1. INTRODUCTION:

In India, medicinal plants form the backbone of several indigenous traditional systems of medicine. Pharmacological studies have acknowledged the value of medicinal plants as potential source of bioactive compounds¹. Phytochemicals from medicinal plants serve as lead compounds in drug discovery and design². Medicinal plants are rich source of novel drugs that forms the ingredients in traditional system of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates, bioactive principles and lead compounds in synthetic drugs³.

WHO, report depicts that more than 80% of world’s population rely on plant based products to meet health care needs. Nearly, 25 to 45% of modern prescriptions contain plant derived lead molecules as a basic source in drug formulations. The value of plant based prescribed drugs in 1990 was estimated at $ 15.5 billion which ahs been on the raise since then. Furthermore, about 42% of 25 top selling drugs marketed world wide are either directly obtained from natural sources or entities derived from plant products⁴.

Furthermore the active components of herbal remedies have the advantages of being combined with many other substances that appear to be inactive. However, these complementary components give the plant as a whole safety and efficiency much superior to that of its isolated and pure active components. Presently, in the developing countries, synthetic drugs are not only expensive and inadequate for the treatment of diseases but are also often with adulteration and side effects⁵. Therefore, there is the need to search for plants and plant derived formulations of medicinal value.

Ashwagandharishta is a polyherbal hydro alcoholic preparation and is used as rasayana. Rasayanas are used to promote health and longevity by increasing defense against disease, arresting the ageing process and revitalizing the body in debilitated conditions⁶. The chief ingredient of Ashwagandharishta is roots of Ashwagandha, Withania somnifera, commonly known for its usefulness in the treatment of hypercholesterolemia, arthritis in combination with other drugs, is also credited to be hypoglycemic and diuretic⁷. The pharmacological effect of the roots of Withania somnifera is attributed to withanolides, a group of steroidal lactones⁸.

Besides Withania roots, the other ingredients of Ashwagandharishta as arjuna (bark of Terminalia arjuna), liquorice (roots of Glycyrrhiza glabra), majith (roots of Rubia cordifolia), rasna (roots of Alpinia chinensis), taj (inner bark of Cinnamomum zeylanicum), nagarmotha (rhizomes of Cyperus rotundus), haritaki (fruits of Terminalia chebula), turmeric (rhizomes of Curcuma longa), nagakesara (stamens of Mesua ferrea) etc. contain a rich quantity of polyphenolic compounds and flavonoids and possess significant antioxidant activity^9-10. Therefore, we undertook the present investigation to evaluate the antimicrobial activity of Ashwagandharishta-T, Ashwagandharishta-M prepared by traditional and modern methods respectivelyand marketed Ashwagandharishta against common human pathogens.

2. MATERIAL AND METHODS:

2.1 Preparation of Ashwagandharishta-T

This was prepared by the method as given in the Ayurvedic Formulary of India⁶. The ingredients of Ashwagandharishta were procured from local market, Jamnagar. Identification of all the individual plant material was done as per Ayurvedic Pharmacopoeia of India. Authentication of all these ingredients was done by Dr. G D Bagchi, Scientist, Department of Taxonomy and Pharmacognosy, Central Institute of Medicinal and Aromatic Plants, Lucknow. Prepared herbarium has been deposited in the CIMAP for future reference.

According to this method, coarsely powdered ashwagandha roots (Withania somnifera) with prescribed ingredients were placed in polished vessel of brass along with prescribed quantity of water (24.576 l), and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one eighths of the prescribed quantity (3.072 l), then the heating was stopped and it was filtered in cleaned vessel and after that honey was added. Then, dhataki flowers (Woodfordia floribunda) and prakshepa dravyas as sonth, marich, pippali, tvak, tejpatra, priyangu and nagakesara were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33^oC±1^oC. After 15 days, completion of fermentation was confirmed by standard tests¹¹. The fermented preparation was filtered with cotton cloth and kept in cleaned covered vessel for further next seven days. Then, the preparation was poured in amber colored glass bottles, packed and properly labeled.

2.2 Preparation of Ashwagandharishta-M

Method of preparation was same as followed with Ashwagandharishta-T only dhataki flowers were replaced with yeast for inducing fermentation¹².

2.3 Antimicrobial Activity Test

Antimicrobial activity of Ashawagandharishta-T, Ashwagandharishta-M and marketed Ashwagandharishta was tested using a modified disc diffusion assay (DDA) method originally described by Baurer (1966)¹³. Test preparations of Ashwagandharishta were dissolved in 20% DMSO treated water. The inoculums for each microorganism were prepared from broth cultures (10⁵ CFU/ml). A loop of culture from the slant stock was cultured in nutrient agar medium overnight and spread with a sterile swab into Petri-plates. Sterile disc (6 mm dia, Hi-media Mumbai, India) impregnated with test preparations (100µl/disc) and Kanamycin (30µg/disc) were placed on the culture plates and incubated for 24h at 37ºC. The solvent (DMSO) loaded disc without test preparations served as control in the study. The results were recorded by measuring the zones of growth inhibition. Clear inhibition zones around discs indicated the presence of antimicrobial activity. All data of antimicrobial activity were taken as average of triplicate.

3. RESULTS:

All types of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M prepared by traditional and modern methods respectively and marketed Ashwagandharishta showed significant antibacterial activity by exhibiting significant zone of inhibition against common human pathogens as Staphylococcus aureus, bacillus subtilis, Salmonella typhii, Escherichia coli and Pseudomonas aeruginosa as shown in Table 1.

Table1. Diameter of Zone of Inhibition (mm) of Ashwagandharishta-T, Ashwagandharishta-M and marketed Ashwagandharishta

Sample	Zone of Inhibition (mm)
Sample	Staphylococcus aureus	Bacillus subtilis	Salmonella typhii	Escherichia coli	Pseudomonas aeruginosa
Ashwagandharishta-T (100µl/disc)	21.42±0.93	24.78±0.49	22.42±1.19	25.62±0.73	24.64±0.49
Ashwagandharishta-M (100µl/disc)	20.68±1.14	22.96±0.57	21.35±0.61	24.48±0.85	22.85±0.74
Marketed Ashwagandharishta (100µl/disc)	20.52±1.26	23.46±0.69	21.42±0.59	23.97±1.16	23.53±1.12
Kanamycin (30µg/disc)	28±1.24	34±0.98	33.14±0.87	34.91±1.42	32.64±0.59
Negative Control (DMSO)	-ve	-ve	-ve	-ve	-ve

All values are shown as mean±SD of three replicates

4. DISCUSSION:

Plants are known to have beneficial therapeutic effects documented in Traditional Indian System of Medicine. Though bioactive products of Ashwagandha and its preparations as Ashwagandharishta have been used in treatment of various aliments since time immemorial, role of phytochemicals in inhibition of growth of microorganisms has gained less prominence¹⁴. In the present study, preparations of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M and marketed Ashwagandharishta exhibited significant antibacterial activity against common human pathogens. Further investigations may lead to the development of naturally derived new antibiotics of high potency.

5. REFERENCES:

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Received on 19.06.2014 Accepted on 29.06.2014

Asian J. Res. Pharm. Sci. 4(3): July-Sept. 2014; Page 115-117