CHAPTER ONE

1.0 Introduction

1.1 Phytochemical

Phytochemicals are biologically active, naturally occurring chemical compounds found in plants,

which provide health benefits for humans as medicinal ingredients and nutrients (Hasler and

Blumberg, 1999). They protect plants from disease and damage, and also contribute to the plant’s

colour, aroma and flavour. Humans have relied mostly on plants for nutritional and medicinal

needs; Herbal plants provide most of the medicinal needs. Important herbal products include

spices, herbal teas, functional foods, medicinal raw materials, essential oils, flavouring and dietary

supplements (Nweze et al., 2004).

The medicinal use of plant is as a result of the phyto-constituents present in them. Some of these

chemicals is bioactive and produce definite physiological and biochemical actions in humans and

animals. They are known as secondary metabolites or phytochemicals and comprise alkaloids,

flavonoids, tannins, phenolics, saponin, steroids, glycoside, and terpenes etc. The use of plants in

traditional medicine is of global interest. Phytochemicals have been isolated and characterized

from fruits such as grapes and apples, vegetables such as broccoli and onion, spices such as

turmeric, beverages such as green tea and red wine, as well as many other sources (Doughari and

Obidah, 2008).

After centuries of empirical use of herbal preparation, the first isolation of active principles

alkaloids such as morphine, strychnine, quinine etc. in the early 19th century marked a new era in

the use of medicinal plants and the beginning of modern medicinal plants research. Emphasis

shifted away from plant derived drugs with the tremendous development of synthetic

pharmaceutical chemistry and microbial fermentation after 1945 (Walton et al., 2003).

1
Plant metabolites were mainly investigated from a phytochemical and chemotaxonomic viewpoint

during this period. Over the last decade, however, interest in drugs of plant and probably animal

origin has grown steadily Utilization of medicinal plants has almost doubled in Western Europe

during that period. Medicinal plants have been the mainstay of traditional herbal medicine

amongst rural dwellers worldwide since antiquity to date. The therapeutic use of plants certainly

goes back to the Sumerian and the Akkadian civilizations in about the third millennium BC.

Hippocrates (ca. 460–377 BC), one of the ancient authors who described medicinal natural

products of plant and animal origins, listed approximately 400 different plant species for

medicinal purposes. Natural products have been an integral part of the ancient traditional

medicine systems, e.g. Chinese, Ayurvedic and Egyptian (Sarker and Nahar, 2007).

This represents about 88 per cent of the world’s inhabitants, who rely mainly on traditional

medicine for their primary health care. According to the World Health Organization, a medicinal

plant is any plant which, in one or more of its organs, contains substances that can be used for

therapeutic purposes, or which are precursors for chemo-pharmaceutical semi synthesis. Such a

plant will have its parts including leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or

seeds, employed in the control or treatment of a disease condition and therefore contains chemical

components that are medically active. These non-nutrient plant chemical compounds or bioactive

components are often referred to as phytochemicals (‘phyto-‘from Greek - phyto meaning ‘plant’)

or phyto-constituents and are responsible for protecting the plant against microbial infections or

infestations by pests (Nweze et al., 2004; Doughari et al., 2009). The study of natural products on

the other hand is called photochemistry.

Phytochemicals have been isolated and characterized from fruits such as grapes and apples,

vegetables such as broccoli and onion, spices such as turmeric, beverages such as green tea and

red wine, as well as many other sources (Doughari and Obidah, 2008; Doughari et al., 2009).

2
The science of application of these indigenous or local medicinal remedies including plants for

treatment of diseases is currently called ethno pharmacology but the practice dates back since

antiquity.

Ethno pharmacology has been the mainstay of traditional medicines the entire world and currently

is being integrated into mainstream medicine. Different catalogues including De Materia Medica,

Historia Plantarum, Species Plantarum have been variously published in attempt to provide

scientific information on the medicinal uses of plants. A single plant may be used for the

treatment of various disease conditions depending on the community. Several ailments including

fever, asthma, constipation, esophageal cancer and hypertension have been treated with traditional

medicinal plants (Cousins and Huffman, 2002; Saganuwan, 2010).

1.1.1 Classes of Phytochemicals

Alkaloids, Flavonoids, Glycosides, Phenolics, Saponins, Tannins, Terpenes, Essentials Oils and

Steroids.

Figure 1: Phytochemical image (A.D.A.M., 2019)

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1.2 Antioxidants

Antioxidants are substances that protect cells from the damage caused by unstable molecules

known as free radicals. Antioxidants interact with and stabilize free radicals and prevent some of

the damage free radicals might otherwise cause. Free radical damage may lead to cancer.

Examples of antioxidants include beta-carotene, lycopene, vitamins C, E, A and other substances

(Sies, 1997).

Antioxidants protect cells against the damaging effects of reactive oxygen species otherwise

called, free radicals such as singlet oxygen, super oxide, peroxyl radicals, hydroxyl radicals and

peroxynite which results in oxidative stress leading to cellular damage (Mattson & Cheng, 2006).

Natural antioxidants play a key role in health maintenance and prevention of the chronic and

degenerative diseases, such as atherosclerosis, cardiac and cerebral ischema, carcinogenesis,

neurodegenerative disorders, diabetic pregnancy, rheumatic disorder, DNA damage and ageing

(Uddin et al., 2008; Jayasri et al., 2009).

Antioxidants exert their activity by scavenging the ‘free-oxygen radicals’ thereby giving rise to a

fairly ‘stable radical’. The free radicals are metastable chemical species, which tend to trap

electrons from the molecules in the immediate surroundings. These radicals if not scavenged

effectively in time, they may damage crucial bio molecules like lipids, proteins including those

present in all membranes, mitochondria and, the DNA resulting in abnormalities leading to

disease conditions (Uddin et al., 2008). Thus, free radicals are involved in a number of diseases

including: tumour inflammation, hemorrhagic shock, atherosclerosis, diabetes, infertility,

gastrointestinal ulcerogenesis, asthma, rheumatoid arthritis, cardiovascular disorders, cystic

fibrosis, neurodegenerative diseases (e.g. parkinsonism, Alzheimer’s diseases), AIDS and even

early senescence (Chen et al., 2006; Uddin et al., 2008).

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The human body produces insufficient amount of antioxidants which are essential for preventing

oxidative stress. Free radicals generated in the body can be removed by the body’s own natural

antioxidant defences such as glutathione or catalases (Sen, 1995). Therefore, this deficiency had to

be compensated by making use of natural exogenous antioxidants, such as vitamin C, vitamin E,

flavones, carotene and natural products in plants (Madsen and Bertelsen, 1995; Rice Evans et al.,

1997; Diplock et al., 1998). Plants contain a wide variety of free radicals scavenging molecules

including phenols, flavonoids, vitamins, terpenoids hat are rich in antioxidant activity (Madsen

and Bertelsen, 1995; Cai and Sun, 2003).

Many plants, citrus fruits and leafy vegetables are the source of ascorbic acid, vitamin E,

caratenoids, flavanols and phenolics which possess the ability to scavenge the free radicals in

human body. Significant antioxidant properties have been recorded in phytochemicals that are

necessary for the reduction in the occurrence of many diseases (Hertog and Feskens, 1993;

Anderson and Teuber, 2001). Many dietary polyphenolic constituents derived from plants are

more effective antioxidants in vitro than vitamins E or C, and thus might contribute significantly

to protective effects in vivo (Rice-Evans and Miller, 1997; Jayasri et al., 2009).

Antioxidants are often added to foods to prevent the radical chain reactions of oxidation, and they

act by inhibiting the initiation and propagation step leading to the termination of the reaction and

delay the oxidation process. Due to safety concerns of synthetic compounds, food industries have

focused on finding natural antioxidants to replace synthetic compounds. In addition, there is

growing trend in consumer preferences for natural antioxidants, all of which has given more

impetus to explore natural sources of antioxidants.

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Methanol extract of Murraya koenigii contains a number of antioxidant compounds which can

effectively scavenge reactive oxygen species including superoxide anions and hydroxyl radicals as

well as other free radicals in vitro. The leaves of Murraya koenigii, an under-utilized and

unconventional part of the plant, contains a good amount of phenolic antioxidants to counteract

the damaging effects of free radicals and may protect against mutagenesis.

Fig 2: Image of the sources of antioxidants (Michael Byrne 2015)

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1.3 Murraya koenigii

Murraya koenigii commonly known as curry plant belongs to the family Rutaceae. The plant is a

medicinal plant and native to India, Sri Lanka, African and other South Asian countries. Curry

leaves have the richest source of carbazole alkaloids such as koenigine and mahanimbine

extracted from the leaves which have been found to demonstrate anti-cancer and anti-oxidant

properties.

The medicinal values of Murraya koenigii are numerous and beneficial to humans, hence this

work seeks to evaluate the phytochemical and antioxidant; vitamin content and minerals elements

present in the plant. The plant is highly valued for its leaves which are used for flavouring and

spicing of food. The curry leaf is believed to have several medicinal properties such as anti-

diabetic, antioxidant, antimicrobial, anti-inflammatory, anti-carcinogenic and hepato-protective

properties.

1.3.1 Taxonomical Status

Murraya koenigii can be botanically classified or identified by the following procedure as seen

below, they are:

a. Kingdom - Plantae

b. Sub-kingdom - Tracheobionta

c. Superdivision - Spermatophyta

d. Division - Magnoliophyta

e. Class - Magnoliospida

f. Subclass - Rosidae

g. Order - Sapindales

h. Family - Rutaceae

i. Genus - Murraya

j. Species - Murraya koenigii L. Spreng.

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1.3.2 Origin of Murraya Koenigii

Curry leaf trees are naturalized in forests and abandoned land throughout the Indian subcontinent

except in the higher parts of the Himalayas. From the Ravi River in Pakistan its distribution

extends eastwards towards Assam in India and Chittagong in Bangladesh, and southwards to

Tamil Nadu in India. The plants spread to Malaysia, Africa and Reunion Island with South Asian

immigrants (Priyanka et al., 2012).

The origin of curry leaves is seen in early 1st to 4th century AD. In Tamil and Kannada literature

it was updated as word ‘kari’ with its uses. The word now popularly used for the Murraya

koenigii is curry leaf which is originated from Tamil word Kari which means as ‘spiced sauce’. In

the early literatures of Tamil and Kannada the use of Murraya koenigii is described as the

flavouring agent for the vegetables. Today Murraya koenigii are grown as the cultivated crop in

India, Sri Lanka, Southeast Asia, Australia, Pacific Islands and Africa as flavouring agent for the

food. (Jane M. et al., 2017). The species name commemorates the botanist Johann König. The

genus Murray commemorates Swedish physician and botanist Johann Andreas Murray who died

in 1791. Hence the botanical name of the curry leaves is Murraya koenigii.

Fig 3: Murraya koenigii image by (Sankar Ganesh et al., 2015)

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1.3.3 Ethanobotany

Murraya koenigii is a plant which has various important uses in the traditional system of medicine

in Eastern Asia. Based on ethanomedicine, Murraya koenigii is used as a stimulant, anti-

dysenteric and for the management of diabetes mellitus. The plant is highly valued for its leaves

an important ingredient in an Indian cuisine to promote appetite and digestion.

The leaves, root and bark are tonic, stomachic and carminative. Leaves are used internally in

dysentery also checking vomiting. Steam distillate of the leaves can be used as stomachic,

purgative, febrifuge and anti-anaemic. Leaves are applied externally to bruises and eruption.

The leaves and roots are bitter, acrid, cooling, anti-helminthic, analgesic, it cures piles, allays heat

of the body, thirst, inflammation and itching. It is also useful in leucoderma and blood disorders.

An infusion of the toasted leaves in used to stop vomiting. The juice of the root is good for pain

associated with kidney.

Fruits are also considered as astringent in Nigeria, crushed leaves are applied externally to cure

skin eruption and to relieve burns. The pastes of leaves are applied externally to treat the bites of

poisonous animals. The plant is credited with tonic and stomachic property. The fruits are known

to have very high nutritional values with many medicinal properties.

The branches of Murraya koenigii are very popular for cleaning the teeth used as datun. It is also

said that the branches of M. koenigii are used to strengthen gums and teeth’s. It has also been used

as an antiperiodic and many a time the powdered dry leaf, mixed with honey and juice of betel

nut, is recommended in the Ayurvedic system of medicine. (Jane et al., 2017).

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1.4 Allium sativum (Garlic)

Plants that possess therapeutic properties or exert beneficial pharmacological effects in the animal

body are generally termed as “medicinal plant”. And It is now being established that the plants

which naturally synthesize and accumulate some secondary metabolites and vitamins, possess

medicinal properties (Sofowora, 1993). Medicinal plants constitute an important natural wealth of

a country. They play a significant role in providing primary health care services to rural people.

They also serve as therapeutic agents as well as important raw materials for the manufacture of

traditional and modern medicine. Substantial amount of foreign exchange can be earned by

exporting medicinal plants to other countries. In this way indigenous medicinal plants play

significant role in the economy of a country (Sofowora, 1993).

Garlic is commonly known as Allium sativum, In Hausa it is called Tafarnuwa, in Yaruba it is

called Ayu and in Igbo it is called Ayu-Ishi (Aliyu, 2006), and also it is a species in the onion

genus “Allium”. Its close relatives include the onion, shallot, leek, chive and rakkyo (Block,

2010). The plant is a member of the Liliaceae family and one of the most popular herbs used

worldwide to reduce various risk factors associated with several diseases (Thomson et al., 2007).

1.4.1 Botanical classification

 Kingdom - Plantae
 Subkingdomn – Trachebionta
 Super Division - Spermatophyta
 Subclass - lilIdae
 Order - lillales
 Family - Allaceae
 Genus - Allium
 Species – sativum

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Fig 4: Bunch of Allium sativum cloves (chengyuz heng, 2020).

1.4.2 Description of the Allium sativum Taxonomy

Formerly classified in the lily (Liliaceae) family, garlic was at one time known as Allium

controversum, which hints at the problems classifying the herb. Most sources recognize one major

hard-neck variety, Allium sativum var. ophioscorodon, and one major soft neck variety, Allium

sativum var. sativum. One other little-known variety, Allium sativum var. pekinense (Peking

garlic) also exists. It belongs to the genus Allium. Recent taxonomy revisions place garlic in the

family Alliaceae, which is made up of approximately 700 Species. A great number of species in

this genus are perennial plants with underground storage organs consisting of bulbs or rhizome.

The most common edible members include chives, (Allium Schoenoprasum L.), leek (Allium

porum L.), and onion (Allium Cepa L.). It belongs to the species Sativum and has the scientific

name Allium Sativum L 2. Allium sativum is a diploid species (2n = 2x = 16) in the subgenus

Allium of the Alliaceae (formerly in the Liliaceae, and then the Amarylidaceae).

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The other cultivated plants in this subgenus are leek, usually tetraploid, or elephant garlic, usually

hexaploid (both Allium Ampeloprasum L.). Leek and garlic have flat, folded leaves. Elephant

garlic and garlic form a bulb, but leek does not. Elephant garlic bulbs consist of 2 to 6 large cloves

and several small cloves, while garlic bulbs usually have more cloves of a relatively consistent

size, especially for bolting types. Bolting garlic, leek, and elephant garlic all have a solid scape,

unlike the hollow scape of the most economically important Allium, onion (Allium cepa L.).

1.4.3 History of Allium sativum

It is certain that Garlic was introduced in India after the writings of Ayurvedic literatures,

otherwise, it would have been referred and mentioned mainly in Atharva veda. This is also to

mention here that Yasti-madhu, Liqorice, Glycyrrhiza glabra, which is also an exotic and

important Ayurvedic drug, has not been mentioned in the Vedic literature. As we have seen in the

first part, (Shah, 2014) that garlic was introduced in India from the Middle-East countries, but

when? No doubt, India was on trade with Mesopotamia and Egypt about 1500-2000 BC. The

utility of garlic as a medicine and condiment was well known to the people of the above stated

countries. We have seen that garlic is not mentioned in Atharva veda and there is no definite time

or period ascertained for its introduction in to India. Shah, N.C., (2014)

 By Arabian Traders

In Later years, before the 1st century, A.D., when the Arabian traders (merchants) began to trade

with India and began to settle down in the western coast of India. Possibly, they brought with

them for their own daily use with their other trade goods to India and in return, traded from India

with crude drugs, silk, spices, etc. The progeny of these traders are still found in India Shah, N.C.,

(2014).

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  The Mohmeddan introduction

Later, the Mohmeddans invaders, 600-700 AD., who were called by the Hindus as Malecha or

'filthy people' and named garlic after their habits as malech-kanda, meaning, 'the tuber of the

Mohmeddans'. Possibly, the the Mohmeddans' had brought garlic with them for their own use.

The Hindus disliked the Mohmeddans', who were cruel and filthy. The Hindus also did not like

their food habits, who often ate cow-meat therefore, they kept them at their arms' length mostly by

the Brahmins and by the orthodox Hindus and both these sects, hated the Mohmeddans and

prohibited their food articles in their own use, like garlic and onion and the Mohmeddans often

slaughtered cows as they were the cow-meat eaters.

Further, garlic and onion has awful smell and as a mark of hatred against the Mohmeddans, both

the commodity was treated as unholy, 'apavitra' by the orthodox Hindus and by the Brahmins. Not

only, the Brahmins but also the Indian Budhists and the Jainis also prohibited garlic and onion in

their food (Shah, 2014).

 Greeks' introduction

Further, we know that garlic is known by various Sanskrit names, however, its name, Yavanpriya,

meaning 'likings of the Greeks' yavan word was used for the Greeks, in general. The Greeks were

the soldiers in the army of Alexander the Great, who invaded India in 325-327 BC. It is possible

that the Greeks would have brought its bulb-lets for their medicinal as well as for culinary purpose

as a spice & condiment and thence, introduced in India). Garlic was used by the ancient Egyptians

as a medicine for the treatment of various diseases. It has been consumed by athletes at the first

Olympic Games in history, for better performance. Garlic is currently starting to be more and

more interesting, although it has been used for thousands of years.

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In Chinese medicine, garlic is used more than 3000 years because it contains biologically active

substances with demonstrated positive effects on human health. In a more general synthesis,

Garlic contains a greater amount of biologically active substances and the chemical composition is

rich and has always been used in use from time memorial (Shah, 2014).

1.5 Aims and Objectives

1.5.1 Aim

The aim of this study is to determine the phytochemical contents and antioxidant activities of

methanol leaf and clove extracts of Murraya koenigii and Allium Sativum cloves.

1.5.2 Objectives

 To collect samples of Murraya koenigii and Allium sativum from the garden and market.

 To transport the collected samples to the laboratory for evaluation.

 To determine the phytochemical components of Murraya koenigii and Allium sativum.

 To determine the antioxidants of Murraya koenigii and Allium sativum.

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 CHAPTER TWO

2.0 LITRATURE REVIEW

2.1 Background study

2.1.1 Murraya koenigii

Murraya koenigii has been mentioned in the traditional medicinal system Ayurveda (Sathyavati et

al., 1987). Bark, root, leaves, fruits and fruit pulp of Murraya koenigii are widely used in the

treatment of diabetes, obesity, vomiting, constipation, indigestion, diarrhoea, dysentery, piles,

nausea, to relieve kidney pain etc. A few reports are available on the scientific probing to validate

the pharmacological properties of Murraya koenigii. Some constituents of Murraya koenigii are

reported to have antifungal activity (Das et al., 1965).

Anti-spasmodic and anti-amoebic activity reported by Bhakuni et al., (1969) and Kong et al.

(1986). Ramsewak, et al. (1999) and Rahman and Gray, 2005 reported antimicrobial activity.

Antitrichomonal activity was reported by Adebajo et al., (2006). The apoptotic activity of

mahanini, pyrayafoline-D and murrafoline-I, corbozole alkaloids from Murraya koenigii in human

myeloid cancer cell line HL-60 have been reported (Roy et al., 004; Ito et al., 2006). The positive

ionotropic effect of Murraya koenigii extracts reported by Shah and Juvekar, (2006).

Since ancient times, plants have been an exemplary source of drugs/medicines. Ayurveda and

other Indian literature mention the use of plants in treatment of various ailments. India has about

45,000 plant species and among them, several thousands have been claimed to possess medicinal

properties. Researches work conducted in the last few decades on plants mentioned in ancient

literature or used traditionally for diabetes have shown experimental or clinical anti-diabetic

activity. Narayana and Sastry (1975) reported the hypoglycemic activity of Murraya koenigii.

The aqueous extract of the leaves of Murraya koenigii after oral as well as intravenous

administration to normal and alloxan diabetic dogs produced the hypoglycemia. Santhakumari et

al., (1987) reported the hypoglycemic activity of crushed leaves of Murraya koenigii in rabbits,

15
human volunteers and alloxan induced diabetic rats. Iyer and Mani (1990) reported that curry

leaves powder supplementation (12g providing 2.5 g fibre) to 30 non-insulin dependent diabetes

mellitus patients for a period of 1 month resulted in the transient reduction in fasting and post-

prandial blood sugar levels.

Khan et al., (1995) reported the hypoglycemic activity of Murraya koenigii and attributed to

increased glycogenesis and decreased glycogenolysis and gluconeogenesis. Methanol extract of

Murraya koenigii leaves are reported to produce hypoglycemia in human volunteers and alloxan

induced rats and rabbits (Bhat, 1995; Rupashree, 1999). Yadav et al., (2002) reported that feeding

of diet containing various doses of curry leaf powder (5, 10 and 15%) to normal rats for 7 days as

well as to mild and moderate diabetic rats for 5 weeks showed varying hypoglycemic and

antihyperglycemic effect.

Bawden et al., (2002) reported the alpha amylase inhibitory activity of cold hexane extract of

Murraya koenigii. Yadav et al., (2004) reported that the Murraya koenigii supplemented diet

could reduce the development of insulin resistance and diabetes. Vinuthan et al., (2005) reported

antidiabetic activity of methanol extract of Murraya koenigii. Kesari et al., (2005) reported the

hypoglycemic effect of aqueous extract of Murraya koenigii. Recently Xie et al., (2006) reported

the hypoglycemic and hypolipidemic activity of Murraya koenigii in ob/ob mice. Arulselvan et

al., (2006) reported the antidiabetic effect of ethanol extract of Murraya koenigii in STZ induced

diabetic rats. Antimicrobial, antioxidant, anticancer and renal protective effects of the plant had

been reported (Ghasemzadeh et al., 2014; Gill and Sharma, 2014; Punuru et al., 2014; Rajendran

et al., 2014; Shekar et al., 2016).

They contain several medicinal properties such as antidiabetic, antioxidant, antimicrobial, anti-

fungal, anti-inflammatory, anti-carcinogenic and hepato protective properties (Al Harbi et al.,

2016). The dried leaves powder has been mixed with honey and juice of betel nut was

16
recommended in the Ayurveda of medicine. Table 1, gives the other benefits of curry leave parts

(Kumari, 2018).

Table 1: Various Ethnobotanical uses of curry leaf (Kumari, 2018)

Part of plant Benefits

Bark and roots Cure eruptions and bites of poisonous animals

Juice of root Cure kidney pain and preventing the premature
greying of hair.
Green leaves  Cure dysentery and diarrhoea
 Infusion of the washed leaves stops
vomiting
 Blood purifier and tonic.
Fresh juice of curry with lime juice and  leaves, with lime juice and sugar
sugar  Treatment of morning sickness, nausea and
indigestion.
 Treatment of renal diseases
 Dropped into the eyes for the prevention of
cataracts.
Leaves and roots  Curing piles, inflammation, itching
 useful in leukoderma and blood disorders.
Tea of curry leaves Treat fever.
 Applied on a burn, bruises, boils and skin
A paste made of curry leaves eruption
 Applied on gums to avoid Pieria
Fresh leaves chewed  Reduce the body weight gain

Khan et al., (1996) reported that Murraya koenigii leaf powder and Brassica juncea seeds

decreased the levels of cholesterol and phospholipids in the dimethyl hydrazine (DMH) induced

colon carcinogenesis experimental animals. Bile acids and neutral sterols in liver and feces,

showed a sharp increase in the groups given Murraya koenigii leaf powder and Brassica juncea

seeds when compared with the control. In a field research work carried out on albino rat, it was
17
observed that the rats fed with Murraya koenigii leaf powder and Brassica juncea seeds, there was

a decrease in the concentration of malondialdehyde, while hydroperoxides and conjugated dienes

were increased in liver and heart.There was increased activity of Superoxide dismutase and

catalase in liver and heart of administered groups. Glutathione levels in liver, heart and kidney

were lowered in rats administered these spices. Glutathione reductase, glutathione peroxidase and

glutathione S-transferase activity showed a sharp increase in the administered group (Khan,

1996).

Addition of Murraya koenigii leaf powder in the high fat diet resulted in reduction of lipid

peroxidation (thiobarbituric acid reactive substances) level to a beneficial extent. Histological

studies also indicated the modulation of hepatic functions to near normal level (Khan et al., 1997).

Carbazole alkaloids isolated from Murraya koenigii are recognized as sources of natural

antioxidants and thus play an important role in the chemoprevention of diseases resulting from

lipid peroxidation (Nakathani, 2000).

Baliga et al., (2003) reported the dose-dependent nitric oxide (NO) scavenging activity of aqueous

leaf extract of Murraya koenigii. This study suggests that Murraya koenigii might be a potent and

novel therapeutic agent for scavenging of (NO) and the regulation of pathological conditions

caused by excessive generation of (NO) and its oxidation product, peroxynitrite. It was suggested

that an aryl hydroxyl substituent on the carbazole rings from Murraya koenigii plays a role in

stabilizing the thermal oxidation and rate of reaction against DPPH (2,2-diphenyl-1-hydrazyl-

hydrate) radical (Tachibana, 2003). Murraya koenigii treatment exerts a therapeutic protective

nature in diabetes by decreasing oxidative stress and pancreatic beta-cell damage (Arulselvan and

Subramanian, 2007).

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2.1.2 Allium sativum (Garlic)

Garlic has been used for centuries in various societies to combat infectious disease. Historically, it

is believed that Louis Pasteur described the antibacterial property of garlic in 1858 for the first

time, although no reference is available (Wang et al., 2010). More recently, garlic has been shown

to be effective against a groups of gram-positive, gram-negative, and acid-fast bacteria (Nervi,

2006). These include Salmonella, Escherichia coli (Adler and Beuchat, 2002), Pseudomonas,

Proteus, Staphylococcus aureus (Cavallito, 1944), Escherichia coli, Salmonella and Klebsiella

(Jezowa and Rafinski, 1966), Micrococcus, Bacillus subtulis (Sharma et al., 1977), Clostridium

(De Witt et al., 1979), Mycobacterium (Delaha and Garagusi, 1985), and Helicobacter (O’Gara et

al., 2000).

It has been documented that garlic exerts a differential inhibition between beneficial intestinal

microflora and potentially harmful enterobacteria (Rees et al., 1993; Nervi, 2006). The

antibacterial activity of garlic is widely attributed to allicin. It is known that allicin has sulfhydryl

modifying activity (Wills, 1956) and is capable of inhibiting sulfhydryl enzymes. Cysteine and

glutathione counteract the thiolation activity of allicin. Garlic extract and allicin have been shown

to exert bacteriostatic effects on some vancomycin-resistant enterococci.

An inhibitory synergism was observed when used in combination with vancomycin (Jonkers et

al., 1999). The antibacterial effect of different concentrations of garlic extract against human

dental plaque microbiota has been shown in in vitro study (Houshmand et al., 2013). The

synergism between ciprofloxacin with garlic extract has been shown, but not between ampicillin

and the garlic extracts (Zain al-abdeen et al., 2013). The cloves of garlic and rhizomes of ginger,

extracted with 95% ethanol, suggested to have anti-bacterial activity against multi-drug clinical

pathogens and can be used for prevention of drug resistant microbial diseases. Pseudomonas

aeruginosa was the most sensitive germ to the mixture (Karuppiah and Rajaram, 2013).

19
Garlic also suggested as a treatment for multi-drug resistant tuberculosis (Dini et al., 2011).

Antifingal activity was first established in 1936 by Schmidt and Marquardt whilst working with

epidermophyte cultures (Lemar et al., 2002). Many fungi are sensitive to garlic, including

Candida (Yousuf, 2011), Torulopsis, Trichophyton, Cryptococcus (Fromtling and Bulmer, 1978),

Aspergillus (Hitokoto et al., 1980), Trichosporon, and Rhodotorula (Tansey and Appleton, 1975).

Garlic extracts have been shown to decrease the oxygen uptake (Szymona, 1952), reduce the

growth of the organism, inhibit the synthesis of lipids, proteins, and nucleic acids (Adetumbi et

al., 1986), and damage membranes (Ghannoum, 1988). A sample of pure allicin was shown to be

antifungal. Removal of the allicin from the reaction by solvent extraction decreased the antifungal

activity (Nervi, 2006; Hughes and Lawson, 1991). Activity has also been observed with the garlic

constituents, diallyl trisulfide, against cryptococcal meningitis (Cai, 1991), ajoene, and against

Aspergillus (Yoshida et al., 1987).

It has been reported that garlic exhibited antifungal effects on two species, the airborne pathogen

Botrytis cinerea and Trichoderma harzianum (Lanzotti et al., 2012). Greater satisfaction with the

use of garlic rather than nystatin was reported by the patients with denture stomatitis (Bakhshi et

al., 2012). Many countries have used garlic extract for clinical treatments, but the untoward

actions of garlic following long-term administration should be fully noted. Even though many

studies on garlic and its derivatives have been performed, the exact biological mechanism of

garlic extract still remains to be elucidated (Qi and Wang, 2003).

In the case of HIV, it is thought that ajoene acts by inhibiting the integrin dependent processes

(Tatarintsev et al., 1992). Allyl alcohol and diallyl disulfide have also proven effective against

HIV infected cells (Shoji et al., 1993). No activity has been observed with allicin or S-allyl

cysteine. It appears that only allicin and allicin-derived substances are active.

20
Taken together, the beneficial effects of garlic extract make it useful in medicine. There are

insufficient clinical trials regarding the effects of garlic in preventing or treating the common cold.

Antiviral properties in comparison with the antibacterial action of garlic, very little work has been

done to investigate its antiviral properties. The few studies have reported that garlic extract

showed in vitro activity against influenza A and B (Fenwick and Hanley, 1985), cytomegalovirus

(Meng et al., 1993; Nai-Lan et al., 1993), rhinovirus, HIV, herpes simplex virus 1 (Tsai et al.,

1985), herpes simplex virus 2 (Weber et al., 1992), viral pneumonia, and rotavirus. Allicin, diallyl

trisulfide and ajoene have all been shown to be active (Hughes et al., 1989; Weber., 1992).

A single trial suggested that garlic may prevent occurrences of the common cold, but more studies

are needed to validate this finding. This trial randomly assigned 146 participants to either a daily

garlic supplement (with 180 mg of allicin content) or a placebo for 12 weeks. The investigation

revealed 24 occurrences of the common cold in the garlic group compared with 65 in the placebo

group, resulting in fewer days of illness in the garlic group compared with the placebo group.

However, claims of effectiveness of garlic on common cold appear to rely largely on poor quality

evidence (Lissiman et al., 2012). In in vivo animal experiments, intravenous administration of

garlic extracts produced slight reductions in both systolic and diastolic pressures (Sial and Ahmed,

1982) and oral ingestion of garlic extract in hypertensive animals brought the blood pressure back

to the normal level (Chandekar and Jain, 1973).

Several clinical studies showed that garlic reduced blood pressure in more than 80% of patients

suffering from high blood pressure (Auer et al., 1989; Konig and Scineider, 1986; Petkov, 1979;

Omar, 2013; Stabler et al., 2012). In one trial, investigation on 47 hypertensive patients showed

that garlic significantly decreased the mean systolic blood pressure by 12 mmHg and the mean

supine diastolic blood pressure by 9 mmHg versus placebo.

The authors stated that garlic was free from side effects and no serious complication was reported

(Auer, 1990). In another study, 200 mg of garlic powder was given three times daily, in addition

21
to hydrochlorothiazide-triamterene baseline therapy, produced a mean reduction of systolic blood

pressure by 10-11 mmHg and of diastolic blood pressure by 6-8 mmHg versus placebo

(Kandziora 1988). However, these data are insufficient to determine if garlic provides a

therapeutic advantage versus placebo in terms of reducing the risk of cardiovascular morbidity in

patients diagnosed with hypertension (Stabler et al., 2012; Banerjee and Maulik, 2002).

A recent increase in the popularity of alternative medicine and natural products has renewed

interest in garlic and their derivatives as potential natural remedies. This review may be useful to

increase our knowledge of garlic therapeutic effects and improve our future experimental and

clinical research plans. Although it is shown that garlic may have a significant clinical potential

either in their own right or as adjuvant therapy in different disorders, however, due to some issues,

such as methodological inadequacies, small sample sizes, lack of information regarding

permissible dose, variation between efficacy and effectiveness trials, the absence of lack of

control groups more standard experiments and researches are needed to confirm the beneficial

effect of garlic in various diseases.

22
 CHAPTER THREE

3.0 Field Work

3.1 Plant Collection

A five hundred and fifty-two grams of Murraya koenigii leaves was purchase from the garden in

Ogun state and a 1.2 kilograms of Allium sativum, was bought from Oyingbo market, Local

Government Area of Lagos, Nigeria. The leaves and rhizomes of this specimens, were botanically

identified and authenticated at the herbarium of the Department of Botany, University of Lagos,

Nigeria. The herbarium number of Murraya koenigii was 8697 and the one for Allium sativum

was 8702 both were identified on the 4th of November, 2020, at exactly 11:25am.

3.2 Laboratory Work

3.2.1 Qualitative Analysis Preparation of the extracts

The samples were transported to the laboratory (university of Lagos department of Biochemistry)

so as to determine and evaluate the contents present in them. The Murraya koenigii leaves was

selected, washed, and the Allium sativum cloves, was pilled and chopped into smaller units, both

were oven dried at a room temperature in the laboratory for three to four weeks (1 month). The

dried samples were individually milled into a powder and the resulting powder was screened with

a sieve having 0.5Mm mesh size. 258.2g of the samples was weighed and was infused into 1litre

of methanol solution the mixture was vigorously stirred and allow to stand for 72hours. The

solution was now filtered using a watsman’s filter and the solvent was then removed and

concentrated at 40degree Celsius using rotary evaporator, the extracts were stored in a refrigerator

at 4degree Celsius until required for usage. The yield of the dried extract, 79.07g was calculated

using the expression as follow;

Yield (%) = weight of dried extract ×100

Weight of sample used

23
3.2.2 Qualitative analysis of phytochemical compounds

Qualitative analysis was carried out to ascertain the presence of the different phytochemical

compounds contained in the extract. Preliminary phytochemical analysis of the extracts was

carried out with reference to the standard methods of Sofowora (1998) and Evans (2009).

Test for Alkaloids

The extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were

used to test the presence of alkaloids using dragendroffs test.

Dragendroff’s Test: Filtrates were treated with Dragendroff’s reagent (solution of Potassium

Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids.

Test for Flavonoids

Lead acetate test: Extracts were treated with few drops of lead acetate solution. Formation of

yellow colour precipitate indicates the presence of flavonoids in the extracts.

Test for Steroids

2ml of acetic anhydride was added to 5mg of the extracts each with 2ml of H2SO4. A colour was

changed from violet to blue or green in some samples indicates the presence of steroids in the

extract.

Test for Terpenoids

Salkowski s test: 5mg of the extract of the leaves, flowers and seeds was mixed with 2ml of

chloroform and 3ml concentrated H2SO4 was carefully added to form a layer. An appearance of

reddish brown colour in the inner face indicates the presence of terpenoids.

24
Test for Phenols

Ferric chloride test: 5ml extracts were treated with few drops of ferric chloride solution.

Formation of bluish black colour indicates that the presence of phenol.

Test for Saponins

0.5mg of the extract was shaken with 5ml of distilled water. Formation of frothing (appearance of

creamy mass of small bubbles) shows that the presence of saponins.

Test for Tannins

A small quantity of extract was mixed with water and heated on a water bath. The mixture was

filtered and Iron (III) chloride was added to the filtrate. Formation of a dark green colour indicates

the presence of tannins.

Test for reducing sugar

Benedict’s test

Equal volume (2ml each) of Benedict’s solution and aqueous extract were mixed in a test tube and

heated in boiling water bath for 10min the changes in colour to yellow, green and red indicates the

presence of reducing sugar.

25
3.3 Quantitative Phytochemical Screening

Estimation of Alkaloids

Total alkaloid was measured using methods as described by Shamsa et al., (2007) with slight

modification. 1ml plant extract, 5 ml pH 4.7 phosphate Buffer was added and 5 ml BCG solution

and the mixture was shake with 4 ml of chloroform. The extracts were collected in a 10-ml

volumetric flask and then diluted to adjust volume with chloroform. The absorbance of the

complex in chloroform was measured at 470 nm against blank prepared as above but without

extract. Atropine is used as a standard (using calibration curve) and compared the assay with

Atropine equivalents.

Preparation of Standard Curve Accurately measure aliquots (0.4, 0.6, 0.8, 1 and 1.2 mL) of

atropine standard solution and transfer each to different separatory funnels. Add 5 mL pH 4.7

phosphate buffer and 5 mL BCG solution. Shake mixture with 1, 2, 3 and 4 mL of chloroform.

The extracts were collected in a 10 mL volumetric flask and then diluted to volume with

chloroform. The absorbance of the complex in chloroform was measured at 470 nm against blank

prepared as above but without atropine.

Estimation of Steroids

Total steroid was measured using methods as described by Salomi et al., (2019) with slight

modification. 1ml of test extract of steroid solution was transferred into 10ml volumetric flasks,

Sulphuric acid (4N, 2ml) and iron (III) chloride (0.5% w/v, 2 ml), were added, followed by

potassium hexacyanoferrate (III) solution (0.5% w/v, 0.5 ml). The mixture was heated in a water-

bath maintained at 70±2 0C for 30 minutes with occasional shaking and diluted to the mark with

distilled water. The absorbance was measured at 780 nm against the reagent blank. Total steroids

in extracts was expressed in terms of cholesterol equivalents (mg of CHO/g of extract).

26
Estimation of Flavonoids

Aluminium chloride colorimetric method (Chang et al.,2002) with some modifications was used

to determine flavonoid contents. Plant extract (1mL) in methanol was mixed with 1ml of

methanol, 0.5 mL aluminium chloride (1.2%) and 0.5 mL potassium acetate (120 mM). The

mixture was allowed to stand for 30 min at room temperature; then the absorbance was measured

at 415 nm. Quercetin was used as standard. Flavonoid content is expressed in terms of quercetin

equivalent (mg g-1 of extracted compound).

Estimation of Total Phenols

The Follins method described by pearson (1979) was used to determine the phenol contents.

1 ml of the extract was placed in a test tube, 2.5ml of 10% Folin-Ciocalteu’s reagent dissolved in

water and 2.5 ml of 7.5% of NaHCO3 aqueous solution were added. The samples were thereafter

incubated in a thermostat at 45oC for 45 min. The absorbance was determined using

spectrophotometer at wave length = 765 nm. The samples were prepared in triplicate for each

analysis and the mean value of absorbance was obtained. The same procedure was repeated for

the standard solution of garlic acid and the calibration line was construed. Based on the measured

absorbance, the concentration of garlic acid equivalent expressed in terms of (mg of GA/g of

extract).

Estimation of Tannins

Tannin contents was determined by the Folin-Denis colorimetric method described by Kirk and

Sawyer (1998). 5g sample was dispersed in 50mls of distilled water and shaken. The mixture was

allowed to stand for 30min at 28oC before it was filtered through what man No. 42 grade of filter

paper. 2mls of the extract was dispersed into a 50ml volumetric flask. Similarly, 2ml standard

tannin solution (tannic acid) and 2ml of distilled water were put in separate volumetric flasks to

serve as standard and reagent was added to each of the flask and the 2.5ml of saturated Na2C03

27
solution added. The content of each flask was made up to 50ml with distilled water and allowed to

incubate at 28oC for 90 min. Their respective absorbance was measured in a spectrophotometer at

765nm using the reagent blank to calibrate the instrument at zero.

DNS reducing sugar assay

This assay is based on the protocol described by Hussain et al., (2018) and miller (1978) with

slight modification. 1ml of the plant samples was taken and 3ml of DNSA was added and boiled

for 10min and absorbance was taken at 540nm. Glucose was used as standard (100mg/ml). DNS

reagent was prepared by mixing 1.6 g NaOH and 1.0 g dinitrosalicylic acid (Sigma) in 70 mL

dH2O and the mixture was heated in boiling water to dissolve. Once dissolved, 3.0 g Na2K

tartrate (Sigma) was added to the solution and swirled until dissolved followed by further addition

of dH2O to make up to 100 mL. The reagent was stored dark at room temperature. 1ml of the

plant samples is taken and 3ml of DNSA was added and boiled for 10min and absorbance was

taken at 540nm. Glucose was used as standard (100mg/ml).

28
3.4 In Vitro Antioxidant Assays

Reducing power assay (Oyaizu, 1986)

The reducing powers was determined according to the method with slight modifications. Reaction

was carried out in a mixture containing 1 ml of sample (25-100 µg/ml), 2.5 ml of 0.1 M sodium

phosphate buffer (pH 6.6) and 2.5 ml of 1%, w/v potassium ferro cyanate [K3Fe(CN)6] by

incubating at 50°C for 20 min. After addition of 2.5 ml trichloroacetic acid (10%, w/v), the

mixture was centrifuged at 5000rpm for 10 min. The upper layer (5 ml) was mixed with 0.5 ml of

fresh FeCl3 (0.1%, w/v), and the absorbance at 700 nm was measured against a blank. Gallic acid

was used as the control.

DPPH radical scavenging assay (Shimada et al.,1992).

Briefly 0.1 Mm solution of DPPH in ethanol was prepared; 1ml of the solution was added to 1 ml

of extract in water at different concentrations (25-100 μg/ml). The mixture was shaken vigorously

and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517

nm by using a UV-Visible Spectrophotometer. Lower absorbance of the reaction mixture

indicated higher free radical scavenging activity. The percent DPPH scavenging effect was

calculated using the following equation:

DPPH Scavenging effect (%) = [(A0-A1)/A0] x 100

Where A0 was the absorbance of the control and A1 was the absorbance in the presence of the

standard sample or extract. The IC50 value represented the concentration of the compounds that

caused 50% inhibition of DPPH radical formation.

29
NITRIC OXIDE (GRIESS REAGENT)

Sodium nitroprusside (10mM) in phosphate buffer saline was mixed with different concentrations

(25-100 µg/ml) of each extract, and incubated for 30min, after incubation period, 0.5ml of Griess

reagent (1% sulfanilamide, 2% H2PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihyrochloride

was added. Absorbance was taken at 550nm and ascorbic acid was used as standard. IC50 which

is an inhibitory concentration of each extract required to reduce 50% of nitric oxide formation was

determined. The same reaction mixture without the extract but equivalent amount of methylated

spirit was used as control.

Nitric oxide (inhibition %) = A (control) – A (sample) x 100

A (control)

30
 CHAPTER FOUR

4.0 Results

Table 2: Result of phytochemical screening (Qualitative Analysis) for the methanol extracts
of Murraya koenigii leaf and Allium sativum cloves (Qualitative Analysis)

Specimens Reducing Saponin Phenol Flavonoid Tannin Alkaloid Steroid Terpernoid

sugar
Murraya + - + + + + + +
koenigii

Allium + - + + + + + +
sativum

Key: + present

- Absent

Table 3: Percentage (%) results of phytochemical parameters of the methanol extract

Murraya koenigii leaf and Allium sativum cloves (Quantitative Analysis)

Specimens Reducing Saponin Phenol Flavonoid Tannin Alkaloid Steroid Terpernoid

sugar (mg/100g) (mg/100g) (mg/100g) (mg/100g) (mg/100g) (mg/100g) (mg/100g)
(mg/100g)
Murraya 17.33 - 87.20 51.59 63.43 101.68 42.34 16.83
koenigii

17.22 - 88.13 51.77 64.11 100.88 42.17 17.44

Allium 6.89 - 86.26 32.70 62.75 98.67 24.79 13.55

sativum

6.78 - 84.78 33.61 61.67 99.12 34.60 14.00

31
 4.1 Antioxidant Essay Results

Table 4: Results of Anti-oxidant activity of Murraya koenigii leaf and Allium sativum cloves
(REDUCING POWER)
Specimens 25µg/ml 50µg/ml 75µg/ml 100µg/ml
Murraya koenigii 0.152 0.177 0.208 0.254

0.154 0.180 0.212 0.251

Allium sativum 0.115 0.132 0.178 0.235

0.120 0.136 0.181 0.232

ASCORBIC ACID 0.242 0.382 0.481 0.624

0.233 0.379 0.485 0.626

Table 5: Results of Anti-oxidant activity of Murraya koenigii leaf and Allium sativum cloves
(DPPH)
Specimens 25µg/ml 50µg/ml 75µg/ml 100µg/ml
Murraya koenigii 20.93 47.41 63.69 81.57

20.21 48.12 62.97 81.40

Allium sativum 26.83 43.65 60.82 78.35

26.30 44.01 61.36 78.89

ASCORBIC ACID 45.71 62.29 78.64 86.39

46.58 61.47 79.57 87.61

32
 Table 6: Results of Nitric oxide of Murraya koenigii leaf and Allium sativum cloves

Specimens 25µg/ml 50µg/ml 75µg/ml 100µg/ml

Murraya koenigii 32.21 49.29 66.55 80.60

33.27 49.64 66.73 81.32

Allium sativum 33.27 59.79 66.01 75.62

34.34 59.61 65.48 75.80

ASCORBIC ACID 47.65 63.51 76.89 85.44

48.13 62.67 75.25 84.38

Table 7: Results of Total Anti-Oxidant Capacity of Murraya koenigii leaf Allium

sativum cloves

Total Antioxidant Capacity

Specimens Total Antioxidant
Capacity mg/100g
Murraya koenigii 71.81

72.96

Allium sativum 57.68

58.09

33
 4.2 Statistical Results

Table 8: Statistical data analysis of results of Murraya koenigii leaves and Allium sativum
roots (Phytochemicals)
Row stats
Murraya koenigii Allium sativum
Mean SD Mean SD
Reducing Sugar 17.28 0.08 6.84 0.08
Phenol 87.66 0.66 85.52 1.05
Flavonoid 51.68 0.13 33.16 0.64
Tannin 63.77 0.48 62.21 0.76
Alkaloid 101.28 0.57 98.90 0.32
Steroid 42.26 0.12 29.70 6.94
Terpenoid 17.14 0.43 13.78 0.32
TAC 72.38 0.81 57.89 0.29

150
M u r r a y a k o e n ig ii
A lliu m s a tiv u m

100
m g /1 0 0 g

50

0
r

C
id
id

in
o

id
a

id

A
g

lo
o

ro

T
u

n
h
S

te
o

lk

e
P

T
v
g

rp
S
A
la
in

e
F
c

T
u
d
e
R

P h y t o c h e m ic a ls

Figure 5: General simple bar chart showing the phytochemical values of the constituent in
Murraya koenigii leaves and Allium sativum roots.
Value expressed as mean + standard error of mean (SEM).

34
 Table 9: The Mean and Standard Deviation of Murraya koenigii leaves
and Allium sativum cloves (antioxidants)
Row stats of Reducing power

Murraya koenigii Allium sativum ASCORBIC ACID

Mean SD Mean SD Mean SD

25 0.153 0.001 0.118 0.004 0.238 0.006

50 0.179 0.002 0.134 0.003 0.381 0.002

75 0.210 0.003 0.180 0.002 0.483 0.003

100 0.253 0.002 0.234 0.002 0.625 0.001

Table 10: The Mean and Standard Deviation of Murraya koenigii leaves and Allium
sativum cloves (antioxidants)
Row stats of DPPH

Murraya koenigii Allium sativum ASCORBIC ACID

Mean SD Mean SD Mean SD

25 20.57 0.51 26.57 0.37 46.15 0.62

50 47.77 0.50 43.83 0.25 61.88 0.58

75 63.33 0.51 61.09 0.38 79.11 0.66

100 81.49 0.12 78.62 0.38 87.00 0.86

35
Table 11: The Mean and Standard Deviation of Murraya koenigii leaves and
Allium sativum cloves (antioxidants)
Row stats of NITRIC

Murraya koenigii Allium sativum ASCORBIC ACID

Mean SD Mean SD Mean SD

25 32.74 0.75 33.81 0.76 47.89 0.34

50 49.47 0.25 59.70 0.13 63.09 0.59

75 66.64 0.13 65.75 0.37 76.07 1.16

100 80.96 0.51 75.71 0.13 84.91 0.75

KEY: SD = Standard Deviation

Value expressed as mean + standard error of mean (SEM).

0 .8
R e d u c in g p o w e r a c t iv ity

A S C O R B IC A C ID
A lliu m s a tiv u m
0 .6
M u r r a y a k o e n ig ii

0 .4

0 .2

0 .0
5 0 5 0
2 5 7 0
1

C o n c e n t r a t io n ( µ g /m l)

Figure 6: simple graph chart showing the anti- oxidant (REDUCING POWER) values of the

constituent Murraya koenigii leaves and Allium sativum cloves extracts.

Value expressed as mean + standard error of mean (SEM).

36
 D P P H S c a v e n g in g a c t iv ity 100
A S C O R B IC A C ID

80 A lliu m s a tiv u m
( % In h ib it io n )

M u r r a y a k o e n ig ii
60

40

20

0
5 0 5 0
2 5 7 0
1

C o n c e n t r a t io n ( µ g /m l)

Figure 7: Simple graph chart showing anti- oxidant (DPPH) values of the constituent in Murraya
koenigii leaves and Allium sativum cloves extracts.
Value as expressed as mean + standard error of mean (SEM).
N itr ic o x id e s c a v e n g in g a c t iv ity

100
A S C O R B IC A C ID

80 A lliu m s a tiv u m
( % In h ib it io n )

M u r r a y a k o e n ig ii
60

40

20

0
0
5

0
2

C o n c e n t r a t io n ( µ g /m l)

Figure 8: Simple bar chart showing anti- oxidant (NITRIC OXIDE) values of the constituent in

Murraya koenigii leaves and Allium sativum cloves extracts.

Value as expressed as mean + standard error of mean (SEM).

37
 CHAPER FIVE

5.0 Discussion

The results of the present study suggested that several phytochemicals are present in Murraya

koenigii leaf and Allium sativum cloves extracts. Phytochemicals give plants their colour, flavour,

smell and are part of a plant`s natural defence system and protect them against herbivorous insects

and vertebrates, fungi, pathogens, and parasites. The phytochemicals saponin, flavonoid, tannin,

reducing sugar, steroid, and terpernoid were present in both Murraya koenigii and Allium sativum

extracts according to this study. The phytochemical contents of the extract of Murraya koenigii

revealed that the Alkaloids was found to be the most abundant phytochemical (101.28 %)

followed by phenol (87.66%), tanin (63.77%) and flavonoids (51.68 %). While the phytochemical

content of the extract of Allium sativum revealed that the Alkaloids was found to be the most

abundant phytochemical (98.90 %) followed by phenol with value of (85.52%), tannin having the

value of (62.21%) and flavonoids with the value of (33.16%).

Also, based on the finding of this study, terpernoid is present in the both the extracts. Terpernoids

have been found to be useful in the prevention and therapy of several diseases, including cancer.

Terpenoids are also known to possess antimicrobial, antifungal, antiparasitic, antiviral, anti-

allergenic, antispasmodic, antihyperglycemic, anti-inflammatory and immunomodulatory

properties. Flavonoids are also present in the extracts as a potent water-soluble antioxidant and

free radical scavenger, which prevent oxidative cell damage and also have strong anticancer

activity. It also helps in managing diabetes induced oxidative stress. Steroids are importance in

pharmacy as they possess compounds like sex hormones and can be used for drug production.

Also Tannin was present in abundance.

38
The growth of many fungi, yeast, bacteria and viruses was inhibited by tannins. The finding of this

study correlate with the finding of Abaoab et al., (2011) which found that garlic clove extract,

possessed a broad spectrum of antimicrobial activity exhibited for both bacteria and fungi due to

presence saponin, tannin, flavonoid and terpernoid. The various pharmacological activities of

these plants have been seen such as activity on Anti diabetic, cholesterol reducing property,

antimicrobial activity, antiulcer activity, Antioxidative property, cytotoxic activity, antidiarrhoea

activity, anti-cancer activity with many other phagocytic activities. The chemical composition of

the Murraya koenigii consists of essential oil alkaloids and terpenoid. Thus Curry leaves merits

further phytochemical, pharmacological, antioxidant and clinical investigations for development

of an effective natural plant. Also the result of this study on Phytochemistry of Allium sativum

supported the study conducted by Deresse D., (2010) who found that garlic extracts exhibited

activity against both gram negative (E. coli, Salmonella sp, and Citrobacter Enterobacter,

Pseudomonas Klebsiella) and gram positive (S. aureus, S. pneumonia, streptococcus and Bacillus

anthrax) due to presence of some phytochemicals such saponin and tannin.

These bioactive principles are believed to be responsible for the observed antimicrobial effect of

the plant extract. Several workers have attributed the antimicrobial effect of plant extract to the

presence of these secondary plant metabolites (Nweze et al., 2004; Gandhiraja et al., 2009). The

presence of active principles in the bulb extract of Allium sativum could be used to establish a

good support for the use of the plant in herbal medicine. This is in agreement with the work of

Rojas et al., (2006) on ten medicinal plants whose work only differs a little as a result of location

of where the plant was cultivated. The Murraya koenigii activities of the plant extract, agree with

the report of Leven et al., (1979). Nwadiaro and Nwachukwu, (2007) linked the antimicrobial

activities of plants to the presence of tannins, alkaloids, flavoinoids and saponins.

39
It has been reported that various plants that are rich in alkaloids tannins and glycosides possess

antimicrobial activity against a number of micro-organisms (Adebajo et al., 1983). This suggests

that the plant extracts used in this study has a general antimicrobial activity.

The antifungal and antibacterial potential of Allium sativum bulb extract, is well pronounced this

is in consonance with the report of Nantitanon et al., (2007) on Hyptis sauveolens. The present

study therefore shows that Allium sativum bulb extract has useful antioxidant properties. Further

work is needed to isolate the active principles from the plant in order to test the specific

antimicrobial activity of the respective phytochemical components.

5.1 Conclusion

Based on the findings of the present study, phytochemical constituents and antioxidants

components of Murraya koenigii leaves and Allium sativum cloves were determined. The

phytochemical components of Allium sativum cloves contain alkaloid, Reducing sugar, Phenol,

saponins, flavonoids, tannin and terpernoids. The results of the antioxidants analyses of the whole

leaves and root bulbs, indicated the presence of considerable amount of antioxidants. The

presence of the phytochemicals has authenticated its usefulness by traditional herbalists in ethno

medicine and potentials in drug formulation and development. This study has shown that Murraya

Koenigii (curry) leaf and Allium sativum cloves, are used as spice and flavouring agent in food

contains substantial amount of phytochemicals. With their contents of anti-oxidants flavonoids,

phenols etc., both specimens possess anti-cancer and cardio-protective agents supporting its use as

medicinal plant.

40
5.2 Recommendation

Further research should be done on isolating each phytochemical and antioxidant present in this
samples so as to know the effect on the human body.
Understanding how this plant extracts works in the human body when taken, is of paramount
importance.
Mechanism of how this plants works in the human body, should be further studied.
Caution with regard to the dose or quantity of this plant extract intake should always be
considered.
The effect of the isolation of each phytochemical and antioxidant present in the samples should be
further researched on the human body so as to know there permissible values.

Knowing the importance of how this plant extracts works in the human body when taken in.

Further study should be done on clinical chemistry on how this plants react with the physiology of
the body.
Consideration should be taken as regards to the caution.

41
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spices on some pathogens. Agriculture and Biology Journal of North America 2(8): 1187-
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A.D.A.M. (2019). Phytochemicals: Medical plus. 2:1

Adebajo A.C., Avoola O.F., Iwalewa E.O., Akindahunsi A.A., Omisore N.O. and Adewunmi C.
(2006). Anti-trichomonal, biochemical and toxicological activities of methanolic extract and
some carbazole alkaloids isolated from the leaves of Murraya koenigii growing in Nigeria.
Phytomedicine 13(4): 246-54.

Adler B.B. and Beuchat, L. (2002). Death of Salmonella, Escherichia coli 0157:H7, and Listeria
monocytogenes in garlic butter as affected by storage temperature. Journal of Food
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