Use of herbal medicines in the developed world continue to rise because they are rich source of novel drugs and their bioactive principles form the basis in medicine,¹ with few adverse reactions. The emergence and dissemination of multidrug resistance bacteria has made chemically synthesized antibiotics ineffective. ² These circumstances have propelled researchers and scientists to explore new antimicrobial substances from various sources such as medicinal plants. ³

Azadirachta indica is an evergreen tree commonly found in the Indian subcontinent, which is famous by the local name ‘Neem’, it has been considered to provide freedom from all diseases, and used for thousands of years in Indian and African continents. Different parts of the plant including flowers, leaves, seeds and bark have been used to treat both acute and chronic human diseases; and used as an insecticide; antimicrobial, larvicidal, antimalarial, antibacterial, antiviral, and spermicidal 4. Neem tree contains large number of biologically active compounds that are diverse and complex. More than 140 compounds including alkaloids, flavonoids, triterpenoids, carotenoids and nimbin have been isolated from different parts of the plant ^{5, 6}. The extracts of the tree (leaves, roots, bark) have been reported to possess anti-viral, anti-fungal, anti-bacterial properties7. The purpose of this study is to investigate the antimicrobial property of neem using solvent extracts method on neem leaves.

The present study is an observational study conducted in the department of Microbiology at AIMS, B.G.Nagara for a period of two months.

Once the desired OD is obtained the culture suspensions were inoculated on to sterile MHA plates prepared earlier using lawn culture method using a sterile cotton Q-tip. The cotton is inserted into the suspension and rotated and compressed against the wall of the test tube to express the excess fluid. To ensure growth is uniform and confluent the cotton tip is streaked in zig-gag fashion then turning the plate by 60º repeating the same 3-4 times to leave no gap. The discs are prepared using Whatman filter paper measuring 6mm in diameter and there sterilized by hot air oven. The discs are then placed at appropriate position with the center disc being the standard antibiotic. With the observation and values obtained from the above MIC experiment suitable six consecutive dilution are taken, such that it includes the MIC concentration. The six chosen concentrations are loaded on to the discs in order of their dilution using micropipette. An extra disc labelled ‘C’ is used as control which is loaded with DMSO and at the center standard disc of Gentamicin GM10 is used as positive control, all the above steps are done under sterile condition to avoid contamination and same is done for each of the chosen organisms. Inoculated MHA plates are then placed inside incubator at 35-37ºC for 24hrs. The results are then read by measuring the zone of inhibition for each of the discs using a scale and the obtained data is tabulated.

In recent years, we have seen antimicrobial resistance rapidly emerge at a global scale and spread from one country to the other faster than previously thought. Multidrug-resistant bacteria are endemic in many parts of the world. There is no question that the widespread use, overuse, and misuse of antimicrobials during the previous years have been associated with the explosion of antimicrobial resistance ¹¹. Use of herbal medicines in the developed world continue to rise because they are rich source of novel drugs and their bioactive principles form the basis in medicine 1. These circumstances have propelled the researchers and scientists to explore new antimicrobial substances from various sources such as medicinal plants. ³

In the present study, leaves of Azadirachta indica also known as ‘Neem’ was taken to test their potential as antimicrobial agent. Solvent extraction was done using three solvents namely Ethanol, Ethyl acetate and Petroleum ether. These solvent extracts were tested against two gram- negative (Escherichia coli and Klebsiella) two gram-positive organisms (Staphylococcus aureus and MRSA) and a fungus (Candida albicans).

The results obtained from minimum inhibitory concentration testing (Table 2) showed that Ethanolic extract of the leaves had more activity against E. coli (16 and 32µg) than the other two solvent extracts, however all the extracts showed equal activity against Klebsiella (32µg). For S.aureus ethyl acetate extract gave the best result (16µg) followed by ethanolic extract (32µg). Whereas for MRSA best results were seen with Ethyl acetate (16 and 32µg) followed by ethanolic extract (32µg). For Candida albicans all showed similar results (64µg) (Figure 2). In a study conducted by Rajaratna Reddy et al, showed that aqueous extracts also have good antimicrobial property After MIC to confirm whether the extract is bacteriostatic or bactericidal MBC was done , then it was confirmed that the extracts had bactericidal action. DPPH assay showed that extracts have antioxidant property and UOM Neem Pet.ether has the highest antioxidant property when compared to other extracts.

Taking MIC values as reference antimicrobial susceptibility testing (AST) was done by disc diffusion method. Alternatively in a study conducted by Mahmood Rasool et al., they used well diffusion method and had achieved good results. The zone of inhibition was measured (Table 3) after incubation the results showed Ethyl acetate extracts showed best result among the three solvents (ZOI 14 and 12mm) followed by Petroleum ether (12 and 13mm) for E. coli, however maximum zone was seen with ethanolic extract for Klebsiella (16mm) followed by Petroleum ether and Ethyl acetate. For S.aureus good results were seen with ethanolic extracts (8 and 6mm) followed by Ethyl acetate (7and 0mm) but Petroleum ether failed to inhibit the organism(0mm). Whereas for MRSA good results were seen with Ethyl acetate and petroleum ether (13&10mm and 13& 8mm) followed by the ethanolic extract (10 and 6mm). For Candida albicans ethanol and Petroleum ether showed better results (6 and 7mm) than ethyl acetate (Figure 4).

From the above data on comparing both sample ‘ACU Neem’ and sample ‘UOM Neem', Sample ACU neem showed better antimicrobial activity against E. coli, UOM Neem showed better antimicrobial activity against Klebsiella spp. For S.aureus both showed almost similar results, but for MRSA sample UOM neem showed slightly better results than ACU Neem. For Candida albicans all extracts showed almost similar results. From this it is evident that site of sample collection also has significant impact on the antimicrobial activity of the extracts. The altitude, cultivar fertility, soil conditions and climatic conditions from where plant sample is collected all have impact on the antimicrobial activity. ¹³

Ethanolic extract showed better results against Klebsiella spp and S.aureus, Ethyl acetate extract showed better results against E.coli and MRSA, whereas both ethanolic and Petroleum ether extract showed activity against C.albicans. 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay showed that all the extracts have antioxidant property and variations were noted in the antimicrobial activity on comparing samples taken from different locations. The antimicrobial property of extracts of different solvents showed variations in their antimicrobial activity against different organisms.

The results obtained from minimum inhibitory concentration testing (Table 2) showed that Ethanolic extract of the leaves had more activity against E. coli (16 and 32µg) than the other two solvent extracts, however all the extracts showed equal activity against Klebsiella spp (32µg). For S.aureus ethyl acetate extract gave the best result (16µg) followed by ethanolic extract (32µg). Whereas for MRSA best results were seen with Ethyl acetate (16 and 32µg) followed by ethanolic extract (32µg). Hence MIC of the leaf extracts depends on the solvent used for extraction and also showed varied antimicrobial property against different organisms.

Along with the above-mentioned extracts other solvent extracts like methanol, aqueous extracts can also be tried and has shown significant results in the past experiments. More microbes can also be included to widen the array and obtain more data. Other factors like temperature and pH changes can be included to find the range where the antimicrobial activity is optimum. Since the extracts were crude and methodological choices were constrained by need for higher technology, the actual results gone using better technology is expected to have better and specific antimicrobial activity. To make things more sophisticated further studies can be conducted to isolate different components of the neem extract and test their actions and mechanisms at molecular level on a higher scale.

The limitation of the present study is that since MDR strains were not used in the present study activity against drug resistance cannot be concluded. Further to improvise the study multiple samples can be tested in future with the same solvent extracts to obtain repeatability. Studies are needed into the bioactive compounds of neem in order to explore its antimicrobial activity. Hence Azadirachta indica can be used treatment of commonly encountered organsims in the hospital because of its antimicrobial and anti-oxidant property. Further studies are required to confirm its antimicrobial properties against multidrug resistant organisms. 14

I acknowledge Dr. M G Shivaramu, principal and Dr. Venkatesha D Head of Dpt of Microbiology, Adichunchanagiri Institute of Medical Sciences, B G Nagara. I express my sincere thanks to Dr. Sudhanva KV whose support and technical guideline helped the present work as its show. Thanks Adichunchanagiri Institute for Molecular Medicine (AIMM) for providing material and equipments