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 has been on the raise since then. Furthermore, about 42% of 25 top selling drugs marketed worldwide 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.

Amritarishta is a polyherbal hydroalcoholic Ayurvedic preparation and is used as antioxidant and advised as a choice of remedy in mostly all types of fevers⁶. The chief ingredient of Amritarishta is guduchi, dried stem of Tinospora cordifolia. The chemical constituents reported from stems of Tinospora cordifolia belong to different classes such as alkaloids as tinosporin^7-8, glycosides as cordifoliosides-A and cordifolioside-B^9-10, steroids as β- sitosterol¹¹, sesquiterpenoid as tinocordifolin¹² and a large amount of phenolic compounds as gallic aciod, ellagic acid, catechin and epicatechin¹³. These compounds have many notable medicinal properties as antidiabetic¹⁴, hepatoprotective¹⁵, antioxidant¹⁶, antimalarial¹⁷, immunomodulatory¹⁸ and antineoplastic properties¹⁹. Therefore, we undertook the present investigation to evaluate the antimicrobial activity of Amritarishta-T, Amritarishta-M prepared by traditional and modern methods respectively and marketed Amritarishta against common human pathogens.

2. Materials and Methods:

2.1 Preparation of Amritarishta-T:

This was prepared by the method as given in The Ayurvedic Formulary of India, Part-I^6. All the ingredients of Amritarishta were procured from local market, Jamnagar while jaggery was procured from local market, Mehsana. Authentication of all the ingredients of Amritarishta 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 Central Institute of Medicinal and Aromatic Plants, Lucknow for future reference. Identification of all the individual plant material was done as per The Ayurvedic Pharmacopoeia of India. Quantity of ingredients taken for the preparation of batch size 3.072 l of Amritarishta has been calculated according to the formula as given in The Ayurvedic Formulary of India, Part-I, 2000.

According to this method, coarsely powdered stems of guduchi (Tinospora cordifolia) with prescribed ingredients as Aegle marmelos (stem bark), Oroxylum indicum (roots), Gmelina arborea (stem bark), Stereospermum suaveolns (stem bark), Premna integrifolia (stem bark), Hedysarum gangeticum (entire plant), whole plant of Paederia foetida, entire plant of Solanum indicum, entire plant of Solanum xanthocarpum and Tribulus terrestris were placed in polished vessel of brass along with prescribed quantity of water (12.288l) and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity(3.072 l) , then the heating was stopped and it was filtered in cleaned vessel and after that jaggery was added and mixed properly. Then, prakshepa dravyas as svet jiraka, raktapuspaka, saptaparni, sunthi, marica, pippali, nagakesara, mustaka, katuka, ativisa and indravaruni in fine powdered form were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33±1°C. After 15 days completion of fermentation was confirmed by standard tests²⁰. The fermented preparation was filtered with cotton cloth and kept in clean covered vessel for further next seven days. Then, when the fine suspended particles settled down, it is strained again and poured in amber colored glass bottles previously rinsed with ethyl alcohol, packed and properly labelled.

2.2 Preparation of Amritarishta-M:

Method of preparation of Amritarishta-M was same as followed with Amritarishta-T only in addition to jaggery, yeast was also added for inducing fermentation²¹.

2.3 Antimicrobial Activity Test

Antimicrobial activity of Amritarishta-T, Amritarishta-M and marketed Amritarishta was tested using a modified disc diffusion assay (DDA) method originally described by Baurer (1966)²². Test preparations of Amritarishta 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 Amritarishta as Amritarishta-T, Amritarishta-M prepared by traditional and modern methods respectively and marketed Amritarishta 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 Amritarishta-T, Amritarishta-M and marketed Amritarishta

Sample	Zone of Inhibition (mm)
Sample	Staphylococcus aureus	Bacillus subtilis	Salmonella typhii	Escherichia coli	Pseudomonas aeruginosa
Amritarishta-T (100µl/disc)	19.89±0.47	24.62±0.57	21.49±1.43	22.64±0.45	22.92±1.26
Amritarishta-M (100µl/disc)	19.24±0.64	23.74±0.42	20.95±0.82	21.94±0.37	22.28±1.19
Marketed Amritarishta (100µl/disc)	18.75±0.72	23.12±0.89	19.78±0.57	21.25±0.49	21.64±0.97
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 Giloe and its preparations as Amritarishta have been used in treatment of various ailments since time immemorial, role of phytochemicals in inhibition of growth of microorganisms has gained less prominence²³. In the present study, preparations of Amritarishta as Amritarishta-T, Amritarishta-M and marketed Amritarishta exhibited significant antibacterial activity against common human pathogens. Further investigations may lead to the development of naturally derived new antibiotics of high potency.

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Received on 22.06.2014 Accepted on 29.06.2014

Asian J. Pharm. Res. 4(2): April-June 2014; Page 114-116