Antimicrobial activity of Ficus benghalensis and Ficus racemosa roots L

Objective: To evaluate the antimicrobial activity of aqueous and ethanolic extracts of Ficus benghalensis and Ficus racemosa against three bacterial strains that is Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumonia. Methods: The different concentrations viz. 25, 50 and 75 mg/ml of aqueous as well as ethanolic extract solutions of roots of Ficus benghalensis & Ficus racemosa were tested against above three bacterial strains. The effects were compared with that of a standard antibiotic-loaded disc, ampicillin (10 mcg/disc) of Himedia Labs. Finally, the plates were incubated with lids closed at 37oC for 24 hours. Discs were observed for zones of inhibition by measuring the Diameter of Inhibition Zone (DIZ) using a ruler. Results: Ficus racemosa ethanolic extract showed maximum inhibition against Staphylococcus aureus when compared with Ficus benghalensis ethanolic extract. However standard drug ampicillin showed maximum antimicrobial activity compared with both the plants. Conclusion: In conclusion, it was clearly observed that ethanolic extract of both the plants were having good antimicrobial activity towards Staphylococcus aureus.


Introduction
Medicinal plants have been used as an exemplary source for centuries as an alternative remedy for treating human diseases because they contain numerous active constituents of therapeutic value [1]. The development of microbial resistance to antibiotics has led the researches to investigate the alternative sources for the treatment of resistant strains [2]. Presently 80 percent of the world population relies on plant derived medicines and serves as first line of defense in maintaining health and combating many diseases [3]. However, their scientific study has been made possible only after the development of microbiology. Natural antimicrobials can be derived from barks, stems, leaves, flowers and fruits of plants, various animal tissues or from microorganisms [4]. Over 50% of all modern clinical drugs are having their origin in natural products [5]. In general, bacteria have the genetic ability to transmit and acquire resistance which is utilized as therapeutic agents [6].
Flavonoid compounds exhibit inhibitory effect against bacteria. Flavonoids, hydroxyl groups on their β-rings are more active against microorganisms and have also been found that the more hydroxylation, the greater the antimicrobial activity [7]. Levels of total phenolics, total flavonol and total flavanoids compounds in F. benghalensis aerial roots in 70 mg/g of extract, 3 mg/g quercetin equivalent and 5 mg quercetin equivalent/gm extract have also been reported [8]. Though, the antimicrobial properties of plants have been investigated time to time by a number of researchers worldwide, but recently it has gained much importance globally after the development of molecular biology. Thus, in the present work, aqueous as well as ethanolic extracts of Ficus benghalensis & Ficus racemosa roots have been investigated for their antimicrobial activities against various microbes and interesting results have been obtained.

Materials and Methods
(i) Plant material:

Aqueous extract:
Fresh roots of Ficus bengalensis & Ficus racemosa were collected and identified by Dr. M.K.Jangid, Taxonomist, Department of Botany, Hemchandra North Gujarat University, India. A voucher specimen has been submitted to the Institute herbarium. The roots were dried and cut into small pieces which were then mechanically crushed. 4 kg of these crushed roots were continuously extracted with distilled water using soxhlet up to 48 h. The extract was filtered and concentrated in rotary evaporator at 35-40°C under reduced pressure to obtain a semisolid material.
Ethanolic extract: 2 kg of fresh roots of Ficus bengalensis & Ficus racemosa were shade dried for one week and then extracted with ethanol (10 lit × 3) for 72 hours. The ethanolic extracts were filtered and concentrated in rotary evaporator under reduced pressure to obtain a semisolid material.
(ii) Bacterial strains, stocks and growth in vitro: Bacterial strains of Klebsiella pneumonia, Staphylococcus aureus and Escherichia coli were clinical isolates obtained from the Department of Microbiology, Vidyabharti Trust College of Pharmacy, Umrakh, India. All cultures were grown at 37ºC in a shaker incubator (190-220 rpm) overnight. Luria Bertani broth (Himedia), Luria Bertani agar (Himedia) and sterile discs as well as Ampicillin disc (Himedia) were used in antimicrobial sensitivity testing.

(iii) Determination of Zone of Inhibition (ZOI):
The freshly prepared inoculum of Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli were swabbed all over the surface of the LB Agar plates. Three sterile discs of 6 mm diameter were placed on the inoculated media with the help of a disc dispenser and were numbered properly. The different concentrations viz. 25, 50 and 75 mg/ml of aqueous as well as ethanolic extract solutions of roots of Ficus benghalensis & Ficus racemosa were poured on the discs with the help of sterilized micropipette. Discs were left for some time till the extract solutions diffused in them. The effects were compared with that of a standard antibiotic-loaded disc, ampicillin (10 mcg/disc) of Himedia Labs. Finally, the plates were incubated with lids closed at 37ºC for 24 hours. Discs were observed for zones of inhibition by measuring the Diameter of Inhibition Zone (DIZ) using a ruler.

Results
Roots of F. benghalensis & F. racemosa aqueous and ethanolic extracts have been screened for their antimicrobial activity and very interesting profiles have been found against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli bacterial strains. Effect of varied concentrations of 25, 50 and 75 mg/ml of aqueous as well as ethanolic extract solutions was studied and comparison with standard drugs, viz. ampicillin was done. Their zones of inhibition (ZOI) can be seen at a glance as follows: The entire data suggested that the ethanolic extract of Ficus bengalensis and Ficus racemosa roots was active against all the bacterial strains against which ampicillin was found to have maximum activity. To the contrary, aqueous extract showed little activity against each bacterial strain except Staphylococcus aureus, depicting that the extract possessed lesser activity than that of the standard drug, Ampicillin. The use of medicinal plants to heal diseases, including infectious ones, has been extensively applied by people worldwide. Data from literature as well as our results reveal the great potential of plants for therapeutic treatment, in spite of the fact that they have not been completely investigated. Therefore, more studies need to be conducted to search for new compounds responsible for their antibacterial activity. Once extracted, and before being used in new therapeutic treatments, they should have their toxicity tested in vivo. Bioassays [9,10]have demonstrated the toxicity of extracts from different plants [11]. It is not surprising that there are differences in the antibacterial effects of plant groups, due to the phytochemical differences between species. To better evaluate the plants growing naturally that are potentially useful resources, additional studies are necessary from both the medicinal and economic stand points [12]. Therefore, our results revealed the importance of F. benghalensis & F. racemosa aqueous as well as ethanolic extracts when associated with antibiotics, to control resistant bacteria, which are becoming a threat to human health. Furthermore, in a few cases, these plant extracts were active against antibiotic resistant bacteria under very low concentration, thus minimizing the possible toxic effects.

Conclusion
The activity was highest against Staphylococcus aureus and lowest against Klebsiella pneumoniae in both the aqueous and ethanolic extracts. The results suggest that Ficus benghalensis roots are very promising not only as antidiabetic [13] and antioxidant agents but also as antimicrobial agent with special reference to Staphylococcus aureus infections as both, aqueous and ethanolic extracts were active against this bacterial strain. This antimicrobial profile of Ficus benghalensis & Ficus racemosa roots can be exploited for developing a novel antimicrobial agent of high potential. Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antibacterial activity assay [14]. Many reports are available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and anti-inflammatory properties of plants [15,16]. Some of these observations have helped in identifying the active principle responsible for such activities and in the developing drugs for the therapeutic use in human beings. Since, most of the plants, rich in flavonoids, possess antimicrobial activity [17], therefore, the presence of flavonoids identified in Ficus bengalensis & Ficus racemosa roots by phytochemical screening might be responsible for its antimicrobial activity.