Antibacterial Potential of the Extracts of the Leaves of Azadirachta indica

Azadirachta indica A. Juss (syn. Melia azadirachta) is well known in India and popularly known as Indian neem. To evaluate antibacterial potential, the agar well diffusion assay was used against Gram-negative and Gram-positive bacteria. Penicillin and Dimethyl sulfoxide were used as positive and negative controls, respectively. Methanol extract showed the highest and chloroform extract showed moderate to good antibacterial activity. Proteus vulgaris and Micrococcus luteus were the most susceptible bacteria while Bacillus subtilis was more resistant bacterium to the hexane, chloroform and methanol extracts of neem. The study recommended for the isolation and separation of bioactive compounds responsible for the antibacterial activity.


Introduction
Plants have been a source of herbal remedies throughout the history of mankind.Various medicinal plants have been used for years in daily life to treat diseases all over the world (Nimri et al., 1999;Saxena, 1997).The wide spread use of herbal remedies and healthcare preparations, such as those described in ancient texts like the Vedas and Bible, has been traced to the occurrence of natural products with medicinal properties.Existence and survival of mankind is impossible without plant kingdom, as plants are the primary procedures and play important role in sustaining the life forms on earth.Search of newer drugs from plants has been rise; since many of the microorganisms are posing series health related disorders.According to recent estimates by the WHO more than 3.5 billion people in the developing world rely on plants as source of medicine for various ailments.Over 20,000 plants have medicinal values and many plants are yet to be explored for their potentials.In addition, many of the existing synthetic drugs cause various side effects.Hence, drug development from plant based compounds could be useful in meeting this demand for newer drugs with minimal side effects (Srivastava et al., 2000).
Azadirachta indica A. Juss (syn.Melia azadirachta) is well known in India and its neighboring countries.It is popularly known as Indian neem (margosa tree) or Indian lilac.It is an evergreen tree, cultivated in various parts of the Indian subcontinent.Every part of the tree has been used as traditional medicine for household remedy against various human ailments.Neem has been extensively used in Ayurveda, Unani and homoeopathic medicine and has become a cynosure of modern medicine.The Sanskrit name of the neem tree is 'Arishtha' meaning reliever of 'sickness' and hence is considered as 'sarbarigabubarini' .The tree is still regarded as 'village dispensary' in India.Chemical investigation on the products of the neem tree extensively undertaken in the middle of the isolation of nimbin, the first bitter compound isolated from neem oil, more than 135 compounds have been isolated from different parts of neem (Ganguli, 2002).Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex (Subapriya and Nagini, 2005).Seeds contain a complex secondary metabolite azadirachtin.The medicinal properties of the plant Azadirachta indica were studied by several workers.They were Antipyretic (Khattak et al., 1985;Okpanyi and Ezeukwk, 1981), anti-malarial effect (Rochankij et al., 1985;Tella, 1977), anti tumour effect (Fujiwara et al., 1982), anti ulcer effect (Pillai and Santhakumari, 1984), anti diabetic effect (Shukla et al., 1973), antifertility effect (Sinha et al., 1984), effect on central nervous system (Singh et al., 1987) cardiovascular effect (Thompson and Anderson, 1978) and Antioxidant activity (Bandyopadhyay et al., 2002;Sultana et al., 2007).Boiled neem leaf water makes an excellent antiseptic to clean wounds, soothes, swellings and eases skin problems (Shahidi Bonjar et al., 2004).Oil from the leaves, seeds and bark possesses a wide spectrum of antibacterial activity action against Gram-negative and Gram-positive microorganisms, including M. tuberculosis and Streptomycin resistant strains (Chopra et al., 1956).In vitro it inhibits Vibrio cholerae, Klebsiella pneumoniae, M. tuberculosis and M. pyrogenes (Satyavati et al., 1976).Rao et al. (1986) reported the antimicrobial activity of the seed oil against a variety of pathogens.The antifungal effect of leaf extract against Alternaria alternata (Bhomick and Choudhary, 1982).Antimicrobial effects of neem extract have been demonstrated against Streptococcus species (Almas, 1999).broth were used for inoculation of the nutrient agar plates.An aliquot (0.02 ml) of inoculums was introduced to molten and cooled to 45°C nutrient agar and placed on petriplate by pour plate technique.The appropriate wells were made on respective agar plate by using cork borer.In agar well diffusion method 0.05 ml of hexane, chloroform and methanol extracts were introduced to their respective wells following an incubation period of 24 to 48 hours at 37°C.Antibacterial activity was evaluated by qualifying inhibition zones (IZ) of bacterial growth surrounding the plant extracts.The entire antibacterial assay was carried out under strict aseptic conditions.Penicillin (5 µg/disc) was used as positive control and DMSO as a negative control.Triplicates were carried out for each extract against each of the test bacterium.The results of screening are shown in the Tab. 1.

Results and discussion
The preliminary screening results of hexane, chloroform and methanol extracts are presented in Tab. 1.All the three extracts of leaves of Azadirachta indica were expressed antibacterial activity at least one bacterium.Methanol extract was the most effective against all the tested bacteria.The chloroform extract showed good to moderate where as hexane extract showed low antibacterial activity.The inhibition zone values were interpreted as sensitive (18 mm), intermediate (14-17 mm) and resistant (<14 mm) (Barry et al., 1970).
Hexane extract of Azadirachta indica found effective against Proteus vulgaris and Micrococcus luteus, while the same bacteria were highly sensitive to chloroform and methanol extracts, respectively.Escherichia coli, Proteus vulgaris, Micrococcus luteus, Enterococcus faecalis and Streptococcus faecalis were showed fairly high degree of sensitivity (IZ=18-33 mm) to both chloroform and methanol extracts.Enterococcus faecalis and Klebsiella pneumoniae were found to be intermediate sensitivity (IZ=14-15 mm) to hexane and chloroform extracts, respectively.Escherichia coli, Klebsiella pneumonia and Streptococcus faecalis were most resistant (IZ=11-13 mm) to hexane extract.Metha-Recently, the antibacterial activity of neem seed oil was assessed in vitro against 14 strains of pathogenic bacteria (Baswa et al., 2001).We planned the present study to find out the antibacterial activity of neem leaves against human pathogenic bacteria, including Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis and Streptococcus faecalis.

Plant material and extracts preparation
The leaves of Azadirachta indica were collected from Kambalalkonda forest in Visakhapatnam district, Andhra Pradesh.The collected leaves were identified and authenticated by a Botanist, Department of Botany, Andhra University.The collected leaves were washed thoroughly with running tap water and finally with sterile distilled water.The material was chopped into small pieces and then air dried on a sterile blotter under shade for 20-30 days.
The completely shade dried material was coarsely powdered and allowed to Soxhlet for successive extraction with hexane, chloroform and methanol.The obtained liquid extracts were subjected to Rotary evaporator and subsequently concentrated under reduced pressure (in vaccum at 40°C).The residues obtained were designed as crude extracts were labeled and stored in refrigerator for further study (Nostro et al., 2006).The dried plant extracted residues obtained were re dissolved in 0.1% Dimethyl sulfoxide (DMSO) to get 100mg/ml concentration and filtration through a 0.45 µm membrane filter and stored in sterile brown bottles in a freezer at 20°C until bioassay.

Test microorganisms
The microorganisms in the present study were including Escherichia coli (ATCC B9637), Klebsiella pneumoniae (MTCC B2405), Proteus vulgaris (MTCC B0426), Micrococcus luteus (MTCC B1538), Bacillus subtilis (MTCC B2274), Enterococcus faecalis (MTCC B0439) and Streptococcus faecalis (MTCC B0459).All the cultures were purchased from microbial type culture collection (MTCC), Institute of Microbial technology (IMTECH), Chandigarh, India.The strains were maintained and tested on nutrient agar (Himedia).Active cultures were generated by inoculating a loop full of culture in separate 100ml nutrient broths and incubating on a shaker at 37°C overnight.The cells were harvested by centrifuging at 4000 rpm for 5 min, washed with normal saline, spun at 4000 rpm for 5 min again and dilute in normal saline to obtain 5x10 5 CFU/ml.

Determination of antibacterial activity
The hexane, chloroform and methanol extracts of Azadirachta indica were screened for antibacterial activity by agar well diffusion method (Perez and Paul Mand Bazeque, 1990) with cork borer of size 6.0 mm.For all bacterial strains, overnight cultures grown in nutrient nol extract of Azadirachta indica showed fairly high degree of sensitivity (IZ=20-33 mm) (Fig. 1) to all tested bacteria, except Bacillus subtilis, which was least susceptible to chloroform and methanol extracts, while did not showed any activity to hexane extract.Penicillin (5 µg) was used as standard antibiotic reference (positive control) exhibited zone of inhibition values range from 11-20 mm against all tested bacteria (Fig. 2).These values were lower than values observed by methanol extract of Azadirachta indica.
Hence methanol extract was exhibited a broad spectrum of antibacterial activity.Negative control, DMSO had no effect on microbial growth.
Proteus vulgaris and Micrococcus luteus belongs to Gram-negative bacteria and the most susceptible bacteria to hexane, chloroform and methanol extracts of Azadirach-ta indica.This selective toxicity could be linked to the differences in the composition of the lipid bilayer for the two strains of bacteria.A greater degree of depolarization and hence increased permeability, was expressed in the lipid bilayer of the Gram-negative bacteria for this cembranoid, because they contain more lipids in their cell walls (Stainer et al., 1986).This depolarization effect is suggested to be associated with hydrogen bonding on the hydroxyl group in the carboxylic functionally situated at the C-19 position in the diterpene ( Jente et al., 1990).
The antibacterial activity of Azadirachta indica might be due to presence of triterpenoids, phenolic compounds, carotenoids, steroids, valavinoids, ketones and tetra-triterpenoids azadirachtin.Earlier studies on Azadirachata claim that a spermicidal fraction of neem oil (NIM-76) is more effective as an antimicrobial agent as compared to the neem oil itself especially its effect is less on Escherichia coli and Klebsiella pneumonia ( Sairam et al., 2000).Antibacterial activity of the extracts of Azadirachta indica was effective on Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis etc. and needs further confirmation ( Jagannadh and Radhika, 2006).Almas (1999) reported antimicrobial activity of neem oil on Escherichia coli and Klebsiella pneumonia with methanol but not with chloroform and hexane extracts as is influenced by pH of the final extract.The present study report also shows very high efficacy of chloroform and methanol extracts on Proteus vulgaris and Micrococcus luteus and to a lesser extent against Escherichia coli, Klebsiella pneumonia, Enterococcus faecalis and Streptococcus faecalis.This study is also needs further study to isolate and purification of bioactive compounds responsible for the antimicrobial activity.

Conclusions
Although this study investigating the in vitro antibacterial activity, the results showed that the extracts from Azadirachta indica leaves possessed good antibacterial activity, confirming the great potential of bioactive compounds and are useful for rationalizing the use of this plant in primary health care.In vivo data may be helpful in determining the real potential usefulness of this plant for the treatment of infectious diseases.