ANTIMICROBIAL ACTIVITY OF CHLOROFORM AND METHANOL EXTRACT OF LENNEA COROMANDELICA Merr.

. The antimicrobial activity of chloroform and methanol extracts of Lennea coromandelica were screened for their was studied against gram positive bacteria strains Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis gram negative bacteria strains Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli and fungal strains such as Candida albicans, C. parapsilosis, C. krusei and C. glabrata using disc diffusion method, determination of Minimum Inhibitory Concentrations (MIC), Minimum Bacterial Concentrations (MBC) and Minimum Fungicidal Concentrations (MFC). The antimicrobial activity for different concentrations like 250 μg, 500 μg and 1000 μg of different solvent extracts of Lannea cormandelica. bacterial strains and recorded in highest mean zones of inhibition ranged from 19.6 mm and Candidal strains and the exhibited the highest mean zones of inhibition ranged from 10.6 mm. Methanol extracts showed the best results as inhibition zones against tested organisms. Results showed also that, the greatest effect was towards Staphylococcus aureus and the lowest was against Candida krusei . The present study reported the great effect of Lannea cormandelica extracts against some of most important pathogens.


INTRODUCTION
Medicinal plants are major source of great economic value in the Indian subcontinent.Nature has bestowed on us a very rich botanical wealth and a large number of diverse types of plants grow in different parts of the country.India is rich in all the three levels of biodiversity, namely species diversity, genetic diversity and habitat diversity.In India thousands of species are known to have medicinal value and the use of different parts of several medicinal plants to cure specific aliments has been in vogue since ancient times (Jigha and Sumitra 2006).A number of new antibiotics have been produced by pharmacological industries but the toxic effects and the global emergence of multi-drug resistant (MDR) of microbes is limiting the effectiveness of these drugs (Hancock, 2005).Medicinal plants have been used to treat human diseases for thousands of years because they have vast and diverse assortment of organic compounds that can produce a definite physiological action on the human body.Most important of such compounds are alkaloids, tannins, flavonoids, terpenoids, saponins and phenolic compounds.Pharmacists are interested in these compounds because of their therapeutic performance and low toxicity (Inayatullah et al., 2012).A number of such compounds have been isolated from plants which could be used for the development of new drugs to inhibit the growth of bacterial and fungal pathogens and to quench ROS with possibly novel mechanisms of action and low toxicity to the host cell (Ahmad and Aqil, 2007).
Lennea coromandelica Merr.Coming under Anacardiaceae family.It is a medium sized deciduous tree.Bark thick, ashy grey.Leaves crowded at the end of branches.Flowers small, greenish yellow in compact fascicles of racemes, at the end of the leafless branches.Drupes, reniform, produced in clusters from the end of leafless branches.The plant used in astringent and stomachic; used as a lotion in impetigenous eruptions, leprous and obstinate ulcers; cures sprains, bruises, skin eruptions, heart diseases, dysentery and mouth sores.In the present investigation, various solvent extracts viz.chloroform and methanol of leaf and bark Lannea coromadelica were studied for its antimicrobial activity against gram positive bacteria viz., Staphylococcus aureus,

Determination of the Minimum Bactericidal Concentration (MBC) and the Minimum Fungicidal Concentration (MFC)
MBC and MFC were determined by plating a loopfull of samples from each MIC assay well with growth inhibition in to freshly prepared MHA for bacteria and SDA for fungal strains.The plates were incubated at 37 °C for 24 h for all bacterial strains, 28 °C for 24-48 h for Candida species.The MBC and MFC were recorded as the lowest concentration of the crude extracts that did not permit any visible bacterial and fungal growth after the period of incubation.

RESULT
In The minimum inhibitory concentration and minimum fungicidal concentration were from 125 to 500 μg/mL and 250 to 1000 μg/mL respectively.The standard drug, Ciprofloxacin (10 μg/disc) was used for bacterial strains and recorded mean zones of inhibition ranged from 27.0 ± 0.5 to 31.5 ± 0.3 mm and Amphotericin B (100 units/disc) was utilized for Candidal strains and the exhibited the mean zones of inhibition ranged from 15.5 ± 0.8 to 17.8 ± 0.2 mm.
The results of antimicrobial activity of chloroform extract of leaves of Lannea coromandelica is presented in Table 1.The mean zone of inhibition for bacteria ranged between 9.0 ± 0.5 and 16.3 ± 0.2 mm on the other hand, for Candidal strains, the mean zone of inhibition values were from 7.2 ± 0.2 to 9.6 ± 0.5 mm.For bacteria, the highest mean zone of inhibition (16.3 ± 0.2 mm) was observed against Staphylococcus aureus.The lowest zone of inhibition (9.0 ± 0.5 mm) was recorded against E. coli.With regard to fungal strains tested, the highest zone of inhibition was recorded against Candida glabrata with a mean zone of inhibition of 9.6 ± 0.5 mm.The lowest zone of inhibition (7.2 ± 0.2 mm) was recorded against Candida krusei.
The antimicrobial activity of methonal extracts of leaves of Lennea coromandelica was carried out and the results is presented in Table 2.The mean zone of inhibition for bacteria ranged from 9.6 ± 0.7 to 19.6 ± 0.2 mm.On the other hands, for Candidal strains, the mean zone of inhibition values was from 7.1 ± 0.5 to 10.6 ± 0.5 mm.For bacteria, the highest mean zone of inhibition (19.6 ± 0.2 mm was recorded against Staphylococcus aureus.E. coli was less susceptible towards the extract in the mean zone of inhibition 9.6 ± 0.7 mm.With regard to fungal strains tested, the highest mean zone of inhibition (10.6 ± 0.5 mm) was observed against Candida glabrata and the lowest mean zone of inhibition (7.1 ± 0.5 mm) was recorded against C. krusei.

Table 1. Antimicrobial activity of chloroform extract of leaves of Lannea coromandelica
a-diameter of zone of inhibition (mm) including the disc diameter of 6 mm b-mean of three assays; ± standard deviation.a-diameter of zone of inhibition (mm) including the disc diameter of 6 mm b-mean of three assays; ± -standard deviation.
The antimicrobial activity of chloroform extract of bark of Lannea coromandelica against tested microbes was shown in Table 3.The results showed mean zone of inhibition for bacteria ranged were 10.0 ± 0.5 to 18.6 ± 0.5 mm.On the other hand, for Candidal strains, mean zone of inhibition were from 7.0 ± 0.5 to 10.3 ± 0.2 mm.For bacteria, the maximum zone of inhibition (18.6 ± 0.5 mm) was observed against Staphylococcus aureus.The minimum mean zone of inhibition (10.0 ± 0.5 mm) was recorded against K. pneumonia.In fungi, the maximum zone of inhibition (10.3 ± 0.2 mm) was observed against Candida glabrata while minimum inhibitory zone (7.0 ± 0.5 mm) was shown against C. albicans.
The antimicrobial activity of methonal extracts of bark of Lennea coromandelica was screened against bacterial and fungal stains and the results is presented in Table 4.The mean zone of inhibition for bacteria ranged between 10.9 ± 0.2 and 18.6 ± 0.2 mm.On the other hand, for inhibition values of fungi were from 7.1 ± 0.2 to 10.9 ± 0.3 mm.For bacteria, the highest mean zone of inhibition (18.6 ± 0.2 mm) was recorded against Staphylococcus aureus.The lowest International Letters of Natural Sciences Vol. 45 inhibitory activity was recorded against P. aeruginosa with mean zone of inhibition 10.9 ± 0.2 mm.With regard to fungal strains tested, the highest mean zone of inhibition (10.9 ± 0.3 mm) was observed against Candida glabrata and the lowest mean zone of inhibition (7.1 ± 0.2 mm) was recorded against C. krusei.All the Candidal strains susceptible with MIC of 500 μg/mL and MFC of 1000 μg/mL.

DISCUSSION
The plants usually involve approaches; ethno botanical approach is one of the common methods that are employed in choosing the plant for pharmacological study.Traditionally plant parts, extracts, infusions etc., were used for treatment of various diseases.The plants have traditionally provided a source of hope for novel drug compounds as plant herbal mixtures have made large contribution to human health and well being the use of plant extracts with known antimicrobial properties can of great significance for therapeutic treatment (Iwu et al., 1999).
The present research work showed the chloroform and methanol extracts of Lennea coromandelica leaf and bark possess antimicrobial activity against most of the tested pathogens.The result of this study showed that the methanol extracts of Lennea coromandelica was more effective than the chloroform extracts demonstrated the highest activity.These results support earlier studies which observed that plant extracts in organic solvent provide more consistent antimicrobial activity (Ahmad et al., 1998).Among the Gram -positive bacteria and Gramnegative bacteria tested against the leaf extract of Lennea coromandelica have most sensitive organism was Staphylococcus aurers.The tested plant extracts were most active against gram positive bacteria than the gram negative bacteria (Ayyappan et al., 2010;Parekh et al., 2005;Rajakaruna et al., 2002).Methanol provided more consistent antimicrobial activity compared to other extracted in chloroform.These activities might depend on the compounds being extracted by each solvent, the polarity of the solvints, and their intrinsic bioactivity.Which have been associated to antibacterial activities and thus have curative properties against pathogens (Nweze et al., 2004).
The antifungal activity of both the extract of Lennea coromandelica leaf and bark extracts were determine against four fungal (Candidal) strains and recorded.The highest antifungal activity was observed in methanolic leaf extracts of Lennea coromandelica against the tested Candidal strains.Chareprasert et al., (2006) isolated endophytes fungi from Tectona grandis L. and Samanea saman Merr., from 37 isolated fungi, 18 shown antimicrobial activity against Bacillus subtilis, staphylococcus arureus, Escherichia coli and three isolates inhibited the growing of Candida albicans.Weber et al., (2007) demonstrated antifungic activity from metabolities of endophyte fungi group ascomicete against Candida albicans.Similar results were observed in previous studies (Josephin Sheeba and Selva Mohan, 2012; Hema et al., 2013;Vinoth and Manivasagaperumal, 2015 ).Further pharmacological evaluations, toxicological studies and possible isolation of the therapeutic drug from the plant are the future challenges.

CONCLUSION
Finaly it can be concluded that methanol extract of Lennea coromandelica had a potential antimicrobial activity against all the microorganisms tested.Based on this study, isolation and identification of antimicrobial compound from methanol extract of Lennea coromandelica will fetch a new natural antimicrobial agent.

STATISTICAL ANALYSIS
All the data of microbial activities were examined as mean ± SD.One-way analysis of variance (ANOVA) was carried out to determine the significant difference (P < 0.005) between the means.The analyses were carried out using SPSS package software, 11.5 (SPSS Inc., Chicago, IL).
International Letters of Natural Sciences Vol. 45 the present study, variuos concentrations of chloroform and methanol extracts of leaves and bark of Lannea cormandelica were tested against gram positive bacteria viz., Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis, gram negative bacteria viz., Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli and fungal strains such as Candida albicans, C. parapsilosis, C. krusei and C. glabrata.The Isolated test organisms exhibited the antimicrobial activity for different concentrations like 250 μg, 500 μg and 1000 μg of different solvent extracts of Lannea cormandelica.

Table 2 . Antimicrobial activity of methanol extract of leaves of Lannea coromandelica
a-diameter of zone of inhibition (mm) including the disc diameter of 6 mm b-mean of three assays; ± -standard deviation.