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  • The Internet Journal of Pharmacology
  • Volume 8
  • Number 2

Original Article

Antibacterial Activity of Ficus lyrata -An In vitro Study

W Rizvi., M Rizvi, R Kumar, A Kumar, I Shukla, M Parveen

Keywords

antibacterial activity, ficus lyrata, flavonoid, triterpenoid

Citation

W Rizvi., M Rizvi, R Kumar, A Kumar, I Shukla, M Parveen. Antibacterial Activity of Ficus lyrata -An In vitro Study. The Internet Journal of Pharmacology. 2009 Volume 8 Number 2.

Abstract


Objective : To evaluate the antibacterial potential of extract and compounds isolated from Ficus lyrata. Materials
and Methods: The aqueous and ethanol extracts of F. lyrata and two pure compounds i.e. Ursolic acid (FL-1) and Acacetin-7-O- neohesperidoside (FL-2) isolated from F. lyrata were tested against several standard bacterial strains by the Kirby Bauer method on Mueller Hinton agar and two fold serial dilution method with saline for determining the MIC. Key Findings : F. lyrata showed potent antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Shigella dysenteriae Shigella boydii, Citrobacter freundii, Proteus vulgaris, Proteus mirabilis, Klebsiella. The aqueous extract was more potent than alcoholic extract. The isolated compounds were more potent and showed an improved spectrum of activity as compared to the crude extract. The minimum inhibitory concentration (MIC) of aqueous extract of F.lyrata and the isolated compounds were found to be significantly low for all the tested bacterial strains. Conclusion : The study suggests that the extracts obtained from the leaves of F. lyrata possess excellent antibacterial activity which could possibly be attributed to the two compounds i.e. FL-1 and FL-2.

 

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:

Plant Moraceae (Ficus lyrata) leaves were collected from University campus and authenticated by Prof. Wazahat Hussain in the department of Botany. A voucher specimen has been deposited in the department no. 215

Extraction procedure:

Alcoholic extract: Air dried and powdered leaves of F. lyrata were thoroughly extracted twice with distilled ethanol which on concentration under reduced pressure gave a reddish brown residue. Yield was 6% w/w.

Aqueous extract: Air dried and powdered leaves of F. lyrata were refluxed with distilled water for 18-24 hours followed by concentration over a water bath. Yield was 4% w/w.

The structure of pure compounds Ursolic acid (FL-1) and Acacetin-7-O- neohesperidoside. (FL-2) were elucidated on the basis of UV, IR, IH-NMR.

Preparation of extract solution for screening of antibacterial activity: 200 mg of aqueous and alcoholic extracts were dissolved in 1ml of sterile distilled water and ethanol respectively. Ursolic acid was dissolved in petrol whereas Acacetin-7-O- neohesperidoside (FL-2) was dissolved in ethanol. Sterile discs of 6 mm diameter were prepared from Whatman filter paper No.1. Each disc was incorporated with 10µl of either extract and isolated pure compounds (200 mg/ml) to give a fixed concentration of 2 mg/disc and left to dry under the laminar flow cabinet overnight. Disc diffusion assay was performed according to NCCLS guidelines using the Kirby Bauer method on Mueller Hinton agar (7).Each extract was tested in quadruplicate. Sterile water impregnated discs served as negative control.

Microorganisms:

Both gram positive and gram negative bacterial isolates were tested against the plant extracts and isolated pure compounds.

Gram positive strains: Staphylococcus. aureus ATCC 25923, Methicillin resistant Staph aureus (MRSA), Coagulase negative staphylococcus ATCC 12228, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, Streptococcus viridans. Prior to testing with the test material these strains were tested against ampicillin, cefaclor, gentamicin, amikacin, erythromycin, clindamycin, oxacillin, and vancomycin to confirm they were susceptible to these drugs, except for MRSA

Gram negative strains: Pseudomonas aeruginosa ATCC 27853, Escherichia. coli ATCC 25922, Klebsiella pneumoniae, ESBL producing Escherichia coli and K. pneumoniae ATCC 700603 Salmonella typhi, Salmonella paratyphi A, Salmonella typhimurium, Vibrio cholerae serotypes and ogawa inaba, Citrobacter freundii, Proteus vulgaris, Proteus mirabilis, Shigella dysenteraie, Shigella boydii. Cefaclor, ceftriaxone, cefotaxime, cefoperazone , cefoperazone sulbactam, amikacin, gentamicin, gatifloxacin, ciprofloxacin and ofloxacin were used to test the susceptibility of the gram negative standard strains.

The inoculum size was adjusted to 0.5 Mc Farland for each of these strains before performing disc diffusion assay. The plates were incubated for 24 hours at 37oC. The antibacterial activity was interpreted according to the zones of inhibition produced around the impregnated discs measured to the nearest millimeter. An inhibition zone of 12 mm or more was considered to indicate good antibacterial activity.

Determination of MIC:

The MIC’s were determined for all strains which showed significant zones of inhibition. MIC was performed using the two fold serial dilution method with saline at a final concentration ranging from 5120 mg/L to 0.025 mg/L. Each extract and isolated compound was tested in duplicate. The bacterial suspension was used as positive control and sterile saline as negative control. Two control plates prepared under the same conditions but without the plant extract were included in every MIC determination as a quality control measure. An overnight broth was prepared by picking several identical colonies. The inoculum when applied to the plate with a steers replicator device contained 103 to 104 colony forming units/spot. The plates were incubated overnight at 37oC. The MIC was determined as the lowest concentration of antibiotic inhibiting visible growth after 18-24 hours. One to three colonies or a faint haze was considered a negative result.

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

We acknowledge Miss Priyamvada Raghav and Dr. Onkar Singh for their technical help in our research work.

References

1. Goldstein FW. Combating resistance in a challenging, changing environment. Clin. Microbiol. Infect 2007;13:2-6.
2. Anonymous..Wealth of India, raw materials: New Delhi: CSIR, 1956
3. Dhar ML et al. Screening of Indian plants for biological activity. Indian Journal experimental biology 1968; 6 (4):232- 247.
4. Basudan OA.et al. A new chromone from Ficus lyrata. Journal of Asian Natural Products Research 2005; 7(1): 81-85.
5. Dhawan B N et al. Screening of Indian plants for biological activity. Indian journal experimental biology 1977;15: 208-219.
6. Mandal SC et al. Studies on antibacterial activity of Ficus racemosa Linn.leaf extract. Phytotherapy Research 2000;14 (4): 278 – 280.
7. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—(Sixth Edition:) Approved Standard M7-A6. Wayne, PA,: NCCLS; 2003.
8. Muñoz-Mingarro D et al. Biological activity of extracts from Catalpa bignonioides Walt. (Bignoniaceae). J Ethonopharmacol 2003;87: 163–7.
9. Coelho de Souza, G. et al. Ethnopharmacological studies of antimicrobial remedies in the south of Brazil. J Ethnopharmacol 2004.; 90 :135–43.
10. Lin, J. et al. Preliminary screening of some traditional Zullu medicinal plants for anti-inflammatory and antimicrobial activities. J.Ethnopharmacol 1999; 68: 267-74.
11. Kowalewski, Z. et al. Antibiotic action of Beta- Ursolic acid. Arch. Immunol. Ther. Exp. ( Warsz.) 1976 ;24 (1): 115-119.
12. Zoletova, N., et al. Preparation of some derivatives of ursolic acid and their antimicrobial activity. Chemical Abstracts 1987; 106: 18867e.
13. Sattar, A.et al. Chemical composition and biological activity of leaf exudates from some Lamiaceae plants. Pharmazie 1995; 50: 62-65.

Author Information

Waseem Rizvi., (M.D)
Department of Pharmacology, J.N. Medical College, A.M.U.

Meher Rizvi, (M.D)
Department of Microbiology, J.N. Medical College, A.M.U.

Rakesh Kumar, (PhD)
Department of Chemistry, J.N. Medical College, A.M.U.

Anil Kumar, (M.D)
Department of Pharmacology, J.N. Medical College, A.M.U.

Indu Shukla, (M.D)
Department of Microbiology, J.N. Medical College, A.M.U.

Mehtab Parveen, (PhD)
Department of Chemistry, J.N. Medical College, A.M.U.

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