STANDARDIZATION OF HINGUVACADI CHURNA - AN AYURVEDIC FORMULATION AND IDENTIFICATION OF PIPERINE AS A MARKER COMPOUND

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1 J. Res. Educ. Indian Med., Vol. XVI (1-2) : (2010) ISSN STANDARDIZATION OF HINGUVACADI CHURNA - AN AYURVEDIC FORMULATION AND IDENTIFICATION OF PIPERINE AS A MARKER COMPOUND V. GAYATHRI DEVI, 1 ANITHA JOHN 1 AND K. GOPAKUMAR 2 Regional Research Institute (Drug Research), Poojapura, Thiruvananthapuram Kerala (India) Abstract: Standardization ensures the quality of medicines and gives authenticity to the medicines prepared by the manufacturers, satisfaction of the prescribing physicians and relief to the consumers. This paper deals with the standardization of an Ayurvedic formulation - Hinguvacadi churna - by carrying out a detailed analysis based on WHO and FDA guidelines and identification of a marker compound piperine in it. Hinguvacadi churna is commonly used in Ayurveda for the treatment of Adhmana, Sula, Gulma, Pandu, Parsvasula, Vastisula, Trikasula, Yonisula, Gudasula, Vibandha, Mutrasanga, Kanthabandha, Hrtroga, Aruci, Pliharoga, Hikka, Svasa, Kasa and Agnimandya. The formulation used for the study was prepared by the Clinical Research Section, Regional Research Institute, Thiruvananthapuram, India. The physico-chemical parameters - Moisture content, Total ash, Acid insoluble ash, Water soluble ash, Water soluble extractives, Alcohol soluble extractives, ph of water extract, Volatile oil, Fibre content, Swelling index, Foaming index and Particle size - were determined. TLC, HPTLC and HPLC profiles and IR fingerprint were taken which contribute to a great extent for standardization. A comparative study on Hinguvacadi churna, Piper nigrum Linn. (fruit) and piperine, which was isolated from the churna, was also carried out using chromatographic and IR spectrophotometric methods. The shelf-life period of the formulation was also studied. Keywords: Quality control, Good manufacturing practices, Physico-chemical analysis, Hinguvacadi churna, Piperine, Marker compound, Vanillin-sulphuric acid, WHO guidelines. Introduction The Indian traditional systems of medicine provide good market potential through sincere and systematic research documentation. The Government of India introduced Good Manufacturing Practices in manufacturing of Ayurvedic medicine to ensure production of good quality medicines for the consumers in the domestic and international market and is given in Quality assurance of Ayurveda, Siddha, Unani and Homoeopathic drugs (2008). The Good Manufacturing Practices are prescribed to ensure that (a) Raw materials used in the manufacture of drugs are authentic, of prescribed quality and are free from contamination, (b) The manufacturing process is, as has been prescribed to maintain the standards, (c) Adequate quality control measures are adopted, (d) The manufactured drug which is released for sale is of acceptable quality, (e) To achieve the listed objective. Each licensee shall evolve methodology and procedures for following the prescribed process of manufacturer of drugs which should be documented as a manual and kept for reference and inspection. The accurate analysis for quality control of all the herbal products is the prime requirement for a safe and effective health management programme. Physico-chemical analysis provides the objective parameters to fix up the standards for quality of raw drugs, intermediate process and materials as well as finished products. Analytical study of the drug also helps to interpret the pharmacokinetic and pharmacodynamic of the same in a suitable manner. It is the need of the present era for a scientific explanation to 1. ARO (Chemistry) 2. Assistant Director (Ayurveda)

2 20 Gayathri et al. understand the concept of Ayurveda and its quality and efficacy. In this paper an attempt was made to evaluate an Ayurvedic formulation, Hinguvacadi churna, physico-chemically. Hinguvacadi churna is commonly used in Ayurveda for the treatment of Adhmana (flatulence with gurging sound), Sula (pain/colic), Gulma (abdominal lump), Pandu (anemia), Parsvasula (intercostals neuralgia and pleurodynia), Vastisula (pain in urinary bladder), Trikasula (pain in sacral region), Yonisula (pain in the female genital tract), Gudasula (pain in the anus), Vibandha (constipation), Mutrasanga (obstruction in urinary tract), Kanthabandha (obstruction in the throat), Hrtroga (cardiac failure), Aruci (tastelessness), Pliharoga (splenic disease), Hikka (hiccup), Svasa (dyspnoea/ asthma), Kasa (cough) and Agnimandya (digestive impairment) (The Ayurvedic Formulary of India, 2003). Churna is a fine powder of one or more drugs. Drugs according to the composition of the churna are collected, dried, powdered individually and passed through sieve number 85 to prepare a fine powder. They are mixed in the specified proportion and stored in well closed container. In the present study, an attempt was also made to isolate a marker compound and piperine was isolated, the main source of which is Piper nigrum Linn. (fruit). A comparative study on Hinguvacadi churna, Piper nigrum Linn. (fruit) and piperine was also carried out using TLC and IR spectrophotometric methods. Materials and Methods I. Preparation of Hinguvacadi churna Hinguvacadi churna is a compound formulation having twenty four ingredients and was prepared by the Clinical Research Section, Regional Research Institute, Poojapura, Thiruvananthapuram (India) by the method described in (Astanga Hridaya Chikitsasthana: 31-32/118). The ingredients of the preparation are given in Table 1. Drugs mentioned in the yoga were cleaned, dried and powdered properly. They were finely powdered and sieved separately. Each one of them was weighed separately and well mixed together to get 500 gm formulation. This was divided into five parts each weighing 100 gm and the samples were kept in airtight containers for analysis. II. Organoleptic characters The organoleptic characters such as colour, touch, taste and odour were noted. III. Physico - chemical parameters Physico - chemical parameters include (a) Moisture content (b) Total ash (c) Acid insoluble ash (d) Water soluble ash (e) Water soluble extractives (f) Alcohol soluble extractives (g) ph of water extract (h) Volatile oil (i) Fibre content (j) Swelling index (k) Foaming index and (l) Particle size. Moisture content was determined using Dean and Stark apparatus using xylene. Volatile oil was determined using Clevenger s apparatus. Total ash, acid insoluble ash, water soluble ash, water soluble extractives and alcohol soluble extractives were determined by standard methods given in The Ayurvedic Pharmacopoeia of India (2007). The particle size (bulk density and tap density) were determined by the method described by Alfred Martin (2005). The other parameters fibre content, swelling index and foaming index were determined using the methods given in WHO guidelines (Quality Control Methods of Medicinal Plant Materials, 1998). IV. Assay of constituents (a) Estimation of tannin content The quantitative analysis of tannin (Sadasivam and Manickam, 1997) was carried out by Folin Dennis method. (b) Estimation of chloride. The churna was made into ash by incinerating a known weight in a silica crucible until it was free from carbon. The ash obtained was dissolved in water and made up to a known volume and used for the estimation of chloride.

3 Phytochemical Standardisation of Hinguvacadi Churna 21 Table 1. Ingredients of Hinguvacadi curna. Sl. No. Sanskrit Name Scientific Name Parts used Quantity 1 Hingu Ferula foetida Regel Exudate 1 part 2 Vacha Acorus calamus Linn. Rhizome 3 Haritaki Terminalia chebula Retz Fruit 4 Pasugandha Gynandropsis gynandra Linn. Plant 5 Dadima Punica granatum Linn. Seed 6 Dipyaka (yavani) Trachyspermum ammi Linn. Fruit 7 Dhanyaka Coriandrum sativum Linn. Fruit 8 Patha Cyclea peltata Cooke. Root 9 Pushkaramoola Inula racemosa Hk.f. Root 10 Sati Kaempferia galanga Linn. Rhizome 11 Hapusa Sphaeranthus indicus Linn. Root 12 Chitraka Plumbago rosea Linn. Root 13 Yavaksara Potassium carbonate impure - 14 Svajikaksara Silicate of alumina, magnesia and - oxide of Iron 15 Sauvarcaka lavana Sodium chloride with sodium - 16 Saindhava lavana Sulphate and sodium hydroxide - 17 Vida lavana Sodium chloride associated with clay and - calcium sulphate. Sodium chloride with traces of sodium sulphate, alumina, magnesia, ferric oxide and iron sulphide 18 Sunthi Zingiber officinale Rosc. Rhizome 19 Maricha Piper nigrum Linn. Fruit 20 Pippali Piper longum Linn. Fruit 21 Ajaji (svetajiraka) Cuminum cyminum Linn. Seed 22 Chavya Piper attenuatum Ham. Stem 23 Tinidika Tamarindus indica Linn. Fruit pulp 24 Vetasamla Vitis carnosa Wall. Fruit Chloride present in the churna was estimated by Volhard s method (Bassett et al., 1978). V. Thin layer chromatographic (TLC) studies TLC profiles of the petroleum ether and alcohol extracts of the churna were taken in different solvent systems using silica gel 60 F 254 pre-coated aluminium sheets. TLC profiles were taken by developing the plates in toluene for petroleum ether extract and in toluene : ethyl acetate (6:1) for alcohol extract. 50 % H 2 SO 4 was used as the spray reagent. VI. High performance thin layer chromatography (HPTLC) HPTLC is a micro analytical separation and determination method which has a wide application in herbal drug analysis. Alcoholic extract of the churna was spotted in the form of bands with Camag microlitre syringe on a precoated silica gel plate 20 x 10 cm with Camag Linomat V applicator. Mobile phase used was toluene : ethyl acetate (1:1). Linear ascending development was done in 10 x 10 cm twin glass chamber saturated with mobile phase.

4 22 Gayathri et al. Densitometric scanning was performed on Camag TLC scanner III in the absorption mode at 254 nm. VII. High performance liquid chromatography (HPLC) HPLC of the alcoholic extract of churna was taken using TRLPA/HPLC analyzer. The experiment was conducted in a C18 column using dichloromethane/chloroform as the mobile phase. The Retention time (R t ) was detected at 254 nm. VIII. Isolation of piperine from Hinguvacadi churna Piperine is reported to be the major compound present in Piper nigrum and Piper longum. Moreover the spot for piperine was observed in the Thin Layer Chromatography of the churna. Hence piperine was selected as the marker compound. 100 gram churna was extracted with 95 % ethyl alcohol in Soxhlet extraction apparatus for two hours (Harborne, 1973). The extract was filtered and concentrated. 100 ml of 10% alcoholic KOH was added and the residue formed was discarded. The solution was left overnight. Yellow needle shaped crystals were formed. The crystals were recrystallised from ethyl alcohol to get pure crystals of piperine. The yield obtained was 0.25%. The purity was checked by measuring its UV spectrum in ethanol (λ max : 245 nm). IX. Identification of the yellow crystals as piperine Melting point was determined in open capillaries. The crystals were tested for alkaloids. IR spectrum of the compound was taken in KBr. The results obtained were compared with that of an authentic sample from Chemistry section, Regional Research Institute, Trivandrum. X. Use of piperine as a marker compound by TLC method The thin layer chromatographic studies of alcohol extracts of the Hinguvacadi churna, Piper nigrum Linn. (fruit) and an alcoholic solution of Piperine were carried out. TLC was developed by spotting alcoholic extracts of Hinguvacadi churna, Piper nigrum and Piperine on silica gel 60 F 254 precoated aluminium plates (Wagner and Bladt, 1996). The solvent system used was toluene : ethyl acetate in the ratio (1:1). The spots were visualised in short UV light and using Vanillin-sulphuric acid reagent. XI. Infra-red (IR) spectrum The IR spectrums of the alcoholic extracts of the churna, Piper nigrum and the marker compound piperine were taken using FTIR impact U10 Spectrophotometer. XII. Shelf-life period The samples were analysed by determining the physico-chemical parameters at an interval of three months for a period of one year. Results Three batches of the churna were prepared as described above and the analysis of different samples of the three batches were carried out and the values fixed within a reasonable range are reported as the standard value. Table 2. Physico-chemical parameters of Hinguvacadi churna. Sl. No. Tests Results 1 Moisture content (%) Total ash (%) Acid insoluble ash (%) Water soluble ash (%) Water soluble extractives (%) Alcohol soluble extractives (%) ph Volatile oil (%) Swelling index ( ml/gm) ml/g 10 Foaming index < Fibre content (%) Particle size (a) Bulk density (b) Tap density g/cm g/cm 3

5 Phytochemical Standardisation of Hinguvacadi Churna 23 Table 3. HPTLC profile of the alcohol extracts of the churna. Peak Starting Maximum Ending Area R f H(AU) R f H(AU) % R f H(AU) F(AU) (%) The organoleptic characters were noted as given below (a) Colour - Yellowish brown (b) Odour - Characteristic smell of hingu (c) Touch - Fine powder (d) Taste - Acrid and saline 2. The physico-chemical parameters were determined by the methods given in materials and methods and the results are given in Table Assay of constituents (%) (a) Tannin (b) Chloride Thin layer chromatography The TLC studies of the petroleum ether and alcohol extracts of the churna were carried out, the number of spots and the R f values obtained are given below. a) Petroleum ether extract Solvent system used Adsorbent - Toluene - silica gel 60 F 254 precoated aluminium sheets Spray reagent - 50% sulphuric acid No. of spots - 5 R f values , 0.31, 0.43, 0.58, 0.92 b) Ethyl alcohol extract Solvent - Toluene: ethyl acetate (6:1) system used Adsorbent - silica gel 60 F 254 precoated aluminium sheets Spray reagent - 50% sulphuric acid Table 4. R f values of piperine at room temperature. Compound R f values Colour detected in UV long Compound isolated 0.93 Yellow Authentic piperine 0.93 Yellow Both mixed together 0.93 Yellow Adsorbent : TLC Silica gel 60 F 254 Aluminium sheets; Solvent system: Ethyl acetate No. of spots - 7 R f values , 0.19, 0.40, 0.59, 0.78, 0.92, HPTLC profile HPTLC profile of the alcohol extracts of the churna is given in Figure 1 and the R f values are given in Table HPLC profile HPLC analysis was carried out as given in materials and methods. The retention times are measured and the values obtained are 3.362, 3.599, and min. The HPLC profile of churna is given in Figure IR spectrum The IR finger printing of the alcoholic extract of the churna is given in Figure 2 and that of Piper nigrum Linn. (fruit) is given in Figure 3. The IR spectral values are as follows: IR, νmax, KBr (Hinguvacadi churna) 3006, 2922, 2852, 1708, 1605, 1512, 1455, 1376, 1250, 1205, 1172, 1118, 1038, 877, 828, 720 cm -1 IR, νmax, KBr ( Piper nigrum Linn., fruit)

IR spectrum of the alcoholic extract of Hinguvacadi churna Figure 3.

IR spectrum of the alcoholic extract of piperine Figure 5.

RT Area % Area Height 1. 3.362 21002223 36.66 2773927 2. 3.599 11282406 19.

6 24 Gayathri et al. Figure 1. HPTLC profile of alcohol extract of Hinguvacadi churna at 254 nm Figure 2. IR spectrum of the alcoholic extract of Hinguvacadi churna Figure 3. IR spectrum of the alcoholic extract of Piper nigrum Linn. (fruit) Figure 4. IR spectrum of the alcoholic extract of piperine Figure 5. Structure of piperine Figure 7. HPLC Profile of Hinguvacadi churna Sl. No. RT Area % Area Height Figure 6. TLC profile of Hinguvacadi churna, Piper nigrum Linn. (fruit), Piperine Stationary phase: Silica gel G 60 F 254 precoated Aluminium plates Mobile phase: Toluene : ethyl acetate (1:1) Detecting reagent: (a) UV short light; (b) Vanillin sulphuric acid reagent (c) UV short light

7 Phytochemical Standardisation of Hinguvacadi Churna 25 Table 5. TLC profile of alcohol extracts Name of extract Hinguvacadi churna Piper nigrum R f values In UV long In UV short Using Vanillin-sulphuric acid 0.84 (light yellow), 0.64 (light violet), 0.61(dark blue), 0.55 (light violet), 0.12 (light brown), 0.08 (brown) 0.92(orange), 0.61(dark blue), 0.31(yellowish brown), 0.22(light yellow), 0.10(orange), 0.04(brown) 0.91 (light violet), 0.80 (violet), 0.74 (violet), 0.60 (violet), 0.41 (light violet), 0.21 (lightviolet), 0.11 (light yellow), 0.05 (brown) 0.91(yellow), 0.76(violet), 0.62(yellow), 0.60(violet), 0.36(light violet), 0.30(light violet), 0.26(light violet), 0.11(light yellow), 0.05(light yellow) 0.95 (light blue), 0.78 (darkbrown), 0.60 (brown), 0.54 (light blue), 0.43 (light blue), 0.37 (light blue), 0.23 (light blue), 0.16 (light blue), 0.07 (light brown) 0.94(light blue), 0.78(light blue, 0.71(light blue), 0.60(brown), 0.30(light brown), 0.17(light blue), 0.05(light brown) Piperine 0.61(dark blue) 0.60(violet) 0.60(brown) Adsorbent : TLC Silica gel 60 F 254 Aluminium sheets; Solvent system: toluene: ethyl acetate (1:1) 3845, 3670, 2692, 2655, 2561, 2545, 2482, 2407, 2212, 2167, 1660, 1093, 904, 495 cm Characterisation of the isolated compound as piperine Melting point of the compound was observed as 128 o C which is the reported value for piperine. The crystals were tested for alkaloids using Mayer s reagent and was found to be positive. The TLC study shows that the R f value and the colour observed in UV light for the compound are the same as that of the authentic sample (Table 4). The IR spectrum (Figure 4) of the compound was taken and the spectral values were compared with that of an authentic sample and found to be identical. IR, νmax, KBr (Piperine) 3933, 3915, 3823, 3680, 3521, 3502, 3438, 3361, 3037, 2904, 2335, 2072, 1851, 1606, 1458, 1240, 997, 827, 673, 607 cm -1. On alkaline hydrolysis piperine (Figure 5) gave a base piperidine and an acid piperic acid. NaOH C 17 H 19 O 3 N + H 2 O C 5 H 11 + C 12 H 10 O 4 Piperine piperidine piperic acid The hydrolysis products of piperine indicate that in piperine molecule the piperidine and piperic acid moieties are linked by an amide type linkage. Piperine is the condensation product of piperic acid and piperidine. 9. Use of piperine as a marker compound The thin layer chromatographic studies of alcohol extracts of Hinguvacadi churna, Piper nigrum Linn. (fruit) and an alcoholic solution of piperine was carried out and the R f values are given in Table 5. TLC profiles are given in Figure 6. Piperine was detected as dark blue in long UV. The spot corresponding to piperine is found in Piper nigrum and the churna. 10. Shelf-life period The physico chemical parameters of Hinguvacadi churna were determined at an interval of three months for a period of one year. The results show that there is no detectable variation observed in the values obtained on analysis of the samples at different periods. Hence the shelf - life period of the churna may be fixed as not less than one year. Discussion The organoleptic and physico-chemical parameters help to a great extent for the purpose of standardization. The total ash is the total

8 26 Gayathri et al. amount of material remaining after ignition. Ash values are helpful to determine the quality and purity of a drug. Acid-insoluble ash measures the amount of silica present, especially as sand and siliceous earth. The test for loss on drying determines both water and volatile matter. Alcohol soluble extractives and water soluble extractives determine the amount of active constituents extracted with solvents from the drug. The TLC profile and R f values obtained are important parameters for standardisation. The spot corresponding to the marker compound - piperine is present in Piper nigrum and Hinguvacadi churna, detecting its presence. HPTLC and HPLC studies of the formulation gave characteristic patterns which can be used to establish the identity of the drug. IR finger printing profile is also informative and serves as additional parameter for fixing standards for the churna. The IR fingerprint values which is specific for a particular compound or extract and gives valuable information about its identity. Conclusion The above discussed parameters - organoleptic, physico-chemical, TLC, HPTLC, HPLC and IR finger printing data can be considered as pharmacopoeial standards and will help us to determine the quality of the churna. These results can be used in turn to check and to ensure the quality of the medicine. Piperine was isolated from the churna as the marker compound and the spots similar to that obtained for piperine (Table 5), can be observed in the chromatogram of Piper nigrum and the churna with the same R f values and colour which confirms the genuineness of the drug. Acknowledgements The authors are thankful to the Director, Central Council for Research in Ayurveda and Siddha, New Delhi for providing necessary facilities to carry out this work. The authors are thankful to the Drug Standardisation Unit, Ayurveda College, Thiruvananthapuram, Kerala (India) for giving facilities for taking the HPTLC. The authors are also thankful to Dr. S. Indirakumari (R.O., Ay.) and Smt. B. Padmaja (A.R.O., Bot.), for their help and support. References 1. Alfred Martin: Physical Pharmacy. London. 4 th edition. pp.444 (2005). 2. Anonymous: Quality assurance of Ayurveda, Siddha, Unani and Homoeopathic drugs. Workshop documents. Department of AYUSH, Govt. of India, June 10-11, 2008, New Delhi. pp (2008). 3. Anonymous: The Ayurvedic Formulary of India. Part I. Ministry of Health and Family Welfare, Govt. of India, New Delhi. pp (2003). 4. Anonymous: The Ayurvedic Pharmacopoeia of India. Part II. (formulations). Volume I. Ministry of Health and Family Welfare, Govt. of India, New Delhi. pp (2007). 5. Bassett, J., Denney, R.C., Jeffery, G.H. and Mendham, J: Vogel s Text Book of Quantitative Analysis. ELBS, London. 4 th edition. pp.342 (1978). 6. Harborne, J.B: Phytochemical Methods. Chapmann and Hall, London. pp.192 (1973). 7. Sadasivam, S. and Manickam, A: Biochemical Methods. Tamil Nadu Agricultural University, Coimbatore. 2 nd edition. First reprint, pp.196 (1997). 8. Vagbhata: Astanga Haridaya. English translation by Prof. K. R. Srikantha, Murthy. Chaukhambha Sanskrit Series, Varanasi. Vol Chikitsasthana: 31-32/118 (1999). 9. Wagner, H. and Bladt S: Plant drug analysis - A Thin Layer Chromatography Atlas. Springer - Verlage, Berlin. pp.3-4, 364 (1996). 10. World Health Organization (WHO): Quality Control Methods of Medicinal Plant Materials. Geneva. pp.28-34, (1998). Address for correspondence: Dr. K. Gopakumar, Assistant Director (Ay.) RRI (Drug Research), Poojapura, Thiruvananthapuram Kerala (India). rritvm1@yahoo.co.in; drgopan2001@yahoo.com 019_2009

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