Introduction

The semitropical climate of India serves as an excellent environment for abundant bacterial growth. Adding to it the large human host population and the easy availability of the entire gamut of antibiotics over the counter, we face not only rampant bacterial infection but also an enormous problem of escalating drug resistance to known antimicrobial drugs.

In such a situation indigenous plants serve as invaluable sources of new antimicrobial agents (1). In the present study we assessed Ficus lyrata (F. lyrata) for its potential antibacterial activity.

Ficus is a broad genus that includes trees, shrubs and often climbers, widely distributed throughout the tropics of both hemispheres but abundantly found in South – East Asia. In India itself 65 species have been identified. Almost all species of Ficus yield latex – containing caoutchouc (2). Many species of Ficus are medicinally important (3). A new chromone, flavonoids, and β-sitosterol were isolated from the leaves of Ficus lyrata (4). Ficus lyrata have been shown to possesses diuretic and CNS depressant activities (5). Mandal, Saha, Pal, have reported antibacterial activity in leaves of Ficus recemosa (6). However, antibacterial activity of F. lyrata have not been reported so far.

Material And Methods

The study was conducted in the Departments of Microbiology, Pharmacology, J.N. Medical College and the Department of Chemistry Aligarh Muslim University, Aligarh, U.P., India.

Plant Material:

Extraction procedure:

Microorganisms:

Determination of MIC:

Results

Disc diffusion assay : The antibacterial properties of aqueous and alcoholic extracts of F. lyrata and isolated pure compounds i.e. Ursolic acid (FL-1) and Acacetin-7-O- neohesperidoside. (FL-2) were tested against twenty standard bacterial strains. F. lyrata and isolated pure compounds showed potent antibacterial activity against P. aeruginosa, S. aureus (Methicillin sensitive, Methicillin resistant) and Coagulase negative Stapylococci, S. dysenteriae, S. boydii, C. freundii, P. vulgaris, P. mirabilis In case of V. cholerae, ogawa and inaba subtypes, Salmonella typhi, paratyphi A, S. typhimurium, E. coli, Klebsiella pneumoniae as well as the ESBL producing strain of E.coli and K. pneumoniae, however the zone of inhibition was less than 12mm. The aqueous extract of F. lyrata was more active against these strains than the alcoholic extract as measured by the size of zones of inhibition.

Figure 1

Table I : Pattern of Antibacterial Activity of crude extract of

Both FL-1 and FL-2 showed improved results in the disc diffusion assay as compared to the crude extract

Figure 2

Table II : Pattern of Antibacterial Activity of Pure compound from

_ Zone of inhibition of > 12 mm was taken as significant

FL-1 : Ursolic Acid, FL-2 : Acacetin-7-O- neohesperidoside.

. It is important to note that the zones of inhibition of Methicillin resistant and ESBL producing strains increased to beyond the significant cut off of 12mm in case of FL-1. Significant increase in zones of inhibition were also noticed against E. coli, K. pneumoniae, V. cholerae, S. typhi, S. Paratyphi A, and S. typhimurium in comparison to the crude extract.

Determination of MIC

MIC values were obtained against the strains which showed significant zones of inhibition (> 12mm) around the discs impregnated with the plant extracts and pure compounds. As expected the MIC’s of the aqueous extract were lower than the alcoholic extracts. The MIC’s of aqueous extracts of F. lyrata were found to be significantly low for all the seven bacterial strains

Figure 3

Table III : Comparative MIC’s of crude extract and Isolated compounds

The MIC’s of C.freundii were the same for both the alcoholic and aqueous extracts. As expected a larger number of different bacterial strains demonstrated lowered MIC’s against FL-1 and FL-2. Of special note are S. typhi, S. Paratyphi A, and S. typhimurium, V. cholerae, K. pneumoniae, ESBL producing K. pneumoniae and E. coli against which the crude extract did not show significant activity. Although both FL-1 and FL-2 had strong activity against the above mentioned bacteria FL-1 was more potent than FL-2. Comparative MICs are shown in table III

Discussion

With ever increasing momentum in the quest for newer antimicrobial agents, to counteract the spiraling bacterial drug resistance plants are being increasingly explored in many parts of the world. These plants may offer a new source of potential activity against infective microorganisms (8,9). We studied the antibacterial properties of F. lyrata. Both aqueous and alcoholic extracts showed antibacterial property. However, aqueous extract was more active. It may be possible that the active principle responsible for antibacterial activity is concentrated more in the aqueous extract as compared to the alcoholic extract. This study shows F. lyrata to have significant antibacterial activity against most of the tested bacterial pathogens. Of these S. aureus and P.aeruginosa are important human pathogens with known potential for drug resistance. C. freundii is an emerging pathogen. It is increasingly been reported as an extended spectrum β lactamase producer. This property renders the penicillin group of drugs ineffective against such bacteria. P. mirabilis and P.vulgaris are again important multi drug resistant pathogens with potential for producing metallo β lactamases and extended spectrum β lactamases, S. dysenteriae and S. boydii are important diarrhea producing pathogens.

It is interesting to note that the aqueous /alcoholic extract was selectively active against some Gram positive and some Gram negative bacteria. F. lyrata was exceptionally active against S. aureus (MRSA, MSSA) and Coagulase negative Staphylococcus but not against S. pyogenes, S. agalactiae, E. faecalis, S. pneumoniae. Similarly it was active against P.aeruginosa, C. freundii, S. dysenteriae, S. boydii, P. mirabilis and P. vulgaris. It had weak activity against E. coli, Klebsiella sp, S. Typhi, S. Paratyphi A, S. Typhimurium and V. cholerae inaba and ogawa subtypes. It overall was found to inhibit more gram negative bacteria than gram positive bacteria. Generally, plant extract are usually more active against gram positive bacteria than gram negative bacteria (10). The MIC values for the seven bacteria tested correlated well with the results obtained using disc diffusion method. The MIC’s of the aqueous extract were extremely low for S. aureus and C. freundii followed by S. dysenteriae and S. boydii, P. mirabilis and P. vulgaris and P. aeruginosa. However, the MIC value of S. aureus was lower than of the gram negative bacteria with the exception of C. freundii which had the same MIC.

To identify the active principle responsible for the antibacterial activity we screened the isolated pure compounds (FL-1 – FL8) from F. lyrata for their inhibitory effect against the growth of various bacterial strains. It was observed that only two compounds . i.e. Ursolic acid (FL-1) and Acacetin-7-O- neohesperidoside (FL-2) showed antibacterial activity. The MICs were significantly lower not only of those bacterial strains sensitive to the crude extract but also to Salmonella Typhi, Salmonella Paratyphi A, Salmonella Typhimurium, Vibrio cholerae, E. coli, K. pneumoniae and ESBL producing E.coli and K. pneumoniae. Thus the range of activity against gram negative bacteria was greatly enhanced on testing with pure compounds. Ursolic acid was more potent than Acacetin-7-O- neohesperidoside. Ursolic acid is a triterpenoid sapogenin from the ursan group whereas Acacetin-7-O- neohesperidoside is a flavonoid glycoside. Antifungal (11) and antimicrobial activity of Ursolic acid have been reported against several strains of Staphylococcus (12). ^{Numerous Ursolic acid containing plants from the Lamiaceae family exhibited antibacterial activity (13). Although antibacterial activity has been shown against Staphylococcus relatively little is known about its effect on other bacteria. Our findings suggest that Ursolic acid has excellent antibacterial activity against several problematic bacteria like MRSA and ESBL producing bacteria, Pseudomonas, Salmonella, Shigella and Vibrio cholerae are other known pathogens with drug resistance. The other pure compound i.e. Acacetin-7-O- neohesperidoside has not yet been explored for such activity which too exhibited significant antibacterial activity in our study.}

Our results show that Ursolic acid and Acacetin-7-O- neohesperidoside contributes significantly to the antimicrobial activity of the crude extract of F. lyrata.

Acknoledgements