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Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(6),1019-1021 Available online through www.phresearchjournal.info
Antimicrobial Potentialities of Mangrove Plant Avicennia marina
Varaprasad Bobbarala*, Varahala Rao Vadlapudi ** and K. Chandrasekhar Naidu** *For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam, A. P-530017, India. ** Department of Botany, Andhra University, Visakhapatnam (A.P.) Received on: 22-02-2009; Accepted on:26-03-2009 ABSTRACT In this present study Antimicrobial activity of aerial parts of Avicennia marina were evaluated against the pathogens belong to aquatic, human and plant origin, Soxhlet extraction method used to get the corresponding extracts of hexane, chloroform and methanol. The antimicrobial activities of the organic solvent extracts on the various test microorganisms, including bacteria and fungi investigated using agar well diffusion technique. The length of inhibition zone was measured in millimeters from the edge of the well to the edge of the inhibition zone. Methanol and chloroform extracts exhibited promising antimicrobial activity than hexane extracts. Among all microorganisms studiedErwinia caratovara and Streptococcus mutans showed the considerable growth inhibition with chloroform and methanolic extracts. Keywords: Phytopathogens, Antimicrobial activity, Folkloric medicine, Acanthaceae INTRODUCTION Natural products can be selected for biological screening based on ethno medical use of plants, because many infectious diseases are known to have been treated with herbal remedies throughout the history of mankind. Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world [1, 2, 3]. It has been reported that the higher plants have shown to be a potential source for the new antimicrobial agents . Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries [5, 6]. Studies on antibacterial and antifungal activity of the plants and their prospects for use in different systems require scientific experimentation. In recent years, drug resistance to pathogenic microorganisms has been commonly and widely reported in literature [7, 8] therefore antimicrobials may have a significant clinical value in treatment of resistant microbial strains . Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, many scientists have recently paid attention to extracts and biologically active compounds isolated from plant species used in herbal medicines . Mangrove and mangrove associates contain biologically active antiviral, antibacterial and antifungal compounds . They provide a rich source of steroids, triterpenes, saponins, flavonoids, alkaloids and tannins. Therefore, it is worth to screen mangrove plants for the presence of new antibacterial compounds to combat the pathogenic bacteria and fungi. A. marina (Forssk.) is commonly known as gray mangrove tree classified in the plant family Avicenniaceae, and is commonly used for ulcers. So the main objectives of this study was to screen antimicrobial activity of the organic solvent extracts of A. marina mangrove plant species against pathogenic and antibiotic *Corresponding author. Tel.: +91-9949129539 E-mail: firstname.lastname@example.org resistant bacterial strains and also to isolate and characterization of chemical components which are responsible for the claimed activity. MATERIALS AND METHODS Plant material and extraction: A. marina is commonly known as gray mangrove and vernacular name is Tella mada belongs to the family Aviceniaceae, grow as a shrub or tree to a height of three to ten metres, or up to 14 meters in tropical regions, growing in the saline intertidal zones of sheltered coast lines. It has been reported to tolerate extreme weather conditions, high winds. The material was taxonomically identified and the Voucher specimen is stored. The aerial plant parts were collected from Coringa Mangrove Wetland, Andhra Pradesh, India. The plant material were dried under shade with occasional shifting and then powdered with a mechanical grinder and stored in an airtight container. The powder obtained was subjected to successive soxhlet extraction with organic solvents with increasing order of polarity i.e. Hexane, Chloroform and Methanol respectively. Test microorganisms: The antibacterial activity of the extracts was assessed against microbial strains of clinical, plant and aquatic origin i.e. Aeromonas hydraophylla (MTCC 646), Alternaria alternate (MTCC 1362), Ustilago maydis (MTCC 1474), Asperigellus niger (MTCC 2723), Acremonium strictum (MTCC 3072), Pencillium expansum (MTCC 2006), Fusarium oxysporum (MTCC 1755), Xanthomonas compestries (MTCC 2286), Erwina caratovara (MTCC 3609), Lactobacillus acidophilus (MTCC 447), Pseudomonas marginalis (MTCC), Pseudomonas syringae (MTCC 1604), Pseudomonas aeruginosa (MTCC 1688), Streptococcus mutans (MTCC 890), Steptococcus salivarious (MTCC 1938) and Staphylococcus aureus (MTCC 96) including both fungi and bacteria were procured from Microbial Type Culture Collection (MTCC), Chandigarh. Active cultures were generated by inoculating a loopful of culture in separate 100mL nutrient/potato dextrose broths and incubating on a shaker at 37oC overnight. The cells were har-
Journal of Pharmacy Research Vol.2.Issue 6.June 2009
Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(6),1019-1021 vested by centrifuging at 4000 rpm for 5 min, washed with normal saline, spun at 4000 rpm for 5 min again and diluted in normal saline to obtain 5 x 105cfu/mL. Determination of antimicrobial activity: The crude extracts of the different plant parts of different species were subjected to antimicrobial assay using the agar well diffusion method of Murray et al.,  modified by Olurinola . 20 ml of nutrient agar was dispensed into sterile universal bottles these were then inoculated with 0.2 ml of cultures mixed gently and poured into sterile petri dishes. After setting a number 3-cup borer (6mm) diameter was properly sterilized by flaming and used to make three to five uniform cups/wells in each petri dish. A drop of molten nutrient agar was used to seal the base of each cup. The cups/wells were filled with 50µl of the extract concentration of 500mg/ml, 250mg/ml and 100mg/ ml and allow diffusing for 45 minutes. The solvents used for reconstituting the extracts were similarly analyzed. The plates were incubated at 37°c for 24 hours for bacteria. The above procedure is allowed for fungal assays but except the media potato dextrose agar instead of nutrient agar and incubates at 25°c for 48 hours. The zones of inhibition were measured with antibiotic zone scale in mm and the experiment was carried out in duplicates. The extracts and the phytochemicals that showed antimicrobial activity were later tested to determine the Minimal Inhibitory Concentration (MIC) for each bacterial and fungal sample Minimum inhibitory concentration (MIC) assays: Based on the preliminary screening chloroform and methanolic extracts were found to have potent antimicrobial activity and Minimum Inhibitory Concentrations (MIC) of the extracts was determined according to Elizabeth, . A final concentration of 0.5% (v/v) Tween-20 (Sigma) was used to enhance crude extract solubility. A series of two fold dilution of each extract, ranging from 0.2 to 100 mg/ml, was prepared. After sterilization, the medium was inoculated with 3ìl aliquots of culture containing approximately 105 CFU/ml of each organism of 24 hours slant culture in aseptic condition and transferred into sterile 6 inch diameter petri dishes and allowed to set at room temperature for about 10 minutes and then kept in a refrigerator for 30 minutes. After the media solidified a number 3-cup borer (6mm) diameter was properly sterilized by flaming and used to make three to five uniform cups/wells in each petri dish. A drop of molten nutrient agar was used to seal the base of each cup. Different plant crude extracts ranging from 0.2 to 100 mg/ml were added to the cups/wells of each petri dish and the control plates without plant extract. Inhibition of organism growth in the plates containing test crude extracts was judged by comparison with growth in blank control plates. The MICs were determined as the lowest concentration of extracts inhibiting visible growth of each organism on the agar plate. Similarly the MICs of methanolic extracts were determined against all other microorganisms. The results were given in Table-1. RESULTS In the present study, chloroform and methanol extract exhibited different degree of growth inhibition against tested bacterial and fungal strains (Table 1). According to Table 1, methanolic extracts of A. marina exhibited considerable antimicrobial activity against tested microbial strains. Methanolic extracts of A. marina showed more inhibition than chloroform extracts of A. marina against S. mutans, E. caratovara, P. aeruginosa and L. acidophilus (Table 1). Whereas chloroform extracts of A. marina exhibited more pronounced inhibition against X. compestris and E. caratovara (Table 1). Among the all pathogens tested S. aureus was the most resistant bacteria and E. caratovara was the most sensitive strain. No inhibition of the growth of pathogens tested was observed in extracts in hexane extract of A. marina. The degree of antimicrobial activity of extracts decreased with time for all tested microbial strains. According to phytochemical screening, mature leaves of A. marina contained alkaloids, steroids and flavonoids. Table 1: MIC assays of chloroform and methanol extracts Avicennia marina
Plant Name Chloroform 100 50 Aeromonas hydraophylla 14 13 Alternaria alternate 11 10 Asperigellus niger 10 10 Acremonium strictum 12 Erwinia caratovara 15 13 Fusarium oxysporum 13 10 Lactobacillus acidophilus 12 12 Pencillium expansum 10 Pseudomonas marginalis 12 13 Pseudomonas syringae 14 13 Pseudomona aeruginosa 11 12 Steptococcus mutans 13 Steptococcus salivarious 12 Stephylococcus aureus 10 Ustilago maydis 12 10 Xanthomonas compestries 15 25 12 8 10 10 Methanol 100 50 19 15 15 10 15 16 14 25 18 17 15 21 20 14 17 17 18 17 23 21 25 21 20 13 14 18 20 14 25 13 10 10 16 10 18 21 19 16 9
Volume per well: 50µl, Borer size used: 6mm, Drug concentration in mg/ ml, Zone of inhibition in mm DISCUSSION The results of the present study clearly showed that mangrove plant A. marina extracts showed antimicrobial activity against tested pathogenic strains including antibiotic resistant strains. The effectiveness of the active compounds present in plant extracts cause the production of growth inhibition zones that appear as clear areas surrounding the wells. Antibacterial activity may be due to active components which are present in plant extracts. However, some plant extracts were unable to exhibit antibacterial activity against tested bacterial strains. These bacterial strains may have some kind of resistance mechanisms e.g. enzymatic inactivation, target sites modification and decrease intracellular drug accumulation  or the concentration of the compound used may not be sufficient. No inhibition was observed with controls, which proves that solvents could not act as antibacterial agents. In almost all tests, crude methanolic extracts showed better inhibition against all tested bacterial strains, indicating that active ingredients in plant materials could be extracted into methanol. However, highest antibacterial activity was observed against Erwinia caratovara . The results obtained from preliminary phytochemical screening are comparable with the results reported earlier by Abeysinghe et al.,  according to the studies the presence of secondary metabolites such as alkaloids, flavonoids and steroids may exert antibacterial activity against tested bacterial strains. Further research is necessary for successful separation, purification and characterization of biologically active compounds using chromatographic methods and spectroscopic techniques. Further studies are being carried out in order to separate the individual components that are present in plant extracts of A. marina using column chromatography.
Journal of Pharmacy Research Vol.2.Issue 6.June 2009
Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(6),1019-1021 REFERENCES
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Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.2.Issue 6.June 2009