International Journal of Drug Development and Research

Key words

oldenlandia biflora, pergularia daemia, antibacterial activity, cup plate method.

Introduction

Plants are the source of most complex organic molecules. These molecules are showing wide structural diversities and are serving as templates for many semi synthetic and synthetic drug molecules. Plant derived drug molecules are frequently showing their role in treating the disease conditions with minimal side effects comparing to the synthetic molecules. Plants have a limitless ability to synthesize aromatic substances mainly secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total [1]. So, still more drug molecules that are hiding in the plants want to be investigated for isolating the new drugs having less toxic and more therapeutic effects their by decreasing the cost of the drugs and increasing the treatment efficiency.

Many antibacterial agents are available in the market, which are developed by the great scientists. But still the microbes are challenging the scientists by developing the resistance to the presently available drugs. Plants are known to produce a variety of compounds to protect themselves against a variety of their pathogens and therefore considered as potential source for different classes of antimicrobial substances [2]. So, our present study was aimed to identify new anti bacterial agent from two selected medicinal plants.

Oldenlandia biflora is a perennial herb belongs to the family rubiaceae. It mainly grows on seaside rocks up to 5-20cm height. The leaves are waxy, 1- 2.5cm long and 7- 12mm wide. It contains white colored flowers [3]. By our ethno- botanical survey we found that this plant was using as folk medicine in treating dysentery, fever and also gastric ulcers.

Pergularia daemia is a perennial twining herb belongs to the family asclepiadaceae. It was mainly found in tropical and sub- tropical areas secreting milky latex. Leaves are thin, broadly ovate and heart-shaped 2-12 cm long, covered with soft hairs. The literature review shows that it was reported to have anti-fertility [4], hepatoprotective [5], wound healing [6], anti diabetic [7] and antiinflammatory activity [8].

Materials and methods

Plant materials

The leaves of the plants are collected from Kakinada, Andhra Pradesh and authenticated by department of pharmacognosy, Koringa College of pharmacy, korangi. The collected leaves are shade dried and powdered. Then the powder was passed through sieve no. 40 and then stored in an air tight container until the use.

Bacterial cultures

Gram- positive bacterial cultures like Bacillus subtilis, Staphylococcus aureus and gram – negative organisms like Pseudomonas epidermis, Escherichia coli, Shigella dysenteriae and Salmonella typhi are used as test organisms. All the test strains were maintained on nutrient agar slopes and were sub cultured once in every two-week.

Extraction of plant materials

The leaf powders of the two plants were extracted successively in soxhlet apparatus, using petroleum ether and ethyl acetate respectively. The aqueous extract was prepared by maceration method. The extracts were distilled, concentrated and dried. These extracts are stored in desecrator at room temperature until the use. At the time of testing, the extracts were reconstituted to a concentration of 100mg/ml in Dimethyl Sulphoxide (DMSO).

Preliminary phytochemical analysis

Qualitative phytochemical analysis of the crude powder of the 12 plants collected was determined as follows: Tannins (200 mg plant material in 10 ml distilled water, filtered); a 2 ml filtrate + 2 ml FeCl3, blue-black precipitate indicated the presence of Tannins. Alkaloids (200 mg plant material in 10 ml methanol, filtered); a 2 ml filtrate + 1% HCl + steam, 1 ml filtrate + 6 drops of Mayor’s reagents /Wagner’s reagent/Dragendroff reagent, creamish precipitate /brownish-red precipitate/orange precipitate indicated the presence of respective alkaloids. Saponins (frothing test: 0.5 ml filtrate + 5 ml distilled water); frothing persistence indicated presence of saponins. Cardiac glycosides (Keller-Kiliani test: 2 ml filtrate + 1 ml glacial acetic acid + FeCl3 + conc. H2SO4); green-blue color indicated the presence of cardiac glycosides. Steroids (Liebermann-Burchard reaction: 200 mg plant material in 10 ml chloroform, filtered); a 2 ml filtrate + 2 ml acetic anhydride + conc. H2SO4. bluegreen ring indicated the presence of terpenoids. Flavonoids (200 mg plant material in 10 ml ethanol, filtered); a 2 ml filtrate + conc. HCl + magnesium ribbon pink-tomato red color indicated the presence of flavonoids [9].

Antibacterial assay

Antibacterial activity of the extracts was determined by cup plate method on nutrient agar medium [10]. Cups are made in nutrient agar plate using cork borer (6 mm) and inoculums containing 108 CFU/ml of bacteria were spread on the solid plates with a sterile swab moistened with the bacterial suspension. Then 50 μl of each extract was placed in the cups made in inoculated plates. 50 μl of control (DMSO) was also maintained with extracts which served as control. After introducing the sample, standard and control in the cups the plates were kept in refrigerator at 4ºC for 2 hrs, for diffusion and then the plates were incubated for 24 h at 37°C. After proper incubation, antibacterial activity was determined by measuring the diameter of the zone of the inhibition around the well and the activity was compared with Ciprofloxacin (10 μg/ml). Three replicates were carried out for each extract against each of the test organism.

Determination of Minimal Inhibitory Concentration (MIC)

MIC was determined by broth dilution method. In this method 0.1ml of standardized suspension of bacteria (108 CFU/ml) was added to each tube containing different concentrations of the extracts (prepared by two fold dilution method) and incubated for 24h at 37°C. The lowest concentration of the tube that did not show any visible growth by macroscopic evaluation was considered as the MIC.

Results and Discussion

The petroleum ether, ethyl acetate and aqueous extracts of oldenlandia biflora and pergularia daemia were examined for possible sources of antibacterial activities and phytochemical constituents. Both the plants were found to contain alkaloids, tannins and flavonoids (Table: I). In addition to these phytochemicals Pergularia daemia also contains cardiac glycosides. Saponins and steroids are found to be absent in both the plant materials.

Both the plants exhibited significant antibacterial activity. All the extracts of Oldenlandia biflora show antibacterial activity but petroleum ether extracts of Pergularia daemia dose not exhibited any activity. Both the plant extracts exhibited more activity against Bacillus subtilis and Staphylococcus aureus. The ethyl acetate extracts of both the plants exhibited more antibacterial activity compared to petroleum ether and aqueous extracts. Pergularia daemia dose not exhibited antibacterial activity against the Shigella dysenteriae.

The MIC of each extract of both the plants was determined by broth dilution method. The ethyl acetate extract of Oldenlandia biflora inhibited the growth of the Bacillus subtilis at a concentration of 125μg/ml. Both the plant extracts exhibited MIC of 500μg/ml against Pseudomonas epidermis, Shigella dysenteriae, Salmonella typhi and Escherichia coli and MIC of 250μg/ml against Bacillus subtilis and Staphylococcus aureus. The petroleum ether extracts of Pergularia daemia dose not exhibited any activity.

Conclusion

From the present experiment it can be concluded that both the plant extracts are exhibiting anti bacterial activity. The plants may be further explored for its phytochemical profile to recognize the active constituent accountable for antibacterial activity.

Tables at a glance

Table 1 Table 2 Table 3

Figures at a glance

Figure 1

References

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