Activity of Amritāriṣṭa against selected Pyrexia Causing Microbes

Transcription

1 Activity of Amritāriṣṭa against selected Pyrexia Causing Microbes Rajni 1 Ramakant Vyas 2 Parimi Suresh 3 A b s t r a c t Āyurveda, the Upaveda of Atharvaveda has emphasized on the principle of protecting the health in a healthy individual and eradicating the disease in the diseased. Living in good health has been the dream of man right from the day one of his existence on earth. Now a day s infectious disease makes a lot of trouble for human being. In order to avoid different infections there are lots of antibiotics which are derived from the microbial sources in synthetic manner. Due to problem like adverse effect, limited life span of antibiotics effects, hence researches are currently underway to look for natural origin. A number of Ayurvedic classical preparations were being used in cases of infections, and they were found to be effective clinically. Amritāriṣṭa is a time tested formulation with proven antipyretic effect. The chief ingredient of Amritāriṣṭa is Gudūci, it is renowned in Ayurvedic therapeutics for its usefulness in the treatment of Jvara (fever). Its therapeutically effectiveness in Jvara may be due to its antibiotic effect on fever causing microbes. The present study was performed to screen the antimicrobial activity of Amritāriṣṭa against selected pyrexia causing microbes using its petroleum ether extract and benzene extract. The extracts were tested against four bacteria through well diffusion assay where standard azithromycin was used. The results showed that both extracts possess good antimicrobial activity against selected test bacteria. The present results therefore offer a scientific basis for traditional use of the various extracts of Amritāriṣṭa. All the extracts possessed antimicrobial potential against all test bacteria which explains that their use in daily life will generate a resistance or immunity to fight against microorganisms. Key words: Āyurveda, Amritāriṣṭa, Gudūci, Antimicrobial activity. 1 P G Scholar, 2 PhD Scholar, 3 Associate Professor, Department of Rasa Shastra and Bhaishajya Kalpana, National Institute of Āyurveda, Jaipur, Rajasthan. CORRESPONDING AUTHOR Dr. RAJNI PG Scholar, Department of Rasa Shastra and Bhaishajya Kalpana, National Institute of Āyurveda, Jaipur, Rajasthan drrajni.kumawat@gmail.com 649

2 INTRODUCTION In Ayurvedic medicine, many medicinal plants are useful in strengthening human health care system and the formulations based on such medicinal plants play an important role in modern medicine [i]. Ayurvedic practitioners also identified a number of herbal preparations for curing various ailments and diseases [ii]. The primary benefits of using plant-derived medicine are relatively safer than synthetic drugs and offer profound therapeutic benefits [iii]. Single and polyherbal preparations have diverse range of bioactive molecules and play a dominant role in the maintenance of human health since ancient times [iv]. More than 00 herbal preparations are sold as dietary supplements or ethnic traditional medicines [v]. With the advancement in Science and Technology, remarkable progress has been made in the field of medicine with the discoveries of many natural and synthetic drugs [vi]. Antibiotics are undeniably one of the most important therapeutic discoveries of the 20 th century that had effectiveness against serious bacterial infections. However, only one third of the infectious diseases known have been treated from these synthetic products [vii]. This is because of the emergence of resistant pathogens that is beyond doubt the consequence of years of widespread indiscriminate use, incessant and misuse of antibiotics [viii,ix]. Antibiotic resistance has increased substantially in the recent years and is posing an ever increasing therapeutic problem. With the augment in the resistance of many microorganisms to the presently used antimicrobials and the high cost of production of synthetic compounds; in addition to many side effects; there is a need to look for the alternatives. Amritāriṣṭa is a poly-herbal hydro-alcoholic Ayurvedic preparation and is used as antioxidant and advised as a choice of remedy in mostly all types of fevers [x]. The chief ingredient of Amritāriṣṭa is Gudūci, dried stem of Tinospora cordifolia. The chemical constituents reported from stems of Tinospora cordifolia belong to different class such as alkaloids as tinosporin [xi - xii], glycosides as cordifoliosides-a and cordifolioside-b [xiii - xiv], steroids as β-sitosterol [xv], sesquiterpenoid as tinocordifolin [xvi] and a large amount of phenolic compounds as gallic aciod, ellagic acid, catechin and epicatechin [xvii]. These compounds have many notable medicinal properties as antidiabetic [xviii], hepatoprotective [xix], antioxidant [xx], antimalarial [xxi], immunomodulatory [xxii] and antineoplastic [xxiii] properties. Therefore, in the present study attempts were made to screen antimicrobial efficacy of Amritāriṣṭa by using its petroleum ether extract and benzene extract against selected test microbes. MATERIAL AND METHODS Collection: Authentic sample: Under the present research work, Amritāriṣṭa was used for antimicrobial activity. This sample was prepared according to the classical reference (Bhaishajya Ratnavali, Jvararogadhikara,5/ ) in the Dept. of Rasashastra and Bhaishajya Kalpana, National Institute of Āyurveda, Jaipur (Raj.). Sources of test organisms: Bacteria-Pure culture of all test organisms, namely Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi were procured from Institute of Microbial Technology (IMTECH), Chandigarh and the stock cultures were maintained at Dept. of Biotechnology, JECRC University, Jaipur. Table no. 1: For the present study following pure bacterial cultures were taken. Sr. no. Species MTCC No. 1 Staphylococcus aureus (S.A.) Escherichia coli (E.C.) Pseudomonas aeruginosa (P.A.) Salmonella typhi (S.T.) 531 The antibacterial Studies were performed at Research Lab, JECRC University,Jaipur. Microbiological techniques adopted: Preparation of Media and Media Plates: For the development of bacteria, Nutrient Agar Medium (NAM) was performed using 20 gm Agar, 5 gm Peptone, 3 gm beef extract and 3 gm NaCl in 1 liter distilled water and then it was sterilized at lbs pressure and 121 C temperature for minutes. Agar test plates were prepared by pouring 20 ml of NAM into the Petri dishes (10 mm) in aseptic conditions. A saline solution was arranged (by mixing 0.8 % NaCl) in distilled water, pursued by autoclaving and the bacterial cultures were maintained on this medium by regular sub-culturing and incubation at 37 C for hours. In the preparation of the test plates, bacteria, 10- ml along with the respective medium was poured into the petri plates and further analysis were performed on the samples. For evaluation of the bactericidal efficacy, a fresh suspension of the test bacteria was prepared in saline solution from a freshly grown Agar slant.

3 Preparation of test extracts: Amritāriṣṭa was used for various solvent extraction using different solvents i.e. petroleum ether (40-60 ⁰C) and benzene. The use of various solvent system is performed for extraction of various bio actives present in Amritāriṣṭa and each secondary metabolite is eluted with solvent specific extraction. Thus, maximum terpenoids were extracted in petroleum ether solution. Thus, various solvents were used for the extraction of different extracts as active rich fractions for evaluation of antimicrobial efficacy. 50 ml of Amritāriṣṭa volume was added to the 100 ml of respective solvent and soxhlet extracted for 6 hours so that the active ingredients were subjected to the relevant solvent. Later, each of the homogenates was filtered and the residue was re-extracted twice for complete exhaustion, the extracts was pooled individually. Further, these solvents were filtered and the filtrate was concentrated to dryness in vacuo and stored at 4 C in a refrigerator, until screened for antibacterial activity. Bactericidal assay: For bactericidal assay in vitro well diffusion method was based on modified methods of Murray et al, 1995 [xxiv] and Gould, 1952 [xxv], because of reproducibility and precision. The different test organisms were proceeded separately using a sterile swab over previously sterilized culture medium plates and the zone of inhibition were measured around sterilized dried well puncture, there was of three different concentration: 25mg of test extract/well 50mg of test extract/well 75mg of test extract/well And azithromycin mg as reference drugs (standard disk) separately. Such treated well was air-dried at room temperature to remove any residual solvent, which might interfere with the determination, sterilized and inoculated. These plates were initially placed at low temperature for 1 hr so as to allow the maximum diffusion of the compounds from the test disc into the agar plate and later, incubated at 37 C for 24 hrs in case of bacteria, after which the zones of inhibition could be easily observed. Replicates of each test extract were examined and the mean values were then referred. The inhibition zone (IZ) in each case was recorded and the activity index (AI) was calculated as compared with those of their respective standard reference drugs (AI = Inhibition Zone of test sample / Inhibition zone of standard). RESULTS The Petroleum ether extract and Benzene extract of Amritāriṣṭa inhibited the growth of all the four selected bacterial strains in a dose dependent manner. Antimicrobial activity of Petroleum ether extract of Amritāriṣṭa: Table no.2: showing zone of inhibition in mm and Activity Index of Pet ether extract of Amritāriṣṭa and standard drug against selected microbes Sr. no. Sample extract Conc. E.C. S.A. S.T. P.A. 1. Pet ether 2. Standard (AZM) 25mg 50 mg 75 mg mg IZ AI IZ AI IZ AI IZ AI While antimicrobial screening of petroleum ether (40-60 o C) extract of Amritāriṣṭa showed that all the concentrations (25mg, 50mg, 75mg) of the sample showed appreciable efficacy against selected test microbes. Further, table no. 2 clearly depicted that maximum efficacy of pet ether extract was against Salmonella typhi (IZ = 29mm; AI = 1.04) with a concentration of 75 mg and is more than standard drug Azithromycin. Even it was also effective against Escherichia coli (IZ =26mm), Staphylococcus

4 aureus (IZ =25mm) and Pseudomonas aeruginosa (IZ = 18mm) Fig 1 Petroleum Ether Extract of Amritarishta EC SA ST PA 25mg 50 mg 75 mg 18 Images showing zone of inhibition of pet ether extract of Amritāriṣṭa

5 Antimicrobial activity of Benzene extract of Amritāriṣṭa: Table no.3: showing zone of inhibition in mm and Activity Index of Benzene extract of Amritāriṣṭa and standard drug against selected microbes Sr. no. Sample extract Conc. E.C. S.A. S.T. P.A. 1. Benzene 2. Standard (AZM) mg 25mg IZ AI mg IZ AI mg IZ AI IZ AI Benzene extract of Amritāriṣṭa at various concentrations (25mg, 50mg, 75mg) of the sample showed appreciable efficacy against selected test microbes. As it is clearly shown in table no.3 that maximum efficacy of Benzene extract of Amritāriṣṭa was against Escherichia coli (IZ = 25 mm; AI = 0.81) with a concentration of 75 mg. Further, it was also effective against Staphylococcus aureus (IZ = 21 mm), Salmonella typhi (IZ = 24 mm) and Pseudomonas aeruginosa (IZ = 12 mm) Fig 2 Benzene Extract of Amritarishta EC SA ST PA 25mg 50 mg 75 mg Images showing zone of inhibition of benzene extract of Amritarishta

6 DISCUSSION Plants are chemical reservoirs which are known to have beneficial therapeutic effects documented in Traditional Indian System of Medicine. Though bioactive products of Gudūci and its preparations as Amritāriṣṭa have been used in treatment of various ailments since time immemorial. Now a day s people are more prone to infectious diseases. There are antibiotics which are used but due to their origination from microbial sources in synthetic manner they show hypersensitivity reaction and many more side effects. So there is a need of the hour for an effective antibiotic with minimum side effects and this can be possible if the drug is of natural origin. As the present study showed petroleum ether extract and benzene extract of Amritāriṣṭa has antimicrobial potential so it can be used as safe and an effective antibiotic. Also mentioned in Bhaishajya Ratnavali that Amritāriṣṭa pacifies all types of fevers. In the present study, Amritāriṣṭa exhibited significant antibacterial activity against common human pathogens causing fever. Further investigations may lead to the development of naturally derived new antibiotics of high potency and lesser microbial resistance. CONCLUSION The present study was performed to screen the antimicrobial activity of Amritāriṣṭa against selected pyrexia causing microbes using its petroleum ether extract and benzene extract. Petroleum ether extract and benzene extract both shows appreciable antimicrobial activity against test microbes. Petroleum ether extract shows maximum activity against Salmonella typhi (IZ = 29mm; AI = 1.04) with a concentration of 75 mg. Benzene extract possess maximum efficacy against Escherichia coli (IZ = 25 mm; AI = 0.81) at 75 mg concentration. **** REFERENCES i. Patwardhan B, Vaidya AD, Chorghade M. Āyurveda and natural product drug discovery. Curr Sci.2003;86: ii. Pandey MM, Rastogi S, Rawat AK. Indian herbal drug for general healthcare: An overview. Int J Alter Med. 2008;6:1 5. iii. Barrett B, Kiefer D, Rabago D. Assessing the risk and benefits of herbal medicine: An overview of scientific evidence. Alter Health Med. 1999;5:40 9. [PubMed] iv. Handa SS. Indian efforts for quality control and standardization of herbal drugs/products. Proceedings of the 1st joint workshop on quality control and standardization of traditional medicine Indo-China experience.2004 v. WHO, General guidelines for methodologies on research and evaluation of traditional medicine. Geneva: World Health Organization; vi. Antioxidant Efficacy of Some Medicinal Plants Against Food Borne Pathogens. Adv. in Bio.Res. 2010; 4 (2): vii. Sharma A. Antibacterial activity of ethanolic extracts of some arid zone plants. Int. J. of Pharm.Tech. Res. 2011; 3(1): viii. Enne VI, Livermore DM, Stephens P, Hal LMC Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction. The Lancet. 2001; 28: ix. Westh H, Zinn CS, Rosdahl VT. An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from hospitals in 14 countries. Microb. Drug Resist. 2004; 10: x. The Ayurvedic Formulary of India, Part-I. 2000, 1st edition, The Controller of Publications, Delhi, xi. Kumar S, Verma NS, Pande D and Srivastava PS. In vitro regeneration and screening of berberinein Tinospora cordifolia.journal of Medicinal and Aromatic Plant Science 2000; 22:61. xii. Biset NG and Nwaiwu J.Quaternary alkaloids of Tinospora species. Planta Medica 1983; 48: xiii. Maurya R, Wazir V, Tyagi A and Kapil RS. Cordifoliosides A and B, two new phenylpropene disaccharides from Tinosporacordifolia possessing immunostimulant activity. Natural Product Letter 1996;8:7-10. xiv. Gangan VD, Pradhan P, Sipahimalani AT and Banerji A. Cordifoliosides A, B,C:Norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 1994;37: xv. Dixit SN and Khosa RL. Chemical investigation of Tinospora cordifolia. Indian Journal of Applied Chemistry 1971;34:46-7. xvi. Maurya R and Handa SS. Tinocordifolin, a sesquiterpene from Tinospora cirdifolia. Phytochemistry 1998;49: xvii. Kidwai AR, Salooja KC, Sharma VN, Siddiqui S. Chemical examination of Tinospora cordifolia. Journal of Science and Indian Research 1949; 8:1-8. xviii. Stanely M, Prince P and Menon VP. Antioxidant action of Tinospora cordifolia root extract in alloxan diabetic rats.phytotherapy Research 2001;: xix. Mehrotra R, Katiyar CK and Gupta AP. Hepatoprotective compositions and composition for treatment of conditions related to hepatitis-b and E infection. US Patent xx. Prince PS and Menon VP. Antioxidant activity of Tinospora cordifolia roots in experimental diabetes. Journal of Ethnopharmacology 1999;65: xxi. Ikram M, Khattak SG and Gilani SN. Antipyretic studieson some indigenous Pakistani medicinal plants. Journal of Ethnopharmacology 1987;19:

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