HPLC and HPTLC Methods by Design for Quantitative Characterization and in vitro Anti-oxidant Activity of Polyherbal Formulation Containing Rheum emodi Wasim Ahmad 1 , Syed Mohammad Arif Zaidi 2 , Mohd Mujeeb 1 , Shahid Hussain Ansari 1 and Sayeed Ahmad 1 * 1 Bioactive Natural Product Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Jamia Hamdard, New Delhi 110062, India, and 2 Department of Surgery, Faculty of Medicine, Jamia Hamdard (Hamdard University), New Delhi 110062, India *Author to whom correspondence should be addressed. Email: sahmad_jh@yahoo.co.in Received 21 April 2013; revised 21 June 2013 Safoof-e-Pathar phori (SPP) is a traditional polyherbal formulation and has been used since long time for its anti-urolithiatic activity. It contains three plant constituents Didymocarpous pedicellata, Dolichous biflorus and Rheum emodi. Emodin and chrysophanic acid were selected as chemical markers for SPP and quantified using sim- ultaneous HPTLC and RP-HPLC methods in R. emodi and in SPP. The simultaneous methods were found linear r 2 5 0.991 in a wide range (10 – 800 ng spot 21 with HPTLC, 5–500 mg mL 21 with HPLC) precise, accurate and robust for both the drugs. Anti-oxidant activity of SPP, R. emodi as well as standard emodin and chrysophanic acid were determined by using DPPH (2,2-diphenyl-1-picryl hydrazyl radical), which showed better activity of R. emodi (IC 50 5 12.27) extract when compared with SPP (IC 50 5 32.99) and standard drugs (IC 50 5 66.81). The robustness of methods were proved by applying the Box–Behnken response surface design software and other valid- ation parameters evaluated were satisfactorily met; hence, the developed method found suitable for application in the quality control of several formulations containing emodin and chrysophanic acid. Introduction Plant-derived products supported by scientific evidence have been gaining user assurance as complementary and alternative medicine (CAM) for prevention and cure of disease. Traditional medicines have been used from ancient times, but they have not been standardized or validated for their efficacy. Trade of herbal products is rising rapidly as a result of popularization of CAM, but they lag behind in the case of the knowledge on active prin- ciples, validation etc. Herbs and herbal medicines are most prone to variation in their phytochemical profile due to variabil- ity in climatic conditions, maturity, post-harvest processing, storage, stability etc. (1). It is extremely important to standardize the formulation and its ingredients, based on marker compounds specific to each plant followed by validation for their efficacy. Rhizomes of Rheum emodi have been used in Unani system of medicine either alone or as an ingredient of many polyherbal formulations for treating various ailments. Safoof-e-Pathar phori (SPP) is an Unani polyherbo-mineral for- mulation and has been used in Unani system of medicine for its anti-urolithiatic activity (2). It is a powdered formulation, which contains six different plant/mineral constituents: Pathar phori (Didymocarpous pedicellata)(3), Kulthi (Dolichous biflorus) (4), Revand chini (Rheum emodi)(5), Namak turb (Raphanus sativus), Jawakhar (Potassium carbonate) and Shora qalmi (Potassium nitrate). Rheum emodi contains a large number of hydroxyanthraqui- none derivatives such as chrysophanic acid, emodin, physcion, aloe emodin, emodin glycoside, rhein etc. (6), which are report- edly known for various biological activities such as anti-oxidant (7), anti-microbial (8), anti-fungal (9), cytotoxicity (10), nephro- protective (11) and anti-viral activities (12). This has also been used as a purgative, stomachic, astringent and for the treatment of chronic bronchitis, asthma and in certain skin diseases (13). The D. pedicellata contains chalcones, flavones and didymo- carpene (14), whereas D. biflorus seeds contain a dipeptide pyr- oglutamyl glutamine as active principal which are rare and not available in pure form for analysis purpose (15). Rheum emodi is a vegetable, however often thought as fruit. It is used in desserts, jams, jellies as well as sauces due to its tart flavor. Its stalks have also been consumed with sugar; however, it may cause poison- ing in people with susceptibility to oxalic acid (16). Anti-oxidants from natural sources play a paramount role in helping endogenous anti-oxidants to neutralize oxidative stress. Several epidemiological, clinical and experimental data sug- gested that plant-based anti-oxidants effects on prevention of chronic diseases (17). Studies have shown that treatment with anti-oxidants prevents calcium oxalate (CaOx) deposition in the kidney and reduce oxalate (Ox) excretion (18). Improved renal anti-oxidant status leads to cell membrane integrity and thus pre- vents the recurrence of renal stone (19). The emodin had shown to be a potent inhibitor of superoxide radicals and probably depends on scavenging hydroxyl radicals (20). There are various analytical methods reported for determin- ation of chrysophanic acid and emodin (the major hydroxyan- thraquinones of R. emodi) in plant extracts such as high-speed counter-current chromatography (21), capillary zone electro- phoresis (22), high-performance thin layer chromatography (23, 24) and most commonly used high-performance liquid chroma- tography (25, 26). However, there has been no report till date for simultaneous determination of emodin and chrysophanic acid content in traditional Unani/Ayurvedic formulations by HPTLC or by using HPLC. Therefore, an attempt has been made for quantitative characterization of traditional Unani formulation (SPP) for their emodin and chrysophanic acid content using developed and validated HPTLC as well as RP-HPLC methods, as per the ICH guidelines with improved range of linearity when compared with the methods reported for extracts due to large variation in contents of multi-component traditional formula- tions. The anti-oxidant potential of traditional polyherbal # The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com Journal of Chromatographic Science 2014;52:911– 918 doi:10.1093/chromsci/bmt123 Advance Access publication August 26, 2013 Article Downloaded from https://academic.oup.com/chromsci/article-abstract/52/8/911/275845 by guest on 28 May 2020