INTRODUCTION:

In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world. Consequently, new infections can occur in hospitals resulting in high mortality. From 1980 to 1990, Montelli¹ documented a high incidence of resistant microorganisms in clinical microbiology in Brazil. Therefore, several medicinal plants have been tried against pathogenic microorganisms.^2,3 Such plants should be investigated to better understand their properties, safety and efficiency.⁴ According to World Health Organization⁵ medicinal plants would be the best source to obtain a variety of drugs.

Withania somnifera (Solanacae) are gaining attention in various field of research, as they are best suited to the present environmental conditions. W. somnifera used for its anti-inflammatory effect⁶, analgesic effect⁷, Antioxidant⁸, memory-improving effects.⁹ It shows relaxant and antispasmodic effects against several plasmogens on intenstinal, uterine, blood vascular, bronchial and tracheal muscles. Withanolides possess remarkable antibacterial, anti-arthritic and immunosuppressive. The anti tumor and radio sensitizing effects of W. somnifera have been studied.^10X

Klebsiella is the genus name for one of these bacteria found in the respiratory, intestinal, and urino-genital tracts of animals and man. When Klebsiella bacteria get outside of the gut, however, serious infection can occur. K. pneumonia more frequently causes lung destruction and pockets of pus in the lung (known as abscesses). The mortality rate for untreated cases is around 90%. There may also be pus surrounding the lung (known as empyema), respiratory infections, such as bronchitis, which is usually a hospital-acquired infection.^11,12 P. merabilis is a rod shaped bacterium causes obstruction and renal failure. It can also cause wound infections, septicemia and pneumonias, mostly in hospitalized patients. A. tumefaciens (Plant pathogen) uses horizontal gene transfer to cause tumors “crown gall disease” in plants. It can be responsible for opportunistic infections in humans with weakened immune system.^13,14

MATERIAL AND METHOD

Experimental design: Crude extracts of flower of W. somnifera (RUBL-20668) were prepared with a series of non polar to polar solvents by hot extraction method¹⁵ in soxhlet assembly. Different extracts were then screened for antimicrobial activity by disc diffusion Assay¹⁶ against a few medically important bacteria, plant pathogen and fungi. The fraction showing best activity was then used for determining of minimum inhibitory concentration (MIC) by tube dilution method¹⁷and minimum bactericidal/fungicidal concentration (MBC/MFC).

Collection of plant material: Flowers of W. somnifera (RUBL-20668) were collected in the month of January from Jaipur district of Rajasthan. Plants samples were identified and deposited in the herbarium, department of botany, university of Rajasthan, Jaipur. The collected plant materials were separately shade dried for one week. Shade dried flower were powdered with the help of grinder. Fine powder of flower was stored in clean container to be used for Soxhlet extraction following the method of Subramanian and Nagarjan¹⁸, in different polar solvents selected.

Extraction procedure: Flowers (10 gm) were sequentially extracted with different solvents (250 ml) according to their increasing polarity (hexane < petroleum ether < toluene < benzene < iso propyl alcohol < chloroform < ethyl acetate < acetone < ethanol < glacial acetic acid < water) by using Soxhlet apparatus for 18 hours at a temperature not exceeding the boiling point of the respective solvent.¹⁹ The obtained extracts were filtered by using Whatman No. 1 filter paper and then concentrated at 40⁰C by using an evaporator.²⁰ The residual extracts were stored in refrigerator at 4⁰C in small and sterile glass bottles. Percent extractive values were calculated by the formula (table 1).

Weight of dried extract

Percent Extracts = ———————————— x 100

Weight of dried plant material

Drugs and chemicals used:

Drugs: Gentamycin (for bacteria) and Ketoconazole (for fungi)

Chemicals: hexane, petroleum ether, toluene, benzene, iso propyl alcohol, chloroform, ethyl acetate, acetone, ethanol, glacial acetic acid and water, Muller-Hinton Agar (MHA) Medium, Nutrient Agar (for bacteria), Sabouraud Dextrose Agar (for fungi).

Micro-organisms: The organisms used in this study were three Gram-negative bacteria and one fungus, viz., Proteus merabilis (MTCC-3310), Klebsiella pnemoniae (MTCC-4030), Agerobacterium tumefaciens (MTCC-431) and Aspergillus niger (MTCC-282). Selected microorganisms were procured from IMTECH, Chandigarh, India. Bacterial strains were grown and maintained on Muller-Hinton Agar Medium²¹, sub cultured regularly (after every 30 days) and stored at 4^oC as well as at –80^oC by preparing suspensions in 10% glycerol.

Screening for antimicrobial activity: Bacterial strains were grown and maintained on Nutrient Agar medium, while fungi were maintained on Sabouraud Dextrose Agar medium (SDA). Disc diffusion assay (DDA) was performed for screening. NA and SDA base plates were seeded with the bacterial and fungal inoculum, respectively (inoculum size 1×10⁸ CFU/ml for bacteria and 1×10⁷ cell/ml for fungi). Sterile filters paper discs (Whatman no. 1, 5mm in diameter) were impregnated with 100 μl of each of the extracts (100 mg/ml) to give a final concentration of 1 mg/disc and left to dry in vaccuo so as to remove residual solvent, which might interfere with the determination. Petri plates were pre-seeded with 15 ml of growth agar medium and 1.0 ml of inoculum.²²Extract discs were then placed on the seeded agar plates. Each extract was tested in triplicate with gentamycin (10mcg/disc) and ketoconazole (10mcg/disc) as standard for bacteria and fungi, respectively. The plates were kept at 4°C for 1 h for diffusion of extract, thereafter were incubated at 37°C for bacteria (24 h) and 27°C for fungi (48 h).²³ The inhibition zones were measured and compared with the standard reference antibiotics.^24-26 Activity Index (AI) for each extract was calculated by the formula (Table 2).

Inhibition Zone of the sample

Activity index (AI) = __________________________

Inhibition Zone of the standard

Broth micro-dilution method: Broth micro-dilution method²⁷ was followed for determination of Minimum inhibitory concentration (MIC) values for each plant extract showing antimicrobial activity against test pathogens. To measure the MIC values, various concentrations of the stock, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029 mg/ml were assayed against the test pathogens. Plant extracts were re-suspended in acetone (which has no activity against test microorganisms) to make 15mg/ml final concentration and then two fold serially diluted; 1 ml of each extract was added to test tubes containing 1 ml of sterile NA media (for bacteria) and SDA (for fungi). The tubes were then inoculated with standard size of microbial suspension (for bacteria 1×10⁸ CFU/ml and 1×10⁷ cell/ml for fungi) and the tubes were incubated at 37°C for 24 h for bacteria and 28°C for 48 h for fungi in a BOD incubator and observed for change in turbidity after 24 h compared with the growth and in controls.²⁸ A tube containing nutrient broth and inoculum but no extract was taken as control. The least extract concentration which inhibited the growth of the test organisms was taken as MIC. Bacterial and fungal suspensions were used as negative control, while broth containing standard drug was used as positive control. Each extract was assayed in duplicate and each time two sets of tubes were prepared, one was kept for incubation while another set was kept at 4°C for comparing the turbidity in the test tubes. The MIC values were taken as the lowest concentration of the extracts in the test tubes that showed no turbidity after incubation.^29-30 The turbidity of the test tube was interpreted as visible growth of microorganisms.

Determination of Minimum bactericidal/fungicidal concentration (MBC/MFC): Equal volume of the various concentration of each extract and nutrient broth mixed in micro-tubes to make up 0.5ml of solution. 0.5ml of McFarland standard of the organism suspension was added to each tube.³¹ The tubes were incubated aerobically at 37^oC for 24 h for bacteria and 28°C for 48 h for fungi. Two control tubes were maintained for each test batch. These include tube-containing extract without inoculum and the tube containing the growth medium and inoculum. The MBC was determined by sub culturing the test dilution on Mueller Hinton Agar and further incubated for 24 h. The highest dilution that yielded no single bacterial colony was taken as the Minimum bactericidal Concentration.³² MBC was calculated for some of the extracts showed high antimicrobial activity against highly sensitive organisms.

Total activity (TA) determination: Total activity is the volume at which the test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the amount of extract from 1 g plant material by the MIC of the same extract or compound isolated and is expressed in ml/g.³³

Extract per gram dried plant part

Total Activity = ____________________________

MIC of extract

Statistical Analysis: Mean value and standard deviation were calculated for each test bacteria and fungi. Data were analyzed by one-way ANOVA and p values were considered significant at p > 0.005.²²

RESULTS

1.Qualitative and quantitative estimation: The preliminary phyto–profiling (Qualitative and quantitative estimation) for the flower of W. somnifera were carried out according to Farnsworth³⁴ wherein the consistency was found to be sticky in all polar solvent extracts and non-sticky in water extracts which are supported by Singariya.^22,28The yield (% w/w) of the extracts was also analyzed wherein the highest yield was recorded for glacial acetic acid extracts (29.33%) and followed water extract (21.95%) (Graph 1).

Graph 1: Total yield (% w/w) of flower extracts of Withania somnifera in different polar solvents

2. Total activity: Total activity indicates the volume at which extract can be diluted with still having ability to kill microorganism. Glacial acetic acid extracts showed high values of TA against all the pathogens which prove the potential to inhibit the growth of the test microorganisms, even at low concentration. Maximum TA values were recorded 1271.20 ml, 317.12 ml, 281.86 ml and 158.65 mg/ml against A. tumefaciens, K. pneumoniae, A. niger and P. merabilis respectively (table 1).

3. Antimicrobial activity: Antimicrobial activity (assessed in terms of inhibition zone in mm and activity index) of the flower of W. somnifera extracts in different polar solvents, tested against selected microorganisms were recorded (Table 2). In the present study total eleven extracts of selected plant were tested for their bioactivity, among which seven extracts showed significant antimicrobial potential against test microbes.

(a) Antibacterial activity: Highest antibacterial activity was recorded for glacial acetic acid extract (IZ-40.17±0.23 and AI-2.009) and iso propyl alcohol extract (IZ-16.67±0.22 and AI-0.834) against A. tumefaciens.

(b) Antifungal activity: Highest antifungal activity was recorded for glacial acetic acid extract (IZ-15.83±0.21 and AI-1.055) against A. niger

4. MIC and MBC/MFC: MIC and MBC/MFC values were evaluated for those extracts, which were showing activity in diffusion assay. The range of MIC and MBC/MFC of extracts recorded was 0.234-15 mg/ml. In the present investigation lowest MIC values 0.234 mg/ml and 0.439 mg/ml were recorded for glacial acetic acid extract against A. tumefaciens and A. niger respectively, followed by 0.938 mg/ml for glacial acetic acid as well as for acetone extract against K. pneumoniae and A. tumefaciens accordingly, indicating significant antimicrobial potential of test extracts. MIC and MBC/MFC values were found equal show bactericidal and fungicidal activity (table 3).

DISCUSSION:

Results of the present study showed that 7/11 extracts tested inhibited the growth of selected bacteria and fungi, indicating bioactive nature of W. somnifera. Glacial acetic acid, iso propyl alcohol and acetone extracts express maximum antimicrobial activities by suppressing the growth of all microbes under investigation. In the present study, most of the extracts were found to be potent inhibitor of tested. Excellent antibacterial and antifungal activities were observed by glacial acetic acid and ethyl acetate extracts were shown by activity index were found higher against microorganisms tested; indicate the bacteriostatic/ fungistatic effects of the extracts. A. tumefaciens bacteria were the most susceptible organisms.

Table 1: Phyto-chemical estimation and Total activity of Flower extracts of Withania somnifera

Solvent	Yield (%)	Color	Consistency	Total activity of flower extracts
Solvent	Yield (%)	Color	Consistency	P. m.	K. p.	A. t.	A. n.
W	21.95	Green	Nonsticky	58.54	-	-	-
AA	29.75	Dark brown	Sticky	158.65	317.12	1271.2	281.86
E	15.04	Yellowish green	Sticky	-	-	-	-
A	3.77	Yellowish green	Sticky	-	10.05	40.20	-
EA	4.83	Yellowish green	Sticky	25.74	-	-	-
C	5.73	Dark green	Sticky	-	-	-	-
IP	6.68	Yellowish green	Sticky	-	17.82	35.65	-
B	3.69	Green	Sticky	-	-	-	-
T	4.02	Yellowish green	Sticky	21.42	-	-	-
PE	2.45	Yellowish green	Sticky	-	-	13.04	-
H	2.61	Yellowish green	Sticky	-	-	-	-

P. m.- Proteus merabilis, K. p.- Klebsiella pnemoniae, A. t.- Agerobacterium tumefaciens, A. n.- Aspergillus niger, H-hexane, PE-petroleum ether, T-toluene, B-benzene, IP-iso propyl alcohol, C-chloroform, EA-ethyl acetate, A-acetone, E-ethanol, AA-glacial acetic acid, W-water.

Table 2: Inhibition Zone (mm) and Activity index (AI) for Flower extracts of Withania somnifera.

z	Bio-activity of Flower extracts of W. somnifera against pathogens
	Proteus merabilis		Klebsiella pnemoniae		Agerobacterium tumefaciens		Aspergillus niger
	IZ	AI	IZ	AI	IZ	AI	IZ	AI
W	9.33±0.28	0.933	-	-	-	-	-	-
AA	9.17±0.25	0.917	16.17±0.24	0.809	40.17±0.23	2.009	15.83±0.2 1	1.055
E	-	-	-	-	-	-	-	-
A	-	-	9.33±0.24	0.467	15.33±0.23	0.767	-	-
EA	12.50±0.64	1.25	-	-	-	-	-	-
C	-	-	-	-	-	-	-	-
IP	-	-	11.67±0.27	0.584	16.67±0.22	0.834	-	-
B	-	-	-	-	-	-	-	-
T	9.17±0.25	0.917	-	-	-	-	-	-
PE	-	-	-	-	10.33±0.23	0.517	-	-
H	-	-	-	-	-	-	-	-

All values are mean±SD, n-3 (p>0.005), IZ-Inhibition Zone (mm±S.D.), AI-Activity index, H-hexane, PE-petroleum ether, T-toluene, B-benzene, IP-isopropyl alcohol, C-chloroform, EA-ethyl acetate, A-acetone, E-ethanol, AA-glacial acetic acid, W-water.

Table 3: MIC and MBC/MFC of Flower extracts of Withania somnifera

S. No.	Polar Solvents	Bio-activity of Flower extracts of Withania somnifera
		P. merabilis		K. pnemoniae		A. tumefaciens		A. niger
		MIC	MBC	MIC	MBC	MIC	MBC	MIC	MFC
1.	W	3.75	7.5	-	-	-	-	-	-
2.	AA	1.875	3.75	0.938	0.938	0.234	0.234	0.439	0.938
3.	E	-	-	-	-	-	-	-	-
4.	A	-	-	3.75	7.5	0.938	0.938	-	-
5.	EA	1.875	1.875	-	-	-	-	-	-
6.	C	-	-	-	-	-	-	-	-
7.	IP	-	-	3.75	7.5	1.875	3.75	-	-
8.	B	-	-	-	-	-	-	-	-
9.	T	1.875	3.75	-	-	-	-	-	-
10.	PE	-	-	-	-	1.875	1.875	-	-
11.	H	-	-	-	-	-	-	-	-

MIC - Minimum inhibitory concentration (mg/ml), MBC - Minimum bactericidal concentration (mg/ml), MFC - Minimum fungicidal concentration (mg/ml), H-hexane, PE-petroleum ether, T-toluene, B-benzene, IP-iso propyl alcohol, C-chloroform, EA-ethyl acetate, A-acetone, E-ethanol, AA-glacial acetic acid, W-water

CONCLUSION:

Flower extracts of W. somnifera under this study not only inhibit the bacterial/fungal growth but the IZ developed, was more or less permanent when compared with the IZ developed by the standard drug used, as after sometime bacterial/fungal colonies could be easily seen in IZ developed by standard drugs. In the light of the fact that microorganism are becoming resistant against the drugs in use, present investigation is of great significance, as far as the future drugs are concerned and uses of selected plants by the pharmaceutical industries for preparing plant based antimicrobials drugs.

ACKNOWLEDGEMENT:

Authors are expressing their thanks to UGC for providing the funds for the project under Dr. D. S. Kothari, Post doctoral fellowship scheme.

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Received on 10.01.2012 Modified on 12.02.2012

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