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E5df876f5e0f28178c70af790e47 Journal of Applied Pharmaceutical Science Vol. 8(07), pp 001-009, July, 2018 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2018.8701 ISSN 2231-3354 Application of Chemometrics for the simultaneous estimation of stigmasterol and β-sitosterol in Manasamitra Vatakam-an ayurvedic herbomineral formulation using HPLC-PDA method Srikalyani Vemuri1, Mohan Kumar Ramasamy1, Pandiyan Rajakanu1, Rajappan Chandra Satish Kumar2, Ilango Kalliappan1,3* 1Division of Analytical Chemistry, Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM Institute of Science and Technology, Kattanku- lathur-603 203, Kancheepuram (Dt), Tamil Nadu, India. 2Clinical Trial and Research Unit (Metabolic Ward), Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM Institute of Science and Tech- nology, Kattankulathur-603 203, Kancheepuram (Dt), Tamil Nadu, India. 3Department of Pharmaceutical Chemistry, SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur-603 203, Kancheepuram (Dt), Tamil Nadu, India. ARTICLE INFO ABSTRACT Article history: A new research method has been developed to approach multiresponse optimization for simultaneously optimizing Received on: 08/05/2018 a large number of experimental factors. LC Chromatogram was optimized using Phenomenex RP C18 column (250 x Accepted on: 15/06/2018 4.6 mm; 5 µm); mobile phase was surged at isocratic mode with a flow of 1.0 mL/min using methanol and acetonitrile Available online: 30/07/2018 (95:5% v/v) at the detection max of 208 nm with the retention time of 16.3 and 18.1 min for Stigmasterol and β-Sitosterol respectively. Amount of Stigmasterol and β-Sitosterol was quantified and found to be 51.0 and 56.3 µg/ mg respectively. The r2 value of 0.9971 and 0.9960 was found in the linear range of 80-130 µg/mL for Stigmasterol Key words: and β-Sitosterol respectively. LOD and LOQ were 6.951, 21.063 µg/mL and 6.048, 18.328 µg/mL for Stigmasterol Chemometrics, Manasamitra and β-Sitosterol respectively. The relative standard deviation for the system and the method precision were found to be vatakam, RP HPLC, 0.94%, 0.40% and 1.51%, 1.1% (≤2%) for stigmasterol and β-Sitosterol. Recovery studies were performed and found Stigmasterol, β-sitosterol. in the range of 95-105% indicates the accuracy of the developed method. The developed chromatographic method is the first report for the concurrent estimation of Stigmasterol and β-Sitosterol in Manasamitra Vatakam. INTRODUCTION single response with varying all the factors at a single approach, the chemometric analysis makes the best choice of separation ICH Q8 (R) defines QbD approach as a “systematic commence for the development that begins with predefined (Mannemala et al., 2016), which helps in hasting the method objectives which conspicuous on the product, process development and extensively explains the chromatographic nature understanding, and process control based on scientific attributes of the eluent. The different approaches to chemometric analysis and quality risk management” (Pande et al., 2017). include the pareto-optimality, path of steepest ascent, Derringer’s Analytical methods are widely used in pharmaceutical desirability function and coerce acceleration of the procedure. development for divergent formulations of all categories which The alley of gradient ascent can be exerted when the obtained involves elution of active analytes and its separation with minimal responses are linear (Sivakumar et al., 2007). resolution criteria (Sivakumar et al., 2007). Optimization of a The potent therapeutic agents are prepared from the traditional plants where herbal drugs and the chemical constituents from the plants form the major traditional claim in Ayurvedic, * Corresponding Author homeopathic, naturopathic and other medicine systems (Benzie Ilango Kalliappan, Division of Analytical Chemistry, Interdisciplinary et al., 2011). Due to the extensive and unknown side effects of Institute of Indian System of Medicine (IIISM), SRM Institute of Science and Technology, Kattankulathur-603 203, Kancheepuram (Dt), Tamil allopathic systems, the herbal drug manufacturers have become Nadu. E-mail: ilangok67 @ gmail.com relatively more as they are obtained from the natural origin © 2018 Srikalyani Vemuri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). 002 Vemuri et al. / Journal of Applied Pharmaceutical Science 8 (07); 2018: 001-009 which is less toxic reactions. Polyherbal formulations (PHF) have Design Expert was extensively used for the Chemometric become the test of time, whereas the vast utilization of herbs has measures, factorial analysis, Perturbation plots and desirability driven the hostile approach. PHF has better acceptability and function calculations using 11.0 version and the MS Excel 2010 compatibility than allopathic formulations, upon selection of high was used for the data analysis. dose, the efficacy and safety increases and the adverse effects can be minimized. Traditional medicine provides an important health Materials care service comparatively to allopathic medicine. In the global Stigmasterol (99% w/w) and β-sitosterol, depicted in market of all the available medical formulation, 25% of the potent Fig. 1 were purchased from M/S Natural Remedies, Bangalore, drugs are been synthesized and prescribed are from plants of India. HPLC grade methanol and acetonitrile were used for the higher therapeutic use (Sahoo et al., 2010). analyses. The mobile phase was vacuum filtered with a 0.45 µm India is rich in ethnic diversity and well-practiced membrane filter. MMV was prescribed by the ayurvedic physician knowledge in herbal medicines. Many classical ayurvedic and was procured from the Ayurvedic pharmacy. The MMV used formulations were texted in most of the contexts like for the analysis further was manufactured by Kottakal. Charakasamhita, Sahasrayogam, and Susrutasamhita. Manasamitra Vatakam (MMV), texted in Sahasrayogam (Krishna Kumar, 2008) ANALYTICAL PROCEDURES a classical Ayurvedic polyherbal formulation available in the market as tablet dosage form is officially texted in Sahasrayogam, Preparation of standard stock solution “Kerala Ayurvedic Pharmacopeia” used for the treatment of The standard with the concentration of 200 µg/mL convulsions, stress, anxiolytic and depression disorders. It helps in using methanol as a diluent was prepared for the construction of providing the treatment for Generalized Anxiety Disorder (GAD) calibration curve for stigmasterol and β-sitosterol, the working and depression on prolonging usage of the drug. MMV is also a standard solution was prepared in the linear range of 80–130 µg/ powerful memory enhancer showing its overall therapeutic effects mL. Both the stock and working standard solutions were stored in on the central nervous system (Goldberg et al., 2013). The major the refrigerator and protected from sunlight. The working standards therapeutic indications include schizophrenia post-traumatic were freshly prepared on the day of validation. The calibration stress disorder, amnesia, Alzheimer’s and cardiac arrhythmia curve reported was taken against peak area vs. concentration (µg/ due to anxiety. Literature survey reveals that MMV has the mL) of the analyte. neuroprotective and antioxidant properties (Thirunavukkarasu et al., 2013). Preparation of sample solution Phytosterols comprise several bioactive properties Accurately weighed about twenty tablets and evenly like deprivation of cholesterol levels, regulating the LDL levels, powdered, from which weighed and transferred 1 g of powder into inhibiting the tumor growth (Naiyer et al., 2017). Literature a 10 mL standard flask. 5 ml of methanol as a diluent was added review explores that the inclusion of phytosterols in the diet and complete extraction was exerted by sonication for about 30 can prevent and reduce the cancerous growth. The commonly min and made to the mark with the diluent. The sample matrix occurring phytosterols in the diet are stigmasterol, β-sitosterol, and prepared was then subjected to prior filtration with Whatman filter campesterol (Scholz et al., 2015). In the extent to the analytical paper and further filtration was done with 0.2 µm membrane filter applications, few chromatographic methods were available for and the filtered solution was injected with a volume of 20 µl for the simultaneous estimation of phytosterols in various herbal LC analysis. formulations (Sandhiya et al., 2015; Nair et al., 2006; Careri et al., 2001), HPLTC method validation of stigmasterol in the leaf Chromatographic parameters and stem of Bryophyllum pinntum (Anjoo et al., 2015). There is The optimized chromatographic measures performed no scientific method available to quantify any of the chemical indulges the eluent composition of MeOH and ACN in the quotient constituents present in MMV. Hence an attempt was made to of 95:5% v/v with a flow rate of 1.0 mL min−1 and degassed the quantify phytosterols present in the formulation and validation as mobile phase for 15 min using ultrasonicator. Phenomenex C18 per ICH Q2b guidelines. column was used as stationary phase. All the determinations were done under ambient temperature conditions (25 ± 2°C) with MATERIALS AND METHODS an injection volume of 20 µL at the detection speck of 208 nm. Experimental conditions The chromatographic conditions were maintained at an ambient temperature. HPLC Instrumentation
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