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1 Available online at Der Pharmacia Sinica, 2012, 3 (1):41-46 ISSN: CDEN (USA): PSHIBD Determination of withanolides from the roots and herbal formulation of Withania somnifera by HPLC using DAD and ELSD detector J. V. Manwar 1, K. R. Mahadik 1*, A. R. Paradkar 2, S. P. Takle 3, L. Sathiyanarayanan 1 and S. V. Patil 3 1 Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune, Maharashtra, India 2 Institute of Pharmaceutical Innovation, and IRC in Polymer Science and Technology, University of Bradford, Bradford, West Yorkshire- BD7 1DP, United Kingdom 3 Department of Alcohol Technology, Vasantdada Sugar Institute, Pune, Maharashtra, India ABSTRACT Roots of Withania somnifera are widely used in Indian herbal formulations. Withaferine A and withanolide A are two pharmacologically important withanolides present in the roots of the plant. HPLC method using DAD and ELSD detector was developed for simultaneous determination of withaferine A and withanolide A from the roots and herbal formulation of the plant. Coarse powder of root was extracted with methanol by shaking for 30 minutes and sonication for 45 minutes. Herbal formulation was first extracted with chloroform and then with ethyl acetate, extracts were combined, evaporated and reconstituted with methanol. Retention time of both the compounds was vary with detector to detector and it was found for withaferine A (3.74, 3.84 min) and withanolide A (4.51, 4.60 min) with DAD and ELSD detector, respectively. Amount of withanolides in each sample were calculated from the regression equations of standard calibration graphs. The results showed that the HPLC method is suitable for the determination of withaferine A and withanolide A from the roots and herbal formulation of W. somnifera. Keywords: Withaferine A, withanolide A, Withania somnifera, HPLC. INTRDUCTIN Withania somnifera (family Solanaceae) is a medicinally important herb used in number of Indian herbal formulations. In India, it is locally known as Ashwagandha and is considered as Indian Ginseng. Roots of the plant are major source of active chemical substances and are traditionally used to cure ulcers, fever, cough, dyspnoea, consumption, dropsy, impotence, rheumatism, toxicosis and leucoderma [1]. These activities are mainly attributed towards the presence of different withanolides mainly withaferine A and withanolide A [2-5]. Ashwagandharishtha is the herbal formulation that contains roots of W. somnifera as a major crude drug along with 26 other herbs. It is mainly recommended for the treatment of impotency. ther uses of the formulation include syncope, epilepsy, cachexia, psychosis, emaciation, piles, digestive impairment, and neurological disorders [6-7]. Quality of the crude drug used in manufacturing of such formulations depends on the amount of active ingredient presents. Literature survey indicated phytochemical variability in marketed preparations of W. somnifera [8]. Therefore, standardization 41

2 of these crude drugs has become necessary before their use for preparing formulations. Moreover, standardization of finished product is of equal importance to be considered in assuming the safety and efficacy product before use. Evaporative Light Scattering detector (ELSD) is a sensitive and a universal detector which eliminates the need of derivatization for low or non UV absorbing compounds [9]. Literature survey shows that the HPLC and HPTLC method is available for the determination of withaferine A from W. somnifera [10-12]. For simultaneous determination of withaferine A with withanolide D, RP-HPLC method is reported [13]. HPLC-UV (DAD)-positive ion electrospray ionization-mass spectrometry is available for determination of withaferine A, 12- deoxywithastramolides, withanolide A using withanone as external standard [14]. This method is costly and time consuming as separation time is 35 minutes. ne TLC method is reported for simultaneous determination withaferine A and withanolide A from different plant parts of W. somnifera. This method is time consuming, non reproducible and requires derivatization of compounds prior to analysis [15]. Both the withanolides are low UV absorbing compounds can be accurately analyzed by ELSD detector even at very low concentration. HPLC is the most commonly used technique for the separation and analysis of pharmaceutical and phytochemical compounds [16-18]. It possesses advantages like high resolving power, speed of separation, accuracy and reproducibility. In the literature, there is no report of HPLC method for the simultaneous determination of withaferine A and withanolide A in roots and herbal formulation of W. somnifera. Therefore, in the present paper we have reported simple, rapid and reproducible HPLC method for the determination of these withanolides from the roots and herbal formulation of W. somnifera. (I) CH 2 H (II) H H H H H Structure of withaferine A (I) and withanolide A (II) H MATERIALS AND METHDS Plant materials and reagents Dried roots of W. somnifera were collected from local market and were authenticated by the Agharkar Research Institute, Pune, India. The roots were first crushed into small pieces and then converted to coarse powder by pulverization. Herbal formulation Ashwagandharishtha (Baidyanath) was procured from local Ayurvedic Pharmacy. Standard drug sample of withaferine A and withanolide A were purchased from Natural Remedies Private Limited, Bangalore, India. All the solvents used in the study were of HPLC grade. Preparation of standard solution Standard withaferine A and withanolide A were dissolved methanol to produce concentration of each 1000 µg/ml (standard stock solution). 1 ml of the resultant solution was further diluted to 10 ml to get a final concentration containing 100 µg/ml of each drug. This concentration was used for the experimental work (standard solution). Extraction of samples The coarse powder of W. somnifera (1g) was extracted with 10 ml of methanol by shaking for 30 minutes and then sonicated (Bandelin Sonorex sonicator) for 45 minutes at room temperature. The extract was concentrated to dryness by removing the solvents using rotary vacuum evaporator (Laborota 4000, Heidolph). The extract was redissolved in 10 ml of methanol, filtered through 0.45 micron filter paper and was used for analysis. For herbal formulation, 10 ml of product was successively extracted thrice with each of 10 ml chloroform and then with each of 10 ml of ethyl acetate. Both the extracts were combined, concentrated to dryness by removing the solvents using rotary vacuum evaporator and redissolved in 10 ml of methanol. The solution was filtered through 0.45 micron filter paper and was used for analysis. 42

3 TLC analysis of withanolides Both the extracts were analyzed by thin layer chromatography using CAMAG HPTLC system comprising of Linomat-IV sample applicator, scanner III with WinCATS software for scanning and documentation. About 5 µl of standard solution, 50 µl of both extracts were spotted separately on TLC plate precoated with silica gel GF 254 (5 x 10 cm). The development of plate and measurement of peak areas were carried out according to procedure given by Sharma [15]. Figure 1. Densitograms of withaferine A (1) and withanolide A (2) in standard solution (A), roots extract (B) and marketed formulation (C). Densitogram of marketed formulation (C) showing masking of withanolides peaks by unknown peak (3) HPLC analysis of withanolides HPLC analysis of withaferine A and withanolide A was carried out on Agilent 1260 Infinity Quaternary LC System consist of diode array detector G4212A (DAD) and 1260 Evaporative Light Scattering detector (ELSD) with auto injector using Nucleosil C 18 (4.6 mm I.D x 250 mm) column. The mobile phase was consisting of acetonitrile and water at the ratio of 60:40 at 35 C, with a flow rate of 1.0 ml/min. The measurements of withanolides with DAD detector were made at 215 nm (Bw 4 nm) and ELSD detector at evaporation temperature 45 C and nitrogen pressure 4 bar. About 4 µl of standard solution and both sample solutions were injected separately and chromatograms were recorded. The amount of withaferine A and withanolide A present in the sample were calculated from respective regression equations. The chromatograms of standard solution, roots and herbal formulation extract are shown in Figure 2 and Figure 3. Validation of proposed method Validation of proposed method was carried out in terms of linearity and range, limit of detection (LD), limit of quantitation (LQ), precision and recovery. Calibration graph, LD and LQ To study the linearity of withanolides, a series of dilutions (60, 80, 100, 120 and 140 µg/ml) were made from the standard stock solution. Calibration graph was plotted as concentration versus peak area response. The limit of detection and limit of quantitation were measured on the basis of signal-to-noise ratios. Precision The precision of method was checked by injecting three consecutive replicates of standard solution. Precision of the method was expressed in terms of %RSD calculated from the measurement of peak area response. 43

4 Recovery study To study the accuracy of method, recovery study was carried out by standard addition method. Accuracy was evaluated in triplicate by adding known concentration of standard solution to the preanalysed sample of roots and herbal formulation. Figure 2. Chromatograms of withaferine A (1) and withanolide A (2) in standard solution by DAD (A) and ELSD detector (B) I II Figure 3. Chromatograms of withaferine A (1) and withanolide A (2) in roots (I) and herbal formulation (II) of W. somnifera by DAD (A) and ELSD detector (B) RESULTS AND DISCUSSIN Thin layer chromatography method reported by Sharma et al. [15] for quantification of withaferine A and withanolide A from roots and other parts of W. somnifera require derivatization of spots using vanillin reagent prior to detection. Peak response is depends upon the various factors like concentration and immersion time of plate in vanillin reagent, drying temperature and waiting time before detection. This whole process affects the sensitivity and reproducibility of method. Most important drawback of this method is it can only use for analysis of withanolides in various parts of plant including roots, but it cannot be used for the analysis of herbal formulation. When extracts of roots and herbal formulation were analysed by this method, it was observed that there was no interference of peaks of withanolides in roots sample, but in case of herbal formulation peaks of both withanolides get masked by big peak of unknown compound (Figure 1). Therefore, attempts were made to develop a simple, rapid and reproducible 44

5 HPLC method for determination of withaferine A and withanolide A from the roots and herbal formulation of W. somnifera using DAD and ELDS detector. Use of ELSD detector eliminates the problem of sensitivity of withanolides which are low UV absorbing. Comparison of peak areas shown by DAD and ELSD detector indicated that withaferine A is near about 1300 time more sensitive to ELSD detector than DAD detector, whereas withanolide A is more than 1900 times more sensitive to ELSD detector than DAD detector. Calibration graphs for withaferine A and withanolide A were obtained by plotting concentration versus peak area response. The graphs were found to be linear over the concentration range 60 to140 µg/ml with high correlation coefficient values ranges from to By DAD detector, observed LD and LQ values were withaferine A (2.29, 6.95 µg/ml) and withanolide A (2.07, 6.27µg/mL), respectively. By ELSD detector, LD and LQ values were withaferine A (6.95, µg/ml) and withanolide A (6.27, µg/ml). Higher LD and LQ values by ELSD detector may be due to the noisy base line. Precision of the method was expressed as %RSD and it was calculated from the peak area response by injecting three consecutive replicates of standard solution. The %RSD found out by DAD and ELSD detector were withaferine A (0.46, 0.93) and withanolide A (0.23, 0.97), respectively. The values lie within a standard limit. The data of validation parameters is given in Table 1. The contents of withanolides in roots extract and herbal formulation were calculated from the regression equations of standard calibration graphs. The %RSD of sample analysis of roots extract and herbal formulation was range from within 0.44 to The percent recovery obtained for withanolides in roots extract and herbal formulation was within the limit of to (Table 2). The results confirm the method is accurate and free from any positive or negative interference of other compounds present. The work demonstrated that proposed HPLC method is suitable for determination of withaferine A and withaferine A and withanolide A from roots and herbal formulation of W. somnifera. Table 1. Validation parameters of HPLC method for withanolides DAD detector ELSD detector Withaferine A Withanolide A Withaferine A Withanolide A Retention time Area Range (µg/ml) Linearity (R 2 ) LD (µg/ml) LQ (µg/ml) Precision Table 2. Results of analysis of withanolides in roots and herbal formulation and recovery study DAD detector ELSD detector Contents %Recovery Contents %Recovery Mean %RSD Mean %RSD Mean %RSD Mean %RSD Roots extract Withaferine A Withanolide A Herbal formulation Withaferine A Withanolide A *contents in µg/ml; n=3 REFERENCES [1] U Devi. Ind. J Pharm Sci, 1996, 34, [2] SK Bhattacharya, KS Satyan, S Ghosal, Ind J Exp Biol, 1997, 35, [3] B Jayaprakasam, YJ Zhang, NP Seeram, MG Nair, Life Sci, 2003, 21,

6 [4] H Ichikawa, Y Takada, S Shishodia, B Jayaprakasam, MG Nair, BB Agarwal, Mol Cancer Ther, 2006, 5, [5] L Misra, P Mishra, A Pandey, RS Sangwan, NS Sangwan, R Tuli, Phytochemistry, 2008, 69, [6] Anonymous, Ayurvedic Pharmacopoeia of India (1 st Edn.). The Controller of Publications, New Delhi, 2001, [7] CP Khare, Indian Medicinal Plants. Springer Science and Business Media, New York, USA, [8] RS Sangwan, ND Chaurasiya, LN Misra, P Lal, GC Uniyal, R Sharma, NS Sangwan, KA Suri, GN Qazi, R Tuli R, Current Sci, 2004, 86, [9] XY Chai, SL Li, P Li, J Chromatogr A, 2005, 1070, [10] N Mahadevan, RP Kasar, T Subburaju, B Suresh, J Sep Sci, 2003, 26, [11] S Bala, R Govindrajan, A Rawat, S Mahrotra, Ind J Pharm Sci, 2004, 66, [12] PS Nayak, S Upadhyay, A Upadhayay, J Plan Chromatogr, 2009, 22, [13] M Ganzera, MI Choudhari, IA Khan, Fitoterapia, 2003, 74, [14] R Khajuria, K Suri, R Gupta, N Satil, M Amina, Suri, G Qazi, J Sep Sci, 2004, 27, [15] V Sharma, AP Gupta, P Bhandari, RC Gupta, B Singh, Chromatogr, 2007, 66, [16] JV Manwar, BV Sonawane, SV Patil, SP Takle, Der Chemica Sinica, 2011, 2, [17] M Sugumaran, M Poornima, Y Yogesh Kumar, S Ramarajasekhar, Der Pharmacia Sinica, 2011, 2, [18] RP Mahor, V Parcha, Y Singh, R Sharma, A Bhandari, Der Chemica Sinica, 2011, 2,

Development and Validation of Stability Indicating HPTLC Method for Estimation of Seratrodast

ARC Journal of Pharmaceutical Sciences (AJPS) Volume 2, Issue 3, 2016, PP 15-20 ISSN 2455-1538 DOI: http://dx.doi.org/10.20431/2455-1538.0203004 www.arcjournals.org Development and Validation of Stability