1. INTRODUCTION:

Ashwagandharishta is a polyherbal hydroalcoholic ayurvedic preparation and is used as rasayana¹. Rasayanas are used to promote health and longevity by increasing defence against disease, arresting the ageing process and revitalizing the body in debilitated conditions². The chief ingredient of Ashwagandharishta is roots of Ashwagandha, Withania somnifera, commonly known for its usefulness in the treatment of hypercholesterolemia, arthritis in combination with other drugs, is also credited to be hypoglycemic and diuretic³. The pharmacological effect of roots of Withania somnifera is attributed to withanolides, a group of steroidal lactones⁴.

Besides withania roots, all the other ingredients of Ashwagandharishta as arjuna (bark of Terminalia arjuna), liquorice (roots of Glycyrrhiza glabra), majith (roots of Rubia cordifolia), rasna (roots of Alpinia officinalis), taj (inner bark of Cinnamomum zeylanicum), nagarmotha (rhizomes of Cyperus rotundus), haritaki (fruits of Terminalia chebula), turmeric (rhizomes of Curcuma longa), nagakesara (stamens of Messua ferrea) etc. contain a rich quantity of phenolic compounds and flavonoids and possess significant antioxidant activity^5-10.

Furthermore, no validated HPTLC method has been reported for the quantification of quercetin and rutin from Ashwagandharishta. Standardization is an important aspect for establishing the quality and efficacy of Ayurvedic formulations or any poly herbal formulation. Therefore, a proper scientific validation as chromatographic fingerprinting is required for quantification of marker compounds for quality control purposes.

2. MATERIALS AND METHODS:

2.1 Preparation of Ashwagandharishta-T

This was prepared by the method as given in the Ayurvedic Formulary of India¹. The ingredients of Ashwagandharishta were procured from local market, Jamnagar. Identification of all the individual plant material was done as per Ayurvedic Pharmacopoeia of India. Authentication of all these ingredients was done by Dr. G D Bagchi, Scientist, Department of Taxonomy and Pharmacognosy, Central Institute of Medicinal and Aromatic Plants, Lucknow. Prepared herbarium has been deposited in the CIMAP for future reference.

According to this method, coarsely powdered ashwagandha roots (Withania somnifera) with prescribed ingredients were placed in polished vessel of brass along with prescribed quantity of water (24.576 l), and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one eighths of the prescribed quantity (3.072 l), then the heating was stopped and it was filtered in cleaned vessel and after that honey was added. Then, dhataki flowers (Woodfordia floribunda) and prakshepa dravyas as sonth, marich, pippali, tvak, tejpatra, priyangu and nagakesara were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33^oC±1^oC. After 15 days, completion of fermentation was confirmed by standard tests¹¹. The fermented preparation was filtered with cotton cloth and kept in cleaned covered vessel for further next seven days. Then, the preparation was poured in amber colored glass bottles, packed and properly labeled.

2.2 Preparation of Ashwagandharishta-M

Method of preparation was same as followed with Ashwagandharishta-T only dhataki flowers were replaced with yeast for inducing fermentation¹².

2.3 Reagents and Materials:

All solvents used were of analytical grade and were purchased from Merck. Quercetin (purity98%) and rutin (purity 98%) were purchased from Yucca Enterprises, Mumbai, India.

2.4 Instrumentation and chromatographic conditions:

Chromatography was performed on 20 x 10 cm HPTLC plates coated with 0.25 mm layers of silica gel 60 F₂₅₄ (Merck, Darmstadt, Germany). Prior to use the plates were washed with methanol and activated at 110ºC for 5 min. Samples were applied as bands 4 mm wide and 6 mm apart by use of Desaga (Ziegel Wiesen, Germany) AS 30 Win sample applicator equipped with a 100 µL syringe. A constant application rate of 10 µL s^-1 was used. The mobile phase for quercetin was toluene-ethyl acetate-methanol-formic acid, 6 + 3 + 0.2 + 0.4 (v/v), while for rutin was ethyl acetate-n-butanol-formic acid-water, 10 + 6 + 2 + 2 (v/v), were used for chromatography. Linear ascending development was performed in a Desaga 20 cm x 10 cm glass twin-trough chamber. Before insertion of the plate, the chamber was saturated with mobile phase vapor for 20 min at room temperature (25 ± 2ºC) and relative humidity 60 ± 5% by lining the TLC chamber on three sides with filter paper, also placed in the mobile phase. The development distance was 8 cm. After development the TLC plates were dried in a current of air by means of an air dryer. Densitometric scanning was performed with a Desaga TLC scanner CD 60 in reflectance absorbance mode at λ = 300 nm for quercetin and λ = 280 nm for rutin controlled by ProQuant software (v1.06; Desaga) resident in the system. The slit dimension was 4 x 0.02 mm and the scanning speed was 100 nm s^-1. The radiation source was a deuterium lamp emitting continuous UV radiation between 190-360 nm. The amounts of the compounds chromatographed were determined from the intensity of diffusely reflected light.

2.5 Preparation of standard stock solutions and calibration plots:

2.5.1 Preparation of standard solution of quercetin:

Stock solution of 1000 µg mL^-1 of quercetin was prepared by dissolving 50 mg of accurately weighed quercetin in methanol and making the volume of solution up to 50 mL with methanol in volumetric flask. The aliquots (0.5 to 2.5 mL) of stock solutions were transferred to 10 mL volumetric flasks and the volume of each was adjusted to 10 mL with methanol, to obtain standard solutions containing 50, 100, 150, 200 and 250 µg mL^-1 of quercetin, respectively. 10µL of each of the standard solutions of quercetin (500-2500 ng spot^-1) were applied as bands 4 mm wide and 6 mm apart in triplicate on a TLC plate using an automatic sample spotter (AS 30 Win). Linear regression data for the calibration plot are listed in Table 1. A good linear relationship between response (peak area) and amount was obtained over the range 500-2500 ng/band.

Table 1 Method validation parameters for the quantification of quercetin and rutin in Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation.

Parameter	Quercetin	Rutin
Instrumental Precision (% RSD, n = 6)	0.61	0.73
Repeatability (% RSD, n = 6)	0.64	0.76
LOD (ng)	180	120
LOQ (ng)	500	400
Linear range (n = 3)	500-2500 ng/band	400-2000 ng/band
Correlation coefficient (r)	0.9996	0.9995
Linearity (Regression equation)	y = 2381x + 2205.2	y = 1065.2x + 2262.1

2.5.2 Preparation of standard solution of rutin:

Stock solution of 800 µg mL^-1 of rutin was prepared by dissolving 40 mg of accurately weighed rutin in methanol and making the volume of solution up to 50 mL with methanol in volumetric flask. The aliquots (0.5 to 2.5 mL) of stock solutions were transferred to 10 mL volumetric flasks and the volume of each was adjusted to 10 mL with methanol, to obtain standard solutions containing 40, 80, 120, 160 and 200 µg mL^-1 of rutin, respectively. 10µL of each of the standard solutions of rutin (400-2000 ng spot^-1) were applied as bands 4 mm wide and 6 mm apart in triplicate on a TLC plate using an automatic sample spotter (AS 30 Win). Linear regression data for the calibration plot are listed in Table 1. A good linear relationship between response (peak area) and amount was obtained over the range 400-2000 ng/band.

2.6 Sample Preparation:

1 g (equivalent to 0.90 mL) of each of the test formulation of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation was dried on water bath for half an hour to remove the alcohol. Then, each of the test sample of Ashwagandharishta was diluted with methanol up to 10 mL and sonicated for 15 min and centrifuged at 3200 rpm to settle down the precipitated sugars. 1 mL of supernatant was passed through 0.45 µm filter (Millipore) and 10µL of each of the test formulation was applied as band on plate for quantification.

2.7 Validation of the method:

ICH guidelines were followed for the validation of analytical methods developed for precision, repeatability and accuracy¹³.

Table 2 Intra-day and Inter-day precision of the HPTLC method.^a)

Marker	Amount [ng/band]	Intra-day precision		Inter-day precision
Marker	Amount [ng/band]	Mean area [AU]	RSD [%]	Mean area [AU]	RSD [%]
Quercetin	500	3383.37	0.67	3382.29	0.77
	1500	5781.48	0.40	5780.74	0.48
	2500	8113.15	0.33	8112.40	0.41
Rutin	400	2687.21	0.78	2686.43	0.86
	1200	3527.19	0.66	3526.37	0.71
	2000	4379.15	0.37	4378.53	0.44

^a) n = 6

Table 3 Results of recovery study of quercetin from Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation (n = 3).

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery [%]	RSD [%]
Ashwagandharishta-T	50	31	99.89	0.99
	100	41	100.08	0.74
	150	51	100.20	0.70
Ashwagandharishta-M	50	29	99.77	1.11
	100	39	100.35	1.05
	150	48	100.14	0.73
Marketed Ashwagandharishta	50	30	99.77	1.08
	100	39	100.08	0.77
	150	49	100.20	0.54

Table 4 Results of recovery study of rutin from Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation (n = 3).

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery [%]	RSD [%]
Ashwagandharishta-T	50	71	100.09	0.36
	100	94	100.11	0.39
	150	117	100.06	0.22
Ashwagandharishta-M	50	66	100.30	0.55
	100	88	100.19	0.37
	150	110	100.09	0.24
Marketed Ashwagandharishta	50	70	99.86	0.52
	100	93	100.07	0.34
	150	116	99.97	0.27

3. RESULTS AND DISCUSSION:

3.1 Selection of the optimum mobile phase:

In an attempt to optimize mobile phase, toluene-ethyl acetate-methanol-formic acid mixtures in different proportions were investigated. Use of toluene-ethyl acetate-methanol-formic acid 6 + 3 + 0.2 + 0.4 (v/v) resulted in sharp, well defined quercetin peaks of R_F0.36 ± 0.02 while solvent system ethyl acetate-n-butanol-formic acid-water 10 + 6 + 2 + 2 (v/v) resulted in sharp rutin peaks of R_F0.59 ± 0.02. Well defined bands were obtained only when the chamber was saturated with the mobile phase for 30 min at room temperature before plate development.

3.2 Validation of the method:

ICH guidelines were followed for the validation of the analytical methods developed. The developed HPTLC method was validated in terms of precision, accuracy, LOD, LOQ and specificity¹³.

3.2.1 Instrumental precision:

Instrumental precision was checked by repeated scanning (n = 6) of the same spot of quercetin (500 ng spot^-1) and rutin (400 ng spot ^-1) and expressed as relative standard deviation (% RSD) as shown in Table 1.

3.2.2 Repeatability:

The repeatability of method was affirmed by analysing 500 ng spot^-1 of quercetin and 400 ng spot^-1 of rutin individually on TLC plate (n = 6) and expressed as % RSD as shown in Table 1.

3.2.3 LOD and LOQ:

The limit of detection and quantification were determined by visual evaluation. The detection and quantification limits obtained by this method for quercetin were 180 and 500 ng, respectively while for rutin detection and quantification limits were 120 and 400 ng respectively as shown in Table 1 which indicates that the sensitivity of the method is adequate.

3.2.4 Intra-day and Inter-day Precision:

The intra-day and inter-day precision of the method were estimated by analysing aliquots of standard solution containing 500, 1500, 2500 ng spot^-1 of quercetin and 400, 1200, 2000 ng spot^-1 of rutin on the same day (intra-day precision) and on different days (inter-day precision) and the results are expressed as % RSD in Table 2 ^14-15.

3.2.5 Specificity:

The specificity of the method was ascertained by analyzing reference standard and samples. The bands for quercetin and rutin from Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation were confirmed by comparing the R_F of the separated bands with those from the standard.

Figure 1: Overlay HPTLC densitogram of quercetin standard

Figure 2: Overlay HPTLC densitogram of quercetin from samples of Ashwagandharishta

a, Ashwagandharishta-T; b, Ashwagandharishta-M; c, marketed Ashwagandharishta

(the mobile phase was toluene-ethyl acetate-methanol-formic acid, 6+3+0.2+0.4)

3.2.6 Recovery:

The pre-analyzed samples of Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation were spiked with 50, 100 and 150% of quercetin standard and the mixtures were analysed again, in triplicate, by the proposed method, to check the recovery of different amounts of quercetin from the Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation. Recovery for quercetin was found in between 99.89-100.20% in Ashwagandharishta-T, 99.77-100.14% in Ashwagandharishta-M and 99.77-100.20% in the marketed formulation of Ashwagandharishta as depicted in Table 3. Similarly, the pre-analyzed samples of Ashwagandharishta-T, M and its marketed formulation were spiked with 50, 100 and 150% of rutin standard and the mixtures were analysed again, in triplicate, by the proposed method, to check the recovery of different amounts of rutin from Ashwagandharishta-T, M and its marketed formulation. Recovery for rutin was found in between 100.06-100.11% in Ashwagandharishta-T, 100.09-100.30% in Ashwagandharishta-M and 99.86-100.07% in marketed Ashwagandharishta as shown in Table 4.

Figure 3: Overlay HPTLC densitogram of rutin standard

Figure 4: Overlay HPTLC densitogram of rutin from samples of Ashwagandharishta

a, Ashwagandharishta-T; b, Ashwagandharishta-M; c, marketed Ashwagandharishta

(the mobile phase was ethyl acetate-n-butanol-formic acid-water, 10+6+2+2)

3.3 Estimation of quercetin and rutin in Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation:

The suitability of the method was examined by estimation of quercetin in Ashwagandharishta-T, M and its marketed formulation. Bands of R_F0.36 ± 0.02 were observed in the densitogram for quercetin standard (Figure 1) while the bands of same R_F were observed in the densitogram obtained from the quercetin isolated from Ashwagandharishta-T, M and its marketed formulation (Figure 2). Similarly, rutin was also estimated in Ashwagandharishta-T, M and its marketed formulation. Bands of R_F0.59 ± 0.02 were observed in the densitogram for rutin standard (Figure 3) while the bands of same R_Fwere observed in the densitogram obtained from rutin isolated from Ashwagandharishta-T, M and its marketed formulation (Figure 4). Quercetin was found to be 0.0021, 0.00192 and 0.00197 %w/w in Ashwagandharishta-T, M and its marketed formulation respectively while rutin was found to be 0.00469, 0.00441 and 0.00464 %w/w in Ashwagandharishta-T, Ashwagandharishta-M and in its marketed formulation respectively as shown in Table 5.

Table 5 Estimation of Quercetin and Rutin from Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation by proposed HPTLC method.

Sample	Quercetin (% w/w)^a)	Rutin (% w/w)^a)
Ashwagandharishta-T	0.0021±0.0002	0.00469±0.0002
Ashwagandharishta-M	0.00192±0.0003	0.00441±0.0002
Marketed Ashwagandharishta	0.00197±0.0001	0.00464±0.0002

^a) Mean ± SD, n = 3

4. CONCLUSION:

This HPTLC technique is found to be precise, specific, robust and accurate and could find application in routine quality-control analysis of Ayurvedic formulations.

5. REFERENCES:

1. The Ayurvedic Formulary of India Part-I. Controller of Publications, Delhi, 2000; 8-9.

2. Kokate CK, Purohit AP and Gokhale SB. Pharmacognosy. 39^th Edition, Nirali Prakashan, Pune, 2007; 604-606.

3. Andallu B and Radhika B. Hypoglycaemic, diuretic and hypocholesterolemic effect of Winter cherry (Withania somnifera, Dunal) root. Indian Journal of Experimental Biology 2000; 38: 607-609.

4. Budhiraja RD and Sudhir S. Review of biological activity of withanolides. Journal of Science and Industrial Research 1987; 46:488.

5. Rahman Z, Kohli K, Khar RK, Lamba HS, Rathore A and Pahwa R. An overview of Terminalia arjuna, chemistry and pharmacological profile. Indian Drugs 2004:641-648.

6. The Ayurvedic Pharmacopoeia of India Part-I, vol. I. First Edn. Government of India, Controller of Publications. Delhi. 1990; 45-48, 113-116, 127-128.

7. Nadkarni AK. Indian Materia Medica. Vol. II. 3^rd revised edition. Popular Prakashan Ltd. Mumbai. 1982; 411-412, 428-428, 582-584.

8. Jadhav PD and Laddha KS. Estimation of gallic and ellgic acid from Terminalia chebula Retz. Indian Drugs. 2004; 41(4): 200-206.

9. Tuba AK and Ilhami Gulchin. Antioxidant and free radical scavenging properties of curcumin. Chemici Biological Interactions 2008; 174: 27-37.

10. Bagul M, Srinivisa H, Anandjiwala S and Rajani M. Phytochemical evaluation and free radical scavenging activity of nagakesara (stamen of Messua ferrae). Indian Drugs 2006. 43(8); 665-670.

11. Mishra S. Bhaisazya Kalpana Vigyan, Chaukambha Surbharati Prakashan. Varanasi. 2005; 253-254.

12. Alam M, Radhamani S, Ali U and Puroshottam KK. Microbiological screening of dhataki flowers. Journal of Research in Ayurveda and Siddha. 1984; 2(4): 371-375.

13. ICH guideline Q2 (R1). Validation of analytical procedures. Methodology, Geneva, 2006.

14. Singh B, Mungara P, Nivsarkar M and Anandjiwala S. HPTLC densitometry quantification of glycyrrhizin, glycyrrhizinic acid, apigenin, kaempferol and quercetin from Glycyrrhiza glabra. Chromatographia 2009; 70: 1665-1672.

15. R.P.W. Scott, Encyclopedia of chromatography, 10^th edn. Marcel Dekker, USA, 2001, pp. 252-254.