BACTERICIDAL EFFECTS OF CRUDE NEEM LEAF (AZADIRACHTA INDICA) EXTRACT ON PATHOGENIC BACTERIA: AN INVITRO STUDY 1*Vaibhavi Subhedar, 1ARohit Subhedar, 2Sudhir Kumar Jain 1 Department of Microbiology and 1APhysiotherapy Bombay Hospital Indore (MP) -India 2 School of Studies in Microbiology -Vikram University Ujjain (MP) 456010 India. Corresponding Author: E.mail Vaibhavisubhedar14@rediffmail.com

ARE WE ENTERING POST-ANTIBIOTIC ERA??!!! ANTIBIOTIC RESISTANCE”: IF NO ACTION TODAY NO CURE TOMORROW. WHO AND CDC HAVE WARNED AGAINST POST-ANTIBIOTIC ERA IN APRIL AND JULY 2014 RESPECTIVELY. HARDLY ANY NEW DRUG DISCOVERY IN PIPELINE WITH RESISTANCE INCREASING TO THE EXISTING DRUGS DAY BY DAY. EVERYDAY WE DELAY IT BECOMES HARDER AND MORE EXPENSIVE TO FIX THIS PROBLEM.

IS TRADITIONAL MEDICINE OF INDIA A BETTER OPTION!!!!!!!!!! DRUG DISCOVERY IS NOT A FAST ENOUGH PROCESS TO OVERCOME THIS CRUNCH WHAT SHOULD BE DONE??????? IS TRADITIONAL MEDICINE OF INDIA A BETTER OPTION!!!!!!!!!! YES………..

IT MAKES IMMENE SENSE TO EXPLORE THE EMPRICAL WISDOM OF ANCIENTS WITH MODERN RESEARCH TECHNOLOGY. ANCIENTS HAVE PRACTICED HERBAL MEDICINE THROUGH THOUSANDS OF YEARS AND EVEN ATTEMPTED SURGERIES. INDIAN MEDICINAL PLANTS ,NEXT ALTERNATIVE FOR TREATMENT OF INFECTIONS!!!! RESONABLE TO TURN TO OUT OWN NATURAL RESOURCES ,SINCE THE APPROVAL OF TRADITIONAL ALTERNATIVE MEDICINE ACT IN 1997. GLOBAL SCENARIO IS NOW CHANGING TOWARDS THE USE OF NON TOXIC PLANT PRODUCTS ACCORDING TO WHO MORE THAN80% OF WORLDS POPULATION RELIES ON TRADITIONAL MEDICINES FOR THEIR PRIMARY HEALTH CARE NEEDS WITH THERAPEUTIC BENEFITS AND RELATIVELY CHEAPER AND SAFER.

ADVANTAGES OF MEDICINAL PLANTS NO OF INDIAN MEDICINAL PLANTS HAVE PROVEN ANTIMICROBIAL ACTIVITY. E.G NEEM, TURMERIC GINGER,GARLIC ETC. TRADITIONAL MEDICINES ARE: ECONOMICAL EASILY AVAILABLE LESS LIKELY TO FORM ALLERGIES LESS TOXIC AND LESSER SIDEEFFECTS LESS RESISTANCE

Indian Name :Neem Tree Scientific Name :Azadirachta Indica In India, Neem is known as “the village pharmacy” because of its healing versatility, and it has been used in Ayurvedic medicine for more than 4,000 years due to its medicinal properties. The seeds, bark and leaves contain compounds with proven antiseptic, antiviral, antipyretic, anti-inflammatory, anti-ulcer and antifungal uses.

WHY NEEM LEAF EXTRACT WAS USED EASILY AVAILABLE. ECONOMICAL PROVEN ANTIMICROBIAL AGENT UPCOMING RESEARCHES ON ITS USE AGAINST PATHOGENIC BACTERIA IF PROVED CAN BE A BEST ALTERNATIVE BOTH AS A PREVENTIVE ANTIBIOTIC AGAINST PATHOGENIC BACTERIA AS WELL AS IN PREVENTING THE TRANSMISSION OF THESE BACTERIA. CRUDE NEEM LEAF EXTRACT CAN BE EASILY EXTRACTED AS WELL AS AVAILABLE COMMERCIALLY. LESS TOXIC AND BACTERIAL RESISTANCE AGAINST IT LESS LIKELY.

NEEM HAS BEEN EXTENSIVELY USED IN AYURVEDA, UNANI AND HOMEOPATHIC MEDICINES AND HAS BEEN A CYNOSURE OF COMPOUNDS. -ALL THE PARTS OF TREE SUCH AS LEAVES, FLOWERS, SEEDS, FRUITS, BARK AND ROOTS ARE USED FOR TREATMENT OF VARIOUS DISEASES. -PRESENT STUDY WAS CARRIED OUT USING CRUDE NEEM LEAF EXTRACT FOR ITS ANTI BACTERIAL EFFECT ON VARIOUS PATHOGENIC BACTERIA FROM CLINICAL SPECIMENS. -SUCH STUDY CAN FURNISH INFORMATION ON THE USE OF MEDICINAL PLANTS.

NEEM RESEARCH A NO OF STUDIES HAVE PROVED ANTIMICROIAL ACTVITY OF NEEM LEAF EXTRACT AGAINST HUMAN PATHOGENIC BACTERIA. NEEM LEAVES HAS ANTIBACTERIAL PROPERTIES AND COULD BE USED FOR CONTROLLING AIRBORNE BACTERIAL CONTAMINATION IN THE RESIDENTIAL PREMISES (Saseed A et.al.,2008,AMEI.Mahmood et al 2010) AQUEOUS EXTRACT OF NEEM LEAVES AS POWERFUL CHEMOTHERAPEUTIC AND ANTIVIRAL AGENTS (Hassan Amer et.al 2010) A NO OF STUDIES ARE STILL IN PIPELINE TO PROVE THE ANTIMICROBIAL EFFECT OF NEEM LEAVES.

AIMS AND OBJECTIVES: THE PURPOSE OF THE PRESENT STUDY WAS TO INVESTIGATE THE POTENTIAL ROLE OF CRUDE NEEM LEAF EXTRACT AS AN ANTIBACTERIAL SUBSTANCE. TO PROVE THE BACTERIOSTATIC AND BACTERICIDAL ACTIVITIES OF CRUDE NEEM LEAF EXTRACT AGAINST DIFFFERENT PATHOGENIC BACTERIA. TO PROVE THE SYNERGISTIC ACTIVITY OF NEEM LEAF EXTRACT WITH SELECTIVE ANTIBIOTICS. TO PROVE THE THE USE OF NEEM LEAF EXTRACT AS A SURFACE DISINFECTANT.

MATERIALS AND METHODS . # STUDY DESIGN: STUDY UTILIZED AN INVITRO EXPERIMENTAL STUDY WITH AQUEOUS AND ALCOHOLIC NEEM LEAF EXTRACT ALCOHOLIC EXTRACT WILL BE PREPARED AS PER THE STANDARD GUIDELINES(MACERATION METHOD). AQUEOUS EXTRACT WAS PREPARED BY THE STANDARD GUIDELINES(RAJA RATAN REDDY ET AL.,2013)

DATA COLLECTION STRATEGY-1 PRIMARY:- IDENTIFICATION AND SOURCING OF NEEM LEAVES AFTER VERIFICATION FROM EXPERTS. . SECONDARY:- ALCOHOLIC EXTRACT OF NEEM LEAVES WILL BE PREPARED AS PER THE STANDARD METHOD(MACERATION METHOD). . AQUEOUS EXTRACT WAS PREPARED BY STANDARD GUIDELINES (RATAN REDDY ET AL., 2013).

INCLUSION CRITERIA: 10 STRAINS EACH OF STAPHYLOCOCCUS AUREUS (METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS MRSA), KLEBSIELLA PNEUMONIAE, PSEUDOMONAS AERUGINOSA AND ESCHERICHIA.COLI WERE INCLUDED IN THE STUDY. ALL THE ABOVE MENTIONED PATHOGENIC BACTERIA ARE RESPONSIBLE FOR CAUSING SERIOUS HOSPITAL ACQUIRED INFECTIONS IN PATIENTS AND ARE VERY RESISTANT TO ANTIBIOTICS. STRAINS WERE OBTAINED FROM CLINICAL SPECIMENS RECEIVED IN THE DEPARTMENT OF MICROBIOLOGY BOMBAY HOSPITAL INDORE. EXCLUSION CRITERIA: NON PATHOGENIC BACTERIA WERE NOT INCLUDED IN THE STUDY.

MATERIALS AND METHODS: Crude aqueous and alcoholic neem leaf extract were prepared as per the standard guidelines. 1) Use as a surface disinfectant: A surface intentionally contaminated with 24 hr broth culture of pathogenic bacteria like Methicillin Resistant Staphylococcus aureus (MRSA), Pseudomonas sp, Klebsiella and E.coli was covered with neem leaf extract. And the efficacy of neem leaf extract was checked by culturing the swabs taken from the contaminated surface treated with neem leaf extract at different intervals of time with half hour intervals. 2) To study synergistic effect of neem leaf extract with different antibiotics: Nutrient agar plates with two different concentrations of neem leaf extract i.e 0.1mg/ml and 0.5mg/ml were prepared and the test organism was layered over it. Antibiotics effective for that particular test strain were placed and the results were noted for increased zone of inhibition to show synergism.

3) To determine the MIC of neem leaf extract for effective bactericidal activity: Serial dilutions of aqueous and alcoholic neem leaf extract were prepared as per the standard guidelines. 10µl broth culture of the test organism was added to each tube and incubated aerobically overnight and the minimum inhibitory concentration was determined by subculturing the stock tubes with test organism on nutrient agar to see visible growth. The concentration of neem leaf extract which did not show any growth was considered as the minimum inhibitory concentration. 4)To compare the efficacy of crude aqueous and crude alcoholic neem leaf extract with surface and hand disinfectants i.e sodium hypochlorite and chlorhexidine respectively: The nutrient agar plate was swabbed with test organism and 4 wells were cut with the help of sterile blade. The first well was filled with alcoholic neem leaf extract, 2nd with aqueous neem leaf extract, 3rd with chlorhexidine disinfectant and 4th with sodium hypochlorite solution. Plates were incubated overnight and the zone diameters were measured.

RESULTS: Pseudomonas aeruginosa was killed after approximately 90 minutes of exposure to crude neem leaf extract (Figure -1). while rest of the bacteria took 60 minutes. Present study also proved a good synergistic activity of neem leaf extract with 0.5g/ml concentration with aminoglycosides like gentamycin, amikacin and flouroquinolones like levofloxacin and ciprofloxacin which commonly prescribed drugs against all the test organisms. Also the neem leaf extract showed good synergism with chloramphenicol, ceftazidime and cefoparazone+sulbactum. Best synergistic activity was found against E. coli. (Table-1). This experiment proved that the minimum inhibitory concentration for mostly all the organisms ranged from 1:4 to 1:8 dilutions per ml (10-2 to 10-3 dilution). (Table-2)

4) This experiment proved that alcoholic neem leaf extract had a better efficacy as compared to aqueous neem leaf extract. Also the zone sizes were compared with chlorhexidine and sodium hypochloride solution, wherein the alcoholic neem leaf extract showed nearly equal zones of inhibition against Acinetobacter and MRSA.

Graph Graph-1 indicates the minimum time taken by different pathogenic bacterias to be killed by the crude neem leaf extract.   TABLE Table-1: Zone of inhibition with neem powder and antibiotic to show synergism for E. coli. Name of Antibiotic Standard Diameter of Inhibition zone (in mm) Neem extract + antibiotic 0.1mg/ml 0.5mg/ml Amikacin 15-16 20 17-18 Levofloxacin 14-16 22 23 Cefoparazone + sulbactum 15-19 20 23 chloramphenicol 18 21 23  

S = Methicillin-resistant Staphylococcus aureus (MRSA) TABLE-2: MIC of neem extract for MRSA strains.   Stock = original concentrated solution without dilution. Dilution S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Stock - 10-1 10-2 10-3 + 10-4 10-5 + = growth - =no growth S = Methicillin-resistant Staphylococcus aureus (MRSA)

CONCLUSION: Under the limitations of this study, it was concluded that Neem leaf extract has a significant antimicrobial effect against various pathogenic bacteria. Microbial inhibition potential of neem leaf extract observed in this study opens perspective for its use in treating various infections. There is possible potentiating of antibacterial effects of some antibiotics against different bacterial infection when co administered with neem extract. Extensive research and standardization of techniques and controlled predetermined combinations of neem extract and these antibiotics could find clinical applications in the treatment of bacterial infections and in prevention of resistance in bacterias. However, preclinical and clinical trials are needed to evaluate biocompatibility and safety before neem can be conclusively recommended as irrigating solution, but invitro observation of neem effectiveness appears promising. As the global scenario is now changing towards the use of non toxic plant products that have traditional medicinal use, extensive research and development work therefore should be undertaken on neem and its products for their better economic and therapeutic utilization.