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HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis

HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis www.ijpsonline.com ed. New York: Marcel Dekker Inc; 2005. ACKNOWLEDGMENTS 3. Panchagnula R. Transdermal delivery of drugs. Indian J Pharmacol 1997;29:140-56. We are highly thankful to Prof. B.G. Shivananda, 4. Naik A, Kalia YN, Guy RH. Transdermal: overcoming the skin’s barrier function. PSTT 2000;3:318-26. Principal, Al-Ameen College of Pharmacy for his 5. Jasti BR, Abraham W, Ghosh TK. Transdermal and Topical drug continuous encouragement, Indian Pharmaceutical delivery systems. In: Ghosh TK, Jasti BR, editors. Theory and Practice Association for IPA-IRF scholarship, 3M India and of Contemporary Pharmaceutics. 1st ed. Florida: CRC Press; 2005. p. 423-53. 3M USA for their support in research work. REFERENCES AAccepted 30 January 2008 Revised 26 July 2007 1. Torotora G. Principles of Anatomy and Physiology. 10th ed. New York: Received 3 October 2006 John Wiely and sons; 2003. Indian J. Pharm. Sci., 2008, 70 (1): 94-96 2. Chein YW. Novel Drug Delivery Systems Revised and expanded. 2nd HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis G. V. SRINIVASAN*, K. P. UNNIKRISHNAN, A. B. REMA SHREE AND INDIRA BALACHANDRAN Phytochemistry Division, Centre for Medicinal Plants Research, Arya Vaidya Sala, Changuvetty, Kottakkal - 676 503, Kerala, India Srinivasan, et al.: HPLC Estimation of berberine in Tinospora species A high-performance liquid chromatographic method for the estimation of berberine in the stem of Tinospora cordifolia (Willd.) Miers. ex Hook.f. and Thoms. and Tinospora sinensis (Lour.) Merrill is described. The dried stems of T. cordifolia and T. sinensis were defatted with petroleum ether (60-80°). The marc was dried and further extracted with methanol. The concentration of berberine in methanol extract was determined using a C-18 reverse phase column with a mobile phase of acetonitrile:water (10:90 v/v) at a fl ow rate of 0.6 ml/min and with UV detection at 266 nm. TLC and HPLC comparison of both the species revealed signifi cant variation in the chemical constitution of the two species. This observation becomes important in the context of the use of T. sinensis in place of the genuine drug T. cordifolia. Key words: Tinospora cordifolia, Tinospora sinensis, berberine, HPLC T. cordifolia (Family: Menispermaceae), known as antileishmanial, antiinflammatory, antiarthritic, 9,14 Amrita (Guduchi) in Sanskrit is a widely used plant analgesic and diuretic properties. The drug is in folk and Ayurvedic systems of medicine. The term reported to possess 20% of the analgesic effect of 10,12 Amrita meaning divine nectar is attributed to this drug sodium salicylate . The plant is used in Ayurvedic in recognition of its capacity to impart youthfulness, Rasayanas to improve the immune system and the 1 11 vitality, and longevity to the consumer . Drug consists body resistance against infections . Amrita is a of the dried stem with bark intact. It is widely used in constituent of several preparations like Amritarishtam, folk and Ayurvedic systems of medicine for its general Dhanvantaram tailam, Cheriya rasnadi kashayam 2 3 4 5 1 tonic, anticancer , antiulcer , antipyretic , antihepatitis , and Valiya marmagulika . T. sinensis (Fam: 6 7 8 immunomodulatory , antioxidant , hypoglycaemic , Menispermaceae) is used almost in the same way antineoplastic, cardiotonic, antibacterial, antimicrobial, as T. cordifolia . However, practitioners consider T. cordifolia as the genuine source for Amrita. *For correspondence E-mail: avscmpr@sify.com Sesquiterpene tinocordifolin , sesquiterpene glycoside 96 Indian Journal of Pharmaceutical Sciences January - February 2008 www.ijpsonline.com tinocordifolioside , an immunologically active resolution of B was obtained at 25 ± 2° using a 17 18 Phenomenex Luna C-18 column (250 × 4.6 mm i.d; arabinogalactan , phytoecdysones viz., ecdysterone 19,20 5 μm) and an isocratic solvent system consisting of and makisterone, Alkaloids viz., berberine and magnoflorine are the major chemical compounds acetonitrile-water in the ratio 10:90 (v/v). The mobile isolated from the stem of T. cordifolia. Magnofl orine, phase was passed through 0.45 μm PVDF filter, berberine, tinosporicide, menispermacide, palmatine, degassed before use. The fl ow rate was kept constant at 0.6 ml/min and the detection was at 266 nm. For (+)- malabarolide and tinosinen I are the major calibration, standard solutions of B were prepared at chemical compounds isolated from the stem of 21-23 T. sinensis . concentrations of 1, 5, 10, 20, 40, 50, 80, 100, 200, 400, 800 and 1000 μg/ml using methanol as solvent. The standard solution was injected in triplicate and A reverse phase HPLC method has been developed the average detector response was measured. The to quantitatively estimate the berberine content in stem extracts were assayed in triplicate and peak the stem of T. cordifolia and T. sinensis. Berberine (B ) is an isoquinoline alkaloid reported to have areas corresponding to B were compared with the antimalarial, antipyretic, antimicrobial, antibacterial, calibration curve and amount of B was determined. antitumour and antiprotozoal (Leishmania ) 13,14 activities . An attempt has also been made to The validated parameters were specifi city, linearity, chromatographically compare the methanolic extracts precision and accuracy according to the ICH of both the species. guidelines . Inter-day reproducibility was verified by analyzing six different concentrations of B , The stem of T. cordifolia and T. sinensis were each injected four times, and determining the collected from the Herb Garden, Arya Vaidya Sala, relative standard deviation (RSD%). The intra-day Kottakkal, Kerala State, India in July 2006 and reproducibility was evaluated by the analysis of voucher specimens were deposited at the Herbarium, B at two different concentrations (80 and 100 Centre for Medicinal Plants Research, Kottakkal, μg/ml), four times a day on seven consecutive Kerala (Voucher No. 02202, 01363, 02426, and days and determining the RSD%. For recovery 03378). The stem was dried in the shade and coarsely studies, 500 μl of methanol solution containing 100 powdered. Powdered material of T. cordifolia and μg ml of B was added to 500 μl of three methanol T. sinensis (5 g) were defatted with 100 ml petroleum extract solutions (10, 20, 40 mg/ml). Recovery was ether in a Soxhlet extractor for 12 h. The marc calculated by comparing the resulting peak area with was air-dried and was further extracted with 50 ml the peak obtained from equal concentrations of B and methanol in a Soxhlet extractor for 12 h at 60°. expressed as percentage of this ratio. The extract was fi ltered and concentrated to dryness under reduced pressure below 60° using a rotary fl ash Thin layer and HPLC analyses were carried out to evaporator. Different concentrations of the residue compare the crude methanolic extracts of T. cordifolia were prepared in methanol and used for HPLC and T. sinensis. Thin layer chromatography was analysis to quantify the berberine content. performed on precoated silica gel 60 F TLC plates (E. Merck) of uniform thickness of 0.2 mm. The Solvents used were of HPLC grade (E. Merck). Test chromatogram was developed up to 80 mm under solutions were filtered through 0.20 μm nylon-6,6 chamber saturation conditions with chloroform- membrane before injection. All analyses were run in methanol, 70:20 (v/v) in a twin trough chamber. For triplicate and averaged. The standard berberine used HPLC analysis the conditions mentioned above were was purchased from Fluka Chemicals, Switzerland. maintained. TLC was done on pre- coated silica gel 60 F plates (E. Merck) of uniform thickness of 0.2 mm. In view of the potential therapeutic importance of the drug Amrita, and considering the degree of A Shimadzu (Kyoto, Japan) HPLC system consisted of adulteration/substitution of the raw materials, a simple LC-10AT VP pump, SPD-M10A VP photodiode array HPLC method was developed and validated in order detector, CLASS-VP 6.12 SP5 integration software to quantify berberine (B ). Satisfactory retention times and a Rheodyne injection valve fitted with a 20 μl and good resolution of B was achieved using reverse injection loop, was used for the analysis. Baseline phase C-18 column eluted with acetonitrile-water January - February 2008 Indian Journal of Pharmaceutical Sciences 97 www.ijpsonline.com (10:90 v/v) at a flow rate of 0.6 ml/min. A sharp inter-day and intra-day reproducibility were found to and symmetric peak for B was obtained, with good 1 be 3.5% and 4.7%, respectively. The concentration of baseline resolution and minimal tailing, thus facilitating B in the stem of T. cordifolia and T. sinensis (dry the accurate measurement of peak area. The HPLC weight basis) was found to be 0.3192% and 0.0967% analysis was carried out in isocratic conditions and a (w/w), respectively (Table 1). retention time of 8.65 min was obtained for standard berberine. Typical HPLC chromatograms of B and 1 Comparative chromatographic studies of the methanol methanol extracts of the stem of T. cordifolia and extract of the two species revealed the presence of T. sinensis are shown in fi gs.1-3. two additional compounds in T. sinensis. These two compounds were isolated by column chromatography The calibration curve for B was found to be linear followed by preparative TLC. HPLC analysis gave over the range 0.1 to 0.01 mg/ml (r = 0.98). The peaks at 24.54 and 28.48 min, in the methanol extract linear regression equation for the calibration curve of T. sinensis. Preliminary studies (UV, FTIR, TLC was y = 123512x -78787, where y is the peak area and derivatisation) indicate that these two compounds ratio of B and x is the concentration of B (μg/ml). 1 1 possess a steroid nucleus. Detailed characterization The average recovery was 99.10 ± 0.32%, while the studies of these compounds are under progress. Ayurvedic practitioners of Kerala use both T. coridifolia and T. sinensis as Amrita. Present study revealed that T. cordifolia and T. sinensis show differences in chemical constituents. The berberine content of the two species show marked variation, T. cordifolia having three times higher berberine concentration. On the other hand T. sinensis contain certain compounds that do not occur in T. cordifolia. Whether they are of any therapeutic significance is not known and hence need to be investigated. Such chemical variation highlights the need for pharmacological standardization and validation. The Fig. 1: HPLC chromatogram of standard berberine. Berberine peak at the retetion time 8.6 min detected at a wavelength analytical conditions presented here are applicable of 266 nm. Retention Time Detector A-266 nm Detector A-266 nm TS TC Retention Time Tinospora sinenfair002 Tinospora cordifair001 80 30 -20 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 Minutes Fig. 2: HPLC chromatogram of methanol extract of T. cordifolia. Fig. 3: HPLC chromatogram of methanol extract of T. sinensis. Peak at the retention time 8.6 min correspond to berberine. Peak at the retention time 8.6 min correspond to berberine. TABLE 1: CONCENTRATION OF BERBERINE IN DIFFERENT SAMPLES Plant drug Concentration of berberine (%) in the samples Sample 1 Sample 2 Sample 3 Average Conc. (%) T. cordifolia 0.3196 0.3188 0.3191 0.3192 T. sinensis 0.0965 0.0971 0.0966 0.0967 a b Methanol extract of the defatted plant drug, concentration of berberine in the dried plant drugs 98 Indian Journal of Pharmaceutical Sciences January - February 2008 mAU 3.051 3.499 4.779 4.523 6.592 8.651 10.933 mAU mAU 2.976 3.285 3.211 3.445 4.768 6.624 7.008 7.211 8.000 8.672 9.643 10.955 19.296 24.544 28.480 mAU www.ijpsonline.com 10. Rajul B, Bhatt JV. Phagocytic coeffi cient as a measure for evaluating with respect to the identification and quantification plant antibiotic. Indian J Pharm 1953;15:309. of B and for the quality checking of raw drugs to 11. Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro B, Ghosh AC. distinguish the two species conveniently. Chemistry and medicinal properties of T.cordifolia (Guduchi). Indian J Pharmacol 2003;35:83-91. 12. Chadha YR, editor. The Wealth of India-Raw Materials. Vol. X. New ACKNOWLEDGEMENTS Delhi: CSIR; 1976. 13. Budavari S, editor. The Merck Index. 13th ed. Whitehouse Station, NJ: Merck and Co. Inc; 2001. We are grateful to Kerala State Council for Science 14. Patani A, editor. Indian Herbal Pharmacopoeia. Revised New ed. Technology and Environment (KSCSTE), Govt. of Mumbai: Indian Drug Manufactures Association; 2002. Kerala for providing fi nancial assistance to carry out 15. Maurya R, Handa SS. Tinocordifolin, a sesquiterpene from T.cordifolia. Phytochemistry 1998;49:1343-6. this work. 16. Maurya R, Dhar KL, Handa SS. A sesquiterepene glycoside from T.cordifolia. Phytochemistry 1997;44:749-50. REFERENCES 17. Chintalwar G, Jain A, Sipahimalani AT, Banerji A, Sumariwalla P, Ramakrishnan R, et al. An immunologically active arabinogalactan from T.cordifolia. Phytochemistry 1999;52:1089-93. 1. Sivarajan VV, Balachandran I. Ayurvedic Drugs and their Plant Sources. 18. Pradhan P, Gangan VD, Sipahimalani AT, Banerji A. Two New Delhi: Oxford and IBH Publishing Co. Pvt. Ltd; 1999. phytoecdysones from T. cordifolia: Structural assignments by 2D NMR 2. Mathew S, Kuttan G. Immunomodulatory and anti-tumour activities of spectroscopy. Indian J Chem Sec B 1997;36:958-62. T.cordifolia. Fitoterapia 1999;70:35-43. 19. Pachaly P, Schneider C. Alkaloids from Tinospora cordifolia. Arch 3. Bairy KL, Roopa K, Malini S, Rao CM. Protective effect of T.cordifolia Pharm (Weinheim Ger) 1981;314:251-6. on experimentally induced gastric ulcers in rats. J Nat Remedies 20. Bisset NG, Nwaiwu J. Quaternary alkaloids of Tinospora species. Planta 2002;2:49-53. Medica 1983;48:275-9. 4. Kumar A, Srivastava S. Study of antipyretic activity of Guduchi 21. Rastogi RP, Mehrotra BN, editors. Compendium of Indian Medicinal Sachitra. Ayurveda 1995;48:289-91. Plants. Vol. 3. PID, New Delhi: CSIR; 1993. 5. Prakash S, Rai NP. Role of T.cordifolia (Willd) Miers (Guduchi) in the 22. Rastogi RP, Mehrotra BN, editors. Compendium of Indian Medicinal treatment of infective hepatitis. J Res Ayurveda and Siddha 1996;17:58- Plants. Vol. 4. PID, New Delhi: CSIR; 1995. 23. Rastogi RP, Mehrotra BN, editors. Compendium of Indian Medicinal 6. Manjrekar PN, Jolly CI, Narayanan S. Comparative studies of the Plants. Vol. 5. PID, New Delhi: CSIR; 1998. immunomodulatory activity of T.cordifolia and T.sinensis. Fitoterapia 24. Swartz ME, Krull IS. Analytical method development and 2000;71:254-7. validation. NY, USA: Marcel Dekker Inc; 1997. 7. Prince PSM, Menon VP. Antioxidant action of T.cordifolia root extract in alloxan diabetic rats. Phytother Res 2001;15:213-8. 8. Prince PSM, Menon VP. Hypoglycaemic and other related action of Accepted 31 January 2008 T.cordifolia roots in alloxan diabetic rats. J Ethnopharmacol 2000;70: Revised 31 July 2007 9-15. Received 11 January 2007 9. Sharma PC, Yelne MB, Dennis TJ, editors. Database on Medicinal Indian J. Pharm. Sci., 2008, 70 (1): 96-99 Plants Used in Ayurveda. Vol.3. New Delhi: CCRAS; 2001. Parenteral Formulation of Zopiclone P. V. SWAMY*, P. SUSHMA, G. CHIRAG, K. PRASAD, M. YOUNUS ALI AND S. A. RAJU Department of Pharmaceutics, HKES’ College of Pharmacy, Sedam Road, Gulbarga - 585105, India Swamy, et al.: Parenteral Formulation of Zopiclone The present study was undertaken with an intention to develop a stable and effective parenteral formulation, containing the drug zopiclone. Since zopiclone is a water insoluble drug, various methods such as co-solvency, pH control and hydrotrophy have been tried in order to enhance its solubility. When all these methods could not give adequate solubility enhancement of the drug, a hydrochloride salt was prepared, and it was found to be thermostable. Various batches of zopiclone hydrochloride injection formulation were prepared in order to assess the infl uence of light, atmospheric oxygen and antioxidant on the stability of the drug and the formulations were also subjected to accelerated stability testing in order to predict approximate shelf-life of the product. Key words: Zopiclone, solubility enhancement, parenteral formulation, shelf-life, accelerated stability *For correspondence E-mail: vspadavala@rediffmail.com January - February 2008 Indian Journal of Pharmaceutical Sciences 99 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Indian Journal of Pharmaceutical Sciences Pubmed Central

HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis

Indian Journal of Pharmaceutical Sciences , Volume 70 (1) – Apr 1, 168

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Pubmed Central
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© Indian Journal of Pharmaceutical Sciences
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0250-474X
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1998-3743
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10.4103/0250-474X.40341
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Abstract

www.ijpsonline.com ed. New York: Marcel Dekker Inc; 2005. ACKNOWLEDGMENTS 3. Panchagnula R. Transdermal delivery of drugs. Indian J Pharmacol 1997;29:140-56. We are highly thankful to Prof. B.G. Shivananda, 4. Naik A, Kalia YN, Guy RH. Transdermal: overcoming the skin’s barrier function. PSTT 2000;3:318-26. Principal, Al-Ameen College of Pharmacy for his 5. Jasti BR, Abraham W, Ghosh TK. Transdermal and Topical drug continuous encouragement, Indian Pharmaceutical delivery systems. In: Ghosh TK, Jasti BR, editors. Theory and Practice Association for IPA-IRF scholarship, 3M India and of Contemporary Pharmaceutics. 1st ed. Florida: CRC Press; 2005. p. 423-53. 3M USA for their support in research work. REFERENCES AAccepted 30 January 2008 Revised 26 July 2007 1. Torotora G. Principles of Anatomy and Physiology. 10th ed. New York: Received 3 October 2006 John Wiely and sons; 2003. Indian J. Pharm. Sci., 2008, 70 (1): 94-96 2. Chein YW. Novel Drug Delivery Systems Revised and expanded. 2nd HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis G. V. SRINIVASAN*, K. P. UNNIKRISHNAN, A. B. REMA SHREE AND INDIRA BALACHANDRAN Phytochemistry Division, Centre for Medicinal Plants Research, Arya Vaidya Sala, Changuvetty, Kottakkal - 676 503, Kerala, India Srinivasan, et al.: HPLC Estimation of berberine in Tinospora species A high-performance liquid chromatographic method for the estimation of berberine in the stem of Tinospora cordifolia (Willd.) Miers. ex Hook.f. and Thoms. and Tinospora sinensis (Lour.) Merrill is described. The dried stems of T. cordifolia and T. sinensis were defatted with petroleum ether (60-80°). The marc was dried and further extracted with methanol. The concentration of berberine in methanol extract was determined using a C-18 reverse phase column with a mobile phase of acetonitrile:water (10:90 v/v) at a fl ow rate of 0.6 ml/min and with UV detection at 266 nm. TLC and HPLC comparison of both the species revealed signifi cant variation in the chemical constitution of the two species. This observation becomes important in the context of the use of T. sinensis in place of the genuine drug T. cordifolia. Key words: Tinospora cordifolia, Tinospora sinensis, berberine, HPLC T. cordifolia (Family: Menispermaceae), known as antileishmanial, antiinflammatory, antiarthritic, 9,14 Amrita (Guduchi) in Sanskrit is a widely used plant analgesic and diuretic properties. The drug is in folk and Ayurvedic systems of medicine. The term reported to possess 20% of the analgesic effect of 10,12 Amrita meaning divine nectar is attributed to this drug sodium salicylate . The plant is used in Ayurvedic in recognition of its capacity to impart youthfulness, Rasayanas to improve the immune system and the 1 11 vitality, and longevity to the consumer . Drug consists body resistance against infections . Amrita is a of the dried stem with bark intact. It is widely used in constituent of several preparations like Amritarishtam, folk and Ayurvedic systems of medicine for its general Dhanvantaram tailam, Cheriya rasnadi kashayam 2 3 4 5 1 tonic, anticancer , antiulcer , antipyretic , antihepatitis , and Valiya marmagulika . T. sinensis (Fam: 6 7 8 immunomodulatory , antioxidant , hypoglycaemic , Menispermaceae) is used almost in the same way antineoplastic, cardiotonic, antibacterial, antimicrobial, as T. cordifolia . However, practitioners consider T. cordifolia as the genuine source for Amrita. *For correspondence E-mail: avscmpr@sify.com Sesquiterpene tinocordifolin , sesquiterpene glycoside 96 Indian Journal of Pharmaceutical Sciences January - February 2008 www.ijpsonline.com tinocordifolioside , an immunologically active resolution of B was obtained at 25 ± 2° using a 17 18 Phenomenex Luna C-18 column (250 × 4.6 mm i.d; arabinogalactan , phytoecdysones viz., ecdysterone 19,20 5 μm) and an isocratic solvent system consisting of and makisterone, Alkaloids viz., berberine and magnoflorine are the major chemical compounds acetonitrile-water in the ratio 10:90 (v/v). The mobile isolated from the stem of T. cordifolia. Magnofl orine, phase was passed through 0.45 μm PVDF filter, berberine, tinosporicide, menispermacide, palmatine, degassed before use. The fl ow rate was kept constant at 0.6 ml/min and the detection was at 266 nm. For (+)- malabarolide and tinosinen I are the major calibration, standard solutions of B were prepared at chemical compounds isolated from the stem of 21-23 T. sinensis . concentrations of 1, 5, 10, 20, 40, 50, 80, 100, 200, 400, 800 and 1000 μg/ml using methanol as solvent. The standard solution was injected in triplicate and A reverse phase HPLC method has been developed the average detector response was measured. The to quantitatively estimate the berberine content in stem extracts were assayed in triplicate and peak the stem of T. cordifolia and T. sinensis. Berberine (B ) is an isoquinoline alkaloid reported to have areas corresponding to B were compared with the antimalarial, antipyretic, antimicrobial, antibacterial, calibration curve and amount of B was determined. antitumour and antiprotozoal (Leishmania ) 13,14 activities . An attempt has also been made to The validated parameters were specifi city, linearity, chromatographically compare the methanolic extracts precision and accuracy according to the ICH of both the species. guidelines . Inter-day reproducibility was verified by analyzing six different concentrations of B , The stem of T. cordifolia and T. sinensis were each injected four times, and determining the collected from the Herb Garden, Arya Vaidya Sala, relative standard deviation (RSD%). The intra-day Kottakkal, Kerala State, India in July 2006 and reproducibility was evaluated by the analysis of voucher specimens were deposited at the Herbarium, B at two different concentrations (80 and 100 Centre for Medicinal Plants Research, Kottakkal, μg/ml), four times a day on seven consecutive Kerala (Voucher No. 02202, 01363, 02426, and days and determining the RSD%. For recovery 03378). The stem was dried in the shade and coarsely studies, 500 μl of methanol solution containing 100 powdered. Powdered material of T. cordifolia and μg ml of B was added to 500 μl of three methanol T. sinensis (5 g) were defatted with 100 ml petroleum extract solutions (10, 20, 40 mg/ml). Recovery was ether in a Soxhlet extractor for 12 h. The marc calculated by comparing the resulting peak area with was air-dried and was further extracted with 50 ml the peak obtained from equal concentrations of B and methanol in a Soxhlet extractor for 12 h at 60°. expressed as percentage of this ratio. The extract was fi ltered and concentrated to dryness under reduced pressure below 60° using a rotary fl ash Thin layer and HPLC analyses were carried out to evaporator. Different concentrations of the residue compare the crude methanolic extracts of T. cordifolia were prepared in methanol and used for HPLC and T. sinensis. Thin layer chromatography was analysis to quantify the berberine content. performed on precoated silica gel 60 F TLC plates (E. Merck) of uniform thickness of 0.2 mm. The Solvents used were of HPLC grade (E. Merck). Test chromatogram was developed up to 80 mm under solutions were filtered through 0.20 μm nylon-6,6 chamber saturation conditions with chloroform- membrane before injection. All analyses were run in methanol, 70:20 (v/v) in a twin trough chamber. For triplicate and averaged. The standard berberine used HPLC analysis the conditions mentioned above were was purchased from Fluka Chemicals, Switzerland. maintained. TLC was done on pre- coated silica gel 60 F plates (E. Merck) of uniform thickness of 0.2 mm. In view of the potential therapeutic importance of the drug Amrita, and considering the degree of A Shimadzu (Kyoto, Japan) HPLC system consisted of adulteration/substitution of the raw materials, a simple LC-10AT VP pump, SPD-M10A VP photodiode array HPLC method was developed and validated in order detector, CLASS-VP 6.12 SP5 integration software to quantify berberine (B ). Satisfactory retention times and a Rheodyne injection valve fitted with a 20 μl and good resolution of B was achieved using reverse injection loop, was used for the analysis. Baseline phase C-18 column eluted with acetonitrile-water January - February 2008 Indian Journal of Pharmaceutical Sciences 97 www.ijpsonline.com (10:90 v/v) at a flow rate of 0.6 ml/min. A sharp inter-day and intra-day reproducibility were found to and symmetric peak for B was obtained, with good 1 be 3.5% and 4.7%, respectively. The concentration of baseline resolution and minimal tailing, thus facilitating B in the stem of T. cordifolia and T. sinensis (dry the accurate measurement of peak area. The HPLC weight basis) was found to be 0.3192% and 0.0967% analysis was carried out in isocratic conditions and a (w/w), respectively (Table 1). retention time of 8.65 min was obtained for standard berberine. Typical HPLC chromatograms of B and 1 Comparative chromatographic studies of the methanol methanol extracts of the stem of T. cordifolia and extract of the two species revealed the presence of T. sinensis are shown in fi gs.1-3. two additional compounds in T. sinensis. These two compounds were isolated by column chromatography The calibration curve for B was found to be linear followed by preparative TLC. HPLC analysis gave over the range 0.1 to 0.01 mg/ml (r = 0.98). The peaks at 24.54 and 28.48 min, in the methanol extract linear regression equation for the calibration curve of T. sinensis. Preliminary studies (UV, FTIR, TLC was y = 123512x -78787, where y is the peak area and derivatisation) indicate that these two compounds ratio of B and x is the concentration of B (μg/ml). 1 1 possess a steroid nucleus. Detailed characterization The average recovery was 99.10 ± 0.32%, while the studies of these compounds are under progress. Ayurvedic practitioners of Kerala use both T. coridifolia and T. sinensis as Amrita. Present study revealed that T. cordifolia and T. sinensis show differences in chemical constituents. The berberine content of the two species show marked variation, T. cordifolia having three times higher berberine concentration. On the other hand T. sinensis contain certain compounds that do not occur in T. cordifolia. Whether they are of any therapeutic significance is not known and hence need to be investigated. Such chemical variation highlights the need for pharmacological standardization and validation. The Fig. 1: HPLC chromatogram of standard berberine. Berberine peak at the retetion time 8.6 min detected at a wavelength analytical conditions presented here are applicable of 266 nm. Retention Time Detector A-266 nm Detector A-266 nm TS TC Retention Time Tinospora sinenfair002 Tinospora cordifair001 80 30 -20 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 Minutes Fig. 2: HPLC chromatogram of methanol extract of T. cordifolia. Fig. 3: HPLC chromatogram of methanol extract of T. sinensis. Peak at the retention time 8.6 min correspond to berberine. Peak at the retention time 8.6 min correspond to berberine. TABLE 1: CONCENTRATION OF BERBERINE IN DIFFERENT SAMPLES Plant drug Concentration of berberine (%) in the samples Sample 1 Sample 2 Sample 3 Average Conc. (%) T. cordifolia 0.3196 0.3188 0.3191 0.3192 T. sinensis 0.0965 0.0971 0.0966 0.0967 a b Methanol extract of the defatted plant drug, concentration of berberine in the dried plant drugs 98 Indian Journal of Pharmaceutical Sciences January - February 2008 mAU 3.051 3.499 4.779 4.523 6.592 8.651 10.933 mAU mAU 2.976 3.285 3.211 3.445 4.768 6.624 7.008 7.211 8.000 8.672 9.643 10.955 19.296 24.544 28.480 mAU www.ijpsonline.com 10. Rajul B, Bhatt JV. Phagocytic coeffi cient as a measure for evaluating with respect to the identification and quantification plant antibiotic. Indian J Pharm 1953;15:309. of B and for the quality checking of raw drugs to 11. Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro B, Ghosh AC. distinguish the two species conveniently. Chemistry and medicinal properties of T.cordifolia (Guduchi). Indian J Pharmacol 2003;35:83-91. 12. Chadha YR, editor. The Wealth of India-Raw Materials. Vol. X. New ACKNOWLEDGEMENTS Delhi: CSIR; 1976. 13. Budavari S, editor. The Merck Index. 13th ed. Whitehouse Station, NJ: Merck and Co. Inc; 2001. We are grateful to Kerala State Council for Science 14. Patani A, editor. Indian Herbal Pharmacopoeia. Revised New ed. Technology and Environment (KSCSTE), Govt. of Mumbai: Indian Drug Manufactures Association; 2002. Kerala for providing fi nancial assistance to carry out 15. Maurya R, Handa SS. Tinocordifolin, a sesquiterpene from T.cordifolia. Phytochemistry 1998;49:1343-6. this work. 16. Maurya R, Dhar KL, Handa SS. A sesquiterepene glycoside from T.cordifolia. Phytochemistry 1997;44:749-50. REFERENCES 17. Chintalwar G, Jain A, Sipahimalani AT, Banerji A, Sumariwalla P, Ramakrishnan R, et al. An immunologically active arabinogalactan from T.cordifolia. Phytochemistry 1999;52:1089-93. 1. Sivarajan VV, Balachandran I. Ayurvedic Drugs and their Plant Sources. 18. Pradhan P, Gangan VD, Sipahimalani AT, Banerji A. Two New Delhi: Oxford and IBH Publishing Co. Pvt. Ltd; 1999. phytoecdysones from T. cordifolia: Structural assignments by 2D NMR 2. Mathew S, Kuttan G. Immunomodulatory and anti-tumour activities of spectroscopy. Indian J Chem Sec B 1997;36:958-62. T.cordifolia. Fitoterapia 1999;70:35-43. 19. Pachaly P, Schneider C. Alkaloids from Tinospora cordifolia. Arch 3. Bairy KL, Roopa K, Malini S, Rao CM. Protective effect of T.cordifolia Pharm (Weinheim Ger) 1981;314:251-6. on experimentally induced gastric ulcers in rats. J Nat Remedies 20. Bisset NG, Nwaiwu J. Quaternary alkaloids of Tinospora species. Planta 2002;2:49-53. Medica 1983;48:275-9. 4. Kumar A, Srivastava S. Study of antipyretic activity of Guduchi 21. Rastogi RP, Mehrotra BN, editors. Compendium of Indian Medicinal Sachitra. Ayurveda 1995;48:289-91. Plants. Vol. 3. PID, New Delhi: CSIR; 1993. 5. Prakash S, Rai NP. Role of T.cordifolia (Willd) Miers (Guduchi) in the 22. Rastogi RP, Mehrotra BN, editors. Compendium of Indian Medicinal treatment of infective hepatitis. J Res Ayurveda and Siddha 1996;17:58- Plants. Vol. 4. PID, New Delhi: CSIR; 1995. 23. Rastogi RP, Mehrotra BN, editors. Compendium of Indian Medicinal 6. Manjrekar PN, Jolly CI, Narayanan S. Comparative studies of the Plants. Vol. 5. PID, New Delhi: CSIR; 1998. immunomodulatory activity of T.cordifolia and T.sinensis. Fitoterapia 24. Swartz ME, Krull IS. Analytical method development and 2000;71:254-7. validation. NY, USA: Marcel Dekker Inc; 1997. 7. Prince PSM, Menon VP. Antioxidant action of T.cordifolia root extract in alloxan diabetic rats. Phytother Res 2001;15:213-8. 8. Prince PSM, Menon VP. Hypoglycaemic and other related action of Accepted 31 January 2008 T.cordifolia roots in alloxan diabetic rats. J Ethnopharmacol 2000;70: Revised 31 July 2007 9-15. Received 11 January 2007 9. Sharma PC, Yelne MB, Dennis TJ, editors. Database on Medicinal Indian J. Pharm. Sci., 2008, 70 (1): 96-99 Plants Used in Ayurveda. Vol.3. New Delhi: CCRAS; 2001. Parenteral Formulation of Zopiclone P. V. SWAMY*, P. SUSHMA, G. CHIRAG, K. PRASAD, M. YOUNUS ALI AND S. A. RAJU Department of Pharmaceutics, HKES’ College of Pharmacy, Sedam Road, Gulbarga - 585105, India Swamy, et al.: Parenteral Formulation of Zopiclone The present study was undertaken with an intention to develop a stable and effective parenteral formulation, containing the drug zopiclone. Since zopiclone is a water insoluble drug, various methods such as co-solvency, pH control and hydrotrophy have been tried in order to enhance its solubility. When all these methods could not give adequate solubility enhancement of the drug, a hydrochloride salt was prepared, and it was found to be thermostable. Various batches of zopiclone hydrochloride injection formulation were prepared in order to assess the infl uence of light, atmospheric oxygen and antioxidant on the stability of the drug and the formulations were also subjected to accelerated stability testing in order to predict approximate shelf-life of the product. Key words: Zopiclone, solubility enhancement, parenteral formulation, shelf-life, accelerated stability *For correspondence E-mail: vspadavala@rediffmail.com January - February 2008 Indian Journal of Pharmaceutical Sciences 99

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