Cissus species FROM NIGERIA: MORPHOLOGICAL AND …

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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


1 Alkaloids


Mayer - - - - - -

Wagna - - - - - -


3 Anthoquinone

glycoside

+ + ++ + + +

4 Flavonoid - - - - - -

5 Saponin - - - - - -

6 Starch + + + ++ ++ ++

7 Steroid ++ ++ ++ ++ ++ ++


9 Reducing sugar - - - - - -

10 Terpenoid - - - - - -

11 Tannin

Condensed - - - +++ +++ +++

hydrolysable - - - +++ +++ +++



32

Table 3: Phytochemical profile of Cissus spp. Roots from Edo and Delta State


1 Alkaloids


Mayer - - - - - -

Wagna - - - - - -


3 Anthoquinone

glycoside

+ + ++ + + +

4 Flavonoid - - - - - -

5 Saponin - - - + + +

6 Starch - - - ++ ++ ++

7 Steroid ++ ++ ++ + + +


9 Reducing sugar - - - - - -

10 Terpenoid + + + + + +

11 Tannin

Condensed - - - + + ++

Hydrolysable + + + - - -



33

Plate 13: Colour of Powdered samples


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


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


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


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


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


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


41

4.5 Leaf anatomy

Plate 14. Leaf TS E1 (x 100)


Plate 16 Leaf TS E3 (x 100)


Plate 18. Leaf TS E2(x 400)


42

Plate 14. Leaf TS D1 (x 100)


Plate 16 Leaf TS D3 (x 100)


Plate 18. Leaf TS D2(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 24 Leaf TS E2(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


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 34 Stem TS E3 (x 40)


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



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



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 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


smooth


veined

Wavy


smooth


veined

Wavy

E1 cordate tapered arced present Glabrous Dull Pinnately

veined

Serrated


veined

Serrated


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


D3 Round-

rectangular


E1 Round-

rectangular

Dark

brown

present umbel yellow berry White &

black

Soft

E2 Round-

rectangular

Dark

brown


black

Soft

E3 Round-

rectangular

Dark

brown


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


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

REFERENCES

Almeida, E.R., Oliveira, J.R.G., Lucena, F.R., Soares, R.P.F. and Couto, G.B.L. (2006). The action of

extract of the dry leaves of Cissus sycioides L. in pregnant rats. Acta Farm Bonaer 25: 421-

424.

Alquini, Y., Bona, C., Bueno, N.C., Cislinski, J., Contin, A., Dunaiski, A. and Segecin, S. (1995).

Anatomia caulinar de quatro espécies do Gênero Cissus (Vitaceae), ocorrentes em Corumbá

(MS)-Brasil. Brazilian Archives of Biology and Technology 38: 815-827.

Angiosperm Phylogeny Group (2016). An update of the Angiosperm Phylogeny Group classification

for the orders and families of flowering plants; APG IV Botanical Journal of the Linnean

Society, 181 (1) 1-20.

Ayodele, D. (2000). Systematic Studies in the Family Polygonaceae Ph.D Thesis, Dept. of Botany,

University of Lagos, 241p.

Ayodele, D. (2005).The morphology and taxonomical significance of pollen in the West African

Polygonaceae. Thaisza Journal of Botany, 15: 143-153.

Ayodele, D. and Olowoudejo, J. D. (1997).Systematic importance of leaf and epidermal characters in

West African species of family Myrtaceae. Boletin da SociedadeBroteriana, 68; 35-75.

Ayodele, D. and Olowoudejo, J.D. (2006).The family Polygonaceae in West Africa. Taxonomic

significance of leaf epidermal characters. South African Journal of Botany, 72; 442-459.

Baig, N. A. (1965). Cytotaxonomy in the genus Cerastium Linn (cited by Isawumi, 1983 Ph.D Thesis,

University of Ife).

Barbosa, W.L., Santos, W.R.A., Pinto, L.N. and Tavares, I.C.C. (2002). Flavonóides de Cissus

verticillata e a atividade hipoglicemiante do chá de suas folhas. Review of Brazilian

Farmacognosy 12: 13-15.

62

Barthlott, W. C., Neinhuis D., Cutler F., Ditsch I.M., Theisenn I. J. and Wilhelmi H. (1998).

Classification and terminology of plant epicuticular waxes. Botanical Journal of the Linnean

Society 126: 237-260.

Beltrame, F.L., Sartoretto, J.L., Bazotte, R.B., Cuman, R.N.E. and Cortez, D.A.G. (2001). Estudo

fitoquímico e avaliação do potencial antidiabético de Cissus sicyoides L. (Vitaceae). Quim

Nova 24: 783-785.

Braga, T.V. (2008). Avaliação da atividade farmacológica de Cissusverticillata Nicolson& C. E. Jarvis

subsp. verticillata como antioxidante, antifúngico, hipoglicemiante e cicatrizante. Ouro

Preto, 175 p. Dissertação de Mestrado, Programa de Pós-graduação em Ciências

Farmacêuticas, Universidade Federal de Ouro Preto.

Brain, K. R. and Turner, T. D. (1975). The Practical Evaluation of Phytopharmaceuticals. Bristol Write

Scentechnica 16:105-106.

Brundet, M. C., Kendrick, B. and Peterson, C. A. (1991). Efficient lipid staining in plant material with

Sudan Red 7B or Fluoral Yellow 008 in polyethylene glycol-glycerol. Biotechnology of

Histochemistry 66: 111-116.

Christenhusz, M. J. and Byng, J. W. (2016). The number of known plants species in the world and its

annual increase. Phytotaxa Magnolia 261 (3): 201-217.

Clifford, H. T. (1965). The classification of the Poaceae (Grasses). Botanical Journal of Linnean

Society 62: 59-67.

Dehgan, B. (1980). Application of epidermal morphology to taxonomic delimitations in the genus.

Jatropha L. (Euphobiaceae). Botanical Journal of Linnean Society 80 (3): 257-278.

Fahn, A. (1970). Secretary tissues in plants. London Academic Press, London. 382pp.

Franceschi, V. R. and Horner, H. T. (1980). The botanical review. Botanic Garden 46: 361-427.

Franceschi, V.R. and Nakata, P.A. (2005). Calcium oxalate in plants formation and function. Annual

Review of Plant Biology 56: 41-71.

63

Friedrich, H. and Bailer, S. (1973). Investigation of the contents of Senna leaves. Plantamedica 23 (1):

74-87.

Garcia, X., Heredia-Cartas, L., Jímenez-Lorenzana, M.E. and Gijón, E. (1997). Vasoconstrictor effect

of Cissus sicioydes on guinea-pig aortic rings. Gennetic and Pharmacology 29: 457-462.

Gbile, Z. (1980). Vernacular Names of Nigeria Plants (Hausa). The Federal Department of Forestry,

Lagos State University, Lagos. 88pp.

Gill, L. S. (1935). Arceuthobium in the United States. Connecticut Academy of Arts and Science

Transactions 32: 11-245.

Hall, J. B., Morton, J. A. and Hooper, S. S. (1976). Application of principal components analysis with

constant character number in a study of the Bulbostylis frimbristylis (Cyperaceae) complex

in Nigeria. Botanical Journal of Linnean Society 73: 33-35.

Howard, R. A. (1979). Anatomy of the dicotyledons. Clarendon Press, Oxford. 88-96pp.

Hutchinson, J. and Dalziel, J. M. (1958). Flora of West tropical Africa Second Edition part 2. Crown

Agents for Oversea Government and Administration, Millbert London. 672-683 pp.

Ibrahim, H., Mdau, B. B., Ahmed, A. and Ilyas, M. (2011). Anthraquinones of Cissus populnea Guill

and Perr. (Amplidaceae). African Journal of Traditional, Complementary and Alternative

Medicines 8(2): 140-143.

Ibrahim, H., Rai P. P. and Bangudu, A. B. (1993). Pharmacognostic studies of the stem bark of Cissus

populnea Guill & Perr. Glimpses in Plant Research 1: 175-180.

Jensen, W. A. (1962). Botanical histochemistry: Principles and Practice. W. H. Freeman and Company,

San Francisco. 218pp.

Johansen, D. A. (1940). Plant microtechnique. McGraw Hill, New York. 388pp.

Kim, S. W., Ban, S. H., Chung, H. G., Choi, D. W., Choi, P. S., Yoo, O. J. and Liu, J. R. (2004).

Taxonomic discrimination of higher plants by pyrolysis mass spectrometry. Plant Cell

Report 22: 519-522.

64

Lizama, R.S., Martinez, M.M. and Pèrez, O.C. (2000). Contribución al estúdio de Cissus sicioydes L.

(Bejuco-ubí). Rev Cubana Farm 34: 120-124.

Lombardi, J. A. (2007). Systematics of Vitaceae in South Africa. Canadian Journal of Botany 85: 712-

721.

Mace, M.E. and Howell, C.R. (1974). Histological and histochemical uses of periodic acid. Stain

Technology 23: 99-108.

Metcalfe and Chalk L. (1950). Anatomy of Dicotyledons. Oxford, University Press, 724.

Metcalfe and Chalk L. (1979). Anatomy of Dicotyledons.Oxford, Clarendon Press.

Metcalfe, C.R. and Chalk, L. (1957). Anatomy of the dicotyledons. Clarendon Press, Oxford. 84pp.

Mueller, J. (1978). New observations of Pollen morphology and fossil distribution of the genus

Sonneratia (Sonneratiaceae). Review of Paleobotany and Palynology 26: 277-300.

O’Brien, T. P. and McCully, M. E. (1981). The study of structure, principles and selected methods.

Termarcarphi Pty Limited, Melbourn. 269pp

Paiva, E. A. S., Buono, F. A. and Lombardi, J. A. (2009). Food bodies in Cissus verticillata (Vitaceae)

ontogenesis, structure and functional aspects. Annals of Botany 103: 517-524.

Pepato, M.T., Baviera, A.M., Vendramine, R.C., Perez, M.P., Kettelhutt, I.C. and Brunetti, I.L. (2003).

Cissus sicyoides (princess vine) in the long-term treatment of streptozotocin-diabetic rats.

Biotechnology and Applied Biochemistry 37: 15-20.

Raju, V. S. and Rao, P. N. (1977). Variation in the structure and development of Foliar Stomata in the

Euphobiaceae. Botanical Journal of the Linnean Society 75: 67-97.

Shellard, E. J. (1979). African medicinal plants. Sofowara A. Ed, Ife University Press, Ile-Ife, Nigeria.

112pp.

65

Silva, L., Onik, G.H., Agripino, D.G., Moreno, P.R., Young, M.C., Mayworm, M.A. and Ladeira, A.M.

(2007). Biciclogermacremo, resveratrol e atividade antifúngica em extratos de folhas de

Cissus verticillata (L.) Nicolson & Jarvis (Vitaceae). Review of Brazilian Farmacognosy

17: 361-367.

Sneath, P. H. A. and Sokal, R. R. (1973). Numerical taxonomy. Freeman W. H. and Company. San

Francisco. 573pp.

Sofowora, A. (1993). Medicinal Plants and Traditional Medicinal in Africa. 2nd Ed. Sunshine House,

Ibadan, Nigeria: Spectrum Books Ltd, Nigeria. 134–156pp.

Sokal, R. R. and Sneath, P. H.A. (1963). Principles of Numerical Taxonomy. Freeman W. H. and

Company, San Francisco. 359pp.

Solereder, H. (1908). Systematic anatomy of the dicotyledons. Clarendon Press, Oxford. 890pp.

Solereder, H. (1908). Systematic anatomy of the dicotyledons. Translated by Boddle L. A. and Fritsch

F. E., revised by Scott D. H., Clarendon Press, Oxford, London. 1183pp.

Sonibare, A. M. (2003). Taxonomic Revision of the Genus Ficus Linn (Moraceae) in Nigeria. Ph.D

Thesis, Dept. of Botany and Microbiology, University of Ibadan. 292pp.

Stance, A. C. (1965). Cuticular studies as an aid to plant taxonomy. The Bulletin of the Museum

(Natural History).Botany series. Trustees of the British Museum (Natural History) 4:1.

Stance, D. (1984). Reading the Past: Current Approaches to Interpretation in Archaeology (3rd ed.).

Cambridge University Press, Cambridge. 103pp.

Stebbins, G. L. and Khush, G. S. (1961).Variation in the organization of the stomatal complex in the

leaf epidermis of monocotyledons and its bearing on their phylogeny. American Journal of

Botany 48: 51-59.

Stessy, T. F. (1970). The genus Acanthosermum (Compositae- Heliantheae – Meamposinnae)

taxonomic changes and generic affinities. Rodora 72: 106-109.

66

Tarkhtajan, A. (1973). The chemical approach of plant classification with special reference to the

higher taxa of Magnoniales In; Chemistry in Botanical Classification. 275-283pp.

Thoebald, W. L., Krahulik, J. L. and Rollins, R. C. (1979). Trichome description and classification. In

Metcalfe CR, Chalk L. Anatomy of the dicotyledons. Clarendon Press, Oxford. 40-53pp.

Trease, G.E. and Evans, W.C. (2002). Pharmacognosy. 15th Ed. Saunders Publishers, London. 2002.

pp.

Triton, A. F. (1986). Stasis, diversity and function in spores based on an electron microscope survey of

pteriodophyta. Pollen and spores; form and function. Blackmore S. and Ferguson I. K. (eds),

Academic Press, London. 233-249pp.

Varela, B. G., Fernandez, T., Ricco, R. A., Zolezzi, P. C, Hajos, S. E., Gurni, A. A., Alvarez, E. and

Wagner, M. L. (2004). Phorandndron Liga (Gjill ex H et al) Eichl (Viscaceae) used in folk

medicine anatomical, phytochemical and immunochemical studies. Journal of

Ethnopharmacology 94: 109-116.

Vianna, G.S. B, Medeiros, A. C. C, Lacerda, A. M. R., Leal, L. K., Vale, T. G. and Matos J. A (2004).

Hypyoglycemic and anti-lipemic effects of the aqueous extract from Cissus sicyoides.

Pharmacology 4: 1-14.

Watson, L. (1962). The taxonomic significance of stomatal distribution and morphology I.

Epacridaceae. New Phytology 61: 36-40.

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