MATERIALS AND METHODS

Collection of plant material: Fresh leaves (5 kg) of C. rumphii were collected during winters (December 2009) from different localities of Aligarh district, U.P., India. Plant samples (Voucher specimen number: AV023, AV2080) were deposited in the Herbarium of Department of Botany, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, U.P., India.

Preparation of non-polar and polar extracts: Shade dried leaves of C. rumphii (1.75 kg) were pulverized in an electric grinder and powder was stored in a desiccator. The grinded material was used for the extraction process. Five hundred grams plant powder was macerated with 95% methanol in a round bottom flask at room temperature for about 24 h. Mother liquor was filtered out and residual plant material was again macerated with 95% methanol for another 24 h. The entire process was repeated four times to ascertain the maximum yield of methanol soluble compounds. The extract was evaporated to dryness at 35°C under reduced pressure using rotary evaporator to get concentrated methanol extract. The extract was freeze-dried to give a final yield of 55 g (11.0%) methanol extract and kept in the chiller at -18°C till further use. Freeze-dried methanol extract was reflexed with light petroleum ether (60-80°C) for 5 h on the hot water bath. Light petroleum ether soluble portion upon filtration was evaporated under reduced pressure and freeze-dried to obtain light petroleum ether extract (LPE) (10.0 g, 2.0%). Light petroleum ether insoluble fraction of methanol extract obtained in above process was further reflexed with benzene for 5 h, filtered and evaporated under reduced pressure and freeze-dried to obtain benzene extract (BZ) (12.5 g, 2.5%). Benzene insoluble fraction was reflexed with ethyl acetate for 5 h, filtered and evaporated under reduced pressure and freeze-dried to obtain ethyl acetate extract (EtOAc) (8.0 g, 1.6%). Ethyl acetate insoluble fraction was further reflexed with methanol for 5 h, filtered and evaporated under reduced pressure and freeze-dried to obtain methanol extract (MeOH) (13.0 g, 2.6%) while methanol insoluble residue was discarded. During the entire successive extraction, each step was repeated five times to ensure maximum yield of every desirable extract.

Five hundred gram plant powder macerated with double distilled water and left for 72 h at room temperature under dark. The flask was then reflexed over hot water bath for 10 h and the mother liquor was filtered. The distilled water was again added reflexed and filtered and the entire process was repeated for four times. The filtrate, thus obtained, was evaporated to complete dryness under reduced pressure and freeze-dried to obtain aqueous extract (AQ) (40.0 g, 8.0%) (Harborne, 1998).

Microorganisms: Leaves extracts were tested for possible antibacterial activity through disc diffusion method against eighteen (18) human pathogenic bacteria viz., Staphylococcus aureus, Staphylococcus aureus ATCC 25953, Staphylococcus albus, Streptococcus haemolyticus Group A, Streptococcus haemolyticus Group B, Streptococcus faecalis, Bacillus subtilis, Escherichia coli, Edwardsiella tarda, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi, Shigella boydii, Shigella dysenteriae, Shigella flexneri and Plesiomonas shigelloides. All bacterial strains taken into consideration were obtained from the bacterial stock, Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh, U.P., India. The bacterial cultures were maintained at 4°C on nutrient agar.

Bacterial susceptibility test: Disc diffusion method (Bauer et al., 1966; Colle and Marr, 1989; Vaghasiya et al., 2007) was used to determine susceptibility of plant extracts. Standardized inoculum (1-2x10⁷ CFU mL^-1 0.5 McFarland standard) was introduced on the surface of the plates containing Mueller Hinton agar (NCCLS, 2004) which was spread evenly with a glass spreader. Paper disc (6 mm) were impregnated with 500 μg mL^-1, 1, 2, 5, 10 and 15 mg mL^-1 plant extracts. The discs were dried in an oven at 37°C and placed on the agar surface with the help of a sterile forceps, aseptically. Finally the discs were pressed with forceps to make complete contact with the surface of the medium. Inoculated petridishes were incubated at 37°C overnight and the inhibition zones were recorded. The experiments were repeated thrice and the mean of the triplicate of the results is summarized in Table 1. Standard sensitivity discs of chloramphenicol (10 μg/disc) (Span Diagnostics Limited, Surat, India) were used to test the sensitivity profile against the reference bacteria. Each experiment was done in triplicate and the mean values are presented. The diameters of the inhibition zones were measured in mm with respect to evaluate antimicrobial activity of all extracts in vitro.

Table 1:	Yields of non-polar and polar freeze dried extracts of the leaves of C. rumphii

RESULTS

Five hundred gram pulverized leaves of C. rumphii produced = 40.0 g (8.0%) freeze-dried AQ extract. Five hundred gram pulverized leaves of C. rumphii yielded = 55.0 g (11.0%) freeze-dried methanol extract. Methanol extract upon successive extraction with non-polar and polar organic solvents at room temperature subsequently yielded LPE = 10.0 g (2.0%), BZ = 12.0 g (2.5%), EtOAc = 8.0 g (1.6%) and MeOH =13.0 g (2.6%) freeze-dried extracts (Table 1).

In the present study, the investigation of antibacterial activity of the aforementioned extracts of the leaves of C. rumphii against seven Gram-positive and eleven Gram-negative bacteria was studied using disc diffusion method. The data pertaining to the antimicrobial potential of non-polar (LPE and BZ) and polar (EtOAc, MeOH and AQ) extracts of the leaves of C. rumphii are presented in Table 2. The results revealed variability in the inhibitory concentrations of each extract for given bacteria. All extracts presented antimicrobial activity to at least four of the tested microorganisms. None of the extracts except EtOAc extract of the leaves of C. rumphii were active against S. typhi while all the extracts were active against S. aureus, S. haemolyticus (group B) and K. pneumoniae; the LPE extract was inactive against S. albus, S. faecalis, B. subtilis, E. coli, S. typhi and S. boydii. BZ extract was found inactive against S. aureus (ATCC 25953), S. haemolyticus (group A), P. vulgaris, P. aeruginosa, S. typhi, S. dysenteriae and S. flexneri, however, EtOAc extract did not show any antibacterial activity against E. tarda, P. vulgaris and S. typhi. AQ extract showed no inhibitory activity against E. coli, P. mirabilis, P. aeruginosa, S. typhi and S. dysenteriae. The most susceptible Gram-positive bacteria was S. albus, inhibition zone = 22 mm (EtOAc extract) and Gram-negative bacteria was S. boydii, inhibition zone = 21 mm (EtOAc extract), respectively. The most resistant bacteria was Gram negative S. typhi strain with almost no antibacterial activity against all extracts except EtOAc extract which displayed 11 mm inhibition zone at 15 mg mL^-1 (Table 2).

Moreover, the EtOAc and MeOH extracts presented the highest activity, i.e., they were able to inhibit 15 (83.33%) types of microorganisms of interest. Furthermore, they also had the highest activity rate against bacteria. On the other hand, AQ extract showed inhibitory activity against 12 (66.66%) types of microorganisms and non-polar extracts (LPE and BZ) revealed antimicrobial activity against 11 (61.11%) microorganisms of interest, respectively. The antibacterial activity was more prominent on the Gram-positive bacteria than the Gram-negative bacteria. Gram-positive bacteria were the most susceptible to growth inhibition by EtOAc and MeOH extracts. The results showed that all the extracts have variable degrees of antibacterial activity and that the inhibition of bacterial growth was dose dependent as inhibitory action of the extracts was found to increase with the increase of concentration against all bacterial strains.

All extracts displayed similar antibacterial activity compared to positive control (Chloramphenicol) though at higher concentration. All extracts up to 1 mg mL^-1 did not exert any antibacterial activity against all bacterial strains.

Table 2:	Antibacterial activity of Cycas rumphii Miq. leaves extracts against some tropical human pathogenic bacteria
Gram positive bacteria: 1. Staphylococcus aureus 2. Staphylococcus aureus ATCC 25953 3. Staphylococcus albus 4. Streptococcus haemolyticus, Group-A 5 and Streptococcus haemolyticus, Group-B 6. Streptococcus faecalis 7. Bacillus subtilis. Gram negative bacteria: 1. Escherichia coli 2. Edwardsiella tarda 3. Klebsiella pneumoniae 4. Proteus mirabilis 5. Proteus vulgaris 6. Pseudomonas aeruginosa 7. Salmonella typhi 8. Shigella boydii 9. Shigella dysenteriae 10. Shigella flexneri 11. Plesiomonas shigelloides. Values are the mean of replication of three experiments; -: no inhibition. Zone of inhibition in mm LPE: Light petroleum ether extract, BZ: Benzene extract, EtOAc: Ethyl acetate extract, MeOH: Methanol extract, AQ: Aqueous extract

Highest antibacterial activity was observed at 15 mg mL^-1 by all extracts against most of the bacterial strains. EtOAc extract exhibited the highest antibacterial activity against most of the bacterial strains in comparison to other extracts of the leaves of C. rumphii.

DISCUSSION

Plants are imperative source of potentially important compounds for the development of new therapeutic agents. Natural products from medicinal plants as secondary metabolites (minor phytoconstituents) have been associated with the conferment of antibacterial activities by many studies (Rios and Recio, 2005; Tamokou et al., 2008; Sule et al., 2011b). The presence of some of such biological active substances in a substantial amount in the explored part of C. rumphii may have bestowed the strong and significant antibacterial activity on the leaves extracts. In this regard, higher concentration of these phytochemicals might have been responsible for a higher degree of inhibition on the bacterial strains.

Formerly, in many studies of similar kinds, polar extracts i.e., acetone, ethanol and methanol have been used as extractants, however this orchestrated strategy could not reveal the furthermost sensitivity in yielding strong antimicrobial compounds that might pave the ways to synthesize new antimicrobial drugs (Cowan, 1999; Ncube et al., 2008). For this reason, in the present study, five different solvents based on their different degrees of polarity as determined by the functional group present, i.e., light petroleum ether, benzene, ethyl acetate, methanol and water were used and taken into account to obtain maximum active compounds in the extracts. It is quite noteworthy to observe that a relationship was detected between the extract solubility and antibacterial activity of different fractions. This suggests that in sequential extraction, maximum biological active compounds were solubilized according to their degree of solubility in polar solvents as EtOAc and MeOH extracts (fractions) displayed the highest antibacterial activity followed by AQ extract, BZ and LPE fractions. Parekh et al. (2005) and Sule et al. (2011a) accounted that methanol is a better solvent for consistent extraction of antimicrobials from medicinal plants. Our results also comply with the same study as polar fractions (EtOAc and MeOH) obtained from methanol extract revealed potent antibacterial activity in comparison to non-polar solvents (LPE and BZ). These results further confirm that significant antibacterial compounds are polar in nature as evidenced by the higher degrees of antibacterial activity of EtOAc and MeOH fractions from the methanol extract of the leaves of C. rumphii.

All the extracts exhibited strong antibacterial activity against Gram-positive bacteria in comparison to Gram-negative bacteria. The susceptibility of Gram-positive bacteria towards various plant extracts than those of Gram-negative bacteria has already been reported previously (Bele et al., 2009; Vaghasiya and Chanda, 2010). This has been attributed to the fact that the Gram-negative bacterial cell wall outer membrane appears to act as a permeability barrier to many substances including amphipathic (cationic, neutral and anionic) broad spectrum antibiotics (Lewis, 2001). The activity of C. rumphii leaves extracts against both Gram-positive and gram-negative bacteria might indicate the presence of broad spectrum antimicrobial compounds. Biological active substances in the form of pure compounds and standardized plant extracts endow infinite prospects for the development of new drugs owing to bountiful unparalleled availability of chemical miscellany (Sati et al., 2011; Cos et al., 2006; Chanda et al., 2011; Sule et al., 2011b). These results explicitly suggest that the bioactive compounds present in polar extracts have good potential for development of novel antibacterial herbal products.

CONCLUSION

Results obtained from our study further confirm the traditional use of C. rumphii in different ailments associated with pathogenic bacteria. Non-polar and polar extracts revealed varying degrees of antibacterial activity. However, polar extracts i.e., EtOAc and MeOH extracts demonstrated most pronounced antibacterial activities against bacterial strains taken into consideration. Hence, EtOAc and MeOH extracts of the leaves of C. rumphii deserve further investigations to develop new antibiotics that may help in combating several bacterial diseases in tropical countries. This is the first ever report on the antibacterial potential of the leaves of C. rumphii.

ACKNOWLEDGMENT

Thanks are due to Department of Science and Technology SERC Division, New Delhi for financial support to the author Dr. Abdul Viqar Khan.