Litsea glutinosa ( Lauraceae ) : Evaluation of its Foliar Phytochemical Constituents for Antimicrobial Activity

The phytochemical investigation of the leaves of Litsea glutinosa revealed the presence of secondary metabolites like alkaloids, anthraquinones, cardiac glycosides, flavonoids, glycosides, phenols, saponins, steroids, tannins, terpenoids, volatile compounds, amino acids and carbohiydrates. The antimicrobial activity and minimum inhibition concentration values were determined for these phytochemical constituents as crude extracts using the agar well diffusion and two-fold serial dilution methods. The results indicated that Bacillus subtilis was the most susceptible bacterium with high inhibition zones for the methanol and chloroform extracts of 31 mm and 26 mm, respectively. The MIC values indicated that extracts possess good antimicrobial activity with significant MIC value against Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus pneumoniae at 31.2 μg/ml concentrations. The extracts showed marked antimicrobial activity against both bacteria and fungi. Among the bacterial strains, gram-positive bacteria were more susceptible than the gram-negative. All the 13 microorganisms tested showed dose dependent susceptibility towards the phytochemicals present in the foliar extracts. The study suggests that Litsea glutinosa leaves possess potent antimicrobial activity and can be a good source for the development of new antibiotics.


Introduction
In India, from ancient times, different parts of medicinal plants have been used to treat the infectious diseases which are the cause of premature deaths to an extent of 50,000 people every day globally (Anonymous, 2000).India is rich in medicinal plant diversity because of different agroclimatic, ecological and edaphic conditions.Medicinal plants are the richest source of natural products for traditional system of medicine, pharmaceutical intermediates and chemical entities for synthetic drugs.Thus, there is a constant need to develop new antimicrobial drugs for the treatment of infectious diseases from the medicinal plants (Ncube et al., 2008).
Litsea glutinosa (Lour.)C.B. Robinson (Lauraceae) is an evergreen or deciduous, that reaches a height of 3-15 meters.This species is native to India, South China to Malaysia, Australia and the western Pacific Islands.It is a medicinal plant known as Indian laural, soft/brown bollygum or beech/bolly beech, bollywood and sycamore.In Telugu, it is called 'narra alagi' or 'narra mamidi'.It is a threatened species due to over exploitation for its bark and considered as endangered species in Philippines (Rabena, 2010).Traditionally, it is considered as promoter of longevity, semen generation and emollient.The sap of fresh bark or its decoction is prescribed as a remedy for diarrhoea, dysentery and rheumatism.The mucilaginous leaves are considered antispasmodic and emollient.In addition, a paste prepared by grinding bark with water is used as a plaster in cases of sprain, bruises, wounds, inflammation, back pain, rheumatic and gouty joints, bone fractures, etc.It has analgesic, antiseptic and emollient effects (Devi and Meera, 2010).Although the most of the antimicrobial activities had been carried out on the bark extract (Mandal et al., 2000;Lohitha et al., 2010;Poornima, 2011;Haque et al., 2014) there are a few studies on the antimicrobial activity on methanol extracts of leaves (Meera and Devi, 2009;Gulzar et al., 2015).However, there are no reports on the antimicrobial activity of L. glutinosa leaves, and their effects on pathogenic fungi and bacteria.Thus, the present study evaluated the antimicrobial activity of hexane, chloroform, methanol and aqueous extracts of L. glutinosa leaves.
The experiment was done three times to minimize the error.After incubation period the antimicrobial activity was evaluated by measuring the inhibition zones by using an antibiotic zone reader scale(HiAntibiotic Zonescale-c).
Sabouraud agar was used to culture the fungi.The inoculated petridishes were incubated at 25 °C for two days for the C. albicans, S. cerevisiae and three days for A. niger.About 500 µg of fluconazole was dissolved in 1 ml of sterile deionized water.About 10 µl of 0.5 mg/ml of fluconazole (equivalent to 5 µg dose) pipette into the wells for comparison with fungal inhibition zones.The bacterial inhibition zones were was compared with tetracycline disc (5 µg /disc) of multidrug disc (Axiom Laboratories Ltd. India).About 50 µl of DMSO was pipette into each well for bacteria and fungi as a negative control.
The extracts that exhibited inhibition zones were subjected to minimum inhibition concentration (MIC) assay by using two-fold serial dilution (Aniel Kumar et al., 2015).A quantity of 0.6 g of each extract was dissolved in 300 ml sterile nutrient broth which yields initial concentration of 2,000 µg/ml.Subsequently, two-fold serial dilution was made from the stock to obtain 1,000, 500, 250, 125, 62.5, 31.2 µg/ml concentrations.One ml of standardized inoculums of each test organism was introduced into each extract nutrient broth mixture and then incubated at 37 0C.The lowest concentration inhibiting growth was regarded as the MIC of the extracts.

Statistical analysis
Each experimental data from triplicates of standard error was subjected to one way ANOVA using Minitab version 15.The significant level of p < 0.001 was used.

Plant material
The leaves of Litsea glutiona were collected from Andhra University campus of a planted tree from Visakhapatnam, Andhra Pradesh, India.Its taxonomic identity was confirmed by Prof. M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam, India.The leaves collected were shade-dried.Then, they were powdered in the mixture grinder and stored in airtight bottles.

Extraction of plant material
The shade dried leaf powder (10 g of each) was extracted with hexane, chloroform, followed by methanol by using sequential extraction method (Aniel Kumar et al., 2010).Thereafter, it was filtered by rotary evaporator at 40 °C to obtain the crude dried extract.Simultaneously, the aqueous extract of the leaves was prepared by adding boiled water to the powdered in a beaker on water bath, with occasional stirring for 4 hours.The aqueous extract was then filtered and centrifuged at 5,000 rpm for 15 min.The supernatant was collected and evaporated to dryness to give the crude dried extract.The extracts were dissolved in DMSO to get the known concentrations of 25 mg/ml, 50 mg/ml and 100 mg/ml.

Microbial strains
The test bacterial and fungal strains used in the study were obtained from Microbial type culture and collection (MTCC), Chandigarh, India.They are Bacillus subtilis MTCC B2274, Enterococcus faecalis MTCC B3159, Escherichia coli MTCC B1560, Klebsiella pneumoniae MTCC B4030, Micrococcus luteus MTCC B1538, Pseudomonas aeruginosa MTCC B2297, Proteus vulgaris MTCC B7299, Staphylococcus aureus MTCC B3160, Streptococcus pneumoniae MTCC B2672, Aspergillus niger MTCC F4325, Candida albicans MTCC F7315 and Saccharomyces cerevisiae MTCC F2567.The bacterial strains were grown in the nutrient broth and maintained on nutrient agar slants at 4 °C whereas the fungal strains were grown in Sabouraud broth and maintained on Sabouraud agar slants C. albicans and S. cerevisiae) and potato dextrose agar slants (A.niger) at 4 °C.

Antimicrobial screening
The antimicrobial activity of hexane, chloroform, methanol and aqueous extracts of leaves of L. glutinosa was determined by agar well diffusion (Aniel Kumar et al., 2014) and agar disc diffusion methods for standard antibiotics tetracycline and fluconazole separately for bacteria and fungi.The lyophilized culture was sub cultured and concentration of working stock culture was assessed as 10 -6 CFU/ml.For susceptibility test, 100 µl of inoculum was mixed with 6 ml of sterilized nutrient agar and poured immediately into the sterile petridishes.The petridishes were left to solidify for 10 minutes.A sterilized 6 mm metal borer was used to make wells in the centre of the divided areas.About 50 µl of each extract was then pipette into the wells.The petridishes were incubated at 28 °C for 24 hours.
In the present study, the extracts of L. glutinosa leaves exhibited the antimicrobial activity against all tested bacteria and fungi except that A. niger was resistant to the hexane and chloroform extracts (Table 2).B subtilis was the most susceptible bacteria with high inhibition zones for the methanol and chloroform extracts of 31 mm and 26 mm, respectively.Aqueous extract showed the high inhibition zone against S. pneumoniae and P. aeruginosa while hexane extract against M. luteus and K. pneumoniae.The fungal strain S. cerevisiae was more susceptible to all extracts than other fungal strains A. niger and C. albicans.The most susceptible gram positive bacterium is B. subtilis for all extracts while the gram-negative bacterium is P. aeruginosa.When the concentration of these extracts was increased, the inhibition zones also increased and it indicated dose dependent susceptibility.
The results of antibacterial and antifungal of the different extracts of L. glutinosa leaves were compared with the standard antibiotics.The extracts showed inhibition zones were similar or more than the antibiotics against in more than 50% of the investigated microbial strains.It is a promising result and suggests that the plant extracts contain certain phytochemical constituents with antimicrobial properties that can be used to develop new drugs for therapy of infectious diseases caused by microorganisms.
The MIC values indicate that the leaf extracts of L. glutinosa possess antibacterial activity against B. subtilis, E. faecalis, S. pneumoniae and P. aeruginosa at 31.2 µg/ml concentrations (Table 3).These bacteria also shown strong MIC values for aqueous and chloroform extracts at 62.5 µg/ml concentration.These results agree with previous studies, ethanol extracts of bark shown antibacterial activity against S. aureus, B. cereus, P. aeruginosa and E. coli (Lohitha et al., 2010), while the same extract active against P. aeruginosa, E. coli, S. aureus but less effective against fungal strains A. fumigates and C. albicans (Poornima, 2011).Ethanolic and water extracts of bark and leaves have antibacterial activity against E. coli, Enterobacter intermedium, Salmonella sp., S. aureus and S. epidermis (Haque et al., 2014), while leaves shown antimicrobial activity against gram-negative S. paratyphi (Gulzar et al., 2015) and methanol extract of bark shown antibacterial activity against 16 microorganisms tested (Mandal et al., 2000).The antimicrobial activity of the extracts on bacteria was more pronounced than on fungi.It could be due to the fungal cell wall which has a complex structure and extensive cross-linking between chitin, glucans and other polymers.It also was observed that gram-positive bacteria were more susceptible than the gram-negative as has been found by Meera and Devi (2009), who studied the methanol extract of L. glutinosa leaves and had the similar result.This difference in the activity may be attributed to the fact that the cell wall in gram-positive bacteria have of a single layer whereas the gram-negative bear multilayered structure along with more lipids.The present study also receives support from Ali et al. (2004).
The antimicrobial activity may be due to the presence of some metabolites like alkaloid, saponins and terpenoids which have been implicated in various biological activities (Thomas et al., 2013) and presently found in all the extracts.The present study suggests that L. glutinosa has great potential as a source of useful bioactive compounds which cure infectious diseases caused by pathogenic bacteria and fungi.

Conclusions
The L. glutinosa leaf extracts showed marked antimicrobial activity against both bacteria and fungi, while in the bacterial strains, gram-positive bacteria were more susceptible than the gram-negative.The extracts showed marked antimicrobial activity against both bacteria and fungi.The antimicrobial activity is dose dependent susceptibility towards the phytochemicals present in the solvent foliar extracts.Therefore, the study shows that L. glutinosa leaf extracts have a broad spectrum of antimicrobial activity and could be useful in antiseptic/disinfectant formulations and chemotherapy.

Table 2 .
The antimicrobial activity of L. glutinosa leaf extracts against the standard drugs a: Each value is the mean of three replicates, with standard deviation; P <0.001 extremely significant when compared to the standard S: Standard (T-Tetracycline; F-luconazole) D: DMSO-: No activity

Table 3 .
MIC values of Litsea glutinosa leaf extracts against the tested microorganisms