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TRACE ELEMENTS CONTENT OF NEEM LEAVES
(AZADIRACHTA INDICA).
BY
AMHANDIN OSAHUME DEBORAH
BC/2006/077
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF NATURAL SCIENCES
CARITAS UNIVERSITY, AMORJI-NIKE, ENUGU,
ENUGU STATE.
AUGUST, 2010
TRACE ELEMENTS CONTENT OF NEEM LEAVES
(AZADIRACHTA INDICA).
BY
AMHANDIN OSAHUME DEBORAH
BC/2006/077
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF NATURAL SCIENCES
CARITAS UNIVERSITY, AMORJI-NIKE, ENUGU,
ENUGU STATE.
A RESEARCH PROJECT PRESENTED IN PARTIAL FULFILMENT
OF THE REQUIREMENT FOR THE AWARD OF BACHELOR OF
SCIENCE (B.Sc.) DEGREE IN BIOCHEMISTRY.
AUGUST, 2010
CERTIFICATION
This is to certify that this project research on ‘Trace Elements Content Of
Neem Leaves (Azadirachta Indica)’ was carried out by Amhandin Osahume
Deborah, under the supervision of Mr. Eze Peter Stephen, Biochemistry
Department, Caritas University, Amorji-Nike, Emene, Enugu State.
………………………………..
MR. EZE-STEPHEN P.E
…………………………
Date
(Project Supervisor)
………………………………...
…………………………
MR. MOSES EZENWALI
(Head of Department)
…………………………………
EXTERNAL SUPERVISOR
Date
………………………...
Date
DEDICATION
This project is dedicated to God Almighty for being the first fruit of my intellect and
making my dream a reality.
ACKNOWLEDGEMENT
My profound and unreserved gratitude goes to my parents, Mr. Dennis Amhandin and
Mrs. Sabina Amhandin for their immense support both spiritually, morally, educationally
and most of all financially.
I am very much grateful to my supervisor, Mr. Eze-Stephen P.E., who not only
contributed immensely but also helped in making this project a great success. My H.O.D.
Mr. Ezewanli Moses and other lecturers in Biochemistry Department are also
acknowledged.
My gratitude also goes to my siblings for their love and concern all the time. To them I
say thank. You all have been great.
Others that I will not forget to mention here are my great friends who have the same mind
set with me and have always been there when I needed them. They really are true friends,
they include Mathias Izuchukwu, Enudi Onyeka, Aroboinosen Usinan, Nneka Chime and
to all my fellow colleagues in biochemistry department.
ABSTRACT
The global scenario is now supporting the development of modern drugs from less toxic
plant products with proven medicinal properties. Each part of the neem plant
(Azadirachta indica A. Juss) reportedly has various medicinal properties and has been in
use in many continents for centuries. In this project work, water extract from Neem
leaves (Azadirachta indica A Juss) with the modern atomic absorption spectrophotometer
was presented. The mineral and chemical properties in the water extract of Neem leaf
were studied. Results showed that the neem leaf contains some essential minerals needed
by the biochemical system. It is obvious that the water extract of Azadirachta indica is
significantly high in sodium ion (Na+) and potassium ion (K+) concentrations which are
responsible in the conduction of impulses along the axons of the central nervous system.
Iron is another element determined. This element is significantly low in the water extract
of the sample used (neem leaf).
TABLE OF CONTENTS
Title page ……………………………………………………………………. i
Certification ………………………………………………………………….ii
Dedication …………………………………………………………………..iii
Acknowledgement …………………………………………………………..iv
Abstract ………………………………………………………………………v
Table of contents ……………………………………………………………vi
CHAPTER ONE
1.0 Introduction ………………………………………………………………1
1.1 Aim of project…………………………………………………………….2
CHAPTER TWO
2.0 Literature review…………………………………………………………3
2.1 History of Neem tree …………………………………………………….3
2.2 Scientific classification …………………………………………………..4
2.3 Description of the Neem tree ………………………………………….....5
2.4 Neem oil ………………………………………………………………….6
2.5 Neem gum ………………………………………………………………..8
2.6 Neem honey ……………………………………………………………...8
2.7 Chemical compounds in Neem …………………………………………..8
2.8 Medicinal properties of Neem ………………………………………….10
2.9 Modern researches on the effect of Neem ……………………………...11
2.9.1 Neem’s effect against microorganisms ………………………………12
2.9.2 Neem’s effect against ailments ……………………….........................14
2.9.3 Toxicity of Neem ……………………………………………………..15
2.9.4 Neem as a vegetable ………………………………………………….16
CHAPTER THREE
3.0 Materials and methods…………………………………………………..17
3.1 Raw material used ………………………………………………………17
3.2 Equipment used…………………………………………………………17
3.3 Sample preparation and extraction ……………………………………..18
3.4 Atomic absorption spectrophotometer determination ………………….18
CHAPTER FOUR
4.1 Results …………………………………………………………………19
CHAPTER FIVE
Discussion and conclusion …………………………………………………23
REFERENCES…………………………………………………………….24
CHAPTER ONE
1.0
INTRODUCTION
Neem (Azadirachta indica A. Juss) is one of the very few trees known in the
Indian subcontinent (Puri, 1999). This tree belonged to Meliceae family, and grows
rapidly in the tropic and semi-tropic climate. It is also observed that this tree could
survive in very dry and arid conditions. (Puri, 1999). The Neem Tree is an incredible
plant that has been declared the Tree of the 21st century by the United Nations (Puri,
1999). In India, it is variously known as ‘Divine Tree’, ‘Life giving tree’, ‘Nature’s
Drugstore’, ‘Village Pharmacy’ and ‘Panacea for all diseases’. It is one of the major
components in Ayurvedic medicine, which has been practiced in India since many
centuries.
Extracts from the Neem tree (Azadirachta indica A Juss) also called ‘Dogonyaro’
in Nigeria are most consistently recommended in ancient medical texts for
gastrointestinal upsets, diarrhoea and intestinal infections, skin ulcers and malaria
(Schmutterer, 1995). All parts of Neem plant such as leaves, bark, flower, fruit, seed and
root have advantages in medical treatment and industrial products. Its leaves can be used
as drug for diabetes, eczema and reduce fever. Barks of Neem can be used to make
toothbrush and the roots has an ability to heal diseases and against insects. (Puri, 1999).
The seed of Neem tree has a high concentration of oil. Neem oil is widely used as
insecticides, lubricant, drugs for variety of diseases such as diabetes and tuberculosis
(Puri, 1999; Ragasa et al., 1996).
India encouraged scientific investigations on neem tree as part of his program to
revitalise India tradition and also increase commercial interest on neem (Stix, 1992) and
presently some authors believe that no other plant or tree in the world has been so
extensively researched or used in all possible capacities so far. In Africa, extracts from
neem leaves have provided various medicinal preparations (Ekanem, 1971; Udeinya,
1993). Neem plant (Azadirachta indica) has been of great benefit in human health due to
its biochemical, pharmacological, and medicinal properties.
1.1
AIM OF PROJECT
Many researches have been carried out on neem plant (Azadirachta indica) and
results have shown that it has both medicinal and pharmacological properties. However,
there are no documented information relating the mineral properties inherent in the leaf
of the plant. Consequently, this work was aimed at determining some minerals present in
the water extract of neem leaf using the modern atomic absorption spectrophotometric
analysis.
CHAPTER TWO
LITERATURE REVIEW
2.1
HISTORY OF NEEM TREE
Neem (Azadirachta indica A. Juss) is thought to have originated in Assam in
northeast India, and Myanmar, where it is common throughout the central dry zone. Later
it became naturally distributed throughout much of the Indian subcontinent, particularly
in drier areas. Neem name was derived from the Sanskrit Nimba, and it was known as the
curer of all illness. The neem tree was intimately connected with the everyday life of
Indians.
The neem tree has been used for more than 4,500 years in the Indian subcontinent. The Indian physicians charaka (2nd century AD) and susruta (4th century AD),
whose books provided the foundation of the Indian system of natural treatment, the
Ayurveda, also mention the tree and its medical use. In Ayurveda the neem tree was
called the ‘Sarva Roga Nivarini’ (one that could cure all ailments and ills). At the
beginning of this century the neem tree was still highly estimed by Indian emigrants and
they took it along to the places where they settled. Thus, the neem tree was introduced in
places like Australia, East and sub- Sahelian Africa, South East Asia, and South America.
Pioneering work in the possible commercial use of Neem oil and cake had been done by
the Indian Institute of Science in Bangalore as early as the 1920s.
Pioneering work in the possible commercial use of Neem oil and cake had been
done by the Indian Institute of Science in Bangalore as early as the 1920s. In the last two
decades research on neem has been intensified and many of the trees agricultural and
medical properties were rediscovered. Today, Neem plays a major role in the rural
industry of India and projects for the commercial use of Neem have been successfully
introduced in other countries. The green pinnate leaves of neem have a very bitter taste
and garlic- like smell.
2.2 SCIENTIFIC CLASSIFICATION
Kingdom:
Plantae
Division:
Magnoliophyta
Order: Sapindales
Family:
Meliceae
Genus:
Azadirachta
Species:
A. indica
Binomial name: Azadirachta indica
2.3 DESCRIPTION OF THE NEEM TREE.
The neem tree (Azadirachta indica), is a tropical evergreen with a wide
adaptability, native to India and Burma, it has been transplanted to Africa, the Middle
East, South America and Australia. It is especially suited to semi-arid conditions and
thrives even in the poorest soil with rainfalls as little as 18 inches (450 mm) per year and
temperatures up to 50° C (120° F). Neem can grow into a big tree to a height of about 20
to 35 m. Its canopy of leaves makes it a useful shade tree. It is planted along roads and
avenues in the towns and villages of India The lifespan of the Neem tree is described to
be anywhere between 150 to 300 years. Its blossoms are small, white flowers with a very
sweet, jasmine-like scent. Its edible fruit is about 3/4 of an inch (2 cm) long, with white
kernels. A neem tree generally begins bearing fruits at three to five years of age, and can
produce up to 50 kg of fruit annually when mature. The pinnate leaves have a very bitter
taste and a garlic-like smell Trunk: The trunk is relatively short, straight and may reach a
diameter of 1.2 m (about 4 feet). It is classified as a bush.
Leaves: The opposite pinnate leaves are 20-40 cm (8 to 16 inch) long, with 20 to 31
medium to dark green leaflets about 3-8 cm (1 to 3 inch) long. The petioles are short.
Very young leaves are reddish to purplish in colour. The shape of mature leaflets is more
or less asymmetric and their margins are dentate with the exception of the base of their
basiscopal half, which is normally very strongly reduced and cuneate or wedge-shaped.
(Ganguli, 2002).
Flowers: The (white and fragrant) flowers are arranged axillary, normally in more-or-less
drooping panicles which are up to 25 cm (10 in.) long. The inflorescences, which branch
up to the third degree, bear from 150 to 250 flowers. An individual flower is 5-6 mm long
and 8-11 mm wide. Protandrous, bisexual flowers and male flowers exist on the same
individual. Flowers are used to make a curry called ugadi pachadi.
Fruit: The fruit is a smooth (glabrous) olive-like drupe which varies in shape from
elongate oval to nearly roundish. The fruit skin (exocarp) is thin and the bitter-sweet pulp
(mesocarp) is yellowish-white and very fibrous. The mesocarp is 0.3-0.5 cm thick. The
white, hard inner shell (endocarp) of the fruit encloses one, rarely two or three, elongated
seeds (kernels) having a brown seed coat (Ganguli, 2002). Seeds usually fall to the
ground and might stay there or be carried away with rain water. Occasionally they are
dispersed away from the parent tree by birds which give them a greater chance of
growing into a healthy new plant. Neem oil is obtained from the seeds.
2.4 NEEM OIL
Neem oil is a vegetable oil pressed from the fruits and seeds of neem plant
(Azadirachta indica). Neem oil is generally light to dark brown, bitter and has a rather
strong odour that is said to combine the odours of peanut and garlic. It comprises mainly
triglycerides and large amounts of triterpenoid compounds, which are responsible for the
bitter taste. It is hydrophobic in nature and in order to emulsify it in water for application
purposes, it must be formulated with appropriate surfactants (Rajeev Seenappa, 2009).
Neem oil also contains steroids (campesterol, beta-sitosterol, stigmasterol) and a
plethora of triterpenoids of which azadirachtin is the most well-known and studied. The
azadirachtin content of neem oil varies from 300ppm to over 2500ppm depending on the
extraction technology and quality of the neem seeds crushed (Puri,1999).
Methods of extraction of neem oil
There are several methods to obtain Neem oil from the seeds like mechanical
pressing, supercritical fluid extraction, and solvent extraction (Puri, 1999). Mechanical
extraction is the most widely used method to extract neem oil from Neem seed. However,
the oil produced with this method usually has a low price, since it turbid and contains a
significant amount of water and metals contents. Extraction using supercritical fluid, the
oil produced has very high purity; however the operating and investment cost is high.
Extraction using solvent has several advantages. It gives higher yield and less turbid oil
than mechanical extraction, and relative low operating cost compared with supercritical
fluid extraction. The oil can also be obtained through pressing (crushing) of the seed
kernel both through cold pressing and through a process incorporating temperature
controls. A large industry in India extracts the oil remaining in the seed cake using
hexane. This solvent-extracted oil is of a lower quality as compared to the cold pressed
oil and is mostly used for soap manufacturing. Neem cake is a by-product obtained in the
solvent extraction process for neem oil. (Puri, 1999).
2.5 NEEM GUM
Neem Gum is a clear, bright and brown-coloured gum obtained from the trunk of
neem. This is as a result of certain metabolic mechanism of plants and trees. The gum is a
multipurpose by product either water soluble or absorbs water to form a viscous solution.
2.6 NEEM HONEY
In India, neem trees are a major source of honey bee forage. Honey obtained from
the Neem tree has more medicinal properties. Neem honey is composed primarily of
water, fructose and glucose (22.88%), sucrose (7.46%), ash (0.06%), free acid (20.8
meg/kg). The honey is light amber in colour and its viscosity is low. The taste is good
although slightly bitter. Neem honey improves eye sight and is harmless for diabetic
patients. It is also used to treat eye disorder by applying as netranjan (eye-liner). It is very
beneficial in care of burning sensation of the body. Since Neem is believed to be a great
blood purifier and good for the eyes, Neem honey is highly valued.
2.7 CHEMICAL COMPOUNDS IN NEEM.
The late Pakistani scientist Salimuzzaman Siddiqui was the first scientist to bring
the plant to the attention of phytopharmacologists. In 1942 while working at the
Scientific and Industrial Research Laboratory at Delhi University, India, he extracted
three bitter compounds from neem oil, which he named nimbin, nimbinin, and nimbidin
respectively (Ganguli, 2002). The seeds contain a complex secondary metabolite
azadirachtin. Several chemical compounds have been identified and scientists feel that
there are many more compounds yet to be identified in neem. Other than sodium,
potassium, salts, it contains chloriphyle, calcium, phosphorus, iron, thiamine,
riboflasium, nicocin, vitamin C, carotene, and oxalic acid. The chemicals classified are:
 Nimbin: anti-inflammatory, anti-pyretic, anti-histamine, anti-fungal
 Nimbidin: anti-bacterial, anti-ulcer, analgesic, anti-arrhythmic, anti-fungal
 Ninbidol: anti-tubercular, anti-protozoan, anti-pyretic
 Gedunin: vasodilator, anti-malarial, anti-fungal
 Sodium nimbinate: diuretic, spermicide, anti-arthritic
 Quercetin: anti-protozoal
 Salannin: insect repellent
 Azadirachtin: insect repellent, anti-feedant, anti-hormonal
Other chemicals that form its therapeutic value are:
 Limonoids
 Terpenoids and steroids
 Tetranortarpenoids
 Fatty acid derivatives like margosinone and margosinolone
 Coumarins like scopoletin, dihydrosocoumarins
 Hydrocarbons like docosane, pentacosane, hetacosane, octacosane etc.
 Sulphur compounds
 Phenolics
 Flavonoglycosides
 Tannins
The highest concentrations of the active ingredients are found in the seed
and oil, however the active ingredients are also found in lesser amounts in the bark
and the leaves.
2.8 MEDICINAL PROPERTIES OF NEEM.
For thousands of years the beneficial properties of Neem (Azadirachta indica A.
Juss) have been recognized in the Indian tradition. Each part of the neem tree has some
medicinal properties (Biswas et al., 2002).
Traditionally Neem was used in Ayurveda for a number of conditions. It is one of
the main ingredients in every blood purification formula used in Ayurveda and it appears
in most diabetic formulas as well. It is also used for arthritis, rheumatism, the removal of
external and internal parasites, including malaria and fevers and as an insect repellent.
Leaf: Leprosy, skin problems, skin ulcers, intestine worms, anorexia, eye problems,
epistaxis, biliousness
Bark: Analgesic, curative of fever
Flower: Elimination of intestine worms, phlegm, bile suppression,
Fruit: Diabetes, eye problem, piles, intestine worms, urinary disorder, wounds, leprosy,
epistaxis
Twig: Asthma, cough, piles, intestine worms, obstinate urinary disorder, phantom tumor,
spermatorrhoea
Gum: Scabies, wounds, ulcer, skin diseases
Seed: Intestine worms and leprosy
Oil: Intestine worms, skin diseases and leprosy
Root: Refrigerant, diuretics
(Sri, 2009).
2.9 MODERN RESEARCHES ON THE EFFECTS OF NEEM.
Neem leaf extract has been prescribed for oral use for the treatment of malaria by
Indian Ayurvedic practitioners from time immemorial. Recently, a clinical trial has been
carried out to see the efficacy of neem extract to control hyperlipidemia in a group of
malarial patients severely infected with P. falciparum. The lipid level, especially
cholesterol, was found to be lower during therapy when compared to non-malaria
patients. Reports are available regarding the use of neem to treat patients suffering from
various forms of cancer. One patient with parotid tumour and another with epidermoid
carcinoma have responded successfully when treated with neem seed oi1 Neem leaf
aqueous extract effectively suppresses oral squamous cell carcinoma induced by 7,12dimethylbenz[a] anthracene (DMBA), as revealed by reduced incidence of neoplasm.
Neem may exert its chemopreventive effect in the oral mucosa by modulation of
glutathione and its metabolizing enzymes.
NIM- 76, a refined product from neem oil, was studied in 10 human volunteers,
where intra-vaginal application before sexual intercourse could prevent pregnancy with
no adverse effect on vagina, cervix and uterus. The data suggested that intrauterine
treatment is safe (Kausik, 2002).
2.9.1 NEEMS EFFECT AGAINST MICROORGANISMS.
There have been a number of clinical studies showing that Neem has significant
effects on several bacterial strains. Among some of the more prominent strains studied
were staphylococcus aureus, streptococcus pyogenes, cornebacterium, E. coli, and
Salmonella typhosa. These bacteria’s can cause meningitis, cystitis, sore throats, typhoid,
blood poisoning, and food poisoning. Neem's ability to exert significant effects over the
above mentioned bacterial strains indicates its ability to resolve the aforementioned
conditions.
There are certain compounds in Neem that demonstrate a unique ability to
surround viruses, which prevents them from causing infection. Neem also inhibits viral
multiplication by interacting with the surface of the cells to prevent the cell from
becoming infected by the virus. Neem has been observed to be effective against a number
of viral pathogens in various clinical studies demonstrating it contains unique properties
to inhibit viral disease. Neem is one of just a few known antiviral agents. Chickenpox,
shingles, herpes, and hepatitis are viral conditions, which have been successfully treated,
in clinical studies by Neem’s therapeutic compounds. The uncomfortable symptoms of
colds and flu’s can be relieved during seasonal changes by the regular consumption of
Neem Leaf capsules, extract, or tea.
India's tropical climate especially in the coastal regions creates the kind of humid
hot house atmosphere that funguses thrive in. Traditionally, in Ayurveda, Neem seed oil,
aqueous extracts of Neem leaf, Neem leaf powder, the smoke from burning dried Neem
leaves, and Neem leaf pastes have been used for the prevention and treatment of fungal
conditions in India. Athlete's foot, ringworm, and Candida, which causes vaginal yeast
infections and thrush, are some of the more common fungi that attack humans. There are
two medicinal compounds in the Neem leaf, gedunin and nimbidol, which have been
clinically proven to control these fungi. Jock itch, other fungi that attacks humans, has
been treated traditionally in India for thousands of years with Neem seed oil and aqueous
extracts of Neem leaf. A clinical study found that smoke from burning dried Neem leaves
exerted an extreme suppression of fungal growth and germination.
Also Ayurveda has recommended the use of Neem to rid the body of all forms of
parasites. Simple aqueous extracts of Neem leaves have been the standard treatment for
external parasitic infestation without any side effects throughout India. Neem’s
effectiveness against parasites is due to compounds that mimic hormones. This activity
interrupts the life cycle of parasites by inhibiting the ability of the parasites to feed, and
preventing parasite eggs from hatching. Neem has demonstrated these effects against lice,
and against itch mites which cause scabies. Neem has both curative and preventative
effects on the Malaria parasite the methods for accomplishing this are as varied as the
active ingredients in Neem. There are two compounds in Neem, which are clinically
proven to be as effective against the malarial parasite as both quinine and chloroquine;
they are gedunin, a limonoid and quercetin, a flavonoid. There is still another study,
which shows that Neem leaf extracts prevent the normal development of the malarial
plasmodia by increasing the state of oxidation in the red blood cells.
2.9.2 NEEM'S EFFECT AGAINST AILMENTS
Studies have shown that neem has effects against certain diseases like eczema,
acne, and some skin problems like dry Skin, wrinkles, dandruff, itchy Scalp, skin ulcers
and warts are other conditions that can be effectively resolved by the use of soaps,
lotions, and creams, containing neem leaf extracts and oil.
In the case of eczema clinical studies demonstrate that even the application of
weaker Neem leaf extracts effectively cured acute conditions of eczema. Using a Soap or
shampoo containing Neem oil can easily relieve the itching and redness of eczema.
Neem effectively kills the bacteria that cause Acne and studies prove that Neem will
reduce inflammation, even the inflammation produced by Acne (Uday Bandyopadhyay,
2002).
Throughout India village people use Neem twigs and leaves to brush their teeth,
and keep their gums free of disease and infection even though they have limited access to
modern dental care. The ancient Ayurvedic practice of using Neem to heal and rejuvenate
gum tissue and to prevent cavities and gum disease is verified in modern clinical studies.
According to the ancient healing system of Ayurveda the bark of the Neem tree
will strengthen an individual’s resistance to disease. Modern clinical studies have
identified a number of compounds in the Neem tree that effectively regulate immune
system functions. There are immunomodulatory polysaccharide compounds, especially
present in Neem bark, that apparently increase antibody production. (Mukherjee, 1996)
2.9.3 TOXICITY OF NEEM
Neem oil is nonmutagenic in the Ames mutagenicity assay. Neem oil has
traditionally been considered to be a relatively safe product in adults. The LD50 of neem
oil is 14 ml/kg in rats and 24 ml/kg in rabbits. In rats, a dose of up to 80 ml/kg caused
stupor, respiratory distress, depression of activity, diarrhea, convulsions and death. Gross
examination of all organs except the lungs was normal after acute dosing.
The seeds of neem, which are poisonous in large doses, resemble the more toxic
drupes of M. Azadarach and are sometimes confused. Severe poisoning in 13 infants who
had received 5 ml to 30 ml doses of margosa (neem) oil has been reported. Toxicity was
characterized by metabolic acidosis, drowsiness, seizures, loss of consciousness, coma
and death in two infants. These infants exhibited Reye's syndrome-like symptoms, with
death from hepatoencephalopathy. Neem oil administered to mice can induce
mitochrondrial injury, resulting in similar hepatic damage. The toxin has not been
identified, but may be a long-chain monounsaturated free acid, to which infants and small
children are particularly vulnera.
2.9.4 NEEM AS A VEGETABLE
The tender shoots and flowers of the neem tree are eaten as a vegetable in India.
Neem flowers are very popular for their use in Ugadi Pachhadi (soup-like pickle), which
is made on Ugadi day in the South Indian States of Andhra Pradesh, Tamilnadu and
Karnataka. A soup like dish called Veppampoo Rasam (Tamil) translated as ‘neem
flower rasam’ made of the flower of neem is prepared in Tamil Nadu. Neem is also used
in parts of mainland Southeast Asia, particularly in Cambodia, Laos (where it is called
kadao), Thailand (where it is known as sadao or sdao), Myanmar (where it is known as
tamar) and Vietnam (where it is known as sau dau and is used to cook the salad: goi sau
dau). Even lightly cooked, the flavour is quite bitter and thus the food is not enjoyed by
all inhabitants of these nations, though it is believed to be good for one's health. Neem
Gum is a rich source of protein. In Myanmar, young neem leaves and flower buds are
boiled with tamarind fruit to soften its bitterness and eaten as a vegetable. Pickled neem
leaves are also eaten with tomato and fish paste sauce in Myanmar.
CHAPTER THREE
MATERIALS AND METHOD
3.1 RAW MATERIAL USED
The raw material used was 20g of Dogonyaro leaf, which was purchased in the
month of July 2010 from Ogbette main market, Enugu State. The sample was identified
to be a neem plant (Azadirachta indica).
3.2 EQUIPMENTS USED
Soxhlet extractor
Atomic absorption spectrophotometer
Ultra violet spectrophotometer
Test tubes
Digital weighing balance
Water bath
Beakers
Weighing balance
Knife
Bowl
Distilled water
3.3 SAMPLE PREPARATION AND EXTRACTION.
The sample of the neem leaves (Azadirachta indica) were purchased in the month
of July 2010, from Ogbette main market, Enugu State. The neem leaves were removed
from the stem and washed thoroughly with clean tap water, cut into slices with a knife for
easier extraction and allowed to dry for some days under room temperature. After proper
drying, 20g of the dried leaves sample was weighed out and pounded.
The extraction of the neem leaves was carried out using 20g of the ground leaves
sample in soxhlet extractor with distilled water in the Department of Biochemistry,
Caritas University, Amorji-Nike, Enugu State. The recycling of the solvent was allowed
to be repeated for complete extraction. The slurry extract were then poured into
evaporating dish to evaporate the solvent in the extract over the water bath at the
temperature of 80oC – 95oC and a yield of 150ml of crude extract was obtained.
3.4ATOMICABSORPTION SPECTROPHOTOMETER DETERMINATION
After extraction the water extract of the neem leaves sample was later read in the
atomic absorption spectrophotometer to determine the actual concentration of mineral
elements present in the leaf extract.
CHAPTER FOUR
4.0 RESULTS.
From the result of the analysis carried out on the water extract of neem leaf
(Azadirachta indica), using the modern Atomic Absorption Spectrophotometric method.
It was observed that neem leaf has a high mineral constituent like calcium ion, potassium
ion and other minerals as shown in the table below.
Table 4.1: Result of water extract and analysis of mineral composition.
Unit of
measurement
Sample
Composition of elements
(mg/g)
materials
Water
extract
Ca
of
neem leaf
Confirmatory
from AAS
Mg
Na
K
Fe
Cu
Zn
Neem Leaf
Mn
Mg/g
readings
0.34 0.03 0.60 0.08 0.06 0.021 18.14 Nil 0.0011
Table 4.2 Absorbance and concentration of 20g of the extract for manganese.
Sample
Pb
Absorbance
0.19Ao
Concentration
0.054
10-3 mg/g
To get concentration equivalent of absorbance:
0.001nm (Ao) → 0.000285mg/ml
If 0.19 → ?
= 0.054mg/ml
Using the formula:
(conc.
dilution factor
0.054
2.5
100)mg/L
100 = 0.14mg/L
0.14 = 0.021mg/150ml
Quantity in mg/g =
= 0.011mg/g
Table 4.3 for Iron
Sample
Absorbance
0.326Ao
Neem Leaf
Using the formula:
(conc.
(1.47
dilution factor
10-3
5
1000)mg/L
1000) mg/L
= 7.35mg/L
= 1.10mg/150ml
Quantity in mg/g = 0.06mg/g
Concentration
1.47
10-3 mg/ml
Table 4.4 for Copper
Sample
Absorbance
0.592Ao
Neem Leaf
Concentration( 10-3)
0.55mgcu/ml
If 0.054Ao → 0.05mgcu/mg
0.592Ao → ?
= 0.55mgcu/mg
How to get copper content in mg/g:
(conc.
dilution factor
0.55 10-3
5
1000)mg/L
1000 = 2.75mg/L
= 0.143mg/150ml
Note: 20g 0f the sample was used, therefore quantity in mg/g
=
= 0.021mg/g.
Table 4.5 for Zinc
The titre value gotten after titration was:
Sample
Titre value
Neem leaf
37.00cm3
(
) mg/ml
Where T.v = Titre value
0.6538 = Constant for Zinc
150 = Vol. of the filtrate
=
= 362.9mg/150ml
Quantity in mg/g = 18.14mg/g.
CHAPTER FIVE
DISCUSSION AND CONCLUSION
The result of the project work is as presented in the Tables in Chapter four. From
the tables, it is obvious that the water extract of Azadirachta indica is significantly high
in sodium ion (Na+) and potassium ion (K+) concentrations. The interpretation of this is
that the continuous intake of the water extract of the neem leaves improve the conduction
of impulses along the axons of the central nervous system. Sodium and potassium ions
are vital electrolytes in the transfer of information along the central nervous system. Lack
of these electrolytes indicates serious deleterious effect to the physiological homeostasis
of higher organisms, especially man. On another note, sodium ions play important role in
the transport of glucose across the plasma membrane. Glucose is the cellular-preferred
metabolite for the generation of energy through the glycolytic pathway. This metabolite
would not cross the cellular membrane barrier unaided. It is, therefore, transported to
where it is metabolized in exchange for sodium ions. This shows that lack of this vital
electrolyte (example sodium ion) could ‘ordinarily’ lead to the starvation of cells.
Iron is another element determined. This element is significantly low in the water
extract of the sample used (neem leaf). Thus, the intake of water extract of Azadirachta
indica leaf should be discouraged for individuals who are convalescing from serious
anaemia and need urgent replenishment of blood – red blood cells. This is evident from
the fact that the human red blood cells is built on the iron. Though iron can be
regenerated or recycled from aged or dead red blood corpuscles, constant intake from
‘outside’ sources (example plant sources) should be encouraged. The water extract of
Azadirachta indica is not a good source.
Besides, certain heavy elements assayed for included lead (Pb) and zinc (Zn). It is
known that these elements are very toxic to the biochemical system and most often can
initiate certain cancers or cause certain deformities. The water extract of Azadirachta
indica was not found to possess any of these heavy elements and as thus could not be
considered as poisonous.
It is therefore recommended that future researchers should study the various
actions of the extract to their effect on the health of the heart; that is if it would lead to the
packing of the heart. The extract should also be purified and graded for further clinical
studies.
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