MATERIALS AND METHODS

Collection and identification of plants: In the present study, the leaves of the medicinal plants (Family) namely, Aegle marmelos (L.) Corr. (Rutaceae), Albizia amara (Roxb.) Boiv. (Fabaceae, Sub-family: Mimosoideae), Cassia auriculata L. (Fabaceae, Sub-family: Caesalpinoideae) and Cissus quadrangularis L. (Vitaceae) (Fig. 1) were collected in and around Dharapuram Taluk, Tiruppur District, South India and the identification was confirmed using standard local floras (Matthew, 1983).

Preparation of crude leaf extracts: The crude extracts of the leaves of all the four plant species were prepared separately using ethanol (95%) and distilled water as described below.

Solvent extraction: The collected leaves of the plants were immediately transported to the laboratory and individually washed with tap water, blotted with filter paper and spread over news paper for air drying under shade. After complete dryness, the leaves of each plant were powdered using a mixer grinder. A known quantity of leaf powder (50 g) of each plant was taken in a 250 mL conical flask and added with 100 mL of ethanol (95%). The ethanol-leaf powder mixtures were kept at room temperature for 48 h and rapidly stirred using glass rod every 8 h.

After 48 h, the extract of each plant was filtered through Whatman No. 1 filter paper to exclude the leaf powder. Then each filtrate was concentrated using vacuum evaporator. A greasy final material (crude ethanolic-leaf extract) obtained for each plant was transferred to screw cap bottles, labeled and stored under refrigerated (4°C) condition till use.

Fig. 1(a-d):	Plants used in the study: (a) Aegle marmelos, (b) Albizia amara, (c) Cassia auriculata and (d) Cissus quadrangularis

Aqueous extraction: For aqueous extraction, 10 g of air-dried powder of each plant leaves was placed in 100 mL distilled water and boiled for 6 h. At 2 h intervals, it was filtered through eight layers of muslin cloth and centrifuged at 5000 rpm for 15 min. The supernatant was collected and concentrated to make the final volume one-fourth of the original volume. A greasy final material (crude aqueous-leaf extract) obtained for each plant was transferred to screw cap bottles, labeled and stored under refrigerated (4°C) condition until use.

Preparation of stock and test solutions: By using digital electronic balance, 200 mg of each aqueous and ethanolic leaf extracts was carefully taken in a standard measuring flask and 5 mL of ethanol was added to dissolve the ethanolic-leaf extract and 5 mL of distilled water for aqueous leaf extract respectively. One to two drops of emulsifier (Triton-X100) were added to completely dissolve both aqueous and ethanol extracts. Then each extract was made up to 200 mL by adding distilled water and stored under refrigerated (4°C) condition till use. This formed the stock solution of 1000 ppm.

Bacterial susceptibility testing
Bacterial culture: Six bacterial species, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi and Bacillus subtilis were used for the antibacterial activity test. The organisms were maintained on agar slope at 4°C and sub-cultured for 24 h before use. These organisms were originally obtained from the Microbial Type Culture Collection (MTTC) of Institute of Microbial Technology (IMTECH), Chandigarh, India.

Antibacterial assay: Standardized inoculums of each bacterium, i.e., 1 to 2x10⁷ CFU (Colony Forming Units)/mL with 0.5 McFarland standard was introduced onto the surface of sterile Muller-Hinton (MH) agar plates and a sterile glass spreader was used for even distribution of inoculums. A sterile paper disc previously soaked in known concentration of extracts (20 μg/mL/disc) was carefully placed at the centre of the seeded and labeled MH agar. Sterile paper discs containing physiological saline alone was served as control. For each test solution, three replicates were maintained. Amoxycillin at 10 μg disc^-1 was used as an antibiotic reference standard. The aqueous and ethanolic test extracts were individually tested at a concentration of 1000 ppm against test organisms. The crude aqueous leaf extracts were mixed in equal proportions in combination of two, three, four, five or six extracts. For comparison, individual plant extracts (aqueous and ethanol) were also tested for antibacterial activity. The same procedure was followed for the preparation of different combinations of aqueous leaf extracts. Whatman No.1 filter paper discs (5 mm diameter) were dipped in each test solution, evaporated to dryness in hot air oven and used for antibacterial assay. The plates were incubated aerobically at 37°C and examined for zone of inhibition after 24 h. Each zone of inhibition was measured with a ruler and compared with the control (Bauer et al., 1966).

RESULTS AND DISCUSSION

In this study, four commonly available medicinal plants used by traditional users in South India were tested against six different bacteria. The result of antibacterial susceptibility testing showed that all the bacteria, S. aureus, B. cereus, P. aeruginosa, E. coli, S. typhi and B. subtilis were highly susceptible to amoxicillin with average diameter zone of inhibitions (ZI) of 2.9, 3.2, 3.1, 2.9, 3.3 and 3.2 cm, respectively (Table 2). Aqueous and ethanolic leaf extracts when tested individually for their antibacterial activity, showed various degrees of activity (Table 3). The ethanolic leaf extracts of C. auriculata showed comparatively a high degree of activity followed by C. quadrangularis and A. marmelos. The diameter of ZI was 1.8 cm for C. auriculata ethanolic extract against B. cereus. The lowest antimicrobial activity was shown by aqueous leaf extract of A. amara (Table 3).

Table 2:	Effect of antibacterial reference standards on selected bacteria

Table 3:	Effect of crude aqueous and ethanolic extracts of selected plant leaves on different bacteria (24 h)
SA: S. aureus, BC: B. cereus, PA: P. aeruginosa, EC: E. coli, ST: S. typhi and BS: B. subtilis. Values are mean of three replicates

Table 4:	Synergistic activity of aqueous and ethanolic extracts of selected plant leaves in combination of two against bacteria
SA: S. aureus, BC: B. cereus, PA: P. aeruginosa, EC: E. coli, ST: S. typhi and BS: B. subtilis; (A) A. marmelos, (B) A. amara, (C) C. auriculata and (d) C. quadrangularis; Values are mean of three replicates

Table 5:	Synergistic activity of aqueous and ethanolic extracts of selected plant leaves in combination of three against bacteria
SA: S. aureus, BC: B. cereus, PA: P. aeruginosa, EC: E. coli, ST: S. typhi and BS: B. subtilis. Values are mean of three replicates

The studies on screening the extracts of specific plant parts alone obtained by many workers fall in line with the present investigation: Different solvent extracts of Zapoteca portoricensis (Agbafor et al., 2011), antibacterial ester from root bark extracts of Vitellaria paradoxa (Garba and Salihu, 2011), methanolic leaf extracts of Anogeissus leiocarpus (Ichor and Ekoja, 2011) and antibacterial activity of Artemisia dracunculus essential oil against multi-drug resistant Acinetobacter baumannii (Jazani et al., 2011).

The antibacterial activities of extracts in combination of two plants showed different degrees of ZI as shown in Table 4. The average diameter above 2.0 cm ZI was observed in the following aqueous and ethanolic leaf extract combinations: A. marmelos +C. auriculata, A. amara +C. auriculata and C. auriculata +C. quadrangularis followed by other combinations. A ZI of 2.7 cm was observed in ethanolic leaf extract combination of C. auriculata +C. quadrangularis against S. aureus (Table 4). The highest ZI of 2.8 cm against S. aureus was observed in ethanolic leaf extract combination of A. amara +C. auriculata +C. quadrangularis (1:1:1) (Table 5). The combination of aqueous extracts of all the four plants in equal proportion showed a maximum of 3.0 cm ZI against B. cereus followed by 2.4 cm ZI against B. subtilis (Fig. 2). These findings are in coherence with the study reported earlier on synergistic activity of six different plants against pathogenic bacteria by Karmegam et al. (2008). Synergistic activity of aqueous and ethanolic extracts of selected plant leaves, in combination of two, three, four, five and six against test organisms ranged from 0-2.8 cm zone of inhibition. The highest ZI of 2.8 cm was observed against S. aureus in ethanolic leaf extract combinations of Balanites aegyptiaca +Lobelia nicotianaefolia (Karmegam et al., 2008). Similarly, Prakash et al. (2006b) reported that the ethanolic leaf extracts of Catharanthus roseus, Lawsonia inermis and Chrysanthemum odoratum showed least activity against methicillin resistant Staphylococcus aureus (MRSA) when used individually. Whereas, the combination of these three plant-extracts exerted a higher activity of 26 mm zone of inhibition followed by C. roseus +L. inermis (2.3 cm) and L. inermis +C. odoratum (2.0 cm) extract combinations against MRSA.

Fig. 2:	Synergistic activity of aqueous and ethanolic extracts of selected medicinal plant leaves, in combination of four, against different bacteria (24 h). Values are mean of three replicates (Refer Tables for abbreviations)

CONCLUSION

The antimicrobial activity of aqueous and ethanolic leaf extracts of A. marmelos, A. amara, C. auriculata and C. quadrangularis showed lower inhibition zones when used alone than that of the extract combinations. The ZI reached 3.0 cm against certain bacteria by the combination of aqueous extracts of all the four plants used in the study, indicating the high potential of combined use of plant extracts against pathogenic microorganisms. There is a possibility of using plant extracts in combinations against pathogenic bacteria as has been observed from the results.