1. INTRODUCTION:

The increasing prevalence of multi drug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics is major threat for human life. The efforts have been made to discover new antimicrobial compounds from various kinds of sources such as micro-organisms, animals, and plants. One of such resources is folk medicines. Systematic screening of them may result in the discovery of novel effective compounds (N. Tomoko et al.¹).

Plants are the rich source of bioactive molecules. Plants, as sources of medicinal compounds, have continued to play a dominant role in the maintenance of human health since ancient times (Farombi EO²). Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in drug development programs in the pharmaceutical industry.

The methanolic extract Boerrhavia diffusa screened against different microorganisms responsible for various infection. Boerrhavia diffusa (Punarnava) belongs to Nyctaginaceae family is an important indigenous medicine for the treatment of dyspepsia, jaundice, enlargement of spleen, abdominal pain, arthritis, and asthma (A.N Sahu et al.)³. Present investigation reports the antimicrobial and cytotoxic activities of Boerrhavia diffusa.

2. MATERIALS AND METHOD:

Plant material: Fresh plant material was collected from NIIST campus, Thiruvananthapuram and were identified and authenticated by Dept. of Botany, University of Kerala, Thiruvananthapuram, India.

Preparation of plant extract: The plant material, stem and leaves were separated and air dried. Dried stem and leaves were powdered and extracted with methanol using a soxhlet apparatus. The extract was filtered and concentrated under vacuum in a rotary evaporator (<50°C). The concentrated extract was then made up to 100 ml with Methanol. For cytotoxic evaluation, whole plant was extracted with methanol as mentioned before. Then the concentrated extract was lyophilized and kept in 4°C until use.

Test microorganism: The strains were obtained from microbial type culture collection, Chandigarh, India. The bacterial strains studied are Escherichia coli (MTCC 739), Staphylococus aureus (MTCC 2940), Micrococcus luteus (MTCC 2470), Bacillus subtilis (MTCC 121), Staphylococcus aureus sub sp aureus (MTCC 96), Pseudomonas aeruginosa (MTCC 2453).

The fungal strains were obtained from Biotechnology division, NIIST (CSIR), Thiruvananthapuram, India. The fungi strains studied are Botrytis cinerea (KACC 43524), Fusarium oxysporum (KACC 42109), Penicillium expansum (KACC 40815), Penicillum chrysogenum (KACC 40399), Rhizoctonia solani (KACC40136), Fusarium moniliforme (KACC 41031), Geotrichum candidum (OKI 605/8402), and Yeast strains studied Candida albicans and Yarrowia lipolytica (KACC 41237).

3. Antimicrobial assay:

Agar well diffusion was employed for the antibacterial assay of plant extract (C. Perez et al.⁴, and B. Bhushan et al.⁵). A loop full of the strain was inoculated in 5-5 ml of nutrient broth in a test tube and incubated on a rotary shaker at 140 rpm for 24 hrs. Mueller Hinton Agar no. 2 was prepared for the study. The test strain (1ml) was inoculated in to the media (inoculum size 10⁸ cells/ml) when the temperature reached 40-42^oC care was taken to ensure proper homogenization. The experiment was performed under strict aseptic condition. After the medium was solidified, a well was made in the plates with the help of a cup-borer (0.85cm). The test compound was introduced in to the well and the plates were incubated for 24 hrs at 37^oC. Microbial growth was determined by measuring the diameter of zone of inhibition. Methanol was used as the control. The control activity was deducted from the test and the result obtained was plotted.

For antifungal activity, agar well bioassay was employed (Linday⁶ and A. Sabitha⁷) using PDA medium (Hi- Media, 39g). Fungi were inoculated and the procedures were similar as mentioned before. The treated and the controls were kept in an incubator at 37 C For 24 hrs to 72 and inhibition zones were measured. Three to four replicates were maintained for each treatment.

4. Cytotoxicity assay:

The Cytotoxicity assays were performed according to MTT method (M. Kawaze et al.⁸). The human colon tumor cells (HCT-116) and Leukemia cells (K-562) were used for the MTT assay. The cells were harvested (1 ×10⁵ cells / well) and inoculated in 96 well microtiter plates. The cells were washed with phosphate buffered saline (PBS) and the cultured cell were than incubated for 72 hrs with the test sample and the final concentration of 25- 800µg/ ml. 100 µl of MTT solution (5mg Ml^-1 in PBS, PH 7.2) is added to each well and plates are incubated for 4 h at 37^oC, after incubation 200µl DMSO was added to each well and the absorbance of each well was measured at 570 nm using a micro plate reader and surviving cell fraction was calculated. The inhibition of cell viability was calculated by means of formula % inhibition = (1- absorbency of treated cells / absorbance of untreated cells) × 100

3. RESULTS AND DISCUSSION:

Antibacterial activity of stem and leaf extract of Boerhaavia diffusa were given in Table. 1. The antibacterial activity of stem extract was relatively low against all six tested bacteria. The low activity of extract at lower concentration may be due to crude nature of the test solution. The highest activity was found at highest concentration (1200 μg) for stem extract to all bacteria tested ranging from 12mm - 17 mm of inhibition zone. Highest activity with stem extract was detected against Pseudomonas aeruginosa, Micrococcus luteus and Escherichia coli with 17, 15 and 14 mm zone of inhibition relatively. In the case of leaf extract, 450-1500 µg concentrations show low activity. But significant antibacterial activity was found at 1800 µg concentration. The test organism which were very sensitive at 1800 µg concentration were Staphylococcus aureus sub sp aureus (inhibition zone 22mm ), Staphylococus aureus (inhibition zone 16mm), and Micrococcus luteus (inhibition zone 13mm) respectively.

Effect of different concentration (300-1800 µg) of Boerrhavia diffusa extract was also tested against seven different fungi and two types of yeast (Table. 2). All the concentration of the test solution inhibited the fungal species with varying degree of sensitivity. The antifungal activity was very less at 300-500 µg for stem extract and 450-750 µg for leaf extracts. The diameter of inhibition zones ranged from 5-13 mm among different fungal species with respective stem and leaf extract. An increased activity was observed with increase in concentration of test solution. The maximum zone of inhibition was found for 1200 and 1800 µg with stem and leaf respectively. Among the test organisms, high inhibition zones were observed in P. expansum (12mm) and F. oxysporum (13mm) with the extract of stem and leaf respectively. Highest zone of inhibition against C.albicans and Y. lipolytica were 11mm in both stem and leaf extracts.

The crude methanolic extract of Boerhaavia diffusa was evaluated for their cytotoxic activity on cancer cell line (HCT-116 and K-562). The extract was tested at the range (25 - 800 µg /ml after 72 h treatment. The result showed that methanolic extract did not have any significant cytotoxicity against both of the cell lines. In HCT-116 cell lines, the percentage of Cytotoxicity is 42.1 % and that in K-562 cell lines is 28.4% at the concentration of 800μg (Fig. 1).

Figure 1. In vitro cytotoxic activity of a methanol extract of Boerrhavia diffusa on Human colon tumor cells (HCT-116) and Leukemia cells (K-562).The values are shown as the mean of triplicate determination with the standard.

Table. 1 Antimicrobial activity of Boerhavia diffusa

Extract	Zone of inhibition (mm)
Extract	Conc. (μg)	*E. coli*	*S .aureus*	*M. .Luteus*	*S. aureus*	*P. areuginosa*	B. subtilis
Stem	300	9	8	9	8	10	9
	500	11	9	11	9	11	11
	1000	12	10	11	9	11	11
	1200	14	12	15	12	17	13
	Control	9	8	9	9	8	9
Leaf	450	8	5	11	11	7	7
	750	8	6	11	11	7	7
	1500	11	8	12	12	9	9
	1800	11	16	13	22	12	12
	Control	8	10	10	11	9	9

*Control used is methanol (100μl)

Extract	Zone of inhibition (mm)
Stem	Conc. (μg)	B. *cinerea*	F. *oxysporum*	P. *expansum*	P. *chrysogenum*	R. *solani*	F. *moniliforme*	G. *candidum*	C. *albicans*	*Y .* *lipolytica*
	300	6	5	8	6	6	8	7	8	6
	500	7	7	11	7	6	8	7	9	8
	1000	8	8	12	7	7	7	7	11	9
	1200	9	9	12	9	7	8	9	11	11
	Control	8	6	9	8	6	7	8	7	9
Leaf	450	7	9	7	7	6	8	7	7	4
	750	7	11	11	8	6	5	7	8	8
	1500	8	11	9	9	7	4	8	8	9
	1800	8	13	9	9	7	8	9	9	11
	Control	7	8	8	8	6	7	8	8	11

Table.2 Anti fungal activity of Boerhavia diffusa

*Control used is methanol (100μl)