phytochemical and pharmacognostic profile of the leaves of leptadenia...
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Journal of Phytomedicine and Therapeutic 2018; 17(2): 189 – 213 Page 196 www.jopat.gov.ng
JOPAT Vol. 17(2) 196 – 213, 2018. ISSN 2636 – 5448
PHYTOCHEMICAL AND PHARMACOGNOSTIC PROFILE OF THE LEAVES OF
LEPTADENIA HASTATA (PERS.) DECNE.
Ibrahim Jemilat A., 1, 2* Gwadabe Ummi S., 2 Fatokun Omolola T1 and Esievo Kevwe B1.
1 Department of Medicinal Plant Research and Traditional Medicine, National Institute for
Pharmaceutical Research and Development (NIPRD), P. M. B. 21, Garki, Abuja, Nigeria.
2 Department Of Biological Sciences, Faculty of Computing and Applied Sciences, Baze
University, Abuja, Nigeria.
ABSTRACT
Objective: This study was designed to evaluate the phytochemical constituents and
pharmacognostic parameters, which could be helpful to ensure the purity and safety of Leptadenia
hastata.
Method: Phytochemical analysis and pharmacognostic studies including microscopy, chemo
microscopy and proximate analysis were conducted.
Result: Leaf epidermal study reveals polygonal cell and irregular cell shape on adaxial and abaxial
surfaces, respectively with striated abaxial cells and anomocytic stomata type. Leaf transverse
section shows two layers of palisade cells, abundant oil globule, starch grains and multicellular
trichomes on both abaxial and adaxial epidermises. Quantitative leaf microscopy reveals stomata
number (16 ±1.0), stomata index (14.97), vein islet number (56.3 ±0.3) and vein termination
number (4.7 ±0.7). Powdered microscopy of leaf revealed the presence of parenchyma cells, spiral
xylem vessels and epidermal cells with anomocytic stomata. Chemomicroscopy indicated
presence of lignin, starch, protein, oil, cellulose and calcium oxalate crystals. The physicochemical
parameters evaluated were: Ash value 13.61%, water and alcohol extractive values 30.125% and
15.23 %, moisture content 6.95%. Preliminary phytochemical investigations showed the presence
of tannins, flavonoids and saponins.
Conclusion: The pharmacognostical and preliminary phytochemical characters observed in the
leaf of Leptadenia hastata could help in the plant identification and provide clues to its
standardization. The information could also be useful in the development of the monograph of the
plant.
Keywords: Leptadenia hastata, pharmacognostic studies, proximate analysis, phytochemical
analysis
Correspondence email- [email protected] ©2017The authors. This work is licensed under the Creative Commons Attribution 4.0 International License
Ibrahim et al
Journal of Phytomedicine and Therapeutic 2018; 17(2): 189 – 213 Page 197 www.jopat.gov.ng
Introduction
Leptadenia hastata (Pers.) Decne.,
(Asclepidiaceae) is a leafy vegetable plant
found in nature as a wild plant. It is a climber
with pale white, soft and grooved stem.
Leave is simple, acuminate and ovate shaped
and exude white latex when crushed. Flowers
are cream or yellowish green and fruits are
dehiscent, two-valved releasing cotton
winged seeds [8]. It is widely distributed in
tropical Africa including northern Nigeria
[15]. The plant is locally known in Nigeria as
Yadiya, Dan- bakwa (Hausa) Kalimbo, Njera
(Kanuri) Isanaje-igbo, Ogbo funfun
(Yorubas) [7, 13].
In Nigeria, the plant is used as a spice and in
preparation of sauces [14] and considered as
a famine food in some parts of Africa [12, 15,
21]. Leptadenia hastata is used in Nigeria
ethnomedicine to manage hypertension,
catarrh and skin diseases [9], gonorrhea and
stomach-ache in children [23].
Pharmacological studies have shown that the
plant has antimicrobial [2], Anti-androgenic
[5], Anti – inflammatory and wound healing
[20] and anti-diabetic activities [6].
The major secondary metabolites present in
L. hastata are chemical constituents isolated
from L. hastata, which include lutein, lupeol,
β- carotene, kidjolanin, cynanforid and
gagaminin [4, 6,19].
The increased use of herbal drugs and
concern over their safety and efficacy has
certainly augmented the need for their
standardization. World health organization
(WHO) set up some guidelines for
standardization of crude drugs which include
morphological, microscopic, physical,
chemical and biological observation. These
standards are crucial in establishing the
quality, identity and purity of crude drugs [1,
17]. Pharmacognostic standards must be set
for every crude drug to be included in an
herbal pharmacopoeia.
The aim of this study was to evaluate the
pharmacognostical parameters of
Leptadenia hastata towards standardization,
monograph development on the plant and
for quality control purposes.
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Figure 1: Photographs of Leptadenia hastata
Key: A: plant showing ovate leaf shape; B: Plant in its habitat showing it habit; C: fruits
Materials and Method
Chemicals, reagents and solvents
All chemicals, reagents and solvents used
during the experimentation were of analytical
grade.
Collection
The fresh leaves of Leptadenia hastata were
collected on 18th of October 2017, from
Airport road Jiwa Abuja. The plant was
identified and authenticated in the herbarium
of National Institute for Pharmaceutical
Research and Development, Abuja, by Dr.
Ibrahim. Voucher specimen was deposited in
the same herbarium with voucher no:
NIPRD/H/6889.
Sample Preparation
Collected fresh leaves were preserved in 50%
ethanol for microscopic study (transverse
sectioning of leaves), while some were dried
in between old newspaper for epidermal
peeling. Some leaves of the plant were also
air dried for a week and powdered using
electrical blender. The powdered sample was
stored in a polythene bags for further use.
Macroscopic / Organoleptic Study
A
B
C
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The leaves were subjected to macroscopic
analysis viz. organoleptic characteristics such
as appearance, taste, colour, odour, shape,
texture, fracture, etc. of the drug. These
parameters are considered to be quite useful
in quality control of the crude drug and were
evaluated as required by WHO guidelines [1,
24].
Epidermal Leaf Microscopy study
Microscopic analysis was carried out on the
upper and lower epidermal surfaces of the
leaf and pulverized leaf samples. The method
of Jemilat et al [16] was used to prepare
epidermal surfaces of the leaf. About 5 mm2
– 1 cm2 leaf fragments were obtained from
the standard median portion of the leaf and
macerated in concentrated nitric acid in petri-
dish for a period of 18 h The appearance of
air bubbles indicated the readiness of the
epidermises to be separated. The fragments
were transferred into water in a petri-dish
with a pair of forceps. The upper, lower
epidermises and mesophyll were separated
and cleaned using forceps and carmel hair
brush. Epidermal peels were stained with
safranin and mounted on a slide with
glycerol. Subsequently, some leaf samples
were cut and soaked in sodium hypochlorite
for clearing to study the venation pattern.
Transverse section of leaves
Sections were manually obtained by
sectioning with a razor blade. The sections
were cleared for some minutes in 3.5%
hypochlorite solution, washed with water and
stained with sudan IV solution. These were
mounted on a slide with glycerol.
Quantitative microscopy
Quantitative examinations such as vein-islet
number, vein-termination number, palisade
ratio, stomatal number and stomatal index
were carried out using standard methods [16].
Chemo-microscopic Studies
Chemo-microscopic studies of the pulverized
leaf was done using reagents and stains like
iodine, concentrated Sulphuric acid,
concentrated hydrochloric acid, ferric
chloride, Sudan III, ruthenium red and
phloroglucinol with conc. HCl (1:1) to test
for the presence of various parameters [1].
Microphotography
Photomicrographs of different sections were
taken using Leica CME microscope with
digital microscope eyepiece attachment and
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Journal of Phytomedicine and Therapeutic 2018; 17(2): 189 – 213 Page 200 www.jopat.gov.ng
Photo Explorer 8.0 SE Basic software at
different magnifications (x100 and x400).
Phytochemical Screening
Standard screening tests of the powdered
leaves sample for the presence of secondary
metabolites such as phenolic compound,
tannins, saponins, flavonoids, cardiac
glycosides, and anthraquinones were done
according to standard methods [22].
Chromatographic fingerprinting
Analytical TLC was done on silica gel plate
. The plant material was extracted
successively with hexane, ethyl acetate and
methanol. The plate was developed, after
spotting the extracts at the origin using
solvent system hexane and ethyl acetate, ratio
4:3. Detection was done in daylight, under
UV366nm and with 10% aqueous H2SO4
spray reagent. Plates were dried at 100°C
after spraying. The different retardation
factors (Rf) of each spot were calculated [18].
Statistical analysis
The data obtained were expressed as mean ±
SEM (standard error of mean), and n
represents the number of replicates in an
experiment.
RESULTS
Macroscopic / organoleptic
characteristics
Leaves of L. hastata are simple, opposite in
arrangement, deciduous and stipulate. Leaf is
ovate with acuminate apex and entire blade
showing parallel venation. Upper surfaces
are dark green, while lower surfaces are light
green in color with a bristle texture (Figure
1). The powdered leaf material is green in
color, with characteristic odour and tasteless.
Microscopic studies
The adaxial epidermis shows abundant non-
glandular trichomes. Epidermal cells are
polygonal in shape, striations were present
but no stomata were observed. The abaxial
epidermis also shows abundant multicellular
trichomes epidermal cells are elongated,
irregularly shaped with wavy outline,
abundant anomocytic stomata type.
Epidermal cells are striated; striations are
more on the subsidiary cells and prismatic
crystals were seen (Fig 2 & 3). The
quantitative leaf microscopy measurements
are shown in Table 1.
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The T/S of the leaf showed a layer of
epidermal cells on both adaxial and abaxial
surfaces, two layers of palisade cells, spongy
mesophyll, abundant oil globules scattered
on the palisade cells. Druses (crystals) are
abundant, especially on the epidermal cells
and multicellular trichomes. Spiral xylem
vessel and large vascular bundle were seen at
the midrib. Cells closer to the palisade cell
also contained starch grains (Fig 4, 5 & 6).
The powdered leaves sample showed
fragment of parenchyma tissues, fragment of
epidermal cells with stomata, different types
of multicellular trichomes, fragments of
spiral xylem vessel, abundant starch grains
and oil globules and prismatic crystals (Fig
7 & 8).
The result of the chemo microscopic
evaluation is shown in Table 2.
Physicochemical parameters
Determination of physicochemical
parameters of a crude drug is essential as it
helps in identification and estimation of
mishandling, adulteration and also in setting
of proper standards. Physicochemical
parameters viz ash values, extractive values,
and moisture content were investigated and
the results are presented in Table 3.
Phytochemical Analysis and Thin Layer
Chromatography (TLC)
The result of phytochemical screening is
presented in Table 4. The Retardation Factor
(Rf) values of spot detected from TLC
analysis of the three extracts (hexane, ethyl
acetate, and methanol extracts) developed in
the solvent system of Hexane: ethyl acetate
(4:3) are presented in Table 5.
Discussion
Some important diagnostic characters that
might be useful in determining authenticity
and identifying adulteration of the crude drug
were observed in this study. Macroscopy
characters, organoleptic and epidermal
characteristic of leaf of Leptadenia hastata
especially the striated abaxial epidermal and
subsidiary cells, prismatic crystals and
different multicellular trichomes are
diagnostic features for the plant. The stomatal
index of 14.97 per mm², vein islet number of
56.3 per mm², and vein islet termination of
4.7 per mm², number of epidermal cells
(adaxial) 90 per mm², number of epidermal
cells (abaxial) 91per mm²provide valuable
information regarding leaf constants which
are useful parameters in standardization.
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Abundance of oil globules scattered on the
palisade cells, abundant druses found all over
the cell especially on the trichomes seen on
transverse section of the leaves are also
diagnostic and can be used to determined
authenticity of the plant even when in
fragments.
Ash values are reliable tools in detecting
adulteration and in establishing the purity of
crude drugs [1]. The ash value of 13.6%
shows that the plants contain low amount of
inorganic and other impurities present along
with the plant. Moisture content of 6.95%
falls within the recommended range of 8 –
14% for vegetable drug - an indication that
the plant can be stored for a long period with
less probability of microbial attack. High
water content promotes the growth of
microorganisms leading to degradation and
spoilage. The report of Fai et al [11] showed
differences in moisture content and ash
content in samples collected in Abuja (North
central) and Gombe state (North East),
Nigeria. The observed differences could be
due to geographical location.
Phytochemical investigations indicated the
presence of carbohydrate, Tannins,
flavonoids, and saponins. Phenols, tannins,
flavonoids, and saponins were also present in
the leaves of Leptadenia hastata collected
from northern part of the country [6].
Presence of these metabolites might be
responsible for various pharmacological
activities of the plant. Tannins have anti-
diarrheal activity and have also been reported
in the treatment of sexually transmitted
diseases; saponins for gastrointestinal
infection; Flavonoids are free radical
scavengers and therefore useful in
management of inflammatory diseases [3, 25,
26]. The alcohol extractive value of 15.3 and
water extractive value of 30.1 signify the
amount of organic constituents presents.
Extractive values are primarily useful for the
determination and evaluation of the chemical
constituents present in the crude drug and
they also help in the estimation of specific
constituents soluble in particular solvents
[10]. Water soluble extractive value of 30.1%
was two times higher than alcohol soluble
extractive value of 15.2% which means
greater yield was obtained using the aqueous
medium than the organic medium.
The thin layer chromatography of various
extracts showed spots which have different
Rf values. Rf value of various spots provide
valuable clue regarding their polarity and
selection of solvents for separation and the
profile gives a preliminary view of the
secondary metabolites that are present. The
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Journal of Phytomedicine and Therapeutic 2018; 17(2): 189 – 213 Page 203 www.jopat.gov.ng
various spots observed on TLC can be used
as finger-prints in the identification of L.
hastata.
Conclusion
The pharmacognostic evaluation of L.
hastata leaf is being reported for the first time
and results from this study have provided
information on the morphological and
anatomical features, and the physicochemical
parameters of L. hastata leaf. These
parameters can be used for identification and
quality control of the plant drug and provide
information, which may be useful in
monograph development on the plant.
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Fig 2 A: Upper Epidermal Layer of Leptadenia hastate, B: Lower Epidermal Layer of Leptadenia
hastate, C: Leaf Surface of Leptadenia hastata showing Vein Islet and Vein Termination
Key: PE: polygonal epidermal cells, IS: irregular shape cells, ST: striations, AS: anomocytic
stomata type, TB: Trichome base, VI: Vein Islet; VT: Vein Termination
AS
ST
TB
IS B
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Fig 3: Trichomes and Prismatic crystals on Epidermal Layers of Leptadenia hastata
Key: TC: Trichomes; PC: Prismatic crystals
Fig 4: Transverse Sections of Leaves of Leptadenia hastata
Key: MR: Midrib; PC: Palisade cells; TR: Trichomes; Pl: Phloem; Xy: Xylem
PC TR
Ph
Xy
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Fig 5: Transverse Sections of Leaves of Leptadenia hastata showing spiral xylem vessels, 2
layered palisade cells and striated trichomes
Key: XV: Xylem Vessels; PC: Palisade Cells; TR: Striated Trichomes; EP: Epidermal
Cells; SG: Starch grains
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Fig 6: Transverse Sections of Leaves of Leptadenia hastata showing mesophyll and palisade
layers
Key: PC: 2 layers of Palisade Cells; MC: Mesophyll cells; OG: Oil globules
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Fig 7: Microscopy of powdered leaves of Leptadenia hastata
Key: PT: Parenchyma tissue; EP: Epidermal cell; ST: Stomata; Star: Starch grains; OG:
Oil globules; XY: spiral xylem vessel
Fig 8: Microscopy of powdered leaves of Leptadenia hastata showing different Trichome
Types (TR)
TR
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Table 1: Quantitative Leaf Microscopy of Leptadenia hastata
* - n = 12, *1 - n =3
Table 2: Chemo-microscopy of the Powdered Leaves of Leptadenia hastata
Test Inference
Lignin +
Cellulose +
Tannins +
Starch +
Oil +
Protein +
Crystal +
Key: + = present; - = absent
Parameters Range (per mm2) Mean ± SEM
Number of adaxial Epidermal cells* 79 – 100 90 ± 1.8
Number of abaxial Epidermal cells* 80 – 103 91 ± 2.5
Stomata number (adaxial) absent absent
Stomata number (adaxial)* 10 – 21 16 ± 1.0
Stomatal index (abaxial)*1 14.97
Vein islet number 56 – 57 56.3 ± 0.3
Vein termination number*1 4 – 6 4.7 ± 0.7
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Table 3: physiochemical parameters of the leaves of Leptadenia hastate
Test Result (%)
Moisture content 6.95
Total ash 13.61
Water extractive value 30.13
Alcohol extractive value 15.23
Table 4: Phytochemical Screening of the Powdered Leaf of Leptadenia hastate
Key: + = Positive; - = Negative
Test Inference
Carbohydrate +
Saponins +
Tannins +
Flavonoids +
Terpenes _
Steroils _
Glycosides _
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Table 5: Retardation Factor (Rf) of Components of Organic extracts of Leaves of
Leptadenia hastata in Solvent System Hexane:Ethylacetate (4:3)
Extract Rf Daylight UV366nm 10% v/v aq
H2SO4
Hexane 0.48
0.67
0.85
0.86
0.89
0.91
Light yellow
-
Green
Yellow
-
-
-
-
Pink
-
Pink
Brown
Light brown
Brown
Brown
Brown
Brown
Ethlyacetate 0.41
0.46
0.52
0.59
0.78
0.84
0.91
Light yellow
Light yellow
Green
Light yellow
Green
Darkgreen
yellow
-
Pink
Green
Pink
Pink
Green
-
Brown
Brown
Brown
Brown
Brown
Green
Brown
Methanol 0.46
0.77
0.85
0.77
Light yellow
-
-
-
-
Pink
-
-
Brown
Brown
Brown
Brown