COMPARATIVE PROXIMATE AND CHLOROPHYLL ANALYSIS OF SOME LEAFY

VEGETABLES.(OHA, UZIZA AND BITTER LEAVES).

TABLE OF CONTENT

INTRODUCTION

1.1 Background of the study

1.2 Aims and objectives of the study
1.3 Scope of the study
1.4 Delimitation of the study
1.5 Significance of the study

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Vegetables Cultivations

2.2 Nutrient Compositions of the Three Vegetables Leave (Oha, Uziza and Bitter leave)

2.2.1 Nutritional composition of Oha Leaves

2.2.2 Nutritional Value of Uziza Leave

2.2.3 Nutritional Value of Bitter leaves

2.3 Phytochemistry

CHATPER THREE

3.0 Methodology

3.1 Materials and Method

3.2 Reagents Name

3.3 Study Area

3.4 Collection of Plant leaves

3.5 Preparation of leaves (Oha Leave, Uziza leave and Bitter leave).

3.6 Proximate Analysis

3.7 Chlorophyll Analysis

3.8 Results and Discussion

CHAPTER FOUR

5.1 Conclusion

5.2 Recommendations

Reference
 CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Green leafy vegetables occupy an important place among food crops as they provide adequate
amounts of vitamins and minerals for humans (Vyankatrao, 2014). They are fresh agricultural
produce having high moisture content and characterized as highly perishable in nature. They also
occupy a vital place in diet because of their colour, flavor, nutrient content and health benefits.

According to Negi and Roy (2010), these vegetables serve as rich sources of β-carotene, ascorbic
acid, iron, zinc, folate and dietary fibre. They are usually consumed as cooked complements to
major staples like cassava, rice, plantain and maize and also used in the preparation of soups and
sauces in Africa. Some are consumed raw, some eaten cooked in order to be edible while others
are dried for shelf-life extension.

Vegetables are the fresh and edible portions of herbaceous plants, which can be eaten raw, or
cooked. Vegetables are valuable in maintaining alkaline reserve of the body. They are valued
mainly for their high carbohydrate, vitamin and mineral contents. Vegetables may be edible
roots, stems, leaves, fruits or seed. Each group contributes to diet in its own way. However, there
are some used and inexpensive leafy vegetables whose nutritive and anti-nutritive potentials are
yet to be adequately studied and utilized. Among these leafy vegetables are the leaves of sweet
potatoes. Sweet potato (Ipomoea batatas) is a herbaceous creeping plant with smooth, lightly
moderate green leaves sometimes with a considerable amount of purple pigmentation especially
along its veins. Its starchy tuberous root is the major economic part of the crop. Occasionally, the
leaves are used as vegetable in yam and cocoyam porridges in some parts of Nigeria particularly
among the Efik-Ibibio people of South-Eastern Nigeria. Islam et al. (2002) further reported that,
Ipomoea batatas leaves as an excellent source of ant oxidative polyphenolics compared to other
commercial vegetables. Though consumed in Asia and some sub-Saharan countries (Duke,
2008), they have generally been regarded as an underexploited green vegetable. Drying is an
important step in maintaining the quality, nutritional value and shelf-life extension of vegetables.
When vegetables are properly dried, they are less susceptible to spoilage caused by the growth of
bacteria, molds and insects (Adeleye, 2018). Plants respond differently to drying. The effect of
drying on plants will depend on the initial moisture content of the product and this varies with
the type of vegetable to be dried. Carried out a study of drying characteristics of some selected
vegetables (okra, onion, red pepper and tomato). It was observed that tomato had maximum
average drying rate of 1.23 kg/hr and onion had the least (0.77 kg/hr). The effect of drying on the
qualities of some selected vegetables was also studied by Awogbemi and Ogunleye (2009) and it
was observed that Bitter leaves had the highest drying rate, followed by Amarantus spp (Tete)
and fluted pumpkin (ugu). It is evident that the changes in moisture content as a function of time
will characterize the drying behavior of the vegetables to be studied. The drying characteristic of
each vegetable is intrinsic and so will behave uniquely during drying (Guine and Fernandes,
2006) which is why there is a need to investigate the comparative effect of drying on the drying
characteristics and nutrient constituents of some common edible leafy vegetables in the country
The objective of this work therefore is to determine the effect of drying on the drying
characteristics, product quality and proximate composition of some selected vegetables in
order to serve as a guide for industrialist and nutritionist in understanding the drying
behaviors of these vegetables so as to maximize nutrient retention.

1.1.1 OHA LEAVE (Pterocarpus mildbraedii)

Harms locally known as “Oha” in the Eastern Nigeria is one of the vegetables consumed widely
in Nigeria. Vegetables are the fresh and edible portions of herbaceous plants, which can be eaten
raw, or cooked. Vegetables are valuable in maintaining alkaline reserve of the body; they are
valued mainly for their high carbohydrate, vitamin and mineral contents. Vegetables are rich
sources of carotene, ascorbic acid, riboflavin, folic acid and minerals like calcium, iron and
phosphorus. In addition, they contain photochemical or anti-nutrients such as phytic acid, tannic
acid and oxalate which reduce their bioavailability.

According to Aletor and Adeogun, some anti-nutritional compounds exhibit protective effects,
thus making them to serve a dual purpose of reducing some essential nutrients and protecting the
body against a number of biochemical, physiological and metabolic disorders. Vegetables may
be edible roots, stems, leaves, fruits or seed. Each group contributes to diet in its own way,
however there are some inexpensive leafy vegetables whose nutritive and anti-nutritive
potentials are yet to be adequately studied and utilized. Among these leafy vegetables are the
leaves of Pterocarpus mildbraedii. The leaves which are used for soup preparation give good
taste and palatability. Pterocarpus mildbraedii is found in Cameroon, Equatorial Guinea, Ghana,
Liberia, Nigeria, Sierra Leone and Tanzania. Pterocarpus mildbreadii Harms is a green leafy
vegetable and Seeds contain three distinct structures. The inside of a seed contains an embryo,
which is a baby plant with a shoot and a tiny root. The two halves of a seed are stored food that
provides the nourishment necessary for seeds to germinate, or begin growing. Surrounding the
seed is a hard, tough seed coat, which protects the seed during dormancy (Anville 2007).

Most seeds contain a built-in food supply called endosperm. The endosperm can be made up of
proteins, carbohydrates and fats. Seeds also contain anti-nutrients in their seed coat. These anti-
nutrients include phytin, lectin, trypsin inhibitor activity, tannin and cyanide. In addition, they
also contain minerals such as sodium,
potassium,calcium,magnesium,phosphorus,zinc,manganese,iron,selenium and copper.(Balogun
2000) Oilseeds are energy dense foods; for example, sesame seeds provide 600kcal or
2470kj/1000g.Although oilseeds contain protein(|14-32g/100g)and carbohydrate(ranging from
less than 1g/100g to more than 34g/100g),most of the food energy they provide is as fat(which
provides 9kcal or 37kj/g).Oilseeds vary widely in their fatty acid composition but tend to be rich
in MUFA(e.g peanut) PUFA(e.g sunflower seeds).Some seed oils contain significant amounts of
EFA, ALNA, an n-3 fatty acid, and linoleic acid(LA),an n-6 fatty acid. From these two fatty
acids, the body can make all the fatty acids it needs. From LA, arachidonic acid can be produced,
and from ALNA the long chain n-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) can be made.
1.1.2 UZIZA LEAVE (Piper guineense)

Piper guineense (uziza) is a local spice that comprises of dillapiol, 5-8% of piperine, elemicine,
10% of myristine and safrole and these chemicals exhibit bactericidal and antimicrobial effects
on certain microorganisms. Useful chemicals have being found embedded in uziza seed and
these chemicals exhibits bactericidial and antimicrobial effects on certain microbes, these effects
is associated with the presence of phytochemicals like flavonoids, alkaloids, tannins, saponins,
phenols, phytates, steroids and essential oil.

Piper guineense is a West African tropical plant. It belongs to the family Piperaceae. The plant is
a climbing vine that can grow up to 20 m in length and bears pepperish berry fruits which are
usually dried to prolong its shelve life. It is commonly known as African black pepper and
popularly called Uziza by the South-Eastern Nigerians and Iyre by the Yorubas. The leaves and
seeds are consumed widely as spice and used in preparation of different dishes. The leaves and
seeds are considered to be medicinal as have been reported in literatures. In some parts of
Nigeria, the seeds and leaves are used in preparation of a popular dish consumed by women after
childbirth, to enhance the contraction of uterine and also to enhance the expulsion of placenta
and other remains from the womb4. They are used for treating rheumatic pains and for weight
control guineense is a spice plant from the family Piperaceae and from genius piper. It is a West
African spice plant commonly called ashanti pepper. It is known as Uziza in Igbo and Iyere in
Yoruba. Other common names are benin pepper, guinea pepper, false cubeb and Kale. The plants
that provide the pepper are vines that grow up to 20m tall climbing up bole of trees by means of
adventitious roots. It is a perennial plant that is characterized by heart shaped leaves and oval,
petiole, alternate and 12cm long. The fruits of P. guineense, Pterocarpus mildraedii and Vernonia
amygdalina occur in clusters, small, reddish or reddish brown when ripe and black when dry.
The plant is native to tropical regions of Central and Western Africa and is semi-cultivated in
countries like Nigeria, where it is found commonly in the southern part. It grows in evergreen
rain forest, forest edges, usually in wet places, gallery forest along rocky rivers of an elevation of
750 – 1680m (Besong et al., 2016). There are more than 700 species of this plant throughout the
tropical and sub-tropical regions of the world. The different parts of the plant have been
characterized and their chemical compositions determined and are used as therapeutic agents in
minor ailments.

According to Daba et al., (2013), Piper guineense is a local spice made of dillapol, 5.8%
piperine, elemicine and 10% myristicine and safrole and these chemicals exhibit bactericidal and
anti-microbial effects on certain microorganisms. In traditional herbal medicine, the seeds of P.
guineense, Pterocarpus mildraedii and Vernonia amygdalina are put into a variety of uses; for
instance in some parts of Nigeria, the seeds are consumed by women after child birth to enhance
uterine contraction for the expulsion of placenta and other remains from the The seed and leaf
extracts are capable of exhibiting a depolarizing neuromuscular activity in a concentration
related manners (Ojinnaka et al., 2016).

P. guineense, Pterocarpus mildraedii and Vernonia amygdalina have nutritional and non-
nutritional factors which are responsible for its aroma, flavour and preservative properties and
proximate analysis of the plant shows that it contains crude protein, fat carbohydrates and
vitamins, the plant contains vitamin C in considerable amount and this could aid the good health
of teeth and gums and also promote healing.

According to Anyanwu and Nwosu, (2004), P. guineense, Pterocarpus mildraedii and Vernonia
amygdalina by its nature is aromatic and carminative and that it is a natural antioxidant, act as
anti-inflammatory, anti-cancer and anti-pyretic agents.

Memudu et al., (2015) studied the effects of dry fruits of P. guineense, Pterocarpus mildraedii
and Vernonia amygdalina on male fertility parameters using adult sprague darhey rats. 200mg/kg
of the extract was given to two groups of the rats for four weeks and eight weeks respectively.
The result showed that the extract improved male reproductive functions, sperm motility, sperm
function and testicular spermatogenesis. In essence, they recommended that the plant should be
used for the treatment of male and female fertility problems especially those associated with
hormonal secretions in humans. Based on the general usefulness and importance of the seeds of
P. guineense, Pterocarpus mildraedii and Vernonia amygdalina, studies were carried out to
determine its proximate and mineral contents in three selected states (Ebonyi, Cross-River and
Delta) in southern parts of Nigeria. Nwosu, (2004),

1.1.3 BITTER LEAVE (Vernonia amygdalina)

Bitter leaf scientifically known as V. amygdalina is one of the most famous plants found in
Africa and Asia. It is the most cultivated species of the genus Vernonia that is about 1,000
species of shrub.V. amygdalina has been the most prominent species in the family of Asteraceae
that had been studied in Normally, V. amygdalina does not produce seeds but its cultivation is
usually done by stem planting and mostly grow in tropical areas. This plant is found majorly
along the drainage, commercial plantation or forest (Yeap et al., 2010).

Vernonia amygdalina is a wooded shrub of about 2 to 10 m height that regenerates rapidly after
planted. The leaves are petiolated in shape with a bitter taste of which its name “Bitter leaf”
spring up. V. amygdalina are being called different local names which vary from country to
country. The bitter taste had been associated with the presence of saponins, alkaloids, tannins,
and glycosides. These made them act as a bittering agent and a hop substitute used for
controlling microbial contamination in beer brewing without reducing the quality of malt. This
plant can be harvested twice per month for the period of seven years. They are popularly used for
food and traditional medicine, their characteristic odor and bitter taste can be reduced either by
washing in several changes of water or by boiling before consumption.

The Medical Traditional Healer Association in Rukararwe, Uganda produced the greenish
powder packed in sachet and consume as tea by patients suffering from malaria, it has also been
reported to be used in soup (Ogbono and Orugbo soups) in Nigeria and Cameron (Ndole dish).
The honey wines called Tei are being produced from the bitter leaf in Ethiopia. The leaves and
roots decoctions have been used in ethnomedicine to treat hiccups, fevers, kidney problems and
stomach disorder.

Several studies carried out on this plant had suggested that it contains different bioactive
compounds, including, flavonoids, saponins, alkaloids, tannins, phenolics, terpenes, steroidal
glycosides, triterpenoids, and several types of sesquiterpene lactones. These bioactive
compounds made them possess different pharmacological properties like antimicrobial, ant
malarial, antithrombotic, antioxidant, anti-diabetic, laxative, hypoglycemic, antihelmintic, anti-
inflammatory, cathartic, anticancer, ant fertility, anti-fungi, antibacterial, and among others.

1.2 OBJECTIVES OF THE STUDY

AIMS

i. To determine the comparative proximate and chlorophyll analysis of three different

vegetables leaves (Oha, uziza and bitter leave).
ii. To evaluate the micronutrient composition and also the nutritional quality of the three
above named leaves,.

OBJECTIVES

i. To compare the proximate and chlorophyll analysis of the three leaves (Oha, uziza, and
bitter leaves, here by determining the best nutrient contents among them.
ii. To know the mineral contents of three different leaves (Oha, uziza and bitter leave).
1.3 SCOPE OF THE STUDY:

The study is set out to determine the proximate and chlorophyll analysis of three major vegetable
leaves with medicinal properties commonly grown in west African. Therefore, the study is
determine to selected peoples who consumed and used this three major leaves as their daily
foods or medicinal. This study was undertaken to estimste mineral content, bioactive
compositions, and antioxidant properties of Oha, Uziza and Bitter leave vegetables.

1.4 DELIMITATION TO THE STUDY

In carrying out this research work the researchers was faced with some constraints such
as lack of readily available materials on the topic under review. As such the researcher had to
carry out investigation by herself and visited the cyber café several times before writing this
project work.

The researcher was also faced with financial constraint during the time of writing this
project work.

Lastly, the researcher was faced with time constraint; this is because the researcher had to
combine the project work with other academic activities such as going to class and also
preparing for the final examination at the institution this reduced the time that was put in by the
researcher in writing the project work.

1.5 SIGNIFICANCE OF THE STUDY

Healthy and nutritional vegetables are not consumed in area where there is minimum rainfall,
this study therefore focuses on the proximate, chlorophyll, chemical and anti-nutritional
composition of these vegetables spices. The nutritional value and anti - nutritional composition
of culinary herbs such as uziza, Oha and Bitter leaves vegetables are very good for the health.

There are also various claims about the usefulness of these spices, especially their use in
fattening homes, and remarkable growth of new born babies whose mothers use these spices
leaves (Piper guineense), Oha leave and bitter leaves are scarcely discussed. Many people
consume only the seedy species and do not concern themselves with their leaves. This study
therefore focuses on the proximate composition and anti nutrient composition of these leaves
spices.
 CHAPTER TWO

LITERATURE REVIEW

2.1 VEGETABLES CULTIVATIONS

According to the World Health Organization (WHO), a matured person should consume daily
400 grams of vegetable to live a healthy life. Similarly, the Government of Nepal suggests a daily
intake of 375 gram of vegetables. However, healthy vegetables are consumed in many parts of
Nigeria, the cultivation of vegetables gas becomes one of the great impact to Nigeria
Agricultural economy, especially the cultivation of Onion has become the most expensive
vegetables been planted, insufficient access to market and seasonal fluctuation of available
vegetable makes it difficult to intake sufficient amount of vegetable for rural households. Thus, a
home garden in rural Nepal is very essential where people can regularly grow vegetables in small
piece of land. However, it is also commonly seen that many rural households do not apply
appropriate cultivation techniques, which result in limited production, and frequent occurrences
of pest and diseases attacks. As a result, it is compellation to enjoy with limited production of
vegetables. Hence, by utilizing effective techniques in home gardens with good seed and
appropriate vegetable cultivation technologies, vegetable production can be significantly
increased. This will enable the rural households to secure more vegetable for their consumption.
At last, it will further contribute to the healthy lives of the rural population. (Yeap et al., 2010).

Vegetables can make a significant difference to smallholder livelihoods. Vegetable production

needs only a small area of land, with minimal capital outlay and can provide access to a valuable
food under subsistence conditions, but also has the potential to provide an initial step towards
establishing an income base for poorer households.
Vegetables form a large and diverse commodity group: although they do not have botanical
features in common, they generally share similarities in cultivation methods. For example,
tomatoes, melon and watermelon are commonly classified as vegetables, although traders and
consumers classify them as fruits (which botanically are correct). (Yeap et al., 2010).

Usually smallholders intensively cultivate vegetables in gardens, and promoting vegetables in

gardens can help smallholders in a number of ways:
However, even though home gardens provide advantages for smallholders, often they are seen as
small and complicated for inclusion in development programmes. This requires appraising
diverse and often location-specific economic, cultural and environmental conditions in
traditional farming systems. However, policy-makers and advisors need to integrate vegetable
gardens into development programmes and provide training and promotion for such initiatives.
(Yeap et al., 2010).

Most vegetables are bulky and perishable, in contrast to staple foods that can be stored. As a
result of improved roads, vegetable production has developed in areas where land and climatic
conditions are good. Improving livelihoods is not only based on increased vegetable production
yields, but also on parallel improvements in associated infrastructure, post-harvest and marketing
activities.

With the idea of vegetables planting in Nigeria, three most common and consumed vegetables
will go into determine the proximate and chlorophyll analysis of them, which are Oha, Uziza,
and Bitter leaves vegetables. (Yeap et al., 2010).

2.2 NUTRIENT COMPOSITIONS OF THE THREE VEGETABLES LEAVE (OHA,

UZIZA AND BITTER LEAVE)

The mineral content of fruit vegetables was done by Mayer, 1997 by comparing the analysis for
the 8 minerals nutrients: sodium (Na), calium(Ca), magnesium (Mg), iron(Fe), Cu and Zn, for
this following research, the nutritional composition of Oha, Uziza and bitter leaves vegetables
will be stated. Nwosu, (2004),

2.2.1 NUTRITIONAL COMPOSITION OF OHA LEAVES

Oha leaves are the leaves from the evergreen tree in Africa. The plant that botanically known as
Pterocarpus mildraedii.Oha leaves are commonly known as Oha leaves in most parts of
Nigeria .Oha leaf is called a medicinal tree in Nigeria that retains its green leaves throughout the
year. It contains many nutrients such as iron calcium, amino acids, iron, potassium, fiber, vitamin
A and C, etc They bring many health benefits for the body.It is used to make various delicious
soup recipes in the southern part of Nigeria. Oha soup is a popular delicious. Nwosu, (2004),
Fig 2.1 Oha leaves

2.2.1.1 BENEFITS OF OHA LEAVES

i. Digestive function.

The first oha leaves benefits for health is for digestive function. Oha leaves are great in
dietary fiber. It helps to improve the digestive function and absorb the nutrient well. Nwosu,
(2004),

ii. Prevent constipation.

The dietary fiber in Oha leaves has the capability to prevent constipation. This fiber helps to
digest the food well so the food residue can be pushed out of the body easily. Nwosu, (2004),

iii. Controls the blood glucose levels.

Oha leaves dietary fiber can absorb and distribute the calorie to be used well in all organs of
the body. It will prevent the accumulation of calorie in the blood that can increase the blood
glucose level. This is the best diet for the diabetic patients to control their blood glucose at a
normal level.
 iv. Helps to treat diarrhea effectively.

Oha leaves contain zinc mineral which has the mechanism in repairing and protecting the
intestinal mucosa. This mechanism will help the intestine can recover quickly so the diarrhea
symptom can be stopped faster.

v. Helps to treat anemia.

Oha leaves are rich in an iron mineral that helps to treat anemia. This mineral plays the
important role in red blood cell formation to create hemoglobin. This health benefit is
supported also by its vitamin C which helps to absorb the iron well.

vi. Boost your immune system.

Oha leaves are rich in vitamin C. This is the best vitamin to boost your immune system. It
will keep the antibody stay alert to fight the microbial.

vii. Help to fight the free radicals.

Oha leaves can fight the free radicals in the body because they contain antioxidant. This
substance will prevent your body from any diseases.

viii. Prevent the cancer cell mutation.

The antioxidant within Oha leaves can prevent the cancer cell mutation. It can inhibit the
tumor growth and spread.

ix. Makes joint healthy and prevent arthritis.

Another important health benefit of Oha leaves is Oha leaves contain manganese mineral
which plays the important role in the cartilage formation and lubricating fluid in the joint.
This mineral will prevent the joint diseases such as arthritis.

x. Helps your heart contract normally.

Potassium mineral in Oha leaves is important to maintain the normal contraction of the heart.
It will make your heart can contract well and the blood can be pumped to all part of the body
effectively.
 xi. Helps the bone formation.

Oha leaves contain calcium which is important in bone formation. This mineral involves also in
the strengthen process of the bone.

xii. Reduce the muscle cramp.

Oha leaves have magnesium mineral which acts as a muscle relaxant. This effect will bring the
Oha leaves have the capability to reduce the muscle cramp.

xiii. Helps the formation of collagen.

Collagen is the important substance to maintain the healthy skin. Oha leaves contain the copper
mineral that helps in the formation of collagen. It is used to create the healthy and young skin.

xiv. Improves the brain function.

Oha leaves contain glutamic acid, a non-essential amino acid substance. This substance is an
essential neurotransmitter that maintains the normal function of the brain. It helps to treat some
brain disorders such as muscular dystrophy, Parkinson disease, or schizophrenia.

xv. Boost the energy especially for athletes.

One of the amino acid in Oha leaves which are called lysine has the capability to boost the
energy, especially for athletes. This substance is needed by the body to maintain the proper
growth and produce the extra energy.

xvi. Builds the body muscle mass.

Oha leaves can help to build the muscle mass through its cysteine amino acid content. Cysteine
helps the body to build protein which is used for the development of muscle mass.

xvii. Controls the normal blood pressure.

Oha leaves have only low sodium content. This is a good diet for persons who have hypertension
problem. In addition, the potassium contained in Oha leaves can also control the normal blood
pressure through its mechanism in regulating the muscle contraction
xviii. Protects the nervous system.

The vitamin B inside Oha leaves is involving to protect the nervous system. It covers the end of
the nerves from any damages and reduces the symptom of numbness or tingling sensation.

xix. Maintains the pH balance in the body.

The green of Oha leaves have the alkaline characteristic which helps to maintain the pH balance
in the body. It will help your body from the excessive acid that can cause several disorders.

xx. Helps to treat malaria.

Oha leaves contain anti-microbial properties that help to treat malaria. This anti-microbial agent
can kill the parasite that can cause malaria.

You can consume this plant as a herb in the hot water or you can cook them as the vegetable for
your various dishes such as salad or soup. The taste is delicious enough to be consumed in
various ways.

2.2.2 NUTRITIONAL VALUE OF UZIZA LEAVE

Piper guineense have nutritional and non-nutritional factors which are responsible for its aroma,
flavor, and preservative properties. The proximate analysis reveals that the plant contains crude
protein, fat, carbohydrate, vitamins and minerals. The essential oil content is high in the range of
0.1 to 5% while the peroxide value and free fatty acid contents are generally low. Nwankwoet al.
also studied Uziza Leaf and found that in addition to the above mentioned, it also contains a high
amount of ash. This implies high mineral content such as calcium, zinc, magnesium, copper and
potassium in the vegetable. The crude fiber content of Uziza leaf was also found to be high, so
consumption of this leaf could aid digestion, absorption of water from the body, bulky stool and
prevent constipation. The fat content of Uziza Leaf was found to be low so could be employed as
part of the weight loss regimen. Also, the protein content of this plant makes it a good source of
plant protein. The study also showed that P.guineense has high carbohydrate content, so its
consumption could provide the body with the energy needed for daily activities Oko also
reported that P.guineense contains vitamin C in considerable amount and this aids the good
health of teeth and gums and also promotes healing. He reported that P.guineense contains
vitamin A and traces of vitamin B1 and B2 which all are important for general good health. It also
contains vitamin E which plays a significant role as an antioxidant. Nwosu, (2004),

Fig 2.2 Piper. guineense leaves

2.2.3 NUTRITIONAL VALUE OF BITTER LEAVE

The analysis had shown that V. amygdalina leaves, stems, and root is enriched with proteins,
fats, fibres, amino acids, minerals vitamins, and carbohydrates (Igile et al., 2012; Alabi and
Amusa, 2005; Owu et al., 2008). The nutritional compositions of V. amygdalina leaf root and
stems varied from one study to another, probably due to different geographical location, genetic,
environmental, harvest conditions, and ecology of the plant. Early studies reported that V.
amygdalina leaves contain carbohydrates (4.31 mg/100 g), proteins (20.2 mg/100 g), lipids (15.0
mg/100 g), acids (10.26 mg/100 g), iodine (35.82 mg/100 g), hydrocyanic acid (0.46 mg/100 g),
total oxalate (0.62 mg/100 g), amino acid, viz: thiamine (170 mg/100 g), pyridoxine (2.6 mg/100
g), ascorbic acid (20.49 mg/100 g), glycine (4.63 mg/100 g), cysteine (1.84 mg/100 g), casein
hydrolysate (96.99 mg/100 g), nicotinamide (1.65 mg/100 g). In another study, there was a
variation in the nutritional values of V. amygdalina leaf, stem and root. (Igile et al., 2012).
Figure: Vernonia amygdalina Source: (Yeap et al., 2010)

TABLE 1: DIFFERENT LOCAL NAMES OF VERNONIA AMYGDALINA

COUNTRY LOCAL NAME(S)

English Bitter leaf
Tanzania Omjunso
Nigeria Onugbo, Ewuro, Etidot,
Ityuna, Oriw, Chusa-doki
Shiwaka
Malaysia South Africa leaf
Rwanda Umubilizi
Cameron Suwaaka
Uganda Labwori, Omubirizi,
Ekibirizi
Ghana Awonoo, Awonwene,
Jankpantire
Congo Mpasi nyioso
Zimbabwe Musikavakadzi
Gabon Ndoki
China Ikaruga
Kenya Olulusia
Ethiopia Grawa, Graw
Source: (Oduah, 2012).

2.3 PHYTOCHEMISTRY
The different parts of the plant have been characterized and their chemical composition
determined. They are used as therapeutic agents in minor ailments. Photochemical are not
vitamins or minerals but are bioactive compound found in plant foods that work with nutrient
and dietary fibers to protect against disease. The presence of photochemical like alkaloids in both
the leaves and seed extracts of (P. guineense, Pterocarpus mildraedii and Vernonia
amygdalina)signified the possession of medicinal properties within the plant. The flavonoids
possess antioxidant, anti-inflammatory, anti-tumor, anti-allergic and antiplatelet properties. They
are also found to have cholesterol lowering ability. Alkaloids which are natural products present
in P. guineense, Pterocarpus mildraedii and Vernonia amygdalina are made up of heterocyclic
nitrogen that has anti-malarial, antihypertensive, antiarrhythmic and anticancer properties.
Alkaloids are being used as CNS stimulant, powerful pain relievers, topical anesthetic in
ophthalmology among others. Tannins are compounds with proline-rich proteins that help to
inhibit the absorption of iron when present in the gastrointestinal lumen thus reducing the
bioavailability of iron due to the presence of compounds that help in the treatment of diseases
like enteritis, gastritis, and esophagitis. Plants that contain tannins as their primary component
are astringent, thus very beneficial for the management of diarrhea, dysentery, inflammation of
the mucous membrane. Saponins have anti-carcinogenic properties and may also play an
important role in antimalarial activity of plants. P.guineense also contains cardiac glycosides in a
significant amount and cardiac glycosides are useful in the management of diseases associated
with the heart. P. guineense, Pterocarpus mildraedii and Vernonia amygdalina contains
dillapiol, 5-8% of piperine, elemicine, 10% of myristicine and safrole and these chemicals
exhibit bactericidal and antimicrobial effects on some micro-organisms. P. guineense,
Pterocarpus mildraedii and Vernonia amygdalina like other members of the piper family
contains 5-8% of the chemical “piperine” which gives them their “heat”. They also contain large
amounts of beta-carophyllene which is being investigated as an anti-inflammatory agent. (Igile et
al., 2012

CHAPTER THREE

METHODOLOGY

3.1 MATERIALS AND METHOD

S/N EQUIPMENT USED Model
1 Thermo-gravimetric analyzer To determine the SDTGA6000
moisture, ash and
volatile matter as
well as calculation
of fixed carbon
value in Oha, uziza
and bitter leaves
2 Bomb calorimeter
3 Ash fusion tester
4. Auto sampling system
5. Dual furnace Used to check
moisture and ash
analyzer
6. Weighing dish
7. Beaker
8. Heating furnace
9 Desiccators Vacuum Using in drying the
solvents

10 Conical flash/glass container For solvent

mixtures

11 Stainless steel knife

12 Laboratory grinder

13 Polythene bags

14 Atomic absorption spectrophometer Buck Scientific

Model 205, East
Norwalk, CT
Table 3.1 Equipments Used

3.2 REAGENTS NAME

 S/N REAGENTS

1. Sulfuric acid

2. Sodium hydroxide

3. Distilled water

4. Acetone tris

5. Nitric acid and perchloric acid

6. Ethanol

7. Hydrochloric acid

Table 3.2 Reagents Igile et al., (2012)

3.3 STUDY AREA

The experiment was conducted in the science laboratory Technology, Departments of

labsci.Waziri Umaru Federal Polytechnic, Birnin Kebbi, Kebbi State.
3.4 COLLECTION OF PLANT MATERIALS

Fresh leaves of Oha, Uziza and Bitter leaves (Peper. guineense, Pterocarpus mildraedii and
Vernonia amygdalin) were bought and collected from new market area, commonly known as
Tsoho Kasuwa Birnin kebbi, Kebbi state, the put into a sepearte polythene bags and labeled
accordingly, Sample A, B and C. they were taken immediately to the laboratory for preparation
and analysis.

3.5 Preparation of leaves (Oha Leave, Uziza leave and Bitter leave).

The three samples leaves (Oha, Uziza and Bitter leaves) were washed with clean water to remove
the dust and dirts. This was macerated and divided into two portions, one part of the bitter leaf
was unwashed and the other part was washed to remove part of the juice that is responsible for
the bitter taste. The extraction was done as follows according to Azu and Onyeagba. Igile et al.,
(2012).

3.6 PROXIMATE ANAYSIS (Moisture, Protein, Ash, Crude Fiber, Fat, Dry Matter, and
Total carbohydrate)

The leaves were individually washed with distilled water and dried in a convection oven (model
OV-160, Gallenkamp BS, England) at 55ºC until a constant weight was obtained. All samples
were ground using a glass mortar to produce a fine composite powder. All chemicals and
solvents used for the assays were of HPLC or analytical grades. Igile et al., (2012).

Determination of moisture, protein, ash, crude fiber, fat, dry matter, and total carbohydrate were
performed at Waziri Umaru Federal Polytechnic science laboratory, Birnin Kebbi State,
Nigeria. Moisture content was determined by drying 2 g of the sample in pre weighed crucible at
105°C for 24 h in a convection oven (model OV-160, Gallen kamp BS, England) and determined
according to standard protocols. Crude protein was determined from total organic nitrogen using
the macro-Kjeldhal method (Koyuncu et al., 2014).

3.6.1 For crude fiber: 1g of the extracted sample was added to 3 mL of H2SO4 followed by
addition of 17 mL of hot 1.25% H2SO4. The solution was boiled for 30 min, allowed to cool and
filtered through filter paper under gravity. The insoluble residue was washed with hot water to
remove the acid. Three-mL of 0.313 MNaOH was added to the residue followed by addition of
17 mL of hot 0.313 MNaOH. The mix was shaken for 30 min and passed through filter paper
under gravity. The residue was washed with 1% HCl followed by washing with boiling water
until no residual acid was present in the final filtrate. The residue was washed with ethanol and
ether before drying in a convection oven (Gallenkamp)at 100°C. Fiber content was calculated by
dividing loss in weight of the sample by the weight of the sample. (Koyuncu et al., 2014).

3.6.2 Fat content: was determined by extracting 2g of the sample with petroleum ether for 12h.
The extract was reduced to half by evaporation and dried at 105°C in convection oven
(Gallenkamp) until a constant weight was obtained. The percent fat content was calculated by
dividing the weight obtained after drying with the weight of the sample for estimation of the fat
content (Anonymous, 2005).

3.6.3 The carbohydrate content: was calculated from the differences in the sum of protein, fat,
moisture, and ash content subtracted from 100 (Anonymous, 2003).

3.6.4 Mineral contents: The leaves were determined using an atomic absorption
spectrophotometer (Buck Scientific Model 205, East Norwalk, CT)(Anonymous, 2005). A 0.5g
amount of crushed composite sample was digested in concentrated nitric acid and perchloric acid
at 115ºC for about 1h to generate the digests solution. An aliquot of the digest solution was used
for determination of calcium, magnesium, sodium, copper, zinc, iron, and phosphorus contents.
Approximately 0.5g of samples was extracted in 5mL of oxalic acid-EDTA. Extracts were
centrifuged at 2991 × g for 15 min. The supernatant was used for determination of ascorbic acid.
Two and half-mL of supernatant was added to a tube containing 2.5 mL of oxalic-EDTA, and 2
mL of 5%ammonium molybdate (w/v), and 1 mL of 5% H2SO4(v/v) added. The mix was
vortexes and incubated at 42°C for 40 min. Absorbance was estimated at760 nm wavelength
using a UV-vis spectrophotometer (model 205, Buck Scientific, East Norwalk, CT). The amount
of ascorbic acid was calculated using a standard curve of L-ascorbic acid.

3.6.5 Total polyphenol content: It was determined from aqueous solutions of each sample
following Singleton et al. (2012). Phenolic content was extracted with 1mL of 100% methanol
for 10 min, re extracted twice and used for analysis. Analiquot of the sample (100 μL) was mixed
with 1.15 mL of distilled water and250 µL of Folin-Ciocalteau’s reagent (1N) and vortexed.
After 3 min incuba-tion 1 mL of 20% Na2CO3was added prior to boiling the mix for 1 min in
awater bath. The solution was cooled, diluted twice, and absorbance was read at725 nm with a
UV-vis spectrophotometer (Buck Scientific). A standard curvewas used for calculation of phenol
concentration in the samples. Singleton et al. (2012).

The extract (1 mL) was incubated at room temperature for 5 min after mixing with 1.7 mL of
30% methanol, 0.3 mL of 0.5 MNaNO2solution and 0.3 mL of 10% AlCl3. With addition of 2.0
mL of 4%NaOH (w/v), absorbance was estimated at 500 nm. The total flavonoid concentration
was calculated from a standard calibration curve. Singleton et al. (2012).

3.6.6 To determine saponins content: The extracts of each sample were boiled together with 20
mL of distilled water in a water bath, and vigorously shaken. Appearance, and persistence, of
frothing before and after warming indicated presence of saponins (Okunlola et al., 2017a).Tannin
was determined by the Folin-Denis colorimetric method. About 0.5g of extracts of each sample
was boiled in 20 mL of water in a test tube and filtered through filter paper under gravity. A few
drops of 0.1% ferric chloride were added. A brownish green or a blue-black coloration indicated
presence of tannins (Okunlola et al., 2017). Means were separated by standard error.

3.6.7 Determination of flavonoids: This was determined according to the method of Harborne.
300 gram of the same was boiled in 150 mL of 2 m HCL solution for 30 min under reflux. It was
allowed to cool and then filtered through What man No 42 filter paper. A measured volume of
the extract was treated with equal volume of ethyl acetate starting with a drop. The flavonoids
precipitated were recovered by filtration using weighed filter paper. The resulting weight
difference gave the weight of flavonoids in the sample.

3.6.8 Determination of carotenoids: A measured weight of each sample was homogenized in

ethanol using a laboratory blender. A 1:10 (1%) mixture was used. The homogenate was filtered
to obtain the initial crude extracts. 20 mL of ether were added to the filtrate to take up the
carotenoids mixed well and then treated with 20 mL of distilled water in a separating funnel. The
other layer was recovered and evaporated to dryness at low temperature (35-50°C) in a vacuum
dessicator. The dry extract was then saponified with 20 mL of ethanolic potassium hydroxide
and left over night in a dark cupboard. The next day, the carotenoid were taken up in 20 mL of
ether and then washed with two portions of 20 mL - distilled water. The carotenoid extract (ether
layer) was dried in a dessicator and then treated with a light petroleum (petroleum spirit) and
allowed to stand overnight in a freezer (-10°C). The next day the precipitation steroid was
removed by centrifugation and the carotenoid extracts was evaporated to dryness in a weighed
evaporation dish, cooled in a dessicator and weighed. The weight of carotenoid was determined
was expressed as a percentage of the sample weight. (Okunlola et al., 2017).

3.7 CHLOROPHYLL ANALYSIS

Chlorophyll was analyzed using method of sims and gamon (200) as follows: 20mg fresh leaf
was grounded and added with 2ml aceton tris (85.15%) then centrifuged at 14000rpm for 30
seconds. One ml supernatant was added with 3ml aceton tris and vortex thoroughly. Absorbance
was measured at wavelengths of 470, 537, 647 and 663nm. The analysis was conducted at the
department of laboratory science and technology, Waziri Umaru Federal Polytechnic, Birnin
Kebbi.
3.8 RESULTS AND DISCUSSION

Proximate and chlorophyll composition of P. guineense, Pterocarpus mildraedii and V----

ernonia amygdalina leaves will be compared on each of the leaves content. On average, the
decreasing order of proximate content of P. guineense leaves was dry matter> carbohydrate>
protein> moisture> crudefiber> ash> fat; that of V. amygdalina was dry matter> protein>
carbohy-drate> fat> ash> crude fiber> moisture. Percent protein content of Pterocarpus
mildraedii leaf was higher than the two leafy vegetables samples.

TABLE 3.3 PROXIMATE ANALYSIS OF piper guineense, Pterocarpus mildraedii and

Vernonia amygdalina leaves

Parameters Piper. guineense Pterocarpus Vernonia amygdalina

mildraedii
Protein (%) ------ ------- -------

Moisture ------- ------- -------

Fats (%) ------- ------- -------

Ash (%) ------- ------- -------

Crude Fiber (%) ------- ------- -------

Carbohydrates ------- ------- -------

(%)

Dry matter (%) ------- ------- -------

Statements of the resulted table
TABLE 3.4 CHLOROPHYLL ANALYSIS AND VITAMIN CONTENTS OF piper
guineense, Pterocarpus mildraedii and Vernonia amygdalina leaves

Parameters Piper. guineense Pterocarpus Vernonia amygdalina

mildraedii
Mg ------ ------- -------

Na ------- ------- -------

Ca ------- ------- -------

Zn ------- ------- -------

Fe ------- ------- -------

Cu ------- ------- -------

Vitamin ------- ------- -------

Statements of the resulted table
 CHAPTER FOUR

5.1 CONCLUSION

5.2 Recommendations

Reference