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8th Sem

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General Introduction:
Neem (Azadirachta indica) is one of the most suitable and valuable tree species found in India. It can grow on wide range of soils upto pH 10 which makes it one of the most versatile and important trees in Indian sub-continent. Due to its multifarious uses, it has been cultivated by Indian farmers since vedic period and it has become now part of Indian culture. In India, it occurs throughout the country and can grow well in every agro-climatic zones except in high and cold regions and dam sites. In fact in India, Neem trees are often found growing scattered in the farmers fields and on the boundaries of fields without much affecting the crops. Farmers practice this system just to meet the local demand for timber, fodder, fuelwood and also for various medicinal properties. Due to its deep tap root system, it does not compete with annual crops for scarce soil moisture. Neem tree can be labeled as wonder tree for its multipurpose uses in real sense. This has been used as a medicinal plant for long time and provides almost all the requirements of rural areas - be the timber, fuel wood, fodder, oil, fertilizers, pest repellant or the ubiquitous 'datun'. Today, it has been recognised as the most potential tree of India due to its ever green nature (deciduous in drier areas) and ability to grow in even the most arid and nutrient deficient soils as well as for its many commercially exploitable by-products and environmentally beneficial characteristics (it has therefore been labelled as tree of the future). If plantation of this tree has to be taken up on large scale, it has to be integrated as an important component of agriculture under various agro-forestry systems. It has been estimated that India's Neembear about 3.5 million tonnes of Kernels every year. From this about 7 lakh tones of oil might be recovered. The annual production in the late 1980's was only around 1.5 lakhs tones. To increase the amount of oil harvesting, Khadi and Village Industries Commission (KVIC) has pioneered various aspects of processing the fruit and seeds of neems over the past two decades. The major difficulty as observed in most of the tree borne oil seeds including neem is that neem fruits must be harvested during the wet season. Without locally available drying facilities the fruit and seeds rapidly deteriorate and become contaminated with aflotoxin. Ideally, the fruits should be depulped without delay and the seeds have to be thoroughly dried. KVIC has popularized simple methods for depulping, drying and decorticating neem products even in the rearmost villages of the country. The sales and turnover of neem seeds in India has been estimated by various agencies. Based on random survey at major neem seeds market by independent agencies the quantity of neem seed sold during 1996 was 5.5 lakh tonnes with turnover of Rs.137 crores.

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(Mrs. Farzana Panhwar, May 2005)

History of Neem-:

Indian scientist started neem research in 1920s. Neems ability to repel insects was first reported in the scientific literature in 1926 and 1929. In 1962, three scientists showing that neem applied to vegetable crops would repel locusts. 1968 United Nations report called neem plantation in northern Nigeria the greatest boon of the century to the local inhabitants. Researchers at the U.S. Department of Agriculture have been studying since 1972. Neems insect-growth-regulation (IGR) effects were independently observed in England and Kenya in 1972. 1975 U.S. Department of Agriculture Research at Beltsville, Maryland and 1919 of its stations across the country embarked on a comprehensive program to study the post-control properties of the various plants. Bangladesh, Burkina-Faso, Chad, Myanmar, Senegal, Thailand and Mali are being assisted by Denmark and France to set-up an International Net Work. New Guinea neem was introduced at the beginning of 1980s. Queensland and New South Wales, in 1990, 16000 tree were planted the pre 1980 neem project was for ornamental or for timber but post 1980 project for pesticide. According to Morgan (1981), there is a marked difference in the yield of Azadiractin from neem ( Azadirachtaindica,A.Juss) seeds from various sources. From seeds from Ghana he obtained 3.5 gm/Kg, and from old Indian seeds less than 0.2 g/kg. 1991, several hundred researchers in at least dozen countries were studing various aspects of neem and its products. In Nicaragua neem trees have been introduced through a cooperative known as COPINIM helped by Germany, Australia and Sweden. In 1995 they produced 200 tons of neem fruit.

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(THE AYURVEDIC PHARMACOPOEIA OF INDIA)

Plant profile

Biological source-: Neem consists of dried leaves of Azadirachta indica, A. Juss Syn.
Meliaazadirachta Linn. (Fam. Meliaceae); a moderate sized to fairly large evergreen tree, attaining a height of 12-15 m with stout trunk and spreading branches, occurring throughout the country up to an elevation of 900 m.

SYNONYMS Sanskrit: Assamese: Bengali: English: Guajarati: Hindi: Kannada: Malayalam : Marathi : Oriya : Punjabi : Tamil : Telugu : Urdu : Arista, Picumarda Mahanim Nim, Nimgach Margosa Trees Limba, Limbado, Limado, Kohumba Nim, Nimb Nimba, Bevu, Oilevevu, Kahibevu, Bevinama Veppu, Aryaveppu, Nimbam, Veppa Balantanimba, Limba, Bakayan, Nim, Kadunimb Nimba Nimba, Bakan, Nim Vemmu, Veppu, Arulundi, Veppan Vemu, Vepa Neem

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TAXONOMIC HIERARCHY OF NEEM


Kingdom: Division: Class: Order: Family: Genus: Species: Other species: Scientific NameFound In: Plantae Magnoliophyta Magnoliopsida Sapindales Meliaceae Azadirachta A. indica A.juss A.azedarac Azadirachta indica Ranthambore National Park, Bandhavgarh national Park, Mrugavani Naional Park, Bannerghata National Park, Sariska

Description-:
Character Colour Odour Taste Leaf Slightly Yellowishgreen indistinct Bitter Bark Grey or dark reddish brown Flower White or pale yellow Honey like scent Bitter Fruit Young- RipenGreen Yellow Young- RipenBitter Sweet

A large evergreen tree, 12 to 18 meter in height and 1.8 to 2.4 meter in girth with a straight bole and long spreading branches forming a broad crown as much as 20 meters across, commonly found throughout greater parts of India. Leaves Compound, alternate, rachis 15-25 cm long, 0.1 cm thick; leaflets with oblique base, opposite, exstipulate, lanceolate, acute, serrate, 7-8.5 cm long and 1.0-1.7 cm wide, slightly yellowish-green;

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Bark Grey or dark reddish brown with numerous and scattered tubercles. The bark exudes a gum known as East India gum. Leaves alternate 20 - 30 cm long, leaflets 8 19 alternate or opposite ovate glossy, bluntly serrate. Flowers : white or pale yellow, small, scented, numerous on long auxiliary panicles, have a honey like scent and attract many bees. Fruit : Fruit is a ovoid bluntly pointed, smooth drupe green when young and turns yellow with a very thin epicarp, mesocarp with scanty pulp and a hard bony endocarp, enclosing one seed. The timber is relatively heavy with a specific gravity varying from 0.56 to 0.85 (average 0.68) when freshly cut, it has a strong smell.

The flowering season of neem varies from place to place. Generally it flowers from January to May and the ripening time of fruits is from May to August. The fruit pulp is edible.

Geographical distribution
It has been widely cultivated in India and African countries. Native of Burma but grown all over India. In India, it occurs throughout the larger parts of the country in the states of Uttar Pradesh, Bihar, West Bengal, Orissa, Delhi, Maharashtra, Gujarat, Andhra Pradesh, And Tamil Nadu.

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CULTIVATION AND COLLECTION

(sales@neemamerica.com)

Neem tree can grow under variousconditions of climate and soil types.The ripe seeds should be grown soon after collection. It can be sown directly or it can also be transplanted. Neem can also be propagated in a variety of other ways.

Propagation Techniques of Neem


The tree can be easily propagated both sexually and vegetatively. It can be planted using seeds, seedlings, saplings, root, suckers, or through tissue culture. However, it is normally grown from seed. Neem seeds are not viable for a very long time. After about two weeks they will no longer germinate. The seeds that are removed from the fruit should be put in water. Those seeds which sink are good ones and should be used. Some studies have shown that they can be used even after many years but only under specific condition and germination capacity is under 50%. Neem cuttings have to be planted as soon as they are cut for the neem to survive. The part that is cut is covered with straw and should be kept wet if it is to be planted after some time.

Preparation of nursery and Sowing


The soil should be dug 30 cm deep. Raised beds of 10m length, 1 m breadth and 15 cm height should be prepared. Farmyard manure(FYM), sand and local soil should be mixed in the ratio 1:1:3. This should be put on top of the soil for a height of 2.5 to 5 cm. Seeds are normally sown in the nursery in the months of July and August. The seeds should be sown at a distance of 15 to 20 cm and a depth of 1 to 1.5 cm, and then watered. Hence they can be sown as soon as they are collected. They germinate in a week's time.After 5 to 6 weeks the seedlings are removed from the nursery and planted in second nursery or in polythene bags. If polythene bags are used for transplanting, the bag should be filled with silt, sand, clay and farmyard manure in the ratio of 1:1:1:1. The seeds can also be sown directly in the polythene
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bags at the rate of 2 per bag. The healthy seedling is maintained and the other one removed. If the seeding are raised in the polythene bag they can be transported for long distances when

required and then transplanted. The polythene bags that are used should be 150-200 gauge in weight. In the nursery a thatch should be provided for seedlings. Transplantation The place where neem seedlings are to be transplanted should be properly ploughed. The seedlings are ready for transplantation when they are six months old (15 to 22.5 cm height). If the seedlings which are kept for a long period the tap roots become very long and it is difficult to take out without breaking of roots. Seedlings which are healthy and not afflicted with any diseases should be chosen. Pits of dimension 30x30x30 cm should be dug at intervals of 3x3 m. The neem seedling should be transplanted during the period of the South East Monsoon. This increase their survival rate. Initially, they should be irrigated once in two or three days interval. After they grow well they can be wanted once in seven to ten days.

Harvesting In most parts of India today, leaves are collected by simply cutting or picking method and separated from leaflet then dried in shade avoiding any contamination .leaves are generally collected when they are neither yellowish nor green. Seeds that
fall on the ground are simply collected. Neem fruit fall coincides with the monsoon. At moisture content above 14%, neem fruits carry the fungus Aspergillus flavus. These funguses under many conditions produce aflatoxins. Aflatoxins are extremely potent among carcinogens known and these could also contaminate seeds inside the fruits. They are extracted and concentrated along with pesticidal ingredients. Seed viability is also lowered. To avoid all this actual harvesting of seeds is recommended rather than a simple collection of seeds. While harvesting neem seeds it must be ensured that the fruit colour is neither greenish yellow nor brownish yellow but plain absolute yellow. Greenish yellow fruit are not fully mature and are low in Azadirachtin content. After indentifying trees which have yellow fruit a tarpaulin cloth or plastic paper is spread under the tree. A stick is used to beat the branches the same way as one would to collect Tamarind. Neem fruits now drop on the tarpaulin or plastic sheet. Since they do not come in contract with soil, there is no fear of fungus attack and aflatoxin development. Seeds thus collected are brought to the shade where one can further work on them. By twisting yellow fruits between index finger and thumb the pulp is removed. After the removal of pulp the seeds should be milky white in colour. The seeds are dried in shade for two or three days. The seeds are turned upside down while they are put to dry. This type of harvesting increase the germinating capacity and viability of the seeds. The seeds also have better bio-efficacy and can be sold at absolute premium price in the markets.

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Traditional Uses:

(P. Sudhir et al )

Various parts of the neem tree have been used as traditional ayurvedic medicine in India from time immemorial. The medicinal utilities have been described, especially for leaf, fruit and bark. Neem oil and the bark and leaf extracts have been therapeutically used as folk medicine to control leprosy, intestinal helminthiasis, respiratory disorders, and constipation and also as a general health promoter. Its use for the treatment of rheumatism, chronic syphilitic sores and indolent ulcer has also been evident. Neem oil finds use to control various skin infections. Bark, leaf, root, flower and fruit together cure blood morbidity, biliary afflictions, itching, skin ulcers, burning sensations and pthysis. Some of the medicinal attributes of various parts of neem as mentioned in ayurveda have been summarized in Table 2. However, apart from these uses, there are several reports on the biological activities and pharmacological actions of neem based on modern scientific investigations. Anti-inflammatory, antipyretic and analgesic activities: The chloroform extract of stem bark is effective against carrageenin-induced paw oedema in rat and mouse ear inflammation. Inflammatory stomatitis in children is cured by the bark extract. Antipyretic activity has been reported in neem oil. A methanol extract of the leaves exerts antipyretic effect in male rabbits. The plant also possesses analgesic activity mediated through opioid receptors in laboratory animals. Anti-inflammatory and antipyretic activities in various extracts have been review. Immunostimulant activity:
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The aqueous extract of neem bark possesses anticomplement activity, acting both on the alternative as well as the classical pathway of complement activation in human serum. Recently, an aqueous extract of stem bark has been shown to enhance the immune response of Balb-c mice to sheep red blood cells in vivo. The aqueous extract of leaf also possesses potent immunostimulant activity as evidenced by both humoral and cellmediated responses. Leaf extract at 100 mg/kg after three weeks of oral administration causes higher IgM and IgG levels along with increased titer of antiovalbumin antibody. Neem oil has been shown to possess immunostimulant activity by selectively activating the cellmediated immune mechanisms to elicit an enhanced response to subsequent mitogenic or antigenic challenge. Hypoglycemic activity: Aqueous extract of neem leaves significantly decreases blood sugar level and prevents adrenaline as well as glucose-induced hyperglycemia. The aqueous leaf extract when orally fed, also produces hypoglycemia in normal rats and decreased blood glucose levels in experimentally-induced diabetes in rats. Aqueous leaf extract also reduces hyperglycemia in streptozotocin diabetes and the effect is possibly due to presence of a flavonoid, quercetin. A significant hypoglycemic effect was also observed by feeding neem oil to fasting rabbits. Recently, hypoglycemic effect was observed with leaf extract and seed oil, in normal as well as alloxan-induced diabetic rabbits. The possible mechanisms underlying the hypoglycemic activity of the aqueous leaf extract have also been discussed. Antiulcer effect: Neem leaf aqueous extract produces antiulcer effect in rats exposed to restraint cold stressor ethanol orally by preventing mucus depletion and mast cell degranulation. An aqueous extract of neem bark has been shown from our laboratory to possess highly potent antacid secretory and antiulcer activity and the bioactive compound has been attributed to a glycoside. Antifertility effect: Neem oil proved spermicidal against rhesus monkey and human spermatozoa in vitro. In vivo studies showed that intravaginal application of neem oil prior to coitus can prevent pregnancy. Antifertility effect of neem oil has also been studied and suggested to be a novel method of contraception. Oral administration of aqueous extract of neem leaf a antifertility effect in mice. Purified neem seed extract (Praneem) has also been demonstrated to abrogate pregnancy in both baboons and bonnet monkeys, when administered orally. From the hexane extract of neem seed, an active fraction containing six components has been found to completely abrogate pregnancy in rodents when given orally up to a concentration of 10%, with no apparent side effect. The effect is possibly due to activation of cell-mediated immune reaction. The mechanism of action of neem oil appears to be non-hormonal, probably mediated through its spermicidal effect and may have fewer side effects than steroidal contraceptives. Antimalarial activity: Neem seed and leaf extracts are effective against malarial parasites. Components of the alcoholic extracts of leaves and seeds are effective against both chloroquin-resistant and
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sensitive strains of malarial parasite. Recently, neem seed extract and its purified fractions have been shown to inhibit growth and development of asexual and sexual stages of drug sensitive and resistant strains of the human malarial parasite P. falciparum. Antifungal activity: Extracts of neem leaf, neem oil and seed kernels are effective against certain human fungi, including Trichophyton, Epidermophyton, Microsporum, Trichosporon, Geotricum and Candida. High antimycotic activity with extracts of different parts of neem has already been reported. Antibacterial activity: Oil from the leaves, seeds and bark possesses a wide spectrum of antibacterial action against Gram-negative and Gram-positive microorganisms, including M. tuberculosis and streptomycin resistant strains. In vitro, it inhibits Vibrio cholerae, Klebsiella pneumoniae, M. tuberculosis and M. pyogenes. Antimicrobial effects of neem extract have been demonstrated against Streptococcus mutans and S. faecalis. NIM-76, a new vaginal contraceptive from neem oil showed inhibitory effect on the growth of various pathogens, including bacteria, fungi and virus. Recently, the antibacterial activity of neem seed oil was assessed in vitro against 14 strains of pathogenic bacteria. Antiviral activity: Aqueous leaf extract offers antiviral activity against Vaccinia virus, Chikungemya and measles virus in vitro. The antiviral and virucidal effects of the methanol extract of neem leaves (NCL-11) have recently been demonstrated against group-B Coxsackie viruses. NCL11 inhibits plaque formation in different antigenic types of Coxsackie virus B at a concentration of 1 mg/ml at 96 h in vitro. Further studies indicated that NCL-11 is most effective in Coxsackie virus B-4 as a virusidal agent, in addition to its interference at the early events of its replication. Anticarcinogenic activity: 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. That neem leaf extract exerts its protective effect in Methyl- N-nitro-N-nitrosoguanidine (MNNG) (a carcinogenicmaterial)- induced oxidative stress has also been demonstrated by the reduced formation of lipid peroxides and enhanced level of antioxidants and detoxifying enzymes in the stomach, a primary target organ for MNNG as well as in the liver and in circulation. Hepatoprotective activity: The aqueous extract of neem leaf was found to offer protection against paracetamol induced liver necrosis in rats. The elevated levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transpeptidase (GGT) indicative of liver damage were found to be significantly reduced on administration of the neem leaf aqueous extract.
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Antioxidant activity: The antioxidant activity of neem seed extract has been demonstrated in vivo during horse grain germination, which is associated with low levels of lipooxygenase activity and lipid peroxides. An antioxidant principle has also been isolated, which is a potent inhibitor of plant lipooxygenases. Table 2. Some medicinal uses of neem as mentioned in ayurveda Part Leaf Medicinal use Leprosy, eye problem,epistaxis, intestinal worms, anorexia, biliousness, skin ulcers. Bark Analgesic, alternative and curative of fever. Flower Bile suppression, elimination of intestinal worms and phlegm. Fruit Relieves piles, intestinal worms, urinary disorder, epistaxis, phlegm, eye problem, diabetes, wounds and leprosy. Twig Relieves cough, asthma, piles, phantom, tumour, intestinal worms, spermatorrhoea, Obstinate urinary disorder, diabetes. Gum Effective against skin diseases like ringworms, scabies, wounds and ulcers. Seed pulp Leprosy and intestinal worms. Oil Leprosy and intestinal worms. Root, bark, leaf, Blood morbidity, biliary afflictions, Flower itching, skin ulcer, burning sensation and fruit together leprosy. __________________________________________________________________________ Effect on central nervous system: Varying degrees of central nervous system (CNS) depressant activity in mice was observed with the leaf extract. Fractions of acetone extract of leaf showed significant CNS depressant activity. Leaf extract up to a dose of 200 mg/kg body weight produces significant anxiolytic activity in rats. The crude ethanol extract of stem bark and root bark showed hypotensive, spasmolytic and diuretic activities.

Reported Pharmacological Activities:1. Anti-tumor and Antiviral activity


(Hassan et al)

The crude acidic extract of leaves and seeds (90.38, 65.38%, respectively) and alkaline extract of seeds (77.61%) possesses very remarkable antiviral activity against herpes simplex virus type 1 compared with acyclovir (54.33%) at concentration 20 g/mL.

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The acidic extract from leaves and neutral extracts from seeds inhibited Ehrlich ascites carcinoma cell line growth and had anticancer activity. IC50 values of acidic extracts from leaves and neutral extracts from seeds were 669.43 and 724.63 g/ml, respectively.

2. Anti-Hepatotoxicity

(Kale et al)

Methods: Hepatotoxicity was induced in rats by combination of isoniazid, rifampicin and pyrazinamide given orally as suspension for 30 days. Treatment groups received AI aqueous leaf extract along with ant tubercular drugs. In the second phase of study the effect of aqueous leaf extract on established hepatotoxicity was studied by giving the extract for 20 days after withdrawal of antitubercular drugs. Liver damage was assessed by biochemical and histological parameters. Conclusion: Aqueous leaf extract(Neem) significantly prevents and reverses the hepatotoxic damage induced by anti- tubercular drugs in rats.
3.

Anti-fertility activity-:

( Indian J. med. Res. 1983)

Oral administration of 20ml of neem oil from day 1 to 10 of pregnancy to female rats, showed on 16th day growth of fetuses retarded in 50% animals. In another group of animals given the same treatment orally but allowed to complete the term, 80% animals did not deliver, indicating foetal reabsortion and 80% anti fertility activity. Intra vaginal administration studies of neem oil in rat suggested that anti-fertility effect was due to absorption of its active constituents through vaginal mucosa, into the circulation.

4. Anti-fungal activity

(Fitoterapia 1986)

Odorous viscous steam distillate from fresh leaves exhibited in vitro antifungal activity (MIC 125g/ml) against Trichophyton mentagrophytes.

5. Oestrogenic activity

( Indian J. Physiol.Pharmcol. 1986)

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Extract of leaves did not show oestrogenic activity in rats, as evidenced by insignificant change in weight of uterus and adrenal, absence of open vagina and epithelial cells in vaginal smear and incomplete development of uterus. 6. Insecticidal activity
( Heterocycles 1986), (J.Chem. soc. Perkin 1 1987)

An acidic fraction from leaves showed insect growth regulating activity in mosquitoes. Plant extract showed insect growth regulating activity against pulse beetle, callasobruchus analis

. 7. Anti-bacterial activity Purified bark extract showed significant anti-bacterial activity. 8. Anti-hyperglycemic

(Phytochemistry 1988)

( Phytother.res. 1989)

Aqueous extract of leaves lowered hyperglycemia in streptozotocine induced diabetes

REPORTED PHYTOCONSTITUENTS-:
Chemical constituents: There are many active compounds found in the neem tree. The most common ones are: Azadirachtin : Provides repellent, anti-hormonal and anti-feedant properties The most common ones are: Azadirachtin: Provides repellant, anti-hormonal and anti-feedant properties Nimbin:Provides anti-inflammatory, anti-pyretic, antihistamine and antifungal properties Nimbidin:Provides antibacterial, anti-ulcer, analgesic, anti-arrhythmic and antifungal properties

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Nimbidol:Provides anti-tubercular, anti-protozoan and antipyretic properties Sodium nimbinate:Provides diuretic, spermicidal and anti-arthritic properties Gedunin:Providesvasodilator, anti-malaria and antifungal properties Salannin:Provides repellant properties Quercetin:Provides properties anti-protozoal, antioxidant, anti-inflammatory and antibacterial

STRUCTURE OF PHYTOCHEMICAL CONSTITUENTS-:

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Objective of the work


The leaves of neem plants can be categorized as the tender young leaves which are astringent, cause vata, good for eye, skin diseases and in leprosy whereas the old leaves are anthelmintic, alexeteric, insecticidal, good in ophthalmic, biliousness and cure ulcers quickly. The leaves are carminative and expectorant, anti-inflammatory, anti-rheumatic, useful in syphilitic sores, earache, boils, in all blood impurities.

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PLAN OF WORK1. PHARMACOGNOSTICAL EVALUATION


I. II. Macroscopic evaluation Microscopic evaluation Leaf section, powder microscopy Leaf stomata

2. PHYSICOCHEMICAL EVALUATION/STUDY
I. Ash Value Total Ash Acid insoluble Ash Water soluble Ash II. Extractive Value Hot Extraction Cold Maceration III. Loss on Drying

IV.

Fluorescence Analysis

3. PHYTOCHEMICAL EVALUATION

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1. PHARMACOGNOSTICAL EVALUATION
(WHO GUIDELINE 1998)

I.

MACROSCOPIC EVALUATION
Leaf Slightly Yellowishgreen indistinct Bitter Bark Grey or dark reddish brown Flower White or pale yellow Honey like scent Bitter Fruit Young- RipenGreen Yellow Young- RipenBitter Sweet

Character Colour Odour Taste

A large evergreen tree, 12 to 18 meter in height and 1.8 to 2.4 meter in girth with a straight bole and long spreading branches forming a broad crown as much as 20 metres across, commonly found throughout greater parts of India. Leaves Compound, alternate, rachis 15-25 cm long, 0.1 cm thick; leaflets withoblique base, opposite, exstipulate, lanceolate, acute, serrate, 7-8.5 cm long and 1.0-1.7 cmwide, slightly yellowish-green;

Bark Grey or dark reddish brown with numerous and scattered tubercles. The bark exudes a gum known as East India gum. Leaves alternate 20 - 30 cm long, leaflets 8 19 alternate or opposite ovate glossy, bluntly serrate. Flowers : white or pale yellow, small, scented, numerous on long axillary panicles, have a honey like scent and attract many bees. Fruit : Fruit is a ovoid bluntly pointed, smooth drupe green when young and turns yellow with a very thin epicarp, mesocarp with scanty pulp and a hard bony endocarp, enclosing one seed. The timber is relatively heavy with a specific gravity varying from 0.56 to 0.85 (average 0.68) when freshly cut, it has a strong smell.

The flowering season of neem varies from place to place. Generally it flowers from January to May and the ripening time of fruits is from May to August. The fruit pulp is edible.

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II.

MICROSCOPIC EVALUATION

Leaf section cutting Equipment The following are required: - a microscope equipped with lenses providing a wide range of magnification and a substage condenser, a graduated mechanical stage, objectives with a magnification of 4x, 10x, and 40x, and colour filters of ground glass, blue-green; high eyepoint eyepieces are preferred for wearers of spectacles; a lamp, either separate or incorporated into the microscope; - a stage micrometer and an ocular micrometer to be inserted into a 6x eyepiece and placed on the diaphragm or, preferably, a micrometer eyepiece; - a set of drawing attachments for the microscope; - a microburner (Bunsen type); - slides and cover-glasses of standard size; - a set of botanical dissecting instruments Microscopical studies of the T.S. and powdred of the leaves was carried out.Microscopic descriptions of tissues are supplemented with micrographs and photographs.The T.S. and powder characterstics of the leaves are shown in the results. Transverse Section Transverse section of leaves was cleared with chloral hydrate, moistened with phloroglucinol solution and few drops of conc. Hydrochloric acid were added. It was allow standing for about 5 minutes in 50% glycerin. Photographs were taken with the use of a digital microscope.

Powder Microscopic Procedure A small amount of powder was moistened with phloroglucinol solution for few minutes and there after few drops of conc. Hydrochloric acid was added to it. It was mixed well and allowed stand for about 5 minutes. It was then amounted in 50% glycerin and observed under microscope. Similarly, the powder was also stained with weak solution of iodine for the identification of starch grains and mounted in 50% glycerin for identification of calcium oxalate crystal..
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III. LEAF STOMATA


In the mature leaf, four significantly different types of stoma are distinguished by their form and the arrangement of the surrounding cells, especially the subsidiary cells (Fig. 2): The anomocytic or ranunculaceous (irregular-celled) type; the stoma is surrounded by a varying number of cells, generally not different from those of the epidermis. The anisocytic or cruciferous (unequal-celled) type; the stoma is usually surrounded by three or four subsidiary cells, one of which is markedly smaller than the others. The diacytic or caryophyllaceous (cross-celled) type; the stoma is accompanied by two subsidiary cells, the common wall of which is at right angles to the stoma. The paracytic or rubiaceous (parallel-celled) type; the stoma has two subsidiary cells, of which the long axes are parallel to the axis of the stoma. In describing an epidermis where certain stomata differ from the predominant type, the term applying to the majority of stomata is used.

Determination of the stomatal index


Place fragments of leaves, about 5 x 5 mm in size, in a test-tube containing about 5 ml of chloral hydrate TS and heat on a water-bath for about 15 minutes or until the fragments are transparent,remove the epidermal cell and transfer on a glass slide or the lower epidermis uppermost, in chloral hydrate TS; place a small drop of glycerol/ethanol TS at one side of the cover-glass to prevent the material from drying. Examine under a microscope with a 40x objective and a 6x eyepiece, equipped with a drawing apparatus. Mark on the drawing paper a cross () for each epidermal cell and a circle (o) for each stoma. Calculate the stomatal index as follows:

stomatal index(SI) = S*100/E+S where, S = the number of stomata in a given area of leaf E= the number of epidermal cells (including trichomes) in the same area of leaf. For each leaf sample, no fewer than ten determinations should be carried out and the average index calculated.
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Types of leaf stoma

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2. PHYSICOCHEMICAL EVALUATION
I. ASH VALUE
The ash remaining following ignition of medicinal plant materials is determined by three different methods which measure total ash, acid-insoluble ash and water-soluble ash. Total ash method is designed to measure the total amount of material remaining after ignition. This includes both "physiological ash", which is derived from the plant tissue itself, and "non-physiological" ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the plant surface. Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth. Water-soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water.

Procedure-: Total ash


Place about 2-4g of the ground air-dried material, accurately weighed, in a previously ignited and tared crucible (usually of platinum or silica). Spread the material in an even layer and ignite it by gradually increasing the heat to 500-600C until it is white, indicating the absence of carbon. Cool in a desiccator and weigh. If carbon-free ash cannot be obtained in this manner, cool the crucible and moisten the residue with about 2 ml of water or a saturated solution of ammonium nitrate R. Dry on a water-bath, then on a hot-plate and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 30 minutes, then weigh without delay. Calculate the content of total ash in mg per g of air-dried material.

Acid-insoluble ash
To the crucible containing the total ash, add 25 ml of hydrochloric acid (~70g/l) TS, cover with a watch-glass and boil gently for 5 minutes. Rinse the watch-glass with 5 ml of hot water and add this liquid to the crucible. Collect the insoluble matter on an ashless filterpaper and wash with hot water until the filtrate is neutral. Transfer the filter-paper containing the insoluble matter to the original crucible, dry on a hot-plate and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 30 minutes, then weigh without delay. Calculate the content of acid-insoluble ash in mg per g of air-dried material.

Water-soluble ash
To the crucible containing the total ash, add 25 ml of water and boil for 5 minutes. Collect the insoluble matter in a sintered-glass crucible or on an ashless filter-paper. Wash with hot water and ignite in a crucible for 15 minutes at a temperature not exceeding 450C.
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Subtract the weight of this residue in mg from the weight of total ash. Calculate the content of water-soluble ash in mg per g of air-dried material.

II.

EXTRACTION METHOD-:

This method determines the amount of active constituents extracted with solvents from a given amount of medicinal plant material. It is employed for materials for which as yet no suitable chemical or biological assay exists. Recommended procedures Hot extraction Place about 4.0g of coarsely powdered air-dried material, accurately weighed, in a glass-stoppered conical flask. Add 100ml of water and weigh to obtain the total weight including the flask. Shake well and allow to stand for 1 hour. Attach a reflux condenser to the flask and boil gently for 1 hour; cool and weigh. Readjust to the original total weight with the solvent specified in the test procedure for the plant material concerned. Shake well and filter rapidly through a dry filter. Transfer 25 ml of the filtrate to a tared flat-bottomed dish and evaporate to dryness on a water-bath. Dry at 105C for 6 hours, cool in a desiccator for 30 minutes, then weigh without delay. Calculate the content of extractable matter in mg per g of air-dried material and %w/w.. This procedure is also performed for alcohol(as a solvent) similarly. Cold maceration Place about 4.0g of coarsely powdered air-dried material, accurately weighed, in a glass-stoppered conical flask. Macerate with 100ml of the solvent specified for the plant material concerned for 6 hours, shaking frequently, then allow to stand for 18 hours. Filter rapidly taking care not to lose any solvent, transfer 25 ml of the filtrate to a tared flat-bottomed dish and evaporate to dryness on a water-bath. Dry at 105C for 6 hours, cool in a desiccator for 30 minutes and weigh without delay. Calculate the content of extractable matter in mg per g of air-dried material. For ethanol-soluble extractable matter, use the concentration of solvent specified in the test procedure for the plant material concerned; for water-soluble extractable matter, use water as the solvent. Use other solvents as specified in the test procedure.

Extraction Of Plant Material Preparation of methanolic and aqueous extract :200g of the air dried and coarsely ground plant material was defatted with petroleum ether (60-80 celcius) by soxhlation for 8 hrs and the solvent free marc was extracted with 90% methanol by soxhlation for 24hrs and extract was concentrated invacuo in a rotary evaporator. The marc was extracted with distilled water by maceration in a round bottom flask for 24hrs. then it was filtered through muslim cloth
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followed by Buchner funnel and the filtrate was collected in a round bottom flask. The process was repeated using marc for another two times and all the three filtrates were collected and combined. The solvent was removed and concentrated in-vacuo in a rotary evaporator. The dried extracts were placed in a desiccator and used for further studies. The colour, consistency and the % yields of the extracts were recorded.

III. Loss on drying (gravimetric determination)


Place about 2-5g of the prepared air-dried material, or the quantity specified in the test procedure for the plant material concerned, accurately weighed, in a previously dried and tared flat weighing bottle. Dry the sample by one of the following techniques: - in an oven at 100-105C; - in a desiccator over phosphorus pentoxide R under atmospheric pressure or reduced pressure and at room temperature. Dry until two consecutive weighings do not differ by more than 5mg, unless otherwise specified in the test procedure. Calculate the loss of weight in mg per g of air-dried material.

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3. PHYTOCHEMICAL EVALUATION

(KOKATE et al)

The petroleum ether extract, 90% methanol extract, and aqueous extract were subjected to preliminary phytochemical screening for the detection of various phytoconstituents such as alkaloids, steroids, terpenoids, flavonoids, tannis and phenolic compounds, saponins, carbohydrates, proteins and amino acids. The following tests were carried out to identify the various phytoconstituents present in petroleum ether extract, 90% methanol extract, and aqueous extract (harborne,1998) and results are expressed in table.

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COLLECTION AND AUTHENTICATION OF PLANT MATERIAL


Fresh leaves were collected from the HERBAL GUARDAN of our college and were identified by Dr. Perwez alam. The leaves of Azadiracta indica were collected and shade dried, coarsely powdered and used for the present study.

1. PHARMACOGNOSTICAL EVALUATION
I. MACROSCOPIC EVALUATION
Character Colour Odour Taste Leaf Slightly Yellowish-green indistinct Bitter

Leaves Compound, alternate, rachis 15-25 cm long, 0.1 cm thick; leaflets withoblique base, opposite, exstipulate, lanceolate, acute, serrate, 7-8.5 cm long and 1.0-1.7 cmwide, slightly yellowish-green.

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II.

MICROSCOPIC EVALUATION

a. Leaf transverse section cutting:

Fig. 1.Xylem, phloem, collenchymas, palisade cells and vessels.

Fig.2. midrib vessels and vascular bundle

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Fig.3. Spongy cells, palisade cells

b. Powder microscop

Fig.1. Starch grains

Fig.2. Calcium oxalate crystals

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Fig.3. Trachome

c. Leaf Stomata

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Fig.1. Stomata

2. PHYSICOCHEMICAL EVALUATION/STUDY SANJAY SINGH B.Pharm Page 32

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I.

Ash value a. Total Ash value of A. indica

S. No.

Wt. of drug (g) 2.0 2.0

Wt. of empty crucible (g)

Wt. of crucible + Wt. of crude drug before ignition (g) 20.91 22.30

Wt. of crucible after ignition (g)

Wt. of total Total ash ash (g) (%w/w)

Mean (%w/w)

1. 2.

18.91 20.30

19.02 20.40

0.11 0.10

5.5 5 5.25

b. Acid insoluble ash value of A. indica

S. No.

Wt. of drug (g) 2.0 2.0

Wt. of empty crucible (g) 18.91 20.30

Wt. of crucible + acid insoluble ash (g)

Wt. of acid insoluble Ash (g) 0.03 0.04

Acid insoluble ash value (%w/w)

Mean (%w/w)

1. 2.

18.94 20.34

1.50 2.00

1.75

c. Water soluble ash value of A. indica S. No. Wt. of drug(g) Wt. of empty crucible (g) Wt. of total ash (g) Wt. of water insoluble ash (g) Wt. of water soluble ash (g) Water soluble ash value (%w/w) Mean (%w/w)

1. 2.

2.00 2.00

17.61 19.18

0.10 0.10

0.5 0.4

0.05 0.06

2.50 3.00 2.75

II.

Extractive value Page 33

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Water extractive value (hot) of leaves of A.indica: Wt. of empty China dish (g) 34.52 34.52 34.52 Wt. of China dish + Wt. of extract (g) 34.67 34.66 34.68 Wt. of extract (g) 0.15 0.14 0.16 Extractive Value (%w/w) 15 14 16 15 Mean (%w/w)

a. S. No. Wt. of drug (g) 4.000 4.000 4.000

1. 2. 3.

b. Water extractive value (cold) of leaves of A.indica: S. No. Wt. of drug(g) 4.000 4.000 4.000 Wt. of empty China dish (g) 34.52 34.52 34.52 Wt. of China dish + Wt. of extract (g) 34.61 34.62 34.61 Wt. of extract (g) 0.09 0.10 0.09 Extractive value (%w/w) 9 10 9 9.33 Mean (%w/w)

1. 2. 3.

c. Methanol extractive value (cold) of leaves of A.indica: S. No. Wt. of drug (g) 4.00 4.00 4.00 Wt. of empty China dish(g) 38.28 38.28 38.28 Wt. of China dish + Wt. of extract (g) 38.37 38.38 38.39 Wt. of extract(g) 0.09 0.10 0.11 Extractive value (%w/w) 9.00 10.00 11.00 10.00 Mean (%w/w)

1. 2. 3.

d. Methanol extractive value (hot) of leaves of A.indica: SANJAY SINGH B.Pharm Page 34

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S.No.

Wt. of drug(g) 4.00 4.00 4.00

Wt. of empty China dish (g) 38.28 38.28 38.28

Wt. of China dish + Wt. of extract (g) 38.39 38.40 38.40

Wt. of extract (g) 0.11 0.12 0.12

Extractive value (%w/w) 11.00 12.00 12.00

Mean (%w/w)

1. 2. 3.

11.66

III.

Loss on drying

S. No.

Weight Of Taken Drug(g) 3.00

1. 2. 3. 4.

Wt. of Petri dish+ drug (Before drying) (g) A 21.38 21.38 21.38 21.38

Wt. of Petri dish+ Drug (After drying) (g) B 21.14 21.04 20.98 20.98

Time interval (min.) 10 10 10 10

A-B (g)

LOD (% w/w)

Mean (%w/w)

0.24 0.34 0.40 0.40

8 11.33 13.33 13.33

11.49

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Florescence analysis
Reagents Drug powder as such Drug + conc. H2SO4 Drug + conc. H2SO4 + Visible Light Greenish brown Brown UV (254 nm) Light brown Greenish brown UV (366 nm) Black Blackish brown

IV.
S. No. 1. 2.

3.

Distilled water Drug + conc. HCl Drug + conc. HCl + Distilled water Drug + conc. HNO3 Drug + conc. HNO3 +

Brown

Dark brown

Black

4. 5. 6. 7. 8. 9. 10.

Dark brown Dark brown Yellowish brown Yellowish brown Light brown Yellowish brown Sodium Brown

Brown Greenish brown Brown Greenish brown Blackish brown Brown Blackish brown

Brownish black Blackish brown Black Black Brownish black Black Brownish black

Distilled water Drug + Methanol Drug + FeCl3 Drug +10%

hydroxide solution

3. Phytochemical screening SANJAY SINGH B.Pharm Page 36

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Chemical tests for glycosides: result + + + S. No. 1 2 3 4 Name of chemical test Anthraquinone (Borntragers test) Cardiac (keller-killiani test) Saponin (Foam test) Flavone (Alkaline reagent test) result + + +

Chemical tests for alkaloids:Name of chemical test Dragendorffs reagent Mayers reagent Hagers reagent Wagers reagent

S. No. 1 2 3 4

Chemical tests for steroidal triterpenoids S. No. 1 2 Name of chemical test Libermann-Burchard test Salkowski test result + +

Chemical test for Amino acids S. No. 1 2 Name of chemical test Millons test Ninhydrin test result + +

Chemical tests for proteins

S. No. 1 2

Name of chemical test Biuret test Millions test

result + +

MATERIAL AND METHODS-:


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Reference-:
1. The Neem Tree (Azadiractin- Indica, A.Juss), a Natural Pesticide Practice in Pakistan by Mrs. Farzana Panhwar, May 2005 2. THE AYURVEDIC PHARMACOPOEIA OF INDIA, PART- I, VOLUME II 3. sales@neemamerica.com 4. Biological action and medicinal properties of various constituent of Azadirachta indica (Meliaceae) an Overview P. Sudhir Kumar1, Debasis Mishra1, Goutam Ghosh1 and Chandra S. Panda2 1 .School of Pharmaceutical Sciences, Siksha O Anusandhan University, Bhubaneswar, Orissa, India 2 .NISER, Institutes of Physics, Bhubaneswar, Orissa, India. 5. Hassan Amer1, Wafaa A. Helmy1*, Hanan A.A. Taie2 1Department of Natural and Microbial Products Chemistry, 2Department of Plant Biochemistry, National Research Center, Dokki, Cairo (EGYPT) *Corresponding Author: ahmed_gallab2000@yahoo.com. 6 B.P. KALE, M.A. KOTHEKAR, H.P. TAYADE, J.B. JAJU, M. MATEENUDDIN Department of Pharmacology, S.R.T.R. Medical College, Ambajogai 43151 7. Indian J. med. Res. 1983, 83, 89,/.1988,88,339. 8. Fitoterapia 1986,57,302. 9. Indian J. Physiol.Pharmcol. 1986,30,118. 10. Heterocycles 1986,24,1319, J.Chem. soc. Perkin 1 1987,1429. 11. Phytochemistry 1988,27,3903. 12. Phytother.res. 1989,330;Chem.abstr. 1989,111,529q. 13. Quality control methods for medicinal plant materials World Health Organization Geneva. 14. KOKATE et al

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