Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241212EnglishN-0001November30HealthcareSIMULTANEOUS SPECTROPHOTOMETRIC ESTIMATION OF THIOCOLCHICOSIDE AND DICLOFENAC POTASSIUM B.P. IN COMBINED DOSAGE FORM BY RATIO DERIVATIVE AND DUAL WAVELENGTH METHOD English0310Choudhari V.P.English Chabukswar A.R.English Savakhande S.N.English Tryambake M.U.English Suryawanshi V.M.English Sayal P.K.EnglishA simple, economical, precise and accurate method for simultaneous determination of Diclofenac potassium (DICLO K) and Thiocholchiside (THIO) in combined dosage form has been developed. The first method is based on Ratio Derivative (Method A) and second method is based on Dual Wavelength (Method B). The amplitudes 268.78 nm and 355.62 nm wavelength were selected to determine THIO and DICLO K respectively, by method A. In method B, two wavelengths were selected for each drug in a way so that the difference in absorbance is zero for another drug. At wavelengths 252.47 nm and 260.74 nm Thiocholchiside have equal absorbance therefore these two wavelengths were used to determine DICLO K and on similar basis 263.22 nm and 301.65 nm were selected to determine THIO in combined formulation. The drugs obey Beer?s law in the concentration range of 12.5-75 μg mL-1 for DICLO K and 2-12μg mL- 1 for THIO by the method A and 25-75 μg mL-1 for DICLO K and 4-12 μg mL-1 for THIO by the method B. The % assay of DICLO K and THIO was found to be in the range 99.95 – 100.29 % by the proposed methods. Recovery was found in the range of 98.6 - 101.0 % for both analytes by both methods. The results of analysis have been validated statistically and recovery studies confirmed the accuracy and reproducibility of the proposed methods which were carried out by following ICH guidelines. EnglishDiclopfenac potassium (DICLO K) Thiocholchiside. (THIO), Ratio Spectra Derivative Spectrophotometry, Dual wavelength Spectrophotometry.INTRODUCTION Diclofenac potassium, potassium [0- (2,6-dichloroanilino) phenyl] acetate, is non-steroidal anti-inflammatory agent. Diclofenac is officially in B.P. where as its potassium salt is official in European pharmacopeia. Other method reported for its estimation are by laser desorption ionisation mass spectra, thermal and fractional analysis, UV Spectrophotometry method, Direct rapid and sensitive HPLC and HPTLC. Thiocolchicoside is (s)-N-[3-(B-Dglucopyranoxyloxy)-5, 6, 7, 9- tetrahydro-1,2-dimethoxy-10- (methylthio) -9-oxobenzo[a]heptalen- 7yl] acetamide (Fig.1). Thiocolchicoside is a synthetic sulphur derivative of colchicoside. Thiocolchicoside has a selective affinity for γ-amino- butyric acid (GABA) receptors and acts on the muscular contracture by activating the GABAnergic inhibitory pathways thereby acting as a potent muscle relaxant4 . For THIO various analytical methods have been reported or its individual estimation and in combined dosage form which includes HPLC, electrophoresis, LC-MS, extractive Spectrophotometry, visible Spectrophotometry, HPLC with electrochemical detection. Two methods have been reported for simultaneous analysis of DICLO K and THIO in its combination which includes TLC- Densitometry and second order derivative Spectrophotometry. Here an attempt has been made to develop simple, rapid and accurate Dual Wavelength and Ratio Derivative spectroscopic methods for simultaneous estimation of DICLO K and THIO from its formulation. The proposed methods are optimized and validated as per International Conference on Harmonization (ICH) guidelines. MATERIALS AND METHODS Instrumentation An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used for Spectrophotometric method. All weighing were done on electronic balance (Model Shimadzu AUW-220D). Spectroscopic grade methanol was used throughout the study. Ultrasonicator (Model 5.5 150H) was used for sample solution preparation. Reagents and chemicals Pure drug sample of DICLO K and THIO were kindly supplied as a gift sample by Zest Pharma, Indore and Glenmark Pharmaceuticals, Sinner, Nasik, respectively. These samples were used without further purification. Tablet formulation manufactured by Sun Pharmaceutical Industries (Lotensyl, Batch No. AD 92286) was purchased from local market containing DICLO K (50 mg) and THIO (10 mg) per tablet. Spectroscopy grade methanol purchased from Merck, Mumbai was used throughout the study. Preparation of Standard Stock Solutions and calibration Curve Standard stock solutions each containing 1000 μg mL-1 of DICLO K and THIO were prepared separately in the methanol. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in methanol. For verification of Beer?s law a series of dilutions in the concentration range of 12.5-62.5 μg mL-1 for DICLO K and 2- 10 μg mL-1 for THIO were prepared in mixture for method A. Solutions in the concentration rang of 25-75 μg mL-1 for DICLO K (series A) and 4-12 μg mL-1 for THIO (series B) were prepared and mixture of both the drugs (series C) in same concentration range was prepared for method B. Preparation of Sample Stock Solution For formulation analysis, twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 50 mg of DICLO K (8 mg of THIO) was weighed and dissolved in 40 mL of methanol with the aid of ultrasonicator for 10 min and solution was filtered through Whatman paper No. 41 into a 50 ml volumetric flask. Filter paper was washed with methanol, adding washings to the volumetric flask and volume was made up to mark. The solution was suitably diluted with methanol to get of 37.5 μg/ml of DICLO K and 6 μg/ml of THIO. The proposed methods were followed for analysis. Procedure Method A: RATIO DERIVATIVE METHOD The method involves dividing the spectrum of mixture by the standardized spectra of each of the analyte and deriving the ratio to obtain spectrum that is dependent of concentration of analyte used as a divisor. Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different DICLO K standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of THIO (6 μg mL-1 ) and the first derivative of these spectra traced, illustrated in Fig 1 . Wavelength 268.78 nm was selected for the quantification of DICLO K in DICLO K + THIO mixture. The ratio and ratio derivative spectra of the solutions of THIO at different concentrations were obtained by dividing each with the stored standard spectrum of the DICLO K (37.5 μg mL-1 ) (Fig. 2). Wavelength 355.62 nm was selected for the quantification of THIO in THIO + DICLO K mixture. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The amount of DICLO K (C DCLO K) and THIO (C THIO) in tablets and capsules was calculated by using equations 1 and 2, respectively. C DICLO K = [Derivative amplitude at 268.78 - (0.0094)] / (1.4434) ... (1) CTHIO = [Derivative amplitude at 355.62 - (-0.0137) ] / (0.0792) ... (2) Method B: DUAL WAVELENGTH METHOD The spectrum of DICLO K shows identical absorbance at 263.22 nm (λ3) and 301.65 nm (λ4) therefore these two wavelengths were selected for the analysis of THIO. All the solutions of series A were scanned to ensure that the difference between λ3 and λ4 is zero. Similarly, the THIO solutions were scanned to determine the two wavelengths, where absorbance is same. These two wavelengths were found to be 252.47 nm (λ1) and 260.74 nm (λ2). All the solutions of series B were scanned to ensure that difference between (λ1) and (λ2) is zero. All the solutions of series c were scanned to verify the results.   Recovery studies The accuracy of the proposed methods was checked by recovery study, by addition of standard drug solution to preanalysed sample solution at three different concentration levels (50 %, 100 % and 150 %) within the range of linearity for both the drugs. The basic concentration level of sample solution selected for spiking of the drugs standard solution was 37.5 μg/ml of DICLO K and 6 μg/ml of THIO for both the methods. Precision of the Method Reparability of the methods was studied by repeating the methods six times. To study intra-day precision, method was repeated 3 times in a day. Similarly the method was repeated on five different days to determine inter-day precision. RESULTS AND DISCUSSION DICLO K and THIO the proposed methods for simultaneous estimation of in combined dosage form were found to be accurate, simple and rapid. The developed methods can be used for routine analysis of two drugs in combined dosage forms. Practically no interference from tablet excipients was observed in these methods. Both the methods are accurate, simple, rapid, precise, reliable, sensitive, reproducible and economical. These methods were validated as per ICH guidelines. The values of % RSD and correlation of coefficient were satisfactory and result of the recovery study indicates that there is no interference due to excipients present in the formulation. Result of formulation analysis and precision study are summarized in table 1 indicated that method is precise. % Recovery (%RSD) for DICLO K by Method A and Method B was found to be in the range of 99.9- 101.0(0.38-1.27) and 99.50-100.6 (0.25- 1.42) respectively and that of THIO by Method A and Method B was found to be in the range of 98.6-100.2(0.82-1.56) and 99.5-100.1(1.10-1.50) (Table 2). CONCLUSION The proposed methods are simple, precise, accurate and rapid for the determination of DICLO K and THIO in combined tablet dosage forms. Analysis of authentic samples containing DICLO K and THIO showed no interference from the common additives and excipients. Hence, recommended procedure is well suited for the assay and evaluation of drugs in pharmaceutical preparations. Thus these methods can be easily and conveniently adopted for routine quality control analysis. ACKNOWLEDGEMENT The authors are thankful to Zest Pharma, Indore and Glenmark Pharmaceuticals for providing gift samples. The authors are thankful to Management of MAEER?s Maharashtra Institute of Pharmacy, Pune for providing necessary facility for the work.   Englishhttp://ijcrr.com/abstract.php?article_id=2205http://ijcrr.com/article_html.php?did=22051. A.H. Beckett, J.B. Stenlake, Practical pharmaceutical chemistry, the Athlone press of university of London, 3rd ed., part two,1976,249. 2. M. Carta *, L. Murru, P. Botta, G.Talani, G. Pietro Sechi, P. De Riu, E. Sanna, G. Biggio, The muscle relaxant thiocolchicoside is an antagonist of GABAA receptor function in the central nervous system, Neuropharmacology 51 (2006) 805-815. 3. W. Schneider, P.H. Degen, Simultaneous determination of diclofenac sodium and its hydroxy metabolites by capillary column gas chromatography with electroncapture detection, Journal of Chromatography A, Volume 217, 6 November 1981, Pages 263-271. 4. Gao, Songmei, Analysis of diclofenac potassium by laser desorption ionization mass spectrum. 5. The Merck index, an encyclopedia of chemicals, drugs and Biologicals, Merck research laboratories, 13th ed., 1997, 3108,9397. 6. A. fini.et al, Thermal and fractional analysis of sodium and potassium salts of Diclofenac. 7. R. K. Prasad*and R.Sharma, Simultaneous estimation and validation of Rabeprazole Sodium and Diclofenac Sodium in capsule dosage form, J. Chem. Pharm. Res., 2010, 2(2): 186-196. 8. Y.K. Agrawal* and K. Shivramchandras, Spectrophotometric determination of diclofenac sodium in tablets, Journal of Pharmaceutical and Biomedical Analysis, Vol. 9, No. 2, 1991, pp. 97-100. 9. N.A. El-Ragehy *, M.M. Ellaithy, M.A. El-Ghobashy, Determination of thiocolchicoside in its binary mixtures (Thiocolchicosideglafenine and Thiocolchicoside-floctafenine) by TLC-densitometry, 58 (2003) 463- 468. 10. Ming-Thau Sheu, Huei-Lan Chou, Ching-Cheng Kao, Cheng-Hsiung Liu, Theodore D. Sokoloski, Dissolution of diclofenac sodium from matrix tablets , International Journal of Pharmaceutics, Volume 85, Issues 1-3, 20 September 1992, Pages 57-63 11. British Pharmacopoeia, London: The Majesty stationary office, 2004; I: 626. 12. M.S. Bhatia, S.R. Dhaneshwar, Simple colorimetric detection of diclofenac sodium from tablets. 13. M.Riegel*, P.P. Ellis, Highperformance liquid chromatographic assay for antiinflammatory agents diclofenac and flurbiprofen in ocular fluids, Journal of Chromatography B, 654 (1994) 140-145. 14. Julio c. Botello and guadalupe pi~rez-caballero*, spectrophotometric determination of Diclofenac sodium with methylene blue, Talanta, Vol. 42, No. I, pp. 105-108, 1995.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241212EnglishN-0001November30General SciencesPHARMACOGNOSTIC AND PHYTOCHEMICAL INVESTIGATION OF CHLOROPHYTUM BREVISCAPUM DALZ. English1124V. N. PatilEnglish Deokule S. S.EnglishChlorophytum breviscapum Dalz. belongs to family Liliaceae and is being used in the indigenous systems of medicine as a galactogogue and aphrodisiac. The drug part is usually used as the white tuberous roots of this plant. The present study includes the macro and microscopic characters, histochemistry and phytochemistry. The phytochemical screening is also confirmed by HPTLC analysis for saponins and stegmasteroids.                             EnglishChlorophytum breviscapum, Pharmacognosy, phytochemical analysis, HPTLC.INTRODUCTION Chlorophytum breviscapum Dalz. belongs to family Liliaceae. In India, it is found to be growing in rain fed areas. The plant generally grows along the forest margins, grassy slopes and rocky places along valleys (between 1300- 2800 m)1 . The plant body is erect up to 1.5- 2 ft height with sheathing leaf base acute to acuminate with entire margin. Tuberous root are slightly broad at the base and gradually tapering at the end. Tubers are oblong, pendulous in the middle again it becomes fibrous and are measuring 8-12 cm long, 1-1.7 cm diameter 2 . There are 17 species of Chlorophytum recorded in India out of these 11 species of Chlorophytum are found to be growing in Maharashtra (3) . For the present investigation Chlorophytum breviscapum is selected as for their correct botanical identification and standardization of drug. The drug part is usually used as the white tuberous roots. The drug is used an aphrodisiac and galactogogue4,5,6 . Review of literature revealed that it is also used as a nutritive and health promoting properties as well as an immunoenhancing, hepatoprotective and antioxidants 7,8,9,10,11. The tubers are also used as a medicinal expectorant in fever, leucorrhoea and also as an aphrodisiac12. MATERIAL AND METHODS Collection and Identification of Plant Materials The plant materials were collected from in and around Pune district of Maharashtra during rainy season. Efforts were made to collect the plants in flowering and fruiting condition for the correct botanical identification. It was identified with help of Flora of The Presidency of Bombay2. The herbariums were prepared and finally authenticated from Botanical Survey of India, Western Circle, Pune (India). The voucher specimen number is PAVICH3/ 2009. Microscopic and Macroscopic evaluation Thin (25p) hand cut sections were taken from the fresh tuberous roots, permanent double stained and finally mounted in Canada balsam as per the plant microtechniques method of Johansen13. The macroscopic evaluation was studied by the following method of Trease and Evans14 and Wallis15. Histochemical study The thin transverse sections of fresh root were taken (about 25p). It was treated with respective reagent for the detection and localization of chemicals in the tissues as per the method of Krishnamurthy16. Phytochemical evaluation: Some materials were dried under the shade so as to avoid the decomposition of chemical constituents, powdered in blender and finally stored in dry air tied containers for phytochemical screening. Ash and percentage extractives were accomplished by following standard pharmacopoeal techniques17. Fluorescence analysis was carried out as per Chase and Pratt18. Qualitative phytochemical test were carried out by standard methods of Harborne19 and Trease and Evans14. Quantitative phytochemical analysis were determined for proteins, carbohydrates and saponins by the methods of Lowry et al., 20; Nelson21 and Obadoni and Ochuko22 respectively. The phytochemical screening was also detected by the High PerformanceThin Layer Chromatography (HPTLC). HPTLC study was carried out on instrument comprising of Linomat 5 for application using Densitometer- TLC Scanner 3 with "WINCATS" software (Camag, Switzerland). These studies were carried out on pre-coated aluminum fluorescent plates (E. Merck). For HPTLC studies, an extract of methanol (25% GR) solvent system was used and after development, plate was scanned at 254 and 366 nm 23, 24, 25. RESULTS AND DISCUSSIONS Macroscopic evaluation (Figure 1 and 2) Herb: 1.5 - 2 ft. in height. Roots: Tuberous root are slightly broad at the base and gradually tapering at the end. Tubers are oblong, pendulous in the middle again it becomes fibrous and are measuring 8-12 cm long, 1-1.7 cm diameter. Leaves: Green, 6 - 9 (10-15 also) in number, slightly thick dark green, flat with undulate margin, apex acuminate, linear, oblong or lanceolate,membranous, shining above with sheathing leaf base. 60 - 66 2.70 - 3.5 cm long. Scape: Unbranched, Naked, 60 - 65 cm long. Flower: White, racemose, clusters of 2 - 3 flowers, 2 - 4 cm long, erect. Bract: Membranous, Ovate- lanceolate, lower bracts 0.5 - 1.5 cm and upper 0.8 cm long. Pedicels: 0.5 - 0.8 cm long, jointed near the top. Perianth: Segments, linear acute, 3 nerved, 0.9 0.3 cm in broad. Stamen: 0.5 - 1 cm long, anther- 0.7 cm long, linear, oblong. Style: 0.6 cm long, slender, stigma minute. Capsule: Globose, 0.8 - 1.2 cm in diameter, 3 winged, emarginate, (0.7 - 0.8 cm in height and 0.8 cm in diameter) Seeds: Black, globose, compressed 0.1 - 0.3 cm diameter, papillose. Microscopic characters The transverse section of root shows a circular in outline. The outermost layer is epidermis. Epidermis consists of uniseriate trichomes. This is followed by a very large zone of cortex. The outermost layer of the cortex just below the epidermis consists of cells which are mostly rectangular, appearing much longer than wide. The rest of the cortical cells are rounded to polygonal parenchymatous and have no intercellular spaces. The innermost layer of the cortex is single layered endodermis. The stellar structure shows that the endodermis is followed by the pericycle layer. Xylem is exarch type. The phloem is group in between the xylem along with the parenchyma. The centre region is occupied by large pith. These are mostly polygonal in shape (Figure 3). Histochemical Screening Histochemical screening showed the presence of starch, protein, fat, saponins, tannin, sugars and alkaloids (Table 1). Phytochemical Study It contains the total ash 12.8 % and acid insoluble ash is 5.1 % (Table 2). The values of percentage extractives were higher in chloroform and lower in benzene solvent (Table 3). Fluorescence analysis was carried out to check the purity of the drug. The powder drug was observed in visible light as Pale white in color. The powder was then observed in ultraviolet light. It was treated with reagent like nitrocellulose, 1 N sodium hydroxide, 1 N sodium hydroxide in nitrocellulose and dry for 30 minutes and then it was observed under ultraviolet light and it emits the color as shown in (Table 4). Qualitative analysis of the root drug indicated the presence of proteins, reducing and non-reducing sugars, saponins, fats, tannin, glycoside and alkaloids in the plant (Table 5). The quantity of proteins is higher than saponins and carbohydrates (Table 6). Saponins are the important chemical and justify the use of tubers of this plant and are used as a well known health tonic, aphrodisiac and galactogogue3,4,6,26. In HPTLC study the methanolic extract is ultrasonic for 15 minutes and filtered. The filtrate is used as an application for saponins and stegmasteroids. For each application 20 p, 10p and 5p extracts were used and loaded on instrument comprising of Linomat 5 for application using Densitometer- TLC Scanner 3 with "WINCATS" software (Camag, Switzerland). These studies were carried out on pre-coated aluminum fluorescent plates (E. Merck). The plates were scanned at 254 and at 366 nm23, 24. Analytical studies (Saponins) The HPTLC analysis showed that, the saponins from the C. breviscapum samples gave light yellow bands in visible light and blue bands after derivatization in fluorescence light. The plates were scanned at 254 and 366 nm. When images were compared with the graph and table values. It showes maximum area 22.99 % at 366nm after derivatization. The table also indicates the starting Rf values and end Rf values (Figure 4; Graph 1; Table 7). Analytical studies (Stegmasteroids) In HPTLC analysis, stegmasteroids revealed white bands in visible light. After derivatization in fluorescence light it showed the dark blue bands. The plates were scanned at 254 and 366 nm. It was covered the area 29.31% at 366nm after derivatization. The tables also indicate the Rf values for all the peaks scanned by "WINCATS" software (Figure 5; Graph 2; Table 8). CONCLUSION The plant C. breviscapum showed the correct taxonomy. The morphological characters and histochemical study with double staining of the root, percentage extractives, fluorescence and ash analysis and the phytochemical screening of the plant is helpful for the standardization of drug. As in case of saponins and stegmasteroids, the peaks are denoted by the Rf values. Finding the over all results of the study of C. breviscapum, these investigations will be useful for the correct botanical identification and authentication of the drug. ACKNOWLEDGEMENT Both the authors would like to express a sincere thank to Head, Department of Botany, University of Pune, Pune-411 007 for encouragement and necessary laboratory facilities and The first author is grateful to authorities of Pune University for providing financial support in the form of research stipend. Englishhttp://ijcrr.com/abstract.php?article_id=2206http://ijcrr.com/article_html.php?did=22061. Hara H. The Flora of Eastern Himalaya. Tokyo University Press, Japan. 1966. p. 407. 2. Cooke T. Flora of Presidency of Bombay. B.S.I. Calcutta. 1958; 3: 280-289. 3. The Wealth of India- A dictionary of Indian raw materials and industrial products,. Revised Edn. Publication and Information Directorate, CSIR. Dr. R.S. Krishnan Marg, New Delhi. 1992. 3 (Ca-Ci): 482-483. 4. Nadkarni AK. K. M. Nadkarni?s Indian Materia Medica. Popular Book Depot., Lamington Road, Bombay, 3rd Edtn. 1927. 1: 208-209. 5. Chopra RN, Nayer SL, Chopra IC. Glossary of Indian medicinal Plants CSIR. New Delhi. 1956. p. 218. 6. Marais W, Reilly J. Chlorophytum and its related Genera (Liliaceae). Kew Bulletin. 1978; 32 (3): 653-663. 7. Govindarajan R, Vijayakumar M, Pushpangadan P. Antioxidant approach to disease management and the role of Rasayana? herbs of Ayurveda. Journal of Ethnopharmacology. 2005; 99: 165- 178. 8. Anonymous. Medicinal Plants more on Safed Musali. Agriculture and Industry Survey. 2001 May. p. 38- 39. 9. Dhuley JN. Effect of some Indian herbs on macrophase functions in Ochratoxin A treated mice. Jour. of Ethnopharmacology. 1997; 58: 15- 20. 10. Nergard CS, Diallo D, Michaelsen TE, Malterud KE, Kiyohara H, Matsumoto T, et al. Isolation, Partial characterization and immunostimulation activity of polysaccharides from Verninia kotschyana Sch. Bip. Ex. Walp. Journal of Ethnopharmacology. 2004; 91: 141-152. 11. Kirtikar KR, Basu BD. Liliaceae: Chlorophytum. In: Kirtikar, K.R.; Basu B.D. (Eds.) Indian Medicinal Plants. L.M. Basu Publishers. Allahabad, India. 1975. 2508-2509. 12. Sreevidya N, Kumar V, Kumar S, Sikarwar RLS. Utilization, depletion and conservation of Safed Musali (Chlorophytum spp.). Journal of non- Timber Forest Products. 2003; 10: 155-157. 13. Johansen DA. Plant Microtechnique, McGraw-Hill Book Co. Inc. New York. 1940. p. 151-154, 182-203. 14. Trease GE, Evans WC. Trease and Evans Pharmacognosy, 15th Ed. W. B. Saunders Edinburgh London, New York, Philadelphia St. Louis Sydney Toronto. 2002. p. 3-4, 528- 533, 538-547. 15. Wallis TE. A Text Book of Pharmacognosy, reprinted edition, Churchill, Livingstone. London. 1967. p. 578 - 617. 16. Krishnamurthy KV. Methods in the Plant Histochemistry, Viswanadhan Pvt. Limited, Madras. 1988. p. 1 - 77. 17. Anonymous. Pharmacopoeia of India, Government of India, Ministry of Health Manager, Publications Delhi, 1st Edn. 1955. p. 370 and 864. 18. Chase CR, Pratt R. Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. Jour. Of Amer. Phar. Asso. (Sci. ed.). 1949; 38: 324-330. 19. Harborne JB. Phytochemical Methods, Chapman and Hall International Edition, London. 2nd edition. 1973. p. 5-8. 20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagent. Jour. of Biol. Chem. 1951; 193: 265-275. 21. Nelson N. A photometric adaptation of the Somogyi method for the determination of Glucose. Jour. of Biol. Chem. 1944; 153: 375-380. 22. Obadoni BO, Ochuko PO. Phytochemical studies and comparative efficacy of crude extracts of some homeostatic plants in Edo and Delta state of Nigeria. Global Jour. Pure Appl. Sci. 2001; 8: 203-208. 23. Wagner H, Baldt S. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer - Verlag, Berlin. 1996. p. 129, 144, 155, 157, 176, 178, 206. 24. Reich E, Schibii A. High Performance- Thin Layer Chromatography for the analysis of medicinal plants, Thieme medical publishers. Inc. 2007. p. 129-160, 206-210, 224-240. 25. Patil VN, Deokule SS. Pharmacognostic standardization of Chlorophytum bharuchae Ans. et al. International Journal of Current Research, 2010, 3, 27-32. 26. Oudhia P. Problem perceived by Safed Musali (Chlorophytum borivilianum) growers of Chhattisgarh (India) region: A study. Journal of Medicinal and Aromatic Plant Sciences. 2001; 22/4A and 23/ 1A: 396-399.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241212EnglishN-0001November30HealthcareMETHOD VALIDATION FOR ULTRAVIOLET SPECTROPHOTOMETRIC DETERMINATION OF DICLOFENAC SODIUM IN HUMAN STRATUM CORNEUM BY SKIN STRIPPING METHOD USING MARKETED DICLOFENAC SODIUM TOPICAL FORMULATIONS English2531S. S. RawatEnglish R. V. MayeeEnglish V. A. ArsulEnglishThe Diclofenac sodium containing marketed gel, an emugel formulation and a spray formulation were applied on the skin. The release of Diclofenac sodium from transdermal gels and spray formulations was studied using the Tape stripping method. Diclofenac sodium exhibited distinct λmax in methanol at 285nm. A good linear relationship (r2=0.9787) was observed between the concentration ranges of 5-25 pg/mL. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.36 and 99.50, 0.31 and 99.60, for D1 formulation, 0.53 and 98.21, 0.45and 98.23 for D2 formulation and 0.54 and 97.76, 0.30 and 97.83 for D3 formulation respectively. The percutaneous absorption of D1 formulation was found to be more than other two formulations. The percentage recovery values indicates that there no interference from the excipients present in the formulation. This demonstrates that the developed spectroscopic method is simple, accurate and reproducible and can be easily used for the routine quality control of Diclofenac sodium in human stratum corneum by tape stripping method. EnglishDiclofenac gel formulations, Diclofenac spray formulations, Tape stripping method, ValidationINTRODUCTION Diclofenac Sodium is sodium 2-[(2, 6- dichlorophenyl)- amino]phenylacetate. It is a non-steroidal anti-inflammatory drug applied to reduce inflammation and as an analgesic reducing pain in conditions such as arthritis or acute injury1 . It is freely soluble in methanol; soluble in ethanol (95%); sparingly soluble in water and in glacial acetic acid; practically insoluble in ether, in chloroform and in toluene2 . Literature survey revealed the availability of method of estimation of drug by Tape stripping method3 . But a quantitative determination method of diclofenac sodium in various gel formulations and spray formulation and their comparison was not validated. The aim of present investigation is to validate UV Spectrophotometric method for diclofenac sodium topical formulations and to develop a quality control tool for diclofenac sodium gel and spray formulation. Experimental Instruments A UV Spectrophotometer (UV-1700): Chemito-Spectroscan UV 2600, Double beam UV Visible Spectrophotometer with a matched pair of 10 mm quartz cells were used for experimental purpose. Materials Diclofenac sodium API was procured as gift sample from Wockhardt Research Centre, Aurangabad. The obtained Diclofenac sodium was dissolve in methanol for experimental purpose. In present study, three commercial products of diclofenac, a commercially available marketed gel formulation (D1) containing diclofenac diethylamine 1% w/w which is equivalent amount of 1.6% w/w Diclofenac sodium, an emugel formulation (D2) containing 1% w/w diclofenac diethylamine which is equivalent amount of 1.6% w/w Diclofenac sodium and a spray formulation (D3) containing 1% w/w diclofenac diethylamine which is equivalent amount of 1.6% w/w Diclofenac sodium has been used for estimation. We compared their percutaneous absorption through skin from one of the topical preparations. The samples were procured from local market, Aurangabad. Preparation of Standard Stock Solution The standard stock solution was prepared by dissolving 0.2gm Diclofenac sodium in 200ml methanol to make final concentration of 100μg/ml. Different aliquots were taken from stock solution and diluted with methanol separately to prepare series of concentrations from 2-24 μg/ml. The λ max for Diclofenac sodium in methanol was found to be 285 nm. The calibration curve was prepared by plotting absorbance versus concentration of Diclofenac sodium4, 6 . Method Three Diclofenac sodium formulations (D1, D2 and D3) were applied on the skin. The diethylamine acts as penetration enhancer. The release of Diclofenac sodium from transdermal gels and spray formulations was studied using the skin stripping method. Tape stripping is a simple and efficient method for the assessment of quality and efficacy of cosmetical and dermatological formulations. The tape stripping method in its standardized form is well-suited to determine the dermatopharmacokinetics of topically applied substances. Before sampling the drug (D1) remaining on the site was removed by three cotton swabs to ensure complete removal of residual drug from the site. The pre-cut (1 sq. cm) adhesion tape was applied on site and mild force was applied to ensure the proper adhesion. The tape was removed and discarded. Eight adhesion tape pieces were applied on the site area in same manner and each tape were removed using forceps so that complete removal of stratum corneum occurs. All 8 samples were collected in a single test tube with 60 ml of methanol in a 200-ml volumetric flask and dilute to volume with methanol. From this solution, suitable aliquots were prepared, then these dilutions were scanned in UV region and absorbances were noted at 285 nm and concentration was determined by linear regression equation. This method was repeated for D2 and D3 samples5, 8, and 9 . RESULTS AND DISCUSSION The tape stripping method for diclofenac sodium topical formulations was validated by studying the following parameters as ICH guide lines (ICH guide lines 1995) for method validation7 . Linearity The linearity of the response of the drug was verified at 1 to 100 μg/ml concentrations, but linearity was found to be between 5-25 μg/ml concentrations. The equation of the calibration curve for Diclofenac sodium obtained y = 0.0038x, the calibration curve was found to be linear in the aforementioned concentrations. The correlation coefficient (r2 ) of determination was R = 0.9787(Table 1) Precision Assay of method precision (intraday precision) was evaluated by carrying out six independent assays of test samples of D1, D2 and D3 formulations. The intermediate precision (interday precision) of the method was also evaluated using two different analysts, systems and different days in the same laboratory. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.36 and 99.50, 0.31 and 99.60, for D1 formulation, 0.53 and 98.21, 0.45and 98.23 for D2 formulation and 0.54 and 97.76, 0.30 and 97.83 for D3 formulation respectively. (Table 2) Accuracy (Recovery test) Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts of the drug in the placebo. The recovery was performed at two levels, 50 and 100% of Diclofenac sodium standard concentration. The recovery samples were prepared in before mentioned procedure. Three samples of each marketed formulation (D1, D2 and D3) were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The result of analysis the recovery studies presented in Table 3and 4. The percentage recovery values indicates that there no interference from the excipients present in the formulation that developed method is found to be sensitive, accurate, precise and most reproducible. CONCLUSIONS The equation of the calibration curve for Diclofenac sodium obtained y = 0.0038, the calibration curve was found to be linear in the aforementioned concentrations. The correlation coefficient (r2 ) of determination was R² = 0.9787. The relative standard deviation (RSD) and assay values obtained by two analysts? shows that there is no major variation in the results. The recovery was performed at two levels, 50 and 100% of Diclofenac sodium standard concentration. The percentage recovery values indicates that there no interference from the excipients present in the formulations. The percutaneous absorption of D1 formulation was found to be more than other two formulations. This demonstrates that the developed spectroscopic method is simple, accurate and reproducible, can be easily used as the routine quality control tool for Diclofenac sodium gel formulations. ACKNOWLEDGEMENTS The authors acknowledge Principal, Dr. Vedprakash Patil Pharmacy College Aurangabad, as well the Principal, Shri Bhagwan College of Pharmacy, N-6, Cidco, Aurangabad, India for providing facilities for conducting research. The authors also thanks to Wockhardt Research Centre, Aurangabad for providing the Diclofenac Sodium API. Englishhttp://ijcrr.com/abstract.php?article_id=2207http://ijcrr.com/article_html.php?did=22071. United States Pharmacopoeia, USP 24/NF 19, US Pharmacopoeial Convention Inc,2000, USA, pp.2149-2152. 2. 2. Indian Pharmacopoeia I (1996), Government of India Ministry of Human and Family Welfare Published by the Controller of Publication, Delhi. 3. 3. U.V.Sera, M.V.Ramana, In vitro skin absorption and drug release - a comparison of four commercial hydrophilic gel preparations for topical use. The Indian Pharmacist, 2006, 73, 356-360 4. 4. Agrawal, YK. and Shivramchandra K, Spectrophotometric determination of diclofenac sodium in tablets. J. Pharm. Biomed. Anal. 1991, 9: 97- 100. 5. Knudsen, EA. Isolation of dermatophytes from foot wear with adhesive tape strips. J, of Medical and veterinary Mycology 1986, 25; 59-61 . 6. Flynn, G.L., Shah, V.P., Tenjarla, S.N., et al., Assessment of value and applications of in vitro testing of topical dermatological drug products. Pharm. Res. 1999, 16, 1325-1330. 7. ICH-Q2R1, Validation of Analytical Procedures: Methodology International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use 1996, Geneva, Switzerland. 8. Pinkus H. Examination of the Epidermis by the strip method of removing horny layers. J Invest Dermatol 1951, 16; 383-386. 9. Mo, Z and Chen Z: UV spectrophotometric determination of compound diclofenac sodium injection. Yaowu Fenxi Zazhi., 1988, 8: 314-316.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241212EnglishN-0001November30HealthcarePHARMACEUTICAL RESEARCH AND DEVELOPMENT -CASE STUDY OF INDIAN PHARMACEUTICAL INDUSTRY English3240Manthan D. JanodiaEnglish D.SreedharEnglish Virendra S.LigadeEnglish Ajay G PiseEnglish Nayanabhirama UdupaEnglishPharmaceutical Research and Development (RandD) is time consuming, risky and costly. It takes roughly about USD 1 billion to develop and commercialize a new drug. Indian pharmaceutical companies were not engaged in basic research and the amount spent on RandD was very low. With adherence to Trade Related Aspects of Intellectual Property Rights (TRIPS) agreement Indian pharmaceutical companies have increase the amount spent on RandD. This study discusses the current RandD scenario in India. EnglishINTRODUCTION The progress of a country depends on its economic, social and technological advancement. It is important for a country to achieve economic and technological progress which may eventually lead to social progress. Instruments of Intellectual Property Rights are considered to achieve economic, social and technological advancement for a country in all aspects. Of all the instruments of Intellectual Property Rights, patent is the most contentious issue which is deliberated in several international fora. Patents are mostly debated for its role in pharmaceutical field. The debate on Intellectual Property Rights is continuing with proponents viewing it as a tool for country?s development which in turn would result in social and economic development; whereas the opponents view it as impeding technological development and social upliftment of people in developing countries. The major implications of the Intellectual Property Rights are debated for pharmaceutical industry where the issue of access and affordability of medicines is a prime concern. Whether patents promote technological, economic and social progress in developing countries is to be ascertained. PATENT SYSTEM IN DEVELOPED AND DEVELOPING COUNTRIES Viewing patent system differently and taking into account the factor that the implications for patents for developed countries and developing countries would be different, developing countries vociferously objected to the adoption of patent system specifically for pharmaceuticals. As developing countries are faced with the problem of increased healthcare burden, availability of affordable medicines is a roadblock for these countries. Developing countries seek alternative strategies to meet the growing healthcare needs of their population. History shows that countries have fine tuned their Intellectual protection regimes according to their developmental requirements (Kumar, 2003). Many of today?s developed countries have a history of excluding pharmaceutical product patents. The examples include Germany until 1968; France, 1960; Japan, 1976; Switzerland until 1977; Italy until 1978; Spain until 1992; Portugal until 1992; Norway until 1992; Finland until 1995, and Iceland until 1997. (Scherer and Watal, 2001; Nogues, 1990). This clearly shows that countries need to adopt a flexible system with regard to patent that would help advancement of country technologically, socially and economically. An ideal regime of Intellectual Property Rights strikes to balance private incentive for innovators and maximizing access to the fruits of innovation for public interest (Mashelkar, 2001). Correa (2000) argues that any IPR system must try to strike a balance between creating incentives for innovators and consumer?s interest. Developed and several developing countries are members of World Trade Organization and have implemented the provisions of Trade Related Aspects of Intellectual Property Rights (TRIPS) agreement in their national laws to provide for patent protection for all fields of technology. Many countries like India and Brazil provided only process patent and not product patent for pharmaceutical products. PHARMACEUTICAL RESEARCH AND DEVELOPMENT Pharmaceutical Industry is research driven and the amount spent on RandD is quite high. It requires an average of 12 years for a single drug to reach market after its extensive evaluation for safety, quality and efficacy. The cost of development to commercialization is estimated at 800 million to 1 billion US dollars according to various reports and it is increasing with regulatory agencies adopting stringent norms for approval of a drug, Out of 10,000 molecules that are preliminary screened for their potential action, only one or two are successfully commercialized after extensive testing and regulatory approval. Companies require regulatory approval at each and every stage of clinical testing to proceed further with research, which again is time consuming and requires substantial financial resources. The financial resources are also required for preclinical and clinical testing of drugs which takes a decade to complete and for about 50% of total cost of drug development. (Grabovski, 2002) RESEARCH and DEVELOPMENT BY INDIAN PHARMA COMPANIES With adherence to TRIPS agreement, significant improvements have been observed among Indian pharmaceutical companies. Amount spent on RandD by companies has increased substantially. Table 1 shows the RandD expenditure as a percentage of sales for a few Indian pharma companies in 2004. It is evident that RandD in Indian pharmaceutical industry is gaining momentum (Sanghi, 2005). In absolute terms, RandD budgets in the Indian pharma industry have risen several times. RandD expenditure by Indian pharmaceutical industry was Rs.220 crores in 1997-1998 which rose to Rs.260 crores in 1998-1999 and Rs.320 crores in 1999-2000 and to Rs.495 Crores in 2005-06. The majority of RandD is conducted by 15 top Indian companies (IBEF, 2006). India?s Pharmaceutical expenditure is shown in the table 2. Since then the amount spent on RandD by Indian pharmaceutical companies have increased manifold. Some of the big companies have started investing 12 to 14 % of their total turnover for RandD activity. This is a progressive sign on the part of Indian pharmaceutical companies. Table 3 show the amount spent on RandD by a few top Indian companies during last five years. It is seen that company like Dr. Reddy?s have spent almost 18% of total sales revenue on RandD during 2004-2005. Many companies started investing heavily during last five years as it became imminent that Indian pharmaceutical companies have to adopt product patent regime and compete with multinational pharmaceutical giants on a level playing field. Though these figures are not comparable with the huge amount spent by MNCs on pharmaceutical RandD, it is a clear indication that expenditure on RandD has substantially increased in recent years. The outcome of increase in RandD is observed as quite a few numbers of patents are filed by Indian pharmaceutical companies in India and the US. PATENTING BY INDIAN PHARMA COMPANIES The amount spent on RandD by Indian pharmaceutical companies is in corroboration with the number of patents filed by some of the Indian pharmaceutical companies with the United States Patents and Trademarks Office (USPTO). The number of patents filed by Indian pharmaceutical companies in the last decade with the USPTO has increased substantially. Though a strict correlation between the amount spent on RandD and number of patents filed cannot be established, it nevertheless gives a positive image of Indian pharmaceutical companies with respect to their commitment for RandD and filing of patent application in India and abroad. By closely looking at the figures in table 2, it is quite noteworthy that before 1995 not a single Indian company had patent in the US. Incidentally, 1995 was the year from which India agreed to grant product patent with respect to all fields of technology, including pharmaceuticals, for a uniform period of 20 years. Even after 1995, the performance of these companies in acquiring US patents was abysmal. Majority of the pharma companies got patents after 2000. Only Council of Scientific and Industrial Research (CSIR), India has applied for and acquired US patents. Even then, the patents granted to CSIR include patents in all classes and not only related to drugs and pharmaceuticals. This apathy towards understanding importance of patent is resolved and the Indian companies are aggressive in their approach for filing and acquiring patents. Indian companies have increased patent filing after 1995. This may be attributed to the fact that the process of acquiring patents takes a few years. One of the plausible reasons could be filing of patents immediately after India adhered to the TRIPS agreement. These companies thought of the inevitable and prepared themselves to face challenges imposed by TRIPS. Among the pharma companies, Ranbaxy Laboratories Ltd is the frontrunner in acquiring US patents, with 77 patents to its credit from 1969 to 2006. Dr Reddy?s comes a close second, with 52 US patents from 1995 to 2006 (Patents for Dr Reddy?s Research Foundation (30 patents) and Dr Reddy?s Laboratories Ltd (22 patents) combined together). The other major pharmaceutical companies have less than 20 patents each. This can be seen in Table 4. It is not known whether these patents are process or product patents (Chaudhary, 2007), as further classification is not provided by USPTO (Janodia, 2008). The list provided here in the table is indicative of companies that have filed patents with USPTO. Many more companies may have filed for more number of patents which are not listed as the list provided by USPTO is till year 2006. It can be safely assumed that many more US patents may be acquired by Indian pharmaceutical companies after 2006. The comparison of CSIR with pharmaceutical companies is not with respect to pharmaceutical patents. CSIR has acquired patents in all fields of technology and not only related to pharmaceutical products (Janodia, 2008). The comparison of patents acquired by Indian pharma companies with CSIR is indicative only. PIPELINE OF MOLECULES OF THE INDIAN PHARMA COMPANIES Despite increase in RandD spend and number of patents, Indian pharmaceutical industry is not able to come out with a new successful drug molecule in the market. Yet the pipeline of a few Indian companies is promising as a few molecules are at certain stage of development. Another striking feature of RandD in India is that Indian pharma companies RandD expenditure is focused towards "global diseases" like obesity, diabetes, hypertension, cancer rather than diseases affecting developing countries. As these therapeutic segments offer more lucrative opportunities, companies are specifically focused on developing molecules for these segments. On the flip side, Indian companies are ignoring the RandD efforts for tropical diseases such as Malaria, Filariasis, Tuberculosis, Leishmaniasis that are more prevalent in India. Following table shows the progress made by Indian pharma companies in terms of New Chemical Entity (NCE) research. CONCLUSION Research and Development is a long and risky business with a huge amount at stake and the chances of success are less. Further, it takes a long time to develop and commercialize a new drug. With challenges posed by the Trade Related Aspects of Intellectual Property Rights (TRIPS) Agreement, Indian pharma companies have geared up themselves. The amount spent on RandD has increased among Indian pharmaceutical companies in recent years and many companies are actively involved in basic research to come out with new drugs. Increase in budget for RandD shows that Indian pharma companies are committed to RandD activities. Englishhttp://ijcrr.com/abstract.php?article_id=2208http://ijcrr.com/article_html.php?did=22081. Kumar, Nagesh, Intellectual Property Rights, Technology and Economic Development - Experiences of Asian Countries, Economic and Political Weekly, 2003, 209-225. 2. Correa Carlos, Integrating Public Health concerns into patent legislation in developing countries, Geneva, South Centre, 2000. (http://www.southcentre.org/publicat ions/publichealth/publichealth.pdf). 3. Scherer F.M., Watal Jayashree, Post Trips Options For Access to Patented Medicines in Developing Countries, CMH Working Paper Series - Paper No.WG4 : 1, June 2001, 4. 4. Julio Nogues, Patents and Pharmaceutical Drugs - Understanding the Pressures on the developing countries, Working Papers, International Economics Department, The World Bank, September 1990, WPS 502, 3. (cited http://wwwwds.worldbank.org/servlet/WDSCon tentServer/WDSP/IB/1990/09/01/00 0009265_3960929170142/Rendered/ PDF/multi0page.pdf date 10-04-06). 5. Grabowski H., Patents, Innovation and Access to New Pharmaceuticals, Journal of International Economic Law, 5(4), 2002: 849-860 6. Sanghi Mahendra K., Pharmaceutical Industry in India - An overview, 2005 7. Pharmaceuticals - A Report by Ernst and Young for IBEF accessed from www.ibef.org on 30-03-2006 8. Mashelkar R.A., Intellectual property rights and the Third World, Current Science, 81(8), 2001; 955. 9. Chaudhuri Sudip, Is Product Patent Protection Necessary in Developing Countries for Innovation? RandD by Indian Pharmaceutical Companies after TRIPS, Indian Institute of Management Calcutta, WPS No. 614/ September 2007; 16. 10. Janodia M.D., Sreedhar D., Ligade V.S., Udupa N., US Patents granted to Indian pharmaceutical companies, Current Science, 95(4), 2008:435-436.