International Journal of Drug Development and Research

Keywords

Antibacterial activity; Garcinia gummi-gutta; Disc diffusion method; Antioxidant property; Minimum inhibitory concentration; Pseudomonas aeruginosa

Introduction

The Genus Garcinia contains around 200 species, where 35 species are found in the two ecosystems of India, the Western Ghats and The Himalayan foot hills [1]. Garcinia gummi-gutta is a most common species found in Western Ghats [1,2]. G. gummi-gutta belongs to the family Guttiferae. It is a hardwood underutilized medicinal fruit crop, where its fruits are used in food preparations, especially in curries (Figure 1a and 1b) [3,4]. Previous studies had shown that, the leaf extract of the plant had shown strong antifungal activity on three fungal species namely Phytopthora sp., Curvularia sp., and Corynesporia sp., which could be used as natural fungicide [5]. G. gummi-gutta had also shown to increase the levels of erythrocytes, leucocytes, thrombocytes, hemoglobin and was found to decrease the level of glucose, total cholesterol and LDL level in catfish. This was due to the presence of a Hypolipidaemic compound, Hydroxy citric acid. It also increased immunity by increasing cellular immunological indicators [6-11].

Materials and Methods

Sample collection

The dried fruits of Garcinia gummi-gutta which was readily available in the shops (Vernacular Name: Kodampuli) was bought from a local merchant in Kerala and the sample was authenticated by a local botanist. The dried fruits were stored in room temperature for future use.

Extract preparation

For the preparation of extract, three different solvents, viz Water, Acetone and Ethanol were used. The dried fruits of G. gummi-gutta were cut into very small pieces using a sterile knife. Six grams of the dried fruits were transferred into separate flasks containing 30 ml of the solvents. The mixture was kept in a shaker at room temperature (30°C) at 170 rpm for 24 hours for continuous shaking. The extracts were collected in sterile falcon tubes in a laminar air flow chamber and were centrifuged at 4900 rpm for 10 minutes to remove the debris. The supernatant containing the extracts were collected in a sterile falcon tube and stored at 4°C for further use.

Antibacterial test assay

Antimicrobial susceptibility test was carried out by the Disc diffusion method [10]. The Petri-dish containing Muller Hinton Agar was plated with 0.1 ml culture of different bacterial strains (E. coli, B. subtilis, S. aureus, P. aeruginosa). The plates inoculated with bacteria, were made in triplicate. The discs containing the G. gummi-gutta extracts were placed on the agar using sterile forceps. Chloramphenicol (30 mcg) was used as positive control. The plates were incubated at 37°C for about 18-24 hours. The diameter of resultant Zone of inhibition was measured in millimeters.

Evaluation of antioxidant property by reducing power assay

Reducing power assay was done by the modified method of Nikhat et al. Initially 1% (w/v) Ascorbic acid and 1% (w/v) G. gummi-gutta extract were prepared in sterile distilled water and then further diluted to a working concentration ranging from 200 to 1000 μg/ml by with phosphate buffer (pH- 6.6). Then 2.5 ml of 1% (w/v) Potassium ferricyanide solution was added. The mixture was incubated at 50°C for 20 minutes in a water bath. 2.5 ml of 10% (w/v) Trichloroacetic acid were added into the mixture followed by centrifugation at 4900 rpm for 12 minutes. The supernatant of the solution was transferred into sterile test tubes and equal volume of distilled water and 1 ml of freshly prepared 1% (w/v) Ferric chloride solution was added. The tests were done in triplicate. The absorbance of the reaction mixture was measured at 700 nm using a UV-Visible Spectrophotometer. The averages of the values were plotted on a graph with O.D in the Y- axis and Concentration in the X-axis. The Absorbance of standard Ascorbic acid solution was compared with G. gummi-gutta extract solution [11].

Minimum inhibitory concentration

Different concentration of G. gummi-gutta extracts (w/v) i.e., 1%, 2%, 5%, 10%, 20%, 30%, 40% and 50% were prepared in sterile distilled water. 1 ml of each of the different concentration of the extract was added to a sterile test tube containing 5 ml of nutrient broth that contained 10-5 cells of E. coli, which was obtained after serial dilution. The Control test tube did not contain G. gummi-gutta extract. The tubes were then incubated at 37°C for about 18-24 h. Based on Spectrophotometric analysis, the lowest concentration of the extract which inhibited the growth of the organism was considered as Minimum Inhibitory Concentration which was compared with Blank and Control. Blank contained only the broth without the inoculum whereas, the Control contained inoculum.

Statistical analysis

The results of the Disc diffusion test were expressed as Mean ± S.D. The statistical significance was measured using one way ANOVA. The ‘P value’ was found to be <0.05, which is considered to be significant.

Results and Discussion

Antibacterial test assay

The different extracts of G. gummi-gutta showed good to moderate Antibacterial effect on all the strains that was tested. Various extract showed different effects on the bacteria, which were summarized in Table 1. For this study two gram positive bacteria (B. subtilis, S. aureus) and two gram negative bacteria (E. coli, P. aeruginosa) were used. Chloramphenicol (30 mcg) was used as a positive control. The main reason to use Chloramphenicol was because of its broadspectrum antibiotic activity and it was seen that number of multi drug resistant bacteria were still sensitive to Chloramphenicol [14]. From the ‘Standard Zone Size Interpretative Chart for Chloramphenicol’, the results were interpreted. For Chloramphenicol (30 mcg), Resistant: 12 mm and less, Intermediate: 13-17 mm and Sensitive: 18 mm and more.

For E. coli, the water extract showed an inhibition zone of about 18 mm (approx), which indicates that the water extract had shown a good Antibacterial activity. The ethanol and acetone extracts which had a zone of about 17 mm and 16.5 mm (approx) respectively, indicates a moderate activity on the microorganisms. For B. subtilis and S. aureus, both the water and ethanol extracts showed a zone of inhibition of about 15.6 mm and 15.3 mm (approx) respectively, indicating a moderate activity on both the organisms, whereas, the acetone extract with an inhibition zone of about 18.3 mm (approx) for both the organisms showed a good Antibacterial activity. For P. aeruginosa - the water, ethanol and acetone extracts showed inhibitory zones of about 41 mm, 44 mm and 38 mm respectively. This indicates that P. aeruginosa is highly sensitive to all the extracts.

In a nutshell, E. coli was sensitive to the water extract. Both B. subtilis and S. aureus, were sensitive to acetone extract, whereas, P. aeruginosa was highly sensitive to all the extracts. As stated by Naveen et al. G. gummi-gutta contains many Antibacterial compounds which could have contributed for the good activity against microbes. Further research is essential to isolate and identify the compound for therapeutic purpose.

Reducing power assay

For the measuring the Reducing power of any compound, the capability of the compound to convert the Ferric ion (Fe+3) to Ferrous ion (Fe+2) was measured. In this assay, Yellow colour of the solution changes to Blue colour as the Ferric ion (red) to Ferrous ion (blue). Absorbance was measured at 700 nm. Higher absorbance indicates that the compound has more Antioxidant property.

In this study, G. gummi-gutta extract was compared with Ascorbic acid (Vitamin C) for determining its Reducing power. From the results obtained, it was found that the G. gummi-gutta extract had a higher Antioxidant property than Ascorbic acid solution. Ascorbic acid was used to test because of its natural Antioxidant potential. The Antioxidant property of the G. gummi-gutta extract and Vitamin C has been summarized in Table 2 and graphically represented in Figure 2.

Minimum inhibitory concentration

The Minimum inhibitory concentration is the minimum concentration of the compound required to inhibit the growth of an organism, which was determined by comparing the growth of the bacteria in the control test tube, with the other test tubes by checking their turbidity in UV- Visible spectrophotometer at 600 nm. The organism tested was E. coli. From the results obtained, the G. gummigutta extract of concentration 1% and 2% showed growth, which indicated that there was no inhibition in the growth. Slight growth was observed at 5% extract concentration. The extract concentrations above 10% showed no growth of E. coli. From the results obtained, it was evident that the MIC of the extract for E. coli was about 10%.

Conclusion

From the above studies we can conclude that G. gummi-gutta showed a good Antibacterial activity on all the organisms tested. It also showed good Antioxidant activity. So G. gummi-gutta can be used as Antibacterial agent and also can be used as Antioxidant in Food and Pharmaceutical industries. Further investigations must be performed to examine the Antibacterial properties in other stains and other pathogenic bacteria. Cytotoxicity will be very minimal or null since G. gummi-gutta is already used in cooking. Since not much research has been done in this area, the other active compounds present, should be examined for the benefit of humans.

Acknowledgements

The authors thank the VIT University for providing the opportunity to carry out the research. They also thank Mrs. Roopa Rachel and Mr. Roshan P Shajan for providing the Garcinia fruits from Kerala for carrying out the research. The authors also thank Ms. Shoba Sundaramoorthy for her constant support throughout the research.

Tables at a glance

Table 1 Table 2

Figures at a glance

Figure 1 Figure 2

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