AN UPDATED REVIEW OF PHARMACOLOGICAL STUDIES ON AZADIRACHTA INDICA (NEEM)

AN UPDATED REVIEW OF PHARMACOLOGICAL STUDIES ON AZADIRACHTA INDICA (NEEM)

Rohit Kumar Bijauliya ^{* 1}, Shashi Alok ¹, Dilip Kumar Chanchal ¹, Monika Sabharwal ² and Man Singh ³

Department of Pharmacognosy ¹, Institute of Pharmacy, Bundelkhand University, Jhansi - 284128, Uttar Pradesh, India.

Society of Pharmaceutical Sciences and Research ², Panchkula - 134112, Haryana, India.

Department of Pharmacy ³, Moti Lal Nehru Medical College, Allahabad - 211001, Uttar Pradesh, India.

ABSTRACT: Neem has become valuable plant in the world which shows the solutions for hundreds to thousands problems. Neem has become important in the global context today because it offers answers to the major concerns facing mankind. Neem has been extensively used in Ayurveda, Unani and Homoeopathic medicine and has become a centre of attraction of modern medicine. Azadirachta indica (neem) is a rapidly growing evergreen well known tree found generally in various regions of world like America, Africa and India. The aim of this review article provides information mainly on various pharmacological activities like anti-inflammatory, antimalarial, anti-bacterial, anti-allergic, antidermatic, antiulcer, antifungal, insecticidal, larvicidal and other pharmacological activities of neem plant and medicinal uses.

Azadirachta indica, Botanical description, Pharmacological activities, Ayurveda, Unani

INTRODUCTION: Medicinal plants are rich source of novel drugs that forms the ingredients in traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates, bioactive principles and lead compounds in synthetic drugs. WHO pointed out that more than 80% of world’s population depends on plants to meet their primary health care needs. However, overexploitation of the selected medicinal plant species lead to the reduction in number of plants in the wild and inclusion of their name in the red data book ¹.

Neem (Azadirachta indica) commonly called ‘Indian Lilac’ or ‘Margosa’, belongs to the family Meliaceae, subfamily Meloideae and tribe Melieae. Azadirachta indica has been used medicinally throughout history by many different cultures. Many compounds have been found in the exudates of the, Azadirachta indica plant that have been used medically by humans. Neem is a member of the Meliaceae family. The only congener is A. excelsa. Its sanskrit name, ‘arishtha’ means ‘reliever of sickness’ and it is considered as the ‘kalpavriksh of kalyuga’. The Persian name of neem is ‘Azad- Darakth- E- Hind’ which means ‘Free tree of India’².

Azadirachta indica is a fast growing evergreen popular tree found commonly in India, Africa and America ³. It has been used in Ayurvedic medicine for more than 4000 years due to its medicinal properties.

Neem is called ‘Arista’ in Sanskrit a word that means ‘perfect, complete and imperi-shable’⁴. Arishtha is the sanskrit name of the neem tree meaning ‘reliver of Sickness’ and hence is considered as ‘Sarbarogaribarini’. The tree is regarded as ‘Village Dispensory’ in India. The importance of the neem tree has been recognised by the US National Academy of Sciences, which publish a report in 1992 entiteled ‘Neem - a tree for solving global problems’⁵. Neem has become important in the global context today because it offers answers to the major concerns facing mankind. Neem (Azadirachta indica) is considered harmless to humans, animals, birds, beneficial insects and earthworms, and has been approved by the US Environmental Protection Agency for use on food crops ⁶. Neem (A. indica) of family meliaceae is evergreen tree of potential medicinal value found in most tropical countries ⁷.

A. indica has complex of various constituents including nimbin, nimbidin, nimbolide, and limonoids and such types of ingredients play role in diseases management through modulation of various genetic pathways and other activities. Quercetin and β-sitosterol were first polyphenolic flavonoids purified from fresh leaves of neem and were known to have antifungal and antibacterial activities ⁸. Numerous biological and pharma-cological activities have been reported including antibacterial ⁹, antifungal ¹⁰ and anti-inflammatory. Earlier investigators have confirmed their role as anti-inflammatory, antiarthritic, antipyretic, hypoglycemic, antigastric ulcer, antifungal, anti-bacterial and antitumour activities ^{11, 12, 13, 14} and a review summarized the various therapeutics role of neem ¹⁵.

Taxonomic Identity: ¹⁶ It has similar properties to its close relative, Melia azederach. The word Azadirachta is derived from the Persian azaddhirakt (meaning 'noble tree’). The taxonomic positions of neem are as follows:

Order            : Rutales

Suborder       : Rutinae

Family           : Meliaceae

Subfamily       : Melioideae

Tribe                : Melieae

Genus               : Azadirachta

Species               : indica

Description: ¹⁷

Tree: The neem tree (Azadirachta indica) is a fast growing (up to twenty feet in three years) tropical evergreen related to mahogany. It will grow where rainfall is as little as 18 inches per year and thrives in areas that experience extreme heat of up to 120ºF. They are reported to live for up to 200 years.

FIG. 1: TREE OF AZADIRACHTA INDICA

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; odour, indistinct; taste, bitter.

FIG. 2: LEAVES OF AZADIRACHTA INDICA

Stem Bark: Bark varies much in thickness according to age and parts of tree from where it is taken; external surface rough, fissured and rusty-grey; laminated inner surface yellowish and foliaceous, fracture, fibrous; odour, characteristic; taste, bitter.

Flower, Fruits and Seeds: The tree is often covered in delicate flowers in the early summer. The flowers (white and fragrant) are arranged axillary, normally more-or-less drooping panicles which are up to 25 cm long. It has a semi-sweet, olive-sized fruit. The seed inside is rich in oil with tremendous medicinal and botanical properties.

The oil is easily obtained by pressing the kernels in a juicer. It generally begins bearing fruit at three to five years, and can produce up to 110 lbs. of fruit annually when mature.

FIG. 3: STEM BARK OF A. INDICA                   

FIG. 4: FLOWERS OF A. INDICA

FIG. 5: FRUITS OF A. INDICA                             

  FIG. 6: SEEDS OF A. INDICA

Pharmacological Activities of Some Neem Compounds: Although a large number of compounds have been isolated from various parts of neem, a few of them have been studied for biological   activity as   shown   in Table 1.

TABLE 1: LIST OF ISOLATED COMPOUND AND PHARMACOLOGICAL ACTIVITIES

Pharmacological Activities:

Anti-inflammatory: Plants or their isolated derivatives are in the practice to treat/act as anti-inflammatory agents. A study result has confirmed that extract of A. indica leaves at a dose of 200 mg/kg, p.o., showed significant anti-inflammatory activity in cotton pellet granuloma assay in rats ⁴³. Other study results revealed that neem leaf extract showed significant anti-inflammatory effect but it is less efficacious than that of dexamethasone ⁴⁴ and study results suggest that nimbidin suppresses the functions of macrophages and neutrophils relevant to inflammation ⁴⁵.

Earlier finding showed immunomodulator and anti-inflammatory effect of bark and leave extracts and antipyretic and anti-inflammatory activities of oil seeds ^{46, 47}. Experimentation was made to evaluate the analgesic activity of neem seed oil on Albino rats and results of the study showed that neem seed oil showed significant analgesic effect in the dose of 1 and 2 mL/kg and oil has dose-dependent analgesic activity ⁴⁸. Results of the study concluded that the treated animals with 100 mgkg⁻¹ dose of Carbon Tetrachloride Extract (CTCE) of A. indica fruit skin and isolated ingredient azadiradione showed significant antinociceptive and anti-inflammatory activities ⁴⁹.

Another study was made to investigate the anti-inflammatory effect of Neem Seed Oil (NSO) on albino rats using carrageenan-induced hind paw edema and results revealed that NSO showed increased inhibition of paw edema with the progressive increase in dose from 0.25 ml to 2 mL/kg body weight. At the dose of 2 ml/kg body weight, NSO showed maximum (53.14%) inhibition of edema at 4^th h of carrageenan injection ⁵⁰. The chloroform extract of stem bark shows effectiveness against carrageenin - induced paw aedema in rat and mouse ear Inflammation. Inflammatory stomatitis in children is treated by the bark extract. Antipyretic activity has been reported in neem oil. A methanol extract of the leaves showed antipyretic effect when it is administrated into male rabbits. Antipyretic and anti-inflammatory activities in various extracts have been reviewed ⁵¹.

Pendse et al., (1977) reported anti-Inflammatory, immunosuppressive and some related pharma-cological actions of the water extract of neem in albino rats and immunosuppressive effect in Albino rabbits. It significantly inhibited acute inflammatory response evoked by carrageenin in a doss of 50 mg/ 100 g given orally and intraperitoneally. In chronic inflammation produced by crctcn-oil in granuloma pouch technique, 20 mg/ 100 g of the water extract significantly inhibited granulation tissue response; the reduction in exudative response and increase in the weight of adrenal glands were not significant. A significant inhibition of primary and secondary phases was observed in adjuvant induced arthritis. It significantly inhibited antibody formation by typhold “H” antigen. Mild analgesic effects of its own as well as potentiation of morphine analgesia were possessed by the extract but it was devoid of antipyretic effect ⁵².

Traditional Indian system of medicine mentions neem (Azadirachta indica) to have many medicinal properties. So the present study was carried out to access the anti-inflammatory effect of neem. Albino rats were used; they were divided into three groups. Control group treated with normal saline, standard treated with Indomethacin and test drug used was neem oil. For acute inflammation; carregennan induced rat paw edema inhibition method and for sub acute inflammation: cotton pellet granulation method. Ulcer index of Indomethacin and test compound were also studied. It is found that neem oil showed significant anti-inflammatory effect in both acute as well as chronic inflammation, it was also found to have low ulcerogenic potential compared to Indomethacin, hence can be safely used as a potent anti-inflammatory agent ⁵³.

Antidiabetic and Antihyperlipaemic: Bopana et al., (1997) reported antidiabetic and anti-hyperlipaemic effects of neem seed kernel powder on alloxan diabetic rabbits. In alloxan diabetic rabbits there was a significant (P<0.001) increase in fasting blood glucose and urine sugar and there was a significant decrease (P<0.001) in body weight and total haemoglobin content. There was a significant increase in body weight and haemoglobin level, and a significant decrease in Fasting Blood Glucose (FBG) and urine sugar in diabetic rabbits treated with NP, glibenclamide, insulin and in combination of NP and glibenclamide.

Though the entire antidiabetic drugs used significantly decreased the FBG levels, combination therapy of NP (250 mg/kg) and glibenclamide (0.25 mg/kg) p to all the other groups. There was a significant (P<0.001) roduced greater reduction in FBG as compared amelioration of body weight and total haemoglobin content in the diabetic phosphatase increased considerably in alloxan diabetic rabbits compared to the normal control. Treatment with various antidiabetic agents in the above experiments significantly reduced the enzyme activity. Treatment of NP with glibenclamide produced a significant (P<0.001) decrease of HMG CoA reductase, alkaline phosphatase and serum acid phosphatise activity when compared to other experimental antidiabetic agents.

Liver glucose 6-phosphatase (G6P) and serum lactate dehydrogenase (LDH) activity significantly (P<0.001) reduced in alloxan diabetic rabbits. On the contrary, hexokinase activity significantly increased by other experimental antidiabetic agents. The most significant (P<0.001) changes were observed in the combination of NP (250 mg/kg) and glibenclamide (0.25 mg/kg). From our experiments we have found out that, though both NP and glibenclamide produced significant fall in lipid parameter and enzyme activities, the changes were more prominent when combination of NP and glibenclamide were used ⁵⁴.

To study the effects of A. indica aqueous leaf extract on the expression of insulin signaling molecules and glucose oxidation in target tissue of high-fat and fructose-induced type-2 diabetic male rat. The oral effective dose of A. indica leaf extract (400 mg/kg body weight [b.wt]) was given once daily for 30 days to high-fat diet-induced diabetic rats. At the end of the experimental period, fasting blood glucose, oral glucose tolerance, serum lipid profile, and the levels of insulin signaling molecules, glycogen, glucose oxidation in gastro-cnemius muscle were assessed. Diabetic rats showed impaired glucose tolerance and impairment in insulin signaling molecules (insulin receptor, insulin receptor substrate-1, phospho-IRS-1Tyr632, phospho-IRS-1Ser636, phospho-AktSer473, and glucose transporter 4 [GLUT4] proteins), glycogen concentration and glucose oxidation. The treatment with A. indica leaf extract normalized the altered levels of blood glucose, serum insulin, lipid profile and insulin signaling molecules as well as GLUT4 proteins at 400 mg/kg b.wt dose. It is concluded from the present study that A. indica may play a significant role in the management of type-2 diabetes mellitus, by improving the insulin signaling molecules and glucose utilization in the skeletal muscle ⁵⁵.

To evaluated in-vivo diabetic murine model, A. indica and B. spectabilis chloroform, methanolic and aqueous extracts were investigated for the biochemical parameters important for controlling diabetes. It was found that A. indica chloroform extract and B. spectabilis aqueous, methanolic extracts showed a good oral glucose tolerance and significantly reduced the intestinal glucosidase activity. Interestingly, A. indica chloroform and B. spectabilis aqueous extracts showed significant increase in glucose-6-phosphate dehydrogenase activity and hepatic, skeletal muscle glycogen content after 21 days of treatment.

In immunohistochemical analysis, we observed a regeneration of insulin-producing cells and corresponding increase in the plasma insulin and c-peptide levels with the treatment of A. indica chloroform and B. spectabilis aqueous, methanolic extracts. Analyzing the results, it is clear that A. indica chloroform and B. spectabilis aqueous extracts are good candidates for developing new neutraceuticals treatment for diabetes ⁵⁶.

To examined the pharmacological hypoglycemic action of Azadirachta indica in diabetic rats. After treatment for 24 h, Azadirachta indica 250 mg/kg (single dose study) reduced glucose (18%), cholesterol (15%), triglycerides (32%), urea (13%), creatinine (23%), and lipids (15%). Multiple dose study for 15 days also reduced creatinine, urea, lipids, triglycerides and glucose. In a glucose tolerance test in diabetic rats with neem extract 250 mg/kg demonstrated glucose levels were significantly less compared to the control group, Azadirachta indica significantly reduce glucose levels at 15^th day in diabetic rats. Azadirachta indica serves as an important alternative source in the management of diabetes mellitus involved in reducing increased blood glucose during diabetes which should be examined further by oral hypoglycemic therapy ⁵⁷.

To evaluated the in-vivo hypoglycemic effect of aqueous leaf extracts of A. indica in alloxan-induced white male albino mice. The blood glucose lowering effect of the extract was intraperitoneally and orally bioscreened in diabetic mice in serial dilutions of the extract at 25 mg/kgbwt, 48.4 mg/kg bwt, 93.5 mg/kgbwt, 180.9 mg/kgbwt and 350 mg/kgbwt. In both routes, the extract lowered blood glucose at all dosages in a dose independent manner. The extracts contained flavonoids, tannins, sterols, saponins, anthraquinones and alkaloids. The antidiabetic activity may be attributable to these phytochemicals present in the plant extract. The study confirms the traditional use of this plant part in the treatment of diabetes mellitus. However, organic solvent extraction of the leaves of this plant should be done to compare effects of both organic and aqueous fractions ⁸⁹.

Antibacterial Activity: Methanolic extract of A. indica (neem) leaves was tested for its antibacterial, antisecretory and antihemarrhagic activity against Vibrio cholera ⁵⁹. The hexane chloroform and methanol extracts of Azadirachta indica were screened for antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis and Streptococcus faecalis. It was reported that methanol extract was the most effective, chloroform moderately effective and hexane extract showed low antibacterial activity ⁶⁰.

Oil extracted from leaves, seeds and bark gives a wide spectrum of antibacterial activity action against gram positive and gram negative microorganisms which including M. tuberculosis and streptomycin resistant strains. The photo-constituents like alkaloids, spooning, steroids, tennis, crude glycosides and flavonoids of neem plants was tested for antibacterial activity against pathogenic strains of E. coli, Corynebacterium bovis and Staphylococcus aureus ⁵⁸. The outcomes were also supported by Hymete et al., (2005) they reported that flavonoids compounds have antimicrobial activity. Hafiza et al., (2002) reported that crude saponins also prevent the growth of the microbes. Metabolic extract and acetonic extracts of leaves of Azadirachta indica were screened for antibacterial activity against two different bacterial strains i.e. E. coli and B. subtilus and it was reported that methanolic plant extracts showed maximum antibacterial activity as compared to acetonic plant extracts ⁶¹.

El-Mahmood et al., 2010 observed the antibacterial effects of crude extracts of neem seed against pathogenic involved in the infection of eyes and ear. The pure, ethanol, acetone and methanol extracts of neem were screened against bacterial strains i.e. E. coli, B. subtitles, Salmonella typhus, Pseudomonas, Staphylococcus aurous, and Klebsiella pneumonia and Staphylococcus epidermitis for various antibacterial activities. They reported that the neem extracts of acetone showed the maximum antibacterial activity as compared to other solvent extracts ⁶². Neem seed oil gives bactericidal activity against 14 pathogenic bacterial strains ⁶³. The solvent and crude aqueous extracts of A. indica (Neem) were screened against 20 pathogenic bacterial strains, wherein crude extracts shows better outcomes ⁶⁴. Ethanolic extracts of neem leaves and stick of neem plant were screened for antibacterial activity on streptococcus mutans and it was reported that neem stick extracts had higher antibacterial properties than the leaves extracts ⁶⁵.

Extracts of neem tree (Azadirachta indica) leaves were tested against Vibrio parahaemolyticus and Vibrio alginolyticus isolated from cultured shrimp. Aqueous extract of neem leaves did not produce any inhibitory zone while the neem juice produced inhibitory zone that showed linear relationship to the concentration of neem juice on both bacteria. The Minimum Inhibitory Concentration (MIC) for V. parahaemolyticus and V. alginolyticus was 3.13 and 6.25%, respectively. The minimum bactericidal concentration (MBC) for Vibrio parahaemolyticus and V. alginolyticus was 12.50 and 25.00%, respectively. It is concluded that neem juice is an antibacterial agent and is useful for inhibition of vibrios in shrimp ⁶⁶.

Methanol extract has the strongest growth inhibitory effect on both standard and clinical isolated strains of P. aeroginosa. Ethyl acetate and ethanol extracts, showing a growth inhibitory effect on both standard and hospital isolated strains of S. aureus. In the case of E. faecalis, ethanol and methanol extracts showed the highest growth inhibitory effect against standard and clinical strains, respectively. According to the MIC index results, the methanol extract has a bactericidal activity against both standard and nosocomial strains of S. aureus and P. aeroginosa and bacteriostatic activity against nosocomial strain of E. faecalis. Ethanol extract showed bactericidal activity against both standard and nosocomial strains of E. faecalis and P. aeroginosa and bacteriostatic activity against nosocomial strain of S. aureus. Ethyl acetate extract had shown bactericidal activity against standard strains of S. aureus and P. aeroginosa and bacteriostatic against nosocomial strain of S. aureus and standard strain of E. faecalis. Neem may be a prospective therapeutic agent to combat antibiotic resistant bacteria ⁸⁸.

Antifungal Activity: The aqueous and ethanolic extracts of Azadirachta indica leaves have been shown to have antidermatophytic activity against dermatophytes from the 88 clinical isolates with the help of agar dilution technique. In these studies, ethanolic extract showed more conspicuous activity as compared to aqueous extract ⁶⁷. Antifungal characteristics was tested using methanolic and acetone extracts of Azadirachta indica against two different fungal strains i.e. Aspergillus niger and Aspergillus fumigatus and it was reported that methanolic plant extract gives maximum antifungal activity as compared to acetonic extracts ⁶¹. The seed and leaf extracts of Azadirachta indica (neem) were screened for antifungal activity against dermatophytes and the Minimum Inhibitory Concentration (MIC) of (Azadirachta indica) neem seed extracts was found to be lower than that of neem leaf when screened against different species of Trichophyton and E. floccosum ⁶⁸.

Antifungal activity of aqueous ethanolic and ethyl acetone extracts of (Azadirachta indica) neem leaves on growth of few human pathogens. Aspergillus flavus, Candida albicans, Aspergillus terreus, Aspergillus fumigates, Aspergillus niger, and Microsporum gypseum in-vitro using different concentration and it was reported that these extracts prevented the growth of the test pathogenic organism and the effect gradually increased with increase in concentration ⁶⁹. Gedunin isolated from neem seed oil has been reported to have antifungal activity ⁷⁰. The compounds of sulphur such as cyclic tetrasulphide and trisulphide isolated from the stem distillate of fresh, matured neem leaves shows antifungal activity against Trichophyton mentagrophytes ⁷¹.

Mohanty et al., (2008) carried out antifungal activity of neem (Azadirachta indica) against Lagenidium giganteum and Metarhizium anisopliae in PYG and Emerson’s YpSs agar media. The minimum inhibitory concentration of neem oil for L. giganteum showed higher than that for M. anisopliae. The minimum fungicidal concentration of neem (Azadirachta indica) oil in PYG medium was lower than in YpSs for both fungi ⁷². Azadirachta indica (neem) leaf extract was taken to test its antifungal activity against three fungal species - Aspergillus flavus, Alternaria solani and Cladosporium. Ethanolic and methanolic extracts in different concentrations (25%, 50%, 75% and 100%) was prepared and tested against test organisms using disc diffusion method. Ketoconazole was used to compare the toxicity of neem leaf extract and its antifungal activity ⁷³.

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 ⁷⁴. 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 N-methyl- N¢-nitro-N-nitroso-guanidine (MNNG) (a carcinogenic material)-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 ^{75, 76}.

We examined the antioxidant system as a possible mechanism through which Neem Leaves Preparation (NLP) exerts its oncostatic potential. Female Swiss Albino mice were inoculated intramuscularly in the right thigh with Ehrlich Ascites Carcinoma (EAC) cells. NLP (500 mg/kg body weight) was injected for 20 days intraperitoneally into mice beginning on day 5 of post-EAC cell inoculation. Tumor growth, lipid peroxidation (LPx), glutathione (GSH) contents and the activity of the antioxidant scavenger enzymes were examined. Results indicated that NLP efficiently suppressed the growth of tumors which was associated with normalization of the LPx levels and augmentation of GSH contents.

NLP enhanced the activity of the endogenous antioxidant scavenging enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione-S-transferase (GST) in liver and tumor tissue. The effect of NLP was more pronounced when treated as early as day 5 of post-tumor cell inoculation. In conclusion, NLP induced oncostatic activity by modulating lipid peroxidation, augmenting the antioxidant defense system and protecting against oxidative stress ⁷⁷.

Antimalarial Activity: Ball shaped wood scrapings which is soaked in 5% neem oil (Azadirachta indica) which is diluted in acetone and in 45 days the breeding of Anopheles stephensi and Aedes aegypti were controlled, when it is placed in water storage over head tanks ¹⁷. Nimbolide isolated from neem extracts shows the antimalarial activity by preventing the growth of plasmodium falciparum ¹⁸. Gedunin isolated from neem seed oil has been reported to show antimalarial activities ¹⁹. Both aqueous and alcohol extracts of bark and leaves of neem are effective antimalarial agents, particularly on chloroquine resistant strains (badam et al., 1987) ²⁰.

This study was designed to know the antimalarial activity of the extract of the neem leaves (Azadirachta indica A. Juss) on the growth stages of P. falciparum FCR-3. The experimental laboratoric study used “post-test only with control design”. RPMI 1640 used as culture medium for cultivation of P. falciparum. Treated drug was the extract of neem leaves dissolved in dimethyl-sulfooxide and prepared into 7 levels concentration (3.125; 6.25; 12.5; 25; 50; 100 and 200 µg/mL). Negative control was culture medium with the malarial parasites. After cultured, synchronized, micromalarial culture were divided into control and treated groups then incubated in CO₂ Candle Jar at 37 ºC for 72 h. Each 8 h the percentage of parasitemia were measured for observing the activity of the extract on the growth stages of P. falciparum. After incubation, supernatant fluid was removed without disturbing the erythrocyte layer. Parasitemia was calculated by made the thin blood smear from the erythrocyte layer and stained with 10% Giemsa for 30 min. The antimalarial activity of the extract was calculated by counted the fifty percent of growth inhibition 50 (IC₅₀) using probit analysis. The result showed that the neem leaves extract can inhibit the growth of P. falciparum FCR-3 on mature schizont stage and the fifty percent inhibitory concentration (IC₅₀) of the extract was 3.86 µg/ml after 32 h incubating. The result indicated that the extract has an antimalarial activity on P. falciparum FCR-3 in -vitro ⁸².

Antiulcer Activity: The antiulcer effect was obtained with nimbidin in preventing acetyl salicyclic acid, indomethacin, serotonin-induced gastric lesions or streets as well as cysteamine induced duodenal ulcers or histamine ⁸³, ⁸⁴. Leaf extract of A. indica (Neem) shows antiulcer effect was reported by Garg et al., and the inhibition of mucus depletion and most cell defragmentation as possible mechanism. Bandyopadhyay et al., isolates the phenolic glycoside as an active constituent, whose characterization and mechanism are under investigation. Therefore, Azadirachta indica offers another option for an effective antiulcer drug and which is safe ⁸⁵.

This study was carried out to evaluate the antiulcer activity of the Aqueous Extract (AE) of the leaves of A. indica in Wistar rats. Gastric ulcerations were induced by pyloric ligation, aspirin, and cold restraint stress. AE was used in doses of 150, 300, and 600 mg/kg body weight per OS. Distilled water served as the control and ranitidine 20 mg/kg body weight intraperitoneal as the reference standard. The Ulcer Index (UI) and Percentage Inhibition (PI) values were determined in each model. The volume of gastric contents, free acidity, total acidity, and pH were measured in the pyloric ligation-induced ulcer model. AE showed a dose-dependent and significant (p < 0.05) decrease in the UI and an increase in the PI in all models employed compared to the control group. AE caused a dose-dependent decline in the gastric content volume, free acidity, and total acidity. The leaves of A. indica possess significant antiulcer activity and act via multiple mechanisms ⁸⁶.

Wound Healing Activity: The wound healing properties in small animal model, the excision and incision wound models were used and water, ethanol-water (1:1, v/v) and ethanol extracts were applied topically (15% w/w in ointment base). In the excision wound model, wound contraction, hydroxyproline content, DNA content, protein content, and nitric oxide levels were estimated after 14 days of topical treatment along with histopathological examinations. In the incision wound model, wound breaking strength was determined after 10 days of topical application of different extracts of AI. The animals treated with water extract of AI exhibited significant increment in rate of wound contraction (93.39%, P < 0.01), hydroxyproline content (13.31 ± 6.65 mg/g of dry tissue, P < 0.001), DNA content (20.99 ± 0.68 μg/100 mg of tissue, P < 0.01), protein content (100.53 ± 7.88 mg/g of wet tissue, P < 0.01) and nitric oxide level (3.05 ± 0.03 mMol/g of tissue, P < 0.001) as well as in wound breaking strength (289.40 ± 29.45 g, P < 0.01) when compared with vehicle control group which was also supported by histopathological studies. The water extract of stem bark of AI possesses significant wound healing property, validating its traditional use ⁸⁷.

Herbal Cosmetics: “The cure of all ailments". Neem's role as a wonder drug is stressed as far back as 4500 years ago. Some of its health restoring benefits. Effective in skin infection, rashes and pimples, immunity booster, anti obesity, blood purifier for beautiful and healthy skin, piles, hair disorder and oral disorders ⁹⁰.

CONCLUSION:  By reviewing the importance of neem tree in national, regional and international perspective there is an urgent need to study its diversity and develop effect measures to store it for present and future use. At the same time it is also necessary to undertake ethnobotanical studies to link its various therapeutic uses with folklore remedies used by tribes in various areas of its occurrence. For the last few years, there has been an increase tendency and attention in neem research. Quite a significant amount of research has already been carried out during the past few decades in exploring the chemistry of different parts of neem. Several therapeutically and industrially useful preparations and compounds have also been marketed, which generates enough encouragement among the scientists in exploring more information about this medicinal plant. Neem is provides various pharmacological activities like anti-inflammatory, antimalarial, antibacterial, anti-allergic, anti-dermatic, anti-ulcer, anti-fungal, insecticidal, larvicidal and other pharmacological activities of neem plant and medicinal uses

ACKNOWLEDGEMENT: The author thankful with our deepest core of heart to Dr. Shashi Alok and Monika Sabharwal, for his valuable guidance.

CONFLICT OF INTEREST: We declare that we have no conflict of interest.

REFERENCES:

1. Ahmedullah M and Nayar MP: Red data book of Indian plants, (Peninsular India), Calcutta: Botanical Survey India. 1999; 4.
2. Bhat SS and Girish K: Neem - A green treasure. Electronic J Bio. 2008; 4: 102-111.
3. Shirish PS: Hepatoprotection study of leaves powder of indica A. juss. International Journal of Pharmaceutical Sciences Review and Research. 2010; 3(2): 37-42.
4. Girish K and Bhat SS: Neem - A green treasure. Electronic Journal of Biology. 2008; 4(3): 102-111.
5. Kausik B, Chattopadhyay I, Banerjee RK and Bandyopadhyay U: Biological activities and medicinal properties of neem (Azadirachta indica). Current Science. 2002; 82: 1336-1345.
6. Bhowmik D, Chiranjib, Yadav J, Tripathi KK and Kumar KPS: Herbal Remedies of indica and its Medicinal Application. J. Chem. Pharm. Res., 2010; 2(1): 62-72.
7. Dhayanithi NB, Kumar TTA and Kathiresan K: Effect of neem extract against the bacteria isolated from marine fish. Journal of Environmental Biology. 2010; 31: 409-412.
8. Govindachari TR, Suresh G, Gopalakrishnan G, Banumathy B and Masilamani S: Identification of antifungal compounds from the seed oil of Azadirachta indica. Phytoparasitica. 1998; 26(2): 109-116.
9. Singh N and Sastry MS: Antimicrobial activity of neem oil. Indian Journal of Pharmacology. 1997; 13: 102-06.
10. Kher A and Chaurasia SC: Antifungal activity of essential oils of three medical plants. Indian Drugs. 1997; 15: 41-42.
11. Bandyopadhyay U, Biswas K and Sengupta A: Clinical studies on the effect of neem (Azadirachta indica) bark extract on gastric secretion and gastroduodenal ulcer. Life Sciences. 2004; 75(24): 2867-867.
12. Sultana B, Anwar F and Przybylski R: Antioxidant activity of phenolic components present in barks of Azadirachta indica, Terminalia arjuna, nilotica and E. jambolana Lam. Trees. Food Chemistry. 2007; 104(3): 1106-114.
13. Ebong AE, Atangwho IJ, Eyong EU and Egbung GE: The antidiabetic efficacy of combined extracts fromtwo continental plants: indica (A. Juss) (Neem) and V. amygdalina (Del.) (African Bitter Leaf). The American Journal of Bioch and Biotech. 2008; 4(3): 239-244.
14. Paul R, Prasad M and Sah NK: Anticancer biology of Azadirachta indica (neem): a mini review. Cancer Biology and Therapy. 2011; 12(6): 467-476.
15. Biswas K, Chattopadhyay I, Banerjee RK and Bandyopadhyay U: Biological activities and medicinal properties of neem. Cur Sci. 2002; 82(11): 1336-1345.
16. Ogbuewu P, Odoemenam VU, Obikaonu HO, Opara MN, Emenalom OO and Uchegbu MC: The growing importance of neem (Azadirachta indica Juss) in agriculture, industry, medicine and environment: A Review. Res J Med Plant. 2011; 5: 230-245.
17. Ayurvedic Pharmacopeia of India, Government of India. 1(2): 131-132.
18. Bhargava KP, Gupta MB, Gupta GP and Mitra CR: Anti-inflammatory activity of saponins and other natural products. Indian J Med Res. 1970; 58(6): 724-730.
19. Pillai NR and Santhakumari G: Anti-arthritic and anti-inflammatory actions of nimbidin. Planta Medica. 1981; 43: 59-63.
20. David and Mediscope SN: Anti-pyretic of neem oil and its constituents. 1969; 12: 25-27.
21. Pillai NR and Santhakumari G: Hypoglycemic activity of Melia azadirachta: (Neem). Indian Journal of Medical Research. 1981; 74, 931-933.
22. Pillai NR and Santhakumari G: Effects of nimbidin on acute and chronic gastro-duodenal ulcer models in experimental animals. Planta Medica. 1984; 50: 143-146.
23. Pillai NR, Seshadri DS and Santhakumari G: Indian Journal of Medical Research. 1978; 68: 169-175.
24. Sharma VN and Saxena KP: Spermicidal action of sodium nimbinate. Indian Journal of Medical Research. 1959; 47: 322-324.
25. Murthy SP and Sirsi M: Pharmacological studies on Melia Azadirachta Indian Journal of Physiology and Pharmacology. 1958; 2: 387-396.
26. Bhide NK, Mehta DJ and Lewis RA: Diuretic actoin of sodium nimbidinate. Indian Journal of Medical Sciences. 1958; 12: 141-145.
27. Jones I, Ley SV, Denholm AA, Lovell H, Wood A and Sinden RE: Sexual development of malaria parasites is inhibited in-vitro by the neem extract azadirachtin and its semi-synthetic analogues. FEMS Microbiol. Lett., 1994; 120(3): 267-273.
28. Sharma VN and Saksena KP: Sodium nimbidinate in-vitro study of its spermicidal action, Indian Journal of Medical Reaserch. 1959; 1038-1042.
29. Rojanapo W, Suwanno S, Somaree R, Glinsukon T and Thebtaranonth Y: Screening of antioxidants from some Thia vegetables and herbs. J. Sci. Thailand. 1985; 11: 177-188.
30. Rochanakij S, Thebtaranonth Y, Yenjal CH and Yuthavong Y: Nimbolide, a constitutes of Azadirachta indica inhibits falciparum in culture. Southeast Asian J. Trop. Med. Public Health. 1985; 16: 66-72.
31. Khalid SA, Duddeck H and Gonzalez-Sierra M: Isolation and characterization of antimalerial agent of the neem tree, indica. Journal of Natural Product. 1989; 52: 922-927.
32. Rao BS, Nazma and Rao MJ: Antifungal activity of gedunin. Curr. Sci., 1977; 46: 714-716.
33. Devakumar C and Dev S: In Neem (eds Randhawa and Parmar, B. S.). Edition 2^nd, 1996: 77-110.
34. der Nat VJM, der Sluis VWG, Hart LA, Disk VH, de Silva KTD and Labadie RP: Planta Med. 1991; 57: 65-68.
35. Ara I, Siddiqui BS, Faizi S and Siddiqui S: Strucurally novel diterpenoid constituents from the stem bark of Azadirachta indica (melieceae). J. Chem. Soc., Perkin Trans., 1989; 2: 343-345.
36. Pant N, Garg HS, Madhusudanan KP and Bhakuni DS: Sulfurous compounds from Azadirachta indica Fitoterapia. 1986; 57: 302-304.
37. Kakai T and Koho JP: Anti-inflammatory polysaccharides from Melia azadirachta. Chem. Abstr., 1984; 100: 91350.
38. Fujiwara T, Takeda T, Ogihara Y, Shimizu M, Nomura T and Tomita Y: Studies on the structure of polysaccharides from the bark of Melia azadirachta. Chem. Pharm. Bull. 1982; 30: 4025-4030.
39. Fujiwara T, Sugishita E, Takeda T, Ogihara Y, Shimizu M, Nomura T and Tomita Y: Further studies on the structure of polysaccharides from bark of Melia azadirachta. Chem. Pharm. Bull. IBID. 1984; 32: 1385-1391.
40. Nat VJM, Kierx JPAM, Dijk VH, de Silva KTD and Labadie RP: J. Ethnopharmacol., 1987; 19: 125-131.
41. Nat VJM, Hart LAT, Sluis VWG, Dijk VH, Berg VAJJ, De Silva KTD and Labadie RP: IBID. 1989; 27: 15-24.
42. Moursi SAH and Al-Khatib IM: Jpn J. Pharmacol., 1984; 36: 527-533.
43. Chattopadhyay RR: Possible biochemical mode of anti-inflammatory action of indica A. Juss. in rats. Indian Journal of Experimental Biology. 1998; 36(4): 418-420.
44. Mosaddek ASM and Rashid MMU: A comparative study of the anti-inflammatory effect of aqueous extract of neem leaf and dexamethasone. Bangladesh Journal of Pharma-cology, 2008; 3(1): 44-47.
45. Kaur G, Alam MS and Athar M: Nimbidin suppresses functions of macrophages and neutrophils: relevance to its antiinflammatory mechanisms. Phytotherapy Research. 2004; 18(5): 419-424.
46. Arora N, Koul A and Bansal MP: Chemopreventive activity of indica on two-stage skin carcinogenesis in murine model. Phytotherapy Res. 2011; 25(3): 408-16.
47. Biswas K, Chattopadhyay I, Banerjee RK and Bandyopadhyay U: Biological activities and medicinal properties of neem (Azadirachta indica). Current Science. 2002; 82(11): 1336-1345.
48. Kumar S, Agrawal D, Patnaik J and Patnaik S: Analgesic effect of neem (Azadirachta indica) seed oil on Albino rats. I J of Pharma and Bio Sci. 2012; 3(2): P222-225.
49. Ilango K, Maharajan G and Narasimhan S: Anti-nociceptive and anti-inflammatory activities of indica fruit skin extract and its isolated constituent azadiradione. Natural Product Research. 2013; 27(16): 1463-1467.
50. Naik MR, Bhattacharya A, Behera R, Agrawal D, Dehury S and Kumar S: Study of anti-inflammatory effect of neem seed oil ( indica) on infected Albino rats. Journal of Health Research and Reviews. 2014; 1(3): 66-69.
51. Biswas K, Chattopadhyay I, Banerjee RK and Bandyopadhyay U: Biological activities and medicinal properties of neem (Azadirachta indica). Current Science. 2002; 82:1336-1345.
52. Pendse VK, Dadhich AP, Mathur PN, Bal MS and Madam BR: Anti-Inflammatory, immunosuppressive and some related pharmacological actions of the water extract of neem giloe (Tinospora cordrfolla): A preliminary report. Ind J Pharmacy. 1977; 9: 221-224.
53. Jagadeesh K, Srinivas K and Shreenivas PR: Anti inflammatory effect of Azadirachta indica (neem) in Albino rats-An experimental study. IOSR Journal of Pharmacy. 2014; 4(1): 34-38.
54. Bopanna KN, Kannan J, Gadgil S, Balaraman R and Rathod SP: Antidiabetic and antihyperlipaemic effects of neem seed kernel powder on alloxan diabetic rabbits. Ind J Pharmacy. 1997; 29: 162-167.
55. Satyanarayana K, Sravanthi K, Shaker IA and Ponnu-lakshmi R: Molecular approach to identify antidiabetic potential of Azadirachta indica. Journal of Ayurveda and Integrative Medicine. 2015; 6(3): 165-174.
56. Bhat M, Kothiwale SK, Tirmale AR, Bhargava SY and Joshi BN: Antidiabetic properties of Azardiracta indica and Bougainvillea spectabilis: In-vivo studies in murine diabetes model. Evidence-Based Complementary and Alternative Medicine. 2011, Article ID 561625, 9. http://dx.doi.org/10.1093/ecam/nep033
57. Dholi SK, Raparla R, Mankala SK and Nagappan K: In-vivo antidiabetic evaluation of neem leaf extract in alloxan induced rats. J of App Pharm Sci. 2011; 01(04): 100-105.
58. Aslam F, Khalil-Ur-rehman, Asghar M and Sarwar M: Pak. J Agri. Sci. 2009; 46(3).
59. Thakurta P and Bhowmik P, Mukharjee S, Hajra TK, Patra A and Bag PK; antibacterial, anti-secretory and anti-hemorrhagic activity of indica used to treat cholera and diarrhea in India. J Ethnopharmacol. 2007; 111(3): 607-12.
60. Koona S and Budida S: Not Sci Boil. 2011; 3(1): 65-69.
61. Surabhilal W, Charan AA and Bind A: International J of Medicine and Pharma Sci (IJMPS). 2013; 3(2): 79-86.
62. Irshad S et al.: Intl R. J of pharmaceuticals. 2011; 01(1): 9-14.
63. Baswa M, Rath CC and Dash SK: Antibacterial activity of karanj (Pongamia pinnata) and neem ( indica) seed oil; A preliminary report. Microbios. 2011; 105: 183-189.
64. Srinivasan, Nathan D and Suresh S: Antibacterial activity of neem (Azadirachta Indica) and Tamarind (Tamarindus indica) leaves. Asian J Microbial Biotechnal Environ Sci. 2001; 3: 67-73
65. Siswomihardjo et al.: Int Chin J Dent. 2007; 7: 27-29.
66. Banerjee S, Kim LM, Shariff M, Khatoon H and Yusoff FM: Antibacterial activity of neem ( indica) leaves on Vibrio spp. isolated from cultured shrimp. Asian Journal of Animal and Veterinary Advances. 2013; 8(2): 355-361.
67. Venugopal PV and Venugopal TV: Antidermatophytic activity of neem (Azadirachta indica) leaves in-vitro. Indian J Pharmacol. 1994; 26: 141-3.
68. Ranganathan S, Balajee MTAM and Raja SM: Anti-dermatophytic activities of Azadirachta indica: An in-vitro and in-vivo Indian Journal of Dermatology 1996; 41(4): 113-117.
69. Mahmood DA, Hassanein NM, Youssef KA, Zeid AMA: Antifungal activity of different neem leaf extracts and the nimonal against some important human pathogens. Braz J Microbial. 2011; 42(3).
70. Rao BS, Nazma and Rao MJ: Antifungal activity of gedunin. Cur. Sci. 1977; 46: 714-716.
71. Pant N, Garg HS, Madhusudanan KP and Bhakuni DS: Sulfurous compounds from Azadirachta indica Fitoterapia. 1986; 57: 302-304.
72. Mohanty SS, Raghavendra K and Dash AP: Influence of growth medium on antifungal activity of neem oil (Azadirachta indica) against Lagenidium giganteum and Metarhizium anisopliae. Mycoscience. 2008; 49: 318-320.
73. Shrivastava DK and Swarnkar K: Antifungal Activity of leaf extract of neem (Azadirachta indica). Int. J. Curr. Microbiol. App. Sci. 2014; 3(5): 305-308.
74. Balasenthil S, Arivazhagan S, Ramachandran CR and Nagini S: J. Ethnopharmacol. 1999; 67: 189-195.
75. Arivazhagan S, Balasenthil S and Nagini S: Cell Biochem. Funct. 2000; 18: 17-21.
76. Arivazhagan S, Balasenthil S and Nagini S: Phytother. Res., 2000; 14: 291-293.
77. Metwally FM, El-Mezayen HA, Moneim AEA and Sharaf NE: Anti-tumor effect of Azadirachta indica (neem) on murine solid ehrlich carcinoma. Academic Journal of Cancer Research. 2014; 7(1): 38-45.
78. Dua VK, Nagpal BN and Sharma VR: Repellant action of neem cream against mosquitoes. Indian J Malarial 1995; 32: 47-53
79. Rojanapo W, Suwanno S, Somaree R, Glinsukon T and Thebtaranonth Y: Screening of antioxidants from some thia vegetables and herbs. J Sci Thailand. 1985; 11: 177-188.
80. Khalid SA, Duddeck H and Gonzalez-Sierra M: Isolation and characterization of antimalarial agent of the neem tree, indica. Journal of Natural Product. 1989; 52: 922-927.
81. Kabeh JD and Jalingo MGDSS: Mini review exploiting neem for improved life. Int J Agribiol. 2007; 9(3).
82. Yusuf H: The antimalarial activity of the extract of the neem leaves ( indica, A. Juss) on P. falciparum in-vitro. Proceedings of the Annual International Conference Syiah Kuala University 2011 Banda Aceh, Indonesia. 2011; 29-30.
83. Pillai NR, Seshadri DS and Santhakumari G: Ind Journal of Medical Research. 1978; 68: 169-175.
84. Pillai NR and Santhakumari G: Effects of nimbidin on acute and chronic gastro-duodenal ulcer models in experimental animals. Planta Medica. 1984; 50: 143-146.
85. Dharmani P and Palit G: Exploring Indian Medicinal plants for antiulcer activity. Indian J Pharmaceutical. 2006; 38 (2): 95-99.
86. Bhajoni PS, Meshram GG and Lahkar M: Evaluation of the antiulcer activity of the leaves of Azadirachta indica: An experimental study. Integr Med Int. 2016; 3: 10-16.
87. Maan P, Yadav KS and Yadav NP: Wound Healing Activity of Azadirachta indica Juss Stem Bark in Mice. Pharmacognosy Magazine.2017; 13(S-2): S316-S320. doi:10.4103/0973-1296.210163.
88. Maleki L, Sadeghian-Rizi T, Ghannadian M, Sanati MH, Shafizadegan S and Sadeghi-Aliabadi H: Antibacterial activity of Azadirachta indica leaf extracts against some pathogenic standards and clinical bacterial isolates. Avicenna J Clin Microb Infec. 2017; e12987.
89. Arika WM, Nyamai DW, Agyirifo DS, Ngugi MP and Njagi ENM: In-vivo antidiabetic effect of aqueous leaf extract of Azardirachta indica, A. juss in alloxan induced diabetic mice. J Diabetic Complications Med 2016; 1(2): 106.
90. Bijauliya RK, Alok S, Kumar M, Chanchal DK and Yadav S: A comprehensive review on herbal cosmetics. Int J Pharm Sci Res 2017; 8(12): 4930-49.
    

    ---

    How to cite this article:

    Bijauliya RK, Alok S, Chanchal DK, Sabharwal M and Singh M: An updated review of pharmacological studies on Azadirachta indica (neem). Int J Pharm Sci Res 2018; 9(7): 2645-55. doi: 10.13040/IJPSR.0975-8232.9(7).2645-55.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---