PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES STANDARDIZATION OF MARKETED DRAKSHASAVA - A POLYHERBAL AYURVEDIC PRODUCT Girish Sailor*, Avinash Seth, Kavita Parmar, Mohini Patel, Purandare Shrirang Department of Pharmacy, Sumandeep Vidyapeeth, Piparia, Vadodara-391760, Gujarat, India ABSTRACT Traditional medicines are effective but the standardization of Ayurvedic formulations is essential in order to assess the quality of drugs. Drakshasava is a marketed ayurvedic herbal formulation included in ayurvedic formulary containing Vitis vinifera as a main ingredient. The present study aims to develop standardization method for Drakshasava. The formulation was analyzed for various parameters include physicochemical parameters such as ph, specific gravity, total solids content, alcohol content, acid value, amount of reducing and non-reducing sugars and phytoconstituents such as total phenolics, total tannins and total alkaloids. Various extracts of Drakshasava have been prepared and evaluated. The obtained value of physicochemical parameters and phytoconstituents could be used to establish and formulate procedures for standardization and quality controlling of these ayurvedic preparations to maintain batch-to-batch consistency. TLC profile of different extract is also useful to standardize the preparation on quantitative basis. Keywords: Ayurvedic Formulation, Total Phenolic, Total Tannin, TLC, Asava. INTRODUCTION Ayurveda is the indigenous system of medicine being practiced for thousands of years. This plant-based system of medicine has already gained worldwide attention due to its safety and efficacy. The Ayurvedic system tauted as an alternative system of medicine has become especially significant in the post-gatt era 1. Ayurvedic system of treatment has been estimated to meet 70-80% of the healthcare needs of India 2. With the growing need for safer drugs, attention has been drawn to their quality, efficacy and standards of the Ayurvedic formulations 3. Ayurvedic pharmacy advocates the use of quality control tests to make sure that the prepared medicines adhere to the standards mentioned in Ayurveda. Most of the tests described in ancient literature appear to be based on observation and seem subjective without valid scientific backing. Hence standardization and development of reliable quality protocols for Ayurvedic formulations using modern techniques of analysis is extremely important 4. Ayurveda is a plant-based system of medicine and consist of www.pharmasm.com IC Value 4.01 363
various Ayurvedic formulations such as solid dosage forms (pills, powders), liquid dosage forms (asavas, aristhas) and semisolid dosage forms (ghritas, avlehas). Ashavas and arishtas are very popular in India, probably due to their taste and alcoholic content in addition to their medicinal uses and physiological importance 5. The formulation is prepared by making a decoction of plants in specified amounts as listed in Ayurvedic Formulary of India (AFI). Crushed jaggery and the flowers of Woodfordia fruticosa are then added and preparation is kept for a specified period of time during which it undergoes fermentation generating alcohol that helps extraction of active principles and also serves as preservative for these formulations. The official Ayurvedic Formulary of India lists thirty-seven ashavas and arishtas 6. Drakshasava is a marketed ayurvedic herbal formulation included in ayurvedic formulary containing Vitis vinifera as a main ingredient. It contains not more than 10 per cent, and not less than 5 per cent of alcohol that is self generated in the preparation over a period of time. It contains about 14 crude drugs in preparation (Table 1). It is one of the most widely used in piles, tastelessness, heart disease, anaemia, bleeding disorder, diseases of abdomen, wound and fever 6. TABLE 1 INGREDIENTS OF DRAKSHASAVA Sanskrit Name Plant Name Part used Quantity Draksa Vitis vinifera Dry Fruits 4.8 kg Jala for decoction Reduced to 49.152 l 12.288 l Sarkara 4.8 kg Madhu Honey 4.8 kg Praksepa Dravyas: Dhataki Woodfordia fruticosa Flower 336 g Jati Jasminum officinale Flower 24 g Lavanga Syzygium aromaticum Flower Bud 24 g Kakkola (Kankola) Piper cubeba Fruit 24 g Lavaliphala Cicca acida Fruit 24 g Candana (Sveta Candana) Santalum album Heart Wood 24 g Krsna (Pippali) Piper longum Fruit 24 g Tvak Cinnamomum zeylanicum Stem Bark 24 g Ela (Suksmaila) Elettaria cardamomum Seed 24 g Patra (Tejapatra) Cinnamomum tamala Leaf 24 g www.pharmasm.com IC Value 4.01 364
Information on the quantitative parameters of asava and arishta to guarantee the quality and the safety of the product to the consumer is less. Therefore, establishing quality and standard parameters like alcohol content, ph, acid value and other constituents of these preparations are highly significant. It is therefore essential, that there are definite and accurate analytical tools to ascertain consistency and quality of Ayurvedic formulations. Thus, the present study aims to develop standardization method for Draksasava. MATERIALS AND METHODS All the chemicals and reagents used were of analytical grade and were purchased from Suvidhinath Laboratories, Vadodara and Chemdyes Corporation, Vadodar. Spectroscopic measurements were made on Shimadzu 1800 double beam UV Visible spectrophotometer. Commercially available brand of (M/S Dabur India Ltd) Drakshasava was procured from local market. Measurements of physicochemical parameters The sample of Drakshasava was analyzed for various parameters such as ph, specific gravity, total solids content, alcohol content, acid value and amount of reducing and nonreducing sugars 7. Preparation of extract Asava or Arishta, 100 ml, was evaporated on a steam bath to about 50 ml, cooled and then diluted to 100 ml with water. This was extracted successively with solvents such as petroleum ether ( 60 80 C), benzene, chloroform, ethyl acetate and ethyl ether. All the extracts were dried under reduced pressure at room temperature 8. Qualitative chemical examination All the extracts were qualitatively evaluated by chemical tests and TLC studies for the presence of various phytoconstituents like alkaloids, carbohydrates, saponins, phenolic compounds and tannins, phytosterols and anthraquinone glycosides. 9 TLC Studies TLC studies of all the extracts were carried out using Silica gel GF-254 as stationary phase and chloroform: ethylacetate (75:25) as mobile phase 10. Spots were observed under UV and visible light. The R f values of the components were recorded. Quantitative measurements of phytoconstituents Estimation of total phenolics www.pharmasm.com IC Value 4.01 365
Prepare a stock solution (1 mg/ml) of the extract in methanol. From the stock solution, take suitable quantity of the extract into 25-ml volumetric flask and add 10 ml of water and 1.5 ml of Folin Ciocalteau reagent. Keep the mixture for 5 min, and then add 4 ml of 20 per cent sodium carbonate solution and make up to 25 ml with double distilled water. Keep the mixture for 30 min and record absorbance at 765 nm. Calculate percentage of total phenolics from calibration curve of gallic acid prepared by using the above procedure and express total phenolics as percentage of gallic acid. 11 Estimation of total tannins Defat 2 g of sample with 25 ml petroleum ether for 12 h. Boil the marc for 2 h with 300 ml of double distilled water. Cool, dilute up to 500 ml and filter. Measure 25 ml of this infusion into 2-litre porcelain dish; add 20 ml Indigo solution and 750 ml double distilled water. Titrate it with 0.1N potassium permanganate solution, 1 ml at a time, until blue solution changes to green. Thereafter add drops wise until solution becomes golden yellow in colour. Similarly, titrate mixture of 20-ml Indigo solution and 750 ml of double distilled water. Calculate the difference between two titrations in ml. 7 Alkaloid content Alkaloids were precipitated in sample using drangendrof reagent and was allowed to stand for 10 min. Later on samples were centrifuged and precipitate was dissolved in concentrated nitric acid. Yellow colour was developed to above solution using 3% thiourea solution. 12 RESULTS AND DISCUSSION Standardization of Drakhshasava as per pharmacopoeia was carried out based on the physicochemical parameters. The marketed sample of Drakhshasava was found to pass all the pharmacopoeial tests (Table 2). www.pharmasm.com IC Value 4.01 366
TABLE 2 PHYSICOCHEMICAL PARAMETERS OF DRAKSHASAVA Physicochemical tests Standard Observed Description Total solid content Clear dark brown liquid without frothing and significant sedimentation; with aromatic odour and sweet taste Not less than 25.0 per cent w/v Dark brown colored fragrant liquid with Sweet taste 17.08% w/v Specific gravity 1.08 to 1.20 gm/mol 1.09 gm/mol Sugars Reducing Non-reducing 18.20 % w/v 0.52% w/v Not less than 16.0 per cent w/v Not more than 0.80 per cent w/v ph 4.0 to 4.5 4.32 Alcohol content 5 to 10 per cent v/v 1.2%v/v Drakhshasava was successively extracted with the various solvents, i.e. petroleum ether, benzene, chloroform and ethyl acetate and TLC studies of all the extracts were carried out. Petroleum ether, benzene, chloroform and ethyl acetate extracts showed three, three, one and two spots, respectively. A summary of the qualitative analysis is given in Table 3. TABLE 3 QUALITATIVE CHEMICAL EVALUATION OF EXTRACT Phytoconstituents Extracts Petroleum ether Benzene Chloroform Ethylacetate Alkaloids - - - - Carbohydrates - - - - Saponin - - - - Phenolic compound - - + + Phytosterols - - + - Tannin compound - - - + (+) Indicates presence, ( ) indicates absence. The chemical evaluation reported here is for the entire extract and not for the individual spot. www.pharmasm.com IC Value 4.01 367
TLC profile of petroleum ether, chloroform and benzene showed different spot at different R f shown in Fig. 1. Petroleum ether, benzene, chloroform and ethyl acetate extracts showed three, three, one and two spots, respectively. Benzene and petroleum ether extract shows good separation and indicates maximum number of components. FIGURE 1 TLC PROFILE OF PETROLEUM ETHER, BENZENE AND CHLOROFOEM EXTRACT Acid value indicates the total acids present in the product. Acid value of Drakshasava was found to be 0.26 M. Acids are produced during preparation (especially in the fermentation process) and storage (oxidation of alcohols) and are responsible for the sour taste of those preparations. Various phytoconstituents like total phenolics, total tannins and total alkaloids estimated in Drakshasava. Total phenolic and total tannin of Drakshasava was found to be 6.34 GAE µg/ml and 1.18%w/v, respectively. Drakshasava does not show any alkaloidal content. The values of phytoconstituents may vary due to variation in geographical conditions of raw materials, different methods of processing. Thus by analyzing different www.pharmasm.com IC Value 4.01 368
batches of asav-arishta one can finalize the limits of different phytoconstituents and can implement in regular quality control methods to maintain batch-to-batch consistency. Therefore it can be concluded that the recorded levels of alcohol, acidity and ph in commercially available Drakshasava could be used to establish and formulate procedures for standardization and quality controlling of these ayurvedic preparations. Various phytoconstituents like total phenolic and total tannin can be useful to maintain batch-tobatch variability. TLC profile of different extract is also useful to standardize the preparation on quantitative basis. REFERENCES 1. Lalla JK, Jolly CI, Hamrapurkar PD: Standardization by GM standards for Ayurvedic medications. 48 th India National Congress, Madras, 1996:14. 2. Viswanathan MV, Unnikrishnan PM, Komatsu K, Fushimi H & Basnet P: A brief introduction to Ayurvedic System of Medicine and some of its problems. Indian J Traditional Knowledge 2003;2:159-169. 3. Agarwal S, Singh RH: Proceedings of International Congress on Ayurveda, 28 30th January 2002:221. 4. Anantanarayana DB, Proceeding of International Congress on Ayurveda, 28 30th January 2002:67. 5. Singh H, Kumar MS, Pande M: Standarization of Aurjunarishta formulation by TLC method. Int J Pharm Sci Rev Res 2010;2:25 8. 6. The Ayurvedic Formulary of India, Part-II, Vol-I, First Edition, Government of India, Ministry of Health and Family Welfare, 2008,39-41. 7. Ayurvedic Pharmacopoeia of India, Part-II, Vol-I. First Edition, Government of India, Ministry of Health and Family Welfare, 2007. 8. Khandelwal KR: Practical Pharmacognosy-Techniques and Experiments. Nirali Prakashan, Edition 9,2002:152. 9. Kokate CK, Purohit AP, Gokhale SB: Pharmacognosy. Nirali Prakashan, Edition 21,2002:108 109 10. Elamthuruthy ET, Shah CR, Khan TA, Tatkeb TA, Gabhe SY: Standardization of marketed Kumariasava an Ayurvedic Aloe vera product. Journal of Pharmaceutical and Biomedical Analysis 2005;37:937 941 www.pharmasm.com IC Value 4.01 369
11. Singleton VL, Orthofer R, Lamuela-Raventos RM: Analysis of total phenols and other oxidation substances and antioxidants by means of Folin-Chiocalteu reagent. Meth Enzymol 1999;99:152 78. 12. Sreevidya N, Mehrotra S. Spectrophotometric method for estimation of alkaloids precipitable with Dragendorff's reagent in plant materials. J AOAC Int 2003;86:1124 For Correspondence: Girish U Sailor Email: sailorgirish@gmail.com www.pharmasm.com IC Value 4.01 370