Phytochemical and Antimicrobial Activity of Digera Muricata (L.) Mart

The successive Soxhlet extract of Digera muricata (L.) Mart. (Amaranthaceae) were extracted using petroleum ether, chloroform, ethanol and distilled water in ascending order of the polarity. These extracts were subjected to screening of preliminary phytochemicals tests. The results indicate the presence of alkaloids, flavonoids, phenols, tannins, terpenes and saponins. These extracts further subjected to the antimicrobial activity. Among the bacteria used, the petroleum ether extract gave highest zone of inhibition at 400 μg/well against V. cholerae. Similarly, in fungi the ethanol extract exhibited highest zone of inhibition at 400 μg/well against Candida albicans.


Introduction
The Digera muricata (L.) Mart (Amaranthaceae) wild edible plant commonly known as 'Cancali soppu'. It is commonly distributed throughout the India. In Ayurveda the herb is considered as a cooling, astringent to the bowels and also used as laxative. The flowers and seeds are used to treat urinary discharges 1 . Boiled root infusion given to mother after child birth for lactation purpose 2 .
The bacterial and fungal diseases are a major threat to human population and domestic animals, the death caused by infectious diseases ranked 5 th , has become the third leading cause of death 3 . Plant derived antimicrobial agents have been largely overlooked. Hence, the D. muricata (L.) Mart. is used in both folk and traditional system of medicine. The present investigation is carried out to rationalize the therapeutic claims as antimicrobial activity using few pathogenic bacteria and fungi.

Experimental
The plant material of Digera muricata was collected from the agricultural field of Humnabad Taluk in Bidar district, Karnataka, India in January 2008 and identified with the help of

Preliminary phytochemical tests
The extracts of Digera muricata were dissolved in their respective organic solvents and were subjected for preliminary phytochemicals test 5 to screen the presence of various secondary metabolites like alkaloids, flavonoids, phenols, glycosides, sterols, saponins, terpenoids, lignin and tannins.

Antimicrobial activity assay
Antimicrobial activity of organic solvent extracts was determined by cup diffusion method on nutrient agar medium 6 , for bacterial and potato dextrose agar (PDA) medium for fungi. Cups were made in nutrient agar plate using sterile cork borer (5 mm) and inoculums containing 10 -6 CFU/mL of bacteria were spread on the solid plates with a sterile swab moistened with the bacterial suspension. Then 50 µL each of all solvent extracts were placed and the cups medium inoculated plates. The treatments also included 50 µL of DMF separately which served as control. Streptomycin sulphate and Nistatin 1 µg/mL at their respective recommended dosage were treated for comparative efficacy. The plates were incubated for 24 and 38 h for bacterial and fungi and temperature maintained 37 0 C and 27 0 C respectively. The zone of inhibition around the wells was measured in mm (millimeter). For each treatment five replicates were maintained. The data was subjected to statistical analysis using SPSS for windows software.

Results and Discussion
The preliminary phytochemical tests result indicate the presence of phenol, flavonoids, alkaloids, terpenes, sterols, tannins, glycosides and lignins test indicate ( Table 1). The quantitative estimation of primary and secondary metabolite results shows higher amount of terpenoids and tannins secondary and carbohydrates from primary metabolites ( Table 2).

Antibacterial study
The organic successive Soxhlet extracts of Digera muricata (L.) Mart. i.e., petroleum ether, chloroform, ethanol and distilled water, have shown significant zone of inhibition of bacterial growth at the concentrations of 200 and 400 µg/well against these bacterial strains (   The above results clearly indicate that, the organic extracts showed maximum activity against all the tested pathogens. However, the poor activity recorded in aqueous extracts. Among the tested organisms V. cholerae shows higher zone of inhibition in petroleum ether extract at 400 µg/ml (19.13 ± 0.38) compared to all the tested extracts and Streptomycin sulphate a standard drug. Similar, activities were reported by Alam et al. 7 , in amaranthaceae and in achyranthes aspera by Sunil et al. 8 , in alternanthera sessile by Lina and Manuel 9 , in Amaranthus hypochondriacus. While, least zone of inhibitions were recorded in aqueous extract. These results indicate the presences of high amount of phytoconstituents in the organic plant extracts are the responsible for the antimicrobial activity.

Antifungal studies
The organic extracts i.e., petroleum ether, chloroform, ethanol and aqueous extracts have exhibited moderate to significant activity at the concentrations of 200 and 400 µg/well against Aspergillus flavus, A. niger, A. terreus and Candida albicans (Table 4).
Among which a good antifungal activity is exhibited by petroleum ether against C. albicans (19.6 ± 0.58) whereas, chloroform extract against the C. albicans (20.32 ± 0.87) and A. flavus (17.06 ± 1.63). However, the aqueous extract has displayed feeble activity against all the fungi.
The results clearly indicate that both the (200 and 400 µg/well) tested concentrations the maximum zone of inhibition were observed in the higher doses (400µg/well) and all the tested fungi and all the extracts. The maximum inhibition was observed against the C. albicans compared to standard (Nystatin). Nystatin is the most commonly used standard antifungal agent (positive control) which has strong affinity towards sterols particularly ergosterols. Similar, reports reported in Amaranthaceae by Quetin -Leclereq et al. 10 The antifungal activity against Candida species reported by Sunil et al. 8 , in Alternathera sessilis.

Conclusion
D. muricata is commonly found as a weed on road side and in agricultural fields throughout India. The wild plant is edible and used in traditional medicine for curing various ailments. The presence of wide range of phytochemical constituents indicates that plant could serve as lead for the development of novel agents for various pathological disorders. However, less information is available regarding chemical constituents and there is a lack of phytochemical studies of Digera muricata plant. So, further in detailed phytochemical study is needed.