Journal of Planar Chromatography 27 (2014) 3, 157–161 DOI: 10.1556/JPC.27.2014.3.2 157 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest Summary A thin-layer chromatography–2,2-diphenyl-1-picrylhdrazyl (TLC– DPPH) method has been developed for the quantification of with- anolides and simultaneous estimation of antioxidant potential of each constituent present in a complex mixture. High-performance thin-layer chromatography (HPTLC) of withanolide-rich fraction of Withania somnifera root was performed on Si 60F 254 HPTLC plates with dichloromethane–toluene–methanol–acetone–diethyl ether (6.5:7:4:1.5:1, v/v) as a mobile phase for the separation of withanolides and on the basis of R F values 0.07, 0.55, 0.61, 0.63, 0.66, and 0.77 for withanoside V, withaferine A, 1,2-deoxywithastramono- lide, withanone, withanolide A, and withanolide B, respectively. The concentrations of withanoside V, withaferine A, 1,2-deoxywithastra- monolide, withanone, withanolide A, and withanolide B were 0.86, 12.9, 1.92, 1.52, 5.24, and 4.52 mg g −1 respectively. A TLC–DPPH rapid test was used to assess the free-radical scavenging activity of constituents in withanolide-rich fraction for the first time. In with- anolides, withanoside V and withanolide B show the highest antiox- idant potential. In unknown compounds, peak No. 17 at R F 0.92 has the highest antioxidant potential and peak No. 16 at R F 0.87 has recorded the lowest antioxidant potential. It is also found that some of the unidentified components had significant antioxidant poten- tial. This technique has several advantages; therefore, this method will be useful for rapidly validating the Ayurvedic products contain- ing W. somnifera. 1 Introduction Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, winter cherry, and Indian ginseng, has been considered as one of the most important plants of the Indian Ayurvedic medicinal system for over 3000 years [1–3]. Throughout the world, W. somnifera is used in the form of decoction, infusion, ointment, powder, and syrup. In India, it is grown widely in the states of Madhya Pradesh, Uttar Pradesh, Punjab, Gujarat, and Rajasthan. Besides, W. somnifera also grows mainly in arid zones of Baluchistan, Pakistan, Af- ghanistan, Sri Lanka, Congo, South Africa, Egypt, Morocco, and Jordan. W. somnifera has several pharmacological activities, viz., antioxidant, antimicrobial, antifungal [4], adaptogenic [5], nootropic [6], cardioprotective [7], anticancer [8], neuroprotec- tive [1], anticonvulsant [9], immunomodulatory [10], apoptic [11], diuretic [12], hepatoprotective [13], and anti-inflammatory features [14, 15]. Pharmacological action also includes physio- logic and metabolic restorations. Thus, it is effective in the treat- ment of arthritis, geriatric condition, behavioral, neurodegener- ative disorders, and stress-related problems [16, 17]. These med- icinal values are mainly attributed to withanolides present in the roots of W. somnifera [18]. Withanolides are biosynthesized by de novo pathway in roots as well as in leaf [19–21]. Quantitative determination of withanolides has been carried out by high-per- formance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) [22–25]. Withanolides are steroidal lactones, and about 35 withanolides have been isolated from the roots of W. somnifera. The levels of withanolides in roots vary with soil nutrient and variety of W. somnifera [26–28]. The multicomponent extracts show better medicinal efficiency than purified components, i.e., the strength of herbal medicine increased with the mixture of secondary metabolites instead of a single component [29, 30]. Keeping this in mind, we prepared a withanolide-rich fraction from roots of W. somnifera. With a view to estimate the antioxidant potential and free-radical scav- enging activity of individual components contained in a com- plex mixture, several workers have attempted a quantitative measurement of free-radical scavenging potential of individual components in complex mixture for plant extracts by thin-layer chromatography–2,2-diphenyl-1-picrylhdrazyl (TLC–DPPH) method which has several advantages, i.e., simplicity, flexibili- ty, high throughput, or direct access to the compounds separated [31–35]. However, as far as we are aware, analysis of W. som- nifera root fraction by TLC–DPPH method has not been attempted so far. In the present communication, we report for the first time an improved TLC–DPPH method for screening bio- logically active compounds in W. somnifera root fraction. Estimation of Antioxidant Potential of Individual Components Present in Complex Mixture of Withania somnifera (Ashwagandha) Root Fraction by Thin-Layer Chromatography–2,2-Diphenyl-1-Picrylhdrazyl Method Santosh T. Devkar, Suresh D. Jagtap, Surendra S. Katyare, and Mahabaleshwar V. Hegde* Key Words Withania somnifera Ashwagandha Withanolide Withaferine Antioxidant potential HPTLC TLC–DPPH S.T. Devkar, S.D. Jagtap, and M.V. Hegde, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Medical College Campus, Pune – 411043, India; and S.S. Katyare, Center for Innovation in Nutrition Health Diseases, IRSHA, Medical College Campus, Pune – 411043, India. E-mail: mahabaleshwarh@yahoo.com