Scientific evaluation of Panchkola curna An Ayurvedic polyherbal drug formulation

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1 Indian Journal of Traditional Knowledge Vol. 11(4), October 2012, pp Scientific evaluation of An Ayurvedic polyherbal drug formulation S Rajput*, MK Tripathi, AK Tiwari, N Dwivedi & SP Tripathi JRD TATA Foundation for Research in Ayurveda & Yoga Sciences, Ayurveda Sadan, Arogyadham,Deendayal Research Institute,Chitrakoot, Satna , MP s: info@chitrakoot.org, rajput_sushma00@yahoo.com Received , revised The present study deals with the Scientific evaluation and Standardization of the Ayurvedic compound formulation following quality control procedures both for the raw materials and the finished product. The obtained values (ranges of physico-chemical parameters) can be adapted to day down new pharmacopoeial standards to be followed for traditional preparation of with batch to batch consistency. The phytochemical constituents found to present in the raw material used for the preparation of facilitate the desirable therapeutic efficacy of the medicinal formulations a whole in elements and also could help in knowing the underlying mechanism of pharmacological action. Keywords: Ayurvedic formulation,, Drug standardization IPC Int. Cl. 8 : A61K 36/00, A61P 1/04, A61P 1/06, A61P 1/12, A61P 7/06 To start with development of standards for the identity, purity and strength of single drugs and formulations at a later stage, assumed importance for the effectiveness of herbal medicines. If the raw materials to be used in a medicine and stage by stage processes of manufacturer standardized the final product namely, the compound formulation could be expected to conform to uniform standards 1. The requirements that the list of ingredients be displayed on the label will enable analysts in important cases to verify label claims and to that extent will bind the manufacture to a true claim. is an Ayurvedic polyherbal formulation has been use in fever, indigestion 2-3, etc. The preparation of is based on traditional methods is accordance with the procedures given in classical texts like Ayurvedic formulary of India I. Due to lack of modern pharmacopoeia standards laid down and followed for processing of, the medicine prepared using traditional methods may not have the desired quality and batch to batch consistency. Hence there is a need for standardization of Ayurvedic curna following Scientific parameters including descriptive taxonomic identification of raw drugs, organoleptic characters, *Corresponding author powder microscopic characteristics, phytochemical analysis, chromatographic pattern and microbial screening. The work was undertaken is the trust as part of a program of testing and validation of traditional practices of using the Ayurvedic medicine. Panchkola curna is traditionally used for treatment of cold and fever. In this connection, standardization of becomes imperative. The current work deals with detailed standardization guidelines involving Good Manufacturing (GMP) prescribed for preparation of Ayurvedic medicines. Standardization guidelines to be followed 4 for herbals products provided by World Health Organization (WHO), and Ayurvedic pharmacopoeia of India have been considered. In the standardization procedures followed in the work, certain in process tests 5 have also been developed and performed. Methodology Method of preparation of the curna All the ingredients were used of Pharmacopoeial quality. These were washed, dried and ground individually passed through 180 µm separately then weighed separately, mixed in specified ratio and passed through 355 µm to obtain a homogenous blend. It was stored in an airtight container to protect

2 698 INDIAN J TRADITIONAL KNOWLEDGE, VOL 11, NO 4, OCTOBER 2012 from light and moisture. Five different samples of (two samples were taken from Chitrakoot Rasshala Pharmacy, Chitrakoot and three samples were prepared at research laboratory Ayurveda Sadan, Chitrakoot) were studied. Each sample of was formulated using five ingredients 3, i.e. Pippalĩ (Piper longum Linn. fruit), Cavya (Piper retrofractum Vahl. stem), Šunthī (Zingiber officinale Roxb. rhizome), Pippalĩmula (Piper longum Linn. stem.), Citraka (Plumbago zeylanica Linn. root.) in equal proportion (Fig. 1). Physicochemical parameters Organoleptic characters, particle size and physicochemical analysis 6-7 of all the samples were carried out. Quantitative analysis for total ash, acid insoluble ash, water soluble ash 8, extractive values in water soluble and alcohol soluble extractive, loss on drying at 105 C and ph of filtrate of 10% w/v aqueous solution were checked in triplicate according to the prescribed Standard methods in Indian Pharmacopoeia. Microscopic characteristics For microscopic analysis 6 a small quantity represent of the curna, along with the genuine samples, i.e. Pippalĩ (Piper longum Linn. fruit), Cavya (Piper retrofractum Vahl. stem), Šunthī (Zingiber officinale Roxb. rhizome), Pippalĩmula (Piper longum Linn. stem.), Citraka (Plumbago zeylanica Linn. root.) was mixed with water, stained with iodine and mounted in glycerin, were used to examine the starch grains and its type. Another small quantity of samples cleared by heating with chloral hydrate solution and mounted in glycerin was used to identify diagnostic microscopic characters of the ingredients. Further, small quantity of the curna cleared with dilute KOH (5%) and was mounted in glycerin; was subjected to microscopic examination. TLC profile For HPTLC, 5 gm coarsely powdered drug in 250 ml stoppered conical flask and extracted with 100 ml ethanol for 24 hrs by maceration technique with occasional shaking. The extract was extracted and volume was raised up to 100ml in a volumetric flask. Twenty five ml extract was taken from the above stock solution and concentrated on a water bath to similarly, methanol extract was prepared for all 5 ingredients, i.e. Pippalĩ (Piper longum Linn. fruit), Cavya (Piper retrofractum Vahl. stem), Šunthī (Zingiber officinale Roxb. rhizome), Pippalĩmula (Piper longum Linn. stem.), Citraka (Plumbago Fig. 1 Photographs of single drugs used in formulation of

3 RAJPUT et al.: SCIENTIFIC EVALUATIOPN OF PANCHKOLA CURNA 699 zeylanica Linn. root.) plant for use as reference. TLC of extracts of all the samples and the reference ingredients was carried out on silica gel 60 F 254 precoated plates (0.2 mm thickness; from Merck India Limited). An Applicator from Camag Linomat-5 (Camag Switzerland: ) was used for band application and photo documentation unit (Camag Reprostar -3: ) was used for documentation of chromatographic fingerprints. The mobile phase used was Toluene: Ethyl acetate: Formic acid (7: 2.5: 0.5). The plate was developed over a distance of 9 cm in a saturated development chamber (Twin trough chamber (10 10 c with SS lid, and visualized under visible light, 254nm and 366nm. After spraying with Anisaldehyde-Sulphuric acid followed by heating at 105 C for 5 min 7. starch grains, pitted parenchymatous cells, aseptate fibres (Pippalimula); polygonal suberized cork, reticulate and simple pitted vessel, polygonal parenchymatous cells filled with starch grains, elongated parenchymatous cells filled with starch grains (Citraka) (Fig. 3). Test for Aflatoxin The 5 sample of were also checked for mycotoxin, i.e. Aflatoxin with standard markers B 1, B 2, G 1 and G 2 9. Test for microbial limits Following tests were carry out as per API 10 to determine the microbial load in five batches of, a formulated compound drug powder of pharmaceutical substances Enumeration of Staphylococcus aureus/gm Enumeration of Salmonella sp./gm Enumeration of Pseudomonas aeruginosa/gm Determination of E.coli Determination of total microbial plate count (TMC) Determination of Yeast and mould. The microbiological tests were determined using specified agar and enrichment media from Himedia and Pvt. Ltd. Mumbai. Result and discussion Brownish, dust coloured powder with a characteristic spicy odour and taste. The powder completely passes through 355 µm and not less than 50 percent through 180 µm. The Panchkola compound formulation powder is characterized by the presence of stone cells, perisperm cells with starch grains, fragments of parenchyma, oil globules (Pippali); parenchymatous cells, reticulate thickening, simple pitted vessel, fibres (Cavya) (Fig. 2); cork cells, fragment of fibres, parenchymatous cell filled with starch grains, pitted, septate fibres with reticulate and spiral thickening, oleo-resin canal (Sunthi); scalariform thickening, Fig. 2 Powder characteristics of Fig. 3 Powder characteristics of

4 700 INDIAN J TRADITIONAL KNOWLEDGE, VOL 11, NO 4, OCTOBER 2012 Physico-chemical data was subjected to various analytical parameters average value of total ash content 5%, acid insoluble ash 4.5%, alcohol soluble extractive 11.5%, water soluble extractive 15.5%, loss and drying at 105 C 8.5% and ph 5.05 (Table 1). High Performance Thin Layer Chromatography (HPTLC) The TLC plate were examine under ultra violet light at 254 nm; at 366 nm; at visible for both before and after derivetisation with Anisaldehyde- sulphuric acid reagent (Figs. 4-6). The R f values and colours of the bands obtained were recorded (Tables 2-4). It shows major spot under ultraviolet light (254nm) R f at 0.34 (green), 0.37 (black), 0.51(grey) and 0.59 (grey), It shows major spot under ultraviolet light (366 nm) R f at 0.17 (blue), 0.33 (blue), 0.39 (blue), 0.73 (light blue), 0.85 (red) and 0.89 (red) under ultraviolet light (366 nm).spray the plate with Anisaldehydesulphuric acid reagent followed by heating at 105 C for about 5 min and observe under ultraviolet light. It shows major R f under ultraviolet light (254nm) at R f 0.34 (sky blue), 0.38 (bluish), 0.45 (white), 0.59 (white) and 0.75 (brown). Parameter Table 1 Quality test for the finished product, Average value Batch-A Batch-B Batch-C Batch- D Batch-E Pippalĩ (Fruit) Cavya (Stem) Visvãhvã Pippalĩmļũla (Root) Citraka (šunthî) Rhizome (Root) Loss on drying at C (%) Total Ash (%) Acid-insoluble ash (%) Alcohol-soluble extractive (%) Water-soluble extractive (%) ph (10%) aqueous solution Note Batch A, B and C - Research laboratory Ayurveda Sadan, Chitrakoot and Batch D and E-Chitrakoot Rasshala Pharmacy, Chitrakoot Table 2 R f values of test solution of at 254 nm (before derivatization) R f value 001A 001B 001C 001D 001E R f 1(black) NA NA NA NA 0.02 R f 2(dark green) NA 0.34 NA NA 0.34 R f 3 (Black) NA NA 0.37 R f 4 (Grey) NA 0.51 NA NA 0.51 R f 5 (Grey) NA NA NA NA 0.59 R f 6(Grey) NA NA 0.95 NA 0.95 NA-Major spot Not Appeared Table 3 R f values in test solution of at 254 nm (after derivatization) R f value 001A 001B 001C 001D 001E R f 1(Red) NA NA NA NA 0.05 R f 2(Brown) NA NA NA NA 0.11 R f 3 (Sky blue) NA NA NA 0.19 NA R f 4 (Sky blue) NA 0.34 NA NA 0.34 R f 5(Blueish white NA 0.38 NA NA NA R f 6(Glowing white R f 7 (Glowing white) R f 8 (Brown)) NA 0.75 NA NA 0.75 NA-Major spot not appeared

f 1(Red) 0.05 0.05 0.05 0.05 0.05 NA NA NA NA 0.05 R f 2(Red) 0.07 0.07 0.07 0.07 0.07 NA NA NA NA 0.07 R f 3( Blue) 0.17 0.17 0.17 0.17 0.17 NA NA NA 0.

73 NA NA R f 7(Red) 0.85 0.85 0.85 0.85 0.85 NA NA NA NA 0.85 R f 8(Red) 0.89 0.89 0.89 0.89 0.89 NA NA NA NA 0.89 R f 9( Light red) 0.95 0.95 0.95 0.95 0.95 NA NA NA NA 0.

4 TLC Finger prints of test solution of at 254 nm (before derivatization) A: Test Solution of Panchkola curna for Batch 001A, B: Batch 001B, C: Batch 001C, D: Batch

5 RAJPUT et al.: SCIENTIFIC EVALUATIOPN OF PANCHKOLA CURNA 701 R f value Table 4 R f values in test solution of at 366 nm (before derivatization) 001A 001B 001C 001D 001E R f 1(Red) NA NA NA NA 0.05 R f 2(Red) NA NA NA NA 0.07 R f 3( Blue) NA NA NA 0.17 NA R f 4 (Blue) NA 0.33 NA NA 0.33 R f 5(Blue) NA 0.39 NA NA 0.39 R f 6(Light blue) NA 0.73 NA NA R f 7(Red) NA NA NA NA 0.85 R f 8(Red) NA NA NA NA 0.89 R f 9( Light red) NA NA NA NA 0.95 NA-Major spot not appeared Fig.4 TLC Finger prints of test solution of at 254 nm (before derivatization) A: Test Solution of Panchkola curna for Batch 001A, B: Batch 001B, C: Batch 001C, D: Batch 001D, E: Batch001E, 1: Pippalĩ mula, 2: Pippalĩ, 3: Citraka, 4: Cavya, 5: Visvãhvã (šunthī) Fig. 6 TLC finger prints in test solution of at 366 mm (before derivatization) Fig. 5 TLC finger prints in test solution of at 254 nm (after derivetisation) Fig. 7 TLC finger prints in test solution for Aflatoxin of at 366 nm(before derivatization) Standard Marker B1, G1, B2 and G2. A:Test Solution of for Batch 001A, B: Batch 001B, C: Batch 001C, D: Batch 001D, E:Batch 001E

702 INDIAN J TRADITIONAL KNOWLEDGE, VOL 11, NO 4, OCTOBER 2012 Fig. 8 Photographs of Microbiological limit test in The Aflatoxin was absent in the formulated (Fig. 7).

6 702 INDIAN J TRADITIONAL KNOWLEDGE, VOL 11, NO 4, OCTOBER 2012 Fig. 8 Photographs of Microbiological limit test in The Aflatoxin was absent in the formulated (Fig. 7). The microbial profile of the was found satisfactory. Total microbial plate count(average 98 cfu/g), Yeast and Moulds (average 24 cfu/g) counts were reported less than the limit set by API 9 and pathogenic bacteria, i.e. Salmonella, Pesudomonas, Staphyllococcus and E.coli were found to be absent (Figs ). Conclusion The Ayurvedic system of medicines is prevalent in India since the Vedic period and as early as the dawn of human civilization. Though, Ayurveda has undergone many changes in the course of its long history, it still remains the mainstay of medical relief to a large section of population of the nation. Due to urbanization and dwindling of forests, the Vaidya by and large is no longer a self contained unit collecting and preparing his own medicines as before. He has now to depend on the newly developed agencies like one collecting and supplying the crude drugs and the other undertaking mass production of medicines in the Ayurvedic Pharmaceutical units run on the commercial scale 11. Thus, the present study revealed that the characteristic microscopical features and distinguished fingerprints in the HPTLC profiles may be utilized as marker parameters for monitoring the quality of the formulation. Hence, the physicochemical parameters, biochemical analysis, HPTLC finger print profiles and the microscopical characteristic together may be used for quality evaluation and the standardization of compound formulations. Spiking of the formulations with the different genuine ingredients further confirms the presence of individual components in them. Acknowledgement Authors are grateful to Dr Bharat Pathak, General Secretary, Deendayal Research Institute, Arogyadham, Chitrakoot, for providing the infrastructure and thankful to Department of AYUSH, Ministry of Health and Family Welfare, Government of India, for financial support under the scheme Centre of Excellence.

7 RAJPUT et al.: SCIENTIFIC EVALUATIOPN OF PANCHKOLA CURNA 703 References 1 Anonymous the Ayurvedic Formulary of India, Part I, (Government of India, Ministry of Health and Family Welfare, New Delhi), 1996, Anonymous, Indian Pharmacopoeia, (Government of India, New Delhi), Anonymous, The Ayurvedic Formulary of India, Part II, (Government of India, Ministry of Health and Family Welfare, New Delhi), 2000, Arora RB, Gupta L, Sharma RC & Gupta SK, Standardization of Indian indigenous drugs and preparations III, Standardization of Yogaraja Guggulu with reference to its anti-inflammatory activity, J Res Indian Med, 8 (1993) Lowry OH, Rosenberger NJ, Farr AL & Randoll RJ, Protein measurement with the Folin Phenol reagent, J Biol Chem, 193 (1951) Patel PM, Patel NM & Goyal RK, Quality Control of Herbal Products, The Indian Pharmacist, 5(45) (2006) Quality Control Manual for Ayurvedic, Siddha & Unani medicines, (Govt of India, Department of AYUSH, Ministry of Health & Family Welfare, PLIM, Ghazibad, UP), 2008, Montgomery R, Determination of glycogen, Arch Biochem Biophys, 67 (1957) Rastogi S, Khatoon S, Rai V, Agnihotri AK, Rawat AKS & Mehrotra S, Evaluation of Ayurvedic compound formulations-naraca Curna, Indian J Tradit Knowle, 42 (2) (2005) The Ayurvedic Pharmacopoeia of India, I (I-VII), (Govt of India, Ministry of Health & Family Welfare, Department of ISM & H, New Delhi), 2000, World Health Organization (WHO), Quality Control Methods for medicinal plants materials, 1998, 63.

Scientific Evaluation of Ayurvedic Compound Formulation Dhanyapanchaka Kvatha Curna

Available online at www.ijpab.com ISSN: 2320 7051 Int. J. Pure App. Biosci. 2 (5): 88-93 (2014) INTERNATIONAL JOURNAL OF PURE & APPLIED BIOSCIENCE Research Article Scientific Evaluation of Ayurvedic Compound