PHYLOGENETIC STUDIES IN THREE AFRICAN NIGHTSHADES (SOLANUM SPECIES) USING MORPHOLOGICAL AND BIOCHEMICAL MARKERS.

BY

ABU, RICHARD ABANTELHE UR201400186

A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES, FACULTY OF PURE AND APPLIED SCIENCES, FEDERAL UNIVERSITY WUKARI, NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A BACHELOR OF SCIENCE DEGREE IN BIOLOGICAL SCIENCES.

FEDERAL UNIVERSITY WUKARI.

SEPTEMBER, 2018.

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DECLARATION

I, Abu, Richard A. with matriculation number UR201400186 do declare that the work in this project report titled “Phylogenetic Studies in Three African Nightshades

(Solanum Species) Using Morphological and Biochemical Markers” was carried out by me in the Department of Biological Sciences, Faculty of Pure and Applied Sciences,

Federal University Wukari. Information derived from literature has been duly acknowledged in the body of work and list of references provided. No part of this project report has been previously presented for another Degree or Diploma in this or any other institution.

ABU, Richard A. ………………………. ……………………

Name of student Signature Date

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CERTIFICATION

This is to certify that this project work titled, “Phylogenetic Studies in Three African Nightshades (Solanum Species) Using Morphological and Biochemical Markers,” was carried out by ABU, Richard Abantelhe (UR201400186) in the Department of Biological Sciences, Faculty of Pure and Applied Sciences under supervision.

Ekong, N. J ______Supervisor Signature/Date

Agere Hemen (Ph.D) ______Head of Department Signature/Date

______External Examiner Signature/Date

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DEDICATION

This work is dedicated to God almighty and to Mrs Regina Abu.

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ACKNOWLEDGEMENTS

My humble appreciation goes to God almighty for his unending grace upon me throughout my academic endeavours and the course of this project work.

My indepth gratitude goes to my Supervisor, Academic Adviser, Level Adviser and Mentor; Mr. Ekong, N. J. for his impact in my life and academics right from my first year in the University and for his rigorous scrutiny of this project work and also putting in his best and resources to ensure its completion.

My immense appreciation goes to the Department of Biological Sciences, Federal University Wukari. The Head of Department, Dr. Agere Hemen and all Departmental lecturers for the knowledge imparted in me throughout my stay in the University. You all are wonderful mentors and I would not have wished for any better family. Thank you all.

Special thanks to my mother; Mrs. Comfort Afebende and Mrs. Regina Abu for their motherly advice and constant intercession towards ensuring that I succeed. To all my family; Dr. Godwin Afebende, Jerry Abu, Judith Ochui, Helen Akobi, Hon and Mrs. Victor Afebende, Gregory Adigeb, Catherine Ubung, Joseph Akomaye, Mrs. Akobi and Victoria Afebende whose constant support and assistance has made my university days memorable.

My profound gratitude goes to my Mentor and Father; Hon and Mrs. Victor A. Agwu for his constant support and inspiration towards the initiation and completion of this program. May God bless you sir.

Not forgetting the efforts of my friends and colleagues; Benson Onesimus, Mrs. Mercy Rimamtsiwe, Vera Oli, my project colleague David Jauro, Tukura Joseph, Samaila Ezekiel, Tony Akwaji, Tawo Julius and all BIOSAN class of 2018. May God see you all through in your future endeavours.

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TABLE OF CONTENTS

TITLE PAGE ……………………………………………………………………………..i DECLARATION ...... ii CERTIFICATION ...... iii DEDICATION ...... iv ACKNOWLEDGEMENTS ...... v TABLE OF CONTENTS ...... vi LIST OF TABLES ...... ix LIST OF FIGURES...... x LIST OF PLATES ...... xi LIST OF ABBREVIATIONS ...... xii LIST OF APPENDICES...... xiii ABSTRACT ...... xiv CHAPTER ONE ...... 1 INTRODUCTION ...... 1 1.1 BACKGROUND TO THE STUDY ...... 1 1.2 STATEMENT OF THE PROBLEM ...... 4 1.4 JUSTIFICATION ...... 5 CHAPTER TWO ...... 7 LITERATURE REVIEW ...... 7 2.1 BRIEF INTRODUCTION OF AFRICAN NIGHTSHADES ...... 7 2.2 ORIGIN AND DISTRIBUTION ...... 9 2.2.1 Solanum scabrum ...... 9 2.2.2 Solanum nigrum ...... 9 2.2.3 Solanum villosum ...... 10 2.3 MORPHOLOGY OF AFRICAN NIGHTSHADES ...... 10 2.3.1 Solanum scabrum...... 10

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2.3.2 Solanum nigrum...... 11 2.3.3 Solanum villosum...... 12 2.4 PHYTOCHEMICAL SCREENING OF SOLANUM SPECIES...... 13 2.5 THIN LAYER CHROMATOGRAPHY IN SOLANUM SPECIES ...... 14 2.6 APPLICATION OF MORPHOMETRIC DATA IN PHYLOGENETIC AND TAXONOMIC ANALYSES ...... 15 2.7 APPLICATIONS OF PHYTOCHEMICALS IN MEDICINE, PHARMACEUTICALS AND DRUG DISCOVERY ...... 16 CHAPTER THREE ...... 18 MATERIALS AND METHODS ...... 18 3.1 PLANT COLLECTION AND IDENTIFICATION ...... 18 3.2 PLANT EXTRACTION ...... 19 3.4 MORPHOLOGICAL CHARACTERISATION ...... 20 3.5 PHYTOCHEMICAL SCREENING ...... 21 3.5.1 Detection of Alkaloids (Mayer‟s Test) ...... 21 3.5.2 Detection of carbohydrates (Fehling‟s Test) ...... 21 3.5.3 Determination of Cardiac Glycoside (Keller Kiliani Test) ...... 21 3.5.4 Determination of Saponins (Froth Test) ...... 22 3.5.5 Detection of phytosterols (Salkowski‟s Test) ...... 22 3.5.6 Detection of phenols (Ferric Chloride Test) ...... 22 3.5.7 Detection of Tannins (Ferric Chloride Test) ...... 22 3.5.8 Determination of Flavanoids ...... 22 3.5.9 Detection of proteins and aminoacids (Xanthoproteic Test) ...... 23 3.5.10 Coumarin Glycosides (Ferric chloride test) ...... 23 3.5.11 Determination of Steroid (Liebermann Burchard Reaction) ...... 23 3.5.12 Determination of Anthraquinones (Borntrager‟s Test) ...... 23 3.6 THIN LAYER CHROMATOGRAPHY ...... 23 3.7.1 Analysis of Morphological data...... 24 3.7.3 Analysis of phytochemical data ...... 25

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CHAPTER FOUR ...... 26 RESULTS ...... 26 4.1 QUALITATIVE MORPHOLOGICAL TRAITS FOR VEGETATIVE STRUCTURES ...... 26 Table 4.1: Qualitative Morphological traits for vegetative structures ...... 27 4.2 QUALITATIVE FOLIAR TRAITS...... 28 Table 4.2: Vegetative morphometric traits (mean ± SE) ...... 29 4.3 PHYTOCHEMICAL SCREENING – ABSOLUTE METHANOL ...... 30 Table 4.3: Phytochemical screening of Methanolic Extract...... 31 4.4: PHYTOCHEMICAL SCREENING USING 80% ETHANOL ...... 33 Table 4.4: Phytochemical screening using 80% Ethanol ...... 34 4.5 CHARACTERISTICS OF SPOTS FROM THIN LAYER CHROMATOGRAPHY 36 Table 4.5: Colour, RF and Abundance of Spots from Thin Layer Chromatography ... 37 4.6: . PAIRED AFFINITY, GROUP AFFINITY, ISOLATION VALUES AND UNIQUE SPOTS...... 39 Table 4.6: Values of Paired Affinity ...... 39 Table 4.7: Group Affinity, Isolation Value and Unique Spots ...... 39 CHAPTER FIVE ...... 40 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ...... 40 5.1 DISCUSSION ...... 40 5.2 CONCLUSION...... 44 5.3 RECOMMENDATIONS ...... 45 REFERENCES ...... 46

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LIST OF TABLES

Table 4.1: Qualitative morphological traits for vegetative structures. ------27

Table 4.2: Vegetative morphormetric traits. ------29

Table 4.3: Phytochemical screening using absolute methanolic extracts. ------31

Table 4.4: Phytochemical screening using 80% ethanolic extracts ------34

Table 4.5: Colour, Retention Factor and abundance of spots from TLC ------37

Table 4.6: Values of paired Affinity. ------39

Table 4.7: Value of Group affinity, Unique spots and Isolation Value. ------39

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LIST OF FIGURES.

Figure 4.1: Dendrogram of phylogenetic relationship using data from absolute methanol phytochemical analysis. ------32

Figure 4.2: Dendrogram of phylogenetic relationship using data from 80% ethanol phytochemical screening. ------35

Figure 4.3: Dendrogram of phylogenetic relationship using data from TLC. ------38

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LIST OF PLATES

Plate 3.1: Solanum nigrum …………………………………………………...18

Plate 3.2: Solanum nigrum …………………………………………………...18

Plate 3.3: Solanum scabrum.…………………………………..……………...19

Plate 3.4: Solanum villosum .………………………………………………...19

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LIST OF ABBREVIATIONS

SC = Solanum scabrum SN = Solanum nigrum SV = Solanum villosum TLC = Thin Layer Chromatography PA = Paired Affinity GA = Group Affinity IV = Isolation Value RF = Retention Factor

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LIST OF APPENDICES

APPENDIX 1: TLC plates and spots revealed ...... 52 APPENDIX 2: ANOVA...... 53

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ABSTRACT

The genus Solanum contains species which have confounding similarities in their facies. This study was done to delineate three African nightshades species from Nigeria using morphological and biochemical markers. The three species were collected, identified and profiled for their macromorphology, phytochemicals as well as chromatographic spots pattern. For the assessment of qualitative macromorphological traits, all taxa were similar except for stem colour, leaf apex, leaf margin and secondary fruit colour. The assessment of morphometric traits revealed significant differences (p < 0.05) for all traits assessed except for number of leaves. Solanum scabrum recorded the highest mean values for plant height (73.00cm), leaf length (15.88cm), leaf width (6.11cm), petiole length (10.68cm), peduncle length (2.88cm), pedicel length (0.99cm) and Leaf size (210.62cm2). Phytochemical analysis was carried out using extracts of ethanol and methanol and revealed array of phytochemicals like alkaloids, phenols, saponins, phenols, flavonoids, carbohydrates, cardiac glycosides, proteins and amino acids, steroids, coumarine glycosides, terpenoids, phytosterols, diterpenes and triterpenes further proving earlier reports that this group of plants are good sources of plants secondary metabolites. The Thin Layer Chromatographic spots profile of the leaf extract revealed phylogenetic affinities among the taxa with the highest Paired Affinity of 58.82% between S. nigrum and S. scabrum. S. nigrum also had the highest Group Affinity value of 208.02 showing it to be more advanced and closely related to the other two species. The Isolation Values and number of unique spots (20% and 3) respectively for each taxon support the placement of these species in the same group. Overall, both markers suggest that within the group, S. scabrum and S. nigrum are closer to each other than they are with S. villosum as supported by the dendrogram. This study affirms that morphological and biochemical markers are veritable tools with huge potentials in delimiting complex taxa such as Solanum.

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CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND TO THE STUDY

The evolution of species especially with reference to lines of descent and relationships among broad groups of organisms has led to diversity among closely related species.

Fundamental to phylogeny is the proposition and universal acceptance that plants and animals of different species descended from common ancestors and are closely related.

Taxonomy, the science of classification is based on phylogeny which entails the evolutionary relationships between and among taxa. In comparing features common to different species, taxonomists try to distinguish between homologies or similarities that arise in response to similar habits and living conditions using qualitative and quantitative morphological, anatomical and biochemical features. Although both qualitative and quantitative markers have been applied in evaluating genetic relationships among taxa, qualitative markers have proven to be more accurate (Olet, 2004; Ojiewo, Mwai,

Abukutsa-Onyango, Agong and Nono-Womdim, 2013).

Chromatographic spots are excellent markers and are much more important than chromosome numbers in taxonomy of plants (Bathia and Pandey, 2003). According to

Alston and Turner (1963), Biochemical methods utilizing secondary metabolites such as

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phenolic substances are being employed to study phylogenetic affinities and species relationship in many plant genera. Such biochemical features have also been employed in evaluating the taxonomy of Traditional African vegetables such as African nightshades

(Ojiewo et al., 2013).

Traditional African vegetables are important nutrient-rich food consumed locally in the sub-Saharan region where many are also utilized for their medicinal properties (Keding,

Wienberger, Swai and Mndiga, 2007). These vegetables are collected from the wild and/or cultivated for consumption or marketing, serving as important income generating opportunities for the typical subsistence farmers (Weinberger and Msuya, 2004). Adapted to the local environment, Traditional African vegetables often provide more sustainable output than exotic crops (Mal, 2007). Efforts are being put in place to increase the farming and marketing of these vegetables in an attempt to alleviate hunger and improve nutrition, and to increase farmer‟s income thereby improving the local and regional economy (Mal, 2007).

African nightshades are among the most popular and as such high priority African traditional vegetables. They represent a wide group of botanically and genetically related plants belonging to approximately 30 species in the genus Solanum of the family

Solanaceae, and are diversely referred to as garden huckleberries, vegetable nightshades,

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edible nightshades, garden nightshades, common nightshades, „S. nigrum complex‟, or

„S. nigrum‟ and related species (Yang and Ojiewo, 2013).

African nightshades are grown in both high and lowland areas in West and East Africa where much variability have been recorded. Reports indicate much diversity exist in the

African nightshades especially with regard to nutritional and pharmacognostic aspects

(Ojiewo et al., 2013). Despite these nutritional and pharmacognostic attributes, Solanum species are also well known to contain toxic alkaloids with glycosides of solasodine and solanidine being the most prominent (Milner, Brunton, Jones, O‟Brien, Collins and

Maguire, 2011). This biosafety concern is associated with the vegetable African nightshade species, as these compounds are known to be present in the fruits (Carle,

1981) and have limited the promotion of their cultivation and marketing.

Botanically, species known as African nightshades belong to the „Solanum nigrum’ complex which include; Solanum scabrum, Solanum villosum, Solanum nigrum, Solanum tanderemotum, Solanum grossidentatum, Solanum florulentum and Solanum americanum

(Ojiewo et al., 2013). Local African names for nightshades include mnavu (Swahili), enab el-dieb (Egypt) brede martin (Mauritius) musaka (Zimbawe) morelle noir (Morocco and Tunisia), Nigerian names include Kumbi (Hausa), Ewa (Igbo), Yoruba (Igbḁ) and

Kuna (Bette).

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1.2 STATEMENT OF THE PROBLEM

The taxonomy of African nightshades is complex due to extensive synonymy, frequent occurrence of spontaneous inter-specific hybrids, existence of polyploidy series, phenotypic plasticity, inconsistent use of many local names and discordant genetic variations (Edmond and Chweya, 1997). Taxonomic complexity, associated with African nightshades, has led to considerable confusion regarding the identification of popular nightshades vegetables. For instance, in most communities where these plants are cultivated and/or consumed they are known by a single name irrespective of their morphological differences.

Also, only about 6% of the available higher plants species have been screened biologically for secondary metabolites which are essential in drugs discovery (Atanasov et al., 2015). For this reason, higher plants remain essential in the search for new medicine particularly with the development of highly sensitive and versatile analytical techniques. Furthermore, the local food scene has exploded in communities around the world and there is constant need to explore and promote indigenous vegetables as alternative to synthesized foods and industrial medicine. Data on African nightshades delineation in this area as well as information on their secondary metabolites is limited, for this reason their exploration has become necessary.

In light of all these, the current study sought to investigate the phylogenetic relationship of the three Solanum species using morphological traits as well as phytochemical

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constituents and silica gel chromatography of their methanolic extracts for taxonomy and breeding purposes.

1.3 AIMS AND OBJECTIVES

The aim of this study is to assess the phylogenetic relationship among the three Solanum species using morphological traits, phytochemical constituents and chromatographic variations for taxonomic purposes.

The specific objectives of this research are to:

i. Compare the morphological traits among Solanum scabrum Mill., Solanum

nigrum L. and Solanum villosum Mill.

ii. Analyze and compare the phytochemical constituents present in the selected

species of the three African nightshades and

iii. Determine the taxonomic relationship between the selected species using the

variations in the Silica Gel chromatographic spots of their secondary metabolites.

1.4 JUSTIFICATION

Africa is richly endowed with plant genetic resources, with many well-adapted indigenous food crops that have long been grown on the continent (Mwai, Abukusta-

Oyango and Oyango, 2007). These crops not only play important roles in the food security of many resource poor farming families, but also have potential value as a genetic resource for the global community and provide alternative medicine. Plants

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contain thousands of secondary metabolites which interact in complex ways to produce therapeutic effects. Today, biomedicine still relies on plants for about 25% of its medicine (Chevallier et al., 2001).

Plants leaves are commonly used in taxonomic analyses and are particularly suitable to landmark based geometric morphometrics (Viscozi and Cardini, 2011). Morphological and biochemical markers have been applied to resolve issues of taxa delineation and delimitation (Ronoh et al., 2017). African nightshades are known to be one of the highly complex taxa with lots of morphological differences from every region of the world and data on these differences are limited in Africa and Nigeria.

It is hoped therefore, that findings from this research will provide information on the relevance of African Nightshades, delineate taxonomic relationships that exist among

African nightshades from this region, provide information on the phytochemicals and secondary metabolites contained in these plants. It is our sincere hope too that the findings herein would form a basis for research on African nightshades from this region and also educate cultivators as well as consumers on the differences that exist among the plants.

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CHAPTER TWO

LITERATURE REVIEW

2.1 BRIEF INTRODUCTION OF AFRICAN NIGHTSHADES

African nightshades are several species of plants belonging to the section Solanum of the family Solanaceae that are commonly consumed as leafy vegetables and herbs (Gaya et al., 2007). African nightshades are grown in both high and lowland areas in West and

East Africa, prominently in Nigeria and Cameroon where immense diversity has been recorded (Ojiewo et al., 2013). Although nightshade family is known to contain some dangerous and poisonous plants, many nutritional and health benefits have been reported for some members of this family Species known as African nightshade include Solanum scabrum, Solanum villosum, Solanum nigrum, and Solanum americanum (Drescher,

Pasquini and Shackuton, 2009). The genus Solanum is the largest and most diverse in the family Solanaceae, and species belonging to African nightshades are placed in the section Solanum which are mostly used as leafy vegetables and herbs in most parts of

West and East Africa (Ojiewo et al., 2013). Unlike other major vegetable crops in the genus Solanum - such as potato, tomato, and eggplant - African nightshades are not widely utilised outside West and East Africa, as they are considered poisonous weeds in many parts of the world. Upon the realization of their rich nutritional value, their low input requirements for growth and cultivation, and their potential in food security, they have become an important food crop in sub-Saharan Africa to generate secure income, primarily for resource poor, rural and peri-urban populations (Poczai and Hyvönen, 2011;

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Weinberger, Pasquini, Abukusta-Onyango 2011). Their cultivation has therefore been promoted by governmental and non-governmental institutions in the last few years in some countries with little of this in Nigeria. Although some cultivars were developed by public and private seed companies working in collaboration with research institutions, most farmers rely on low-quality seed lots from local markets, or self-harvested seeds, leading to low and varying yields (Mwai et al., 2007). Therefore, vegetable nightshades have attracted the interest of the research community, and are among the top priority

African indigenous vegetables identified for cultivar improvement and development to increase their market share (Edmond and Chewya, 1997; Mwai et al., 2007).

Taxonomical classifications of African Nightshades species are as outlined below:

Kingdom – Plantae Subkingdom – Tracheobionta Superdivision – Spermatophyta Division – Magnoliophyta Class – Magnoliopsida Subclass – Asteridae Order – Solanales Family – Solanaceae Genera – Solanum Species – Solanum scabrum; S. nigrum, S. villosum. (United States Department of Agriculture (USDA), 2018)

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2.2 ORIGIN AND DISTRIBUTION

2.2.1 Solanum scabrum

Solanum scabrum occurs as a cultivated vegetable from Liberia to Ethiopia, and south to

Mozambique and South Africa. It is very common in lowland as well as highland regions in West and East Africa. It is also reported from the Réunion Island and may well occur on other Indian Ocean islands, where its status needs to be confirmed. The wide range of diversity of Solanum scabrum found especially in Nigeria and Cameroon suggests that its origin is likely to be in the warm humid forest belt of West and Central Africa. Outside

Africa, Solanum scabrum can be found in Europe, Asia (India, China and the

Philippines), Australia, New Zealand, North America and the Caribbean (Muthomi and

Musyimi 2009).

2.2.2 Solanum nigrum

Solanum nigrum is one of the largest and most variable species of the genus Solanum also known as the Solanum nigrum complex is distributed from temperate to tropical regions and from sea level to high altitudes of 3500 metres. S. nigrum appears to be distributed throughout the world and on one or more members of the complex probably occur in every nation from Finland in the Northern hemisphere to New Zealand in the Southern hemisphere. Solanum nigrum itself is predominantly Eurasian species which does not naturally occur in South America (Jagatheeswari, Bharathi and Ali, 2013). In Africa,

Solanum nigrum is distributed from Botswana, Egypt, Ethiopia, Ghana, Kenya, Lesotho,

Mauritius, Morocco, Namibia, South Africa, Cameroon, Nigeria and a host of others.

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2.2.3 Solanum villosum

Solanum villosum is believed to have originated in Eurasia, and is sometimes considered to have a southern European origin. It is widespread, but absent in Central and South

America, and New Guinea. It has been introduced in North America and Australia. In

Africa it is recorded from Tunisia, Algeria and South Africa, and from many countries of tropical Africa, including Central Africa from Burundi, in East Africa from Sudan,

Ethiopia, Somalia, Kenya, Uganda and Tanzania, and in southern Africa from Zambia and Angola. In West Africa Solanum villosum has been recorded only from Nigeria

(Edmond and Chewya, 1997).

2.3 MORPHOLOGY OF AFRICAN NIGHTSHADES

2.3.1 Solanum scabrum.

Plants of this species grow up to the height of 2 m, are erect and have branching pattern which could be primary, secondary or even tertiary. Stem and node color range from green to purplish green to purple with medium to prominent wings that are prominently dented; glabrous to sparsely pubescent with short glandular, flattened or intermediate hairs where present (Mwai et al., 2007). Leaves are ovate with entire to sinuate margins and acute (occasionally obtuse) apices. Leaves colour range from light green to green, dark green and greenish purple, and vein colour ranges from light green to purple; glabrous to sparsely pubescent with short, glandular, flattened to intermediate hairs where present; blade length equal to blade width and 2 times, 3 times or more times longer than petiole length, petiole winged half-way (Mwai et al., 2007). Fruiting peduncle facing

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upward, are sparsely to densely pubescent and three times longer than fruiting pedicel.

Fruiting pedicel are mostly erect or reflexed while the inflorescence is cymose to umbellate with many flowers ranging between 8 and 20. Flowers are large about 0.16 mm, corolla white or light purple and fused below half-way, petal length 2 times petal width whereas anthers are yellow or brown with styles not exserted or exserted either clearly or just beyond the anthers, either curved or straight; mature sepals: rounded in shape, reflexed away from berries, sepal length equal to width. Mature berries are large about 0.16 mm, slightly flattened, dark purplish black, without stones, shiny and remain on plant when fully ripe. Chromosome counts indicate that this species is a hexaploid with chromosome number being 2n = 72 (Mwai et al., 2007).

2.3.2 Solanum nigrum.

Plants grow up to 1m high with widely spreading tertiary level branches. Stems are purple with purple nodes, small to medium sized wings either prominently or finely dented, glabrous to sparsely pubescent with short appressed hairs when present. Leaves are ovate or lanceolate with entire sinuate or sinuately dentate, finely lobed margins and acuminate to acute apices; grayish green to purple with greenish purple to purple veins; glabrous or sparsely pubescent with short, glandular appressed hairs when present.

Fruiting peduncles are horizontal or facing upward, sparsely to densely pubescent and about 3 times the pedicel length while fruiting pedicels are reflexed. Inflorescences are either cymose or a mixture of cymes and extended forked cymes on same plant, few

(below 7) too many (above 20) flowered. Flowers are 11 to 14 mm; white corolla: fused

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either at the base or below halfway; petal length 2 to 3 times petal width; yellow anthers; styles: either exserted just beyond or not exserted, either curved or straight; mature sepals: triangular ovate or broadly triangular, usually reflexed away from (occasionally adherent to) berries, sepal length equal to or 2 times longer than wide. Mature berries are

8 to 12 mm slightly flattened to rounded, to dark-purple to black, usually dull, remaining on plant when fully ripe. Stone cells: absent. Cytology is given by 2n = 72, hexaploid

(Mwai et al., 2007).

2.3.3 Solanum villosum.

Plants grow up to 1 m high, spreading or erect with short branches to 3rd level. The Stem is green with node color ranging from green to purplish green to purple; small to medium sized wings (1-2mm) either finely dented or not dented; glabrous to sparsely pubescent with short, appressed to intermediate hairs where present. The Leaves are lanceolate to ovate with entire, sinuate, sinuate-dentate or dentate margins that may have clearly defined lobes or none, leaf apex acuminate to acute, light green to green lamina with light green or green veins; blade length either half as long or equal to blade width, and ranges from half as long to 3 times longer than petiole length; petiole winged all the way or half- way. Fruiting peduncle is facing upward, sparsely to densely pubescent, 3 times or longer than fruiting pedicel. Fruiting pedicel reflexed. Inflorescence: cymose, occasionally cymes mixed with forked cymes on same plant, few (7) to many flowered (above 20).

The Flowers are small (<10 mm), corolla: white fused either halfway or below half-way, petal length to width ratio ranging between 1 and 3; anthers: yellow; style: not exserted or

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exserted just beyond the anthers, straight where exserted; mature sepals: lanceolate

(occasionally triangular-ovate), reflexed away from berries, sepal length 2 to 4 times sepal width. Mature berries are 7 to 9 mm, higher rounded; orange; dull; remaining on plant when fully ripe. Stone cells: absent. Cytologyis given as 2n = 48, tetraploid (Mwai et al., 2007).

2.4 PHYTOCHEMICAL SCREENING OF SOLANUM SPECIES.

Different authors have over the years carried out phytochemical screening on species of the genus Solanum. Shalom (2011) working on the nutritional and phytochemical screening on Solanum aethiopicum and Solanum macrocarporn fruits reported a significant presence of phytochemicals such as alkaloids, saponins, flavonoids, tannins, ascorbic acid in both species with terpenoids, in trace amount. Steroids were present in

Solanum aethiopicum fruits but absent in Solanum macrocarporn fruits. The results of

Venkatesh, Kalsivani and Vidya (2014) on phytochemical analysis of Solanum villousm using different reagents and extraction methods yielded alkaloids, flavonoids, saponins, phenols, tannins, carbohydrates, glycosides, proteins and amino acids in aqueous, ethanol, methanol, chloroform and ethyl acetate extracts.

Sango, Marugu and Zimudi (2016) presented findings on their study to assess the phytochemical profile of Solanum nigrum and inferred that alkaloids, steroids, cardiac glycosides, saponins, phenols, tannins, terpenoids and coumarin glycosides were present in the methanolic extract. The result of the phytochemical analysis of Solanum nigrum

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carried out by Rajathi, Modilal, Anandan, Sindhu and Logeshwari (2015) also revealed the presence of Alkaloids, Terpenoids, Flavonoids, Saponins, Steroids and Phenols in the ethanolic extract. Another result presented by Pronob and Islam (2012) on the phytochemical analysis of the aqueous extract of Solanum nigrum revealed the presence of phytochemicals such as Alkaloids, Saponins, Tannins, Flaronoids and Proteins.

The result of Djaafar and Ridlia (2014) on the phytochemical analysis of Solanum nigrum also revealed the presence of Alkaloids, Saponins, Tannins, Glycosides, Coumarin,

Terpenoids, and Flavonoids. Another phytochemical analysis on the methanolic extract of

„Solanum nigrum complex‟ (S. nigrum, S. scabrum and S. villosum) was carried out by

Lexa, Murigi and Matasyoh (2014) and they observed phytochemicals like Saponins,

Flavonoids, Steroids, Glycosides, Terpenoids, Phenols, Alkaloids, and concluded that S. nigrum complex is rich in many phytochemical compounds.

2.5 THIN LAYER CHROMATOGRAPHY IN SOLANUM SPECIES

Information on the evaluation of Solanum species using Thin Layer Chromatography is limited. De Britto, Gracelin and Kumar (2011) carried out study on the separation of antibacterial constituents from five Solanum species using TLC including Solanum nigrum. The result of the chromatogram seen under visible light revealed 7 bands with the highest spot having a Refractive (RF) value of 0.97 with orange colour and the least spot having an RF of 0.12 with light grey colour in the methanolic extract using a solvent system of n-Hexane: Ethyl acetate: Methanol in the ratio 6:2:2.

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The work of Rashid, Ghulam, Muzaffa and Adil (2017) on Thin Layer Chromatography profiling of the medicinal plant Solanum nigrum using different solvent systems revealed

12 spots in Toluene: Ethyl acetate: Acetic acid (36:12:5), 7 spots in n-Hexane: Ethyl acetate: Acetic acid (31:14:5), 5 spots in n-Hexane: Ethyl acetate: Formic acid (31:14:5),

2 spots in Chloroform: Methanol: Water (7:4:1) and no spots revealed in solvents system

Ethyl acetate: Acetic acid: Water (5:1:1) and Chloroform: Methanol: Acetic acid and concluded that the best solvent system for TLC evaluation of S. nigrum is Toluene:

Ethyle acetate: Acetic acid.

2.6 APPLICATION OF MORPHOMETRIC DATA IN PHYLOGENETIC AND

TAXONOMIC ANALYSES

Taxonomy relies greatly on morphology to discriminate between and among groups.

Computerized geometric morphometric methods for quantitative shape analysis measure, test and visualize differences in form in a highly effective, reproducible, accurate and statistically powerful way. Plant leaves are commonly used in taxonomic analyses and are particularly suitable to landmark based geometric morphometrics (Viscozi and

Cardini, 2011). Different studies have utilized Morphological data in resolving taxonomic complexities.

The delineation of the taxa Ophioglossum, which had previously been complicated using conventional methods, has been successfully resolved with the use of morphometric data by Magrini and Scoppola (2010). Cortinhas, Erban and Carperta (2015) also used

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morphological data, reproductive and karyological data to resolve taxonomic complexities in the halophyte (Limonium vulgare) and other related species and concluded that discriminant analysis using morphometric variables reliably assigned individuals in natural populations to their respective groups. The relationship among five species of Acalypha (Euphorbiaceae) has also been described using phytochemical and quantitative morphological parameters by Soladoye, Sonibare and Rosanwo (2008), the result of the Principal Component Analysis (PCA) and Cluster Analysis showed that two of the nine quantitative traits account for the differences among the taxa.

2.7 APPLICATIONS OF PHYTOCHEMICALS IN MEDICINE,

PHARMACEUTICALS AND DRUG DISCOVERY

Phytochemical screening of vegetables is essential because it often has been used for the maintenance of health and prevention of chronic discoveries (Aggarwal and Shishovia,

2006). Plants contain a large range of phytochemicals belonging to different chemical families and possessing distinct biological activities (Rizvi, Gamal and Salem, 2009). The attraction of pharmaceutical companies and researchers for development of bioefficacies and the provision of knowledge for the advancement of phytomedicine has been stepped up in recent times (Irshad, Ahmad, Goel and Rizvi, 2010).

Various natural phytochemicals such as polyphenols, flavonoids, alkaloids, carbohydrates and essential oils have received increased attention due to the considerable biological benefits (Georgieni, 2014; Xiao, 2015). Evidence based on epidemiological data have

16

showed that natural bioactive compounds play an important role in the prevention and management of Modern diseases such as cardiovascular diseases, diabetes, cancer and

Alzheimer‟s diseases (Andre-Marobela et al., 2013; Xiao and Shao, 2013; Xiao, 2015).

These phytochemicals, although developed by plants for protection, have become responsible for the medicinal properties possessed by the plants (Kessler, Ubead and

Jung, 2003). Tannins are active against diarrhoea and respiratory disorders while flavonoids have anti-cancer, anti-inflammatory, anti-viral and anti-oxidant potentials

(Gertrude 2006; Matasyoh, Murigi and Matasyoh, 2014; Balch and Balch 2000).

Saponins are anti-cancer agents whose mode of action is the interference with the cholesterol-rich membranes of cancer cells (Assiak, Onigemo, Oluyemi and Tijani, 2001;

Dong, Zhoa, Leung and Tsim, 2005). Saponins are also anti-viral, antifungal and anti- inflammatory in their activity (Al-Bayati and Al-Mola, 2008). Steroids have found

Pharmaceutical applications where they are used in the manufacture of sex hormones that bring about hormonal balance in lactating and expectant mothers as well as stimulate libido in men (Victor, Siebert and Wyyk, 2008).

Cardiac glycosides are used in the treatment and prevention of Cardiac disorders in

(Matasyoh et al., 2014). Alkaloids have antimicrobial properties (Oguanwenno, Idowu,

Innocent, Esan and Oyelana, 2007), anticancer, anti-inflammatory effected and are important immune boosters (Jeffery and Harbone 2008). Proteins have bioactive effect against certain ailments like Kwashiorkor and enhance growth and development of individuals.

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CHAPTER THREE

MATERIALS AND METHODS

3.1 PLANT COLLECTION AND IDENTIFICATION

The plant materials; Solanum scabrum and Solanum nigrum were collected from Kurmi

Local Government Area of Taraba State between latitude 7050‟N and 9046‟E while

Solanum villosum was collected from Obudu Local Government Area of Cross River

State between latitude 6033‟N and 904‟E. The plants samples were identified by the researchers and authenticated by Esimekhuai, D. P. O of the University of Ibadan herbarium and assigned herbarium specimen numbers UIH-22695, UIH-22696 and UIH-

22697 for Solanum scabrum, Solanum nigrum and Solanum villosum respectively.

Plate I: Solanum nigrum Plate II: Solanum nigrum leaves with fruits

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Plate III: Solanum scabrum

Plate IV: Solanum villosum

3.2 PLANT EXTRACTION

For phytochemical screening, samples were collected, washed in running tap water and air-dried at room temperature for two weeks. Dried samples were pulverized in aseptic conditions to obtain a fine dry powder. The samples were stored in air tight polythene bags at room temperature and later extracted separately with absolute Methanol and 80%

19

Ethanol. The extracts were concentrated using the water bath maintained at a temperature of 600C.

For Thin Layer Chromatography (TLC), young leaves from the apical part of the plants of same age were collected, washed and air-dried at room temperature for one week as recommended by Bathia and Pandey (2003). Dried sample were pulverized in crucibles and extracts were obtained with absolute methanol for TLC analysis.

3.3 STUDY AREA

The study was carried out in Federal University Wukari, Taraba State between latitude 7050‟37”N and longitude 90 46‟30‟‟E. The morphological study was carried out in the Biological garden of the Department of Biological Sciences while the phytochemical analysis and TLC was carried out in the Biology and Chemistry Laboratories respectively.

3.4 MORPHOLOGICAL CHARACTERISATION

A descriptor list was designed initially for the morphological characteristics and scoring was done based on that descriptor list. The descriptors include leaf length, leaf width, leaf size, petiole length, flower peduncle length, flower pedicel length, number of leaves/per plant, number of flowers per plant for quantitative morphological traits while for qualitative morphological traits, growth habit, branching pattern, stem colour, leaf shape, leaf apex, leaf margin, leaf arrangement, leaf colour, foliage cover, flower colour and arrangement of petals, primary fruit colour, and secondary fruit colour was determined.

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3.5 PHYTOCHEMICAL SCREENING

Concentrated methanolic extracts were redissolved using distilled water to the required concentrations for phytochemical analysis. The following standard tests were carried out following the procedures of Sofowora (1982) and Harborne (2005) with slight modifications.

3.5.1 Detection of Alkaloids (Mayer’s Test)

Two millitres each of the filtrates were treated with 1ml Mayer‟s reagent (Potassium

Mercuric Iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids.

3.5.2 Detection of carbohydrates (Fehling’s Test)

Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were used to test for the presence of carbohydrates. Filtrates were hydrolysed with dilute hydrochloric acid, neutralized with sodium hydroxide and heated with Fehling‟s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars.

3.5.3 Determination of Cardiac Glycoside (Keller Kiliani Test)

Three millilitres of the extracts was added with 1 ml of acetic acid followed by the addition of 3 ml of 10% ferric chloride and a few drops of concentrated sulphuric acid on the sides of the test tubes. A brownish ring and green blue precipitate at the bottom of the test tube confirmed the presence of cardiac glycosides.

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3.5.4 Determination of Saponins (Froth Test)

To determine Saponins, 2 ml distilled water was added to 3 ml of the extracts in a test tube and the solution was vigorously shaken before some drops of olive oil were added.

The formation of stable foam was taken as an indication for the presence of Saponins.

3.5.5 Detection of phytosterols (Salkowski’s Test)

Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden yellow colour indicates the presence of triterpenes.

3.5.6 Detection of phenols (Ferric Chloride Test)

Extracts were treated with 3 to 4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols.

3.5.7 Detection of Tannins (Ferric Chloride Test)

About 0.5 g of the dried powdered samples was boiled in 20 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black coloration.

3.5.8 Determination of Flavanoids

A millilitre of 10% ammonia and 1 ml of concentrated sulphuric acid was added to 3 ml of extract. Disappearance of yellow color indicated the presence of flavonoids.

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3.5.9 Detection of proteins and aminoacids (Xanthoproteic Test)

The extracts were treated with few drops of conc. Nitric acid. Formation of yellow colour indicates the presence of proteins.

3.5.10 Coumarin Glycosides (Ferric chloride test)

To 3 ml of the concentrated alcoholic extract of a drug few drops of alcoholic Ferric chloride solution was added. Formation of a deep green colour, which turned yellow on addition of conc. Nitric acid, indicated the presence of coumarin glycosides.

3.5.11 Determination of Steroid (Liebermann Burchard Reaction)

3 ml of the extract was added with 1 ml of chloroform and a few drops of concentrated

Sulphuric acid along the sides of the test tubes. Formation of a reddish brown precipitate at the bottom of the test tubes indicated the presence of steroids.

3.5.12 Determination of Anthraquinones (Borntrager’s Test)

To 3 ml extract dilute sulphuric acid was added, boiled and filtered. To the cold filtrate equal volume of chloroform was added. The organic layer was separated and ammonia was added. The presence of Anthraquinones was observed with the appearance of pink or red colour.

3.6 THIN LAYER CHROMATOGRAPHY

A chromatographic plate was prepared with silica gel 60 F254 coated on a glass sheet of

1 mm thick. Methanolic extracts of the plant samples were applied at the starting point of the plate. The developed plate was then dipped in the solvent containing a mixture of n-

23

Hexane: Ethyl acetate: Acetic acid in a ratio 6:2:2 and allowed to develop chromatogram.

The chromatogram was first observed under visible light and then treated with ammonia vapour and finally with iodine vapour in silica gel as recommended by Trease and Evans

(1989) to distinguish between the spots. Distinct colours appeared under visible light and ammonia vapour in case of some phenolic spots while the spots of other phenolic compounds became obvious after treatment with iodine vapour in silica gel. The visible spots were traced, recorded and scored. The relative distance travelled by the spots also known as the Retention Factor (RF) was used as a basis for comparison of various phenolic compounds obtained. On the basis of colour and position, spots assumed to be identical in two or more species were assigned the same number. Data from the chromatography were subjected to numerical taxonomic treatment such as Paired

Affinity, Group Affinity and Isolation Value as an aid to the establishment of relationships between the phenolics in the selected Solanum species.

3.7 DATA ANALYSIS

3.7.1 Analysis of Morphological data

The One-way Analysis of Variance (ANOVA) was carried out to compare means for significant differences at p = 0.05 probability level while the Duncan‟s mean Post-hoc test was employed to separate significant means using the Statistical Package for Social Science (SPSS) software version 20.

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3.7.3 Analysis of phytochemical data

The method described by Ellison, Aston and Turner (1962) was adopted to make the suitable comparisons in the form of qualitative relationships. Selected species were compared on the ground of their biochemical affinities. Values of paired affinity (PA), group affinity (GA) and Isolation value (IV) was calculated using the following formulas:

(1)

GA = Total PA +100 (2)

(3)

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CHAPTER FOUR

RESULTS

4.1 QUALITATIVE MORPHOLOGICAL TRAITS FOR VEGETATIVE

STRUCTURES

Table 4.1 shows data for qualitative morphological traits for the vegetative structures of the Solanum species used in this study. Solanum scabrum has bushy growth habit with diffused branches and light green stems. The leaf shape is ovate with pointed leaf apex and entire leaf margin while leaf colour is light green with relatively very good foliage cover. Petals are white and touching while sepals are predominantly yellow. Unripe fruits are green but purple when ripe or fully developed.

Solanum nigrum has bushy growth habit with diffused branches and purple stem. Leaf shape is ovate; leaf apex is blunt with entire leaf margin and dark green in colour. Foliage cover is good; petals are white and touching while sepals are yellow. Fruit is green at developmental stage and becomes dark green when ripe and fully developed.

Solanum villosum also has bushy growth habit with diffused branching pattern and green stem. Leaf is seriated with pointed apex and lobed margin. Leaf colour is dark green with very good foliage cover. Petals are white and touching with while sepals are yellow.

Unripe fruits are green but orange when ripe or fully developed.

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Table 4.1: Qualitative Morphological traits for vegetative structures SN TRAIT SC SN SV 1 Growth habit Bushy Bushy Bushy 2 Branching pattern Diffused Diffused Diffused 3 Stem colour Light green Purple Green 4 Leaf shape Ovate Ovate Ovate 5 Leaf apex Pointed Blunt Pointed 6 Leaf Arrangement Alternate Alternate Alternate 7 Leaf margin Entire Entire Lobed 8 Leaf colour Green Dark green Dark green 9 Foliage cover Very good Very Good Very good 10 Petal colour White White White 11 Sepals colour Yellow Yellow Yellow 12 Arrangement of petals Touching Touching Touching 13 Primary fruit colour Green Green Green 14 Secondary fruit colour Purple Purple Orange Foliage cover: 0-25 leaves-poor; 26-50 fair; 51-75-good; 75 and above- very good. Growth habit: primary branches-; secondary branches-; tertiary branches- Bushy.

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4.2 QUALITATIVE FOLIAR TRAITS.

Table two shows measurements for qualitative foliar traits of the selected species used in the study. For the number of leaves on each species, there was no significant difference at p ≥ 0.05. For height of the plants, there was no significant difference between S. scabrum and S. villosum but S. nigrum differs significantly (p ≤ 0.05) from the other two taxa.

There was statistical difference for leaf length between the three taxa when compared with S. scabrum (15.88 ± 0.58) higher than S. villosum (13.43 ± 0.40) and S. nigrum (8.03

± 0.15) respectively (p ≤ 0.05). For leaf width, at p ≤ 0.05 there was also was significant difference between the three taxa with S. scabrum (6.11±0.31) having the highest followed by S. nigrum (4.17±0.13) and then S. villosum (2.94±0.18).

For petiole length, there was no significant difference between S. scabrum (10.68±0.41) and S. villosum (10.21±0.28) but S. nigrum (5.41±0.07) differed significantly from the other two taxa at p ≤ 0.05. The result for leaf size shows that there was no significant difference between S. scabrum and S. villosum but S. nigrum shows statistical difference from the other two at p ≤ 0.05. They were statistical differences (p ≤ 0.05) for flower peduncle length with S. scabrum (2.88) having the highest followed by S. nigrum (1.80) and S. villosum (0.27). For flower pedicel length, at p ≥0.05 there was significant difference between S. scabrum (0.99), S. villosum (0.74) and S. nigrum (0.48).

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Table 4.2: Vegetative morphometric traits (mean ± SE) Taxa NL PH (cm) LL (cm) LW (cm) LP (cm) LS (cm2) PL (cm) PEL (cm)

SC 96.80 ± 3.00a 73.00±5.59a 15.88±0.58a 6.11±0.31a 10.68±0.41a 210.62±7.59a 2.8760±0.08a 0.9920±0.37a

SN 98.40 ± 3.78a 31.20±0.58b 8.03±0.15c 4.17±0.13b 5.41±0.07b 74.18±9.65b 1.7960±0.08b 0.4840±0.12c

SV 100.60±4.53a 34.60±1.29b 13.43±0.40b 2.94±0.18c 10.21±0.28a 165.35±3.69a 0.02720±0.05c 0.7453±0.28b

NL=No of leaves per plant; PH= Plant height; LL = Leaf length; LW= Leaf width; LP= petiole length; LS= leaf size; PL= flower peduncle length; PEL= flower pedicel length. Similar alphabets on the same column indicate no significant difference.

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4.3 PHYTOCHEMICAL SCREENING – ABSOLUTE METHANOL

The result presented in Table 4.3 shows the phytochemicals and their abundance for the three species under study. Alkaloids are present (+) in S. nigrum, moderate (++) in in S. scabrum and high in S. villosum (+++); Flavonoids are present in S. villosum, moderate in

S. scabrum and high in S. nigrum. Saponins are high in S. nigrum but moderate in S. scabrum and S. villosum; Phenols are present in S. scabrum, moderate in S. nigrum and high in S. villosum; Tannins are present in S. scabrum, moderate in S. nigrum and high in

S. villosum; Proteins are moderate in S. villosum and high in S. scabrum and S. nigrum.

Phytosterols are present in S. scabrum and S. villosum and moderate in S. nigrum;

Coumarins are present in S. scabrum and S. nigrum but high in S. villosum; Cardiac glycosides are present in S. scabrum, moderate in S. nigrum and high in S. villosum.

Carbohydrates are high in all taxa while Anthraquinones are absent in all taxa.

Figure 4.1 shows the phylogenetic relationship among the three Solanum species studied using a Dendrogram drawn with UPGMA technique based on Euclidean distances. There were two major branches with Solanum scabrum and Solanum nigrum clustering at the

Euclidean distance of 3.4.

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Table 4.3: Phytochemical screening of Methanolic Extract. S/N PHYTOCHEMICALS SC SN SV

1 Alkaloids ++ + +++

2 Flavonoids ++ +++ +

3 Saponins ++ +++ ++

4 Phenols + ++ +++

5 Tannins + ++ +++

6 Proteins +++ +++ ++

7 Steroids + + ++

8 Anthraquinones - - -

9 Phytosterols + ++ +

10 Coumarins + + +++

11 Cardiac Glycosides + ++ +++

12 Carbohydrate +++ +++ +++

+ present; ++ moderately present; +++ highly present; - absent.

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Figure 4.1: Dendrogram of phylogenetic relationship among the three Solanum species drawn with UPGMA technique based on Euclidean distances using phytochemicals revealed from Methanolic extracts.

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4.4: PHYTOCHEMICAL SCREENING USING 80% ETHANOL

Table 4.3 shows phytochemicals and their abundance for the three species using 80% ethanolic extracts. Alkaloids are moderately present in S. scabrum and S. nigrum but high in S. villosum; Flavonoids, Saponins, Coumarine, Amino acids and Terpenoids were recorded very high (+++) in all three species. Tannins are high in S. scabrum but moderate in S. nigrum and S. villosum while Diterpenes were recorded high in S. scabrum and S. villosum but moderately high in S. nigrum. Steroids were recorded present (+) in all species, Triterpenes moderately high (++) in S. scabrum but present (+) in S. nigrum and S. villosum while Carbohydrates were moderate (++) in S. villosum but absent in S. scabrum and S. nigrum. Proteins, Anthraquinones, Glycosides, Anthocyanins and Smedins were recorded ansent in all species.

Figure 4.2 shows the phylogenetic relationship among the three Solanum species studied using a Dendrogram drawn with UPGMA technique based on Euclidean distances. There were two major branches with Solanum scabrum and Solanum nigrum clustering at the

Euclidean distance of 1.7.

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Table 4.4: Phytochemical screening using 80% Ethanol SN TEST SC SN SV 1 Alkaloids ++ ++ +++ 2 Flavonoids +++ +++ +++ 3 Saponins +++ +++ +++ 4 Phenols +++ +++ +++ 5 Tannins +++ ++ ++ 6 Proteins - - - 7 Steroids + + + 8 Anthraquinones - - - 9 Phytosterols + + + 10 Coumarine +++ +++ +++ 11 Glycosides - - - 12 Carbohydrate - - ++ 13 Triterpenes ++ + + 14 Amino Acids +++ +++ ++ 15 Diterpenes +++ ++ +++ 16 Terpenoids +++ +++ +++ 17 Anthocyanins - - - 18 Smedins - - - + present; ++ moderately present; +++ highly present; - absent

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Figure 4.2: Dendrogram of phylogenetic relationship among the three Solanum species drawn with UPGMA technique based on Euclidean distances using data from phytochemicals revealed from 80% ethanolic extract.

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4.5 CHARACTERISTICS OF SPOTS FROM THIN LAYER

CHROMATOGRAPHY

Table 4.5 shows the number of chromatographic spots, colour and RF for all three taxa. In

Solanum scabrum (SC), eight chromatographic spots were revealed with highest RF of

0.98 and the least RF of 0.16. The colours observed for the spots were yellow and green. In

Solanum nigrum (SN), nine chromatographic spots were revealed with the highest RF of

0.98 and the least RF of 0.11. The colours observed were yellow and green for all spots. In

Solanum villosum (SV), seven chromatographic spots were revealed with highest RF of

0.91 and the least RF of 0.35. The colours observed were yellow and green.

Figure 4.3 shows the phylogenetic relationship among the three Solanum species studied using a dendrogram drawn with UPGMA technique based on Euclidean distances. There were two major branches with Solanum scabrum and Solanum nigrum clustering at the

Euclidean distance of 4.7.

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Table 4.5: Colour, RF and Abundance of Spots from Thin Layer Chromatography SPOT COLOUR RF SC SN SV

1 Yellow 0.11 - ++ -

2 Yellow 0.16 ++ - -

3 Green 0.35 - - ++

4 Green 0.41 - - ++

5 Yellow 0.42 - ++ -

6 Green 0.49 - ++ -

7 Green 0.54 ++ - -

8 Yellow 0.62 ++ - -

9 Yellow 0.70 +++ +++ +++

10 Green 0.76 +++ +++ +++

11 Green 0.78 _ ++ ++

12 Yellow 0.85 ++ ++ ++

13 Yellow 0.91 - - ++

14 Green 0.93 ++ ++ -

15 Yellow 0.98 ++ ++ -

+ present; ++ moderately present; +++ highly present; - absent

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Figure 4.3: Dendrogram of phylogenetic relationship among the three Solanum species drawn with UPGMA technique based on Euclidean distances using chromatographic spots of leaves phenolics

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4.6: PAIRED AFFINITY, GROUP AFFINITY, ISOLATION VALUES AND UNIQUE SPOTS.

Tables 4.6 and 4.7 shows the values of Paired Affinity (PA), Group Affinity (GA),

Isolation Value (IV) and unique spots for each taxon. The Paired Affinity between S. scabrum and S. nigrum was 58.82%. S. nigrum and S. villosum has PA of 50% while between S. scabrum and S. villosum the PA was 40%. The PA values were supported by the GA which is 198.00 for S. scabrum, 208.02 for S. nigrum and 180.00 for S. villosum.

The three species all had 3 unique spots and 20% Isolation Values.

Table 4.6: Values of Paired Affinity

Taxa SC SN SV SC - - -

SN 58.82% - -

SV 40.00% 50.00% -

Table 4.7: Group Affinity, Isolation Value and Unique Spots Taxa Group Affinity Unique Spots Isolation Value

SC 198.00 3 20%

SN 208.02 3 20%

SV 190.00 3 20%

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CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 DISCUSSION

Taxonomy relies on morphology to discriminate between species of the same group

(Viscozi and Cardini, 2011). Taxonomic resolution is not only effective for biodiversity conservation and opportunities that might occur from these genetic resources but also gives distinction to individual species or groups. In the present study, the taxonomy of three

African nightshades from Nigeria was assessed using morphological data. The results from the assessment of morphological traits in the three species were comparable with those obtained by Mwai et al. (2007) for the same traits. For the qualitative foliar morphology of the taxa in this study, all traits were similar to the report of Mwai et al. (2007). However, the S. villosum in this study lacked erect glandular hair which agreed with the report of

Mwai et al., (2007) who reported two species of S. villosum, one with erect glandular hair and the other, a subspecies without this trait. This corroborated the description of a non- hairy S. villosum subspecies miniatum (Bernh. ex Wild) by Edmonds (1997). Although the results of the quantitative morphological traits varied from those earlier reported by Mwai et al. (2007) for the same traits, they were still comparable since the ranges overlapped in most calses. The slight variations could have resulted from differences in environmental factors like soil fertility, moisture, planting season and climate between locations. This position is also supported by Ojiewo et al. (2013) who stated that the morphology of

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African nightshades varies from region to region and among species due to environmental factors.

The phytochemical screening of methanolic and ethanolic extracts of S. scabrum, S. nigrum and S. villosum was also evaluated in this study. For the methanolic extract, the presence and abundance of alkaloids, flavonoids, saponins, phenols, tannins, cardiac glycosides, proteins, coumarins, steroids, phytosterols, carbohydrates, triterpenes, amino acids, diterpenes and terpenoids in the two extracts as recorded corroborated earlier reports. Reports of Venkatesh et al. (2014), Modilal et al. (2015), Sango et al. (2016) and

Lexa et al. (2014) all agree that the three Solanum species are good sources of plants secondary metabolites. These phytochemicals, although developed by plants for protection, have been reported to hold vast potentials for drug development as well as other industrial applications (Kessler et al., 2003). Tannins are known active agents against diarrhoea and respiratory disorders while flavonoids are vital for their anti-cancer, anti-inflammatory, anti-viral and anti-oxidant potentials (Balch and Balch, 2000, Gertrude, 2006 and

Matasyoh et al., 2014). Saponins are known for their anti-cancer properties whose mode of action in the interference with the cholesterol-rich membranes of cancer cells is well established (Assiak et al., 2001; Dong et al., 2005). Saponins are also anti-viral, antifungal and anti-inflammatory in their activity (Al-Bayati and Al-Mola, 2008). Steroids have found pharmaceutical applications where they are used in the manufacture of sex hormones that bring about hormonal balance in lactating and expectant mothers as well as stimulate libido in men (Victor et al., 2008). Cardiac glycosides are used in the treatment

41

and prevention of Cardiac disorders (Matasyoh et al., 2014). Alkaloids have antimicrobial properties (Oguanwenno et al., 2007), anticancer, anti-inflammatory effected and are important immune boosters (Jeffery and Harbone 2008). Proteins have bioactive effect against certain ailments like Kwashiorkor and enhance growth and development of individuals.

Thin Layer Chromatography carried out to determine phylogeny of the species under study showed nine spots for Solanum nigrum, eight spots for Solanum scabrum and seven spots for Solanum villosum. However, for S. nigrum, the numbers of spots were different from the seven spots reported by De-Britto et al. (2011) as well as Rashid et al. (2017) for the same solvents system but different ratios of constituents. The differences in spot between the studies could have stemmed from the fact that the number of phenolic spot revealed during TLC is dependent on the solvent system used as well as the concentration of constituent solvents as posited by De-Britto et al. (2011). Chromatographic spots are considered excellent markers and are much more important than chromosome number in plants taxonomy (Grants, 1968; Bathia and Pandey, 2003). In the present study, the three common spots in all three taxa appeared to be the characteristic spots for these species.

Spots 14 and 15 were common among S. scabrum and S. nigrum which shows closer relationship among the two species when comparing the three (Table 4.5). Paired Affinity of 50% and above was considered as an indication of close relationship between two species. In this regard, the PA of 58.82% between S. scabrum and S. nigrum shows close relationship, the PA of 50.00% between S. villosum and S. nigrum also shows close

42

relationship between the two while the PA of 40.00 between S. scabrum and S. villosum shows distant relationship. The data for Paired Affinity was supported by the Group

Affinity on the basis of which it could be said that S. scabrum is more closely related to S. nigrum than it is to S. villosum while S. villosum is more closely related to S. nigrum than it is to S. scabrum. This result also further affirms the assertion of Ojiewo et al. (2013) that

S. scabrum, S. nigrum and S. villosum are rightly placed in the same family and that S. nigrum is the oldest and ancestor of the nightshade group.

The chromatographic spots and data from phytochemical analysis was further subjected to phylogenetic studies using dendrogram drawn with UPGMA technique based on Euclidean distances. The results showed two major branches with S. scabrum and S. nigrum clustering at different points which further supports their closer relationship with each other than with S. villosum. The observed differences may be due to the action of evolutionary forces which act to mould variations within and between populations. These differences could be mutation, selection, gene flow or genetic drift.

Mutation is the ultimate source of all genetic variability in a population so it is probable that the major cause of these differences would have arose through mutation. The reason for these differences could also be the case of divergence when members of the same species become adapted differently to their environment and accumulate variations over time. Gene flow could have given rise to interspecific hybridization which has been reported in Solanum species. It is probable that selection could have led to these variations

43

since S. scabrum and S. nigrum grow or are cultivated together and thus selected for in most areas where they are found over S. villosum due to preference and the fact that their vegetative structures are very similar. This would seem to limit gene transfer between them and S. villosum due to the impacts of geographical isolation and probably drift.

5.2 CONCLUSION

African nightshades are one of the most widely consumed indigenous vegetables in Sub-

Sahara Africa with high potentials for food and medicine. The taxonomy of these nightshades group of the genus Solanum is not well known due to taxonomic complexities.

This study was designed to delineate and delimit three African nightshades from Nigeria and as well explore their biochemical capabilities. The results from the study have shown that African Nightshades even though variable in some characteristics, show similarities in many other characters as mostly seen in their leaves, fruits and flowers. Solanum nigrum and S. scabrum share the most of these characteristics. S. scabrum grows taller and has wider leaves than S. nigrum but their flowers and fruit show no differences at all conditions. S. villosum differs slightly from the other two species in their leaves and secondary fruit colours which are lobed and orange respectively.

The result also shows that African nightshades are good sources of plants secondary metabolites. These phytochemicals, though developed by plants to help in their adaptation, overcome predators and help in their evolutionary advancements, can be utilised by man for food, medicine and income. All the phytochemicals identified in this study have found applications in pharmaceuticals or phytomedicine for the treatment of several diseases.

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More so, the result from their phylogenetic relationship assessed using chromatographic spots and phytochemicals have supported the claim that S. scabrum is more closely related to S. nigrum than S. villosum and further affirms the claim that S. nigrum is the common ancestor of the African nightshades group. The result also shows that the three species are a group of related species rightly placed in the same group and family.

5.3 RECOMMENDATIONS

The information from the morphological analysis in this research should be useful in planning crosses for hybrid and line development among Solanum species. This is because genetic variability is considered the reservoir that plants breeders fall upon in their continuous strive to develop improved varieties and hybrids. Findings also show that

African Nightshades are rich sources of plants secondary metabolites and for this reason, the researcher recommend their utilization for food, alternative medicine as well as improving their cultivation since these plants are well adapted to the local environment.

The complexities associated with African nightshades cannot be resolved by a single research, for this reason, it is recommended that further studies should involving other species of Solanum from Nigeria should be carried out to further delineate the nightshades group.

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APPENDIX 1: TLC plates and spots revealed

SC SN SV

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APPENDIX 2: ANOVA

Descriptives 95% Confidence Interval for Mean Std. Std. Lower Upper Minimu Maximu N Mean Deviation Error Bound Bound m m LEAF LENGTH SC 25 15.8800 2.88487 .57697 14.6892 17.0708 10.10 21.00 SN 25 8.0360 .75712 .15142 7.7235 8.3485 6.70 9.20 SV 25 13.4280 2.02804 .40561 12.5909 14.2651 7.20 18.30 Total 75 12.4480 3.88575 .44869 11.5540 13.3420 6.70 21.00 PETIOLE SC 25 10.6760 2.06282 .41256 9.8245 11.5275 7.60 14.50 LENGTH SN 25 5.4120 .33828 .06766 5.2724 5.5516 4.90 5.90 SV 25 10.2160 1.38072 .27614 9.6461 10.7859 8.40 15.20 Total 75 8.7680 2.78903 .32205 8.1263 9.4097 4.90 15.20 LEAVE WIDTH SC 25 6.1120 1.53007 .30601 5.4804 6.7436 3.50 9.00 SN 25 4.1680 .63752 .12750 3.9048 4.4312 3.20 5.10 SV 25 2.9360 .92416 .18483 2.5545 3.3175 1.90 5.00 Total 75 4.4053 1.70309 .19666 4.0135 4.7972 1.90 9.00

Test of Homogeneity of Variances Levene Statistic df1 df2 Sig. LEAF 9.620 2 72 .000 LENGTH PETIOLE 15.818 2 72 .000 LENGTH

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LEAF LENGTH

LEAVE 9.306 2 72 .000 WIDTH

ANOVA Sum of Squares df Mean Square F Sig. LEAF LENGTH Between Groups 805.119 2 402.560 92.836 .000 Within Groups 312.208 72 4.336 Total 1117.327 74 PETIOLE Between Groups 424.998 2 212.499 101.576 .000 LENGTH Within Groups 150.626 72 2.092 Total 575.623 74 LEAVE WIDTH Between Groups 128.199 2 64.100 53.393 .000 Within Groups 86.438 72 1.201 Total 214.638 74

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PETIOLE LENGTH Subset for alpha = 0.05 TAX Subset for alpha = 0.05 TAXA N 1 2 A N 1 2 3 a Tukey HSD SN 25 5.4120 Tukey SN 25 8.0360 a SV 25 HSD10.2160 SV 13.428 25 SC 25 10.6760 0 Sig. 1.000 .502 SC 15.880 a 25 Duncan SN 25 5.4120 0 SV 25 10.2160 Sig. 1.000 1.000 1.000 SC 25 Duncan10.6760a SN 25 8.0360 Sig. 1.000 .265 SV 13.428 25 Means for groups in homogeneous subsets are displayed. 0 a. Uses Harmonic Mean Sample Size = 25.000. SC 15.880 25 0 Sig. 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 25.000. LEAF WIDTH TAX Subset for alpha = 0.05 A N 1 2 3 Tukey SV 25 2.9360 a HSD SN 25 4.1680 SC 25 6.1120 Sig. 1.000 1.000 1.000 Duncana SV 25 2.9360 SN 25 4.1680 SC 25 6.1120 Sig. 1.000 1.000 1.000

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Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 25.000.

Descriptives 95% Confidence Std. Interval for Mean Deviatio Lower Upper Maximu N Mean n Std. Error Bound Bound Minimum m PLANT HEIGHT S 12.5100 5 73.0000 5.59464 57.4668 88.5332 51.00 81.00 C 0 S 5 31.2000 1.30384 .58310 29.5811 32.8189 30.00 33.00 N S 5 34.6000 2.88097 1.28841 31.0228 38.1772 32.00 39.00 V T o 20.7965 t 15 46.2667 5.36964 34.7499 57.7834 30.00 81.00 2 a l NO OF S 105.147 5 96.8000 6.72309 3.00666 88.4522 88.00 104.00 LEAVES/PLANT C 8 S 108.884 5 98.4000 8.44393 3.77624 87.9155 88.00 108.00 N 5 S 10.1390 113.189 5 100.6000 4.53431 88.0107 88.00 112.00 V 3 3

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T o 103.073 t 15 98.6000 8.07819 2.08578 94.1264 88.00 112.00 6 a l LEAF SIZE S 21061.80 1698.10 759.4156 18953.32 23170.2 23716. 5 19321.00 C 00 503 6 41 759 00 S 7417.800 2157.88 965.0347 4738.434 10097.1 9801.0 5 5184.00 N 0 327 4 0 660 0 S 16534.80 8269.85 3698.391 6266.418 26803.1 22201. 5 1932.00 V 00 554 83 1 819 00 T o 15004.80 7496.07 1935.477 10853.61 19155.9 23716. t 15 1932.00 00 381 94 27 873 00 a l

Test of Homogeneity of Variances Levene Statistic df1 df2 Sig. PLANT HEIGHT 4.229 2 12 .041 NO OF .971 2 12 .406 LEAVES/PLANT LEAF SIZE 3.501 2 12 .063 ANOVA Sum of Mean Squares df Square F Sig.

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PLANT HEIGHT Between 5388.933 2 2694.467 48.549 .000 Groups Within 666.000 12 55.500 Groups Total 6054.933 14 NO OF Between 36.400 2 18.200 .249 .784 LEAVES/PLANT Groups Within 877.200 12 73.100 Groups Total 913.600 14 LEAF SIZE Between 482953590 241476795 2 9.541 .003 Groups .000 .000 Within 303722126 25310177. 12 Groups .400 200 Total 786675716 14 .400

Multiple Comparisons Mean 95% Confidence Interval (I) (J) Difference Lower Upper Dependent Variable TAXAS TAXAS (I-J) Std. Error Sig. Bound Bound PLANT HEIGHT Tukey SC SN 41.80000* 4.71169 .000 29.2299 54.3701 HSD SV 38.40000* 4.71169 .000 25.8299 50.9701 SN SC -41.80000* 4.71169 .000 -54.3701 -29.2299 SV -3.40000 4.71169 .756 -15.9701 9.1701 SV SC -38.40000* 4.71169 .000 -50.9701 -25.8299 SN 3.40000 4.71169 .756 -9.1701 15.9701

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NO OF Tukey SC SN -1.60000 5.40740 .953 -16.0262 12.8262 LEAVES/PLANT HSD SV -3.80000 5.40740 .766 -18.2262 10.6262 SN SC 1.60000 5.40740 .953 -12.8262 16.0262 SV -2.20000 5.40740 .913 -16.6262 12.2262 SV SC 3.80000 5.40740 .766 -10.6262 18.2262 SN 2.20000 5.40740 .913 -12.2262 16.6262 LEAF SIZE Tukey SC SN 13644.0000 3181.8345 22132.700 .003 5155.2993 HSD 0* 1 7 SV 3181.8345 13015.700 4527.00000 .361 -3961.7007 1 7 SN SC - - 3181.8345 13644.0000 .003 22132.700 -5155.2993 1 0* 7 SV - - 3181.8345 9117.00000 .035 17605.700 -628.2993 1 * 7 SV SC - - 3181.8345 .361 13015.700 3961.7007 4527.00000 1 7 SN 9117.00000 3181.8345 17605.700 .035 628.2993 * 1 7 *. The mean difference is significant at the 0.05 level.

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PLANT HEIGHT

NO OF LEAVES/PLANT Subset for alpha Subset for TAXA = 0.05 alpha = S N 1 2 TAXA 0.05 Tukey SN 5 31.2000 a S N 1 HSD SV 5 34.6000

Tukey SC 5 96.8000 SC 5 73.0000 a HSD SN 5 98.4000 Sig. .756 1.000

SV 5 100.6000 Duncana SN 5 31.2000 Sig. .766 SV 5 34.6000 Duncana SC 5 96.8000 SC 5 73.0000 SN 5 98.4000 Sig. .484 1.000 SV 5 100.6000 Means for groups in homogeneous subsets Sig. .517 are displayed. Means for groups in homogeneous a. Uses Harmonic Mean Sample Size = subsets are displayed. 5.000. a. Uses Harmonic Mean Sample Size = 5.000. LEAF SIZE Subset for alpha = TAXA 0.05 S N 1 2 Tukey SN 7417.80 5 HSDa 00 SV 16534.80 5 00 SC 21061.80 5 00 Sig. 1.000 .361

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Duncana SN 7417.80 5 00 SV 16534.80 5 00 SC 21061.80 5 00 Sig. 1.000 .180 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 5.000.

Descriptives 95% Confidence Interval for Mean Std. Std. Lower Upper Minimu Maximu N Mean Deviation Error Bound Bound m m FLOWER PEDICEL SC 25 .9920 .18690 .03738 .9148 1.0692 .70 1.30 LENGTH SN 25 .4840 .06245 .01249 .4582 .5098 .40 .60 SV 25 .7600 .13844 .02769 .7029 .8171 .60 1.00 Total 75 .7453 .25002 .02887 .6878 .8029 .40 1.30 FLOWER SC 25 2.8760 .38000 .07600 2.7191 3.0329 2.10 4.00 PEDUNCLE SN 25 1.7960 .30616 .06123 1.6696 1.9224 1.30 2.60 LENGTH SV 25 .2720 .07371 .01474 .2416 .3024 .10 .40 Total 75 1.6480 1.11153 .12835 1.3923 1.9037 .10 4.00

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Test of Homogeneity of Variances Levene Statistic df1 df2 Sig. FLOWER 16.759 2 72 .000 PEDICEL LENGTH FLOWER PEDUNCLE 8.096 2 72 .001 LENGTH

ANOVA Sum of Mean Squares df Square F Sig. Between 3.234 2 1.617 83.634 .000 Groups Within 1.392 72 .019 Groups Multiple Comparisons

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Total Mean 95% Confidence Interval (I) (J) Difference Std. Lower Upper Dependent Variable TAXA TAXA (I-J) Error Sig. Bound Bound FLOWER PEDICEL Tukey SC SN .50800* .03933 .000 .4139 .6021 LENGTH HSD SV .23200* .03933 .000 .1379 .3261 SN SC -.50800* .03933 .000 -.6021 -.4139 SV -.27600* .03933 .000 -.3701 -.1819 SV SC -.23200* .03933 .000 -.3261 -.1379 * SN4.626 74.27600 .03933 .000 .1819 .3701 FLOWER Tukey SC SN 1.08000* .08059 .000 .8871 1.2729 PEDUNCLE HSD SV 2.60400* .08059 .000 2.4111 2.7969 LENGTH SN SC -1.08000* .08059 .000 -1.2729 -.8871 SV 1.52400* .08059 .000 1.3311 1.7169 SV SC -2.60400* .08059 .000 -2.7969 -2.4111 SN -1.52400* .08059 .000 -1.7169 -1.3311 *. The mean difference is significant at the 0.05 level. FLOWER PEDICEL LENGTH FLOWER Between 527.05 85.582 2 42.791 .000 PEDUNCLE Groups 2 LENGTH Within 5.846 72 .081 Groups Total 91.427 74

FLOWER PEDICEL LENGTH TAX Subset for alpha = 0.05 A N 1 2 3 Tukey SN 25 .4840

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HSDa SV 25 .7600 SC 25 .9920 Sig. 1.000 1.000 1.000 Duncana SN 25 .4840 SV 25 .7600 SC 25 .9920 Sig. 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 25.000.

FLOWER PEDUNCLE LENGTH TAX Subset for alpha = 0.05 A N 1 2 3 Tukey SV 25 .2720 a HSD SN 25 1.7960 SC 25 2.8760 Sig. 1.000 1.000 1.000 Duncana SV 25 .2720 SN 25 1.7960 SC 25 2.8760 Sig. 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 25.000.

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Descriptives NO OF FLOWERS/PLANT 95% Confidence Interval for Mean Std. Std. Lower Upper Minimu Maximu N Mean Deviation Error Bound Bound m m SC 123.000 5 9.27362 4.14729 111.4853 134.5147 108.00 132.00 0 SN 5 71.6000 1.14018 .50990 70.1843 73.0157 70.00 73.00 SV 5 79.4000 5.45894 2.44131 72.6218 86.1782 72.00 85.00 Total 15 91.3333 24.11480 6.22642 77.9790 104.6877 70.00 132.00

Test of Homogeneity of Variances NO OF FLOWERS/PLANT Levene Statistic df1 df2 Sig. 3.787 2 12 .053

ANOVA NO OF FLOWERS/PLANT Sum of Mean Squares df Square F Sig.

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Between 7672.933 2 3836.467 98.287 .000 Groups Within 468.400 12 39.033 Groups Total 8141.333 14

Multiple Comparisons Dependent Variable: NO OF FLOWERS/PLANT 95% Confidence Mean Interval (I) (J) Difference Std. Lower Upper TAXAS TAXAS (I-J) Error Sig. Bound Bound Tukey SC SN 51.40000* 3.95137 .000 40.8583 61.9417 HSD SV 43.60000* 3.95137 .000 33.0583 54.1417 SN SC -51.40000* 3.95137 .000 -61.9417 -40.8583 SV -7.80000 3.95137 .161 -18.3417 2.7417 SV SC -43.60000* 3.95137 .000 -54.1417 -33.0583 SN 7.80000 3.95137 .161 -2.7417 18.3417 *. The mean difference is significant at the 0.05 level.

NO OF FLOWERS/PLANT Subset for alpha TAXA = 0.05 S N 1 2 Tukey SN 5 71.6000

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HSDa SV 5 79.4000 SC 123.000 5 0 Sig. .161 1.000 Duncana SN 5 71.6000 SV 5 79.4000 SC 123.000 5 0 Sig. .072 1.000 Means for groups in homogeneous subsets are displayed. .

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