. A. Khan, M. P.
Z. Khan and G. Yaseen.2014. An Ethnobotanical study of Medicinal Plants in high
mountainous region of Chail valley (District Swat- Pakistan). J Ethnobiol
Ethnomed.,10:36
Ahmad, M., Z. A. Kaloo, B. A. Ganai, H. A. Ganaie and S. Singh, 2016a. Phytochemical
Screening of Meconopsis aculeata Royle an Important Medicinal Plant of
161
Kashmir Himalaya: A Perspective. Res. J. Phytochem., 10: 1-9. DOI:
10.3923/rjphyto.2016.1.9
Ahmed A.B., D. Dippriya and R. Sengupta. 2016b. Comparative antipyretic activity of
ethanolic extracts of some species of Cynodon in rabbits. J Pharmacogn
Phytochem; 5(6): 361-365
Ahmed A.U. 2011.An overview of inflammation: mechanism and consequences. Front
Biol., 6(4):274–281.
Ahmed, F. and A. Urooj. 2011. Pharmacognostical studies on Ficus racemosa stem bark.
Pharmacognosy J., 3 (19): 19-24.
Ahmed, S., M. Sultana, M.M.A.U. Hasan, I. Azhar. 2011a. Analgesic and antiemetic
activity of Cleome viscosa L. Pak. J. Bot., 43, 119-122.
https://doi.org/10.13140/RG.2.2.26544.25602
Ahmed, Z.B., M. Yousfi, J. Viaene, B. Dejaegher, K. Demeyer, D. Mangelings, Y. Vander
Heyden. 2017. Seasonal, gender and regional variations in total phenolic,
flavonoid, and condensed tannins contents and in antioxidant properties from
Pistacia atlantica ssp. leaves. Pharm Biol. 55(1):1185-1194. doi:
10.1080/13880209.2017.1291690.
Ahongshangbam S.K., & G. A. S. Devi.2017. Proximate analysis and mineral (elemental)
composition of certain spices of Manipur, India. Int. Res. J. Pharm., 8 (1):1-5.
Ajaib, M., Z. Ashraf, F. Riaz and M. F. Siddiqui.2014. Ethnobotanical studies of some
plants of Tehsil Kharian, District Gujrat. FUUAST J. Biol., 4(1): 65-71.
Ajuru, M.J., L. F. Williams, G. Ajuru.2017. Qualitative and Quantitative Phytochemical
Screening of Some Plants Used in Ethnomedicine in the Niger Delta Region of
Nigeria. J Food Sci Nutr., 5(5): 198-205.doi: 10.11648/j.jfns.20170505.16.
Akbar, S., U. Hanif, J. Ali, S. Ishtiaq.2014. Pharmacognostic studies of stem, roots and
leaves of Malva parviflora L. Asian Pac J Trop Biomed., 4(5): 410-415.
162
Akhtar, M.F., A. Saleem, A. Sharif, B. Akhtar, M.B. Nasim, S. Peerzada, M. Raza,H. Ijaz,
S.Ahmed, M. Shabbir,S. Ali,Z. Akbar,and S.S Ul- Hassan. 2016. Genotoxic and
cytotoxic action potential of Terminalia citrina, a medicinal plant of
ethnopharmacological significance. EXCLI J., 15: 589–598. doi: 10.17179/excli
2016-551.
Akindahunsi, A. A. and S. O. Salawu. 2005. Phytochemical screening and nutrient-
antinutrient composition of selected tropical green leafy vegetables. Afr. Jour.
Biotechn., 4 (6): 497-501.
Alam, F. and Q. N. Saqib. 2015. Pharmacognostic standardization and preliminary
phytochemical studies of Gaultheria trichophylla. Pharm Biol, Early Online: 1–8
DOI: 10.3109/13880209.2014.1003355
Alam, F., and Q. N. Saqib. 2017. Evaluation of Zanthoxylum armatum Roxb for in vitro
biological activities. J Tradit Complement Med., 7(4): 515–518. doi:
10.1016/j.jtcme.2017.01.006
Alam, M.K., S. Ahmed, S. Anjum, M. Akram, S. M.A. Shah, H. M. Wariss, M. M. Hasan
and K. U. Ghani. 2016. Evaluation of antipyretic activity of some medicinal plants
from Cholistan desert Pakistan. Pak. J. Pharm. Sci., 29(2):529-533.
Alamgir A.N.M. (2017a) Origin, Definition, Scope and Area, Subject Matter, Importance,
and History of Development of Pharmacognosy. In: Therapeutic Use of Medicinal
Plants and Their Extracts: Volume 1. Progress in Drug Research, vol 73. Springer,
Cham. DOI https://doi.org/10.1007/978-3-319-63862-1_2
Alamgir A.N.M. 2017b. Introduction. In: Therapeutic Use of Medicinal Plants and Their
Extracts: Volume 1. Progress in Drug Research, vol 73. Springer, Cham. DOI
https://doi.org/10.1007/978-3-319-63862-1_1
Aline, R.M., B.B. Aline, N.S. Anielca, A.D.G. Beatriz. 2013. Aerial stem and leaf morpho
anatomy of some species of Smilax. Braz J Pharmacog., 23:576–84.
163
Al-Saidy, H.A.M., Y. Mwaffek, N. Abdala. 2014. Using of Plant Extracts for Cinnamon,
Syzygium and Thyme to Degradation of Aflatoxin B1. J. Nat. Sci. Res., 14 (17):1-
7.
Altınyay, C., I. Suntar, I., L. Altun, H. Keles ,̧ E.K. Akkol. 2016. Phytochemical and
biological studies on Alnus glutinosa subsp. glutinosa, A. orientalis var. orientalis
and A. orientalis var. pubescens leaves. J. Ethnopharmacol., 192: 148–160.
Amina, L. R., J. Murimboh, N. M. Hassan, R. Mandal, M. E. B. Younes, C. L. Chakrabarti
and M. H. Back. 2003. Kinetic Speciation of Co(II), Ni(II), Cu(II), and Zn(II) in
Model Solutions and Freshwaters: Lability and the d Electron Configuration.
Environ. Sci. Technol., 37 (1): 68-74.
Amri, O., A. Zekhnini, A. Bouhaimi, S. Tahrouch, A. Hatimi. 2018. Anti-inflammatory
Activity of Methanolic Extract from Pistacia atlantica Desf. Leaves. Pharmacogn
J., 10(1): 71-76.
Anal, J. M. H., P. Chase. 2016. Trace elements analysis in some medicinal plants using
graphite furnace-atomic absorption spectroscopy. Environ. Eng. Res., 21(3): 247-
255.
Anjum, S., Z.A. Bazai, S. Rizwan, C. Benincasa, K. Mehmood, N. Siddique, G. Shaheen,
Z. Mehmood, M. Azam, A. Sajjad.2019. Elemental Characterization of Medicinal
Plants and Soils from Hazarganji Chiltan National Park and Nearby Unprotected
Areas of Balochistan, Pakistan. J Oleo Sci., 68(5):443-461. doi:
10.5650/jos.ess19004.
Ankita, J., R.S. Chauhan. 2012. Evaluation of anticancer activity of Chinopodium album
leaves in BHK-21 cells. Int. J. of Univ. Pharma. Bio Sci., 1(2) 92-102.
Anonymous. 2001. Dietary reference intakes for vitamin A, vitamin K, Boron, Chromium,
Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and
Zinc, Food and Nutrition Board, Institute of Medicine. Washington D.C.: National
Academy Press, pp. 290- 442.
164
Anonymous.2004. Potassium. Dietary Reference Intakes for Water, Potassium, Sodium,
Chloride and Sulfate. Washington, D. C.: National Academies Press, pp.173-246.
Ansari, M. M., J. Ahmad and S. H. Ansari. 2006. Pharmacognostic evaluation of the stem
barks of Balanitesaegyptica Delile “Hingot”. Hamdard Medicus., 50 (1): 82-94.
Antia, B. S., E. J. Akpan, P. A. Okon and I. U. Umoren. 2006. Nutritive and antinutritive
evaluation of sweet potatoes (Ipomoea batatas) leaves. Pak. Jour. Nut., 5 (2): 166-
168
Anwar, F. and U. Rashid. 2007. Physico-chemical characteristics of Moringa oleifera seeds
and seed oil from a wild provenance of Pakistan. Pak. J. Bot., 39 (5): 14431453.
AOAC (Association of Official Analytical Chemists). 2000. Official methods of analysis.
Gaithersburg, MD, Washington, USA.
AOAC., 2003. Official Methods of Analysis of the Association of Official's Analytical
Chemists. 17th Edn., Association of Official Analytical Chemists, Arlington,
Virginia.
Aronoff, D. M., E. G. Neilson. 2001. Antipyretics: Mechanisms of action and clinical use
in fever suppression. Am. J. Med., 2001, 111(4):304-315.
Arredondo, M. and T. M. Nunez. 2005. Iron and Copper metabolism. Mol. Aspects Med.,
26: 313-327.
Artun, F.T., A. Karagoz, G. Ozcan, G. Melikoglu, S. Anil, S. Kultur, N. Sutlupinar. 2016.
In Vitro Anticancer and Cytotoxic Activities of Some Plant Extracts on HeLa and
Vero Cell Lines. JBUON., 21(3): 720-725.
Arullappan, S., Muhamad and Z. Zakaria. 2013. Cytotoxic Activity of the Leaf and Stem
Extracts of Hibiscus rosa sinensis (Malvaceae) against Leukaemic Cell Line (K-
562).Trop J Pharm Res.,12 (5): 743-746.
165
Arulmozhi, P., S. Vijayakumar, T. Kumar.2018. Phytochemical analysis and antimicrobial
activity of some medicinal plants against selected pathogenic microorganisms.
Microb Pathog. 123:219-226. doi:
10.1016/j.micpath.2018.07.009.
Ascensao, L., L. Mota and M. D. M. Castro.1999. Glandular Trichomes on the Leaves and
Flowers of Plectranthus ornatus: Morphology, Distribution and Histochemistry.
Annals of Botany., 84: 437–447.
Ashburner, K. and H.A. McAllister. 2013. The Genus Betula: A Taxonomic Revision of
Birches. Kew Publishing, Kew.
Ashfaq, K., B. A. Choudhary, M. U. Sajid, N. Hussain, M. A. Ghaffari, W. Sarwar and
M. Manzoor.2016. Antipyretic, analgesic and anti-inflammatory activities of
methanol extract of root bark of Acacia jacquemontii Benth (Fabaceae) in
experimental animals. Trop J Pharm Res.,15(9): 1859-1863.
Asquith, T. N. and L. G. Butler. 1986. Interaction of condensed Tannins with selected
Proteins. Phytochem., 25 (7): 1591-1593.
Attanayake, A. P. and K.A.P.W. Jayatilaka. 2016. Evaluation of antioxidant properties of
20 medicinal plant extracts traditionally used in Ayurvedic medicine in Sri Lanka.
Indian J Tradit Knowle., 15 (1):50-56.
Atta-ur-Rahman, M.I. Choudhary, J.T. William. 2001.Bioassay techniques for drug
Development; Harward academic Publisher: pp.67-68.
Aviala, J., P. K. Arthala, S. Yeturi, D.V.R. Said-Gopal and S. Dasari. 2016. Inhibition of
Aspergillus flavus growth and detoxification of Aflatoxin B1 by the medicinal plant
leaf extract- Cymbopogon citratus., Int. J. Clin. Biol. Sci.1(1):11-23.
Ayatollahi, A. M., M. Ghanadian, S. Afsharypuor, S. Siddiq and S. M. Pour-Hosseini.
2010. Biological screening of Euphorbia aellenii. Iranian J. Pharm. Res., 9 (4):
429-436.
166
Ayaz, F., N. Kucukboyacı, B. Bani, B. Sener, M. I. Choudhary. 2018a. Phytotoxic,
Cytotoxic and Insecticidal Activities of Chrysophthalmum dichotomum Boiss. and
Heldr. Indian J Pharm Educ Res., 52(3): 467-471.
Ayaz, F., N. Kucukboyaci, B. Bani, B. Sener,M. I. Choudhary. 2018b. Phytotoxicity,
Toxicity on Brine Shrimp and Insecticidal Effect of Chrysophthalmum gueneri
Aytac & Anderb. Growing in Turkey. Turk J Pharm Sci.,15(3):382-385; DOI:
10.4274/tjps.88700
Ayers, E., E. Cameron, R. Kemp, H. Leitch, Mollison A, MuirI, Reid H, Smith D, Sproat
J. 2001. Oral lesions in sheep and cattle in Dumfries and Galloway. Vet Rec., 148,
720–723.
Azhagumadhavan, S., S. Senthilkumar, M.Padma, P.Sasikala, T. Jayaseelan and S.
Ganesan. 2019.A Study on Establishment of Phytochemical Analysis of Quality
Parameters and Fluorescence Analysis of Costus spicatus-rhizome extract
Medicinal Plants a Well Known Tropical Folklore Medicine. J. drug deliv. ther.,
9(1-s):240-243.
Baiano, A., M.A. del Nobile. 2015. Antioxidant compounds from vegetable matrices:
Biosynthesis, occurrence, and extraction systems. Crit. Rev. Food Sci. Nutr., 56:
2053–2068.
Baloch, T.A., S. K. Leghari, M. Shafi, M. I. Khatttak and M. Asrar. 2019. Biological and
pharmacological studies of Heliotropium dasycarpum Ledeb.FUUAST J. Biol.,
9(1): 21-27.
Balunas, M.J., Kinghorn, A.D.2005. Drug discovery from medicinal plants. Life Sci., 78,
31-41.
Bano, A., M. Ayub, S. Rashid, S. Sultana and H. Sadia. 2013. Ethnobotany and
conservation status of floral diversity of Himalayan range of Azad Jammu and
Kashmir Pakistan. Pak. J. Bot., 45(SI): 243-251.
167
Barkatullah and M. Ibrar. 2011. Ethnobotanical profile of plants of Malakand Pass Hills,
District Malakand, Pakistan. Afr. J. Biotechnol.,10(73):16521-16535.
Barkatullah, M. Ibrar and N. Muhammad. 2011. Evaluation of Zanthoxylum armatum DC
for in-vitro and in-vivo pharmacological screening. Afr. J. Pharm. & Pharmacol.,
5 (14): 1718-1723.
Barkatullah, M. Ibrar, M. Nafees, A. Rauf, H. Khan. 2015a. Cytotoxic, acute toxicity and
phytotoxic activity of Callicarpa macrophylla in various models. Americ J Biomed
life Sci., 3(2-1): 1-4.
Barkatullah, M. Ibrar, N. Muhammad and A. Rauf.2013. Antipyretic and Antinociceptive
Profile of Leaves of Skimmia laureola. Middle-East J. Sci. Res. 14 (8): 1124- 1128.
DOI:10.5829/idosi.mejsr.2013.14.8.7395.
Barkatullah, M. Ibrar, N. Muhammad, A. Rauf, Nasruddin, H. khan, J. Ali.2015b.
Evaluation of Zanthoxylum armatum its toxic metal contents and proximate
analysis. J. Phytopharmacol., 4(3): 157-163.
Barnes P.J. 2009. Targeting the epigenome in the treatment of asthma and chronic
obstructive pulmonary disease. Proc Am Thorac Soc.,6(8):693–696.
Basic, N., E. Selimovic, F. Pustahija.2014. Morphological identification of nothospecies
Alnus X pubescens tausch. and their new localities in central Bosnia. Works of the
Faculty of Forestry University of Sarajevo, 1: 15-24.
Beard, J. L. 2001. Iron biology in immune function, muscle metabolism and neuronal
functioning. J. Nutr., 131: 568–579.
Begum, F. & N. Samajpati. 2000. Mycotoxins production on rice, pulses and oilseeds.
Naturwissenschaften, 87:275-277.
Behera, P.C. and M. Ghosh. 2018.Evaluation of antioxidant, antimicrobial, and anti
urolithiatic potential of different solvent extracts of Aerva lanata Linn flowers.
Phcog Mag., 14(53):53-57. http://www.phcog.
com/text.asp?2018/14/53/53/225666
168
Behera, S.K., A. Leelaveni, C. Mohanty. 2019. Phytochemical screening of some
ethnomedicinal plants of Kandhamal District of Odisha, India. Int. Res. J. Pharm.,
10 (1):190-193.
Bellik, Y., L. Boukraa, H.A. Alzahrani, B.A. Bakhotmah, F. Abdellah, S.M. Hammoudi
and M. Iguer-Ouada.2013. Review Molecular Mechanism Underlying Anti-
Inflammatory and Anti-Allergic Activities of Phytochemicals: An Update.
Molecules, 18: 322-353.
Berridge, M.V., P.M. Herst, A.S.Tan. 2005.Tetrazolium dyes as tools in cell biology: New
insights into their cellular reduction. Biotechnol Annu Rev. 2005; 11:127-52.
Bhanushali, M., V. Bagale, A. Shirode, Y. Joshi and V. Kadam. 2010. An in-vitro toxicity
testing – a reliable alternative to toxicity testing by reduction, replacement and
refinement of animals. Int J of Adv in Pharm Sci.; 1: 15–31.
Bhat, J.U., Q. Nizami, S. Parray, M.Aslam, N. Fahamiya, A. Siddique, M. Mujeeb, R.
Khanam and S.T. Ahmad.2012. Pharmacognostical and phytochemical evaluation
of Melissa parviflora and HPTLC finger printing of its extracts. Nat. Prod. Plant
Resour., 2 (1):198-208
Biju, J., C.T. Sulaiman, G. Satheesh, and V.R.K. Reddy. 2014. Total phenolics and
flavonoids in selected medicinal plants from Kerala. Int. J. Pharma. Pharm. Sci. 6:
406-408.
Bikovens, O., L. Roze, A. Pranovich, M. Reunanen and G. Telysheva. 2013. Chemical
Composition of Lipophilic Extractives from Grey Alder (Alnus incana). Bio
Resources,8(1):350-357.
Birben, E., U.M. Sahiner, C. Sackesen, S. Erzurum, O. Kalayci. 2012. Oxidative stress and
antioxidant defense. World Allergy Organ J., 5(1):9-19.
doi:10.1097/WOX.0b013e3182439613.
Bisht, A.S., S. Kandwal, D. Kanan, D. Som. 2014. Anticancerous and
antiproliferative/cytotoxic activity of curcuma pseudomontana (hill turmeric)
169
collected from the sub Himalayan region of Uttrakhand, India. Asian J Plant Sci
Res., 4(6):25-31.
Bisht, D., M. Gupta, S. Srivastava, B. Datt and A. K. S. Rawat. 2011. Comparative
Pharmacognostic Evaluation of Three species of Swertia L. (Gentianaceae).
Pharma. J., 3 (19): 7-12.
Blokhina,O., E. Virolainen, K.V. Fagerstedt. 2003. Antioxidants, oxidative damage and
oxygen deprivation stress: a review. Ann Bot., 91 (Spec No):179-94.
Bola, O.O., O.D. Omowunmi, and O. Adelaja.2017. Trace Elements and Antioxidants in
Some Medicinal Plants. Res Rev Biosci., 12(1):111.
Boldt, K.M., & B. Rank. 2010. Stomata dimorphism in dicotyledonous plants of temperate
climate. Feddes Repertorium, 121:167–183 DOI: 10.1002/fedr.201000023
Boseila, A.A.H. 2011. Preliminary in vitro study for using aqueous cinnamon extract
against foot-and-mouth disease virus. New York Sci. J., 4(11): 59-63.
Boseila, A.A.H. and Hatab, E.M.A. 2011. Evaluation of the antiviral activity of sage leaves
(Salvia officinalis) extract against foot-and-mouth disease virus. Agricultural
Relations (FAR),Egypt,3-5October.p301-308.
Bravo, L. 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional
significance. Nutr Rev.,56:317–333.
Bukhsh, E., S. A. Malik and S. S. Ahmad. 2007. Estimation of nutritional value and trace
elements content of Carthamus oxycantha, Eruca sativa and Plantago ovate. Pak.
J. Bot., 39 (4): 1181-1187.
Bulent, K., A.D.W. Dobson, I. Var. 2006. Strategies to Prevent Mycotoxin Contamination
of Food and Animal Feed: A Review.Crit Rev Food Sci Nutr, 46: 593–619.
170
Butt, M.A., M. Ahmad, A. Fatima, S. Sultana, M. Zafar, G. Yaseen, M.A. Ashraf, Z.K.
Shinwari and S. Kayani. 2015. Ethnomedicinal uses of plants for the treatment of
snake and scorpion bite in Northern Pakistan. J. Ethnopharmacol, 168: 164-181.
Cardellina, J. H. 2002. Challenges and opportunities confronting the botanical dietary
supplement industry, J. Nat. Prod., 65 (7): 1073–1084.
Carr, D. J. & S. G. M. Carr. 1990: Transverse and longitudinal gradients in stomatal size
in dikotyledons. Cytobios., 61: 41– 61.
Cassidy, A., B. Hanley, R.M. Lamuela-Raventos. 2000. Isoflavones, lignans and stilbenes:
origins, metabolism and potential importance to human health. J Sci Food Agric.
80:1044–1062.
Chaffey, N. J. 2001. Putting plant anatomy in its place. Trends in Pl. Sci., 6: 439-440.
Chanda, S. 2014.Importance of pharmacognostic study of medicinal plants: An
overview.J Pharmacogn Phytochem. 2(5): 69-73.
Chatterjee, M.N., R. Shinde. 1995. Text book of medical biochemistry. (2nd edtn),
Jaypee Brother Medical Pub Ltd, New Delhi, India, 811-846.
Chattopadhyay, D. and T.N. Naik. 2007. Antivirals of ethnomedicinal origin: structure–
activity relationship and scope. Mini Rev. Med. Chem., 7:275–301.
Chaudhari, R.K., and N. O. Girase.2015. Determination of soluble extractives and physico-
chemical studies of bark of Sesbania sesban (L) Merr. J Chem Pharm
Res.,7(8):657-660
Chauhan, P.P., A. Nigam, V.K. Santvan. 2014. Ethnobotanical survey of trees in Pabbar
valley, Distt. Shimla, Himachal Pradesh. Life sci. leafl., 52: 24-39.
Chiu, Y.J., T.H. Huang, C.S. Chiu, T.C. Lu, Y.W. Chen, W.H. Peng, C.Y. Chen. 2012.
Analgesic and Antiinflammatory Activities of the Aqueous Extract from
Plectranthus amboinicus (Lour.) Spreng. Both In Vitro and In Vivo. Evid Based
171
Complement Alternat Med., 2012: 508137 .http://dx.doi.org/
10.1155/2012/508137.
Choi, J.G., M.W. Lee, S.E. Choi, M.H. Kim, O.H. Kang, Y.S. Lee, H.S. Chae, B.Obiang-
Obounou, Y.C. OH, M.R. Kim. et al. 2012a. Antibacterial activity of bark of Alnus
pendula against methicillin-resistant Staphylococcus aureus. Eur. Rev. Med.
Pharmacol., 16: 853–859.
Choi, S.E., K.H. Park, M.H. Kim, J.H. Song, H.Y. Jin, M.W. Lee. 2012b.
Diarylheptanoids from the bark of Alnus pendula Matsumura. Nat. Prod. Sci.,
18:106–110.
Choi, S.E., K.H. Park, M.S. Jeong, H.H. Kim, D.I. Lee, S.S. Joo, C.S. Lee, H. Bang,
Y.W. Choi, M.K.Lee, S.J. Seo, M.W.Lee. 2011. Effect of Alnus japonica extract
on a model of atopic dermatitis in NC/Nga mice. J Ethnopharmacol., 136(3):406-
413. http://dx.doi.org/10.1016/j.jep.2010.12.024
Choi, S.I., J. S. Lee, S. Lee, J.H. Lee, H. S. Yang, J. Yeo, J.Y. Kim, B.Y. Lee, I.J. Kang
and O.H. Lee. 2018. Radical scavenging-linked anti-adipogenic activity of A.
firma extracts. Int J Mol Med. 41: 119-128.
Chungsamarnyart, N., T. Sirinarumitr, W. Chumsing, W. Wajjawalku. 2007. In vitro study
of antiviral activity of crude extracts against the foot and mouth disease virus.
Kasetsart. J. (Nat. Sci.), 41(5):97-103.
Croteau, R., Kutchan, T.M. and Lewis, N.G. (2000) Natural Products (Secondary
Metabolites). Biochemistry and Molecular Biology of Plants, Rockville, MD:
American Society of Plant Physiologists; pp. 1250– 1318.
Dahija, S., J. Cakar, D.Vidic, M. Maksimovic and A. Paric. 2014. Total phenolic and
flavonoid contents, antioxidant and antimicrobial activities of Alnus glutinosa (L.)
Gaertn., Alnus incana (L.) Moench and Alnus viridis (Chaix) DC. extracts, Nat.
Prod. Res., 28:2317-2320. DOI: 10.1080/14786419.2014.931390.
172
Daoud, H.M. and E. M. Soliman. 2015. Evaluation of Spirulina platensis extract as natural
antivirus against foot and mouth disease virus strains (A, O, SAT2). Vet World,
8(10): 1260–1265. doi: 10.14202/vetworld.2015.1260-1265.
Davies, G. 2002. Foot and mouth disease. Res Vet Sci., 73, 195–199.
Dellai, A., H.B. Mansour, A. Clary-Laroche, M. Deghrigue, A. Bouraoui. 2012.
Anticonvulsant and analgesic activities of crude extract and its fractions of the
defensive secretion from the Mediterranean sponge, Spongia officinalis. Cancer
Cell Intl., 12:15. https://doi.org/10.1186/1475-2867-12-15
Derkach, T., V. Khomenko.2018. Elemental Composition of the Medicinal Plants
Hypericum perforatum, Urtica dioica and Matricaria chamomilla Grown in
Ukraine: A Comparative Study. Pharmacogn J., 10(3):486-491
Deshpande,T.M.andS.R. Chaphalkar. 2013.Antiviral activity of plant extracts against
FMDV in vitro a preliminary report. Int. J. Inst. Pharm. Life Sci., 3(4): 1-18.
Dibarka, M. 2011. Identification of some ethno medicine for treatment of FMD in polasara
block, Ganjam, India. J.res. bio., 1(7):543-549.
Dilawar, S., A. Shah, Shahnaz, H.U. Khan, S. Imad and K. Shirin. 2018. Elemental
Analysis in the Indigenous Medicinal Plant Moringa oleifera and their Association
with Ameliorative Activity. J.Chem.Soc.Pak., 40(5):834-839
Dinic, J., M. Novakovic, A. Podolski-Renic, S. Stojkovic, B. Mandic, V.Tesevic, V. Vajs,
A. Isakovic, M. Pesic.2014. Antioxidative activity of diarylheptanoids from the
bark of black alder (Alnus glutinosa) and their interaction with anticancer drugs.
Planta Med., 80:1088–1096.
Dinic, J., T. Ranđelovic, T. Stankovic, M. Dragoj, A. Isakovic, M. Novakovic, M. Pesic.
2015. Chemo-protective and regenerative effects of diarylheptanoids from the bark
of black alder (Alnus glutinosa) in human normal keratinocytes. Fitoterapia,
105:169–176.
173
Dollear, F.G., G.E. Mann, L.P. Codifer, H.K. Gardner, S.P. Koltun, H.L.E. Vix. 1968.
Elimination of aflatoxins from peanut meal. J. Am. Oil Chem. Soc., 45:862.
Donsbach, K. and A. Ayne. 1982. The physiological function of minerals in man. In:
chelated minerals nutrition in plants, animals and man. (Eds.): A de Wayne Charles,
C. Thomas publ., Springfield, pp. 247-257.
Dos -Reis, L. F.C., C.D. Cerdeira, B.F. De-Paula, J.J. Silva, L.F.L. Coelho, M.A. Silva,
V.B.B. Marques, J.K. Chavasco and G. Alves-Da-Silva, G. 2015. Chemical
characterization and evaluation of antibacterial, antifungal, anti-mycobacterial, and
cytotoxic activities of Talinum paniculatum. Rev. Inst. Med. Trop. Sao Paulo,
57(5): 397-405. http://dx.doi.org/10.1590/S0036-46652015000500005
Ebana, R.U.B. and V.E. Madunagu. 1993. Antimicrobial effect of Strophanthus hispidus
and Secamone afzelii on some pathogenic bacteria and their drug resistant strains.
Niger J. Bot., 6, :27–31.
Eom, H.J., K. S. Chang, H. Kim and C. S.Chang .2011. Notes on a new overlooked taxon
of Alnus (Betulaceae) in Korea.Forest Sci Technol., 7(1): 42–46.
Erdtmann, G. 1943. Introduction into pollen analysis. Chronica Botanica Co, Waltham,
MA. Leopold, E. B., J. Birkebak, L. Reinink-Smith, A.P. Jayachandar, P. Narváez,
S. Zaborac-Reed.2012. Pollen morphology of the three subgenera of Alnus.
Palynology, 36(1), 131-151.
Evans, W. C. 2002. Pharmacognosy. 15th ed. English Language Book, Society Baillere
Tindall, Oxford University Press.
Fahmy, N. M., E. Al‐Sayed, M. M. Abdel‐Daim, A. N. Singab. 2017. Anti-Inflammatory
and Analgesic Activities of Terminalia Muelleri Benth. (Combretaceae). Drug Dev.
Res., 78: 146–154.
Faitanin, R.D., J. V. D. Gomes, P. M. Rodrigues, L. F. T. De-Menezes, A. C. Neto, C.R.
R. Goncalves, R. R. Kitagawa, D. Silveira and C. M. Jamal. 2018. Chemical study
and evaluation of antioxidant activity and α-glucosidase inhibition of Myrciaria
174
strigipes O. Berg (Myrtaceae). J Appl Pharm Sci., 8(03):120-125; DOI:
10.7324/JAPS.2018.8317.
FAO, Manuals of Food Quality Control 10: Training in Mycotoxins Analysis; FAO food
and nutrition paper; Food and Agriculture Org. Rome, 1990; pp 54-58.
Fapohunda S. O., A. Esan, A. Alabio, O. Adebote and O. Kolawoleet al. 2014. Treated
melon seeds and Aflatoxin Profile in Relation to Blood Parameters in Exposed
Mice. J. Microbiol. Res. Rev., 2:40-47.
Ferdous, M, R. Rouf, J.A. Shilpi, S.J. Uddin. 2008. Anti-nociceptive activity of the
ethanolic extract of Ficus racemosa Lin. (Moraceae). Orient Pharm Exp Med., 8
(1), 93-96. http://dx.doi.org/10.3742/OPEM.2008.8.1.093
Fisher, D.E. 1994. Apoptosis in cancer therapy: crossing the threshold. Cell, 78: 539-542.
Fluck, H. 1973. Medicinal Plants and Their Uses, W. Feulsham and Comp Ltd, New York
.pp. 7–15
Gaichu, D.M., A. M. Mawia, G.M. Gitonga, M. P. Ngugi, D.N. Mburu. 2017.
Phytochemical screening and antipyretic activities of dichloromethane-methanolic
leaf and stem bark extracts of Ximenia americana in rat models. J Herbmed
Pharmacol.,6(3):107-113.
Gan, T. J. 2010. Diclofenac: an update on its mechanism of action and safety profile.
Curr Med Res Opin., 26(7):1715-31. doi: 10.1185/03007995.2010.486301.
Gao Y, F. Liu, L. Fang, R.Cai, C. Zong, and Y. Qi. 2014. Genkwanin Inhibits
Proinflammatory Mediators Mainly through the Regulation of miR-101/MKP-
1/MAPK Pathway in LPS-Activated Macrophages. Plos one., 9(5): e96741.
Garcia, D., A. J. Ramos, V. Sanchis and S. Marin. 2011. Effect of Equisetum arvense and
Stevia rebaudiana extracts on growth and mycotoxin production by Aspergillus
flavus and Fusarium verticillioides in maize seeds as affected by water activity. Int.
J. Food Microbiol., 153:21–27.
175
Garrett W.S, J.I. Gordon, L.H. Glimber. 2010. Homeostasis and inflammation in the
intestine. Cell.,140(6):859–870.
Gayathri, V. and D. Kiruba. 2015.Fluorescence analysis of two medicinal pgillants –
Psidium guajava L and Citrus aurantium. IJPSR., 6(3): 1279-1282.
Gilani, A.H and K. H. Janabaz. 1993. Protective effect of Artemisia scoparia extract
against acetaminophen-induced hepatotoxicity. Gen Pharmacol., 24(6):1455-8.
Gokhale, S. B., C. K. Kokate and A. P. Purohit. 2008. A text book of Pharmacognosy.
Nirali Prakashan, Punne India. pp 12.
Gomathi, V., B. Jayakar, R. Kothai and G. Ramakrishnan. 2010. Antidiabetic activity of
leaves of Spinacia oleracea Linn. in Alloxaninduced diabetic rats. J. Chem. Pharm.
Res., 2 (4): 266-274.
González-Hernández, M.P., E.E. Starkey and J. Karchesy.2000. Seasonal Variation in
Concentrations of Fiber, Crude Protein, and Phenolic Compounds in Leaves of Red
Alder (Alnus Rubra): Nutritional Implications for Cervids. J Chem Ecol., 26(1):
293-301. https://doi.org/10.1023/A:1005462100010
Gonzalez-Hernandez, M.P., J. Karchesy and E.E. Starkey. 2003. Research observation:
Hydrolyzable and condensed tannins in plants of northwest Spain forests. J. Range
Manage.56: 461-465.
Gorsi M.I., M. Abubakar and M.J.Arshed. 2011. Epidemiology and Economic Aspects of
Foot and Mouth Disease in District Sahiwal, Punjab, Pakistan. YYU Vet Fak Derg.,
22: 159- 162.
Gotep, J.G., O.O. Oladipo, M.S. Makoshi, E.T. Doku, T. M. Asala, M.M. Eki, H.B. Yusuf,
B.O. Akanbi, S. Isa, B.B. Dogonyaro, P.A. Okewole, A.A. Atiku, M.S. Ahmed and
C.I. Nduaka. 2018. Toxicological Evaluation of Euphorbia hirta on Baby Hamster
Kidney (BHK-21) Cells and in Albino Rats. EJMP., 25(2): 1-12.
Govaerts, R. 2014. The World Check list of Selected Plant Families.
http://www.kew.org/wcsp/America. New York (NY): MacMillan Publishing Co.
176
Goyal, K. K., B. N. S. Kumar, K. Mruthunjaya, Mythreyi and S. N. Yoganarasimhan. 2011.
Pharmacognostical studies of stem bark of Careya arborea Roxb. Inter. J. G.
Pharmacy., 5 (1): 6-11.
Grover, N., V. Patini. 2013. Phytochemical characterization using various solvent extracts
and GC-MS analysis of methanolic extract of Woodfordia fruticosa (L.) Kurz.
Leaves. Int J Pharm Pharm Sci.,5(4):291-295.
Gul, S., M. Safdar. 2009. Proximate composition and mineral analysis of Cinnamon. Pak
J Nut.,8 (9): 1456-1460.
Gupta, D., A. Goel, A.K. Bhatia.2010. Studies on antiviral property of Acacia nilotica. J.
Environ. Res. Develop. 2010, 5(1), 141-152.
Gutierrez, Y.I., R. Scull, L. Monzote, K. M. Rodriguez, A. Bello and W. N. Setzer.2018.
Comparative Pharmacognosy, Chemical Profile and Antioxidant Activity of
Extracts from Phania matricarioides (Spreng.) Griseb. Collected from Different
Localities in Cuba.Plants.,7:110; doi:10.3390/plants7040110
Hajare, S.S., S.H. Hajare, A. Sharma. 2005. Aflatoxin inactivation using aqueous extract
of Ajowan (Trachyspermumammi) seeds. J Food Sci., 70: 29-34.
Hajjaj, G., A. Bahlouli, K. Sayah, M. Tajani, Y. Cherrah, A. Zellou. 2017. Phytochemical
screening and in vivo antipyretic activity of the aqueous extracts of three Moroccan
medicinal plants. Pharm. Biol. Eval., 4(4): 188-192. DOI:
http://dx.doi.org/10.26510/2394-0859.pbe.2017.30
Hameed, I., G. Dastagir and F. Hussain. 2008. Nutritional and elemental analyses of some
selected medicinal plants of the family Polygonaceae. Pak. J. Bot., 40 (6): 2493-
2502.
Hammond, G.B., I.D. Fernandez, L.F.Villegas, A.J.Vaisberg.1998. A survey of traditional
medicinal plants from the Callejon de Huaylas, Department of Ancash, Peru. J
Ethnopharmacol., 61: 17-30.
Harnborne, J. B. 1998. Phytochemical methods (3rd Edn). Chapman and Hall, New York.
177
Harper, M. E., J. S. Willis and J. Patrick. 1997. Sodium and chloride in nutrition. In:
Handbook of nutritionally essential minerals. (Eds.): B.L. O'Dell and R.A. Sunde.
New York: Marcel Dekker, pp. 93-116.
Harris, E. D. 1997. Copper. In: O‟Dell BL, Sunde RA, eds. Handbook of Nutritionally
Essential Mineral Elements. New York: Marcel Dekker. pp. 231–273.
Hassan, Z.U., M.Z. Khan, M.K. Saleemi, A. Khan, I. Javedet al. 2012. Immunological
responses of male White Leghorn chicks kept on ochratoxin A (OTA)-
contaminated feed. J Immunotoxicol 9: 56-63.
Havsteen, B. H. 2002. The biochemistry and medical significance of the flavonoids.
Pharmacol. Therapeutics, 96: 67–202.
Hays V.W., M.J.Swenson .1985. Minerals and Bones. In: Dukes‟ Physiology of Domestic
Animals (10th edtn), pp. 449-466.
Hazrat, A., M. Nisar, J.Shah and S. Ahmad. 2011. Ethnobotanical study of some elite plants
belonging to Dir, Kohistan valley, Khyberpukhtunkhwa, Pakistan. Pak. J. Bot.,
43(2): 787-795.
Hijazi, M.A., A. El-Mallah, M. Aboul-Ela and A. Ellakany.2017. Evaluation of Analgesic
Activity of Papaver libanoticum Extract in Mice: Involvement of Opioids
Receptors. Evid Based Complement Alternat Med.,2017: 8935085.
Holmes R.A., R.S. Boston, G. A. Payne. 2008. Diverse inhibitors of aflatoxin biosynthesis.
Appl. Microbiol. Biotechnol., 78: 559-572.
Hseu, Z. 2004. Evaluating heavy metal contents in nine composts using four digestion
methods. Bioresource Technol., 95: 53–59.
Hu, W.C., M.H. Wang. 2011. Antioxidative activity and anti-inflammatory effects of
diarylheptanoids isolated from Alnus hirsuta.J. Wood Sci., 57:323–330.
Huang, D.J., B.X. Ou, R.L. Prior. 2005. The chemistry behind antioxidant capacity assays.
J Agric Food Chem., 53:1841-1856.
178
Huang, X., W. Gao, W. Zhao, T. Zhang and J. Xu. 2010. Flavone and steroid chemical
constituents from rhizome of Paris axialis. Zhongguo Zhong Yao Za Zhi., 35 (22):
2994-2998.
Huang, Z. L. and M. L. Failla. 2000. Copper deficiency suppresses effector activities of
differentiated U937 cells. J. Nutr., 130: 1536-1542.
Hunt, J.R.1994. Bioavailability of Fe, Zn and other Trace Minerals forVegetarian Diets.
Am. J. Clin. Nutr., 78, 633-39.
Hussain, F., I. Hameed, G. Dastagir, S. Nisa, I. Khan, B. Ahmad.2010b. Cytotoxicity and
Phytotoxicity of some selected medicinal plants of family Polygonaceae. Afr. J.
Biotechnol., 9(5):770-774.
Hussain, J., F. U. Khan, Riazullah, Z. Muhammad, N. Rehman, Z. K. Shinwari, I. U. Khan,
M. Zohaib, Imad-ud-din and S. M. Hussain. 2011b. Nutrient evaluation and
elemental analysis of four selected medicinal plants of Khyber Pakhtoon khwa,
Pakistan. Pak. J. Bot., 43 (1): 427-434.
Hussain, J., N. U. Rehman, A. L. Khan, M. Hamayun, S. M. Hussain and Z. K. Shinwari.
2010a. Proximate and essential nutrients evaluation of selected vegetables species
from Kohat region, Pakistan. Pak. J. Bot., 42 (4): 2847-2855.
Hussain, M. S., S. Fareed and M. Ali. 2011. Preliminary phytochemical and
pharmacognostical screening of the Ayurvedic drug Hygrophila auriculata (K.
Schum) Heine. Pharmacon Jour., 3 (23): 28-40.
Ibrar, M., F. Hussain and A. Sultan. 2007. Ethnobotanical studies on plant resources of
Ranyal Hills, District Shangla, Pakistan. Pak. J. Bot., 39 (2): 329-337.
Ibrar, M., I. Ilahi and F. Hussain. 2003. Hypoglycemic activity of Hedera helix L. leaves
and possible mechanism of action. Pak. J. Bot., 35 (5): 805-809
Idris, O.A., O. A. Wintola and A. J. Afolayan. 2019. Comparison of the Proximate
Composition, Vitamins (Ascorbic Acid, α-Tocopherol and Retinol), Anti- Nutrients
(Phytate and Oxalate) and the GC-MS Analysis of the Essential Oil of
179
the Root and Leaf of Rumexcrispus L. Plant., 8(3), 51;
https://doi.org/10.3390/plants8030051
Ilyas, M., R. Qureshi, Z.K. Shinwari, M. Arshad, S.N. Mirza and Z.U. Haq, 2013.Some
ethnoecological aspects of the plants of Qalagai hills, Kabal valley, Swat, Pakistan.
Int. J. Agric. Biol., 15: 801–810.
Indrayan, A.K., S. Sharma, D. Durgapal, N. Kumar, M. Kumar. 2005. Determination of
nutritive value and analysis of mineral elements for some medicinally valued plants
from Uttaranchal. Curr Sci., 89: 1252-1255.
Iniaghe, O. M., S. O. Malomo and J. O. Adebayo. 2009. Proximate Composition and
Phytochemical Constituents of Leaves of Some Acalypha Species. Pak. J. Nutr., 8
(3): 256-258.
Iram, W., T. Anjum, M. Iqbal, A. Ghaffar, M. Abbas and A.M. Khan. 2016. Structural
analysis and biological toxicity of aflatoxins B1 and B2 degradation products
following detoxification by Ocimum basilicum and Cassia fistula aqueous extracts.
Front. Microbiol. 7:1105. doi: 10.3389/fmicb.2016.01105.
Ishtiaq, M., Hanif, W., Khan, M.A., Ashraf, M., Butt, A.M., 2007. An ethnomedicinal
survey and documentation of important medicinal folklore food phytonyms of flora
of Samahni Valley, (Azad Kashmir) Pakistan. Pak J Bio Sci., 10 (13):2241– 2256.
Ishtiaq, S., U. Hanif, M. Ajaib, S. Shaheen, M. S. K. Afridi and M. F. Siddiqui.2018.
Pharamcognostical and physicochemical characterization of Amaranthus
Graecizans subsp. Silvestris: an anatomical perspective. Pak. J. Bot., 50(1): 307-
312.
Jadhav, S.W., R. B. Jadhav, S. Rao .2018. Microscopic and physicochemical evaluation of
Lagerstroemia lanceolate Wall leaves. Sch. Acad. J. Pharm.,7(7):291-299.
180
Jadid, N., B.A. Arraniry, D. Hidayati, K.I. Purwani, W. Wikanta, S.R.Hartanti, R.Y.
Rachman.2018. Proximate composition, nutritional values and phytochemical
screening of Piper retrofractum vahl. fruits. Asian PacJ Trop Biomed., 8:37-43.
Jan, A.K., A. Hazrat, S. Ahmad, T. Jan and G. Jan. 2019.Invitro antifungal, antibacterial,
phytotoxic, brine shrimp, insecticidal activities and composition of essential oil of
Tagetes minuta from Dir-Kohistan, Pakistan. Pak. J. Bot., 51(1): 201-204; DOI:
10.30848/PJB2019-1(19).
Jan, G., M. A. Kahan, M. Ahmad, Z. Iqbal, A. Afzal, M. Afzal, G. M. Shah, A. Majid,
M. Fiaz, M. Zafar, A. Waheed and F. Gul. 2011. Nutritional analysis,
micronutrients and chlorophyll contents of Cichorium intybus L. Jour. Med. Pl.
Res., 5 (12): 2452-2456
Jarald, E. E. and S. E. Jarald. 2007. A text book of pharmacognosy and phytochemistry
(1st Edn). CBS publishers and distributors, New Delhi, India.pp.6
Jassim, S.A. A., M.A. Naji. 2003. Novel antiviral agents: a medicinal plant perspective. J
Appl Microbiol., 95: 412–427. pmid:12911688.
Jayathilake, C., V. Rizliya and R. Liyanage. 2016. Antioxidant and free radical scavenging
capacity of extensively used medicinal plants in Sri Lanka. Procedia Food Sci.,6:
123–126.
Jin, W.Y., F.X. Cai, M.K. Na, J.J. Lee, K.W. Bae.2007. Diarylheptanoids from Alnus
hirsuta inhibit the NF-kB activation and NO and TNF-α production. Biol. Pharm.
Bul.,30:810–813.
Jin-Mu Y., K. Mi-Sun, L. Eun-Hee, W. Dae-Han, L. Jai-Kyoo, C. Kwang-Ho, H. Seung-
Heon, K. Hyung-Min. 2003. Induction of apoptosis by Paljin -Hangahmdan on
human leukemia cells. J Ethanopharmacol., 88: 79-83.
Jothi, G., K. Keerthana, G. Sridharan.2019. Pharmacognostic, physicochemical, and
phytochemical studies on stem bark of Zanthoxylum armatum DC. Asian J Pharm
Clin Res., 12 (2):470-474.
181
Joy, P. P., J. Thomas, S. Mathew and B. P. Skaria. 1998. Medicinal plants. Aromatic and
medicinal plants research station, Kerala Agricultural University Kerala, India. pp.
2
Kabir, S. A. K.Khanzada, M. Baloch, A. R.Khaskheli, W. Shaikh. 2015. Determination of
Total Protein Conents from Medicinal Plants (Zygophyllaceae) Famaily Found in
Pakistan. Sindh Univ. Res. Jour (Sci. Ser.).,47 (1):41-44.
Kadam, V.B., R.K. Momin, M.S.Wadikar, S. B. Andhale.2013. Determination of acid
insoluble ash values of some medicinal plants of genus Sesbania. JBPR., 2 (5):31-
34
Kaplan, D. R. 2001. The science of plant morphology: definition, history, and role in
modern biology. Amer. Jour. Bot., 88: 1711-1741.
Kasthuri, O. R., B. Ramesh. 2018. Physicochemical and Fluorescence Analysis of leaves
of Alternanthera brasiliana (L). Kuntze and Alternanthera bettzickiana (Regel)
Voss. Int. J. Pharm. Sci. Rev. Res., 51(1): 66-71.
Kato, K., S.Terao, N.Shimamoto, M. Hirata.1988. Studies on scavengers of active oxygen
Species. 1.Synthesis and biological activity of 2-Oalkylascorbic acids. J. Med.
Chem., 31:793-798.
Kayani, S., M. Ahmad, M. Zafar, S. Sultana, M.P.Z., Khan, M.A. Ashraf, J. Hussain and
G. Yaseen. 2014. Ethnobotanical uses of medicinal plants for respiratory disorders
among the inhabitants of Gallies–Abbottabad, Northern Pakistan. J.
Ethnopharmacol, 156: 47-60.
Kerr, J.R.F., A.H. Wyllie, A.R. Currie.1972. Apoptosis: a basic biological phenomenon
with wide ranging implications in tissue kinetics. Br J Cancer., 26: 239-257.
Khan, H., M. Saeed, A.H. Gilani, M.A. Khan, I. Khan, N. Ashraf. 2011. Antinociceptive
activity of aerial parts of Polygonatum verticillatum: Attenuation of both peripheral
and central pain mediators. Phytother Res. 25(7):1024-1030.
https://doi.org/10.1002/ptr.3369
182
Khan, M. A., H. Khan, S. Khan, T. Mahmood, P.M. Khan, A. Jabar. 2009. A.
Antiinflammatory, analgesic and antipyretic activities of Physalis minima Linn. J.
Enzyme Inhib. Med. Chem. 24(3),632-637.
Khan, M.A., M.A. Khan, G. Mujtaba and M. Hussain. 2012. Ethnobotanical study about
medicinal plants of Poonch valley Azad Kashmir. J. Anim. Plant Sci., 22(2): 493-
500.
Khan, M.P.Z., M. Ahmad, M. Zafar, S. Sultana, M.I. Ali and H. Sun. 2015. Ethnomedicinal
uses of Edible Wild Fruits (EWFs) in Swat Valley, Northern Pakistan. J
Ethnopharmacol., 173: 191-203
Khan, M.Q. and Z.K. Shinwari. 2016. The ethnomedicinal profile of family Rosaceae; a
study on Pakistani plants. Pak. J. Bot., 48: 613-620.
Khan, M.T., L. Ahmad, W. Rashid. 2018. Ethnobotanical documentation of traditional
knowledge about medicinal plants used by indigenous people in the Talash valley
of Dir lower, Northern Pakistan. J Intercult Ethnopharmacol.,7(1): 8–24
Khanum, S., S. Sarwar and M. S. Islam. 2019. In vivo Neurological, Analgesic and in vitro
Antioxidant and Cytotoxic Activities of Ethanolic Extract of Leaf and Stem Bark
of Wedelia chinensis. Bangladesh pharm. J., 22(1): 18-26.
Khurm, M., B. A. Chaudhry, M. Uzair and K.H. Janbaz. 2016. Antimicrobial, Cytotoxic,
Phytotoxic and Antioxidant Potential of Heliotropium strigosum Willd. Medicines,
3(20) :1 -12 ;doi:10.3390/medicines3030020
Kim, H.J., S.H. Yeom, M.K. Kim, I.N. Paek, M.W. Lee. 2005. Nitricoxide and
prostaglandin E2 synthesis inhibitory activities of diarylheptanoids from the barks
of Alnus japonica Steudel. Arch. Pharm. Res.,28:177–179.
Kim, Y.S., M.R. Young, G. Bobe, N.H. Colburn, J.A. Milner.2009. Bioactive food
components, inflammatory targets, and cancer prevention. Cancer Prev. Res., 2:
200–208.
183
Kinghorn, A. D. 2002. The role of pharmacognosy in modern medicine. Informa
healthcare, 3 (2): 77-79.
Klein, J., M. Hussain, M. Ahmad, M. Afzal, S. Alexandersen. 2008. Epidemiology of foot-
and-mouth disease in Landhi Dairy Colony, Pakistan, the world largest buffalo
colony. Virol J., 5: 53.
Knight-Jones T. J and J. Rushton. 2013. The economic impacts of foot and mouth disease-
what are they, how big are they and where do they occur? J. Prev. Vet.
Med.,112:161-173.
Knobloch, E., Kvacek, Z. (1976): Miozne Blatterfloren vom Westrand der Bohmischen
Masse. – Rozpr. Ustr. Ust. geol., 42: 1–31.
Kochhar, A., M. Nagi and R. Sachdeva. 2006. Proximate Composition, Available
Carbohydrates, Dietary Fibre and Anti Nutritional Factors of Selected medicinal
plants. Trad. Med. Pl. J. Hum. Ecol., 19 (3): 195-199.
Kokate, C. K. 1994. Practical Pharmacognosy. 4th Edn. Vallabh Prakashan, New Delhi:
120- 156.
Kopaei, M.R., A. Shakiba, M. Sedighi, and M. Bahmani. 2017. The Analgesic and Anti-
Inflammatory Activity of Linumusitatissimum in Balb/c Mice. J Evi Based
Complementary Altern Med., 22(4):892-896.
Kosala, K., M. A. Widodo, S. Santoso, S. Karyono. 2018. In vitro and in vivo Anti-
inflammatory Activities of Coptosapelta flavescens Korth Root‟s Methanol
Extract. J Appl Pharm Sci., 8(09):42-48.
Kostic, D.A., D.S. Dimitrijevic, S.S. Mitic, M.N. Mitic, G.S. Stojanovic and A.V.
Zivanovic, 2013. Phenolic content and antioxidant activities of fruit extracts of
Morus nigra L (Moraceae) from Southeast Serbia. Trop. J. Pharmac. Res., 12: 105-
110.
Krishnaiah, D., R. Sarbatly, R.A. Nithyanandam. 2011. Food and Bioprod Process, 89(3),
217–233.
184
Kuete, V., E. J. Seo, B. Krusche, M. Oswald, B. Wiench, S. Schroder, H. J. Greten, I. S.
Lee, and T. Efferth. 2013. Cytotoxicity and Pharmacogenomics of Medicinal Plants
fromTraditional Korean Medicine. Evid Based Complement Alternat Med.
2013:14.
Kumar M, A.Shete, Z. Akbar . 2010. A Review on Analgesic: From Natural Sources. Int J
Pharm Biol Arch., 1, 95-100.
Kumar N.S., A. Nusrath, D. Ramadas.2018. Quantitative analysis of chemical constituents
in medicinal plant Coleus aromaticus extracts. Int J Res Med Sci.,6(3):1002-1005
Kumar, A., K. Agarwal, A.K. Maurya, K. Shanker, U. Bushra, S.Tandon, D.U. Bawankule
.2015b. Pharmacological and phytochemical evaluation of Ocimum sanctum root
extracts for its antiinflammatory, analgesic and antipyretic activities. Pharmacogn
Mag. 11(42): 217-224. http://dx.doi.org/10.4103/0973-1296.157743
Kumar, B. J. R. and C. P. Kiladi. 2009. Preliminary Phytochemical and Pharmacognostic
Studies of Holoptelea integrifolia Roxb. Ethnobotanical Leaflets, 13: 1222-1231.
Kumar, S. and P. Kashyap.2015. In-Vivo Anti-Inflammatory Activity of an Methanolic
Extract of Fraxinus Micrantha. ARC j. pharm. sci., 1(1):1-4.
Kumar, S., S. D. Sharma, N. Kumar. 2015a. Ethnobatanical Study of Some Common
Plants From District Hamirpur of Himachal Pradesh (India). Int. J. Adv. Res.,
3(2):492-496.
Kumar, V., Z. A. Bhat, D. Kumar, M. Y. Shah, I. A. Chashoo and N. A. Khan. 2011.
Physicochemical and Preliminary Phytochemical Studies on Petals of Crocus
sativus „Cashmerianus‟. Pharmacon Jour., 3 (23): 46-49.
Kumari, A., A.K. Parida, J. Rangani and A. Panda. 2017a. Antioxidant Activities,
Metabolic Profiling, Proximate Analysis, Mineral Nutrient Composition of
Salvadora persica Fruit Unravel a Potential Functional Food and a Natural Source
of Pharmaceuticals. Front. Pharmacol., 8:61.
185
Kumari, K., H. Biswas, A. Aloria. 2017b. Analgesic Activity of Quisqualisindica. The
pharm. Chem. J., 4(1):1-8.
Kumbhar, R.R. and A.G. Godghate.2015. Physicochemical and quantitative phytochemical
analysis of some medicinal plants in and around Gadhinglaj. Int J Sci Environ
Technol., 4(1): 172 – 177.
Kuo, C.H., C.W. Lee, Y.C. Lai, S.S. Lee. 2008. Determination of Oregonin in Alnus plants
and biological samples by capillary electrophoresis. J Pharm Biomed Anal. 47:195–
200. https;//dx.doi.org/10.1016/j.jpba.2007.12.012
Lai, Y.C., C.K.Chen, W.W. Lin, S.S. Lee.2012. A comprehensive investigation of anti-
inflammatory diarylheptanoids from the leaves of Alnus formosana.
Phytochem.,73: 84–94.
Larson, R.A. 1998. The antioxidants of higher plants. Phytochem., 27:969–978.
Laurence D.R, P.N. Benneth, M.J.Brown. 1997. Clinical Pharmacology. 8th edn.
Edinburgh: ChurchHill Livingstone.
Lee, C.J., S.S. Lee, S. C. Chin, F.M. Ho, W. W. Lin. 2005. Oregonin inhibits
lipopolysaccharide-induced iNOS gene transcription and upregulates HO-1
expression in macrophages and microglia. Br. J. Pharmacol., 146:378–388.
Lee, I.R., J.Y. Song, Y.S. Lee. 1992. Cytotoxicity of folk medicine in murine and human
cancer cells. Saendyak Hakhoechi, 23:132– 136.
Lee, L. S., J.J. Dunn, A.J. Delucca, & A. Ciegler.1981. Role of lactone ring of aflatoxin
B1 in toxicity and mutagenicity. Experientia. 37: 16–7.
Lee, M., M.K. Lee, Y.C. Kim, S.H. Sung. 2011. Antifibrotic constituents of Alnus firma
on hepatic stellate cells. Bioorg. Med. Chem. Lett., 21:2906–2910.
Lee, M.A. H.K. Lee, S.H. Kim, Y.C. Kim, S.H. Sung. 2010.Chemical constituents of Alnus
firma and their inhibitory activity on lipopolysaccharide-induced nitric oxide
production in BV2 microglia. Planta Med., 76:1007–1010.
186
Leong, L.P., G. Shui. 2002. An investigation of antioxidant capacity of fruits in Singapore
markets. Food Chem., 76:69–75
Leon-Gonzalez, A.J., N. Acero, D. Munoz-Mingarro, M. Lopez-Lazaro, M., C. Martín-
Cordero. 2014. Cytotoxic activity of hirsutanone, a diarylheptanoid isolated from
Alnus glutinosa leaves. Phytomed., 21: 866–870.
Leopold, E. B., J. Birkebak, L. Reinink-Smith, A.P. Jayachandar, P. Narváez, S. Zaborac-
Reed.2012. Pollen morphology of the three subgenera of Alnus. Palynology, 36(1),
131-151.
Li, S., H.Y. Tan, N. Wang, Z.J. Zhang, L. Lao, C.W. Wong and Y. Feng. 2015. The role of
oxidative stress and antioxidants in liver diseases. Int. J. Mol. Sci., 16: 26087–
26124.
Lim, S.S., M.Y. Lee, H.R. Ahn, S.J. Choi, J.Y. Lee, S.H. Jung. 2011. Preparative isolation
and purification of antioxidative diarylheptanoid derivatives from Alnusjaponica
by high-speed counter-current chromatography. J. Sep. Sci., 34: 3344–3352.
Liu, A.L. and G.H. Du. 2012. Antiviral properties of phytochemicals. In: Patra A.K, Ed.
Dietary phytochemical and microbes. New York: Springer; pp. 93–126.
Loganathan, V., M.D. Kania-kumari, P. Silva-kumar. 2017. A Study of the Physico-
Chemical and Phytochemical Parameters of Leaves of Mallotus rhamnifolius.
IJPPR., 9(6):858-863.
Loi, M.C., F. Poli, G. Sacchetti, M.B. Selenu, M.Ballero. 2004. Ethnopharmacology of
Ogliastra (Villagrande Strisaili, Sardinia, Italy), Fitoterapia., 75: 277-295.
Lokhande, R., P. Singare and M. Andhale. 2010. Study on Mineral content of Some
Ayurvedic Indian Medicinal Plants by Instrumental Neutron Activation Analysis
and AAS Techniques. Health Sc. Jour., 4 (3): 157-168.
Lucarini, R., M.G. Tozatti, M.L.A. Silva, V.M.M. Gimenez, P.M. Pauletti, M. Groppo,
I.C.C. Turatti, W.R. Cunha and C.H.G. Martins. 2015. Antibacterial and anti-
187
inflammatory activities of an extract, fractions, and compounds isolated from
Gochnatia pulchra aerial parts. Braz J Med Biol Res., 48(9): 822–830. doi:
10.1590/1414-431X20154410
Ludwiczuk, A., A. Saha, T. Kuzuhara, Y. Asakawa.2011. Bioactivity guided isolation of
anticancer constituents from leaves of Alnus sieboldiana (Betulaceae), Phytomed.,
18(6): 491-498.
Lulekal, E., Z. Asfaw, E. Kelbessa and P. V. Damme. 2013.Ethnomedicinal study of plants
used for human ailments in Ankober District, North Shewa Zone, Amhara
Region.Ethiopia. J. Ethnobiol. Ethnomed., 9:63. doi: 10.1186/1746-4269-9-63.
Lungu, L., C. Popa, J. Morris and M. Savoiu. 2011. Evaluation of phytotoxic activity of
Melia azedarach L. extracts on Lactuca sativa L. Romanian Biotech. Letters, 16(2):
6089 -6095.
Lupu, A, Popescu, T. 2013. The noncellular reduction of MTT tetrazolium salt by TiO2
nanoparticles and its implications for cytotoxicity assays. Toxicol in Vitro., 2:1445-
50.
Madhu, M., V. Sailaja, T.N.V.S.S. Satyadev, M.V. Satyanarayana.2016. Quantitative
phytochemical analysis of selected medicinal plant species by using various
organic solvents. J Pharmacogn Phytochem.,5(2): 25-29
Mah, S.H., S.S. Teh, G.C. Lian -Ee. 2019. Comparative studies of selected Calophyllum
plants for their anti-inflammatory properties. Phcog Mag.,2(15):135-139.
Mahoney, N. and R.J. Molyneux. 2004. Phytochemical inhibition of aflatoxigenicity in
Aspergillus flavus by constituents of walnut (Juglans regia)., J.Agric. Food Chem.
52:1882–1889.
Mai, D.H. & H. Walther, 1988. Die pliozänen Floren von Thüringen, Deutsche
Demokratische Republik. – Quartärpaläontologie, 7: 55-297.
188
Mandal, M., D. Misra, N. N. Ghosh, V. Mandal. 2017. Physicochemical and elemental
studies of Hydrocotyle javanica Thunb. for standardization as herbal drug. Asian
Pac J Trop Biomed., 7(11): 979–986.
Mann, G.E., L.P. Codifer, H.K. Gardner, S.P. Koltun, F.G. Dollear.1 970. Chemical
inactivation of aflatoxins in peanut and cottonseed meals.J Am Oil Chem Soc.,
47(5):173-6.
Manouze, H., O. Bouchatta, A.C. Gadhi, M. Bennis, Z. Sokar and S. Ba-M‟hamed. 2017.
Anti-inflammatory, Antinociceptive, and Antioxidant Activities of Methanol and
Aqueous Extracts of Anacyclus pyrethrum Roots. Front. Pharmacol. 8:598.
Maobe, M.A., E. Gatebe, L. Gitu and H. Rotich. 2012. Profile of heavy metals in selected
medicinal plants used for the treatment of Diabetes, Malaria and pneumonia in Kisii
region, Southwest Kenya. Global J. Pharmacol., 6(3): 245-251.
Markert, B. 1994. „Plants as biomonitors–Potential advantages and problems‟, in: D.C.
Adriano, Z.S. Chen and S.S. Yang (eds), Biogeochemistry of Trace Elements.
Science and Technology Letters. Northwood, NY, pp. 601–613.
Martin, J.D.W., P.A. Mayers, V.W. Rodwell, D.K. Granner .1985. Harper‟s Review of
Biochemistry. (20th edtn) Lange Medical Publications, California: 651-660.
Mary, T.A., E. Edmund, M. K. Priscilla, B. E. Mariam, M. L. K. Merlin, W. Eric. 2019.
Pharmacog-nostic Studies of the Leaves, Stem and Root of Capparis erythrocarpos
Isert (Capparaceae). Pharmacog J.,11(1):112-8; DOI
:10.5530/pj.2019.1.19.
Mate, G.S., N.S. Naikwade, C.S.Magdum,A.A. Chowki and S.B. Patil.2008. Evaluation of
Antinociceptive Activity of Cissus quadrangularis on Albino Mice. Int J Green
Pharm., 2:118–121.
189
Mathur, J., P. Khatri, K. C. Samanta, A. Sharma and S. Mandal. 2010. Pharmacognostic
and preliminary phytochemical investigations of Amaranthus spinosus (Linn.)
leaves. Int. J. Pharm. Sci., 2(4): 121- 124.
Matthew, S., A. K. Jain, M. James, C. Matthew and D. Bhowmik.2013. Analgesic and
Anti-Inflammatory Activity of Kalanchoe Pinnata (Lam.) Pers., 1(2): 24-28.
Mau, J.L., M.B. Miklus and R.B. Beelman, 1999. Shelf life studies of foods and beverages
charalambous E.d. Chem. Biol. Phys. Nutr. Aspect., 57: 475-477.
Mazid A, B.K.Datta, L.Nahar, A. Rashid, S.C. Bachar, S.A.M. Khairul-Bashar, S.D.
Sarkar. 2010. Analgesic and diuretic properties of santalone from Polygonum
flaccidum. Phytother Res., 24(7),1084-1087. https://doi.org/10.1002/ptr.3053
McElwain J.C. &W.G. Chaloner. 1995. Stomatal density and index of fossil plants track
atmospheric carbon dioxide in the Palaeozoic. Annals of Botany., 76(4): 389-395.
Mehta, A., R.C. Rana, Y. P. Sharma and P. Thakur.2018. Physico-chemical analysis of
some temperate Himalayan Swertia species. J. Pharmacog. Phytochem., 7(3):
156-159.
Mekaway, A.A.I., M. M. Mokhtar, M.F. Rasha.2009. Antitumor and antibacterial activities
of [1-(2-Ethyl, 6-Heptyl) Phenol] from Cuminum cyminum seeds. J. Appl. Sci. Res.,
5(11):1881-1888.
Mendez-Albores, A., J.C. Del-Rio-Garcia, & E. Moreno-Martinez. 2007. Decontamination
of aflatoxin duckling feed with aqueous citric acid treatment.Anim Feed Sci
Technol., 135:249-262.
Mendez-Albores, A., J.C. Del-Rio-Garcia, & E. Moreno-Martinez. 2007. Decontamination
of aflatoxin duckling feed with aqueous citric acid treatment. Anim Feed sci Tech.,
135:249-262.
Merrill, A. L. and B. K. Watt. 1973. Energy value of foods, basis and derivation. United
States Department of Agriculture Handbook 74. USDA, Washington, DC.
190
Miliauskas, G., P. R. Venskutonis, and T. A. Van Beek. 2004. Screening of radical
scavenging activity of some medicinal and aromatic plant extracts. Food Chem.,
85: 231-237
Ming, D. S., B. J. Hillhouse, E. S. Guns, A. Eberding, S. Xie, S. Vimalanathan and G. H.
N. Towers. 2005. For Bioactive compounds from Rhodiola rosea
(Crassulaceae). Phytotherapy Res., 19 (9): 740-743.
Mohamed, G.A., S.R.M. Ibrahim, E.S. Elkhayat, S.A. Ross, H.M. Sayed, S.A.M. El-
Moghazy, M.A. El-Shanawany. 2015. Blepharisides A and B, new flavonol
glycosides from Blepharis ciliaris growing in Saudi Arabia. Phytochem Lett.,
11,177 182. http://dx.doi.org/10.1016/j.phytol.2014.12.018.
Mohammed, S., F. A. Manan. 2015.Analysis of total phenolics, tannins and flavonoids
from Moringa oleifera seed extract. J. Chem. Pharm. Res., 7(1):132-135.
Mohlenbrock, R. H., "Flowering Plants: Smartweeds to Hazelnuts" (2009). Illustrated
Flora of Illinois. 13. http://opensiuc.lib.siu.edu/siupress flora of illinois/13.
Moncada, S., R.M. Palmer, E.A. Higgs. 1991. Nitric oxide: physiology, pathophysiology,
and pharmacology. Pharmacol Rev., 43,109–142.
Morris, M. J., S. E. Na and A. K. Johnson. 2008. Salt craving: The psychobiology of
pathogenic sodium intake. Phys. & Behavior, 94: 709-721.
Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. of Immunol Methods, 65(1-2), 55-63.
Muhammad, N., M. Saeed and H. Khan. 2012. Antipyretic, analgesic and antiinflammatory
activity of Viola betonicifolia whole plant. BMC Complem. Altern. Med.,12: 59.
https://dx.doi.org/10.1186/1472-6882-12-59.
Murray, R.K., D.K. Granner, P.A. Mayes, V.W. Rodwell. 2000. Harper‟s Biochemistry,
(25th edtn), McGraw-Hill, Health Profession Division, USA.
191
Nadgir, S.V., H. R. Hensler, E. R. Knowlton, C. R. Rinaldo, G. Rappocciolo, F. J.
Jenkins.2013. Fifty Percent Tissue Culture Infective Dose Assay for Determining
the Titer of Infectious Human Herpesvirus 8.J Clin Microbiol. 51: 1931–1934. doi:
10.1128/JCM.00761-13.
Naithani, R., L.C. Huma, L.E. Holland, D.Shukla, D.L. McCormick,R.G. Mehta,
R.M.Moriarty. 2008. Antiviral activity of phytochemicals: A comprehensive
review. Mini Rev. Med. Chem., 8:1106–1133.
Najafi, S. and S. S. Deokule. 2010. Pharmacognostic study of Tylophora dalzellii Hook. f.
J. Med. Pl. Res., 4 (5): 403-406.
Najafi-momen, R., M. Torabi-goudarzi, A. Bahonar, H. Akbari, M. Darabi. 2011.Clinical
evaluation of the effect of Myrtle oil on the oral lesions of FMD in cattle. J. medic.
Plants., 10(38): 135-141.
Naseem, R., K. Mahmud and M. Arshad. 2006. Chemical composition and antibacterial
activity of Crotalaria burhia from Cholistan Desert, Pakistan. Hamdard Medicus,
49 (4): 49-52.
Nasim, M.J., M.H.H.B. Asad, A. Sajjad, S.A. Khan, A. Mumtaz, K. Farzana, Z. Rashid and
G. Murtaza. 2013. Combating of scorpion bite with Pakistani medicinal plants
having ethno-botanical evidences as antidote. Acta Pol Pharm.,70 (3):387-394.
Naz, S., M.A. Anjum, S.A.H. Naqvi, B. Siddique and M.A. Zulfiqar. 2018. Assessment of
Proximate, Nutritional and Mineral Contents in Some Traditional Vegetables
Consumed in Multan, Pakistan. "Pak. J. Agric. Sci., 31(4): 375-381.
Neffati, N., Z. Aloui, H. Karoui, I. Guizani, M. Boussaid & Y. Zaouali. 2017.
Phytochemical composition and antioxidant activity of medicinal plants collected
from the Tunisian flora. J Nat Prod Res., 31(13):1583-1588.
Neves, J.M., C. Matos, C. Moutınho, G. Queıroz and L.R. Gomes. 2009.
Ethnopharmacological notes about ancient uses of medicinal plants in Tras-os-
Montes (northern of Portugal). J Ethnopharmacol., 124: 270-283.
192
Nilam, R., P. Jyoti and C. Sumitra. 2018. Pharmacognostic and phytochemical studies of
Ipomoea pes-caprae, a halophyte from Gujarat. J Pharmacogn Phytochem., 7(1):
11-18.
Nisar, M., S. A. Tariq, I. K. Marwat, M. R. Shah and I. A. Khan. 2009. Antibacterial,
antifungal, insecticidal, cytotoxicity and phytotoxicity studies on Indigofera
gerardiana. J. Enzy. Inhi. &Med. Chem., 24 (1): 224-229.
Noman, A., I. Hussain. Q. Ali, M. A. Ashraf and M. Z. Haider.2013. Ethnobotanical studies
of potential wild medicinal plants of Ormara, Gawadar, Pakistan. Emir. J. Food
Agric., 25 (10): 751-759; doi: 10.9755/ejfa.v25i10.16401
Novakovic, M., M. Pesic, S. Trifunovic, I. Vuckovic, N. Todorovic, A. Podolski-Renic, J.
Dinic, S. Stojkovic, V. Teševic and V. Vajs, et al. 2014. Diarylheptanoids from the
bark of black alder inhibit the growth of sensitive and multi-drug resistant non-small
cell lung carcinoma cells. Phytochem., 97: 46–54.
Novakovic, M., M. Stankovic, I. Vuckovic, N. Todorovic, S. Trifunovic, V. Tesevic, V.
Vajs, S. Milosavljevic. 2013. Diarylheptanoids from Alnus glutinosa bark and their
chemoprotective effect on human lymphocytes DNA. Planta Med., 79: 499– 505.
O‟Rourke C., M. Byres, A.Delazar, Y.Kumarasamy, L. Nahar, F. Stewart, S.D. Sarker.
2005. Hirsutanonol, oregonin and genkwanin from the seeds of Alnus glutinosa
(Betulaceae). Biochem Syst Ecol.,33: 749–
752.http://dx.doi.org/10.1016/j.bse.2004.10.005
Ogidi, O., N. G. Esie and O. G Dike. 2019. Phytochemical, proximate and mineral
compositions of Bryophyllum Pinnatum (Never die) medicinal plant. J
Pharmacogn Phytochem., 8(1): 629-635.
Oguntibeju, O.O. 2018. Medicinal plants with anti-inflammatory activities from selected
countries and regions of Africa. J Inflamm Res.,11: 307–317.
193
Okokon, J.E., P.A. Nwafor. 2010. Antiinflammatory, analgesic and antipyretic activities
of ethanolic root extract of Croton zambesicus. Pak J Pharm Sci., 23(4): 385-392.
Ondua, M., E.M. Njoya, M.A. Abdalla, L.J. McGaw. 2019. Anti-inflammatory and
antioxidant properties of leaf extracts of eleven South African medicinal plants used
traditionally to treat inflammation. J Ethnopharmacol., 234:27-35; doi:
10.1016/j.jep.2018.12.030.
Pagare, S., M. Bhatia, N. Tripathi, S. Pagare, and Y.K. Bansal.2015. Secondary metabolites
of Plants and their Role: Overview., Curr. Trends Biotechnol. Pharm. 9 (3) 293-
304.
Pal, R. S., Y. Pal, P. Wal and A. Wal.2018. Pharmacognostic Evaluation of Roots of
Benincasa Hispida (Thunb.) Cogn. (Cucurbitaceae). TOPSJ. 11:1-6
Palumbo, J. D., T.L. Keeff, and N.E. Mahoney. 2007. Inhibition of ochratoxin A
production and growth of Aspergillus species by phenolic antioxidant compounds.
Mycopathologia,164: 241–248.
Pandey, S. C., H. Zhang, A. Roy and K. Misra. 2006. Central and medial amygdaloid brain-
derived neurotrophic factor signaling plays a critical role in alcoholdrinking and
anxiety-like behaviors. J. Neurosci., 26: 8320–8331.
Park, D., H.J. Kim, S.Y. Jung, C.S. Yook, C. Jin and Y.S. Lee. 2010. A new
diarylheptanoid glycoside from the stem bark of Alnus hirsuta and protective
effects of diarylheptanoid derivatives in human HepG2 cells. Chem. Pharm. Bull.,
58: 238–241.
Parvez, M., B. Ahmad, F. Hussain, J. Ali, S. Muhammad, S. Abbas, S. Hassan, I. Ahmad.
2014. Phytotoxicity Study of Euphorbia granulata Forssk against Lemna minor and
Radish Seeds. Life Sci J., 11(6s):30-34.
194
Patel, S.S. & J. K. Savjani. 2015. Systematic review of plant steroids as potential anti-
inflammatory agents: Current status and future perspectives. J
Phytopharmacol.,4(2): 121-125.
Patil, V. S., K. S. Rajput, N. P. Malpathak. 2016. Comparative study on morpho-anatomy
of leaf, stem and root of Boerhaavia diffusa L. (Nyctaginaceae) and its adulterant
plants. Braz. J. Pharm. Sci.,52(3):433-442. http://dx.doi.org/10.1590/S1984-
82502016000300009
Patra, A., S. Jha, P. N. Murthy, A. Vaibhav, P. Chattopadhyay, G. Panigrahi and D. Roy.
2009. Anti-Inflammatory and Antipyretic Activities of Hygrophila spinosa
T. Anders Leaves (Acanthaceae). Trop. J. Pharma. Res., 8 (2): 133-137.
Phanse, M.A., M.J. Patil, K. Abbulu, P.D. Chaudhari and B. Patil.2012. In-vivo and in-
vitro screening of medicinal plants for their anti-inflammatory activity: an
overview. J. Appl. Pharm. Sci.,02 (06): 19-33.
Pie, S.J. and N.P. Manadhar 1987. Source of some local medicines in the Himalayan
Regions. Himalayan Ecosystem, pp.77-112.
Pohl, P., A. Dzimitrowicz, D. Jedryczko, A. Szymczycha-Madeja, M. Welna and P.
Jamroz.2016. The determination of elements in herbal teas and medicinal plant
formulations and their tisanes. J. Pharm. Biomed. Anal.,130: 326-335.
Pokala, N., N. Alasyam, K. Rasama. 2019. Evaluation and comparison of antipyretic
activity of aqueous leaf extracts of Vitex negundo and Andrographis paniculata in
rabbits. Natl J Physiol Pharm Pharmacol., 9(6):556-561.
Poljsak, B., D. Suput, I. Milisav. 2013. Achieving the balance between ROS and
antioxidants: When to use the synthetic antioxidants. Oxid. Med. Cell. Longev.,
2013: 956792.
Pool, I., J. D. B. Weyfrs, T. Lawson & J. A. Ravfn. 1996. Variations in stomatal density
and index: implications for palaeoclimatic reconstructions. Plant Cell
Environ.,19:705-712.
195
Prakash, B., P. Singh, P.K. Mishra and N.K. Dubey. 2011b. Safety assessment of
Zanthoxylum alatum Roxb. essential oil, its antifungal, antiaflatoxin, antioxidant
activity and efficacy as antimicrobial in preservation of Piper nigrum L. fruits. Int.
J. Food Microbiol., 153: 183–191.
Prakash, B., R. Shukla, P. Singh, A. Kumar, P.K. Mishra and N.K. Dubey. 2010. Efficacy
of chemically characterized Piper betle L. essential oil against fungal and aflatoxin
contamination of some edible commodities and its antioxidant activity. Int. J. Food
Microbiol., 142: 114–119.
Prakash, B., R. Shukla, P. Singh, P.K. Mishra, N.K. Dubey and R.N. Kharwar. 2011a.
Efficacy of chemically characterized Ocimum gratissimum L. essential oil as an
antioxidant and a safe plant based antimicrobial against fungal and aflatoxin B1
contamination of spices. Food Res. Int. 44:385–390.
Prasad, A. S. 1982. Clinical, Biochemical and Nutritional Aspects of Trace Elements.
Alan R. Liss, Inc, New York.
Priyadarshi, A., R. Kumari, A. K. Sharma and M. L. Jaiswal. 2016. Preliminary
Pharmacognostic and Phytochemical Investigation of Blepharis sindica-T. Anders
Seeds. Anc Sci Life.,36(2):78-83. doi: 10.4103/asl.ASL_29_16
Qi, H.M., Q.B. Zhang, T.T. Zhao, R. Chenc, H. Zhang and X.Z. Niu. 2005. Antioxidant
activity of different sulfate content derivatives of polysaccharide extracted from
Ulva pertusa (Chlorophyta) in vitro. Int. J. Biol. Macromol., 37:195–199.
Radulovic, N., G. Stojanovic and R. Palic. 2006. Composition and antimicrobial activity
of Equisetum arvense L, essential oil. Phytother. Res., 20: 85–88.
Raj-Narayana K, M.S. Reddy, M.R. Chaluvadi, D.R. Krishna. 2001. Bioflavonoids
classification, pharmacological, biochemical effects and therapeutic potential.
Indian J Pharmacol, 33:2-16.
196
Raju M.R., P.V.M. Rao, T.S. Reddy, M.K. Raju, J.S.B. Rao and C. R. Venkatasubramani.
2016. Elemental analysis of medicinal plants from different sites by instrumental
neutron activation analysis. Int. J. Bioassays, 5(3): 4892-4896.
Ramadurga, B., R. K. Jat, S. Badami. 2019. Pharmacognostic Evaluation and Antimicrobial
Activity of Root of Careya arborea. Pharmacogn J.,11(3): 608- 612.
Rangari, V. D. 2002. Pharmacognosy and phytochemistry. Volume-1. Career publications,
Maharashtra, India.pp.1
Ranjith, D. 2018. Fluorescence analysis and extractive values of herbal formulations used
for wound healing activity in animals. J. Med. Plants Stud., 6(2):189-192.
Rao, M.R.K., S. S. Kumar. 2017. Preliminary phytochemical analysis of herbal plant
Hygrophila auriculata. Indo Am. J. P. Sci., 4(12):4580-4583.
Raquibul-Hasan, S.M., M.M. Hossain, R. Akhtar, M. Jamila, M.E.H. Mazumder and
M.A. Alam et al. 2010. Analgesic Activity of the Different Fractions of the Aerial
Parts of Commenila benghalensis Linn.Int. J. Pharmacol.,6(1):63–67;
http://dx.doi.org/10.3923/ijp.2010.63.67
Raskin, I., D.M. Ribnicky, S. Komarnytsky, N. Ilic, A. Poulev abd N. Borisjuk et al. 2002.
Plants and human health in the twenty-first century. Trends Biotechnol.,20(12):
522–531 pmid:12443874.
Raters M, R. Matissek. 2008.Thermal stability of aflatoxin B1 and ochratoxin A.
Mycotoxin Res.,24(3):130–4.
Ravi, S., S. Ashokkumar, K. Mallika, P. Kabilar, P. Paneerselvam and M. Gayathri. 2011.
Morphological, micro and macro nutrient analysis of the medicinal plant glory lily
(Gloriosa superba L.). J. Experim. Sci., 2 (6): 04-06.
Reddy, M. and A. A. Chaturvedi. 2010. Pharmacognostical studies of Hymenodictyon
orixence (Roxb.) Mabb. leaf. Int. J. Ayurveda Res., 1 (2): 103-5.
197
Reed, L.J. and H. Muench, 1938. A simple method of estimating fifty per cent
endpoints.Am J Epidemiol., 27: 493-497.
Rehman, A. and M. Adnan (2018). Nutritional potential of Pakistani medicinal plants and
their contribution to human health in times of climate change and food insecurity.
Pak. J. Bot., 50(1): 287-300.
Ren, X., T. He, Y. Chang, Y. Zhao, X. Chen, S. Bai, L. Wang, M. Shen, G. She. 2017.The
Genus Alnus, A Comprehensive Outline of Its Chemical Constituents and
Biological Activities. Molecules, 22:1383.
http://dx.doi.org/10.3390/molecules22081383.
Rezk, A., A. Al- Hashimi, W. John, H. Schepker, M. S. Ullrich and K. Brix. 2015.
Assessment of cytotoxicity exerted by leaf extracts from plants of the genus
Rhododendron towards epidermal keratinocytes and intestine epithelial cells. BMC
Complement Altern Med. 15:364; DOI 10.1186/s12906-015-0860-8.
Ruba, A. A., V.R. Mohan.2016. Pharmacognostical Studies and Phytochemical
Investigation of Andrographis echioides (L). Nees (Acanthaceae).
IJPPR.,8(6):941-948.
Saadullah, M., B.A. Chaudary, M. Uzair. 2016.Antioxidant, phytotoxic and antiurease
activities, and total phenolic and flavonoid contents of Conocarpus lancifolius
(Combretaceae). Trop J Pharm Res; 15(3):555-561.
Sadeghi, Z., J. Valizadeh, O. A. Shermeh and M. Akaberi. 2015. Antioxidant activity and
total phenolic content of Boerhavia elegans (choisy) grown in Baluchestan, Iran.
Avicenna J Phytomed., 5(1): 1–9.
Saeed, M., H. Khan, M. A. Khan, F. U. Khan, S. A. Khan and N. Muhammad. 2010.
Quantification of various metals and cytotoxic profile of aerial parts of
Polygonatum verticillatum. Pak. J. Bot., 42 (6): 3995-4002.
Saifullah, W., Samiullah, N. Khan, Attiq-ur-Rehman, P.W. Mengal, A. Baqi and A. Manan.
2019. Profiling of various elements in Haloxylon griffithii and
198
Convolvulus leiocalycinus using atomic absorption spectroscopy and flame
photometry. Pure Appl Biol., 8(20):1535-1542.
Saiki, M., M.B. Vasconcellos, J.A. Sertie. 1990. Determination of inorganic components
in Brazilian medicinal plants by neutron activation analysis. Biol Trace Elem Res.,
26-27: 743-50.
Sajid, M., C. Yan, D. Li, S. B. Merugu, H. Negi and M. R. Khan.2019. Potent anti-cancer
activity of Alnus nitida against lung cancer cells; in vitro and in vivo studies.
Biomed Pharmacother., 110: 254–264.
Sajid, M., M. R. Khan, S. A. Shah, M. Majid, H. Ismail, S. Maryam, R. Batool, T.
Younis.2017. Investigations on anti-inflammatory and analgesic activities of Alnus
nitida (Spach) Endl. stem bark in Sprague Dawley rats. J. Ethnopharmacol.,
198:407–416.http://dx.doi.org/10.1016/j.jep.2017.01.041
Sajid, M., M.R. Khan, N.A. Shah, S.A. Shah, H. Ismail, T. Younis, Z. Zahra. 2016.
Phytochemical, antioxidant and hepatoprotective effects of Alnus nitida bark in
carbon tetrachloride challenged Sprague Dawley rats. BMC Complem. Altern.
Med., 16:268.
Samapundo, S., B. De-Meulenaer, D. Osei-nimoh, Y. Lamboni, J. Debevere and F.
Devlieghere. 2007. Can phenolic compounds be used for the protection of corn
from fungal invasion and mycotoxin contamination during storage? Food
Microbiol., 24(5):465–473.
Sandosskumar, R., M. Karthikeyan, S. Mathiyazhagan, M. Mohankumar and G.
chandrasekar et al. 2007. Inhibition of Aspergillus flavus growth and detoxification
of aflatoxin B1 by the medicinal plant Zimmu (Allium sativm L. X Allium cepa L.).
World J Microbiol Biotechnol., 23: 1007-1014.
Sanjeeva K. A., R.J. Raveendra, M.G.V. Rama. 2014. Preliminary phytochemical and
standardization parameters of Ipomoea quamoclit Linn whole plant- an
ethnomedicinally important plant. Int J Pharm Sci., 6(7):162-16.
199
Sarkar, A., V. D. Tripathi, R. K. Sahu, E. M. Faller. 2017. Pharmacognostic and
Preliminary Phytochemical Evaluation of Centipeda minima and Bauhinia
purpurea Leaves.UK. J. Pharm. Biosci., 5(2), 08-16.
Sarker, S.D. 2012.Pharmacognosy in modern pharmacy curricula. Phcog. Mag.,8: 91-
92.
Sati, S.C., N. Sati, O.P. Sati.2011. Bioactive constituents and medicinal importance of
genus Alnus. Phcogn Rev., 5(10):174-183. http://dx.doi.org/10.4103/0973-
7847.91115
Saxena, A., D. Yadav, A.K. Maurya, A. Kumar, S. Mohanty, M.M. Gupta, M.C. Lingaraju,
M.I. Yatoo, U.S. Thakur, D.U. Bawankule. 2016. Diarylheptanoids from Alnus
nepalensis attenuates LPS-induced inflammation in macrophages and endotoxic
shock in mice. Int. Immuno pharmacol., 30 :129–136.
Selvakumar, S., B. Sarkar. 2017. In Vitro Cytotoxicity Analysis of Chloroform Extract of
Novel Poly Herbal Formulation. IJPPR., 9(2):193-196
Selvam, A. B. D. 2010. Is the term substitution relevant to Pharmacognosy and/ or
vegetable crude drug industry. Pharmacon. Res., 2 (5): 323-324.
Selvi, A.T., G.S. Joseph and G.K. Jayaprakasha. 2003. Inhibition of growth and aflatoxin
production in Aspergillus flavus by Garcinia indica extract and its antioxidant
activity. Food Microbiol., 20: 455–460.
Serkedjieva, J., S. Ivancheva. 1999. Antiherpes virus activity of extracts from the
medicinal plant Geranium sanguineum L. J. Ethnopharmacol., 64 :59–68
Serkedjieva, J.2003. Influenza virus variants with reduced susceptibility to inhibition by a
polyphenol extract from Geranium sanguineum L. Pharmazie., 58(1):53-7.
Shah, A., A. Niaz, N. Ullah, A. Rehman, M. Akhlaq, M. Zakir and M. S. Khan. 2013.
“Comparative Study of Heavy Metals in Soil and Selected Medicinal Plants”, J.
Chem., 2013:1-5.https://doi.org/10.1155/2013/621265
200
Shah, A.H., S.M. Khan, A.H. Shah, A. Mehmood, I.U. Rahman and H. Ahmad. 2015a.
Cultural uses of plants among Basikhel tribe of District Tor Ghar, Khyber
Pakhtunkhwa, Pakistan. Pak. J. Bot., 47: 23-41
Shah, S.M., Z. Ahmad, M. Yaseen, R. Shah, S. Khan, S. M. Shah, B. Khan. 2015b.
Phytochemicals, in vitro antioxidant, total phenolic contents and phytotoxic activity
of Cornus macrophylla Wall bark collected from the North-West of Pakistan. Pak
J Pharm Sci., 28(1):23-8.
Shaheen, H. and Z.K. Shinwari. 2012. Phyto diversity and Endemic richness of Karambar
Lake Vegetation from Chitral, Hindukush- Himalayas. Pak. J. Bot., 44(1): 17-21.
Shaheen, H., R.A. Qureshi and Z.K. Shinwari. 2011. Structural Diversity, Vegetation
Dynamics and Anthropogenic Impact on Lesser Himalayan Subtropical Forests of
Bagh District, Kashmir. Pak. J. Bot., 43(4): 1861-1866.
Shaikh, R. U., M. M. Pund and R. N. Gacche. 2016. Evaluation of anti-inflammatory
activity of selected medicinal plants used in Indian traditional medication system
in vitro as well as in vivo. J Tradit Complement Med., 6(4): 355–361. doi:
10.1016/j.jtcme.2015.07.001
Shailendra, S., B. Anil, C. Deepak, S. Rambabu. 2014. Pharmacognostical and
Phytochemical analysis of leaf part of the Medicinal Herb: Zaleya govindia. Int. J.
Drug Dev. & Res. 6 (1): 153-159.
Shakiba, Y., S. E. Rezatofighi, S.M. S. Nejad, M. R. Ardakani. 2018. Inhibition of Foot-
and-Mouth Disease Virus Replication by Hydro-alcoholic and Aqueous-Acetic
Acid Extracts of Alhagi maurorum., Iran. J. Pharm. Sci., 14 (1): 85-96.
Shanker, G.R. 1989. Studies on natural variation in Alnus nitida (Spach) Endl. and A.
nepalensis D. Don. PhD thesis university of horticulture and forestry. Solan,
India.
Sharif, A., M. F. Akhtar, B. Akhtar, A. Saleem,M. Manan, M. Shabbir,M. Ashraf,S.
Peerzada,S. Ahmed and M. Raza. 2017. Genotoxic and cytotoxic potential of
201
whole plant extracts of Kalanchoe laciniata by Ames and MTT assay. EXCLI J.,
16: 593–601;doi: 10.17179/excli2016-748.
Sharma, A., A. Kumar. 2016. Pharmacognostic studies on medicinal plants: Justicia
adhatoda. World J of Pharma Res.,5 (7):1674-1704.
Sharma, K.R., M. Agrawal, F.M. Marshall. 2009.Heavy metals in vegetables collected
from production and market sites of a tropical urban area of India. Food Chem.
Toxicol.,47, 583-591.
Shaw, K., L.Stritch, M. Rivers, S. Roy, B.Wilson and R. Govaerts. 2014a. The Red List of
Betulaceae. BGCI. Richmond. UK.
Shaw, K., S. Roy, B. Wilson. 2014b. Alnus nitida. The IUCN Red List of Threatened
Species.2014e.T194659A2356455.http://dx.doi.org/10.2305/IUCN.UK.20143.RL
TS.T194659A2356455.en.
Sheth, K., E. Bianchi, R. Wiedhopf, J.R. Cole. 1973. Antitumor agents from Alnus oregona
(Betulaceae). J. Pharm. Sci., 62:139–140.
Shinwari, S., M. Ahmad, Y. Luo and W. Zaman. 2017. Quantitative analyses of medicinal
plants consumption among the inhabitants of Shangla- Kohistan areas in Northern-
Pakistan.Pak.J.Bot.,49(2):725-734.
Shinwari, Z. K. and M. Qaiser. 2011. Efforts on Conservation and sustainable use of
medicinal plants of Pakistan. Pak. J. Bot., 43:5-10.
Shinwari, Z.K. 1996. Ethnobotany in Pakistan: Sustainable and participatory approach. In
Proceedings Ethnobotany and its application to conservation, p. 14-25,
Ethnobotany and its application to conservation. NARC, NARC, Islamabad,
Pakistan.
Shojaii, A., M. Motaghinejad, S. Norouzi and M. Motevalian.2015. Evaluation of Anti-
inflammatory and Analgesic Activity of the Extract and Fractions of
Astragalushamosus in Animal Models. IranJPharmRes., 14 (1): 263-269.
202
Shrikumar, S., U. Maheshwari, A. Sughanti, T.K. Ravi.2006. WHO guidelines for
standardization of herbal drugs.Pharminfo.net., 2:78-81.
Shrivastava, S. and S. Leelavathi. 2010. Preliminary phytochemical evaluation of leaf
extracts of Catunaregum spinosa thunb. Intern. J. Pharma. Sci. Rev. & Res., 3 (2):
114-118.
Shruthi, S. D., Y.L. Ramachandra, S.P. Rai, P.K. Jha.2010. Pharmacognostic evaluation of
the leaves of Kirganelia reticulate Bail. The Asian and Aust. J.Plant Sci. Biotec., 4
(1): 62-65.
Shukla, A., S. Vats, R. K. Shukla, D. Painuly, A. Porval, R. Tyagi. 2016. Phytochemical
Evaluation, Proximate Analysis and Biological Activity of Reinwardtia indica
Dum. Leaves. IJPPR., 8(5):750-755.
Siddiqui, I.N., V.U. Ahmad, A. Zahoor, A. Ahmed, S.S. Khan, A. Khan,Z. Hassan.2010.
The two diarylheptanoids from Alnus nitida. Nat. Prod. Commun., 5: 1787–1788.
Sigel, I. E. 1978. The development of pictorial comprehension. In B. S. Randhawa & W.
E. Coffman (Eds.), Visual learning, thinking, and communication, New York:
Academic Press. pp. 93–111.
Silva J. D. A., M. G. P. Nascimento, L. G. Grazina, K. N. C. Castro, S. J. Mayo and I. M.
Andrade.2015. Ethnobotanical survey of medicinal plants used by the community
of Sobradinho, Luís Correia, Piauí, Brazil.J. Med. Plants Res., 9(32): 872-883.
Sindhu, Z. U. D., Z. Iqbal, M. N. Khan, N. N. Jonsson and M. Siddique, 2010.
Documentation of ethno-veterinary practices used for treatment of different
ailments in selected a hilly area of Pakistan. Int. J. Agric. Biol., 12: 353–358.
Singh A, A. Singh, O. Chouhan, G.P.Tandi, M. Dua, A. Gehlot. 2016. Anti-inflammatory
and analgesic activity of aqueous extracts of dried leaves of Murrayakoenigii Linn.
NatlJPhysiolPharmPharmacol., 6:286–290.
203
Siriwardhana, N., K.W. Lee, Y.J. Jeon, S.H. Kim and J.W. Haw, 2003. Antioxidant activity
of Hizikia fusiformis on reactive oxygen species scavenging and lipid peroxidation
inhibition. Food Sci. Technol. Int., 9: 339-346.
Smillie, T.J, I.A. Khan. 2010. A Comprehensive Approach to Identifying and
Authenticating Botanical Products. Clin Pharmacol Ther., 87(2):175-186.
Sodipo, O. A., M. A. Akanji, F. B. Kolawole and A. A. Odutuga. 1991. Saponin is the active
antifungal principle in Garcinia kola heckles seed. Biosci. Res. Commun., 3: 171.
Soetan, K.O., C.O. Olaiya, O.E. Oyewole. 2010. The importance of mineral elements for
humans, domestic animals and plants: A review. Afr J Food Sci., 4: 200-222.
Springfield, E.P., P.K.F. Eagles, G. Scott. 2005. Quality assessment of South African
Hertbal Medicines by means of HPLC fingerprinting. J. Ethnopharmacol., 101(1-
3), 75-83.
Srirama, R., B. T. Ramesha, G. Ravikanth, R. Shaanker, K. N. Ganeshaiah. 2007. Are
plants with anti-cancer activity resistant to crown gall? A test of hypothesis.
Current Sci., 95 (10), 99-106.
Stevic, T., K. Savikin, G. Zdunic, T. Stanojkovic, Z. Juranic, T. Jankovic, N. Menkovic.
2010. Antioxidant, cytotoxic, and antimicrobial activity of Alnus incana (L.) ssp.
incana Moench and A. viridis (Chaix) DC ssp. viridis extracts. J Med Food, 13(3):
700–704.
Streeten, D.H., Williams, E.M.V. 1952. Loss of cellular potassium as a cause of intestinal
paralysis in dogs. J Physiol., 118: 149-170.
Sugumaran, M. and T. Vetrichelvan. 2008. Studies on some Pharmacognostic Profiles of
Bauhiniapurpurea Linn. Leaves (Caesalpinaceae). Ethnobotanical Leaflets, 12:
461-468.
204
Sultan, S., M. Akram, H.M. Asif, K. Ahmad, J. Rehman, N. Akhtar and A. Hussain. 2018.
Antipyretic activity of hydro-methanolic extract of Trachyspermum ammi Linn.
seeds in rabbits. Pakistan J Sci.,70(2):109-112.
Sultana, S., H. M. Asif, N. Akhtar, K. Ahmad. 2015a. Medicinal plants with potential
antipyretic activity: A review. Asian Pac J Trop Dis., 5(Suppl 1): S202-S208.
Sultana, S., H.M. Asif, N. Akhtar, K. Ahmad. 2015b. Medicinal plants with potential
antipyretic activity: A review. Asian Pac J Trop Dis., 5(Suppl 1): S202-S208.
Sultana, S., M. A. Khan, M. Ahmad, A. Bano, M. Zafar and Z. K. Shinwari.2011.
Authentication of herbal medicine Neem (Azadirachta Indica A. JUSS.) by using
taxonomic and pharmacognostic techniques. Pak. J. Bot., 43: 141-150.
Sundar, R.A., R. C. Habibur. 2018. Pharmacognostic, Phytochemical and Antioxidant
Studies of Gardenia latifolia Aiton: An Ethnomedicinal Tree Plant. IJPPR,
10(5):216-228
Sunitha, L.P., N. Sathyanarayana, V. C. Suresh, S. Sreeramanan and R. Xavier. 2018.
Phytochemical and Antioxidant analysis of the leaf extract of Malaysian Medicinal
Plant Abroma augusta., Indian J Pharm Sci. 80(1):192-198.
Swiatek, L., B. Rajtar, K. Pawlak, A. Ludwiczuk, K. Głowniak and M. Polz-Dacewicz.
2013. In vitro evaluation of cytotoxicity of n-hexane extract from Alnus sieboldiana
male flowers on VERO and HEK293 cell lines. J Pre Clin Clin Res., 7(2) :107–
110.
Taesotikul. T., A. Panthong, D. Kanjanapothi, R. Verpoorte and J. J. C. Scheffer. 2003.
Anti-inflammatory, antipyretic and antinociceptive activities of Tabernaemontana
pandacaqui Poir. J Ethnopharmacol., 84: 31-35.
Taia, W. K. 2005. Modren trends in plant texanomy. Asian J. Pl. sci., 4 (2): 184-206.
Tambe, D.V., R. S. Bhambar. 2014. Estimation of Total Phenol, Tannin, Alkaloid and
Flavonoid in Hibiscus Tiliaceus Linn. Wood Extracts. Research and Reviews: J
Pharmacogn Phytochem.2(4):41-47.
205
Tapiero, H., K.D. Tew, B.G. Nguyen, G. Mathe. 2002. Polyphenols: do they play a role in
the prevention of human pathologies? Biomed Pharmacother. 56:200–207.
Tareen, N.M., M.A. Saeed-ur-Rehman, Z.K. Shinwari and T. Bibi. 2016. Ethnomedicinal
utilization of wild edible vegetables in district Harnai of Balochistan province
Pakistan. Pak. J. Bot., 48: 1159-1171.
Tatiya A, S. Surana, S. Bhavsar, D. Patil and Y.Patil . 2012.Pharmacognostic and
preliminary phytochemical investigation of Eulophia herbacea Lindl. Tubers
(Orchidaceae). Asian Pac J Trop Dis., 2(Suppl 1): S50-55.
Taylor, R.B., O. Shakoor, R.H. Behrens, M. Everard, A.S. Low, J. Wangboonskul, R.G.
Reid, J.A. Kolawole.2001. Pharmacopoeial quality of drugs supplied by Nigerian
pharmacies. Lancet., 357(9272):1933-1936.
Teerthe, S., B. R. Kerur. 2017. Elemental analysis of medicinal plants from North
Karnataka region by AAS method. Int. J. Res. Ayurveda Pharm. 8 (3):104-108.
DOI: 10.7897/2277-4343.083153.
Tiwari, P., B. Kumar, M. Kaur, G. Kaur and H. Kaur. 2011. Phytochemical screening and
Extraction: A Review. Int. Pharm. Sciencia., 1(1):98-106.
Tolo, F. M., G.M. Rukunga, F.W. Muli, E.N.M. Njagi, W. Njue and K. Kumon et al. 2006.
Anti-viral activity of the extracts of a Kenyan medicinal plant Carissa edulis
against herpes simplex virus. J Ethnopharmacol., 104: 92-99.
Tolouee, M., S. Alinezhad, R. Saberi, A. Eslamifar, S.J. Zad, K. Jaimand, J. Taeb, M.B.
Rezaee, M. Kawachi, and M. Shams-ghahfarokhi et al. 2010. Effect of Matricaria
chamomilla L. flower essential oil on the growth and ultrastructure of Aspergillus
niger van Tieghem. Int. J. Food Microbiol. 139, 127–133.
Tripathi, I.P., C. Mishra. 2015. Phytochemical Screening of Some Medicinal Plants of
Chitrakoot Region. Indian J Appl Res.,5(12): 56-60.
Tripathi, K. D. 2013. Essentials of Medical Pharmacology. 7th Ed. Jaypee Brothers
Medical Publishers (P) Ltd, New Delhi. P.02.
206
Tuncturk, M., T. Eryigit, N. Sekeroglu, F. Ozgokce.2015. Determination of nutritional
value and mineral composition of some wild Scorzonera species.Am. J. Essent. Oil.
Nat. Prod., 3(2): 22-25.
Tung, N.H., H.J. Kwon, J.H. Kim, J.C. Ra, J.A. Kim, Y.H. Kim. 2010a. An anti- influenza
component of the bark of Alnus japonica. Arch. Pharm. Res., 33:363– 367.
Tung, N.H., S.K. Kim, G.C. Ra, Y.Z. Zhao, D.H. Sohn, Y.H. Kim. 2010b. Antioxidative
and hepatoprotective diarylheptanoids from the bark of Alnus japonica. Planta
Med., 76: 626–629.
Turnlund, J. R. 2006. Mineral bioavailability and metabolism determined by using stable
isotope tracers. J. Anim. Sci., 84(Suppl): 73-78.
Uddin, S., M. Ali, S. Ali, S. Shah, S. Ullah and Z. Hussain. 2016. Ethnobotanical studies
of weeds in District Mardan, Pakistan. Pak. J. Weed Sci. Res., 22(3): 481-490.
Umeti, C.C., F. D. Onajobi, E. M. Obuotor, G.N. Anyasor, E. B. Esan. 2019. Anti-
inflammatory properties and gas chromatography-mass spectrometry analysis of
ethyl acetate fraction of Crateva adansonii DC leaves. A J Physiol Biochem
Pharmacol., 9(1):9–20; 10.5455/ajpbp.20181226090820.
Underwood, E. J. 1977. Trace Elements in Human and Animal Nutrition.4th edition.
Academic Press., New York.
Upreti, K., A. Semwal, K. Upadhyaya, M. Masiwal. 2013. Pharmacognostical and
Phytochemical Screening of Leaf Extract of Zanthoxylum armatum DC. Int. j. herb.
med., 1(1):6-11.
Vagnozzi, A., D.A. Stein, P.L. Iversen and E. Rieder. 2007. Inhibition of foot-and mouth
disease virus infections in cell cultures with antisensemorphlino oligomers. J.
Virol.,81(21):11669-11680.
Van-Arman, C. G., D. Armstrong, D.H. Kim. 1985. Antipyretics. Pharmacol. Ther.,
29(1):1-48.
207
Velazhahan R. 2017.Bioprospecting of Medicinal Plants for Detoxification of Aflatoxins.
Int J Nutr Pharmacol Neurol Dis., 7(3) :60-3.
Velazhahan, R., S. Vijayanandraj, A.Vijayasamundeeswari, V. Paranidharan, R.
Samiyappan and T. Lwamoto et a.l2010. Detoxification of aflatoxins by seed
extracts of the medicinal plant, Trachyspermum ammi (L.) Sprague ex Turrill -
Structural analysis and biological toxicity of degradation product of aflatoxin G1.
Food Control., 21(5):719-725.
Venkataswamy, R., H.M. Mubarack, A. Doss, T.K. Ravi and M. Sukumar. 2010.
Ethnobotanical study of medicinal plants used by Malasar tribals in Coimbatore
district of Tamil Nadu (South India). Asian J. Exp. Biol. Sci., 1: 387-392.
Vijayanandraj, S., R. Brinda, K. Kannan, R. Adhithya, S. Vinothini, K. Senthil, R. R.
Chinta V. Paranidharan, R.Velazhahan. 2014. Detoxification of aflatoxin B1 by an
aqueous extract from leaves of Adhatoda vasica Nees. Microbiol Res. 169 (4):294-
300.
Vitalini, S., M. Iriti, C. Puricelli, D. Ciuchi, A. Segale, & G. Fico. 2013. Traditional
knowledge on medicinal and food plants used in Val San Giacomo (Sondrio, Italy).
An alpine ethnobotanical study. J Ethnopharmacol. ,145 (2): 517–529
Vlietinck A.J., D.A.Vanden Berghe.1991. Can ethnopharmacology contribute to the
development of antiviral drugs? J Ethnopharmacol.,32(1-3):141–153.
pmid:1652667
Wadhwa, N. 2015. Textbook of Pediatric Gastroenterology, Hepatology and Nutrition:
Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India.
Wagacha, J.M. and J.W. Muthomi. 2008. Mycotoxin problem in Africa: Current status,
implications to food safety and health and possible management strategies. Int. J.
Food Microbiol. 124, 1–12.
Wallis, T. E. 1985. Text book of pharmacognosy. 5th ed. CBS Publisher and Distributors,
New Delhi.
208
Wallis, T. E. Text Book of Pharmacognosy; CBS publishers, Delhi, 2005; pp 572- 575.
Wang, F. et al. 2011.Structure elucidation and toxicity analysis of the radiolytic products
of aflatoxin B1 in methanol-water solution. J Hazard Mater. 192: 1192–1202.
Wang, F., Y.Li, Y.J. Zhang, Y. Zhou, S. Li, H.B. Li. 2016. Natural products for the
prevention and treatment of hangover and alcohol use disorder. Molecules, 21, 64.
Wang, Y.C., Chuang Y, Hsu H. 2008. The flavonoid, carotenoid and pectin content in peels
of citrus cultivated in Taiwan. Food Chem. 106:277–284.
Waweru, W.R., L. O. Osuwat and F. K. Wambugu. 2017a. Phytochemical Analysis of
Selected Indigenous Medicinal Plants used in Rwanda. J Pharmacogn Phytochem;
6(1): 322-324.
Waweru, W.R., L. O. Osuwat, C.W. Mureithi.2017b. Analgesic and anti-Inflammatory
activity of Tradescantia fluminensis leaves extract. J Phytopharmacol., 6(1): 34-
37.
Weston, L.A. 1996. Utilization of allelopathy for weed management in agroecosystems.
Agron J., 88: 860-866.
WHO, 1992. Cadmium. Environmental Health Criteria, vol. 134, Geneva WHO–World
Health Organization, Tradi tional Medicine, (1998) Avai labl e at:
www.who.int/medi acentre/factsheets.2003/ fs134/en > access in: 07/ 08/2003.
WHO, World Health Organization.1979. Environmental Health Criteria, Safety evaluation
of certain food additives. pp. 1-127.
WHO–World Health Organization, Traditional Medicine, (1998) Availabl e at:
www.who.int/medi acentre/factsheets.2003/ fs134/en > access in: 07/ 08/2003.
Wild, C.P., S.M. Shrestha, W.A. Anwar, R. Montesano.1992. Field studies of aflatoxin
exposure, metabolism and induction of genetic alterations in relation to hepatitis B
virus infection and hepatocellular carcinoma in the Gambia and Thailand.
ToxicolLett., 1992; 64-65 Spec No: 455461
209
William, C.D.2000. Integrative Plant Anatomy. Elsevier Inc.
Williams J.E. 2001. Review of antiviral and immunomodulating properties of plants of the
Peruvian rainforest with a particular emphasis on Una de Gato and Sangre de
Grado. Altern Med Rev., 6(6): 567–579. pmid:11804547.
Williams, J.H., T.D. Phillips, P.E. Jolly, J.K. Stiles, C.M. Jolly, D. Aggarwal.2004. Human
aflatoxicosis in developing countries: a review of toxicology, exposure, potential
health consequences, and interventions. Am J Clin Nutr., 80:1106-1122.
Womack E.D., A.E. Brown, D.L. Sparks.2014. A recent review of non-biological
remediation of aflatoxin-contaminated crops. J Sci Food Agric., 94(9):1706–14.
World Health Organization (WHO). 2006.Mycotoxins in African foods: Implications to
food safety and health. AFRO Food Safety Newsletter. World Health Organization
Food safety (FOS), Issue No. 2 July 2006. http://www.afro.who.int/des
Worobiec, G., A.Szynkiewicz. 2007. Betulaceae leaves in Miocene deposits of the
Bełchato´w Lignite Mine (Central Poland). Rev Palaeobot Palynol.,147(1–4):28–
59
WU, F. 2006.Mycotoxin reduction in Bt corn: Potential economic, health, and regulatory
impacts. ISB News Report, September 2006.
Wu, S.B., C. Long, E.J. Kennelly. 2013. Phytochemistry and health benefits of jaboticaba,
an emerging fruit crop from Brazil. Food Res Int., 54(1):148-159.
Xu, D.P., Y. Li, X. Meng, T. Zhou, Y. Zhou, J. Zheng, J.J. Zhang and H.B. Li. 2017.
Review Natural Antioxidants in Foods and Medicinal Plants: Extraction,
Assessment and Resources. Int. J. Mol. Sci.,18, 96; doi:10.3390/ijms18010096.
Yaseen, G., M. Ahmad, M. Zafar, S. Sultana, S. Kayani, A. A. Cetto, S. Shaheen.2015b.
Traditional management of diabetes in Pakistan: Ethnobotanical investigation from
traditional health practitioners. J Ethnopharmacol., 174:91-117.
210
Yaseen, G., M. Ahmad, S. Sultana, A.S. Alharrasi, J. Hussain and M. Zafar. 2015a.
Ethnobotany of medicinal plants in the Thar desert (Sindh) of Pakistan. J
Ethnopharmacol, 163: 43-59.
Younus, I., A. Siddiq, H. Ishaq, L. Anwer, S. Badar and M. Ashraf. 2016. Evaluation of
antiviral activity of plant extracts against foot and mouth disease virus in vitro. Pak.
J. Pharm. Sci., 29(4):1263-1268.
Yu, Y.B., H. Miyashiro, N. Nakamura, M. Hattori, J. C. Park. 2007. Effects of triterpenoids
and flavonoids isolated from Alnus firma on HIV-1 viral enzymes. Arch Pharm
Res., 30: 820-826. https://doi.org/10.1007/BF02978831
Zafar, M., M. A. Khan, M. Ahmad, G. Jan, S. Sultana, K. Ullah, S. K. Marwat, F. Ahmad,
A. Jabeen, A. Nazir, A. M. Abbasi and Z. Zr-Ullah Z. 2010. Elemental analysis of
some medicinal plants used in traditional medicine by atomic absorption
spectrophotometer (AAS). J. Med. Pl. Res., 4 (19): 1987-1990.
Zain-Ullah, M. K. Baloch, I. B. Baloch and F. Bibi. 2013. Proximate and Nutrient Analysis
of Selected Medicinal Plants of Tank and South Waziristan Area of Pakistan.
Middle East J. Sci. Res., 13 (10): 1345-1350.
Zarnowski, R and Y. Suzuki. 2004. Expedient Soxhlet extraction of resorcinolic lipids from
wheat grains. Jour. Food Comp. & Anal., 17 (5): 649-663.
Zhang, Y.J., R.Y.Gan, S. Li , Y. Zhou, A.N. Li, D.P.Xu and H.B.Li.2015. Antioxidant
phytochemicals for the prevention and treatment of chronic diseases. Molecules,
20: 21138–21156.
Zhao, H., L.Z.Zhao, T. Jiang, X.F.Li, H. Fan, W.X. Hong, Y. Zhang, Q. Zhu, Q. Ye, Y.G.
Tong et al.2014. Isolation and characterization of dengue virus serotype 2 from the
large dengue outbreak in Guangdong, China in 2014. Sci China Life Sci, 2014, 57:
1149–1155, doi: 10.1007/s11427-014-4782-3
211
Zhao, X.M., L.Y. Qu, Z.Yan, Y.Yang, Y.Ma, Z.L.Dong , Z.M. Li , M.W.Li, X. Wang, F.
Jiao.2019. Chinese native medicinal plant Euphorbiaceae show antitumor and
anti-oxidant features in lewis lung cancer-bearing mice. Phcog Mag., 15:459-65.
Zhou, X., Y. Zhang, Y. Li, X. Hao, X. Liu and Y. Wang. 2012. Azithromycin
Synergistically Enhances Anti-Proliferative Activity of Vincristine in Cervical and
Gastric Cancer Cells. Cancers, 4(4):1318-1332; doi:10.3390/cancers4041318.
Zhou, Y., J. Zheng, S. Li, T. Zhou, P. Zhang, H.B. Li. 2016. Alcoholic beverage
consumption and chronic diseases. Int. J. Environ. Res. Public Health,13: 522.
Zihad, S.M.N.K., N. Bhowmick, S.J. Uddin, N. Sifat, M.S. Rahman, R. Rouf, M.T. Islam,
S. Dev, H. Hazni, S. Aziz, E.S. Ali, A.K. Das, J.A. Shilpi, L. Nahar and
S.D. Sarker.2018. Analgesic Activity, Chemical Profiling and Computational
Study on Chrysopogon aciculatus. Front. Pharmacol. 9:1164
212
APPENDICES
Appendix 1). One-way ANOVA followed by Tukey’s multiple comparison test for the effect of different
Concentrations of A. nitida bark(B), leaf(L), staminate catkin (SC) and pistillate cone (PC) extracts on number
of acetic acid induced writhings in mice.
ANOVA table SS DF MS F (DFn, DFd) P value
Treatment (between columns) 5396 13 415.1 F (13, 70) = 256.7 P < 0.0001
Residual (within columns) 113.2 70 1.617
Total 5509 83
Alpha 0.05
Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Adjusted P Value
Saline 10(ml/kg) vs. Aspirin 10(mg/kg) 26.50 23.95 to 29.05 Yes **** < 0.0001
Saline 10(ml/kg) vs. B50 13.50 10.95 to 16.05 Yes **** < 0.0001
Saline 10(ml/kg) vs. B100 20.50 17.95 to 23.05 Yes **** < 0.0001
Saline 10(ml/kg) vs. B200 27.67 25.12 to 30.22 Yes **** < 0.0001
Saline 10(ml/kg) vs. L50 13.17 10.62 to 15.72 Yes **** < 0.0001
Saline 10(ml/kg) vs. L100 21.50 18.95 to 24.05 Yes **** < 0.0001
Saline 10(ml/kg) vs. L200 28.17 25.62 to 30.72 Yes **** < 0.0001
Saline 10(ml/kg) vs. SC50 5.500 2.950 to 8.050 Yes **** < 0.0001
Saline 10(ml/kg) vs. SC100 9.833 7.283 to 12.38 Yes **** < 0.0001
Saline 10(ml/kg) vs. SC200 12.17 9.616 to 14.72 Yes **** < 0.0001
Saline 10(ml/kg) vs. PC50 12.67 10.12 to 15.22 Yes **** < 0.0001
Saline 10(ml/kg) vs. PC100 15.50 12.95 to 18.05 Yes **** < 0.0001
Saline 10(ml/kg) vs. PC200 19.17 16.62 to 21.72 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. B50 -13.00 -15.55 to -10.45 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. B100 -6.000 -8.550 to -3.450 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. B200 1.167 -1.384 to 3.717 No ns 0.9431
Aspirin 10(mg/kg) vs. L50 -13.33 -15.88 to -10.78 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. L100 -5.000 -7.550 to -2.450 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. L200 1.667 -0.8838 to 4.217 No ns 0.5814
Aspirin 10(mg/kg) vs. SC50 -21.00 -23.55 to -18.45 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. SC100 -16.67 -19.22 to -14.12 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. SC200 -14.33 -16.88 to -11.78 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. PC50 -13.83 -16.38 to -11.28 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. PC100 -11.00 -13.55 to -8.450 Yes **** < 0.0001
Aspirin 10(mg/kg) vs. PC200 -7.333 -9.884 to -4.783 Yes **** < 0.0001
B50 vs. B100 7.000 4.450 to 9.550 Yes **** < 0.0001
B50 vs. B200 14.17 11.62 to 16.72 Yes **** < 0.0001
B50 vs. L50 -0.3333 -2.884 to 2.217 No ns > 0.9999
B50 vs. L100 8.000 5.450 to 10.55 Yes **** < 0.0001
B50 vs. L200 14.67 12.12 to 17.22 Yes **** < 0.0001
B50 vs. SC50 -8.000 -10.55 to -5.450 Yes **** < 0.0001
B50 vs. SC100 -3.667 -6.217 to -1.116 Yes *** 0.0003
B50 vs. SC200 -1.333 -3.884 to 1.217 No ns 0.8612
B50 vs. PC50 -0.8333 -3.384 to 1.717 No ns 0.9968
B50 vs. PC100 2.000 -0.5504 to 4.550 No ns 0.2876
B50 vs. PC200 5.667 3.116 to 8.217 Yes **** < 0.0001
B100 vs. B200 7.167 4.616 to 9.717 Yes **** < 0.0001
B100 vs. L50 -7.333 -9.884 to -4.783 Yes **** < 0.0001
B100 vs. L100 1.000 -1.550 to 3.550 No ns 0.9832
B100 vs. L200 7.667 5.116 to 10.22 Yes **** < 0.0001
B100 vs. SC50 -15.00 -17.55 to -12.45 Yes **** < 0.0001
B100 vs. SC100 -10.67 -13.22 to -8.116 Yes **** < 0.0001
B100 vs. SC200 -8.333 -10.88 to -5.783 Yes **** < 0.0001
B100 vs. PC50 -7.833 -10.38 to -5.283 Yes **** < 0.0001
B100 vs. PC100 -5.000 -7.550 to -2.450 Yes **** < 0.0001
B100 vs. PC200 -1.333 -3.884 to 1.217 No ns 0.8612
B200 vs. L50 -14.50 -17.05 to -11.95 Yes **** < 0.0001
B200 vs. L100 -6.167 -8.717 to -3.616 Yes **** < 0.0001
B200 vs. L200 0.5000 -2.050 to 3.050 No ns > 0.9999
B200 vs. SC50 -22.17 -24.72 to -19.62 Yes **** < 0.0001
B200 vs. SC100 -17.83 -20.38 to -15.28 Yes **** < 0.0001
213
B200 vs. SC200 -15.50 -18.05 to -12.95 Yes **** < 0.0001
B200 vs. PC50 -15.00 -17.55 to -12.45 Yes **** < 0.0001
B200 vs. PC100 -12.17 -14.72 to -9.616 Yes **** < 0.0001
B200 vs. PC200 -8.500 -11.05 to -5.950 Yes **** < 0.0001
L50 vs. L100 8.333 5.783 to 10.88 Yes **** < 0.0001
L50 vs. L200 15.00 12.45 to 17.55 Yes **** < 0.0001
L50 vs. SC50 -7.667 -10.22 to -5.116 Yes **** < 0.0001
L50 vs. SC100 -3.333 -5.884 to -0.7829 Yes ** 0.0017
L50 vs. SC200 -1.000 -3.550 to 1.550 No ns 0.9832
L50 vs. PC50 -0.5000 -3.050 to 2.050 No ns > 0.9999
L50 vs. PC100 2.333 -0.2171 to 4.884 No ns 0.1076
L50 vs. PC200 6.000 3.450 to 8.550 Yes **** < 0.0001
L100 vs. L200 6.667 4.116 to 9.217 Yes **** < 0.0001
L100 vs. SC50 -16.00 -18.55 to -13.45 Yes **** < 0.0001
L100 vs. SC100 -11.67 -14.22 to -9.116 Yes **** < 0.0001
L100 vs. SC200 -9.333 -11.88 to -6.783 Yes **** < 0.0001
L100 vs. PC50 -8.833 -11.38 to -6.283 Yes **** < 0.0001
L100 vs. PC100 -6.000 -8.550 to -3.450 Yes **** < 0.0001
L100 vs. PC200 -2.333 -4.884 to 0.2171 No ns 0.1076
L200 vs. SC50 -22.67 -25.22 to -20.12 Yes **** < 0.0001
L200 vs. SC100 -18.33 -20.88 to -15.78 Yes **** < 0.0001
L200 vs. SC200 -16.00 -18.55 to -13.45 Yes **** < 0.0001
L200 vs. PC50 -15.50 -18.05 to -12.95 Yes **** < 0.0001
L200 vs. PC100 -12.67 -15.22 to -10.12 Yes **** < 0.0001
L200 vs. PC200 -9.000 -11.55 to -6.450 Yes **** < 0.0001
SC50 vs. SC100 4.333 1.783 to 6.884 Yes **** < 0.0001
SC50 vs. SC200 6.667 4.116 to 9.217 Yes **** < 0.0001
SC50 vs. PC50 7.167 4.616 to 9.717 Yes **** < 0.0001
SC50 vs. PC100 10.00 7.450 to 12.55 Yes **** < 0.0001
SC50 vs. PC200 13.67 11.12 to 16.22 Yes **** < 0.0001
SC100 vs. SC200 2.333 -0.2171 to 4.884 No ns 0.1076
SC100 vs. PC50 2.833 0.2829 to 5.384 Yes * 0.0163
SC100 vs. PC100 5.667 3.116 to 8.217 Yes **** < 0.0001
SC100 vs. PC200 9.333 6.783 to 11.88 Yes **** < 0.0001
SC200 vs. PC50 0.5000 -2.050 to 3.050 No ns > 0.9999
SC200 vs. PC100 3.333 0.7829 to 5.884 Yes ** 0.0017
SC200 vs. PC200 7.000 4.450 to 9.550 Yes **** < 0.0001
PC50 vs. PC100 2.833 0.2829 to 5.384 Yes * 0.0163
PC50 vs. PC200 6.500 3.950 to 9.050 Yes **** < 0.0001
PC100 vs. PC200 3.667 1.116 to 6.217 Yes *** 0.0003
214
Appendix. 2). Inhibition of edema volume by various concentrations of bark(B), leaf (L), staminate catkin (SC) and pistillate cone
(PC) extracts of A. nitida at different intervals of time (hours).
Edema volume ( EV) =Paw volume after administration of carrageenan (PVA)- Paw volume after administration of carrageenan (PVI) S.
NO. Samples EV /1hr EV/2hr EV/3hr EV/4hr EV5hr
S.
NO Samples EV /1hr EV/2hr EV/3hr EV/4hr EV/5hr
1
Saline (-VE
Control) 9 SC50
MEAN 0.124 0.124667 0.111667 0.111667 0.113333 MEAN 0.111667 0.11 0.093333 0.091667 0.093333
SEM 0.004131 0.003494 0.003085 0.004029 0.004233 SEM 0.003085 0.003666 0.002116 0.003085 0.004233
2
Diclofenac
(+VE
Control) 10 SC100
MEAN 0.095 0.045 0.021667 0.018333 0.03 MEAN 0.11 0.108333 0.093333 0.09 0.093333
SEM 0.006215 0.005019 0.006033 0.006033 0.005796 SEM 0.002592 0.004029 0.003346 0.002592 0.004233
3 B50 11 SC200
MEAN 0.113333 0.085 0.061667 0.063333 0.065 MEAN 0.106667 0.106667 0.091667 0.091667 0.096667
SEM 0.002116 0.004298 0.005447 0.003346 0.005019 SEM 0.004233 0.004233 0.004029 0.001673 0.004233
4 B100 12 PC50
MEAN 0.106667 0.068333 0.043333 0.04 0.045 MEAN 0.108333 0.106667 0.09 0.091667 0.095
SEM 0.002116 0.004029 0.006693 0.003666 0.003429 SEM 0.001673 0.003346 0.00449 0.004029 0.005019
5 B200 13 PC100
MEAN 0.105 0.043333 0.025 0.02 0.025 MEAN 0.101667 0.1 0.086667 0.085 0.09
SEM 0.002245 0.002116 0.002245 0.002592 0.005019 SEM 0.004791 0.002592 0.004233 0.003429 0.002592
6 L50 14 PC200
MEAN 0.101667 0.068333 0.048333 0.046667 0.058333 MEAN 0.1 0.095 0.065 0.063333 0.07
SEM 0.003085 0.001673 0.003085 0.003346 0.006566 SEM 0.002592 0.002245 0.003429 0.002116 0.003666
7 L100
MEAN 0.098333 0.063333 0.033333 0.035 0.038333
SEM 0.004791 0.003346 0.003346 0.003429 0.001673
8 L200
MEAN 0.105 0.021667 0.023333 0.026667
SEM 0.003429 0.004791 0.004029 0.003346 0.002116
215
Appendix. 3). One-way ANOVA with Dunnett’s multiple comparison test for differences in inhibition of
edema volume by various concentrations of bark (B), leaf (L), staminate catkin (SC) and pistillate cone (PC)
extracts of A. nitida after one hour.
ANOVA table SS DF MS F (DFn, DFd) P value
Treatment (between columns) 0.004121 13 0.0003170 F (13, 70) = 4.128 P < 0.0001
Residual (within columns) 0.005375 70 7.678e-005 Total 0.009495 83
Alpha 0.05
Dunnett's multiple
comparisons test Mean Diff. 95% CI of diff. Significant? Summary Adjusted P Value
Saline
vs. Diclofenac 0.0290 0.01450 to 0.04350 Yes **** < 0.0001
Saline
vs. B 50 0.01067 -0.003835 to 0.02517 No Ns 0.2666
Saline
vs. B100 0.01733 0.002831 to 0.03184 Yes * 0.0107
Saline
vs. B200 0.0190 0.004498 to 0.03350 Yes ** 0.0039
Saline
vs. L50 0.02233 0.007831 to 0.03684 Yes *** 0.0004
Saline
vs. L100 0.02567 0.01116 to 0.04017 Yes **** < 0.0001
Saline
vs. L200 0.0190 0.004498 to 0.03350 Yes ** 0.0039
Saline
vs. SC50 0.01233 -0.002169 to 0.02684 No Ns 0.1373
Saline
vs. SC100 0.0140 -0.0005020 to 0.02850 No Ns 0.0641
Saline
vs. SC200 0.01733 0.002831 to 0.03184 Yes * 0.0107
Saline
vs. PC50 0.01567 0.001165 to 0.03017 Yes * 0.0273
Saline
vs. PC100 0.02233 0.007831 to 0.03684 Yes *** 0.0004
Saline
vs. PC200 0.0240 0.009498 to 0.03850 Yes *** 0.0001
216
Appendix. 4). One-way ANOVA with Dunnett’s multiple comparison test for differences in inhibition of
edema volume by various concentrations of bark (B), leaf (L), staminate catkin (SC) and pistillate cone
(PC) extracts of A. nitida after 2 hours.
ANOVA table SS DF MS F (DFn, DFd) P value
Treatment (between columns) 0.05815 13 0.004473 F (13, 70) = 57.14 P < 0.0001
Residual (within columns) 0.005480 70 7.829e-005
Total 0.06363 83
Alpha 0.05
Dunnett's multiple
comparisons test Mean Diff. 95% CI of diff. Significant? Summary Adjusted P Value
Saline
vs. Diclofenac 0.07967 0.06502 to 0.09431 Yes **** < 0.0001
Saline
vs. B 50 0.03967 0.02502 to 0.05431 Yes **** < 0.0001
Saline
vs. B100 0.05633 0.04169 to 0.07098 Yes **** < 0.0001
Saline
vs. B200 0.08133 0.06669 to 0.09598 Yes **** < 0.0001
Saline
vs. L50 0.05633 0.04169 to 0.07098 Yes **** < 0.0001
Saline
vs. L100 0.06133 0.04669 to 0.07598 Yes **** < 0.0001
Saline
vs. L200 0.07633 0.06169 to 0.09098 Yes **** < 0.0001
Saline
vs. SC50 0.01467 2.324e-005 to 0.02931 Yes * 0.0494
Saline
vs. SC100 0.01633 0.001690 to 0.03098 Yes * 0.0207
Saline
vs. SC200 0.0180 0.003357 to 0.03264 Yes ** 0.0080
Saline
vs. PC50 0.0180 0.003357 to 0.03264 Yes ** 0.0080
Saline
vs. PC100 0.02467 0.01002 to 0.03931 Yes *** 0.0001
Saline
vs. PC200 0.02967 0.01502 to 0.04431 Yes **** < 0.0001
217
Appendix. 5). One-way ANOVA with Dunnett’s multiple comparison test for differences in inhibition of
edema volume by various concentrations of bark (B), leaf (L), staminate catkin (SC) and pistillate cone
(PC) extracts of A. nitida after 3 hours.
ANOVA table SS DF MS F (DFn, DFd) P value
Treatment (between columns) 0.07600 13 0.005846 F (13, 70) = 56.32 P < 0.0001 Residual (within columns) 0.007267 70 0.0001038
Total 0.08327 83
Alpha 0.05
Dunnett's multiple
comparisons test Mean Diff. 95% CI of diff. Significant? Summary Adjusted P Value
Saline
vs. Diclofenac 0.0900 0.07314 to 0.1069 Yes **** < 0.0001
Saline
vs. B50 0.0500 0.03314 to 0.06686 Yes **** < 0.0001
Saline
vs. B100 0.06833 0.05147 to 0.08520 Yes **** < 0.0001
Saline
vs. B200 0.08667 0.06980 to 0.1035 Yes **** < 0.0001
Saline
vs. L50 0.06333 0.04647 to 0.08020 Yes **** < 0.0001
Saline
vs. L100 0.07833 0.06147 to 0.09520 Yes **** < 0.0001
Saline
vs. L200 0.0900 0.07314 to 0.1069 Yes **** < 0.0001
Saline
vs. SC50 0.01833 0.001471 to 0.03520 Yes * 0.0259
Saline
vs. SC100 0.01833 0.001471 to 0.03520 Yes * 0.0259
Saline
vs. SC200 0.0200 0.003138 to 0.03686 Yes * 0.0116
Saline
vs. PC50 0.02167 0.004804 to 0.03853 Yes ** 0.0049
Saline
vs. PC100 0.0250 0.008138 to 0.04186 Yes *** 0.0008
Saline
vs. PC200 0.04667 0.02980 to 0.06353 Yes **** < 0.0001
218
Appendix. 6). One-way ANOVA with Dunnett’s multiple comparison test for differences in inhibition of
edema volume by various concentrations of bark (B), leaf (L), staminate catkin (SC) and pistillate cone
(PC) extracts of A. nitida after 4 hours. ANOVA table SS DF MS F (DFn, DFd) P value
Treatment (between columns) 0.07822 13 0.006017 F (13, 70) = 83.40 P < 0.0001
Residual (within columns) 0.00505 70 7.214e-005 Total 0.08327 83
Alpha 0.05
Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary Adjusted P Value
Saline
vs. Diclofenac 0.09333 0.07928 to 0.1074 Yes **** < 0.0001
Saline
vs. B50 0.04833 0.03428 to 0.06239 Yes **** < 0.0001
Saline
vs. B100 0.07167 0.05761 to 0.08572 Yes **** < 0.0001
Saline
vs. B200 0.09167 0.07761 to 0.1057 Yes **** < 0.0001
Saline
vs. L50 0.0650 0.05094 to 0.07906 Yes **** < 0.0001
Saline
vs. L100 0.07667 0.06261 to 0.09072 Yes **** < 0.0001
Saline
vs. L200 0.08833 0.07428 to 0.1024 Yes **** < 0.0001
Saline
vs. SC50 0.0200 0.005943 to 0.03406 Yes ** 0.0014
Saline
vs. SC100 0.02167 0.007609 to 0.03572 Yes *** 0.0004
Saline
vs. SC200 0.0200 0.005943 to 0.03406 Yes ** 0.0014
Saline
vs. PC50 0.0200 0.005943 to 0.03406 Yes ** 0.0014
Saline
vs. PC100 0.02667 0.01261 to 0.04072 Yes **** < 0.0001
Saline
vs. PC200 0.04833 0.03428 to 0.06239 Yes **** < 0.0001
219
Appendix. 7). One-way ANOVA with Dunnett’s multiple comparison test for differences in inhibition of
edema volume by various concentrations of bark (B), leaf (L), staminate catkin (SC) and pistillate cone
(PC) extracts of A. nitida after 5 hours.
ANOVA table SS DF MS F (DFn, DFd) P value
Treatment (between columns) 0.07128 13 0.005483 F (13, 70) = 49.00 P < 0.0001
Residual (within columns) 0.007833 70 0.0001119
Total 0.07911 83
Alpha 0.05
Dunnett's multiple comparisons
test Mean Diff. 95% CI of diff. Significant? Summary Adjusted P Value
Saline
vs. Diclofenac 0.08333 0.06583 to 0.1008 Yes **** < 0.0001
Saline
vs. B50 0.04833 0.03083 to 0.06584 Yes **** < 0.0001
Saline
vs. B100 0.06833 0.05083 to 0.08584 Yes **** < 0.0001
Saline
vs. B200 0.08833 0.07083 to 0.1058 Yes **** < 0.0001
Saline
vs. L50 0.0550 0.03749 to 0.07251 Yes **** < 0.0001
Saline
vs. L100 0.0750 0.05749 to 0.09251 Yes **** < 0.0001
Saline
vs. L200 0.08667 0.06916 to 0.1042 Yes **** < 0.0001
Saline
vs. SC50 0.0200 0.002492 to 0.03751 Yes * 0.0166
Saline
vs. SC100 0.0200 0.002492 to 0.03751 Yes * 0.0166
Saline
vs. SC200 0.01667 -0.0008409 to 0.03417 No ns 0.0704
Saline
vs. PC50 0.01833 0.0008258 to 0.03584 Yes * 0.0352
Saline
vs. PC100 0.02333 0.005826 to 0.04084 Yes ** 0.0032
Saline
vs. PC200 0.04333 0.02583 to 0.06084 Yes **** < 0.0001
220