TRACE ELEMENTS CONTENT OF NEEM LEAVES (AZADIRACHTA INDICA)
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).
   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 sub- continent. 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 technoloy 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 7,12-dimethylbenz[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.