PHARMA SCIENCE MONITOR

Transcription

1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES ANTIBACTERIAL ACTIVITY OF EUGENIA JAMBOLANA PLANT LEAVES EXTRACT Jeethu Anu Mathews, Karthikeyan.M and A.Annamalai* Department of Biotechnology, School of Biotechnology and Health Sciences, Karunya University, Coimbatore , Tamil Nadu, India. ABSTRACT Plants have provided mankind with herbal remedies for many diseases for many centuries and even today. A major part of the total population in developing countries still uses traditional folk medicine obtained from plant resources. They continue to play a major role in primary healthcare as therapeutic remedies in developing countries. Eugenia jambolana commonly known as a Jamun have promising therapeutic value with its various phytochemical constituents such as tannins, alkaloids, steroids, flavonoids, terpenoids, fatty acids, phenols, minerals, carbohydrates and vitamins. The present study was done to determine the antibacterial activity of Eugenia jambolana leaves extract against clinical isolates of some bacteria using well diffusion method. The extracts showed inhibitory activity against clinical isolates of gram negative bacteria such as Salmonella typhi, Shigella dysentriae, Klebsiella pneumonia, Pseudomonas, Escherichia coli and gram positive bacteria such as Bacillus subtilis and Staphylococcus aureus. The results showed that the acetone extracts was more effective than the other solvent extracts. The acetone extracts of Eugenia jambolana showed best activity against Salmonella typhi with a minimum inhibitory concentration (MIC) of 10 mg/ml. Keywords: Eugenia jambolana, antibacterial activity, MIC. INTRODUCTION India is one of the nations blessed with a rich heritage of traditional medical systems and rich biodiversity to complement the herbal needs of the treatment administered by these traditional medical systems. The recognized Indian Systems of Medicine are Ayurveda, Siddha and Unani, which use herbs and minerals in the formulations. India, which has 15 agro-climatic zones, plant species of which are reported to have medicinal properties varying degrees. The World Health Organization (1980) has also recommended the evaluation of the effectiveness of plants in conditions where there is lack of safe synthetic drugs. [1] During the last ten years pace of development of new antimicrobial drugs has slowed down while the prevalence of resistance has increased astronomically. [2] The IC Value

2 problem of microbial resistance of growing and the outlook for the use of antimicrobial drugs in the future is still uncertain. Therefore, actions must be taken to reduce this problem, such as controlling the use of antibiotics and carrying out research for the better understanding of the genetic mechanism of resistance. This prompted us to evaluate plants as the source of potential chemotherapeutic agent, antimicrobial agent and their ethno medicinal use. [3] Syzygium cumini (L.) synonyms such as Syzygium cumini (L.) Druce, Eugenia jambolana Lam.,Syzygium jambolanum DC. [4] belonging to the family Myrtaceae, is a large evergreen tree up to 30 m height and a girth of 3.6 m with a bole upto 15 m found through India upto an altitude of 1,800 m [5]. Most of the plant parts of E. jambolana are used in traditional system of medicine in India. Entire plant of Syzygium cumini such as seed, fruit, leaves, flower, bark used in folk medicine. Charaka used seeds, leaves and fruits in decoctions for diarrhoea and the bark as an astringent. Sushruta prescribed the fruit internally in obesity, in vaginal discharges and menstrual disorders, cold infusion in intrinsic haemorrhage. [6] The bark is astringent, its juice is given ( ml) doses in chronic diarrhoea, dysentery, menorrhagia. Decoction of the bark is an efficacious mouth-wash and gargle for treating spongy gums, stomatitis, relaxed throat and other diseases of mouth. Bark also used for inflammation of skin. [7] The bark is used in dyeing and tanning and for colouring fishnets. According to Ayurveda, its bark is acrid, sweet, digestive, astringent to the bowels, anthelmintic and in good for sore throat, bronchitis, asthma, thirst, biliousness, dysentery, blood impurities and to cure ulcers. [8] The juice of Jambu, Amra and Amalaka leaves mixed with goat milk and honey prescribed in diarrhoea with blood. [6] Leaf juice is taken orally to treat diabetes. The juice is taken mixed with milk every morning. Fresh leaf juice is taken orally for stomach pain. [9] A syrup prepared from the juice of the ripe fruit is a very pleasant drink. Syrup or vinegar prepared from the ripe fruit is useful in spleen enlargement and efficient astringent in chronic diarrhea. Hot water extract of dried fruits is used for stomach ulcers, [10] reduce acidity and for diabetes.[11] Eugenia jambolana has pharmacognosy, phytochemistry, pharmacology and clinical uses. Phytochemical extracts from Eugenia jambolana have been known for their IC Value

3 antiviral, antibacterial, antifungal, anti- HIV, anti-tumor, antioxidant, anti inflammatory activities. They are used in the management of diabetes, to strengthen teeth and gums, to treat leucorrhoea, stomachalgia, fever, gastropathy, strangury, dermopathy, constipation and to inhibit blood discharges in the faeces. Having known the bioactive potency of Eugenia jambolana this study was done to explore the antibacterial activity leaves of Eugenia jambolana. MATERIALS AND METHODS Collection of Plant Material Fresh leaves of Eugenia jambolana free from disease were collected from Karunya University campus. The leaves were washed thoroughly two to three times with running water and then dried under shade and grinded into powder form. Solvent Extraction Thoroughly washed fresh leaves of the plant material were dried in shade and powdered with the help of a grinder. 25 grams of shade dried powder was filled in the thimble and extracted successively with 250 ml of solvents such as water, methanol, acetone, ethyl acetate and benzene using soxhlet apparatus for hours at their respective boiling points such as 100, 65, 56, 72 and 80ºC. The extracts were then vacuum dried using rotary vacuum evaporator to evaporate the solvents and to yield a semisolid mass whose mass was then determined. The extracts were then stored in a refrigerator at 4ºC until further use. Well Preparation The prepared extract was weighed and mixed with known volume of water, methanol, acetone, ethyl acetate and benzene. Wells were punched on the petri plate and the extract of different concentrations such as 25mg/ml, 50mg/ml, 75mg/ml and 100mg/ml were added. Chloramphenicol (30mg/disc) was used as positive control against all the microorganisms except Pseudomonas aeruignosa it showed a range of zone of inhibition from mm. And Imipenum was used against Pseudomonas aeruignosa which gave a zone of inhibition of 26 mm. The respective solvents are used as negative control. Microorganisms IC Value

4 The gram negative bacteria such as Salmonella typhi, Pseudomonas Escherichia coli, Klebsiella pneumoniae and Shigella dysentriae and gram positive bacteria such as Bacillus subtilis, and Staphylococcus aureus were obtained from Microbiology lab of KMCH, Coimbatore. All the test strains were maintained on nutrient agar slopes (Hi-Media) and were sub cultured once in every two-week. These bacteria served as test pathogens for antibacterial activity assay. Antibacterial Test Preparation of Culture Medium and Inoculation The petri plates and Muller Hinton Agar medium were sterilized for 20 minutes at 120ºC. The rest of the procedure was carried out in laminar air flow. Approximately 20ml of the media was poured into the sterile petri plates and allowed to get solidified. After the media gets solidified the bacterial organisms were swabbed on the medium using sterile cotton swabs. [12] AGAR WELL DIFFUSION METHOD Microorganisms were inoculated over the dried surface of Muller Hinton Agar plate by spreading with the swab over the entire surface of the sterile agar plate. This procedure was repeated two or more times and the plates were rotated at 60º each time to ensure an even distribution of inoculums. Appropriate wells were punched evenly (not closer than 24mm from centre to centre) on the surface of the agar plate. The extracts of different concentrations were added into the wells carefully using micropipettes. The plates were then incubated at 35ºC within 15 mins after the extracts were added. The plates were incubated aerobically. After 16 to 18 hours of incubation each plate was tested for activity by checking the zone of inhibition. Minimum Inhibitory Concentration (MIC) of Leaves Extracts The MIC method was applied on extracts that proved their high efficacy against microorganisms by the disc diffusion method. The MIC of the plant extracts was determined on Muller Hinton Agar medium by means of well diffusion method. A lawn of culture was made on Muller Hinton Agar plates and to the wells different concentrations of plant extracts such as 5, 10, 15 and 20 mg/ml were added. The plates were then incubated at 37ºC for 24 hrs. The lowest concentration inhibiting the visible growth of the organism was considered as MIC. IC Value

5 RESULTS AND DISCUSSION In the present study, the antibacterial activities were performed with water, methanol, acetone, ethyl acetate and benzene extracts of the leaf of Eugenia jambolana. The study was made against five gram negative pathogenic bacteria and two gram positive bacteria using the well diffusion method. The aqueous, methanolic, acetonic, and ethyl acetate extracts showed activity against human pathogens namely Salmonella typhi, Shigella dysentriae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas, Bacillus subtilis and Staphylococcus aureus. The activity of Syzygium cumini against human pathogens was in all extracts except for benzene which showed negligible no sensitivity against human pathogens (Table 1). TABLE 1: ANTIBACTERIAL ACTIVITY OF VARIOUS SOLVENT EXTRACTS OF E. JAMBOLANA Sl.No. Microorganisms Water Methanol Acetone Ethyl acetate Benzene 1. Salmonella _ paratyphi 2. Shigella dysentriae _ 3. Escherichia coli _ 4. Klebsiella _ pneumoniae 5. Pseudomonas _ 6. Bacillus subtilis _ 7. Staphylococcus aureus _ The zones of inhibitions were produced by water, methanol, acetone and ethyl acetate extract except benzene against all the test organisms. Acetone extracts were more active than the other extracts against all the microorganisms. The zones of inhibition were ranging from 5-25mm in diameter. The highest zone of inhibitions (25mm) noted in acetone extract against some of the selected typhoid causing microorganism such as Salmonella typhi in 100mg/ml concentration (Table 2). The extracts of higher plants can be very good source of antibiotics against various bacterial pathogens.[13] Plant having antimicrobial compounds have enormous therapeutic potential as they can act without any side effect as often found with synthetic antimicrobial products. IC Value

6 TABLE 2: ANTIBACTERIAL ACTIVITY OF DIFFERENT SOLVENT EXTRACTS USING WELL DIFFUSION METHOD Solvent Microorganisms Zone of inhibition Control mg/ml 25 mg/ml 50 mg/ml 75 mg/ml 100 mg/ml (mm) (mm) (mm) (mm) (mm) Salmonella typhi Shigella dysentriae Escherichia coli Klebsiella pneumonia Water Pseudomonas Bacillus subtilis Staphylococcus aureus Methanol Acetone Ethyl acetate Salmonella typhi Shigella dysentriae Escherichia coli Klebsiella pneumonia Pseudomonas Bacillus subtilis Staphylococcus aureus Salmonella typhi Shigella dysentriae Escherichia coli Klebsiella pneumonia Pseudomonas Bacillus subtilis Staphylococcus aureus Salmonella typhi Shigella dysentriae Escherichia coli Klebsiella pneumonia Pseudomonas Bacillus subtilis Staphylococcus aureus The MIC values confirmed the significant activity against the tested microorganisms (Table 3). The MIC values of Syzygium cumini ranged from 5 to 25 mg/ml for acetone extracts. The MIC value were found to be 10 mg/ml against IC Value

7 Salmonella typhi, 20mg/ml against Shigella dysentriae, Klebsiella pneumonia, E.coli and Staphylococcus auerus 25 mg/ml against Bacillus subtilis and Pseudomonas. TABLE 3: MINIMAL INHIBITORY CONCENTRATION OF LEAVES EXTRACTS WITH ANTIBACTERIAL ACTIVITY (MG/ ML) Microorganisms Acetone Salmonella typhi 10 Shigella dysentriae 20 Escherichia coli 20 Klebsiella 20 pneumonia Pseudomonas 25 Bacillus subtilis 25 Staphylococcus 20 aureus Most antimicrobial medicinal plant are more effective against gram-positive than gram-negative bacteria. [14,15] However, the current findings show a remarkable activity against gram-negative bacteria including multi-resistant gram-negative strains. The antimicrobial activity of the Eugenia jambolana leaves extracts may be due to tannins and other phenolic constituents. Eugenia jambolana is known to be very rich in gallic and ellagic acid polyphenol derivatives.[16,17] Also, acylated flavonol glycosides, kaempferol, myricetin and other polyphenols were isolated from Eugenia jambolana leaves. [18,19] Tannins are considered nutritionally undesirable because they precipitate proteins, inhibit digestive enzyme and affect the absorption of vitamins and minerals. However, some kinds of tannins can reduce the mutagenicity of a number of mutagens and display anticarcinogenic, antimicrobial and antioxidant activities. [17] The result obtained in this study suggests a potential application of Eugenia jambolana leaves for treatment of skin wounds, typhoid and further investigations should be conducted in order to explore their applications. Other medicinal plants containing phenolic compounds, including tannins, as major constituents are used topically for care and repair of skin wounds. [18] The advantage of the use of topical antimicrobials is their ability to deliver high local concentrations of antibiotic irrespective of vascular supply. IC Value

8 Further benefits include the absence of adverse systemic effects, and a low incidence of resistance. [19] Antibiotic-resistant bacteria continue to emerge rapidly, constituting a problem of increasing significance in dermatology. Common pathogenic bacteria, such as Staphylococcus aureus and Pseudomonas which are predominant organisms in both leg ulcers and superficial wounds, showed increased resistance to commonly used antibiotics. The potential of Eugenia jambolana leaves hydroalcoholic extract against multi-resistant and standard strains of Staphylococcus aureus and Pseudomonas may be explored in order to develop a topic antimicrobial therapy to promote skin wounds healing. CONCLUSION Eugenia jambolana, the versatile traditional medicinal plant of India, is the rich source of bioactive compounds with diverse chemical structure. As of now, little work has been done on the biological activity and plausible medicinal applications and hence extensive information is needed to exploit the bioactive principles of Eugenia jambolana for therapeutic utility. In the present study antibacterial activity of Eugenia jambolana extracts towards drug resistant/ clinically significant microbes has been investigated. Further investigation may lead to the discovery of new drugs of high potency. ACKNOWLEDMENTS The authors convey their thanks to the HOD and the Director, School of Biotechnology and Health Sciences, Karunya University, Coimbatore for providing lab facilities. REFERENCES 1. Upadhayay V P and Pandey K: In Ayurvedic approach to diabetes mellitus and its management by indigenous resources. Diabetes Mellitus in Developing Countries, Interprint, New Delhi, 1984; Akinpelu DA and Onakoya ZTM: Antimicrobial activities of medicinal plants used in folklore remedies in South-Western. Afr. J. Biotechnology 2006;. 5: IC Value

9 3. Prashanth KN, Neelam S, Chauhan S, Harishpadhi B, and Ranjani M: Search for antibacterial and antifungal agents from selected Indian medicinal plants. Journal of Ethanopharmacology 2006; 107: Duke James A. Handbook of Medicinal Herbs, 2nd edition, CRC Press, Londan, 2006; The Ayurvedic Pharmacopoeia of India, Part-I, 1st edition, Vol-II, The controller of publications, Delhi, 1999; Khare CP. Encyclopedia of Indian Medicinal plants. Springer-Verlag Berlin Heidelberg, New York, 2004; PDR for Herbal Medicines, 2nd edition, Thomson, Medical Economics, Montvale, 2000; Kirtikar KR and Basu BD. Indian Medicinal Plants, Vol-II, Periodical Experts, New Delhi, 1975; Bhandary MJ, Chandrashekar KR and Kaveriappa K:. Medical ethnobotany of the siddis of Uttar Kunnada district, Karnataka, India. Journal of Ethnopharmacology 1995; 47( 3): Deka L, Majumdar R and Dutta AM; Some Ayurvedic important plants from District Kamrup ( Assam ), Ancient Sci Life 1983;. 3(2): Jain SR and Sharma SN: Hypoglycemic drugs of Indian indigenous origin. Planta Medica 1967; 15 (4 ): S. Shyamala Gowri and Vasantha K: Phytochemical Screening and Antibacterial Activityof Syzygium cumini (L.) (Myrtaceae) Leaves Extracts. International Journal of PharmTech Research 2010; 2 (2): Fridous AJ, Islam SNLM and Faruque ABM: Antimicrobial activity of the leaves of Adhatoda vasica, Calatropis gigantium, Nerium odoratum and Ocimum sanctum. Bangladesh J. Bot 1990; Lin J, Opoku AR, Geheeb-Keller M, Hutchings AD, Terblanche SE, Jagar AK and Van Staden J: Preliminary screening of some traditional zulu medicinal plants for anti inflammatory and antimicrobial activities. Journal of Ethanopharmacology 1990; 68: IC Value

10 15. Scrinivasan D, Nathan S, Suresh T and Perumalsamy O: Antimicrobial activity of certain Indian medicinal plants used in folkloric medicine. Journal of Ethanopharmacology 1989; Chattopadhyay D. Sinha BK and Vaid L.K: Antibacterial activity of Syzygium species. Fitoterapia 1998; 69: Chung KT, Wei C and Johnson MG: Are tannins a double edged sword in biology and health. Trends food Science and Technology 1990;. 9: Mahmoud II, Marzouk MSA, Moharram FA, El- Gindi MR and Hassan AMK: Acylated flavonol glycosides from Eugenia jambolana leaves. Phytochemistry 1990; 58: Timbola AK, Szpoganicz B, Branco A, Monache FD and Pizzolatii MG: A new flavonol from leaves of Eugenia jambolana. Fitoterapia 2002; 73: Shay J P, Komorov S A, Fells S S, Meranze D, Grunstein M & Simpler H, A simple method for For Correspondence: Dr. A. the Annamalai uniform production of gastric ulceration in the rat, Gastroenterology, 5 (1945) aannamalai2001@yahoo.com IC Value