cissus species from nigeria: morphological and …
TRANSCRIPT
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Cissus species FROM NIGERIA: MORPHOLOGICAL AND
ANATOMICAL CHARACTERISTICS.
TAXONOMIC IMPLICATIONS
BY
MR VICTOR IROWA UWAGBOE
PG/LSC/8701675
A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF
PLANT AND BIOTECHNOLOGY
FACULTY OF LIFE SCIENCE, UNIVERSITY OF BENIN, BENIN CITY
IN PARTIAL FULFILMENT OF THE REQUIRMENT FOR THE
AWARD OF MASTERS IN SCIENCE DEGREE IN BIOSYSTEMATICS.
MAY 2019
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CERTIFICATION
This is to certify that this project work was written by Mr. Victor Irowa Uwagbo in the
Department of Plant Biology and Biotechnology, Faculty of Life Sciences as part of the
requirements for the award of M.Sc Biosystematics in the University of Benin, Benin City.
____________________ __________________
Prof. M. E. Osawaru Date
(Project Supervisor)
_____________________ _________________
Dr. H.O. Shittu Date
(Postgraduate Coordinator)
___________________________ ___________
Prof. (Mrs.) F.I. Okungbowa Date
(Head of Department)
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DEDICATION
This work is dedicated to God Almighty and to the memory of my immediate younger
brother, Late Paul Uwagboe, who died on the 8th of November, 2015..May his soul rest in
perfect peace. Amen.
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ACKNOWLEDGEMENT
I sincerely want to thank the Almighty God for giving me good health to
painstakingly undertake the rigors of this work. I want to express my appreciation to my
supervisor, Prof. M. E. Osawaru, the Head of Department, Prof. (Mrs.) F.I. Okungbowa and
all other members of the Department of Plant Biology and Biotechnology for their
encouragement and care shown to me. To my household, I thank you all for being there for
me.
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TABLE OF CONTENT
Cover page ii
Certification iii
Dedication iv
Acknowledgement v
Table of content Vi
Abstract viii
Chapter one
1.0 Introduction 1
1.1 Aims and objectives 4
Chapter two
2.0 Literature review 5
2.1 Taxonomic affinity 5
2.2 Palynology 6
2.3 Phytochemistry 7
2.4 Numerical taxonomy 8
CHAPTER THREE
3.0 Materials and methods 10
3.1 Materials 10
3.2 Methods 10
3.2.1 Field collection 10
3.2.2 Phytochemical screening 10
3.2.3 Preparation of plant sample 11
3.3 Proximate analysis 12
3.3.1 Determination of crude lipid content by soxhlet method 12
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3.3.2 Determination of crude fibre 12
3.4 Pigment extraction for β–carotene analysis 13
3.4.1 Measurement of absorbance 14
3.5 Anatomy of E1(Idogbo), E2 (Ebo), (Usen), D1 (Sapele), D2 (Ughelli) and D3
(Ekpan)
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3.5.1 Leaf anatomy 15
3.5.2 Stem anatomy 15
3.5.3 Root anatomy 15
3.6 Micromorphology 16
3.7 Macromorphology 16
3.8 Statistical analysis 16
Chapter Four
4.0 Results 17
Chapter Five
5.0 Discussion 45
5.1 Conclusion 49
5.2 Recommendation 50
References 51
LIST OF FIGURES
Figure 1: Dendogram of replicated samples similarities in cluster analysis 45
Figure 2: Dendogram of sample differences 46
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LIST OF PLATES
Plate 1-6: Collection from Delta State 18
Plate 7 – 12: Collection from Edo State 19
Plate 13: Colour of Powdered samples 23
Plate 14-15: Leaf summary 31 – 33
Plate 26 – 37: Stem summary 34-35
Plate 38 – 43: Root summary 36
Plate 44-46: Morphology of leaves collected in Edo State 37
Plate 47-55: Morphology of leaves collected in Delta State 38-39
Plate 56: Morphology of leaves collected in Edo State. 40
Plate 57. Morphology of leaves collected in Delta State 41
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LIST OF TABLES
Table 1: Phytochemical profile of Cissus spp. Leaves from Edo and Delta State 20
Table 2: Phytochemical profile of Cissus spp. Stem from Edo and Delta State 21
Table 3: Phytochemical profile of Cissus spp. Root from Edo and Delta State 22
Table 4: Crude lipid for samples from Edo State 24
Table 5: Crude lipid for samples from Delta State 25
Table 6: Crude fibre for samples from Edo State 26
Table 7: Crude fibre for samples from Delta State 27
Table 8: Beta-carotene for stem samples from Edo State and Delta State 28
Table 9: Beta-carotene for root samples from Edo State and Delta State 29
Table 10: Beta-carotene for leaves samples from Edo State and Delta State 30
Table 11: Qualitative leaf macro-morphological characters of specimen 42
Table 12: Qualitative stem and carpological macro-morphological characters of specimen 43
Table 13: Qualitative macro-morphological characters of specimen 44
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ABSRACT
Cissus species that are of the same type exist in Nigeria, these are identified as Cissus
populnea in most literatures of medicinal plant. Cissus populnea Guill & Perr. This is
commonly known as “Okoho” by the Idomas, Igbo and Igala tribes of Nigeria. The
Hausa tribe in Nigeria call it “Lututuwa”. A total of six (6) specimens were collected
in Edo and Delta States. The collected species from Edo State were tagged E1, E2 and
E3 as they were collected from three different locations in Edo State namely; Idogbo,
Ebo and Usen. The specimen from Delta State were tagged D1, D2 and D3 as they
were collected from three different locations in Delta States namely; Sapele, Ughelli
and Ekpan. The phytochemistry, morphological and anatomical properties of the
specimens were subjected to analysis. The colour of the powdered samples of the
plant collected from across Delta and Edo State in Nigeria showed variations, their
micro-morphological, morphological and anatomical characteristics exposed
sufficient variations that are enough to separate them into their various taxon. Though
their photochemistry was almost similar with few variations but the carpological
characteristics of collection from Delta State which were tagged D1, D2 and D3
confirmed that they are all Cissus populnea Guill. & Perr. The carpological
characteristics of collection from Edo State which were tagged E1, E2 and E3 show
that they are all Cissus rubginosa (Welw. Ex Baker) Planch.
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CHAPTER ONE
INTRODUCTION
Cissus species Linn belongs to the family Amplidaceae synonym Vitaceae. The family
Vitaceae (Grape family) are family of dicotyledonous flowering plants with 14 genera and
910 known species (Christenhusz and Byng, 2016). Sometimes the name appears as
Vitidaceae but Vitaceae is a conserved name and therefore has priority over both Vitidaceae
and the other name Ampelidaceae which is sometimes found in the older literatures. In the
cronquist system, the family was placed near the family Rhamnaceae in order Rhamnales but
in the APG III system (2009) onwards, the family is placed in its own order, Vitales.
Molecular phylogenetic studies put the Vitales as the most basal clade in the rosids and the
genera was increased by 3 bringing it to a total of 177 genera which are Acareosperma,
Ampelocissus, Ampelopsis (pepper-vine), Cayratia, Cissus (treebind, treebine), Clematisissus,
Cyphostamma, Leea, Nekemias, Nothocissus, Pathenocissus, Pterisanthes, Pterocissus,
Rhoicissus, Tegrastigma, Vitis (grape) and Yua (Angiosperm Phylogeny Group, 2016). The
family is economically important as the berries of Vitis species, commonly known as grapes
are important fruit crop and on fermentation produce wine. The plant species is a semi woody
liana 9 to 13 m high, 0.6 to 0.9 m in diameter when completely matured. It is widely
dispersed throughout the west tropical Africa to some part of east Africa with abundant
representation of this plant in the following countries; Togo, Mali, Guinea-Bissau, Guinea,
Senegal, Niger, Nigeria, Sudan, South Sudan, Uganda, Ghana, Gambia, Benin, Nigeria,
Central African Republic, Democratic Republic of Gongo, Chad, Ivory Coast, Tanzania
(Hutchinson and Dalziel, 1958).
Many genera are vines or climbing shrubs, rarely erect shrubs or small trees. Stems are hard,
woody with nodes, often swollen or jointed. Tendrils are leaf opposed and considered as the
metamorphosized epical parts of the synpodial stem by Eichler. Leaf alternate but lower leaf
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are sometimes opposed, simple or compound, pinnately or pammately. In this inflorescent,
flowers are borne opposite the leaf in rascemoscely, paniculate manner. Flowers are usually
bracteolate, actinomophic, bi-sexual or unisexual and hypogenous. The calyx are very small,
fused, cupula or valvate. Corolla are free or fused, cardicous and fall easily when the flower
opens. Androecium are usually five to six while the gynoecium are syncarpeous with axial
placentation. Fruits and seeds, the fruits are usually berry with watery juice and the seed with
copious endosperm. Pollination is mostly entomophilious and majority of the dry seeds are
dispersed by winds.
It is commonly called as “Okoho” by the Idomas, Igbo and Igala tribes of Nigeria; “Dafara”
(Kano, Zaria); “latutuwa” (Katsina) by the Hausa language of different states and towns in
northern Nigeria (Gbile, 1980); “Ajara” or “Orogbolo” by the Yoruba tribes of northern
Nigeria. The stem back and fruits are economically important in Delta, Benue and Kaduna
State where they are used in the preparation of soup, bean cake and Bambara nut cake
(Akara). The roots or stem are used in building (Irvine, 1961). Ethno-medicinal uses of the
plant include treatment of sore breast, indigestion, tuberculosis, diarrhea, venereal diseases,
intestinal parasites, oedema and eye problems resulting from attack of black cobra
(Najanigricollis) (Irvine, 1961). The plant is also used as cathartic, aphrodisiac and antidote
to arrow wounds. The Parthenocis sustricuspideta and severel cissus are cultivated as Drina
mental vine on wath. It has a lot of uses which are majorly from northern Nigeria, aside from
south eastern Nigeria (Igbo Land), where it is called “Okoho” same name with the Igala and
Idoma tribe of Nigeria. No other part of southern Nigeria has a comprehensive documented
name or uses for the plant. Although some people from Edo, particularly Okpilla has
reportedly claimed that they have seen the plant growing in their community some years back
and this might be a consequence of them shearing same boundary with Delta state which
gives them almost same vegetation zone with the state.
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Cissus has the following taxonomic classification.
According to Cronquist system;
Kindgom: Plantae
Phylum: Tracheophyta
Class: Magnoliopsida
Order: Rhamnales
Family: Vitaceae
Genus: Cissus Linn
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1.1 AIM AND OBJECTIVES
AIM
This project is aimed primarily to determine the similarity and difference in Cissus spp
collected in different locations across two states in Nigeria Edo State and Delta State. It also
aimed to provide a clear description that will separate the plant species mistakenly identified
as Cissus pulpunea in most Nigeria phytomedicinal literature into it various taxa.
OBJECTIVES
With the use of phytochemistry, morphology and anatomical analysis to separate the various
collections of Cissus species collected from Edo and Delta States into their various taxa.
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CHAPTER TWO
LITERATURE REVIEW
2.1 Taxonomic affinity
Originally floral characters were considered to provide the most valuable indicators for
taxonomic affinity/closeness because they are considered to be relatively constant with little
or no variation. This remained so forsome time up till date because floral characters are the
most prominent and reliable sources in botanical descriptions and diagnosis. However, leaf
characters have good advantages over floral characters in terms of taxonomic marcers which
are generally present in the leaves of the plant for greater part of its lifespan. Also leaves are
valuable in making taxonomic statement about sterile specimens, archeological remains,
fossils, drugs etc (Stance, 1984). Morphologically and anatomically leaf characters are the
most widely used of all the non-reproductive organ characters and important attention is paid
to them now especially aimed at knowing whether their micro-characters are more
conservative and of good taxonomic indicators than the macro-characters (Stance, 1984).
Anatomical features have contributed immensely to taxonomic families. The taxonomic
significance of stomata distribution and morphology was exemplified in the family
Epacridacead by Watson (1962), where he found out that members of the tribe Styphelieae
possess only adaxial stomata on the sepals while in the tribe Epacrideae, they are found on
the abaxial. And this agrees with the Bentham’s primary division of the family based on
ovary and fruits characters (Sonibare, 2003). Stomata characters, if properly interpreted, can
be of potential taxonomic value. It has been established that the development patterns of
stomata are consistent in monocotyledons and therefore of useful diagnostic feature for the
group (Stebbins and Khush, 1961). Taxonomic values of stomata and epidermal characters
among same monocotyledons have been demonstrated by several authors (Raju and Rao
(1977), Ayodele and Olowokudejo (1997), Ayodele and Olowokudejo (2006). Epidermal
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morphology was also used by Dehgan (1980) for taxonomic delimitation of the genus
Jatropha L. and he noted that all the species in the genus have paracytic stomata except J.
fremontiodes Standl, Barthlott et al. (1998) investigated the systematic significance of
epicuticular waxes using major groups of angiosperms and all the genera of gymnosperms
and they discovered that epicuticular waxes exhibited great micro-morphological diversity in
the groups.
Information on anatomy of the genus Cissus is very little but some aspects related to the
morphology and anatomy of Cissus verticillata are found in the works of Solereder (1908),
Metcalfe and Chalk (1957) and in more specific works such as that of Lizama et al. (2000) on
the leaf blade anatomy of Cissus verticillata. Some anatomical characters of the stem such as
the presence of a starch sheath, secretory cavities and fibers containing starch grains, and the
presence of type of trichomes, were shown as important for taxonomy of C. spinosa
cambessedes, C. ulmifolia (Baker) Planchon, C. erosa (Richard) and C. Sicyoides L (Alquini
et al., 1995). On the abaxial face of the leaves of the C. verticillata occur secretory structures
which have been described as pearl glands or pearl bodies. These structures have been
recently studied and constituted food bodies (Paiva et al., 2009). Given that the anatomy is an
important taxonomy parameter for the certification and quality control of medicinal plants,
and for the localization of secretion and accumulation sites of biological active compounds, it
is necessary to investigate the vegetative organs of species with therapeutic potential (Paiva
et al., 2009).
2.2 Palynology
Palynology meaning the science of pollen and spore and it deals mainly with the walls of
pollen and spores. Data in terms of shape, size, apertural configuration and surface
ornamentation from pollen might be very useful taxonomically (Gill, 1935). Pollen
morphological data have been used at all levels of taxonomic hierarchy either suggesting
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relationship or to determine variation within taxa. Mueller (1978) used pollen to determine
variation within a species and below species level. From the pollen shape, aperture, surface
ornamentation and wall structure, the 250 genera of Pteriodophyta were divided into five
main spore types and the groupings agrees with the classification of the genera based on the
entire plant morphological characters (Triton, 1986). West African species of Polygonaceae
were also groped into three based on pollen characters which correspond with the
morphological delimitation of the genera in the family (Ayodele, 2005).
2.3 Phytochemistry
Shellard (1979) opined that of all living organisms (e.g. plants) are unique in as much as the
need for their existence are very simple. Chemical substances such as carbon (IV) Oxide and
oxygen from the air and mineral salts dissolved in water, in soil or in aqueous surrounding,
the plant convert these substances in the process called oxidation to substances called
secondary metabolites. Secondary metabolites are protein, glycosides, phenolics, steroids,
saponins, terpenes, alkaloids and other chemical substances. Secondary metabolites differ
from one plant to another which led to a very wide variety of chemical compounds being
produced in the plant kingdom. The compounds have limited distribution within a plant and a
particular compound may be present in only a few species or varieties of the same species.
Takhtajan (1973) classified secondary metabolites as one of the compounds that have
taxonomic relevance. Also the medicinal value which most plants exhibit show is attributed
to the various secondary metabolites found in them. The biological activity of Cissus
pulpunea as a potential medicinal plant, used in the treatment of diabetes mellitus type 2 has
been found in the numerous literature (Garcia et al., 1997; Garcia et al., 2000; Beltrame et
al., 2001; Barbosa et al., 2002; Pepato et al., 2003; Viana et al., 2004; Almeida et al., 2006;
Silva et al., 2007; Braga, 2008). The effectiveness of the plant as an anti-inflammaroty,
antiepileptic, antihypertensive, antipyretic and antirheumatic has also been emphasized
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(Garcia et al., 2000; Almeida et al., 2006; Braga, 2008). Phytochemical data showed that
leaves of C. verticillata contain tannins, reducer compounds, steroids and triterpenes,
aminoacids, fatty compounds and flavonoids, more specifically kaempferol, luteolin and
luteoline-3-sulfate, which are all related to the biological activity of the plant (Lizama et al.,
2000; Barbosa et al., 2002).
2.4 Numerical taxonomy
The analysis of various types of taxonomic data by mathematical or computerized methods is
called numerical taxonomy or taximetrics. This approach of systematic involve the numerical
evaluation of the similarities or affinities between taxonomic units and the arrangements of
these units into taxa on the basis of their affinities.
It is aimed at overcoming some of the faults inherent in orthodox taxonomy (Baig, 1965),
where intuitive methods are used and which depend on the ability of the mind to recognize
swiftly the overall similarities in morphology. Because of the amount of data generated
during taxonomic study, the need for a method that will be able to order the bulky data to
make meaningful and greater precision was necessary. Sokal and Sneath (1963) described
numerical taxonomy as the numerical evaluation of the affinity or similarities between
taxonomic units and the ordering of these units into taxa on the basis of these affinities.
Numerical taxonomy lessens the difficulties encountered in orthodox taxonomic methods
when trying to interpret variety of data from different sources e.g. morphology, anatomical,
chemistry, physiology etc (Sneath and Sokal, 1973). Reports from various studies have
supported the fact that numerical taxonomy when applied enhanced taxonomic understanding
of the groups of taxa under study. The classification of the family poaceae (Clifford, 1965)
and taxonomic changes made in the genus Acanthospermum Schrank (Stessy, 1970) were
made through the application of numerical methods. In Nigeria, Hall et al. (1976) used
numerical methods to elucidate the position of members of the Bulbostylis Kunth and
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Fimbristylis Vahl (Cyperaceae). Dendrograms from clustering analysis and principal
component analysis were used to deduce affinity in the genus Ficus Linn (Sonibare, 2003)
and to make taxonomic inference for West African species of Polygonaceae (Ayodele, 2000).
In other studies, data generated from pyrolysis mass spectrometry for six species of higher
plants was subject to PCA and canonical variate analysis (CVA) and CVA separated these
species from one another while the dendrogram generated from the CVA grouped the plants
in an order that is in agreement with the known taxonomy of the plants at variety level (Kim
et al., 2004).
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CHAPTER THREE
MATERIALS AND METHOD
3.1 Materials
Beakers, Test tubes, pipette, burette, round bottom flask, soxhlet extractor, water bath, oven,
photospectometer, muffle furnace, photographic microscope, centimeter rule, Glacial acetic
acid and Conc. H2SO4, acetic anhydride (500 ml). lead acetate, ferric chloride (100 g),
sodium potassium tartarate (10 g), Ammonia solution (500 ml), chloroform, Brady’s regent,
conc. HCL, Fehling solution (A & B), Iodine solution, Wagner’s regent, Maeyer’s reagent
and Dragendorff’s reagent.
3.2 Method
3.2.1 Field Collection
Specimen of the cordate Cissus spp were collected from three different locations in Edo and
Delta State respectively in June and October. Voucher specimens were prepared for the
collections made and deposited in Pax Herbal herbarium according to standard herbarium
procedure. The plant specimens collected from Edo State were tagged E1 (Idogbo), E2(Ebo)
and E3 (Usen) while the specimens from Delta as B1 (Sapele), B2 (Ughelli) and B3 (Ekpan).
The different locations visited and the collections were as shown in the plant 1 – 12.
3.2.2 Phytochemical Screening
Phytochemical screening for various constituents such as alkaloids, anthraquinones,
flavonoids, phlobatanin, saponins, sterols, tannins and glycosides was carried out using
standard methods as described by Trease and Evans (2002) and Sofowara (1993). Each of the
test were qualitatively expressed as negative (-) or positive (+).
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3.2.3 Preparation of plant sample
The leaves were air-dried for twelve days at room temperature and the stems were peeled and
air-dried for twenty-three days at room temperature. Both leaves and the stems were
powdered using wooden mortar and pestle. 100 g each of the six powdered samples of the
plants were macerated in 1000 ml of distilled water. Each extract was filtered and
concentrated to 100 ml in a water bath at a temperature of 55oC. A portion of the extract was
used for phytochemistry as shown below.
Test for alkaloids: To 2 ml of extract, was added 2 ml of 1 % HCL. Then introduced into
water bath for 10 minutes, cool and centrifuge. To 1 ml of filtrate, was added 6-drops of;
• Mayer’s reagent – white to buff creamy precipitate will occur.
• Dragendorff’s reagent – orange to red precipitate will be seen.
• Wagner’s reagent – reddish brown precipitate will be seen.
Test for saponins: To 0.5 ml of the extract, 5ml of distilled water was added and shaken
vigorously for a stable persistent froth.
Test for phenolic compounds: To 2 ml of the extract, 2 ml 1 % Ferric chloride solution was
introduced. The formulation of brownish-green precipitate indicates the presence of
condensed tannin while bluish-black precipitate indicates the presence of hydrolysable
tannin.
Test for flavonoids: To 2 ml of the extract, 5 ml of the dilute ammonia solution was added
and then 1ml of concentrated Tetraoxosulphate (VI) acid. The appearance of a yellow colour
indicates presence of flavonoids.
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Test for cardiac glycosides (Keller-Killani test): 5 ml of the extract was treated with 2ml of
glacial acetic acid containing 1ml of ferric chloride solution. This was underplayed with 1ml
of concentrated sulphuric acid. A brown ring at the interface indicates a deoxysugar
characteristic of cardenolides. A violet ring may appear below the brown ring while in the
acetic acid layer a greenish ring may form just gradually throughout thin layer.
Test for steroid: 1 ml of the sample + 0.5 ml acetic acid anhydride + cooled in ice + 0.5 ml
chloroform + 1 ml concentrated H2SO4 added carefully with pipette. A reddish brown ring
formed at the separation level between the two liquids.
Test for Phlobatanins: To 2 ml of the extract, 2 ml of 1% HCL was added and then
introduced into water-bath at 90oC for 10min. The formation of red residues at the base of the
test tube indicates the presence of Phlobatanin.
Polysaccharides/Starch: To 2 ml filtrate, 6 drops of iodine solution was added. Formulation
of blue-black precipitates indicate the presence of starch.
Reducing sugars: To 2 ml of the extract, 5ml of Fehling solution was added and then
introduced into water-bath for 10 min. Red coloration revealed the presence of reducing
sugar.
Terpenoids: To 2 ml filtrate, 6 drops of Brady’s reagent was added. Yellow or orange
coloration revealed the presence of Terpenoids.
3.3 Proximate analysis
3.3.1 Determination of crude lipid content by soxhlet method: A clean dried 500 cm3
round bottom flask containing few anti-bumping granules was weighed (W1) with 300 cm3
petroleum ether (40-60oC) for extraction poured into the flask filled with soxhlet extraction
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unit. The extractor thimbe weighing 20 grams was fixed into the soxhlet unit. The round
bottom flask and a condenser were connected to the soxhlet extractor and cold water
circulation was connected. The heating mantle was switched on and the heating rate adjusted
until the solvent was refluxing at a steady rate. Extraction was carried out for 6 hrs. The
solvent was recovered and the oil dried in an oven set at 70oC for 1hr. The round bottom flask
and oil was then weighed (W2). The lipid content was calculated thus:
Crude lipid content = W2 – W1 x 100
Weight of sample
3.3.2 Determination of crude fibre: The sample (2 g) was weighed into a round bottom
flask, 100 cm3 0.25M Tetraoxosulphate (VI) acid solution was added and the mixture boiled
under reflux for 30min. The hot solution was quickly filtered under suction. The insoluble
matter was washed several times with hot water until it was acid free. It was quantitatively
transferred into the flask and 100 cm3 of hot 0.31M Sodium Hydroxide solution was added.
The mixture boiled under reflux for 30 min and filtered under suction. The residue was
washed with boiling water until it was base free, dried to constant weight in an oven at
100oC, cooled in desiccators and weighted (C1). The weighted sample (C1) was then
incinerated in a muffled furnace at 550 oC for 2 hrs, cooled in a desiccators and reweighed
(C2).
Calculation: The loss in weight on incineration: C2 – C1
Crude fibre = C2 – C1 x 100 .
Weight of original sample
3.4 Pigment extraction for β-carotene analysis
This was done according to the method of the Association of Official Analytical Chemists
(AOAC, 1980). In to a conical flask containing 50 ml of 95% ethanol, 10 g of the macerated
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sample was placed and maintained at a temperature of 70-80 oC in a water-bath for 20 mins
with periodic shaking. The supernatant was decanted, allowed to cool and its volume was
measured by means of a measuring cylinder and recorded in initial volume. The ethanol
concentration of the mixture was brought to 85 % by adding 15 ml of distilled water and it
was further cooled in a container of ice water for about 5mins. The mixture was taken into a
separating funnel and 25ml of petroleum ether (pet-ether) was added and the cooled ethanol
was poured over it. The funnel was swirled gently to obtain a homogenous mixture and it was
later allowed to stand until two separate layers were obtained. The bottom layer was run off
into a beaker while the top layer was collected in to a 250 ml conical flask. The bottom layer
was transferred into the funnel and re-extracted with 10 ml pet-ether for 5-6 times until the
extract became fairly yellow. The whole pet-ether was collected in the 250ml conical flask
and transferred into a separating funnel for re-extraction with 50ml of 80 % ethanol. The final
extract was measured and poured into sample bottles for further analysis.
3.4.1 Measurement of absorbance
The absorbance of the extracts was measured with the use of a spectrophotometer (model
22UV/VIS) at a wavelength of 436 nm. A cuvette containing pet-ether (blank) was used to
calibrate the spectrophotometer to zero point. Samples of each extract were placed in cuvettes
and readings were taken when the figure in the display window became steady. The operation
was carried out 5 times for each sample and the average readings were recorded. The
concentration of β-carotene was calculated using Bear-Lamberts law, which states that the
absorbance (A) is proportional to the concentration (C) of the pigment, as represented by the
equation; A ∞ L (if concentration (C) is constant); A=ECL, C=A/EL.
Where C = concentration of carotene, A = absorbance, E = extinction coefficient, L =
thickness of cuvettes (Path length) = 1 cm, E of β-carotene = 1.25 x 104 µg/l
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3.5 Anatomy of E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli), D3
(Ekpan)
3.5.1 Leaf Anatomy
The leaf transverse section was washed in water and soaked in 15% sodium hypochloride for
2mins. They were later washed in water and stained in sudan IV for 4mins. They were
mounted in glycerol on a slide with the edges of the cover slip ringed with nail varnish to
prevent desiccation. The slides were observed using photographic light microscope.
Photographs of the leaf anatomy are shown in plates 27 – 38.
3.5.2 Stem Anatomy
The stem transverse cross section of about 15 - 20 µm were made by freehand sectioning
from young stem of each of the specimen. The sections were raised in water and later
transferred to 2% Safranin and 3% methylene blue which were used to stain and counter stain
the sections for the anatomical structure to be clearly visible. Excess stain is washed off in
acetone. They were mounted in glycerol on a slide with the edges of the cover slip ringed
with nail varnish to prevent desiccation. The slides were observed using photographic light
microscope. Photographs of the stem anatomy are shown in plate 39-50.
3.5.3 Root anatomy
The transverse cross section of about 15-20 µm were made by freehand sectioning from
young root of each of the specimen. The sections were raised in water and transferred to 2%
neutral red and 3% methylene blue which were used to stain and counter stain the sections for
the anatomical stricter to be clearly visible. Excess stain is washed off in acetone. They were
mounted in glycerol on a slide with the edges of the cover slip ringed with nail varnish to
prevent desiccation. The slides were observed using photographic light microscope and a
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smart phone to make photographs of specimens. Photographs of the root anatomy are show in
plates 31 – 56.
3.6 Micromorphology of E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli),
D3 (Ekpan)
The epidermal peel was prepared using forceps and a razor blade to scrape until a clear
portion of the upper and lower epidermis was obtained. A clear portion of the epidermis was
obtained by boiling the leaf in carbon tri-hydride for 1min. Photographs of the micro-
morphological characters are shown in plates 15 -26.
3.7 Macromorphology of E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2
(Ughelli), D3 (Ekpan)
A digital camera was used to get the photographs of the specimens. Both vegetative and
reproductive characteristics were assessed in the six different plant specimens. Measurement
of diagnostic characters of the specimen was taking with centimeter rule. Characters assessed
in all the specimens were leaf shape, margin, apex type, texture, base type, surfaces, length,
width, blade length, petiole length, venation type. Statistical analysis {minimum (mean
+standand dev.), maximum} was calculated for all characters assessed and principle
component analysis was used to show their relationships. Photographs of the morphological
characters are shown in plates 13 and 14.
3.8 Statistical analysis
Data were analyzed using descriptive statistics in Microsoft excel package, clustering
analysis and principal component analysis were carried out in past statistic package isng pair
group algorithm by Bray Curtis and Euclidian.
27
CHAPTER FOUR
RESULTS
4.1 Field collection
A total of six (6) specimens were collected from Edo and Delta State. The specimen from
Edo State are E1 (Idogbo), E2 (Ebo), and E3 (Usen) while the specimen from Delta State are
D1 (Sapele), D2 (Ughelli) and D3 (Ekpan). The specimens were all lianas with vase
similarities in leaves and stem structure due to visual appreciation but highly distinct in fruit
types. As shown in the following plates.
28
Plate 1, 2 & 3 showing the plant specimen collected from the locations in plate 4, 5 & 6 in Edo State.
Plate 4 Plate 6 Plate 5
29
Plate 7, 8 & 9 showing the plant specimen collected from the locations in plate 10, 11 & 12 in Delta
State
D1 D2 D3
30
4.2 Phytochemistry
The phytochemistry of the collections show the amount of secondary metabolites present in
the collections from Edo and Delta State qualitatively as shown in Tables 1-3.
Table 1: Phytochemical profile of Cissus spp. Leaves from Edo and Delta State
S/N Phytochemicals E1 E2 E3 D1 D2 D3
1 Alkaloids
Dragendoff - - - - - -
Mayer - - - - - -
Wagna - - - - - -
2 Cardiac glycoside - - - - - -
3 Anthoquinone
glycoside
+ + ++ + + +
4 Flavonoid +++ +++ +++ - - -
5 Saponin +++ + +++ + + ++
6 Starch - - - ++ ++ ++
7 Steroid ++ ++ ++ - - -
8 Phlobatanin - - - - - -
9 Reducing sugar ++ ++ ++ ++ ++ ++
10 Terpenoid ++ ++ ++ ++ ++ ++
11 Tannin
Condensed + + + + + +
hydrolysable - - - - - -
Key: + present; ++ moderately present; +++ abundantly present; - absent
E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli) and D3 (Ekpan).
31
Table 2: Phytochemical profile of Cissus spp. Stem from Edo and Delta State
S/N Phytochemicals E1 E2 E3 D1 D2 D3
1 Alkaloids
Dragendoff - - - - - -
Mayer - - - - - -
Wagna - - - - - -
2 Cardiac glycoside - - - - - -
3 Anthoquinone
glycoside
+ + ++ + + +
4 Flavonoid - - - - - -
5 Saponin - - - - - -
6 Starch + + + ++ ++ ++
7 Steroid ++ ++ ++ ++ ++ ++
8 Phlobatanin - - - - - -
9 Reducing sugar - - - - - -
10 Terpenoid - - - - - -
11 Tannin
Condensed - - - +++ +++ +++
hydrolysable - - - +++ +++ +++
Key: + present; ++ moderately present; +++ abundantly present; - absent
E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli) and D3 (Ekpan).
32
Table 3: Phytochemical profile of Cissus spp. Roots from Edo and Delta State
S/N Phytochemicals E1 E2 E3 D1 D2 D3
1 Alkaloids
Dragendoff - - - - - -
Mayer - - - - - -
Wagna - - - - - -
2 Cardiac glycoside - - - - - -
3 Anthoquinone
glycoside
+ + ++ + + +
4 Flavonoid - - - - - -
5 Saponin - - - + + +
6 Starch - - - ++ ++ ++
7 Steroid ++ ++ ++ + + +
8 Phlobatanin - - - - - -
9 Reducing sugar - - - - - -
10 Terpenoid + + + + + +
11 Tannin
Condensed - - - + + ++
Hydrolysable + + + - - -
Key: + present; ++ moderately present; +++ abundantly present; - absent
E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli) and D3 (Ekpan).
33
Plate 13: Colour of Powdered samples
E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli) and D3 (Ekpan).
D1 D2 D3
E1 E2 E3
34
4.3 Proximate analysis
Table 4: Crude lipid for samples from Edo State
E1 % Lipid in 2g E2 % Lipid in 2g E3 % Lipid in 2g
Stem 5.5.
5.8±0.173
5.2
5.6±0.231
5
5.1±0.1
Root 6.1
5.5
4.6±0.2603
6
4.1
4.3±0.153
5.3
4.2
4.4±0.185
Leaves 6.1
3.9
4.5±0.328
4.6
3.5
3.8±0.2027
4.8
3.1
3.5±0.296
5 4.2 4.1
35
Table 5: Crude lipid for samples from Delta State
D1 % Lipid in 2g D2 % Lipid in 2g D3 % Lipid in 2g
Stem 9.2.
10±0.43
9.5
10±0.393
6.4
9±1.354
Root 10.7
7.8
8.5±0.433
10.8
7.1
10±0.392
11
7.1
7.5±0.231
Leaves 9.3
8
7.3±0.202
8.2
6.3
6.6±0.145
7.9
6.9
7.2±0.24
7.6 6.8 7.7
Key for measurement = Minimum, Mean ± Standard error and Maximum
36
Table 6: Crude fibre for samples from Edo State
E1 % fibre in 2g E2 % fibrein 2g E3 % fibre in 2g
Stem 0.4
0.41±0.0088
0.4
0.41±0.0088
0.39
0.42±0.0145
Root 0.43
0.29
0.32±0.0145
0.43
0.27
0.3±0.0153
0.44
0.3
0.32±0.0145
Leaves 0.34
0.2
0.24±0.019
0.32
0.21
0.25±0.023
0.35
0.22
0.24±0.012
0.27 0.29 0.26
37
Table 7: Crude fibre for samples from Delta State
D1 % fibre in 2g D2 % fibre in 2g D3 % fibre in 2g
Stem 0.4
0.4167±0.00881
0.4
0.3057±0.01326
0.41
0.4303±0.01155
Root 0.43
0.3
0.34±0.0208
0.45
0.31
0.33±0.0145
0.45
0.31
0.33±0.0153
Leaves 0.37
0.21
0.22±0.0067
0.36
0.2
0.23±0.0145
0.36
0.21
0.225±0.0104
0.23 0.25 0.25
Key for measurement = Minimum, Mean ± Standard error and Maximum
38
Table 8: Beta-carotene for stems of samples from Edo and Delta State
E1 E2 E3 D1 D2 D3
Absorbance (nm) 0.47
0.48±0.00577
0.49
0.61
0.64±0.01764
0.67
0.43
0.44±0.0088
0.46
0.121
0.12133±0.00033
0.122
0.121
0.12266±0.00088
0.124
0.123
0.182±0.0295
0.212
Concentration (g/l) 25500
25766.7±145.296
26000
18700
19800±556.77
20500
37200
28200±550.75
39100
102000
102333±333.3
103000
101000
102000±577.35
103000
58300
73166±14419
102000
39
Table 9: Beta-carotene for rootsof samples from Edo and Delta State
E1 E2 E3 D1 D2 D3
Absorbance (nm) 0.29
41800±814.45
0.31
0.34
0.35±0.00577
0.36
0.33
0.34±0.00577
0.35
0.22
0.23±0.00577
0.24
0.24
0.25±0.00577
0.26
0.24
0.24333±0.0033
0.25
Concentration (g/l) 40300
41800±814.45
43100
43700
35600±624.5
46800
35700
36600±665.83
37900
55000
38866.7±16699
56800
48100
49100±550.75
50000
52100
52466.6±366.6
532000
40
Table 10: Beta-carotene for leaves of samples from Edo and Delta State
E1 E2 E3 D1 D2 D3
Absorbance (nm) 0.34
0.35±0.0067
0.36
0.34
0.36±0.0115
0.38
0.33
0.35±0.0115
0.37
0.35
0.36±0.00577
0.37
0.35
0.36±0.00577
0.37
0.34
0.35±0.00577
0.36
Concentration (g/l) 34700
35566.7±633.33
36800
32900
33800±519.61
34700
33800
36033.3±1197.68
37900
33700
34400±650.64
0.00577
33800
35800±1184.62
37900
34600
35000±351.18
35700
Key for measurement = Minimum, Mean ± Standard error and Maximum
41
4.5 Leaf anatomy
Plate 14. Leaf TS E1 (x 100)
Plate 15. Leaf TS E2 (x 100)
Plate 16 Leaf TS E3 (x 100)
Plate 17. Leaf TS E1 (x 400)
Plate 18. Leaf TS E2(x 400)
Plate 19. Leaf TS E3 (x 400)
42
Plate 14. Leaf TS D1 (x 100)
Plate 15. Leaf TS D2 (x 100)
Plate 16 Leaf TS D3 (x 100)
Plate 17. Leaf TS D1 (x 400)
Plate 18. Leaf TS D2(x 400)
Plate 17. Leaf TS D3 (x 400)
43
Plate 14 – 25. Transverse section shows similar pattern of arrangement in the cells and tissue structures of E1
(Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli) and D3 (Ekpan).
Plate 20. Leaf TS E1 (x 100)
Plate 21. Leaf TS E2 (x 100)
Plate 22 Leaf TS E3 (x 100)
Plate 23. Leaf TS E1 (x 400)
Plate 24 Leaf TS E2(x 400)
Plate 25 Leaf TS E3 (x 400)
44
4.5.1 Stem anatomy
Plate 26 – 31. Transverse section shows solenestele-syphonostele arrangement of the vascular bundles with
empty space at the middle of the stem occupying the pit, shows the same pattern of arrangement in the stem of
E1 (Idogbo), E2 (Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli) and D3 (Ekpan).
Plate 26.Stem TS D1 (x 40)TS E
Plate 27.StemD2 (x 40)
Plate 28StemTSD3 (x 40)
Plate 29. Stem TS D1 (x 100)
Plate 30StemTSD2(x 100)
Plate31Stem TS D3 (x 100)
45
Plate 32 – 37 has syphonostele arrangement of vascular bundles and pit covered with fibres and parenchyma
cells. Arrow head shows sieve cells in the phloem of E1 (Idogbo), E2 (Ebo), and E3 (Usen).
4.5.2 Root anatomy
Plate 32.Stem TS E1 (x 40)
Plate 33.Stem TS E2 (x 40)
Plate 34 Stem TS E3 (x 40)
Plate 35.Stem TS E1 (x 100)
Plate 36Stem TS E2(x100)
Plate 37Stem TS E3 (x100)
46
Plate 38 – 43 shows similarities in the root of all the collections from both Edo and Delta: E1 (Idogbo), E2
(Ebo), E3 (Usen), D1 (Sapele), D2 (Ughelli) and D3 (Ekpan).
Plate 38.Root TS D1 (x 40)
Plate 39.Root TS D2 (x 40)
Plate 40Root TS D3 (x 40)
Plate 41.Root TS E1 (x 40)
Plate 42Root TS E2(x 40)
Plate 43Root TS E3 (x 40)
47
4.6 Micromorphology photomicrograph
The photomicrograph was taken at a magnification of x100.
Plate 44Adaxia photomicrograph of D1
Plate 45Adaxia photomicrograph of D2
Plate 46Adaxia photomicrograph of D3
48
The photomicrograph was taken at a magnification of x100.
Plate 44-49. The adaxia and abaxia leaf surface of D1 (Sapele), D2 (Ughelli) and D3 (Ekpan) all showed a
anomocytic type of stomata (Plate) as indicated by single arrow head and a conspicuous nucleus double arrow
head containing starch grain in the parenchyma cell.
Plate 47Abaxia photomicrograph of D1
Plate 48Abaxia photomicrograph of D2
Plate 49Abaxia photomicrograph of D2
49
The adaxia leaf surface of E1 (Idogbo), E2 (Ebo), E3 (Usen) have no trichomes and stomata as shown in plate
50-52. The abaxia surface of E1, E2 and E3 have many scale-trichomes with glandula head (as indicated by
single arrow) which has a stomatal base as shown in Plate 53-55.
Plate 50 Adaxia photomicrograph of E1
Plate 51 Adaxia photomicrograph of E2
Plate 52 Adaxia photomicrograph of E3
Plate 53 Abaxia photomicrograph of E1
Plate 54 Abaxia photomicrograph of E2
Plate 55Abaxia photomicrograph of E3
50
4.6.1 Macromorphology
Plate 56. Morphology of leaves collected in Edo State.
Key:= E1 UPLS – Upper leaf surface of plant collected from Idogbo, E2 LPLS – Lower leaf surface of plant
collected from Idogbo; E2 UPLS – Upper leaf surface of plant collected from Ebo community, E2 LPLS – Lower
leaf surface of plant collected from Ebo community,E3 UPLS – Upper leaf surface of plant collected from Usen,
E3 LPLS – Lower leaf surface of plant collected from Usen.
E1 UPLS E2 UPLS
E3 UPLS
E1LPLS E2LPLS E3LPLS
51
Plate 57. Morphology of leaves collected in Delta State.
Key:=D1 UPLS – Upper leaf surface of plant collected from Sapele, D1 LPLS – Lower leaf surface of plant
collected from Sapele; D2 UPLS – Upper leaf surface of plant collected from Ughelli, D2 LPLS – Lower leaf
surface of plant collected from Ughelli; D3 UPLS – Upper leaf surface of plant collected from Ekpan, D3 LPLS
– Lower leaf surface of plant collected from Ekpan
D3 UPLS D3LPLS
D2 UPLS D2LPLS
D1 UPLS D1LPLS
52
Table 11: Qualitative leaf macro-morphological characters of the specimen
Leaf
shape
Leaf apex Leaf base petiole Upper
leaf
surface
Lower leaf
surface
Leaf
venation
Leaf
margin
D1 cordate tapered arced present Dull &
smooth
Glabrescent Pinnately
veined
Wavy
D2 cordate tapered arced present Dull &
smooth
Glabrescent Pinnately
veined
Wavy
D3 cordate tapered arced present Dull &
smooth
Glabrescent Pinnately
veined
Wavy
E1 cordate tapered arced present Glabrous Dull Pinnately
veined
Serrated
E2 cordate tapered arced present Glabrous Dull Pinnately
veined
Serrated
E3 cordate tapered arced present Glabrous Dull Pinnately
veined
serrated
53
Table 12: Qualitative stem and carpological macro-morphological characters of the specimen
Stem shape Mature
stem
colour
Stem
tendril
Flower
type
Flower
colour
Fruit type Fruit
colour
Seed
texture
D1 Round-
rectangular
white present umbel yellow drupe Green Hard
D2 Round-
rectangular
white present umbel yellow drupe Green Hard
D3 Round-
rectangular
white present umbel yellow drupe Green Hard
E1 Round-
rectangular
Dark
brown
present umbel yellow berry White &
black
Soft
E2 Round-
rectangular
Dark
brown
present umbel yellow berry White &
black
Soft
E3 Round-
rectangular
Dark
brown
present umbel yellow berry White &
black
soft
54
Table 13: Quantitative macro-morphological characters of the specimen
Leaf length (cm) Leaf width (cm) Blade length
(cm)
Petiole length
(cm)
Flower length
(cm)
Peduncle length
(cm)
Pedicels length
(cm)
D1 26
26.3±0.17
27
21
21.28±0.16
21.9
15.8
15.92±0.08
16.2
10.2
10.44±0.12
10.8
6.8
7.04±0.08
7.3
2.4
2.72±0.10
2.9
3.1
3.36.3±0.12
3.7
D2 18.4
18.84±0.13
19.2
13.5
13.84±0.18
14.4
11.3
11.74±0.12
12.0
7.0
7.1±0.03
7.2
4.9
5.14±0.08
5.3
2.0
2.14±0.05
2.3
2.0
2.1±0.06
2.3
D3 16.5
16.76±0.08
17.0
11.4
11.7±0.11
12.0
11.6
12.0±0.11
12.2
4.3
4.76±0.14
5.1
3.9
4.34±0.13
4.6
1.7
1.9±0.05
2.0
1.3
1.46±0.06
1.6
E1 22.9
23.22±0.12
23.5
18
18.28±0.10
18.5
15
15.16±0.10
15.5
7.8
8.06±0.09
8.3
6.4
6.72±0.14
7.1
2.3
2.54±0.12
3
2.1
2.24 ±0.05
2.4
E2 19.8
21.26±0.39
22
14.3
16.18±0.54
17.4
10.7
11.56±0.25
12.2
9.1
9.7±0.25
10.5
5
5.68±0.26
6.4
1.7
1.84±0.05
2
2.4
2.88±0.16
3.4
E3 19.6
20.36±0.26
21
13.2
13.88±0.19
14.3
11.2
12.8±0.83
16
7.8
8.36±0.15
8.6
5.8
6.12±0.10
6.4
1.7
1.76±0.02
1.8
3.1
3..24±0.05
3.4
Key for measurement = Minimum, Mean ± Standard error and Maximum
55
Figure 1: Dendogram of replicated samples similarities in cluster analysis
The figure shows 3 distinct groups of sample clusters the group from Idogbo and Ebo are 0.92% similar to their
morphological characteristics. The clusters from Ekpan is approximately 0.87% similar to Ebo, Usen and Ekpan.
56
Figure 2: dendogram of sample differences
Key: E1 (Idogbo), E2(Ebo), E3 (Usen), D1(Sapele), D2(Ughelli) and D3(Ekpan)
Sample from Delta (D1 6.6.%) is far distantly related to samples from Ebo (E2) and Ekpan (D3). The difference
between the quantitative values of Sapele (D1 4.6%) and Idogbo (E1) is not much as compared to Usen (E3) and
Ughelli (D2).
D1 D2 D3
57
CHAPTER FIVE
DISCUSSION
In their qualitative phytochemistry, anthraquinone glycoside, saponin and reducing sugar was present in the
leaves of E1 (Idogbo), E2(Ebo), E3(Usen), D1(Sapele), D2(Ughelli) and D3 (Ekpan).Alkaloid, cardiac glycoside,
pholbatanin and hydrolysable tannin was absent in the leaf of E1, E2, E3, D1, D2 and D3 which mark some sign
of similarity in the genus level. Flavonoid present in E1, E2, E3 but absent in D1, D2 and D3. Starch absent in
E1, E2, E3 but present in D1, D2 and D3 (Table 1).
Phytochemistry of the stem confirms alkaloid, cardiac glycoside, flavonoid, saponin, phlobatannin, reducing
sugar and terpinoid to be absent in E1 (Idogbo), E2 (Ebo), E3 (Usen), D1(Sapele), D2(Ughelli) and D3 (Ekpan).
Anthroquinone glycoside, start and steroid were present in E1, E2, E3, D1, D2 and D3. Condensed and
hydrolysable tannin was confirmed to be abundant in D1, D2 and D3 (Table 2).
Phytochemistry of the root confirmed alkaloid, cardiac glycoside, flavonoid, phlobatanin and reducing sugar to
be absent in E1 (Idogbo), E2 (Ebo), E3 (Usen), D1(Sapele), D2(Ughelli) and D3 (Ekpan). While terpinoid,
steroid and anthroquinone glycoside was present in E1, E2, E3, D1, D2 and D3.Saponin, starch and condensed
tannin confirmed present in E1, E2, E3 but absent in D1, D2 and D3. Hydrolysable tannin was present in E1, E2,
E3 but absent in D1, D2 and D3 (Table 3).
Colour of good samples show dark brown for E1 (Idogbo), E2 (Ebo), E3 (Usen) and brown for D1(Sapele),
D2(Ughelli) and D3 (Ekpan) (Plate 13)
The proximate analysis of the plant shows crude lipid to be high in samples from Delta State with the highest
value of D1 stem (10% in 2g) and D2 stem and root (10% in 2g) than samples from Edo State with the highest
value in E1 stem (5.8% in 2g) – (Table 4 & 5).
The crude fibre was slightly higher in collections from Delta State with D1 stem (4.3% in 2g) then collection
from Edo State with E3 stem (4.2% in 2g) which represent the highest fibre content for both states (Table 6 & 7).
The concentration of beta-carotene was high in collections from Delta State with D1 stem (1.02333 x 105 µg/l)
(Table 8) than collection from Edo State with E1 root (4.1800 x 105 µg/l). Table 8,9& 10).
58
The leaf anatomy of E1 (Idogbo), E2 (Ebo), E3 (Usen), D1(Sapele), D2(Ughelli) and D3 (Ekpan) all show
isobilateral arrangement of leaf tissues (Plate 14 – 25) while the stem anatomy D1(Sapele), D2(Ughelli) and D3
(Ekpan) shows solenostele-syphonostel arrangement of vascular bundles with hole at the pit position (Plat 26 –
31) but E1 (Idogbo), E2 (Ebo), E3 (Usen) shows syphonostele arrangement of vascular bundles and pit covered
with fibres and parenchymatous cells (Plate 32 – 37). These characteristics can be used to identify the species
and delimit their species boundary, though their root anatomy shows similar characteristics (Plate 38 – 43).
Their micromorphological characteristics of the leaves show the presence of trichomes in E1 (Idogbo), E2 (Ebo),
E3 (Usen) but D1(Sapele), D2(Ughelli) and D3 (Ekpan) has no trichomes. E1, E2 and E3 has no visible stomata
as observed in the photomicrograph (Plate 50 – 55) but D1, D2 and D3 has stomata (Plate 44-49).
59
5.2 CONCLUSION
The morphological, anatomical and phytochemical characteristics of E1 (Idogbo), E2 (Ebo), E3 (Usen), D1
(Sapele), D2 (Ughelli) and D3 (Ekpan) has been used to delimit the cordate Cissus collection from different
locations in Edo and Delta States of Nigeria into two taxon at the species level. Though they share the
characteristic draw property which most people use for the identification of Cissus populnea and also share basic
similarities in anatomical and phytochemical characteristics but the carpological characteristics of D1 (Sapele),
D2 (Ughelli) and D3 (Ekpan) shows that they are all Cissus populnea (Guill. &Perr. – Plates 7, 8 & 9) and the
carpological characteristics of E1, E2 and E3 show that they are all Cissus rubiginosa (Welw ex Baker & Planch
– Plate 1).
60
5.2 RECOMMENDATION
I humbly recommend that plant scientists should always consider the carpological characteristics for uncommon
plant before naming them. Also to ascertain the proper identity of a specimen, it should be compared with a
complete herbaria collection.
61
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