In vitro Antibacterial Activity Analysis of Leaves of Limonia acidissima

The present study was carried out the antibacterial activity and phytochemical screening of the hexane, chloroform and methanol extracts of leaves of Limonia acidissima. The antibacterial activity was evaluated against four Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris) and five Gram-positive bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Streptococcus pneumoniae) by agar well diffusion method. Methanol extract showed good antibacterial activity with the high inhibition zones while chloroform extract exhibited mild to moderate activity and hexane extract was found to be less active. Phytochemical screening revealed the presence of various secondary metabolites like steroids, alkaloids, phenols, flavonoids, coumarins, saponins, tannins and triterpenoids. The results of the present study suggest that leaves of Limonia acidissima can be used to treating infectious diseases caused by E. coli, P. vulgaris and S. pneumoniae.


Introduction
Limonia acidissima Linn is an important medicinal plant and is a moderate-sized deciduous tree grown throughout tropical and temperate regions of the world. Tree with rough, spiny bark. Leaves are pinnate, with 5-7 leaflets, with a citrus scent when crushed. Leaves of the plant are used for vitiated conditions of vata and pita (Kirtikar and Basu, 2005). The decoction of the leaves used in the treatment of constipation, vomiting, cardiotonic, diuretic (Tarakeswara Naidu et al., 2012). It has been reported that plant contains flavanoids, glycosides, saponins, tannins (Saima et al., 2000), coumarins (Ghosh et al., 1982) and tyramine derivatives (Parthasarathi et al., 1991). Also the presence of alkaloids, steroids, glycosides, phenols, gum and mucilage, fixed oils and fats, resins and tannins (Thomas and Ponnammal, 2005). The leaves have hepatoprotective activity (Kamat et al., 2003), antimicrobial (Bandara et al., 1990) antifungal (Khewkhom et al., 2008;Adikaram et al., 2007) astringent, anti-inflammatory (Kim et al., 2009) and insulin secretogouge activities (Gupta et al., 2009). As the plant is reported to have various medicinal uses, authors have attempted to study the antimicrobial activity of the leaves.

Materials and methods
The present study was done in the Department of Botany, Andhra University, Visakhapatnam, Andhra Pradesh, India. Leaves of Limonia acidissima were collected from University premises.

Chemicals, Media and Antibiotic
Hexane, chloroform, methanol and Dimethyl sulphoxide (DMSO), were obtained from Rankem company, India. Nutrient broth and Nutrient agar were obtained from Hi-media, Mumbai, India. The antibacterial agent Ciprofloxacin was obtained from Axiom Laboratories Ltd., India.

Extraction procedure
Limonia acidissima leaves washed thoroughly under running tap water, dried on paper towel, then shade dried and finally crushed to fine powder in mixture grinder. The dried powder of the leaves was allowed to Soxhlet for sequential extraction with hexane, chloroform and methanol. The liquid extract was collected, filtered and evaporated to dryness using Rotary evaporator (Heidolph, Heizbad, Laborota 4001, Germany 2002). A semisolid or dried crude extracts of leaves so obtained was re-suspended in an inert solvent, DMSO. The extracts were dissolved in DMSO to reach final concentrations (50 mg/ml, 75 mg/ml and 100mg/ml), and were kept in refrigerator till used.

Antimicrobial activity
The antimicrobial activity of extracts of Limonia acidissima leaves was determined by using agar well diffusion technique (Aniel Kumar et al., 2010a). Nutrient agar plates were each seeded with 0.1 ml of an overnight culture of each bacterial (equivalent to 10 7 -10 8 CFU/mL) strain. The 24 hrs broth culture of each bacterium used to seed sterile molten nutrient agar at 45 ° C, allowed to solidify at room temperature and well made by sterile standard cork borer and 50 μl solution of extract added to each well. Then bacterial plates incubated at 37 ° C for 24 hrs after which diameter of zones of incubation were measured (mm) by using HiAntibiotic ZoneScale-C (Himedia). Each assay was performed in at least triplicate and mean values (± standard error) are reported. Standard antibiotic strip of Ciprofloxacin (5 μg/disc) for each bacteria along with DMSO were used as positive and negative controls, respectively.
Minimum inhibitory concentration (MIC) was determined by the broth dilution method (Aniel Kumar et al., 2010b). A quantity of 0.6g of each extract was dissolved in 300 ml sterile nutrient broth, which yields an initial concentration of 2000 μg/ml. subsequently, two-fold serial dilution was made from the stock to obtain following concentrations 1000, 500, 250, 125, 62.5 μg/ml. Different concentrations of leaf extract in hexane, chloroform and methanol was tested separately for each bacterium and inhibition zone of microbial growth in the plates containing test solutions was judged by comparison with blank control plates. MIC is defined as the lowest concentration of test samples that result in a complete inhibition of visible growth. Experiments were carried out in triplicate.

Results and Discussion
The inhibition zones and MIC values of leaves of Limonia acidissima are presented in Tab. 1, Tab. 2, respectively. Methanol extract showed the high antibacterial activity while chloroform extract showed mild to moderate activity and hexane extract found to less effective. Methanol extract showed the highest zone of inhibition against Proteus vulgaris followed by E. coli and S. pneumoniae, while chloroform and hexane extracts against had the high zone of inhibition Proteus vulgaris followed by S. pneumoniae. Hexane extract did not shown inhibition values against M. luteus, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus whereas chloroform extract against Bacillus subtilis. On comparison standard antibiotic ciprofloxacin, methanol extract exhibited higher inhibition zones against E. coli, P. vulgaris, S. pneumoniae and similar value against E. faecalis while chloroform extract showed higher activity against Proteus vulgaris. DMSO, a negative control did not show any inhibition zones and indicated that it is not interfering zone formation.
The low MIC values of methanol extract were <62.5 μg/ml against Proteus vulgaris and S. pneumoniae while 62.5 μg/ml against E. coli; 125 μg/ml against M. luteus, S. aureus. Chloroform extract showed low MIC against Proteus vulgaris, S. pneumoniae, it was 62.5 μg/ml whereas hexane extract found to be low MIC against E. coli, S. pneumoniae and it was 250 μg/ml.
Plants are important sources of potentially useful substances for the development of new therapeutic agents. Various phytochemical compounds which are naturally present in plants as secondary metabolites have been implicated in plants as the conferment of antibacterial activities (Kishor Naidu et al., 2013). The inhibition of bacterial growth in vitro by the extracts of Limonia acidissima plant parts such as bark, leaf, rind, pulp and seed showed the presence of alkaloids, flavonoids, steroids, saponins, glycosides, phenols, gum and mucilage, fixed oils and fats, resins and tannins (Thomas and Ponnammal, 2005).
Methanol proved as the most effective solvent for extracting broad spectrum of antimicrobial compounds from plants (Vlachos et al., 1996). It is worth mentioning to note that a correlation was observed between the extract solubility and antibacterial activity of different fractions. This suggests that in sequential extraction, maximum antibacterial compounds were soluble in polar solvent as methanol extract displayed the highest antibacterial activity followed by chloroform and hexane extracts. These results further confirm that significant antibacterial compounds are polar in nature as evidenced by the higher degree of antibacterial activity of methanol extracts of Limonia acidissima leaves. These active compounds may act alone or in combination to inhibit bacterial growth and conferred the strong antibacterial activity.
Earlier, it was reported that methanolic extracts of L. acidissima plant parts were tested against Escherichia coli and Staphylococcus aureus showed varying degrees of antimicrobial activity (Thomas and Ponnammal, 2005). Stem bark had antimicrobial activity against Staphylococcus aureus, Escherichia coli, Enterobacter eloacae, Klebsiella erogenes, Aspergillus niger and Candida albicans (Rahman Tab. 1. Antimicrobial activity of extracts of L. acidissima leaves Naidu G.K. et al. / Not Sci Biol, 2014, 6(2) and Gray, 2002) whereas Chloroform extract of the leaves exhibited antifungal activity (Khewkhom et al., 2008) and in the present study stated that Proteus vulgaris was the most sensitive to the hexane, chloroform and methanol extracts of L. acidissima leaves and suggest that extracts of L. acidissima may be used to treat human diseases caused by microorganisms. Therefore, L. acidissima may be a future drug candidate to prove its efficacy as a preventive and therapeutic agent against E. coli, P. vulgaris and S. pneumoniae. Therefore, further studies needed to isolation and identification of compounds responsibility for antibacterial activity.

Conclusion
In conclusion, the results provide a scientific base for the traditional use of L. acidissima as an antimicrobial agent. It is suggested that, L. acidissima may possess promising therapeutic action in the treatment of infectious diseases caused by the species like E. coli, P. vulgaris and S. pneumoniae. Further work will emphasize the isolation and characterization of active compounds responsible for antibacterial activity.