CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Phytochemicals are the chemicals that are produced by plants. These are produced by the

plant’s primary and secondary metabolism. These phytochemicals help the plant by protecting

them from disease and damage caused by environmental hazards like pollution, UV, stress and

draught. They are also used as traditional medicine and as poisons from ancient days.

Phytochemicals are not the essential nutrients because there is no proof established that they

cause any possible health effects in humans and they are known to have roles in the protection of

human health. More than 4,000 phytochemicals have been catalogued and are classified by

protective function, physical characteristics and chemical characteristics (Balamurugan et al.,

2019).

Oxidative reactions are common in living systems and these reactions usually occur

during cellular metabolism and respiration that produce free radicals such as reactive oxygen and

reactive nitrogen species (Victor et al., 2006). These radicals often initiate chain reactions that

are often toxic to the cells causing several deteriorating effects such as DNA damage, lipid

peroxidation, tissue injury and protein degradation which are major contribution to a number of

diseases like cancer, arthritis, neurodegenerative disorders, atherosclerosis and aging (D’Angio

and Finkelstein, 2000). The harmful effects of these free radicals causing potential biological

damage is termed oxidative stress and occur when the production of these free radicals

overwhelm the body’s ability to defend against them (Kovacic and Jacintho, 2001). The body is

endowed with a number of antioxidant defense mechanisms such as superoxide dismutase,

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thioredoxin, catalase, glutathione, uric acid, ascorbic acid that help mop-up free radicals and

terminate the chain reactions they initiate (Cadenas, 1997). In recent time, there have been

increasing interests in natural antioxidants that may oppose these free radicals with a view of

lowering the risk of diseases associated with them. Of such reservoirs of natural antioxidants are

products of plant origin.

1.2 PHYTOCHEMICALS

Phytochemicals are referred to as plant (phyto) chemicals which consist of wide variety

of compounds that occur naturally in plants. They are produced from plant through primary or

secondary metabolism. They generally have biological activity in plant host and play a role in

plant growth or defense against pathogens, competitors or predators. Phytochemicals stimulate

the immune system, slow the growth rate of cancer cells, and prevent DNA damage that can lead

to cancer and other diseases. Many phytochemicals are antioxidants protecting the cell of the

body from oxidative damage from water, food, and the air (Ashley et al., 2016).

Phytochemicals are bioactive compounds found in plants that work with nutrients and

dietary fiber to protect against diseases. They are non-nutritive compounds. These

phytochemicals are the secondary metabolites present in smaller quantities in higher plants and

they include alkaloids, steroids, flavonoids, terpenoids, tannins and many others (Peteros, 2010).

Many phytochemicals have antioxidant activity and reduce the risk of many diseases. It is

crucial to know the type of phytochemical constituent, thus knowing the type of biological

activity which might be exhibited by the plant. The importance of medicinal plants and the

contribution of phytomedicine to the well-being of a significant member of the world's

population have attracted interest from diverse disciplines (Agbafor and Nwachukwu, 2011).

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 Critical reviews of studies available in the literature support the concept that diets high in

fruits and vegetables reduce the risk of hypertension, CHD, stroke, and other diseases evidenced

by dose-response relationships. Several research groups have confirmed the critical role that can

be played by phytochemicals in reducing the risk for several diseases such as cancer and

inflammatory conditions (Boeing, 2012). For example, recent research has cited various effects

of phytochemical consumption on cancer prevention, reduction in stroke risk, and Type 2

Diabetes prevention. Proposed mechanisms of action for these findings include inhibition of lipid

oxidation, lipid-lowering effects, hypoglycemic- and insulin-lowering effects, antioxidant

activity, anti-inflammatory activity, and anti-proliferative or apoptotic cell death activity

(Thangapazham, 2016).

1.2.1 CLASSIFICATION OF PHYTOCHEMICALS

Phytochemicals are classified as primary or secondary metabolites depending on their

role in plant metabolism. Primary metabolites include the common sugars, amino acids, proteins,

purines and pyrimidines of nucleic acids, chlorophylls e.t.c while Secondary metabolites are the

remaining plant chemicals such as alkaloids, terpenes, flavonoids, tannin, steroids, flavonoids,

saponins, phenolics, glucosides e.t.c (Hahn 1998 and Ramawat et al., 2009).

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Table 1.1: Occurrence and role of major classes of phytochemicals

Class of phytochemicals Occurrence as natural Role in health care

product (%)
Phenolics 45 Anti-oxidants, anti-cancerous,
cytotoxicants, anti-microbials
and vasodilating

Terpenoids and steroids 27 Anti-microbials, detoxifying

agents, strengthners, anti-
rheumatics, anti- malaria,
hepaticidal

Alkaloid 18 Neuropharmaceuticals, anti-

cancerous, sedatives, anti-
microbials, insecticidal

Other chemicals 10 Anti-inflammatory,

Immunostimulating
(Deepak et al., 2016).

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 Phenol: Phenolic compounds represent the largest category of phytochemicals and they

are most widely distributed in the plant kingdom. Phenols are hydroxyl group (OH) containing

class of chemical compounds where the (OH) group is bonded directly to an aromatic

hydrocarbon group. Phenol (C6H5OH) is considered the simplest class of this group of natural

compounds. Being a secondary metabolite, they have an important role as defense compounds

and they exhibit several properties beneficial to humans and its antioxidant properties are

important in determining their role as protecting agents against free radical-mediated disease

processes (Walton et al., 2003).

 Alkaloid: Alkaloids are natural products that contain heterocyclic nitrogen atoms and are

always basic in character. The name ‘alkaloids’ was derived from the ‘alkaline’ nature and it was

used to describe any nitrogen-containing base compound (Muller-Harvey, 1999). Almost all the

alkaloids have a bitter taste. For example, the alkaloid ‘quinine’ is one of the bitter tasting

substances known and is significantly bitter (1x10-5) at a molar concentration (Mishra, 1989).

 Terpenoid: This class comprises of natural products that was derived from five-carbon

isoprene units. Most of the terpenoids have multi cyclic structures that differ from one another by

their functional groups and basic carbon skeletons. These types of natural lipids can be found in

every class of living things and therefore considered as the largest group of naturally occurring

secondary metabolites (Elbein and Molyneux, 1999). Many of these are commercially interesting

because of their use as flavours and fragrances in foods and cosmetics (Harborne and Tomas-

Barberan, 1991). Terpenes are widespread in nature, mainly in plants as constituents of essential

oils. Their building block is the hydrocarbon isoprene, CH2=C(CH3)-CH=CH2.

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 Saponin: Many saponins are known to be anti-microbial, to inhibit mould, and to protect

plants from insect attack. Saponins may be considered a part of plants’ defense systems, and as

such have been included in a large group of protective molecules found in plants named

phytoanticipins or phytoprotectants (Lacaille-Dubois and Wagner, 2000). Saponin mixtures

present in plants and plant products possess diverse biological effects when present in the animal

body.

 Flavonoid: Flavonoids are the largest group of plant phenols and also the most studied

one (Dai and Mumper, 2010). More than 4,000 flavonoids have been recognized, many of which

occur in vegetables, fruits and beverages like tea, coffee and fruit drinks (Pridham, 1960). The

flavonoids appear to have played a major role in successful medical treatments in ancient times,

and their use has persisted up to now. Most flavonoids occur naturally associated with sugar in

conjugated form and may be characterized as monoglycosidic, diglycosidic etc. Flavonoids have

gained recent attention because of their broad biological and pharmacological activities. The

flavonoids have been reported to exert multiple biological properties including anti-microbial,

cytotoxic, anti-inflammatory and anti-tumor activities; but the best-described property of almost

every group of flavonoids is the capacity to act as powerful antioxidants (Shirsat et. al., 2012;

Teiten et. al., 2013) which can protect the human body from the dangerous free radicals and

reactive oxygen species (ROS).

1.3 ANTIOXIDANTS

Antioxidants are molecules that fight free radicals in human body. Free radicals are

compounds that can cause harm if their levels become too high in human body. These free

radicals are the causative agent of some illnesses such as diabetes, heart disease and cancer.

6
Human body has its own antioxidant defenses to keep free radicals in check. Antioxidants are

substances that protect our body cells against the effects of free radicals. Free radicals are among

the molecules produced when human body system breaks down food and they are released by

environmental exposures like tobacco smoke and radiation. Environmental agents initiate free

radical generation, which leads to different complications in the body. The toxicity of lead,

pesticides, cadmium, ionizing radiation, alcohol, cigarette smoke, UV light and pollution may all

be due to their free radical initiating capability. Free radicals can damage cells, and may play a

role in heart disease, cancer and other diseases. Oxidative damage can lead to a break down or

even hardening of lipids, which is the major composition of all cell wall. In addition, other

biological molecules including ribose nucleic acid (RNA), deoxyribosenucleic acid (DNA) and

protein enzymes are also susceptible to oxidative damage.(source:

http://en.wikipedia.org/wiki/antioxidant).

Antioxidants cause protective effect by neutralizing free radicals which are toxic

byproducts of natural cell metabolism. The human body naturally produces antioxidants but the

process is not 100% effective in case of over whelming production of free radicals and that

effectiveness also declines with age (Sies, 2009). Increasing the anti-oxidant intake can prevent

diseases and lower health problems. Research is increasingly showing that antioxidant rich foods

and herbs have health benefits. Medicinal herbs are the richest sources of antioxidants

compounds (Sies et al., 1992).

1.3.1 CLASSIFICATION OF ANTIOXIDANTS

Antioxidants are classified based on solubility in solvent and source

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i. Solubility in solvents: Antioxidants are either hydrophobic or hydrophilic depending on

their solubility in either water or lipid. Lipophilic when they are soluble in lipids and

hydrophilic when soluble in water. Another important application of antioxidants is their

inclusion in food products (like edible oil, flour) to serve as preservative by prevent

deterioration caused by air (oxidation).

ii. Sources: classifications of antioxidant based on their sources are natural antioxidant and

synthetic antioxidants.

a. natural antioxidants: they are naturally occurring antioxidants which are gotten from

living organisms. Natural antioxidants can also protect the human body from free radicals

and retard the progress of many chronic diseases as well as lipid oxidation in foods

Examples are ascorbic acid, tocopherols, carotenoids, polyphenol, glutanione.

b. synthetic antioxidants: they are referred to as man-made antioxidants which are not

gotten from living organisms. Many synthetic antioxidants are characterized by a better

antioxidant activity than natural antioxidants and they are more easily available.

Examples are butylated hydroxyl toluene (BHT), propyl gallate (PG) and butylated

hydroxylanisole (BHA). They are added to food products in order to prevent undesirable

deterioration. However, it has been discovered that synthetic antioxidants are

carcinogenic and that they promote tumors, these lead to the search for natural

antioxidants. (source: http://en.wikipedia.org/wiki/antioxidant).

1.4 DIFFERENCES BETWEEN PHYTOCHEMICALS AND ANTIOXIDANTS

Antioxidants and phytochemicals are naturally occurring families of healthful substances.

Phyto means plant which means phytochemicals are present only in plants, whereas antioxidants

8
may be derived from animal sources as well. Antioxidants may be vitamins, minerals,

carotenoids (lycopene) or other compounds. They play a crucial role in the body metabolism,

protecting cells from unstable and potentially harmful molecules known as free radicals.

Phytochemicals have antioxidant abilities, but some have additional powers as well. For

example, the ability to produce enzymes that limit the action of carcinogens to damage a cell's

DNA, fighting inflammation, ability to limit the development of blood vessels that contains

tumors. (source:www.foodcate.com/community/post/Antioxidants-vs-phytochemicals-what-s-

the-difference%3F/587A3402-2BF7-EFC0-4517 88C398C8445D).

1.5 STATEMENT OF PROBLEM/ JUSTIFICATION

Research shows that Moringa oleifera and Musa paradisiaca claim to have a lot of

economic value such as medicinal and nutritional values. These claims have not been clearly

justified. This research and experiment is therefore centered on investigating, analyzing and

justify this claims. And also to investigate the phytochemical constituent and antioxidant

properties which has been linked to the economic values of these particular plants.

1.6 AIM AND OBJECTIVES OF STUDY

The aim of this research work is to investigate the phytochemical constituents and

antioxidant properties of moringa plant and plantain flower extract and their solvent extracts.

The objectives of this research work are as follows:

1. to obtain extracts from moringa (leaf, seed, coat, pod) and plantain flower using six

solvents (methanol, ethanol. acetone, chloroform, ethyl acetate and water);

2. to determine the percentage yield of extract in each of the solvents.

3. to investigate the qualitative phytochemical screening of the extracts;

9
4. to investigate the antioxidant properties of the extracts and the raw samples and

5. to compare the antioxidant properties of the extract with that of the raw samples.

CHAPTER TWO

LITERATURE REVIEW

2.1 MORINGA PLANT

2.1.1 DESCRIPTION, ECOLOGY AND CULTIVATION OF MORINGA PLANT

Moringa oleifera is a fast-growing, deciduous tree that can reach a height of 10–12 m

(32–40 ft) and trunk diameter of 45 cm (1.5 ft). The bark has a whitish-grey color and is

surrounded by thick cork. Young shoots have purplish or greenish-white, hairy bark. The tree has

an open crown of drooping, fragile branches, and the leaves to build up feathery foliage of

tripinnate leaves. The flowers are fragrant and hermaphroditic, surrounded by five unequal,

thinly veined, yellowish-white petals. The flowers are about 1.0–1.5 cm (1/2 in) long and 2.0 cm

(3/4 in) broad. They grow on slender, hairy stalks in spreading or drooping flower clusters,

which have a length of 10–25 cm (Parotta, 1993).

Flowering begins within the first six months after planting. In seasonally cool regions,

flowering only occurs once a year between April and June. In more constant seasonal

temperatures and with constant rainfall, flowering can happen twice or even all year-round.The

fruit is a hanging, three-sided brown capsule of 20–45 cm size, which holds dark brown, globular

seeds with a diameter around 1 cm. The seeds have three whitish papery wings and are dispersed

by wind and water (Parotta, 1993).

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 Plate 2.1: Moringa leaf
(Source: https://en.m.wikipedia.org/wiki/Moringa_oleifera).

11
 Plate 2.2: Blended moringa leaf
(Source: https://en.m.wikipedia.org/wiki/Moringa_oleifera).

12
 Plate 2.3: Moringa seeds
(Source: https://en.m.wikipedia.org/wiki/Moringa_oleifera).

13
 Plate 2.4: Blended moringa seeds
(Source: https://en.m.wikipedia.org/wiki/Moringa_oleifera).

14
 Plate 2.5: Moringa coat
(Source: https://en.m.wikipedia.org/wiki/Moringa_oleifera).

15
 Plate 2.6: Moringa pods
(Source: https://en.m.wikipedia.org/wiki/Moringa_oleifera).

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 Moringa oleifera Lam. (Moringaceae) is a tree growing up to 5-12 m with an open

umbrella shaped crown native to India, Africa, Arabia, Southeast Asia, South America, and the

Pacific and Caribbean Islands (Iqbal and Bhanger, 2006). It tolerates a wide range of soil

conditions, but prefers a neutral to slightly acidic (pH 6.3 to 7.0), well-drained, sandy or loamy

soil. In waterlogged soil, the roots have a tendency to rot. Moringa is a sun- and heat-loving

plant, and does not tolerate freezing or frost. Moringa is particularly suitable for dry regions, as it

can be grown using rainwater without expensive irrigation techniques. The species is cultivated

in many tropical and sub-tropical regions worldwide, where it is known by various vernacular

names as horseradish tree, drumstick tree, bean oil tree, miracle tree, and “Mother’s Best Friend"

(Osayemwenre, 2015).

17
2.1.2 CLASSIFICATION OF MORINGA PLANT

The scientific classification of moringa plant is shown below:

Table 2.1: Scientific classification of Moringa oleifera

Kingdom Plantae

Class Tracheophytes

Order Brassicales

Family Moringaceae

Genus Moringa

Species M. oleifera

(Source: https://en.m.wikipedia.org/wiki/Moringa_oleifera).

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2.1.3 TRADITIONAL USES OF MORINGA PLANT

Traditionally, the bark, sap, roots, leaves, seeds and flowers of moringa are used in

traditional medicine. Research has examined how it might affect blood lipid profiles and insulin

secretion. Extracts from leaves contain various polyphenols, which are under basic research to

determine their potential effects in humans. Despite considerable preliminary research to

determine if moringa components have bioactive properties, there is no high-quality evidence to

indicate that it has any effect on health or diseases (Sreelatha and Padma. 2009). Various parts

of the plant are used culturally for its nutritional and medicinal values. The leaves can be eaten

fresh, cooked, or stored as a dried powder for later use as a food flavoring or additive (Lockett et

al., 2000; Arabshahi et al., 2007, Abdulkarima et al., 2007) and also as animal feed (Sarwatt et

al., 2002; Soliva et al., 2005; Makkar et al., 2007). The immature seed pods, called "drumsticks",

are commonly consumed in South Asia. They are prepared by parboiling, and cooked in a curry

until soft. The seed pods/fruits, even when cooked by boiling, remain particularly high in vitamin

C (which may be degraded variably by cooking) and are also a good source of dietary fiber,

potassium, magnesium, and manganese (Osayemwenre, 2015).

2.1.4 IMPORTANCE OF MORINGA PLANT

Moringa plant has so many importance and benefits to prevent and cure diseases. The leaves,

pods, coats and seeds of the moringa tree are rich in antioxidants, amino acids, vitamins, and

minerals. Some of the importance of moringa plant are as follows:

a. Anti-inflammatory activity: Moringa extracts contain compounds that help the immune

system of humans and animals to heal and repair damaged tissues by the defending the

system against foreign invaders, such as viruses and bacteria.

19
b. Anti-cancer activity: Moringa extracts contain compounds that suppress the development

of cancer cells. It slows the growth of pancreatic cancer cells and helps chemotherapy work

c. Antioxidant activity: Research shows that antioxidant rich plants (e.g. moringa) have

health benefits. Moringa extracts are rich source of antioxidants that can prevent diseases

and lower health problems.

d. Antibacterial activity: The antibacterial properties of moringa extracts help inhibit the

growth of various pathogens; and it fight against bacterial diseases.

e. Antidepressant activity: Moringa extracts helps in treating depression, anxiety and

fatigue.

f. Cardio-protective activity: Moringa extracts help to lower cholesterol and improve heart

health. It has cardio-protective benefits and it also prevent age related heart and vascular

disorder.

g. Anti-hypertensive activity: Moringa extracts contain compounds that help to stop arteries

from thickening, which can cause blood pressure to rise.

h. Anti-diabetic activity: Moringa help to fight diabetes by balancing blood sugar and

reducing related complications. It also help to fight diabetes by reducing insulin

resistance, a condition where cells in the body are less able to absorb blood glucose.

i. Improving eye health: Moringa extracts contains eyesight-improving properties due to its

high antioxidant levels. Moringa extracts stop the dilation of retinal vessels thereby

prevent the thickening of capillary membranes, and inhibit retinal dysfunction. (Source:

https://en.m.wikipedia.org/wiki/Moringa_oleifera).

20
2.1.5 SIDE EFFECTS OF TAKING MORINGA

Some of the side effects of taking moringa are as follows:

a. moringa possess anti-fertility qualities and is therefore not recommended for pregnant

women;

b. moringa has shown to be effective at lowering blood pressure. Taking moringa alongside

other drugs that lower blood pressure may result in it becoming too low and

c. compounds in the Moringa leaf may aid the thyroid function, however it should not taken

in combination with other thyroid medication.

(Source:https://en.m.wikipedia.org/wiki/Moringa_oleifera).

2.2 DESCRIPTION, ECOLOGY AND CULTIVATION OF PLANTAIN

Plantain, Musa paradisiaca (syn. Musa sapientum) is an herbaceous perennial belonging

to the family Musaceae. Plantains are distinguished from bananas by their fruit which, although

morphologically very similar to bananas, are actually, longer, firmer and possess a higher starch

content and thicker skin than their sweeter relative. Like banana, the plant is tall and tree-like

with a sturdy pseudostem and large broad leaves arranged spirally at the top. The leaves are large

blade with a pronounced central midrib and obvious veins. They can reach up to 2.7 m (8.9 ft) in

length and up to 0.6 m (2.0 ft) in width. Each pseudostem produces a group of flowers from

which the fruits develop in an hanging cluster. In commercial plantations, the parent plant dies

after harvest and is replaced with a daughter plant. However, a plantation can grow for 25 years

or more if managed properly. The trees can reach heights between 2 and 9 m (6.6-29.5 ft). The

plantain plant is a gigantic herb that springs from an unground stem. Most varieties are 3-10

metres (10-33 feet) tall and have a conical false ‘trunk’ formed by the leaf sheaths of long

21
spirally arranged leaves. The fruit, which is green to brown-yellow, is typically larger than the

common banana and is borne in brunches. (source: https://plantainvillage.psu.edu).

Plantain tree is a very useful tree for mankind starting from the fruit, leaf, stem, flower,

everything is beneficial. Among all, plantain flower gains more importance. Plantain flower has

exclusive health-enhancing characteristics. Plantain flower is rich in anti-oxidants, helps to

ward/get rid of infections. They are neutral anti-depressant. It prevents cancer and heart disease.

It is also beneficial to diabetes patient. Musa species is one of the well-known plants of the

musaeae family that have been used in traditional medicine since hundred years to alleviate

various diseases and health problems. Active constituent presence in the plants materials might

be responsible to the beneficial of human health. The most important of these bioactive

compounds of plants are alkaloids, flavonoids, tannins and phenolic compounds (Atindehou et

al., 2002; Edeoga et al., 2005). It is estimated that more than 8,000 phenolics, 25,000 terpenoids

and 12,000 alkaloids have been identified in plants (Saeed et al., 2005), but most of them still

remain unknown and need to be qualified for health benefits (Liu, 2003). These compounds were

known to possess various bioactivities such as antioxidant, antimicrobial, antivirus and

anticancer. Due to their potential to overcome health problems, plant based products have been

produced in industries as botanical drugs, dietary supplements and functional foods.

Recently, little work has been done on phytochemistry specifically on plantain flower of

musa sp. Previous studies done have been focused on other parts of the plant for instance banana

pulps and peels. It has been shown unripe plantain flour contained various phytochemical

compounds such as phenolic, tannins, flavanoids, saponins and alkaloids (Eleazu et al., 2010).

Most scientists have on investigating bioactive secondary metabolites in order to discover new

medicinally and commercially plant drugs.

22
Plate 2.7: Plantain flower

23
 Primary cultivation of moringa plant originated in Southeast Asia while secondary

cultivation originate in West Africa. Other regions with plantain crops include the Southern

United States, the Caribbean, Central America, Bolivia, Peru, Ecuador, Colombia, Southern

Brazil, the Canary Islands, Madeira, Egypt, Cameroon, Nigeria, Uganda, Okinawa, Kerala and

Taiwan. Farmers grow plantains as far north as Northern California and as far south as KwaZulu-

Natal. (source: www.britannica.com/plant/plantain).

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2.2.1 CLASSIFICATION OF PLANTAIN

The scientific classification of plantain is shown below:

Table 2.2: Scientific classification of Musa paradisiaca

Kingdom Plantae

Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta

Class Liliopsida

Subclass Zingiberidae

Order Zingiberales

Family Musaceae

Genus Musa

Species Paradisiaca

(source: https://plantainvillage.psu.edu)

2.2.2 TRADITIONAL USES OF PLANTAIN FLOWER

25
 Ethno-medicinal survey around the world revealed that the flowers of musa sp have been

used to treat many illnesses. Musa flowers have been traditionally used to alleviate menorrhagia,

dysentery, diabetes mellitus (Singh, 1986), heart pain, diarrhea, stomach cramps and infantile

malnutrition (Leonard, 1998). It was reported that the extracts of the flowers possess medicinal

properties for illness such as diabetes mellitus (Alarcon-Aguilara et al., 1998; Pari and Uma-

Maheswari, 1999), anaemia (Pari and Uma-Maheswari, 1999) and malaria (Bagavan et al.,

2010).

2.2.3 IMPORTANCE OF PLANTIAN FLOWER

Plantain flower is useful in many ways to human. It serves the health benefits which are

as follows:

a. curing infection because it possesses compounds which help to prevent pathogenic

growth;

b. overcoming diabetes and anemia by boiling so that it reduces the level of blood sugar

and raise hemoglobin in the body as it is rich in fiber and iron which assists the body in

red blood cell production;

c. the antioxidant property possessed in the plantain flower reduces free radical activity. It

also treats health problems like cancer and premature aging;

d. rich source of vitamin C, A, E, fiber and potassium which are the sources of healthy

nutrient;

e. they are rich in soluble and insoluble fiber. A soluble fiber allows the food to pass easily

through the digestive tracts as it dissolves in water and forms a gel. Insoluble fiber assist

26
 the bulk to undigested products as it is not dissolved in water. Both of it boost the

healthy digestion and absorption of food;

f. they are high source of tannins, acids, flavonoids and other antioxidants which helps to

counteract free radicals and eliminates the oxidative damage which leads to cancer and

heart disease and

g. they boost mood and reduce anxiety because they have high level of magnesium.

(source:https://inksofeverything.home.blog/2020/01/29/plantain-flower-the-known-the-

unknown-benefits/).

27
 CHAPTER THREE

MATERIALS AND METHODS

3.1 SOURCES OF MATERIALS

Moringa pod were gotten from Ayeyemi, Ondo town. Moriga seed, leaf, coat and

plantain flower was collected from Igoba, Akure. All chemicals used for the experiment were of

analytical grade gotten from Mackintech enterprises at Akure, Ondo State.

3.2 METHODS

3.2.1 PREPARATION AND EXTRACTION OF MORINGA (PODS, LEAVES, COATS

AND SEEDS) AND PLANTAIN FLOWER.

The samples were plucked by hand separately, rinsed in water then cut into smaller

pieces and air-dried. The dried samples were ground into powdery form using electric blending

machine and sieved. The powdery samples were packed into different plastic containers which

were properly labeled and kept prior to extraction.

20 g of each dried powdery sample was weighed into six (6) cleaned and dried reagent

bottles; and 200 mL of each solvent (methanol, acetone, water, chloroform, ethyl acetate,

ethanol) was separately added to each bottle and was left for 72 hours during which was

intermittedly shaken. The mixture was filtered. The extracts were evaporated to dryness by

pouring the extract into weighed petridish and kept in a dessicator to allow the solvents to

evaporate. Weight of extract obtained was used to calculate the percentage yield of extract in

each solvent (Arawande and Abitogun 2009).

28
 COLLECTION OF PLANT MATERIAL

RINSING

DRYING

GRINDING

SIEVING

POWDERY SAMPLE

MACERATION

EXTRACTION
(ACETONE, CHLOROFORM, METHANOL, ETHANOL, ETHYLACETATE AND WATER)

Figure 3.1: Preparation and extraction of moringa plant and plantain flower extract

29
3.2.2 Phytochemical screening

The phytochemicals that were qualitatively determined are standard methods of

phytochemicals as described by (Trease and Evans, 1989; Evans, 2002 and Sofowora, 2008)

were adopted.

3.2.2.1 Test for tannins:

About 0.2 g of the extract was taken and 2 mL of 10 % ferric chloride was added. Color

changes into blue black which indicates the presence of Tannin.

3.2.2.2 Test for alkaloids (Wagner’s test):

About 0.2 g of the extract was hydrolyzed by 1 % hydrochloric acid; six drops of

Wagner’s reagent were added. Color changes into brown red/orange precipitate which indicates

the presence of alkaloids.

3.2.2.3 Test for saponins:

About 0.2 g of the extract was added with 5 mL of distilled water, it was shaken for 30

seconds and foam was formed, the presence of foam indicates presence of saponins.

3.2.2.4 Test for terpenoids (Salkowski test):

About 3 mL of chloroform was added to about 0.2 g of the extract and then concentrated

sulphuric acid was added from sides of the test tube. Then presence of reddish brown color

appears at the interface which indicates the presence of terpenoids in extract.

30
3.2.2.5 Test for Cardiac glycosides (Keller - Killiani test):

About 0.2 g of the extract was taken and then 1 mL of glacial acetic acid was added and 1

mL of ferric chloride was added, then 1 mL concentrated sulphuric acid was added from the

sides of test tube, green/blue precipitate occur which indicates the presence of cardiac

glycosides.

3.2.2.6 Test for Steroids (Lierbermann-Burchardt test):

In about 0.2 g of the extract, 1 mL chloroform was added then 3 mL acetic anhydride was

added from sides of the test tube, then two drops of concentrated sulphuric acid was added. It

shows dark green color which indicates the presence of steroids.

3.2.2.7 Test for flavonoids:

About 0.2 g of the extract was taken; dilute sodium hydroxide was added to create intense

yellow color, which on addition of concentrated hydrochloric acid turns into colorless which

indicates the presence of flavonoids.

3.2.2.8 Test for reducing Sugars (Fehling’s Test):

About 0.2 g of the extract was shaken with distilled water and filtered. The filtrate was

boiled with drops of Fehling solution A and B for few minutes. An orange red precipitate

indicated the presence of reducing sugar.

31
3.2.2.9 Test for Phlobatanins:

About 0.2 g of the extract was added with distilled water then shaken and filtered, then 2 mL of

2 % hydrochloric acid was added and boiled, Red colored developed which indicate the presence

of Phlobatannins.

3.2.2.10 Test for phenol:

2 mL of distill water followed by few drops of 10 % ferric chloride was added to about

0.2 g of the extract. Formation of blue or green color occurred which indicates the presence of

phenol.

3.2.2.11 Test for volatile oil:

0.1 mL dilute sodium hydroxide and small quantity of dilute hydrochloric acid was added

to about 0.5 g of the extract, the solution was shaken. White precipitate was formed which

indicates the presence of volatile oil.

3.2.2.12 Test for quinone:

To about 0.2 g of the extract, 1 mL of concentrated sulphuric acid was added. Formation

of red color indicates presence of quinone.

3.2.3 Determination of Antioxidant property:

3.2.3.1 Total Flavonoid:

0.1 g of the extract was weighed into a sample bottle; 10 mL of 80 % methanol was

added and allowed to soak for 2 hours. 0.4 mL of the solution was measured into a 10 mL

volumetric flask, 1.2 mL of 10 % sodium hydroxide, 1.2 mL of 0.2M concentrated sulphuric acid

32
and 3 mL of 3M sodium nitrate was added. 4.2 mL of distilled water was used to make it up. The

absorbance was read using 6850 UV spectrophotometer at wavelength 325 nm. (Mahajan and

Badujar 2008).

Concentration∈(mg/l) x volume of sample x DF

Total Flavonoid (mg/100g) =
Sample weight

3.2.3.2 Ferric Reducing Antioxidant Power (FRAP):

0.1 g of the extract was weighed into a sample bottle; 10 mL of 80 % ethanol was added.

2.5 mL sodium phosphate buffer (0.2M PO2, pH 6.6) and 2.5 mL 1% potassium ferricyanide was

added and incubated at 50˚C for 20 minutes. 2.5 mL of TCA (trichloroacetic acid) was added to

stop the reaction. 2.5 mL of the aliquot was taken and diluted with 2.5 mL distilled water and

0.5 mL 0.1 % ferric chloride was added and allowed to stand for 30 minutes in the dark for color

development. The absorbance was read using 6850 UV spectrophotometer at wavelength 700 nm

(Alachaher, et al. 2018).

Absorbance−Intercept x volume of extract x 100 × DF

FRAP (GAE) = 6
Slope of standard × sample weight × 10

DF: Dilution factor. If not diluted, then DF = 1

3.2.3.3 Total Phenol:

0.1 g of the extract was weighed into a sample bottle; 10 mL of distilled water was added

to dissolve. 1 mL of the solution was pipette into a test tube and 0.5 mL 2N Folin-Ciocalteu

reagent and 1.5 mL 20 % sodium carbonate solution was added. The solution was allowed to

stand for 2 hours and the absorbance was read using a 6850 UV spectrophotometer at

33
wavelength 765 nm. Garlic acid solution was used as standard viz 0.5 mg, 1 mg, 2 mg, 4 mg, 6

mg, 8 mg and 10 mg. (Hagerman, et al. 2000).

Concentration∈(mg/l) x volume of sample x DF

Phenol content mg/100g =
Sample weight

DF: Dilution factor. If not diluted, then DF = 1

3.2.3.4 DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging:

0.1 g of the extract was weighed into a sample bottle and 10 mL of ethanol was added,

stirred for 15 minutes and allowed to stand for 2 hours. 1.5 mL of the extract was pipette into a

test tube and 1.5 mL of DPPH solution was added. The 6850 UV spectrophotometer was zeroed

with ethanol as the blank solution. The absorbance/ optical density of the control (DPPH

solution) was read. The absorbance of the test sample was read at 517 nm. (Teraos, et al. 1988)

Absorbance of control – Absorbance of test sample

DPPH Scavenged % = x 100
|of |control x sample weight

DF: Dilution factor. If not diluted, then DF = 1

3.2.3.5 Iron (Fe2+) Chelation Assay

0.1 g of the extract was weighed into a sample bottle, 150 µL of 500µM FeSO4 was

added. 168 µL of 0.1M Tris-HCl (pH 7.4) and 218µL of saline solution was added. 100µL of

the solution was taken and incubated for 5 minutes, before addition of 13 µL of 0.25 % 1,10-

phenanthroline. The absorbance was read using 6850 UV spectrophotometer at wavelength 510

nm. (Oboh and Omoregie, 2011)

Absorbance of control− Absorbance of exract

% inhibition = Absorbance of exract
× 100

CHAPTER FOUR

34
 RESULTS AND DISCUSSION

4.1 EXTRACTIVE VALUES OF MORINGA PLANT AND PLANTAIN FLOWER

The extractive value (% yield) of moringa leaf, moringa pod, moringa coat, moringa seed

and plantain flower using acetone, chloroform, ethanol, ethylacetate, methanol and water are

contained in Table 4.1. The selection of solvent system largely depends on the specific nature of

the bioactive compound being targeted. Also, different solvent systems are available to extract

the bioactive compound from natural products. Extraction efficiency is affected by the chemical

nature of phytochemicals, the extraction method used, sample particle size, the solvent used, as

well as the presence of interfering substances. Under the same extraction time and temperature,

solvent and composition of sample are known as the most important parameters (Alachaher, et

al. 2018). The result showed that the percentage yield of moringa leaf extract was 11.095±0.805

in ethylacetate, 9.291±2.234 in ethanol, 8.497±0.713 in water, 8.108±2.219 in methanol,

7.039±2.084 in acetone and 3.055 ± 1.735 in chloroform. The percentage yield of moringa pod

extract was 2.146±0.000 in methanol, 1.347±0.000 in ethanol, 1.047±0.000 in ethylacetate,

0.898±0.000 in chloroform, 0.850±0.000 in acetone and 0.199±0.000 in water. The percentage

yield of moringa coat extract was 8.709±0.414 in acetone, 3.333±0.108 in ethanol, 3.160±2.731

in methanol, 3.062±0.534 in ethylacetate, 2.149±1.799 in water and 1.451±0.502 in chloroform.

The percentage yield of moringa seed extract was 30.151±1.221 in acetone, 29.997±1.558 in

ethylacetate, 27.725±2.714 in ethanol, 26.190±3.452 in methanol, 23.942±2.955 in chloroform

and 21.417±10.455 in water. While the percentage yield of plantain flower extract was

16.026±0.000 in water, 13.850±0.000 in ethylacetate 6.216±0.000 in ethanol, 3.825±0.000 in

methanol, 1.690±0.000 in acetone and 1.347±0.000 in chloroform. In all the solvents used for

extraction, it was observed that the extractive value (%) was highest in moringa seed and least in

35
moringa pod. Extractive value (%) of moringa leaf and moringa coat was higher than that of

plantain flower using acetone, chloroform as solvents. However, the extractive value (%) of

moringa leaf and plantain flower was higher than moringa coat using ethanol, ethylacetate,

methanol and water. According to Alachaher, et al. 2018, there are quite number of factors in

which extraction of bioactive compounds depends.

Table 4.1: Extractive value of solvent extracts of moringa plants and plantain flower

36
Solvents *Extractive value (%)

Moringa Moringa Moringa Moringa seed Plantain

flower
leaves pod coat

Acetone 7.039±2.084 0.850±0.000 8.709±0.41 30.151± 1.221 1.690±0.000

4
Chloroform 3.055±1.735 0.898±0.000 1.451±0.50 23.942± 2.955 1.347±0.000
2
Ethanol 9.291±2.234 1.347±0.000 3.333±0.10 27.725± 2.714 6.216±0.000
8
Ethyl 11.095±0.805 1.047±0.000 3.062±0.53 29.997± 1.558 13.850±0.000
4
acetate

Methanol 8.108±2.219 2.146±0.000 3.160±2.73 26.190± 3.452 3.285±0.000

1
Water 8.497±0.713 0.199±0.000 2.149±1.79 21.417±10.45 16.026±0.000
9 5
Note: * = Results are expressed as mean value ± mean deviation

37
4.2 QUALITATIVE PHYTOCHEMICAL SCREENING OF SOLVENT EXTRACTS OF

MORINGA SEED

Qualitative phytochemical screening of solvent extracts of moringa seed is presented in

Table 4.2. Phytochemical screening of six different solvent extract of moringa seed were

examined. The solvents are acetone, chloroform, ethanol, ethyl acetate, methanol and water.

While the phytochemicals considered were alkaloid, flavonoid, saponin, cardiac glycoside,

reducing sugar, tannin, quinone, volatile oil, phenol, terpenoid, phlobatannin and steroid. In all

the solvents used for extraction of moringa seed, it was observed that alkaloid and quinone were

present while tannin, phenol and phlobatannin were absent. Flavonoid was present in ethanol and

ethyl acetate extracts. Saponin was present in acetone, chloroform, ethanol, ethyl acetate and

water extracts. Cardiac glycoside was absent in all extracts except chloroform extract. Reducing

sugar was present in acetone, ethanol, ethyl acetate and water extracts. Volatile oil was present in

all extracts except water extract. Terpenoid was present in ethanol and water extracts. Steroid

was present in acetone, ethanol and ethyl acetate extracts. Among the twelve phytochemicals

examined for moringa seed extract, ethanol had eight, ethylacetate had seven, acetone had six,

chloroform and water had five while methanol had six present in them

38
 Table 4.2: Qualitative phytochemical screening of solvent extracts of moringa seed

Parameter Solvent extract

Acetone Chloroform Ethanol Ethyl acetate Methanol Water
Alkaloid + + + + + +
Flavonoid - - + + - -
Saponin + + + + - +
Cardiac Glycoside - + - - - -
Reducing Sugar + - + + - +
Tannin - - - - - -
Quinone + + + + + +
Volatile oil + + + + + -
Phenol - - - - - -
Terpenoid - - + - - +
Phlobatannin - - - - - -
Steroid + - + + - -
(+): positive = present (-): negative = absent

39
4.3 QUALITATIVE PHYTOCHEMICAL SCREENING OF SOLVENT EXTRACTS OF

MORINGA COAT

The qualitative phytochemical screening of moringa coat which was carried out in six

different solvent extracts was examined and shown in Table 4.3. The phytochemicals considered

were alkaloid, flavonoid, saponin, cardiac glycoside, reducing sugar, tannin, quinone, volatile

oil, phenol, terpenoid, phlobatannin and steriod while the solvents used were acetone,

chloroform, ethanol, ethyl acetate, methanol and water. In all the solvents used for extraction of

moringa coat, it was detected that alkaloid was present while cardiac glycoside, phenol and

phlobatannin were absent. Flavonoid and saponin was present in methanol and water extracts.

Reducing sugar was absent in all the extracts except acetone extracts. Tannin was present only in

methanol extracts. Quinone was present only in chloroform extracts. Volatile oil was present in

acetone, chloroform and methanol extracts. Terpenoid was present in ethanol and ethyl acetate

extracts. Steroid is present chloroform, ethanol and ethyl acetate extracts. Among the twelve

phytochemicals considered for moringa coat extracts, methanol had five, chloroform and water

had four while acetone, ethanol and ethylacetate had three present in them.

40
 Table 4.3: Qualitative phytochemical screening of solvent extracts of moringa coat

Parameter Solvent extract

Acetone Chloroform Ethanol Ethyl acetate Methanol Water
Alkaloid + + + + + +
Flavonoid - - - - + +
Saponin - - - - + +
Cardiac Glycoside - - - - - -
Reducing Sugar + - - - - -
Tannin - - - - + -
Quinone - + - - - -
Volatile oil + + - - + -
Phenol - - - - - -
Terpenoid - - + + - -
Phlobatannin - - - - - -
Steroid - + + + - -
(+): positive = present (-): negative = absent

41
4.4 QUALITATIVE PHYTOCHEMICAL SCREENING OF SOLVENT EXTRACTS

OF MORINGA POD

Qualitative phytochemical screening of six different solvent extracts of moringa pod were

examined and shown in Table 4.4. The solvents used are acetone, chloroform, ethanol, ethyl

acetate, methanol and water. The phytochemicals considered were alkaloid, flavonoid, saponin,

cardiac glycoside, reducing sugar, tannin, quinone, volatile oil, phenol, terpenoid, phlobatannin

and steroid. In all the solvents used for extraction of moringa pod, it shows the presence of

alkaloid while cardiac glycoside, tannin, phenol and phlobatannin were absent. Flavonoid was

absent in all except acetone extract. Saponin was present in acetone, ethanol and methanol

extracts. Reducing sugar was present in all except acetone and ethylacetate extract. Quinone was

absent in all except methanol extracts. Volatile oil was present acetone, ethanol and ethyl acetate

extracts. Terpenoid was present in all the six extracts except chloroform extracts. Steroid was

present in all the six extracts except water extracts. Amidst the twelve phytochemicals observed,

acetone, ethanol and methanol extracts had six; ethylacetate had four while chloroform and water

had three phytochemicals present in them.

42
 Table 4.4: Qualitative phytochemical screening of solvent extracts of moringa pod

Parameter Solvent extract

Acetone Chlorofor Ethanol Ethylacetate Methanol Water
m
Alkaloid + + + + + +
Flavonoid + - - - - -
Saponin + - + - +
Cardiac Glycoside - - - - - -
Reducing Sugar - + + - + +
Tannin - - - - - -
Quinone - - - - + -
Volatile oil + - + + - -
Phenol - - - - - -
Terpenoid + - + + + +
Phlobatannin - - - - - -
Steroid + + + + + -
(+): positive = present (-): negative = absent

43
4.5 QUALITATIVE PHYTOCHEMICAL SCREENING OF SOLVENT EXTRACTS

OF MORINGA LEAF

Qualitative phytochemical screening of solvent extracts of moringa leaf is presented in

Table 4.5. Phytochemical screening of six different solvent extracts of moringa leaf was

examined. The solvents are acetone, chloroform, ethanol, ethyl acetate, methanol and water.

While the phytochemicals considered were alkaloid, flavonoid, saponin, cardiac glycoside,

reducing sugar, tannin, quinone, volatile oil, phenol, terpenoid, phlobatannin and steroid. In all

the solvents used for extraction of moringa leaf, it was detected that alkaloid and flavonoid in all

while quinone, phlobatannin and volatile oil were absent. Saponin was present in ethyl acetate,

methanol and water extracts. Cardiac glycoside and phenol were present in acetone, ethanol,

ethyl acetate and methanol. Reducing sugar was present in ethanol, ethyl acetate, methanol and

water extracts. Tannin was present in acetone and ethanol extracts. Terpenoid was present only

in water extract. Steroid was present in acetone, chloroform, ethyl acetate and methanol extracts.

Among all the phytochemicals examined for moringa leaf extract, ethylacetate and methanol had

seven, acetone and ethanol had six, water had five while chloroform had three phytochemicals

present in them.

44
 Table 4.5: Qualitative phytochemical screening of solvent extracts of moringa leaf

Parameter Solvent extract

Acetone Chloroform Ethanol Ethyl acetate Methanol Water
Alkaloid + + + + + +
Flavonoid + + + + + +
Saponin - - - + + +
Cardiac Glycoside + - + + + -
Reducing Sugar - - + + + +
Tannin + - + - - -
Quinone - - - - - -
Volatile oil - - - - - -
Phenol + - + + + -
Terpenoid - - - - - +
Phlobatannin - - - - - -
Steroid + + - + + -
(+): positive = present (-): negative = absent

45
4.6 QUALITATIVE PHYTOCHEMICAL SCREENING OF SOLVENT EXTRACTS

OF PLANTAIN FLOWER

The qualitative phytochemical screening of plantain flower which was carried out in six

different solvent was shown in Table 4.6. The solvent used were acetone, chloroform, ethanol,

ethyl acetate, methanol and water. While the phytochemicals considered were alkaloid,

flavonoid, saponin, cardiac glycoside, reducing sugar, tannin, quinone, volatile oil, phenol,

terpenoid, phlobatannin and steroid. In all the solvents used for extraction of plantain flower,

alkaloid and terpenoid was present in all while cardiac glycoside, tannin, phenol, phlobatannin

was absentnegative in all. Flavonoid was absent in all except methanol extract. Saponin and

volatile oil was absent in all except acetone extract. Reducing sugar was present only in acetone,

chloroform and water extract. Quinone was present only in ethyl acetate extract. Steroid was

present in ethanol and ethyl acetate extract. Amidst all the phytochemicals considered for

plantain flower extract, acetone extract had six; ethylacetate had four while chloroform, ethanol,

methanol and water had three phytochemicals present in them.

Considering the number of phytochemicals present in moringa plant extract, it was noted

that moringa leaf and seed were richer in bioactive compounds (phytochemicals) than in moringa

pod and moringa coat.

46
 Table 4.6: Qualitative phytochemical screening of solvent extract of plantain flower

Parameter Solvent extract

Acetone Chloroform Ethanol Ethyl acetate Methanol Water
Alkaloid + + + + + +
Flavonoid - - - - + -
Saponin + - - - - -
Cardiac Glycoside - - - - - -
Reducing Sugar + + - - - +
Tannin - - - - - -
Quinone - - - + - -
Volatile oil + - - - - -
Phenol - - - - - -
Terpenoid + + + + + +
Phlobatannin - - - - - -
Steroid + - + + - -
(+): positive = present (-): negative = absent

47
4.7 ANTIOXIDANT PROPERTIES OF MORINGA PLANT AND PLANTAIN

FLOWER.

Antioxidant activities were carried out on the first two solvent extracts with the highest %

yield in comparison with the plant raw sample.

4.7.1 Total Phenol

The amount of total phenol obtained in the raw sample and various extract of plant

samples was shown in figure 4.3. For moringa leaf, the concentration (mg/100g) ranges from

0.18 - 0.35with ethanol extract having the highest concentration of 0.35mg/100g, followed by

ethylacetate extract with concentration of 0.25mg/100g while the powdered sample has the least

concentration of 0.18mg/100g. For moringa seed, the concentration (mg/100g) ranges from 0.18

- 0.35 with ethanol extract having the highest concentration of 0.35mg/100g, followed by

ethylacetate extract with concentration of 0.25mg/100g while the powdered sample has the least

concentration of 0.18mg/100g. For moringa pod, the concentration (mg/100g) ranges from 0.06-

0.21with ethylacetate extract having the highest concentration of 0.21mg/100g, followed by

acetone extract of 0.12mg/100g while the powdered sample has the least concentration of

0.06mg/100g. For moringa coat, the concentration (mg/100g) ranges from 0.03 - 0.12 with

acetone extract having the highest concentration of 0.85mg/100g, followed by powdered sample

of 0.06mg/100g while the methanol extract has the least concentration of 0.03.mg/100g. For

plantain flower, the concentration (mg/100g) ranges from 0.06 - 0.17with water extract having

the highest concentration of 0.17mg/100g, followed by powdered sample of 0.09mg/100g while

the ethylacetate extract has the least concentration of 0.06mg/100g.

48
 PS: Powdered sample;
0.4 EL: Ethanol leaf extract;
EAL: Ethylacetate leaf extract
AS: Acetone seed extract;
EAS: Ethylacetate seed extract
MP: Methanol pod extract;
EL EP: Ethanol pod extract;
MC: Methanol coat extract;
0.35 AC: Acetone coat extract;
EAPF: Ethylacetate plantain
flower extract;
WPF: Water plantain flower.

0.3

EAL
0.25
Total Phenol (mg/100g)

EAS
0.2
PS
WPF

0.15
EP
AS AC

0.1
PS

EAPF
PS PS
0.05 MP
PS
MC

0
MORINGA LEAF MORINGA SEED MORINGA POD MORINGA COAT PLANTAIN
FLOWER

Figure 4.1: Total phenol content of raw samples and solvent extracts of moringa plant and
plantain flower

49
 Phenolic compounds have been reported to protect the human body from free radicals,

whose formation is associated with the normal natural metabolism of aerobic cells. The

antiradical activity of total phenols and flavonoids is principally based on the structural

relationship between different parts of their chemical structure (Singleton, et al. 1999). Among

the plant samples, it was noticed the solvent extracts possess more phenolic content than the raw

samples. Acetone, ethanol, ethylacetate and water are good solvents for extracting phenolic

content from moringa plant and plantain flower. The extracts with high phenol content are said to

possess compound that can protect the body from free radicals.

50
4.7.2 Total Flavonoid

The amount of total flavonoid content obtained in the raw samples and different solvent

extract of moringa plant and plantain flower was presented in Figure 4.2. For moringa leaf the

concentration(mg/100g) of total flavonoid ranges from 0.12 - 0.33 with ethanol extract having

the highest concentration of 0.33mg/100g, followed by ethylacetate extract with concentration of

0.27mg/100g while the powdered sample has the least concentration of 0.12mg/100g. For

moringa seed, the concentration (mg/100g) ranges from 0.36 - 0.39 with the powdered sample

having the highest concentration of 0.39mg/100g, followed by ethylacetate extract with

concentration of 0.38mg/100g while the acetone extract has the least concentration of

0.36mg/100g. For moringa pod, the concentration (mg/100g) ranges from 0.04 - 0.08 with

methanol extract having the highest concentration of 0.08mg/100g, followed by ethanol extract

with concentration of 0.06mg/100g while the powdered sample has the least concentration of

0.04mg/100g. For moringa coat, the concentration (mg/100g) ranges from 0.05 - 0.52 with

methanol extract having the highest concentration of 0.32mg/100g, followed by acetone extract

with concentration of 0.26mg/100g while the powdered sample has the least concentration of

0.05mg/100g. For plantain flower the concentration (mg/100g) ranges from 0.03 - 0.04 with the

powdered sample and the water extract having the same concentration of 0.04mg/100g while the

ethylacetate extract has the least concentration of 0.03mg/100g. The biological functions of

flavonoid include protection against allergies, inflammation, free radicals, ulcers and tumors

(Hour, et al. 1980). Flavonoids represent the most common and widely distributed groups of

plant phenolic. They are potent water-soluble antioxidants and free radical scavengers which

prevent oxidative cell damage, have strong anti-cancer activity and protects against all stages of

carcinogenesis (Hour, et al. 1980 and Sodipo and Akinyi, 2000).

51
 PS: Powdered sample;
0.45 EL: Ethanol leaf extract;
EAL: Ethylacetate leaf extract
AS: Acetone seed extract;
EAS: Ethylacetate seed extract
MP: Methanol pod extract;
EP: Ethanol pod extract;
0.4 PS MC: Methanol coat extract;
AC: Acetone coat extract;
EAS EAPF: Ethylacetate plantain
flower extract;
AS WPF: Water plantain flower.

0.35
EL
MC
Total flavonoid content (mg/100g)

0.3

EAL
AC

0.25

0.2

0.15

PS

0.1

MP
EP
0.05 PS
PS PS WPF
EAPF

0
MORINGA LEAF MORINGA SEED MORINGA POD MORINGA COAT PLANTAIN
FLOWER

Figure 4.2: Total flavonoid content of raw sample and solvent extract of moringa plant and
plantain flower.

52
Among the moringa plant, it was observed that moringa seed, moringa leaf and moringa coat

extracts possess high flavonoid content than moringa pod and also plantain flower possess low

flavonoid content. In this study, it was detected that moringa leaf, coat and seed are good plant

samples that can be used for the above flavonoid functions. Comparing the raw sample and their

solvent extracts, it was observed that solvent used for extraction has a significant effect on total

flavonoid content for some of the plant samples. For example among the plant samples and their

solvent extracts, only the raw sample of moringa seed has higher flavonoid content than its

solvent extracts.

53
4.7.3 Ferric Reducing Antioxidant Power (FRAP)

The results in Figure 4.3 show the concentration of the raw sample and the extracts in

Garlic Acid Equivalent (GAE). For moringa leaf, ethylacetate extract exhibited the highest

reducing power of concentration of 0.87GAE followed by ethanol extract which has

concentration of 0.80GAE while the powdered sample has the least concentration of 0.31GAE.

For moringa seed, ethylacetate extract exhibited the highest reducing power of concentration of

0.55GAE followed by acetone extract which has concentration of 0.55GAE while the powdered

sample has the least concentration of 0.29GAE. For moringa pod, methanol extract exhibited the

highest reducing power of concentration of 0.53GAE, followed by ethanol extract of 0.42GAE

while the powdered sample has least concentration of 0.25GAE. For moringa coat, methanol

extract exhibited the highest reducing power of concentration of 0.85GAE followed by acetone

extract of 0.64GAE while the powdered sample has the least concentration of 0.32GAE. The

reducing capacity of a compound may serve as an important indicator of its potential antioxidant

activity (Ho et al., 2012). The reducing properties are generally associated with the presence of

reductones i.e. reducing agent (Krishnamoorthy et al., 2011).

54
 1 PS: Powdered sample;
EL: Ethanol leaf extract;
EAL: Ethylacetate leaf extract
AS: Acetone seed extract;
EAS: Ethylacetate seed extract
MP: Methanol pod extract;
0.9 EAL
EP: Ethanol pod extract;
MC: Methanol coat extract;
MC AC: Acetone coat extract;
EAPF: Ethylacetate plantain
flower extract;
EL WPF: Water plantain flower.
0.8

0.7
AC

0.6
EAS
AS MP
FRAP (GAE)

0.5
EAPF
EP

0.4

PS PS

0.3 PS PS

PS
WPF

0.2

0.1

0
MORINGA LEAF MORINGA SEED MORINGA POD MORINGA COAT PLANTAIN FLOWER

Figure 4.3: Ferric Reducing Activity Power (FRAP) of raw sample and solvent extracts of
moringa plant and plantain flower

55
 Antioxidant action of reductones is based on the breaking of the free radical chain by

donating a hydrogen atom (Gordan, 1990). The antioxidant activity of these plant samples may

be due to the presence of polyphenols which may act as reductones (reducing agent) to convert

free radicals into more stable products and terminate free radical chain reaction. Similar trend of

observation on polyphenolic constituents’ dose dependent reducing power activity has been

reported for several other plant extracts (Zhu et al., 2002; Amarowicz et al., 2004). It was

observed that among the plant samples, their solvent extracts exhibit high reducing power than

their raw samples which means that solvents has a significant effect on the extractability of

antioxidant. Moringa leaf and moringa coat possess high reducing power than moringa seed,

moringa pod and plantain flower.

56
4.7.4 DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging

The radical scavenging activity of moringa plant and plantain flower was measured using

the DPPH radical assay. The percentage DPPH scavenging activity of the raw samples and

various extract of moringa plant and plantain flower were shown in Figure 4.4. For moringa leaf,

powdered sample showed the highest activity of 89.25% followed by ethanol extract which

63.10% while ethylacetate extract has the lowest activity of 57.45%. For moringa seed,

powdered sample showed the highest activity of 79.81% followed by ethylacetate extract which

has 77.43% while acetone extract showed the lowest activity of 71.86%. For moringa pod,

methanol extract showed the highest activity of 96.82% followed by powdered sample which has

83.75% while ethanolic extract showed the lowest activity of 81.95%. For moringa coat, acetone

extract showed the highest activity of 95.20% followed by powdered sample which has 85.07%

while methanol extract showed the lowest activity of 72.13%. For plantain flower, powdered

sample showed the highest activity of 88.35% followed by ethylacetate extract which has

86.71% while water extract showed the lowest activity of 65.81%. Among the plant samples, it

was observed that 75% of the plant raw samples have high antioxidant activity than their solvent

extracts. The raw samples and extract that has high antioxidant activity may contain many

phenolic compounds that contributed to their antioxidant activity. The antioxidant activity of

DPPH from plant sample extracts depends on the solvent used in the extraction. The different

compounds can be extracted with different solvent due to different solubility.

57
 PS: Powdered sample;
120 EL: Ethanol leaf extract;
EAL: Ethylacetate leaf extract
AS: Acetone seed extract;
EAS: Ethylacetate seed extract
MP: Methanol pod extract;
EP: Ethanol pod extract;
MC: Methanol coat extract;
AC: Acetone coat extract;
EAPF: Ethylacetate plantain
flower extract;
100 MP WPF: Water plantain flower.
MC

PS PS EAPF
PS PS
EP
PS
80 EAS

AS AC

WPF
EL
DPPH (%)

60 EAL

40

20

0
MORINGA LEAF MORINGA SEED MORINGA POD MORINGA COAT PLANTAIN FLOWER

Figure 4.4: DPPH content of raw samples and solvent extract of moringa plant and plantain
flower.

58
4.7.5 Iron (Fe2+) chelation assay

The reducing power of moringa plant and plantain flower and their solvent extract were

assessed based on their ability to reduce Fe 3+ to Fe2+ and the results are presented in Figure 4.5.

The reducing power which is a novel antioxidant defense mechanism was determined by

measuring the percentage iron chelating of the raw samples and solvent extracts of moringa plant

and plantain flower. In this investigation, for moringa leaf, it revealed the concentration (%)

ranges from 4.99 - 24.95 with the highest concentration in raw sample 24.95% followed by the

ethylacetate extract 14.37% and the least in the ethanol extract 4.99%. For moringa seed, the

concentration (%) ranges from 22.36 - 39.72 with the highest concentration in acetone extract

39.72% followed by the ethylacetate extract 38.12% and the least in the raw sample 22.36%. For

moringa pod, the concentration (%) ranges from 28.14 - 36.73 with the highest concentration in

methanol extract 36.73% followed by the ethanol extract 32.14% and the least in the raw sample

28.14%. For moringa coat, the concentration (%) ranges from 11.18 - 44.91 with the highest

concentration in acetone extract 44.91% followed by the raw sample 30.34% and the least in the

methanol extract 11.18%. For plantain flower, the concentration (%) ranges from 24.75 - 22.56

with the highest concentration in water extract 22.56% followed by the ethylacetate extract

22.55% and the least in the raw sample 24.75%. The high content of reducing power explains the

medicinal importance and usefulness of plant samples. This assay just indicates how easily a

given antioxidant donates electrons to reactive free radicals species, thus promoting the

termination of free radical chain reactions.

59
 PS: Powdered sample;
50 EL: Ethanol leaf extract;
EAL: Ethylacetate leaf extract
AS: Acetone seed extract;
EAS: Ethylacetate seed extract
MP: Methanol pod extract;
AC EP: Ethanol pod extract;
45 MC: Methanol coat extract;
AC: Acetone coat extract;
EAPF: Ethylacetate plantain
flower extract;
WPF: Water plantain flower.
AS
40
EAS
MP

35
EP

PS
30
PS
Iron chelating (%)

PS PS
25
PS EAPF WPF

20

15 EAL

MC

10

EL
5

0
MORINGA LEAF MORINGA SEED MORINGA POD MORINGA COAT PLANTAIN FLOWER

Figure 4.5: Iron chelating assay of raw sample and solvent extracts of moringa plant and plantain
flower.

60
 The ability of the antioxidant to reduce Fe 3+ to its more active Fe2+ form might also be an

indicator of its ability to act as a pre-oxidant in the system (Zhan-Wu et al. 2011). Among the

plant samples, it was observed that moringa seed, moringa pod and moringa coat raw sample and

their solvent extract shows possess higher concentration than the other plant samples and their

extracts. Among the solvents, acetone and methanol tends to be good solvents for extracting.

61
 CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

In all the solvents used for extraction, it was observed that the extractive value (%) was

highest in moringa seed and least in moringa pod. Extractive value (%) of moringa leaf and

moringa coat was higher than that of plantain flower using acetone, chloroform as solvents.

However, the extractive value (%) of moringa leaf and plantain flower was higher than moringa

coat using ethanol, ethylacetate, methanol and water.

The qualitative screening of the plant samples and their solvent extracts shows some

powerful and important phytochemicals of pharmaceutical and medicinal importance. This study

shows out of the six solvents used for extraction, acetone, ethanol, ethylacetate and methanol are

good solvents for extracting phytochemicals. Among the phytochemicals considered, it was

observed that in plant samples, moringa seed and leaf are richer in bioactive compounds and

exhibit higher phytochemicals than moringa coat pod and plantain flower. It can be concluded

that moringa seed and leaf are more medicinally important and can be useful in management and

treatment of diseases.

The result of this study provides good information to prove the usefulness of the moringa

plant and plantain flower extracts as a potential source of natural antioxidants. Among the plant

samples, moringa leaf and moringa seed raw samples and their solvent extracts possess high total

flavonoid, total phenol and FRAP content than other plant samples and their extracts. Moringa

pod, moringa coat and plantain flower has high DPPH and iron chelating content than other plant

samples and their extracts. This research shows that the plant raw samples and their extracts

62
possess reasonable observation that it can serve as source of natural antioxidants. It was observed

that only 24% of the raw samples has higher antioxidant properties than their solvent extracts.

This shows that solvent plays a significant role in extracting antioxidant activity in plant

samples. These findings confirmed that these plant samples have great potential in health-related

area by preventing or treating diseases caused by the oxidative stress and might be extensively

used for the treatment of degenerative diseases.

5.2 CONTRIBUTION TO KNOWLEGDE

This research work proposes to contribute or advance the knowledge/research on moringa

plant and plantain flower located in Ondo state. It also aims to reduce pollution in the

environment by increasing the evaluation of some of these plant samples that are regarded as

wastes thereby turning these waste samples to wealth. This work also tends to increase the

cultivation/farming of these moringa plant and plantain flower on a large scale which thereby

increases job opportunities.

5.3 RECOMMENDATION

Moringa plant and plantain flower may possess some potential use in pharmaceutical,

cosmetic, and food products. Ethanol, ethylacetate and methanol have proven to be best solvents

for extracting these plant samples. The antioxidant potential of these solvent extracts on stability

of edible oils can be investigated. The effect of the extracts of these plant samples and synthetic

antioxidants such as with propylgallate(PG), butylated hydroxyanisole (BHA) and butylated

hydroxytoluene (BHT) on edible oils stored at room temperature can be further investigated.

Anti-microbial and anti-fungal properties of moringa plant and plantain flower extracts

can be further investigated to ascertain their potency against microbes and fungus.

63
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APPENDIX I

PREPARATION OF 500 mL 10% FERRIC CHLORIDE (FeCl3)

10
×500
100

= 50 g

50 g of FeCl3 was weighed, dissolved and made up to mark in a 500ml standard flask.

APPENDIX II

PREPARATION OF 500 mL 1% HYDROCLORIC ACID (HCl)

1
×500
100

= 5 mL

5 mL of HCl was measured into a 500ml standard flask and made up to mark.

APPENDIX III

PREPARATION OF 500 mL 2% HYDROCLORIC ACID (HCl)

2
×500
100

= 10 mL

10 mL of HCl was measured into a 500ml standard flask and made up to mark.

APPENDIX IV

PREPARATION OF DILUTE HYDROCHLORIC ACID (2M HCl)

Using dilution formula = C1V1 = C2V2

Concentration of HCl = 25M

25M × V1 = 2M × 500

V1 = 40

40 mL of HCl was measured and make up to mark with distilled water in 500 mL volumetric
flask.

73
APPENDIX V

PREPARATION OF DILUTE SODIUM HYDROXIDE (2M NaOH)

Molar mass of NaOH = 40g

Molarity = 2M

Mole = molarity × volume (dm3)

500
= 2×
1000

=1

Mass of NaOH = mole × molar mass

= 1× 40 g

= 40 g

40 g of NaOH was weighed in 500 mL volumetric flask and made up to mark with distilled
water.

APPENDIX VI

PREPARATION OF WAGNERS REAGENT

2 g of iodine was dissolved in 12.5 g of potassium iodide and 250 mL of distilled water was
added to produce solution.

APPENDIX VII

EXTRACTION VALUE (% YIELD) OF MORINGA LEAVES IN ETHANOL

Weight of powder moringa leaf = 20.020g

Weight of petridish = 39.160g

Weight of petridish + ethanol dry extract = 40. 99g

Weight of ethanol extract = 40.990 – 39.160 = 1.830g

74
 weight of extract
% yield of ethanol moringa leaf extract = ×100
weight of samples

1.830
= 20.020
×100

=9.291%

APPENDIX VIII

EXTRACTION VALUE (% YIELD) OF MORINGA LEAVES IN ETHYL ACETATE

Weight of powder moringa leaf = 20.020g

Weight of petridish = 37.580g

Weight of petridish + ethyl acetate dry extract = 39.640g

Weight of ethyl acetate extract = 39.690 – 37. 580 = 2.060g

weight of extract
% yield ethyl acetate moringa leaf extract = ×100
weight of samples

2.060
= 20.020
×100

=10.290%

APPENDIX IX

EXTRACTION VALUE (% YIELD) OF MORINGA PODS IN ETHANOL

Weight of powder moringa pod = 20.050g

Weight of petridish = 31.300g

75
 Weight of petridish + ethanol dry extract = 31.570g

Weight of ethanol extract = 31.570- 31.300 = 0.270g

weight of extract
% yield ethanol moringa pod extract = ×100
weight of samples

0.270
= 20.050
×100

=1.347%

APPENDIX X

EXTRACTION VALUE (% YIELD) OF MORINGA PODS IN METHANOL

Weight of powder moringa pod = 20.040g

Weight of petridish = 35.220g

Weight of petridish + methanol dry extract = 35.650g

Weight of methanol extract = 35.650 – 35.220 = 0.430g

weight of extract
% yield methanol moringa pod extract = ×100
weight of samples

0.430
= 20.040
×100

=2.146%

APPENDIX XI

EXTRACTION VALUE (% YIELD) OF MORINGA COAT IN ACETONE

76
 Weight of powder moringa coat = 20.020g

Weight of petridish = 32.520g

Weight of petridish + acetone dry extract = 33.120g

Weight of acetone extract = 33.120- 32.520 = 0.600g

weight of extract
% yield acetone moringa coat extract = ×100
weight of samples

0.660
= 20.020
×100

=2.997%

APPENDIX XII

EXTRACTION VALUE (% YIELD) OF MORINGA COAT IN METHANOL

Weight of powder moringa coat = 20.03g

Weight of petridish = 29.830g

Weight of petridish + methanol dry extract = 31.010g

Weight of methanol extract = 31.010 – 29.830 = 1.180g

weight of extract
% yield methanol moringa coat extract = ×100
weight of samples

1.180
= 20.030
×100

=5.891%
77
APPENDIX XIII

EXTRACTION VALUE (% YIELD) OF MORINGA SEED IN ACETONE

Weight of powder moringa seed = 20.050g

Weight of petridish = 31.00g

Weight of petridish + acetone dry extract = 37.29g

Weight of acetone extract = 37.29 – 31.00 = 6.29g

weight of extract
% yield acetone moringa seed extract = ×100
weight of samples

6.29
= 20.050
×100

=6.29%

APPENDIX XIV

EXTRACTION VALUE (% YIELD) OF MORINGA COAT IN ETHYL ACETATE

Weight of powder moringa seed = 20.060g

Weight of petridish = 31.20g

Weight of petridish + ethyl acetate dry extract = 37.30g

Weight of ethyl acetate extract = 37.30 – 31.20 = 6.10g

weight of extract
% yield ethyl acetate moringa seed extract = ×100
weight of samples

78
 6.10
= 20.030
×100

=30.439%

APPENDIX XV

EXTRACTION VALUE (% YIELD) OF PLANTAIN FLOWER IN ETHYL ACETATE

Weight of powder plantain flower = 20.000g

Weight of petridish = 36.21g

Weight of petridish + ethyl acetate dry extract = 38.98g

Weight of ethyl acetate extract = 38.98 – 36.21 = 2.770g

weight of extrtact
% yield ethyl acetate plantain flower extract = ×100
weight of samples

2.770
= 20.020
×100

=13.850%

APPENDIX XVI

EXTRACTION VALUE (% YIELD) OF PLANTAIN FLOWER IN WATER

Weight of powder plantain flower = 20.090g

Weight of beaker = 94.15g

Weight of beaker + water dry extract = 97.36g

79
 Weight of water extract = 97.36 – 94.15 = 3.210g

weight of extract
% yield water plantain flower extract = ×100
weight of samples

3.210
= 20.090
×100

=16.026%

APPENDIX XVII

EXTRACTIVE VALUES OF MORINGA PLANT AND PLANTAIN FLOWER

Solvents Moringa Moringa pod Moringa coat Moringa seed Plantain

flower
leaves

1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd
extra extra extra extra extrac extrac extra extrac extra extra
ction ction ction ction tion tion ction tion ction ction
yield yield yield yield yield yield yield yield yield yield
Acetone 9.123 0.85 0.000 8.295 9.123 28.93 31.37 0.000
4.955 0 0 2 1.690
Chlorofor 0.89 0.000 1.953 0.949 26.89 20.98 0.000
1.321 4.790 8 7 7 1.347
m

Ethanol 13.75 1.34 0.000 3.225 3.441 25.01 30.43 0.000

9 9.291 7 1 9 6.216
Ethyl 11.89 10.29 2.14 0.000 2.528 3.595 28.43 31.55 13.85 0.000
9 0 6 8 5 0
acetate

Methanol 10.23 1.04 0.000 0.249 5.891 22.73 29.64 0.000

7 5.889 7 8 1 3.285
Water 0.19 0.000 3.947 0.350 31.87 10.96 16.02 0.000
9.210 7.784 9 1 2 6

80
 APPENDIX XVIII

ANTIOXIDANT PROPERTIES OF MORINGA PLANT AND PLANTAIN FLOWER.

Parameter Moringa leaf Moringa seed Moringa pod Moringa coat Plantain flower

PS EL EAL PS AS EAS PS MP EP PS MC AC PS EAPF WPF

Total 0.12 0.33 0.27 0.39 0.36 0.38 0.04 0.08 0.06 0.05 0.32 0.26 0.04 0.03 0.04
flavanoids
(mg/100g)

FRAP 0.32 0.80 0.87 0.29 0.54 0.55 0.25 0.53 0.42 0.32 0.85 0.64 0.29 0.47 0.22

(GAE)

Total 0.18 0.35 0.25 0.06 0.12 0.21 0.04 0.05 0.13 0.06 0.03 0.12 0.09 0.06 0.17
phenol
(mg/100g)

DPPH (%) 89.25 63.10 57.45 79.81 71.86 77.43 83.75 96.82 81.95 85.07 72.13 95.20 88.35 86.71 65.81

Iron 24.95 4.99 14.37 22.36 39.72 38.12 28.14 36.73 32.14 30.34 11.18 44.91 24.75 22.55 22.56
chelating
(%)

81