1

EFFECTS OF DRYING CONDITIONS ON THE PHYTOCHEMICALS,

ANTIOXIDANTS AND MINERAL CONTENTS OF BITTER LEAF

(Vernonia amygdalina)

BY

ABAIRE OLAWALE JEREMIAH

[100401001]

A RESEARCH PROJECT REPORT SUBMITTED TO THE

DEPARTMENT OF BIOCHEMISTRY, FACULTY OF SCIENCE,

ADEKUNLE AJASIN UNIVERSITY, AKUNGBA AKOKO, ONDO

STATE.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF BACHELOR OF SCIENCE (B.Sc.) HON. DEGREE IN

BIOCHEMISTRY.

FEBRUARY, 2015

CERTIFICATION

2

This is to certify that this research project work was carried out and presented

by ABAIRE OLAWALE JEREMIAH (100401001), except for references to

other people’s work which have been duly acknowledged. This work was

submitted to the Department of biochemistry, Faculty of Science, Adekunle

Ajasin Akungba-Akoko, Ondo state.

Student Date

Prof. A.O. Onigbinde Date

(Supervisor)

Prof. A.O Onigbinde Date

(HOD Biochemistry)

DEDICATION

3

This report is dedicated to the Almighty God, the source of my

wisdom and knowledge. To my parents, Mr. and Mrs. Abaire and to my

siblings.

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ACKNOWLEDGEMENT

Glory to God almighty for the inspiration and initiative he has given

to me for the successful completion of this project work. My gratitude goes to

everyone who in one way or the other contributed to the success of this project

work.

I most acknowledge my supervisor, the HOD of this noble department,

Prof. A.O. Onigbinde for his guidance, fatherly care and ever supportive role

he played during this research project.

I also appreciate my lecturers; Prof. Omueti, Dr. Olusola, Dr.

Elekofehinti, Dr. Saliu, Dr. Shodehinde, Dr. Adedeji, Dr. Abigor, Mr. Fakoya

(Course Adviser), Mr. Adeniran, Mr. Ogunwa, Mrs Odunbanjo and our lab

technologists; Mrs. Oyewale, Mr. Fesobi, and Mrs. Ejelonu for the knowledge

they impacted me with and their contributions during the course of this study.

I cannot but acknowledge my parents, Mr. and Mrs. Abaire, for their

immense contribution both in cash and in kinds. I also appreciate my siblings,

Abaire Oluwatosin, Abaire Oluwakemi and Abaire Oluwatobi, for their love

and care. I also appreciate my friend and confidant, Olawade David.

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I won’t but say a big thank you to my project colleague for their

cooperation, most especially Adewunmi Olabode. I say thank you to Adeyemi

Olaoluwa, Samuel, A.y and my entire course mate (2013/2014 graduates).

To all my neighbours at Emirate villa; David, Victoria, Festus, Joke,

Mercy, James and D sax, I say thank you to you all.

My appreciation also goes to every member of the Redeemed Christian

Fellowship (RCF), for their cares and prayers for me. My gratitude goes to the

workers and excecutives (2013/2014 Excos) of RCF.

Thank you and God bless you all.

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TABLE OF CONTENT

TITLE PAGE…………………………………………………………………i

CERTIFICATION……………………………………………………………ii

DEDICATION………………………………………………………………..iii

ACKNOWLEDGEMENT………………………………………………….iv-v

TABLE OF CONTENT………………………………………………. vi- ix

LIST OF TABLES………………………………………………………….x

LIST OF FIGURES…………………………………………………………x

ABSTRACT………………………………………………………………….xi

CHAPTER ONE

1.0 INTRODUCTION…………………………………………………....1

1.1 LITERATUREREVIEW……………………………………….…….3

1.2 IMPORTANCE AND USES OF BITTER LEAF…..……….……….5

1.3 HEALTH AND NUTRITIONAL BENEFITS OF BITTER LEAF.....6

1.4 BIOACTIVE CONSTITUENTS OF BITTER LEAF…….……….....8

1.5 BODY CALMING EFFECTS OF BITTER LEAF………….……....13

1.6 DRYING METHODS OF BITTER LEAF (Vernonia amygdalina)...15

CHAPTER TWO

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2.0 MATERIALS AND METHODS ………………………………........18

2.1 PROXIMATE ANALYSIS…………………...…………………......18

2.2 DETERMINATION OF MOISTURE CONTENT.....................……19

2.3 DETERMINATION OF ASH CONTENT…………………..............20

2.4 DETERMINATION OF CRUDE FIBRE……………………..…….20

2.5 DETERMINATION OF CRUDE PROTEIN ………………..……..21

2.5.1 Digestion Stage………………….…………………………...22

2.5.2 Distillation Stage……………………..………………..……..23

2.5.3 Titration Stage…………………………………..…..………..23

2.6 DETERMINATION OF FAT CONTENT…………………….…….25

2.7 DETERMINATION OF CARBOHYDRATE CONTENT….............25

2.8 PHYTOCHEMICAL ANALYSIS………………………….……….25

2.8.0 Test for Alkaloids……………………………...………...…..26

2.8.1 Test for Saponins……………………………...…………..…27

2.8.2 Test for Tannins…………………………………….………..27

2.8.3 Test for Phlobatanins……………………………….………..27

2.8.4 Test for Antraquinones…………………....………………....27

2.8.5 Cardiac Glycoside Test………………………………………27

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2.8.6 Test for Flavonoids………………………………………..…28

2.8.7 Test for Steroids…………………………………………..…28

2.8.8 Test for Terpenoids………………………….……………....29

2.8.9 Cardenolide test………………...…………………………...29

2.9 DPPH SCAVENGING ACTIVITY………………………………...29

2.9.0 Chemicals………………………..………………………….29

2.9.1 Plant Materials and Extraction Procedures……………...….29

2.10 METAL ANALYSIS………………….………..…..........................30

2.11 Digestion of Samples for Metal Analysis………………………......30

2.12 Determination of Metals………………,,...…………………………31

CHAPTER THREE

3.0 RESULTS…………………………………………………………..32

CHAPTER FOUR

4.0 DISCUSSION AND CONCLUSION………………………..….....40

4.1 PROXIMATE………………………………………….……...…....40

4.2 PHYTOCHEMICAL…………………...….……………………….46

4.3 ANTIOXIDANT…………………..…………………………..……47

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4.4 METALS….……………………………...……………….…….......49

4.5 CONCLUSION….……………………………,,………………..….51

REFRENCES:…………………………………………,,,……….….…….52

APPENDIX:……………………………………………..….……………...67

LIST OF TABLES

Table 1: Proximate Composition (g%) of fresh, oven dried, freeze dried and

sun dried Bitter leaf.

Table 2: Trolox Antioxidant Activity.

Table 3a: Showing the absorbance of fresh and dried samples of Bitter leaf.

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Table 3b: Absorbance and the calculated concentration of the fresh and the

dried samples of Vernonia amygdalina.

Table 4: Preliminary phytochemical screening of oven dry, sun dry, freeze

dry and fresh bitter leaf (V. amygdalina).

Table 5: Mineral Composition (ppm) of dried bitter leaf

LIST OF FIGURES

Figure 1: Showing Bitter leaf shrub tree.

Figure 2: Showing Bitter leaf Juice extract.

Figure 3: Proximate Analysis of fresh and dried Vernonia amygdalina

extract.

Figure 4: Trolox Standard Curve.

Figure 5: Absorbance versus concentration of Bitter leaf

ABSTRACT

Vernonia amygdalina (Bitter Leaf) is a common African vegetable which is

used as spices for delicacies and also for medicinal purposes. The objective of

this study was to find out the effects of sun drying, oven drying and freeze

drying techniques on the phytochemical, antioxidant and mineral constituent

of bitter leaf, which was evaluated using standard analytical procedures. For

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the phytochemical screening, phytochemical compounds such as saponins,

alkaloids, tannins, phlobatannins, anthraquinone, steroids, flavonoids,

terpenoids, cardenolides and cardiac glycoside were determined in the

aqueous extracts of the dried and the fresh bitter leaf samples using standard

methods. The aqueous extract of the bitter leaf samples showed positive

results for some of the phytochemicals, but negative result for others. The

antioxidant assay of the methanolic extract of the bitter leaf samples showed

that the oven dried extract had a high level of 1, 1-diphenyl-2-picrylhydrazyl

(DPPH) free radical scavenging activity than any of the other extracts. Results

concluded that high temperature enhances the antioxidant properties of this

vegetable and the presences of some active phytochemicals in the dried

samples may be responsible for the medicinal purposes of the plant.

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CHAPTER ONE

1.0 INTRODUCTION

The fresh edible portions of herbaceous plant which traditional African

societies have always exploited are the vegetables. They are considered as

natural gift given by the Almighty God to human beings, as a source of

nutrient, food security and income generation.

Bitter leaf (Vernonia amygdalina) is a shrub tree of 2-5 meters, which

grows under a range of ecological zones in Africa and produces large mass of

forage and it is also drought resistant. The leaves are green with a

characteristic odour and a bitter taste (Bonsi et al., 1995a). Bitter leaves are

used for human and animal consumption. For human consumption, it is

washed to get rid of the bitter taste and used as vegetable. It stimulates the

digestive system as well as helps to reduce fever. They are also used as local

medicine or herb against parasites. The plant is well known for its anti-

diabetic and anti-hypertensive properties, and also used in the treatment of

headache and fever (Oboh, 2003). A preliminary phytochemical screening of

bitter leaf indicated the presence of saponins, tannins, terpenes, alkaloids and

steroid (Igile et al., 1995).The leaves of bitter leaf are bitter; however after

soaking the leaves in water and cooking, local people use it in soup and stew

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as a strength given tonic. The boiling and cooking has been found to reduce

secondary plant compounds and makes the feed more palatable (Oboh, 2005)

However, the effort to ascertain the effects of different drying methods

of bitter leaf was borne out of the benefits of using this vegetable either in its

fresh form or in its dry form, probably due to its scarcity or absence in the dry

season or to ascertain its nutrient retention ability. This is why we saw the

need to analyze the nutrient of this leafy vegetable after being subjected to

various drying techniques, so as to know if there is any significant difference

in its nutrient composition based on drying. The result of this analysis could

be used to advise on the best drying method which can guarantee nutrient

retention. This idea is in conformity with the objectives and findings of some

other researchers.

Minerals are the naturally occurring inorganic elements which

constitute only a small amount of nutrients with definite chemical

compositions. Bitter leaf is an excellent source of minerals that contributes to

the recommended daily allowances of the essential nutrients required for

normal metabolic activities of the body tissues. However, protein,

carbohydrate, crude fibre, crude protein and crude fat which are also present

in bitter leaf also contribute to the recommended daily allowance.

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Fresh bitter leaf, among other vegetables has a high moisture content

which ranges between 60-75%. Therefore, there is a need for its conservation

and preservation against spoilage. In Nigeria today, we are bedeviled by

constant power failure, which make refrigeration of vegetables and vegetable

products difficult, hence there is the need to explore an alternative way for

preserving them. This study therefore examine the effects of three drying

methods on the nutrient and mineral composition of bitter leaf and the best

drying method most suitable for the preservation and the conservation of the

nutrients in this vegetable.

1.1 LITERATURE REVIEW

Several vegetable species are found in tropical and subtropical regions

of the world, where they are used partly as condiments or spices in human

diets or as a supplementary feeds to livestock (Aletor and Adeogun, 1995).

Vegetables have been discovered to have almost all of the mineral and organic

nutrients established as essential for human nutrition.

Vernonia amygdalina, commonly called bitter leaf because of its bitter

taste is one of the common vegetables found in Africa. Vernonia amygdalina

is the most widely cultivated species of the genus Vernonia which has about

1,000 species of shrubs (Muanya, 2013). It belongs to the family Astaraceae.

It is a shrub or small tree of 2-5 meters and is vegetatively cultivated by stem

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cutting at an angle of 450 and popular in most of West Africa countries

including Nigeria, Cameroon, Gabon and Congo Democratic Republic. Water

is a major factor responsible for the growth of this plant. Thus, high yield can

be obtained during rainy season (Kayode, 2004). Bitter leaf is a short cycle

crop which can be harvested twice per month for up to seven years. Planting

bitter leaf can be easy because it is compatible with any type of crop and can

be planted in a variety of arrangements (Biggelaar and Gold, 1996). Bitter leaf

grows under a range of ecological zones in Africa and produces large mass of

forage and it is drought resistant. Vernonia amygdalina was named after an

English Botanist, named Williams Vernon.

Figure 1: Showing bitter leaf shrub tree

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1.2 IMPORTANCE AND USES OF BITTER LEAF

Due to the bitterness of bitter leaf, it can be used as a bittering agent, a

hop substitute and for the control of microbial contamination in beer brewing

without affecting the quality of malt. In Ethiopia, it is used to make honey

wine called Tei (Babalola and Okoh, 1996; Biggelaar and Gold, 1996;

Eleyinmi et al., 2004; Okoh et al., 1995; Uraih and Anetekhai, 1991).

However, after soaking the leaves in water and cooking, local people

use it in soup and stew as a strength given tonic. The boiling and cooking has

been found to reduce secondary plant metabolites and makes the feed more

palatable (Oboh, 2005). It is therefore of importance to consider the effect of

these treatments (i.e. cooking and boiling) on the nutritional properties of the

bitter leaf.

Bitter leaves are used for human and animal consumption. For human

consumption, it is washed to get rid of the bitter taste and used as vegetables.

They are consumed as cooked complements to major staple foods such as

cassava, pounded yam, guinea corn, maize, millet, rice and plantains. It is also

considered very useful because of its high medicinal value, as the juice

extracted from the leaves is wholly applied to fresh wound or cuts in some

rural community (Adanlawo and Dairo, 2006).

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1.3 HEALTH AND NUTRITIONAL BENEFITS OF BITTER LEAF

Bitter leaf stimulates the digestive system as well as helps to reduce

fever. It is also used as local medicine or herb against parasites. Previous

studies have reported that it possess anti-microbial, anti-diabetic, anti-

malarial, anti-parasitic, insecticidal, anticancer, anti- inflammatory, antipyretic,

analgesic, anti-helminthic hepatoprotective, antioxidative and hypolipidaemic

effects among others (Atangwho, et al., 2010; Yeap et al., 2010, Ho et al.,

2012).A preliminary phytochemical screening of bitter leaf indicated the

presence of saponins, tannins, terpenes, alkaloids, steroid and flavonoids

which has antioxidant properties(Igile et al.,1995).

Furthermore, it is well known that proteins are of great importance to

health, and are often deficient in the diets of people in developing countries,

especially those in the vulnerable groups, such as nursing mothers, expectant

mothers, weaning and pre-school children (Fasuyi and Aletor,

2005).However, since the last world war, emphasis has been placed on the

need to increase the dietary protein, particularly by the use of locally grown

vegetables which are rich in protein, as a result of the realization that the diet

in underdeveloped countries is chronically low in protein leading to

malnutrition and wide spread deficiency diseases. Therefore, nutritionist are

researching on suitability of vegetables that has promising values as a means

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of replacing proteins from animal sources which are very expensive and

economically unviable (Oke, 1973). Vernonia amygdalina leaves, with just a

little amount of processing can be classified as healthy food because it

promotes the healthy development of the body. As a result, it serves well as a

low cost and readily available source of important nutrients to humans (Ojiako

and Nwanjo, 2006). Its high content of crude protein has made it a good

source of protein. V. amygdalina leaves, when added to soybean meal is the

best infant weaning food which helps to gain weight (Agbede et al., 2007).

Figure 2: Showing bitter leaf juice extract

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1.4 BIOACTIVE CONSTITUENT OF BITTER LEAF

Phytochemical screening of Vernonia amygdalina leaves revealed the

presence of tannins, phlobatannins, flavonoids, steroids, terpenoids, saponins

and cardiac glycosides, which are the most important bioactive constituents of

medicinal plants. These bioactive constituents of Vernonia amygdalina have

also curative active principles such as lovastatin antilipidaemia, Pleurotin

antibiotics and beta-glucan polysaccharides with heavy molecular weight and

immunomodulator- immunostimulant properties. Polysaccharides stimulate

the immunological system using three mechanisms: interferon production,

excitation of complement chains and the activation of macrophages, inducing

organism’s defense. This bitter leaf when consumed daily prevents

oncogenesis and metastasis in cancer cases; therefore, it’s used as a

coadjunctant therapy in chemotherapy treatments.

V. amygdalina was found to contain 21 to 23% of dry matter (Fafunso

and Bassir, 1976; Ifon and Bassir, 1980). Out of the dry matter, it contained

6.5 to 29.2% of crude fibre, ranging from the fresh leaves to the dried ones

(Alabi et al., 2005a; Antia et al., 2006; Ifon and Bassir, 1979; 1980; Oboh,

2006; Okoli et al., 2003a). Higher hemicellulose was found in the dry than the

fresh leaves of V. amygdalina (Bonsi et al., 1995a; b; Okoli et al., 2003a). V.

amygdalina contains crude protein (17 to 33 g/100g DW) (Ifon and Bassir,

20

1980; Mekoya et al., 2008; Oboh, 2006; Okoli et al., 2003a) and fat (2 to 15

g/100g DW with 24.54% saturated and 65.45% polyunsaturated. Oleic acid

was the major monounsaturated fatty acids (Alabi et al., 2005a; Eleyinmi et

al., 2008; Ifon and Bassir, 1980; Oboh, 2006). Due to its high content of crude

protein, it was found to be a good source of protein. High amount of protein is

essential for animal growth and increased milk production (Oke, 1965;

Tangka, 2003). High ash content (10 to 13 g/100g DW) (Alabi et al., 2005a;

Faboya, 1983; Ifon and Bassir, 1979; Ifon and Bassir, 1980; Mekoya et al.,

2008; Oboh, 2006; Okoli et al., 2003a) reflected the useful mineral contents

(calcium, chlorine, chromium, copper, Iron, potassium, iron, magnesium,

manganese, nickel, phosphorus, potassium, sodium, sulphur and zinc) that are

present in this plant (Alabi et al., 2005a; Faboya, 1983; Gbaruko and Friday,

2007; Ifon and Bassir, 1979; 1980; Oboh, 2006). Ash content of bitter leaf

contained high amount of nitrogen, phosphorus and other types of

exchangeable bases (Calcium, Magnesium, Sodium and potassium)

(Enikuomehin et al., 1998). High concentration of sulphur is important for

detoxification of cyanide while low sodium content is suitable for obese

patients (Ifon and Bassir, 1979). The nutritive values of young and mature

leaves did not differ significantly (Akachuku, 2001).

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However, vitamin C concentration was affected by the soil properties

(regular use of fertilizers). Loss of 60% of vitamin C in the plant is due to

exposure to sunlight and very high temperature. Keeping in the refrigerator

can effectively prevent the loss. Thus, photo-oxidation was identified as the

major contributor for the loss of vitamin C (Faboya, 1990). Washing and

cooking was found to further reduce 40 to 77% of vitamin C content (Ejoh et

al., 2003; Fafunso and Bassir, 1976).These suggest that bitter leaf should be

consumed immediately after harvesting or must be kept in a refrigerator

before processing.

V. amygdalina contains Stigmastane-type steroid glucoside

compounds. One of the major steroid glucoside compounds that have been

identified from V. amygdalina leaves is the vernoniosides. These glucoside

compounds can be isolated from the leaf, stem, pith and root parts of the plant

(Huffman, 2001). Among all other vernonioside, Vernonioside B1 was found

in higher concentrations in the leaves than in the stem and much more

abundant in the pith of the plant (Huffman et al., 1993; Koshimizu et al.,

1994; Ohigashi et al., 1994). Besides, this compound was also identified to be

responsible for the removal of parasites in primates who sucked the young

pith of V. amygdalina, for the control of gastrointestinal illnesses (Huffman et

al., 1993; Igile, 1995b; Koshimizu et al., 1994).

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Bitter leaf is well known for its bitter taste. Vernoniosides A1, A2, A3

and A4, were found to be part of the constituents in contributing to this

characteristic while vernoniosides B1, B2 and B3, did not show any bitter

taste (Jisaka et al., 1992; Jisaka et al., 1993a; Koshimizu et al., 1994;

Ohigashi et al., 1991a; Osinubi, 2007). The vernoniosides B were found to

lack a free hydroxyl end at their C-16, as was present in vernoniosides A.

Hydroxylation at C-16 of these steroid Glucosides was therefore hypothesized

to play an important role in causing bitterness to this plant (Jisaka et al., 1992;

1993a; 1993b; Ohigashi et al., 1991).

Another major group of bioactive compounds that has been isolated

from bitter leaf are the sesquiterpene lactones, consisting of vernodalin,

vernolide, vernolepin, vernomenin, vernomygdin, vernolic, vernodalol,

hydroxylvernolide, 11,13-dihydrovernodalin, 11,13- dihydrovernorodeline,

4,15-dihydrovernodalin, 7,24(28)-stigmastadien-3_-ol and

1,2,3,15,11,13,2’,3’- octahydrovernodalin.

Sesquiterpene lactones can be isolated from the leaf stem, pith and root

of V. amygdalina with the exception of vernodalin which could not be isolated

from the pith of the plant. Vernodalin was also found to be more concentrated

in young leaves than young stems (Huffman et al., 1993; Ohigashi et al.,

1994).

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Previous studies showed that vernodalin possess antitumor activity

against human nasopharynx carcinoma, KB and mouse leukemia P-388 and L-

1210 cancer cell lines (Jisaka et al., 1993 b; Kupchan et al., 1969). This

compound also showed in vitro insecticidal activity against African

armyworm (Ganjian et al., 1983), anti- bacterial effect towards B. subtilis

(Jisaka et al., 1993 b) and Micrococcus luteus as well as antileishmanial

activity on Leishmania infantum (Koshimizu et al., 1994).

Vernolide is mono-hydroxylated as compared to vernodalol which has

2 hydroxyl groups. Vernolide is therefore less polar and more lipophilic than

vernodalol. Lipophilicity was found to be the major influence affecting

fungicidal activities of these two compounds. This was confirmed by Erasto et

al.,(2006) whereby vernolide possessed stronger inhibition against A. flavus,

Mucor hiemalis, Fusarium oxysporum, Penicillium notatum and A. niger than

vernodalol at concentrations ranging from 0.05 to 0.5 mg/ml. Vernolide also

possess antibacterial activity against the gram positive bacteria B. cereus, B.

subtilis, Staphylococcus epidermidus, S. aureus, M. kristinae, M. Luteas and

Streptococcus pyrogens and the gram negative bacterium Salmonella pooni

(Erasto et al., 2006; Jisaka et al., 1993b).

Vernolepin showed antiplatelet effect in rabbits through the inhibition

of arachidonic acid, ADP and collagen induced platelet aggregation, as well as

24

by interference with ATP-release without the inhibition of cyclooxygenase or

lipoxygenase. In addition, vernolepin also demonstrated a time dependent

biphasic enhancement/ inhibition feature of coaxial stimulation and

antagonism against histamine in guinea pig ileum (Laekeman et al., 1983;

1985). On the other hand, vernolepin also showed cytotoxic activity against P-

388 and L-1210 mouse leukemic cancer cell lines and antibacterial effect

against B. subtilis and M. lutea (Jisaka et al., 1993b).

1.5 BODY CALMING EFFECT OF BITTER LEAF

In addition, of the numerous benefits of bitter leaf is the calming effect

of the leaf extract and leaf powder on a subject’s body and outlook. This

calming effect is produced by the relaxation effects of the extract on the

skeletal, cardiac and smooth muscles all over the body. The energy-

moderation effects of bitter leaf extract on the cardiac, smooth and skeletal

muscles that control the activities of the human body resulted in smooth low

energy- functioning of all the organs of the body. This action of bitter leaf

extract was responsible for its calming effects on the muscular activities of the

subject’s body.

The effects of V. amygdalina leaf extract on the muscles of the body

were augmented by the excess fat elimination and the anti-obesity effects of

the extract. The antioxidant effects of V. amygdalina leaves extract account

25

for the energy-lowering effects of the extract that produced the calming of the

body(Mucimapura et al., 2010; Balasubramanyam et al., 2006; Akbari-

Asbagh et al., 2010; Al-Attar, 2010a). Part of the lowering of the energy

demands of the body by bitter leaf extract occurred by inhibitory lipolytic

action of the extract in adipose tissues. These lipolytic and excess fat

elimination properties of bitter leaf are carried out by x2 receptor inhibition

resulting in inhibition of the accumulation of cyclic AMP. This lipolytic and

excess fat elimination effect thus disallows the huge energy yields which

would otherwise have accrued to the body by normal β -oxidation of fats in

the body. This antagonism of the energy yield from fat metabolism is largely

responsible for the calming effect of bitter leaf extract on all the tissues of the

body including the brain. A similar β-oxidation stimulation effect of bitter leaf

occurs in the mitochondria of cardiac, smooth and skeletal muscle cells of the

whole body to antagonize their energy generation process. This action

accounts for the calming of the muscular activities of the body. The

generalized calming of the body by bitter leaf extract (and by implication

bitter leaf soup) was produced by a combination of the excess body fat

lipolytic/elimination effects of the extract; its hypoglycaemic effects and its

inhibitory energy generation antagonism effects on all muscles of the body.

26

Soursop plant extracts employ similar energy generation antagonism actions

to execute their medicinal effects.

V. amygdalina leaf extract produced its calming effects on the body by

its action as an inhibitory first messenger that inhibited x2-adrenoceptors by

occupation of β3-adreceptors on membranes of adipose tissues, smooth,

cardiac and skeletal muscles and other tissues of the body to cause the

inhibition of the accumulation of cyclic AMP. V. Amygdalina leaf extract

acted on the above mentioned β3adrenoceptores I as an inhibitory G1- protein

to bring about the body calming effect on the body.

1.6 DRYING METHODS OF BITTER LEAF (Vernonia amygdalina)

Drying is the oldest method of preserving food. Throughout history,

the sun, the wind and hot air have been used to remove water from fruits,

grains, and vegetables. According to Harrison and Andres (2000), drying

removes moisture from food, the food becomes smaller and lighter in weight.

When the food is ready for use, the water is added back. However, drying

removes moisture from food sample, therefore bacteria, yeast and mold cannot

grow on and spoil the food sample. Drying also slows down the action of

enzymes but does not inactivate them (Harison and Andress, 2000). Dried

foods are tasty, nutritious, lightweight, easy-to prepare, and easy-to-store and

27

use. The nutritional value of food is minimally affected depending on the

drying technique used.

Vitamin A is retained during all of the different drying technique

expect during sun drying, because vitamin A is light sensitive and is denatured

due to excessive exposure to sunlight. Vitamin C is destroyed by exposure to

heat. Dried fruits and vegetables are high in fibre and carbohydrates and low

in fat, making them healthy food choices (Medeiros and Ramlay, 2009).

Complete drying is important since food that are not completely dried are

susceptible to mold and may still harbour harmful pathogens that could cause

food borne illness (Medeiros and Ramlay, 2009). The best temperature for

drying vegetables to preserve carotenoids and vitamin C is at 45°C. Higher

temperatures result in higher loses in sun drying and oven drying (Ejoh et al.,

2005). Sun drying vegetables is an inexpensive and effective method of

preserving surplus micronutrient-rich foods. Sun drying is the most widely

used method of drying agricultural produce in most of the developing

countries of the tropical region (Hassan et al., 2007).

The protein content of the bitter leaf was markedly enhanced by drying

and it range from 7.30% in fresh sample to 17.30% in the dried samples. The

foaming capacity of freeze dry sample is higher than that of sun dry. Freeze

drying has significantly higher (at P<0.05 level) foaming capacity and protein

28

solubility but lower water absorption capacity, solubility and bulk density

(loosed and packed) than the sun dry sample. The result of this study suggests

that drying improves the concentration of both the organic and mineral

constituent and could be useful in preservation.

29

CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 PROXIMATE ANALYSIS

The following analysis was carried out on the leaf extract in duplicate.

Reagent

All reagents used were of analytical grade. These are; 0.1M HCl, 40%

NaOH, Drangendroff’s reagent, Ferric Chloride, Ammonia Chloride, 100%

methanol, methyl orange, Chloroform and 1,1-Diphenyl -2-

Picrylhydrazyl(DPPH).

Materials

Blender, knife, freeze drier, oven, beaker, conical flask, foil paper and

UV spectrophotometer.

Collection of Samples

Sample of the vegetable was randomly collected from local farms in

Akungba Akoko area of Ondo state, Nigeria. Bitter leaf sample collected was

divided into four portions and were designated as follows:

1. Fresh sample.

2. Sun dry sample at ambient temperature for 7 days.

3. Oven dry sample at 650c for 3 hours.

4. Freeze dry sample at standard temperature and pressure.

30

2.2 DETERMINATION OF MOISTURE CONTENT

This is one of the most important and widely used measurements in

samples that absorbs and retain water. Chemical analyses of the leaf extract

were made in dry matter basis.

DRYING METHOD (INDIRECT DISTILLATION): This is considered to be

the most reliable method of moisture content determination.

5g of each sample was weighed in a clean-dried evaporating dish and

weighed as W1,the evaporating dish with the samples reweighed as W2.The

dishes were transferred into a dessicator immediately after each weighing

until all the weighing are completed to prevent absorption of moisture from

atmosphere. The dishes and contents were transferred from the dessicators

into the oven at about 1050c for about 3hours after which they are removed,

cooled in a dessicators and weighed. They were later returned back to the

oven and re-dried for further 30minutes, cooled and weighed. The process of

heating, cooling and weighing continued until a constant weight was obtained

and recorded as W3.

% Moisture content= W2-W3 ×100%

Weight of sample used

31

2.3 DETERMINATION OF ASH CONTENT

A crucible was washed, rinsed with distilled water and dried in the

oven flow to cool in a dessicator. The weight of empty crucible (W1) was

measured using a weighing balance. The sample was added and the weight

was taken (W2), and then transferred into a muffle furnace maintained at

5500c for about 3 hours. The ash obtained plus crucible was allowed to be

cooled in a dessicator and weighed (W3).

% Ash content=Weight of ash (g) × 100

Weight of sample

2.4 DETERMINATION OF CRUDE FIBRE

This is the substance that remains after the defatted sample has been

treated with dilute acid and base respectively. It was originally thought to be

the indigestible portion of man’s food since it contains cellulose, which can

only be digested by ruminants and non- ruminants to a considerable extent.

Crude fibre gives a distinction between the most digestible and least digestible

carbohydrate. Boiling the sample with acid, and then with base (NaOH) to

neutralize the acid dissolves starch and protein. The residue of cellulose and

lignin are wasted, dried and weighed. The residue is ashed and weighed;

weight of ash is subtracted from the weight of the residue.

32

2g of the defatted samples were weighed into a 500ml conical flask

and recorded as W1. 200ml of boiling 1.2% H2S04 was added. The solution

were boiled gently for 30minutes under specified conditions, filtered through

a muslin cloth stretched over 9cm3 buchner funnel and scraped back into the

flask with scapula 200ml of boiling 1.25%w/v NaOH was added and solutions

were built gently for 30 minutes. These were again washed thoroughly with

hot distilled water and were rinsed once with 10%v/v HCL and twice

industrial 100% Ethanol. The residues were rinsed finally three times with

petroleum ether. Residues were transferred into preweighed crucible labeled

W2 and dried in an oven at 1050c, cooled in desicator and weighed W3. These

were later transferred into a muffle furnace at about 3000c for about 30

minutes, removed from furnace and then cooled in a desicator, incinerated and

weighed as W4.

% Crude fibre = W3-W4 × 100%

W2-W1

W2-W1= is the weight of sample used.

2.5 DETERMINATION OF CRUDE PROTEIN

This is the equivalent to the amount of Nitrogen absorbed through

qualitative analysis. The Kjeldahl procedure is used. Amino acid is forced out

of solution by distillation, and NaOH is added to neutralize the H2SO4 which

33

forms ammonium sulphate. The ammonia produced is then measured

accurately by titration with HCL using mixed indicator (bromo cresol green

and methyl red).

The protein content of the sample can be determined by multiplying

the percentage of nitrogen by a factor (6.25) since the average protein contains

approximately 16% Nitrogen (AOAC. 1990). This is equivalent to the

amount of NH3 absorbed through qualitative analysis.

About 5g of the samples were weighed and digested with concentrated

using a digestion catalyst (selenium) to convert organic nitrogen to NH4 ions.

Alkalis were added which liberated NH4. Resulting solutions were distilled

into excess of boric acid solution. The distillates were then titrated with 0.1M

HCl to determine the NH3 absorbed in the boric acid (Pearson, 1976). This

method of determining nitrogen is divided into three steps.

2.5.1 Digestion Stage

About 1.0g of each of the samples was weighed into kjeldahl digestion

flasks with selenium added as catalyst. 10cm3 of concentrated H2SO4 was

added and mixtures were digested on an electro-thermal heater until clear

solutions were obtained. The flasks were allowed to cool after which the

solutions were diluted with distilled water and made up to 100cm3 using

standard flasks.

34

2.5.2 Distillation Stage

It involves steam distillation of the cooled, diluted samples to which

40% of NaOH solution is added to make alkaline. Three drops of mixed

indicator, which composes 0.016g methyl red and 0.083g- bromocresol green

in 100cm3 alcohol, were added to the receiver flask containing 10cm3 of 20%

Boric acid solution; samples turn pink. The distillation is carried out with all

punched corks closed with the end of the condenser below the surface of the

receiving flask containing the boric solution. As distillation continues till the

distillate is about 20ml after which the delivery end of the condenser is rinsed

with distilled water (Pearson, 1976).

(NH3)2 SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

The received NH3 forms a complex with boric acid.

NH3 + H3BO3 NH4H2BO2

HBO2 +NH3 NH4+ +BO2

2.5.3 Titration Stage

The received NH3 and boric acid are titrated with standard 0.1m HCl

solution. The Colour changes from pink to blue.

% Nitrogen can be calculated as:

35

%Nitrogen = titre value 0.1× 0.014× 100 ×50/5

Weight of the sample used

%Crude protein =% Nitrogen ×6.25

2.6 DETERMINATION OF FAT CONTENT

Mixtures of various glycerides of fatty acids that are soluble in certain

organic solvents are called fats. The soxhlet extractor with a suitable solvent

like petroleum ether or diethyl ether amongst others is used for the extraction.

Continuous extraction of the fat content with the solvent can be done in

separating funnel (AOAC, 1990).

A dried thimble is previously weighed as W1. 3g of powdered sample

is put into a fat free extractor thimble inserted into extractor chamber. A

soxhlet extractor is used and the set up is careful checked to make sure that all

joints are tight including the 500cm3 and bottom flask filled with petroleum

ether up to three quarter level. Heat is applied using water bather. The boiling

and siphoning process continues until the fat content has been removed

noticeable by a wireless liquid siphoning back. The thimble containing the

sample W2 is oven dried then cooled in a dessicator and the weight of the

sample is W3. The difference in weight of the sample is the fat content.

%fat= W2-W3 × 100%

W2-W1

36

NOTE: During analysis of the samples for fat, the fat content of the

fresh sample was converted from its dry basis to wet using the formular:

% of Wet Basis = (100-Moisture Content) ×%fat

100

2.7 CARBOHYDRATE CONTENT

The most common approach of carbohydrate content of food is used. It

is calculated as the difference between the total predominant content in

percentage and 100%.

% Carbohydrate=100% - (%ash+%crude protein+%fat+%crude

fiber+%moisture).

2.8 PHYTOCHEMICAL ANALYSIS

Collection of Samples

Sample of the vegetable was randomly selected from local farms in

Akungba Akoko area of Ondo state, Nigeria. All samples were randomly

collected aseptically in a sterile foil paper and a sterilized container which are

tied and labeled appropriately in readiness for phytochemical and nutritional

analysis.

37

Preparation of Sample for Analysis

200g each of the bitter leaf was subjected to sun drying at ambient

temperature for 7 days, oven drying at 650C for 3 hours, and freeze drying

technique using lafreez FD-12-MR at a standard pressure and temperature for

2 days. Each of the dried samples was blended to powder. However, 200g of

the freshly plugged leaf was also blended. 20g each of the dried powder and

the freshly blended leaf were subjected to crude aqueous extraction method,

by adding 200ml 0f distilled water to the four samples each in an air tight

conical flask. The samples in the air tight conical flask were then soaked for

24 hours before it was filtered using watman filter paper No.1. 1ml each of the

filtrate was arranged in test tubes in readiness for phytochemical analysis.

The phytochemical components of the powdered plant leaves were

analyzed according to the method described by Trease and Evans (1989).

2.8.0 Test for Alkaloids

1ml of each of the filtrate was stirred with 5ml of 1% aqueous HCl on

a steam bath and a few drops of Dragendroff”s reagent was later added. A

reddish-brown precipitate indicates the presence of alkaloids (Kumar et al.

2009).

38

2.8.1 Test for Saponins:

About 1ml of the filtered plant extract was put in a test tube and 2ml of

distilled water was added and shaken vigorously. Formation of frothing or

foam which persisted on warming was taken as preliminary evidence for the

presence of saponins.

2.8.2 Test for Tannins:

2ml of distilled water was added to about 1ml of the filtrate in a test

tube.1ml of ferric chloride reagent was the added. A blue- black precipitate

was taken as an evidence of the presence of tannins.

2.8.3 Test for Phlobatanins:

Deposition of a red precipitate when an aqueous extract of the plant

was boiled with 1ml of 1% aqueous hydrochloric acid was taken as evidence

for the presence of phlobatannins.

2.8.4 Test for Anthraquinones:

1ml of each of the plant filtrate was shaken with 2ml benzene and 2ml

of 10% ammonia solution was added. The mixture was shaken and the

presence of a pink red or violet colour in the ammoniacal (lower) phase

indicates the presence of free anthraquinone.

2.8.5 Cardiac Glycoside Test:

The following tests were carried out to test for cardiac glycoside:

39

- Lieberman’s Test: 1ml of acetic anhydride was added to 1ml of the

filtrate and cooled well in ice. Sulphuric acid was then carefully added.

A colour change from violet to blue to green indicated the presence of

a steroid nucleus (i.e aglycone portion of the cardiac glycoside).

- Salkowski Test: 2ml of chloroform was added to 1ml of the filtrate,

and then sulphuric acid was carefully added to form a lower layer. A

reddish brown colour at the interface indicated the presence of a

steroidal ring.

2.8.6 Flavonoids Determination:

2ml of dilute ammonia solution was added to the aqueous filtrate of

the sample followed by addition of concentrated H2SO4. A yellow

colouration observed indicate the presence of flavonoids. The yellow

colouration disappears on standing (sofowora, 1993).

2.8.7 Steroids:

4ml of acetic anhydride was added to 1ml of the sample, with 2ml

H2SO4. There was a colour change from violet to blue or green in some plant

indicate the presence of steroids.

40

2.8.8 Terpenoids:

1ml of the sample was mixed with 2ml of chloroform and 1ml of

concentrated H2SO4 to form a layer. A reddish-brown colour at interface was

seen this showed the presence of terpenoids

2.8.9 Cardenolide test:

To 1ml of the samples, 2ml of glacial acetic acid containing one drop

of ferric chloride solution was added. This was then layered with 1ml of

concentrated sulphuric acid. A brown ring obtained at the interface indicated

the presence of a deoxy sugar characteristic of cardenolides. A violet ring may

appear below the brown ring while in the acetic acid layer; a greenish ring

may form just above the brown ring and gradually spread throughout the layer

(Trease and Evans, 1978)

2.9 DPPH SCAVENGING ACTIVITY

2.9.0 Chemicals

1,1-diphenyl-2-picryl-hydrazyl (DPPH·), 100% methanol and trolox

equivalent(ascorbic acid).

2.9.1 Plant Materials and Extraction Procedures:

Methanolic extraction was used. 10g each of the dry powdered bitter

leaf sample as well as the fresh sample were soaked in 100ml of 100%

methanol for 24 hours. Each sample was then filtered over whatman No.1

41

filter paper.0.004g of DPPH was dissolved in 100ml of the methanol. Then,

0.9ml of the solution was added to 0.1ml of each of the filtrate, which was

used for antioxidant capacity test. The mixture was shaken vigorously and

allowed to stand at room temperature for 30 minutes. Then the absorbance

was measured at 517 nm in a spectrophotometer. Lower absorbance of the

reaction mixture indicated higher free radical scavenging activity (Gulcin and

Ak, 2008). Trolox equivalent was used as a reference standard for this

analysis.

2.10 METAL ANALYSIS

2.11 Digestion of Samples for Metal Analysis:

The vegetable samples were weighed to determine the fresh weight

and dried in an oven at 80oC for 72 hours to determine their dry weight. The

dry samples were crushed in a mortar and the resulting powder digested by

weighing 0.5g of oven-dried ground and sieve (<1mm) into an acid washed

porcelain crucible and placed in a muffle furnace for four hours at 500OC. The

crucibles were removed from the furnace and cooled. 10ml of 6M HCl was

added covered and heated on a steam bath for 15minute. Another 1ml of

HNO3 was added and evaporated to dryness by continuous heating for one

hour to dehydrate silica and completely digest organic compounds. Finally,

5ml of 6M HCl and 10ml of water were added and the mixture was heated on

42

a steam bath to complete dissolution. The mixture was cooled and filtered

through a Whatman no.1 filter paper into a 50ml volumetric flask and made

up to mark with distilled water.

2.12 Determination of Metals

Determination of Sodium (Na), Calcium (Ca), Iron (Fe), Copper (Cu),

Zinc (Zn), Magnesium (Mg) and Potassium (K) were made directly on final

solution of the sun dried samples using Perkin- Elmer Analyst 300 Atomic

Absorption Spectroscopy (AAS).

43

CHAPTER THREE

3.0 RESULTS

The proximate analysis of the fresh, oven dried, freeze dried and sun

dried samples of the bitter leaf is presented in Table 1 below. The table

showed significant difference in the composition (g%) of moisture, ash, crude

fibre, crude protein, fat and carbohydrates of each of the samples.

Table 1: Proximate Composition (g%) of fresh, oven dried, freeze dried and

sun dried samples of bitter leaf (Vernonia amygdalina).

Sample Moisture Ash Crude Fibre

Crude Protein

Fat Carbohydrates

Fresh 72.13±0.83 2.23±0.13 1.52±0.12 7.32±1.15 3.38±0.46 13.44±1.77

Oven dry

9.55±0.07 11.21±0.26 8.07±0.72 17.25±1.16 11.34±0.05 42.59±0.68

Freeze dry

7.50±0.18 11.48±0.03 14.11±0.40 11.60±1.10 10.56±0.33 44.76±0.96

Sun dry

8.50±0.37 14.23±0.59 8.83±0.75 9.45±0.0 9.74±0.35 49.26±0.56

44

0

10

20

30

40

50

60

70

80

Moisture Ash Crude Fibre Crude

Protein

Fat CHO

Fig 3: Proximate analysis of fresh and dried Vernonia

amygdalina leaves extract

Fresh

Oven dry

Freeze

dry

Sun dry

45

Table 2 below shows the trolox standard antioxidant activity, in terms

of its concentration and its absorbance measured in a UV spectrophotometer.

Table 2: Trolox Standard Antioxidant Activity

Trolox Concentration(mg/100g) Absorbance (517nm)

8.0 0.067

6.4 0.089

4.8 0.304

3.3 0.338

1.6 0.426

0 0.680

46

Fig 4 below shows a downward slope of a straight line curve of the

absorbance versus the concentration of the trolox standard.

y = -0.0736x + 0.6129

R² = 0.9346

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8 10

Ab

sorb

an

ce (

51

7n

m)

Concentration mg/100g

Fig 4: Trolox Standard Curve

47

Table 3a: Showing the absorbance of fresh and dried samples of Bitter leaf

(Vernonia amygdalina).

Samples

DPPH(ml) Amount(ml) Absorbance(517nm)

Fresh 0.9 0.1 0.270

Freeze dried 0.9 0.1 0.214

Sun dried 0.9 0.1 0.166

Oven dried 0.9 0.1 0.057

Table 3b: Absorbance and the calculated concentration of the fresh and the

dried samples of Vernonia amygdalina.

Samples Absorbance(517nm) Concentration(mg/100g)

Fresh 0.270

4.669

Freeze dried 0.214 5.420

Sun dried 0.166 6.072

Oven dried 0.057 7.553

Blank 0 8.327

Control 0.320 3.980

48

Fig 5 below shows a straight line curve of the absorbance against the

calculated concentration of the bitter leaf samples.

y = -0.0736x + 0.6129

R² = 0.9346

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8 10

Ab

sorb

an

ce (

51

7n

m)

Concentration mg/100g

Fig 5: Absorbance versus concentration of Bitter leaf (Vernonia amygdalina)

49

Table 4 below shows the phytochemical analysis of the aqueous

extracts of four samples of Vernonia amygdalina, which all tested positive to

saponins, alkaloids, phlobatannins and terpenoids. However, the freeze dry

sample tends to indicate abundance of saponins, while the fresh leaves show

abundance of tannins.

Table 4: Preliminary Phytochemical Screening of oven dry, sun dry freeze

dry and fresh Bitter Leaf (Vernonia amygdalina) extract.

S/NO PHYTOCHEMICALS FRESH

SAMPLE

SUN DRY

SAMPLE

OVEN DRY

SAMPLE

FREEZE

DRY

SAMPLE

1 SAPONINS

+ + + ++

2 ALKALOIDS

+ + + +

3 TANNINS

++ + + _

4 PHLOBATANNINS

+ + + +

5 ANTHRAQUINONE

+ _ _ _

6 STEROIDS

_ _ _ _

7 FLAVONOIDS

_ _ _ _

8 TERPENOIDS

+ + + +

9 CARDENOLIDES

_ _ + _

10 CARDIAC

GLYCOSIDE

-STEROID NUCLEUS

_ _ _ _

11 CARDIAC

GLYCOSIDE

-STEROIDAL RING

+ + + +

+: PRESENT; ++: VERY PRESENT; —: ABSENT

50

Table 5 below shows the mineral composition of the sun dried sample

of bitter leaf. While some of the minerals are in traces, some are very

abundant. The bitter leaf seems to contain more of potassium (K) than any

other mineral elements.

Table 5: Mineral composition (ppm) of dried of Bitter leaf

Sample Na K Ca Fe Cu Zn Mg

Sun dry 8.6 916 70.5 2.2 0.07 0.05 27.52

51

CHAPTER FOUR

4.0 DISCUSSION AND CONCLUSION

4.1 PROXIMATE ANALYSIS

The dominant constituent of the fresh bitter leaf (Vernonia

amygdalina) was moisture, with a mean value of 72.13±0.83%. The amount

of moisture of the fresh bitter leaf in this study is lower than the value of

moisture published by Sobowale, et al.,(2011) in a study of some Nigeria

leafy vegetable which includes Ocinum gratissimum (scent leaf) having the

highest moisture content of 84.00% followed by Bitter leaf with a value of

82.12% and Telfairia. occidentalis with 80.88%. It has been reported that

fresh fruits and vegetables contain as high as 75% water consistent with the

range of moisture level obtained in this study. High moisture content reduces

the shelf life of food substances (Ruberto and Baratta, 2000). Removal of

moisture results in increased concentration of nutrients (Morris, et al., 2004).

The high moisture content of the fresh bitter leaf suggest that the leaf cannot

be stored for long without spoilage, since a higher water activity could

enhance microbial action that leads to spoilage, hence the need for

preservation by drying.

The ash content for the fresh and the three dried samples of Vernonia

amygdalina leaves in this study ranges from 2-14.23%, based on the drying

52

method used. These values are higher compared to 1.8% reported for sweet

potatoe leaves (Asibey-Berko and Taiye, 1999) but lower than 19.61% in

Amaranthus hybridus leaves (Nwaogu et al., 2000).

Ash is the inorganic residue remaining after the water and organic

matter have been removed by heating in the presence of oxidizing agents,

which provides a measure of the total amount of minerals within a food

(McClement, 2003). Higher ash content predicts the presence of an array of

mineral elements as well as high molecular weight elements (Onot et al.,

2007). A study by Nnamani et al., (2009) on Zanthoxylum zanthoxyloides,

Vitex doniana and Adenia cissampeliodes stated that ash content of the test

vegetables ranged from 8.10 - 6.30 %. For this present study, the fresh, oven

dried, freeze dried and sun dried V.amygdalina leaves had its ash content

mean values to be 2.23±0.13%, 11.21±0.26%, 11.48±0.03% and 14.23±0.59%

respectively. These values are higher than that of Cleome gynandra L (spider

flower), which has a total ash content of 2.1-3.0 % (Chweya and Mnzava,

1997).The fresh bitter leaf in this study had the lowest ash content, probably

due to the abundance of moisture in it. However, for the dried ones (i.e. oven

dried, freeze dried and sun dried bitter leaf), the ash content tends to fall

within the same range. Moreover, the sun dried leaves had the highest mean

53

value for ash, 14.23±0.59%. This shows that the dried leaves, especially the

sun dried ones could contains much mineral elements than the fresh bitter leaf

The result for the crude fibre content shows that crude fibre was more

abundant in the dried bitter leaf than in its fresh ones. Mean value for crude

fibre present in the fresh leaves was 1.52±0.12%, whereas, the oven dried, sun

dried and freeze dried bitter leaf have a mean value of crude fibre as

8.07±0.72%, 8.83±0.75% and 14.11±0.40% respectively. The crude fibre

content for Vernonia amygdalina leaves in this report is high compared to

1.3% Tribubus terretris leaves (Hassan and Umar, 2006). This result is also

similar to a study conducted on fresh and dried samples of T. occidentalis and

T. triangulare ( Orhuamen et al., 2012). The dried V.amygdalina leaves have

high level of dietary fibre which varied significantly by the drying technique

used. The freeze dried bitter leaf contains the highest amount of crude fibre.

Craplet and crainplet-Meuner and Tanya et al.,(1979) affirmed that leafy

vegetables are rich in dietary fibre.

High level of dietary fibre in leafy vegetables are advantageous for

their active role in the regulation of intestinal transit, increasing dietary bulk

and increasing faeces consistency due to their ability to absorb water. Dried

fruits and vegetables are high in fibre and carbohydrates and low in fat,

making them healthy food choices (Medeiros and Ramlay, 2009). Dietary

54

fibre helps to reduce serum cholesterol level, risk of coronary meat disease,

colon and breast cancer and hypertension (Ganong, 2003).

The proportion of the crude protein content of fresh, oven dried, freeze

dried and sun dried V. amygdalina leaves are 7.32±1.15%, 17.25±1.16%,

11.60±1.10% and 9.45±0.0% respectively. The crude protein content of the

fresh and the dried V. amygdalina leaves are higher than protein content of

Momor-dica foecide (4.6%) leaves consumed in Nigeria and Swaziland but

lower than those of I. batatas (24.85% DW), Amaranthus candatus (20.5%

DW), Piper guineeses (29.78% DW) and T. triangulare (31.00% DW).

In this study, the fresh V.amygdalina leaves has the lowest crude

protein content compared to the oven dry V.amygdalina leaves, which has the

highest value for crude protein (17.25±1.16%),although it has been reported

that heat denatures protein (Morris et al., 2004). Since heat denatures protein,

the reason for high protein in the oven dried bitter leaf could be because of the

type of protein present in it or ecological factor before the leaf was harvested.

This calls for a further study on the protein constituent of V. amygdalina

leaves. According to Pearson 1976, plant food that provides more than 12% of

its calorific value from protein is considered good source of protein. This

therefore makes Vernonia amgdalina leaves a good source of crude protein,

meeting the nutritional needs of adults, pregnant and lactating mothers,

55

especially when oven dried at a moderately high temperature as seen in this

study. The protein content can be boosted by mixing this leaves concentrate

with foods that are richer in protein.

Proteins may be categorized based on factors such as solubility and

shape. They are broadly divided in two groups namely: simple and conjugated

proteins. Simple proteins consist of only amino acids as building blocks

whiles conjugated proteins contain amino acids but in addition, a non-protein

or prosthetic group which may be glycoprotein, lipoprotein, chromoprotein

(Osei, 2003). Vegetable proteins are increasingly becoming more important in

that they supply high quality protein especially the essential amino acids

precursors.

The values of the crude fat present in the three dried V. amygdalina

leaves; oven dried (11.34±0.46%), freeze dry (10.56±0.33%) and sun dried

(9.74±0.35%) were higher when compared to those of Talinum triangulare

(5.90%), Baseila alba (8.71%), Amaranthus hybridus (4.80%), Calchorus

africanum (4.20%) (Ifon and Bassir, 1979; Akindahunsi and Salawu, 2005).

The values of the crude fat present in the dried V. amygdalina leaves in this

study were closely related, showing that there was no signifant difference in

the amount of fat present in the leaves, despite the different drying techniques

used. However, the fresh bitter leaf has a fat content of 3.38±0.46%, which is

56

higher when compared with some Nigeria pumpkins (Cucurbita spp) in a

study by Aruah, et al., (2011).

Dietary fats function in the increase of palatability of food by

absorbing and retaining flavours (Anita et al., 2006). A diet providing 1- 2%

of its caloric of energy as fat is said to be sufficient to human beings as excess

fat consumption is implicated in certain cardiovascular disorders such as

atherosclerosis, cancer and aging (Antia et al., 2006). Therefore, V.

amygdalina leaves should be moderately consumed as excess consumption of

it could lead to cardiovascular diseases.

The mean value of the carbohydrate content of the fresh V. amygdalina

leaves (13.44±1.77%), oven dried (42.59±0.68%), freeze dried (44.76±0.96%)

and sun dried (49.26±0.56%), were all lower in values compared to some

leafy vegetables like Tribulus terrestris 55.67% and 54.20% reported for

water spinach leaves (Asibey-Berko and Tayie, 1999). In a study of the

proximate composition of Acalypha hispida leaves, carried out by Iniaghe, et

al., (2009), the carbohydrate content of the Acalypha hispida leaves (44.48%)

is close to that of the oven dried and freeze dried V. Amygdalina leaves but a

little bit lower than the sun dried ones. The fresh bitter leaf in this study, has

the lowest Carbohydrate content (13.44±1.77%), but very high when

compared to the carbohydrate contents of Indian spinach (7.52%), scent leaf

57

(1.22%), Amaranthus hybridus(3.36%) and Telfaira occidentalis(1.16%)

(Asaolu, et al., 2012). This signifies that V. amygdalina leaves is rich in

carbohydrate and hence a good source of energy.

4.2 PHYTOCHEMICALS

Phytochemicals are natural bioactive compounds from plants with

general benefits to human health.

The result obtained from the phytochemical screening of fresh, oven

dried, sun dried and freeze dried Vernonia amygdalina leaves extract

contained saponins, alkaloids, terpenoids and glycoside steroidal ring.

Steroidal compounds are of importance due to their relationship with some

compounds such as sex hormones (Okwu, 2001). Cardiac glycosides,

terpenoids and alkaloids have been reported to exert inhibiting activity against

most bacteria (Camacho-Corona et al., 2008; Al-Bayati and Suleiman, 2008).

The presence of alkaloid may be responsible for the analgesic use of this leafy

vegetable by rural communities in south-south Nigerian. However, steroids,

flavonoids, and glycoside steroidal nucleus were not detected in any of the

four V. amygdalina leaves in this study.

Tannins were present in the fresh and sun dried bitter leaves but absent

in the oven dried and freeze dried ones. Anthraquinones was only present in

the fresh bitter leaf but absent in all of the three dried samples. Cimanga et al.

58

(2004) reported the presence of anthraquinone in whole plants of Vernonia

amygdalina. This also relate to a study by Ade-Ademilua Omobolanle

Elizabeth (2013) on anthraquinone in V. amygdalina plant which is present at

a low temperature but destroyed by heat. Anthraquinones serves as laxatives

(Patel et al., 1989). Cardenolides was present in the oven dried bitter leaf

only. The saponin content of the freeze dried bitter leaf was higher than the

others due to its high frothing capacity. Furthermore, higher saponin content

of the freeze dried bitter leaf can be attributed to its high crude fibre content,

as seen in this study. Tannin was abundant in the fresh bitter leaf. Tannin has

been described as an anti-nutrient. It is associated with lower nutritive value

of protein foods (Akwaowo et al., 2000). Nworgu et al., (2007) reported the

reduction of tannin content of Telfaria occidentalis on soaking.

4.3 ANTIOXIDANT

Primary sources of naturally occurring antioxidants are whole grains,

fruits and vegetables. Plant sourced food antioxidants like vitamin C, vitamin

E, carotenes and phenolic acids, which have been recognized as having the

potential to reduce disease risk. Most of the antioxidant compounds in a

typical diet are derived from plant sources and belong to various classes of

compounds with a wide variety of physical and chemical properties. The main

characteristic of an antioxidant is its ability to scavenge free radicals. Highly

59

reactive free radicals and oxygen species are present in biological systems

from a wide variety of sources. These free radicals may oxidize nucleic acids,

proteins, lipids or DNA and can initiate degenerative disease. Antioxidant

compounds scavenge free radicals such as peroxide, hydroperoxide or lipid

peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative

diseases. There are a number of clinical studies suggesting that the

antioxidants in fruits, vegetables, tea and red wine are the main factors for the

observed efficacy of these foods in reducing the incidence of chronic diseases

including heart disease and some cancers. The free radical scavenging activity

of antioxidants in foods has been substantially investigated and reported in

literature by Miller and Rigelhof et al., (2000).

The DPPH (1, 1-diphenyl-2 picrylhydrazyl) free radical scavenging

ability of fresh and dried V. amygdalina leaves extract are presented in Table

3a and 3b. The results revealed that DPPH radical scavenging ability of these

leaves extracts significantly increased with drying.

DPPH radical scavenging of bitter leaf extracts were 0.27nm for fresh

leaves, 0.214nm for freeze dry leaves, 0.166nm for sun dry leaves and

0.057nm for oven dry leaves. However, the fresh bitter leaf has the highest

value for DPPH radical scavenging while the oven dried bitter leaf has the

lowest. As shown in table 4, DDPH scavenging activity was significantly

60

correlated to its concentration (r2=0.9346). This assertion agrees with several

results, where correlation were establish between the total phenol content of

some plant foods and their antioxidant capacity (Crozier et al., 1997; Zhang

and Hamauzu, 2004; Ismail et al., 2004; Sahlin et al., 2004; Stewart et al.,

2000; Turkmen et al., 2005).The concentration of the bitter leaf extracts was

inversely proportional to its absorbance. This signifies that the fresh bitter leaf

contains the least antioxidant with its concentration as 4.66mg/100g while the

oven dried leaves is rich in antioxidant with its concentration as 7.55mg/100g,

followed by sun dried (6.07mg/100g) and freeze dried (5.42mg/100g). High

temperature however, enhances the antioxidant of V. amygdalina leaves in this

study.

4.4 METALS

The mineral compositions (ppm) of the sun dried bitter leaf in this

study are shown below:

Sample Na K Ca Fe Cu Zn Mg

Sample 8.6 916 70.5 2.2 0.07 0.05 27.52

The Na/K ratio in the body is of great concern for prevention of high

blood pressure. Na/K ratio less than one is recommended (www.nap.edu FND

2002). Hence, consumption of sun dried bitter leaf would probably reduce the

risk of high blood pressure because; its Na/K is less than one. This is in

correlation to a study of A. asper (Jimoh, Adedapo, Aliero A.A, Koduru S.

61

and Afolayan A. J, 2010). The calcium (Ca), iron (Fe) and magnesium (Mg)

of V.amygdalina leaves in this study was lower than that of the V.amygdalina

leaves in the Comparative studies on the protein and mineral composition of

some selected Nigerian vegetables, by Omale James and Ugwu Chidiebere

Emmanuel (2010). However, the mineral content of the bitter leaf in this study

is far higher than some other vegetables studied by Omale James and Ugwu

Chidiebere Emmanuel (2010).

Minerals are important for vital body functions such as acid-base and

water balance. Calcium is one of the largest mineral present in the structure of

the body and in bones. Na and K are used as an electron carrier in the body.

Iron (Fe) is an important constituent of Hemoglobin. Vegetables contribute

these minerals and enhance their availability in daily life. However, Cu and

Zn present in this study are in traces.

62

4.5 CONCLUSION

This study revealed that the various drying techniques, namely, sun

drying, freeze drying and oven drying would significantly increase the

nutrient, mineral, and the antioxidant properties of Vernonia amygdalina

(bitter leaf). High moisture in the fresh leaves reduces its nutrient

concentration and hence makes it susceptible to spoilage by microbial

activities. Drying of the bitter leaf at moderate temperature increases and

preserves it nutrient. However, the sun dried and the oven dried V.amygdalina

leaves seem to have the highest nutrient retention ability. High temperature

enhances the antioxidant activity of the V.amygdalina leaves in this study.

Some of the phytochemicals present in this bitter leaf varies with drying.

Anthraquinones seems to be destroyed by heat whereas; cardenolide seems to

be stimulated by oven drying.

Further studies are recommended to uncover the reason(s) behind non

definite approach to nutrients’ retention, especially in the oven dried bitter leaf

probably by carrying in-depth research.

63

REFERENCES:

Adanlawo A and Dairo F, (2006). Nutrition Status of some Nigeria Green

Vegetables. Pakistan Journal of Nutrition;3: 2-3.

Ade-Ademilua Omobolanle Elizabeth, (2013). Shade Conditions Prevents

Anthraquinone Production in leaves of Vernonia Amygdalina Plants.

International Journal of Science and Nature; Vol.4: 713-715 ISSN

2229 – 6441.

Agbede JO, Adegbenro M, Aletorb O, Mohammed A, (2007). Evaluation of

the nutrition value of Vernonia amygdalina leaf protein concentrates

for infant weaning foods. Acta Alimentaria; 36: 387-393.

Akachuku C.O, (2001). Growth of bitter leaf (Vernonia amygdalina, Del.

compositeae) and the nutritive values of its processed and unprocessed

leaves. Discovery and Innovation; 13: 227-233.

Akbari-Asbagh, F., E. Mostafavi, K. Hamdi, O. Azmodeh and A.

Ghasemynejad et al., (2010). Relation of serum and semen

malondialdehyde and total anti-oxidants with sperm parameters in

infertile men. American Journal of Immunology; 6: 43-49.

Akindahunsi A.A, Salawu S.O, (2005). Phytochemical screening and nutrient-

anti-nutrient composition of selected tropical green vegetables. African

Journal of Biotechnology; 4: 497-501.

64

Akwaowo, E. U., Ndon, B.A. and Etuk, E. U. (2000). Minerals and

antinutrients in fluted pumpkin (Telfaria occidentalis Hook F.). Food

Chemistry; 70: 235-240.

Alabi I.D.A., Onibudo M.Z., Amusa N.A., (2005a). Chemicals and Nutritional

Composition of Four Botanicals with Fungitoxic Properties. World

Journal of Agricultural Science; 1:84-88

Al-Attar, A.M., (2010a). Physiological study on the effect of Acalypha

wilkesiana leaves extract on streptozotocin- induced experimental

diabetes in male mice. American Medical Journal; 1: 51-58.

Al-Bayati, F.A., Sulaiman, K.D. (2008). In vitro antimicrobial activity of

Salvadora persica L. extracts against some isolated oral pathogens in

Iraq and Turkish Journal of Biology; 32: 57-62.

Aletor V.A., Adeogun O.A. (1995). Nutrients and antinutrient components of

some tropical leafy vegetables. Food Chemistry 54: 375-379.

Antia BS, Akpan EJ, Okon PA, Umoren IU (2006). Nutritive and nutritive

evaluation of sweet potatoes (Ipomoea batatas) leaves. Pakistan

Journal Nutrition; 5: 166-168.

AOAC, (1990). Official methods of analysis. Association of Official

Analytical Chemists, Washington D.C.; 15th Ed.

65

Asaolu S.S, O. S. Adefemi, I. G. Oyakilome, K. E. Ajibulu & M. F Asaolu.

(2012). Proximate and Mineral Composition of Nigerian Leafy

Vegetables. Journal of Food Research; Vol. 1, No. 3; Published by

Canadian Center of Science and Education, ISSN 1927-0887, E-ISSN

1927-0895.

Asibey-Berko E, Tayie E (1999). The antibacterial properties of some plants

found in Hawaii. Ghana Journal of Science; 39: 91-92.

Atangwho, I. J.,Ebong, P.E., Eyong,E. U and Egbung G. E.(2010). Combined

extracts of Vernonia amygdalina and Azadirachta indica may

substitute insulin requirement in the management of type 1 diabetes.

Research Journal of Medical Science; 5: 35-39.

Babalola GO, Okoh AI (1996). Assessment of Vernonia amygdalina as a

substitute for hop brewing. Tech Q. Master Brew Association. Am.; 33:

44-46.

Balasubramanyam, A., R.V. Sekhar, F. Jahoor, H.J. Pownall and D. Lewis,

(2006). Pathophysiology of adipocyte defects and dyslipidemia in HIV

lipodystrophy: New evidence from metabolic and molecular studies.

American Journal of Infectious Diseases; 2: 167-172.

66

Biggelaar CD, Gold MA (1996). Development of utility and location indices

for classifying agroforestry species: The case of Rwanda Agro forestry.

System; 34: 229-246.

Bonsi M.L.K, Osuji P.O, Tuah A.K (1995a). Effect of supplementing teff

straw with different levels of leucaena or sesbania leaves on the

degradabilities of teff straw, sesbania, leucaena, tagasaste and vernonia

and on certain rumen and blood metabolites in Ethiopian Menz sheep.

Animal Feed Science Technology; 52: 101-129.

Camacho-Corona, M.D.R., Ramirez-Cabrera, M.A., Gonzalez-

Chaisiwamongkol and B. Sripanidkulchai, (2010). Morus alba

enhanced functional recovery after sciatic nerve crush injury.

American Journal of Agricultural Biological Science; 5: 294-300.

Chweya, J. A. and Nameus A. M. (1997). Cat’s whiskers (Cleome gynandra

L.). Promoting the conservation and use of underutilized and neglected

crops.Institute of plant Genetic and crop Plant Research,

Gatersleben/International Plant Genetic Resource Institute, Rome,

Italy.

Crozier A, Lean M E J, McDonald MS,Black C (1997). Quantitative analysis

of the flavonoid content of commercial tomatoes, onions, lettuce, and

celery. Journal of Agricultural Food Chemistry; 45: 590-595.

67

Ejoh RA, Djuikwo VN, Tanya AN (2003). Effect of food preparation

techniques on the nutritional profile of four species of Vernonia. Food,

Nutrition and Health Proceedings Papers,

http://foodafrica.nri.org/nutrition/nutritionpapers.html

Ejoh RA, Tanya AN, Djuikwo VN, Mbofung CM, (2005). Effect of

processing and preservation on the iron and vitamin A (total

carotenoid) levels of some species of Vernonia. Sciences des Aliments;

25: 185-192.

Eleyinmi AF, Amoo IA, Oshodi AA, Hezekiah A, (2004). Evaluation of the

hopping potential of blends of Vernonia amygdalina, Garcinia kola,

and Gongronema latifolium on sorghum lager beer quality and

acceptability. MBAA TQ, 41: 403-407.

Elizabeth Oluwatoyin Orhuamen, Olorunmaiye, Kehinde Stephen, Adeyemi,

Christiana Oreoluwa (2012). Proximate Analysis of Fresh and Dry

Leaves of Telfairia occidentalis (Hook.f.) and Talinum triangulare

(Jacq.) Willd. Croatian Journal of Food Technology, Biotechnology

and Nutrition; 7: 188-191.

Enikuomehin OA, Ikotun T, Ekpo EJA (1998). Evaluation of ash from some

tropical plants of Nigeria for the control of Sclerotium rolfsii Sacc. on

wheat (Triticum aestivum L.), Mycopathoologia; 142:81-87.

68

Erasto P, Griersoon DS, Afolayan AJ (2006). Bioactive sesquiterpene lactones

from the leaves of Vernonia amygdalina. Journal of

Ethnopharmacology; 106: 117-120.

Faboya OOP (1983). The mineral content of some green leafy vegetables

commonly found in the Western part of Nigeria. Food Chemistry; 12:

213-216.

Faboya O (1990). The effect of process handling condition on the ascorbic

acid content of green leafy vegetables. Food chemistry; 38: 297-303.

Fafunso M, Bassir O (1976). Effect of cooking on the vitamin C content of

fresh leaves and wilted leaves. Journal Agricultural Food Chemistry;

24: 354-355.

Fasuyi AO and Aletor VA (2005). Varietal Composition and Functional

Properties of Cassava (Manihot esculenta, Crantz) Leaf Meal and Leaf

Protein Concentrates. Pakistan Journal of Nutrition; 4: 43-49.

Ganjian I, Kubo I, Fludzinski P (1983). Insect antifeedant elemanolide

lactones from Vernonia amygdalina. Phytochemistry; 22: 2525-2526.

Gauong WF (2003). Review of medical physiology. 21st edition, Mc Graw

Hill, companies inc. New York; 316- 318,514.

69

Gbaruko BC, Friday OU (2007). Bioaccumulation of heavy metals in some

fauna and flora. International Journal Environmental Science and

Technology; 4: 197-202.

Harrison J. A. and Andress E. L. (2000). Preserving food: Drying Fruits and

Vegetables. University of Georgia cooperative Service, Page 2.

Hassan S.W., Umar R.A., Maishanu H.M., Matazu I.K., Faruk U.Z. and Sani

A.A. (2007). The Effects of Drying Method on the Nutrients and Non-

nutrients Composition of Leaves of Gynandropsis gynandra

(Capparaceae). Asian Journal of Biochemistry 2; (5): 349-353, ISSN

1815-9923.

Herrera, J. (2008). Activity against drug resistant-tuberculosis strains of plants

used in Mexican traditional medicine to treat tuberculosis and other

respiratory diseases. Phytotheraphy Research; 22:82-85.

Ho, C.K., D. B Stephenson, M. Collins, C.A.T Ferro, and S. j. Brown, (2012):

Calibration strategies: a source of additional uncertainty in climate

change projections. Bull. Amer. Met. Soc; 93, 21-26.

http://foodafrica.nri.org/nutrition/nutritionpapers.html.

Huffman MA, Nishida T, Kaji M, Koshimizu K (1993). Bitter steroid

glucosides,Vernoniosides A1, A2 and A3 and realted B1 from a

70

possible medicinal plant, Vernonia amygdalina, used by wild

chimpanzees. Tetrahedron; 48: 625-632.

Huffman MA (2001). Self-medicative behavior in the African great apes: an

evolutionary perspective into the origins of human traditional

medicine. Bioscience; 51: 651–661.

Ifon ET, Bassir O (1979). The nutritive value of some Nigerian leafy green

vegetables- Part 1: Vitamin and mineral contents. Food Chemistry; 4:

263-267.

Ifon ET, Bassir O (1980). The nutritive value of some Nigerian leafy green

vegetables- Part 2: The distribution of protein, carbohydrates

(including ethanol soluble simple sugars), crude fat, fibre and ash.

Food Chemistry; 5: 231-235.

Igile, G.O., Oleszek, W. and Jurzysta, M. (1994). Flavonoids from Vernonia

amygdalina and their antioxidant activities. Journal of Agriculture,

Food and Chemistry; 42: 2445–2448.

Igile G. O., Fafunso M., Fasanmade A., Burda S., Jurzysta M., and Oleszek

W., (1995). Toxicity of Vernonia amygdalina (Compositeae) Leaves,

Extracts and Purified.

Iniaghe O.M, S.O. Malomo S.O and Adebayo J.O(2009). Proximate

Composition and Phytochemical Constituents of Leaves of Some

71

Acalypha Species. Pakistan Journal of Nutrition; 3: 256-258, ISSN

1680-5194

Jimoh F.O., Adedapo A. A., Aliero A.A., Koduru S. and Afolayan A.

J.(2010). Evaluation of the Polyphenolic, Nutritive and Biological

Activities of the Acetone, Methanol and Water Extracts of Amaranthus

asper. The Open Complementary Medicine Journa; 2, 7-14.

Jisaka M, Ohigashi H, Takegawa K, Huffman MA, Koshimizu K (1993b).

Antitumoral and antimicrobial activities of bitter sesquiterpene

lactones of Vernonia amygdalina, possible medicinal plant used by

wild chimpanzees. Bioscience, Biotechnology and Biochemistry;57:

833–834.

Kayode J (2004). Eco-physiological and conservation studies on Vernonia

amygdalina in Ekiti State, Nigeria. Pakistan Journal Science

Industrial. Research; 47: 227-230.

Koshimizu K (1993a). Steroid glucosides from Vernonia amygdalina, a

possible chimpanzee medicinal plant. Phytochemistry; 34: 409-413.

Koshimizu K, Ohigashi H, Huffman MA, Nishida T, Takasaki H (1993).

Physiological activities and the active constituents of potential

medicinal plants used by wild chimpanzees of the Mahale Mountains,

Tanzania International Journal of Primatology; 14: 345-356.

72

Koshimizu K, Ohigashi H, Huffman MA (1994). Use of Vernonia amygdalina

by wild chimpanzee: Possible roles of its bitter and related

constituents. Physiology and Behavior, 56: 1209-1216.

Laekeman GM, De Clerck F, Vlietinck AJ, Herman AG (1985). Vernolepin:

an antiplatelet compound of natural origin. Naunyn- Schmiedeberg’s

Archives Pharmacology; 331: 108-113.

Mcclements D. J. (2003). Analysis of Food Products. Chenoweth Lab, Room

238. Www-Unix.Oit.Umass.Edu/~Mcclemen/581rheology.Html

Medeiros L. and Remley D. (2009). Drying fruits and vegetables. Ohio State

University Extension;

Morris, A., Barnett A. and Burrows O. (2004). Effect of processing on

nutrient content of foods. Cajarticle; 37: 160-164.

Mucimapura, S., J. Wattanathorn, S. Thongrong, K. Chaisiwamongkol and B.

Sripanidkulchai, (2010). Morus alba enhanced functional recovery after

sciatic nerve crush injury. American Journal Agricultural Biological Science;

5: 294-300. DOI: 10.3844/ajabssp.2010.294.300

Munaya, C. (2013). Bitter leaf-based extracts cures hepatitis co- inferation and

others. The Guardian Newspaper; July, 25, 2013.

Nnamani, C. V., Oselebe, H. O. and Agbatutu, A. (2009). Assessment of

nutritional values of three underutilized indigenous leafy vegetables of

73

Ebonyi State, Nigeria African Journal of Biotechnology; Vol. 8: pp.

2321-2324.

Nwaogu LA, Ujowundu CO and Mgbemena AI (2000). Studies on the

Nutritional and Phytochemcial Composition of amaranthus Hybrides

Leaves. Journal of Bio-research. 4(1): 28 31.

O.M. Iniaghe, S.O. Malomo and J.O. Adebayo (2009). Proximate

Composition and Phytochemical Constituents of Leaves of Some

Acalypha Species. Pakistan Journal of Nutrition 3; ISSN 1680-5194,

256-258.

Obi, RK., Iroagba, II. and OA. Ojiako. (2006). Virucidal potential of some

edible Nigerian vegetables. African Journal of Biotechnology. 5:1785-

1788.

Oboh,G; (2003): Hemolytic effect of saponin extract from vernonia

amygdalina (bitter leaf) on human erythrocyte. Applied Natural

Science Research. 1(14); 25-29

Oboh G, Akindahunsi AA (2004). Change in the ascorbic acid, total phenol

and antioxidant activity of some sun-dried green leafy vegetables in

Nigeria. Nutritional Health 18: 29-36.

74

Oboh G (2005). Effect of blanching on the antioxidant property of some

tropical green leafy vegetables. Lebensm Wiss Technology 38: 513-

517.

Oboh, G (2006). Nutritive value and haemolytic properties (in vitro) of the

leaves of Vernonia amygdalina on human erythrocyte. Nutritional.

Health; 18: 151-160.

Oboh, F.O.J. and Masodje, H.I. (2009). Nutritional and antimicrobial

properties of Vernonia amygdalina leaves. International Journal of

Biomedical and Health Sciences; 5: 51-57.

Oke OL (1965). Chemical studies of some Nigerian vegetables. Experimental

Agriculture; 1: 125-129.

Okoh IA, Babalola G.O, Ilori M.O (1995). Effect of methanol extract of

Vernonia amygdalina on malting and brewing properties of sorghum.

Technical Quarterly Master Brewers Association of Americans, 32:

11-14.

Okoli IC, Ebere CS, Uchegbu MC, Udah CA, Ibeawuchi II (2003b). A survey

of the diversity of plants utilized for small ruminant feeding in south-

eastern Nigeria. Agriculture, Ecosystem and Environment; 96:147-

154.

75

Okwu, D.E., (2001). Evaluation of the chemical composition of indigenous

spices and flavouring Agents. Global Journal of Pure and Applied

Science; 7: 455-459.

Omale J., Ugwu C. E. (2011). Comparative studies on the protein and mineral

composition of some selected Nigerian vegetables. African Journal of

Science; 5(1):22-25

Onot O. E., Umoh I. B. And Eka O. U. (2007). Effect of a Typical Rural

Processing Method on The Proximate Composition and Amino Acid

Profile of Bush Mango Seeds (Irvingia gabonensis).Agriculture,

Nutrition And Development; Volume 7 No. 1. 2007.

Osei S. (2003). Animal Nutrition-Lecture Notes. University of Education,

Winneba. Faculty of Agriculture, Mampong-Ashanti.

Osinubi AAA (2007). Effects of Vernonia amygdalina and chlorpropamide on

blood glucose. Medical Journal of Islamic World Academy Science;

16: 115-119.

Miller, H.E., Rigelhof, F., Marquart, L., Prakash, A., and Kanter, M. (2000).

Journal of Am. Coll. Nutr. 19(3), 312S-319S.

Pearson D (1976). Chemical analysis of foods. 7th ed. London, churchchill,

Livingstone, pp. 218-336. Pakistan Journal of Nutrition 8; (3): ISSN

1680-5194; 256-258.

76

Pieters, L., Totte, J., Vlietink, A. J. (2004). In vitro antiplasmodial activity of

extracts fractions of seven medicinal plants used in the Democratic

Republic of Congo. Journal of Ethnophamacology; 93, 27–32.

Ruberto, G. and M.T. Baratta, (2000). Antioxidant activity of selected

essential oil components in two lipid model systems. Food Chemistry;

69: 167-174.

Sahlin E, Savage GP, Lister CE, (2004). Investigation of the antioxidant

properties of tomatoes after processing. Journal of Food Composition

and Analysis; 17: 635-647.

Sobowale, S.S; Olatidoye, O.P; Olorode, O.O, and Akinlotan, J.V (2011).

Nutritional Potentials and Chemical Value of Some Tropical Leafy

Vegetables Consumed In South West Nigeria. Journal of Sciences and

Multidisciplinary Research; Volume 3: 55-65.

Sofowora, L. A. (1993). Medicinal Plants and Traditional Medicine in Africa.

Ibadan: Spectrum Book Ltd, pp. 55 – 71

Stewart AJ, Bozonnet S, Mullen W, Jenkins GI, Michael EJ, Crozier A

(2000). Occurrence of flavonols in tomatoes and tomato-based

products. Journal of Agricultural Food Chemistry; 48: 2663-2669.

Swee Keong Yeap, Wan Yong Ho, Boon Kee Beh, Woon San Liang, Huynh

Ky, Abdul Hadi Noaman Yousr and Noorjahan Banu Alitheen (2010).

77

Vernonia amygdalina, an ethnoveterinary and ethnomedical used

green vegetable with multiple bioactivities. Journal of Medicinal

Plants Research; Vol. 4(25), pp. 2787-2812, 29; ISSN 1996-0875.

Tangka JK (2003). Analysis of the thermal energy requirements for the

extraction of leaf protein concentrate from some green plants. Biosyst.

Eng; 86: 473-479.

Trease, G.E. and W.C. Evans, (1989). Pharmacognosy; 2nd Edn, Braille

Tiridel and Macmillan publishers.

Turkmen N, Sari F, Velioglu Y (2005). The effect of cooking methods on total

phenols and antioxidant activity of selected green vegetables. Food

Chemistry; 93: 713-718.

Uraih N, Anetekhai WE (1991). Isolation and Utilization of Indigenous Yeast

Strain for Brewing of Millet Beer Oyokpo. Acta Biotechnologica; 11:

31-37.

Zhang D, Hamauzu Y (2004). Phenolics, ascorbic acid, carotenoids and

antioxidant activity of broccoli and their changes during conventional

and microwave cooking. Food Chemistry; 88: 503-509.

78

APPENDIX:

The fat content of the fresh sample was converted from its dry basis to

wet, which was carried out in duplicate using the formular:

% of Wet Basis = (100-Moisture Content) ×%fat

100 = (100-71.54) ×11.20

100

=3.1875%

= (100-72.72) ×13.02

100

=3.551%

Therefore, mean value = (3.1875 + 3.551) %

2

=3.38%

Trolox Standard Antioxidant Activity

Trolox Concentration(mg/100g) Absorbance (517nm)

8.0 0.067

6.4 0.089

4.8 0.304

3.3 0.338 1.6 0.426

0 0.680

Determination of Crude Protein:

Distillation stage

(NH3)2 SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

79

The received NH3 forms a complex with boric acid.

NH3 + H3BO3 NH4H2BO2

HBO2 +NH3 NH4+ +BO2

2.5.3 Titration Stage

The received NH3 and boric acid are titrated with standard 0.1m HCl

solution.

NH4H2BO3 + HCl H3BO3 + NH4Cl

BO2 + H3O+ HBO2+ H2O

Colour changes from pink to blue

% Nitrogen can be calculated as:

%Nitrogen = titre value 0.1× 0.014× 100 ×50/5

Weight of the sample used

%Crude protein =% Nitrogen ×6.25

Reagents:

1. 0.1M HCl

2. 40% NaOH

3. Methyl red

4. Bromocresol green

5. Drangendroff’s reagent

6. Ferric Chloride

7. Ammonia Chloride

8. Concentrated Sulphuric acid

80

9. 100% methanol

10. Chloroform

11. 1,1-Diphenyl -2-Picrylhydrazyl(DPPH)