Development and Validation of HPTLC Method for Quantification of Gallic acid and Ellagic acid in Ashwagandharishta

Preeti Tiwari¹* and Rakesh K. Patel²

¹Department of Pharmacognosy, Shri Sarvajanik Pharmacy College, Mehsana-384001, Gujarat, India.

²Head of Department of Pharmacognosy, Shri S. K. Patel College of Pharmaceutical Education and Research, Kherva-382711, Gujarat, India

*Corresponding Author E-mail: preetitiwari198311@yahoo.com

ABSTRACT:

Ashwagandharishta is a polyherbal hydro-alcoholic formulation and is used as immunomodulator to promote the health and longevity by increasing defence against disease and also known for its usefulness in the treatment of hypercholesterolemia. A simple, precise, robust and accurate HPTLC method has been established for the determination of gallic acid and ellagic acid in Ashwagandharishta–T and Ashwagandharishta-M prepared by traditional and modern methods respectively and also in its marketed formulation. The developed HPTLC method was validated in terms of precision, accuracy, LOD, LOQ and specificity. The amount of gallic acid in Ashwagandharishta-T, M and its marketed formulation was found to be 0.0281, 0.0279 and 0.0280 % w/w respectively while ellagic acid was found to be 0.0191, 0.0189 and 0.0188 % w/w respectively. This is the first report for quantification of gallic acid and ellagic acid in Ashwagandharishta by HPTLC. Furthermore, no TLC densitometric methods have been reported for the quantification of gallic acid and ellagic acid from Ashwagandharishta.

KEYWORDS: Ashwagandharishta, HPTLC, Validation, Gallic acid, Ellagic acid

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 gallic acid and ellagic acid 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. Gallic acid (purity98%) was purchased from SD fine, Mumbai while ellagic acid (purity 97%) was 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 gallic acid was toluene-ethyl acetate-formic acid-methanol, 6 + 6 +1.2 + 0.25 (v/v), while for ellagic acid toluene-ethyl acetate-formic acid-methanol, 9 + 9 + 3 + 0.6 (v/v) was 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 = 290 nm for gallic acid and λ = 285 nm for ellagic acid 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 amount of the compounds chromatographed was determined from the intensity of diffusely reflected light.

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

Parameter	Gallic acid	Ellagic acid
Instrumental Precision (% RSD, n = 6)	0.54	1.08
Repeatability (% RSD, n = 6)	0.58	1.15
LOD (ng)	50	60
LOQ (ng)	150	200
Linear range (n = 3)	150-750 ng/band	200-1000 ng/band
Correlation coefficient (r)	0.9996	0.9996
Linearity (Regression equation)	6715.2	2233.3

2.5 Preparation of standard stock solutions and calibration plots:

2.5.1 Preparation of standard solution of gallic acid:

Stock solution of 300 µg mL^-1 of gallic acid was prepared by dissolving 15 mg of accurately weighed gallic acid 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 15, 30, 45, 60 and 75 µg mL^-1 of gallic acid, respectively. 10µL each of the standard solutions of gallic acid (150-750 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 150-750 ng/band.

2.5.2 Preparation of standard solution of ellagic acid:

Stock solution of 400 µg mL^-1 of ellagic acid was prepared by dissolving 20 mg of accurately weighed ellagic acid 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 20, 40, 60, 80 and 100µg mL^-1 of ellagic acid, respectively. 10µL each of the standard solutions of ellagic acid (200-1000 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 200-1000 ng/band.

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 [%]
Gallic acid	150	2913.9	0.55	2912.1	0.64
	450	4910.6	0.41	4909.5	0.48
	750	6952.9	0.34	6951.2	0.42
Ellagic acid	200	1410.8	1.15	1409.7	1.34
	600	2275.4	0.90	2274.0	1.13
	1000	3197.6	0.60	3196.2	0.69

^a) n = 6

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

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery[%]	RSD[%]
Ashwagandharishta-T	50	422	100.10	0.72
	100	562	99.94	0.54
	150	702	99.81	0.43
Ashwagandharishta-M	50	420	100.24	0.71
	100	559	100.12	0.45
	150	698	100.05	0.44
Marketed Ashwagandharishta	50	419	99.92	0.72
	100	562	100.30	0.57
	150	701	100.19	0.43

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 method developed for precision, repeatability and accuracy¹³.

3. RESULTS AND DISCUSSION:

3.1 Selection of the optimum mobile phase:

In an attempt to optimize mobile phase, toluene-ethyl acetate-formic acid-methanol mixtures in different proportions were investigated. Use of toluene-ethyl acetate-formic acid-methanol 6 + 6 + 1.2 + 0.25 (v/v) resulted in sharp, well defined gallic acid peaks of R_F0.49 ± 0.02 while solvent system toluene-ethyl acetate-formic acid-methanol 9 + 9 + 3 + 0.6 (v/v) resulted in sharp ellagic acid peaks of R_F0.46 ± 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 Results of validation of the method:

ICH guidelines were followed for the validation of the analytical method. 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 gallic acid (150 ng spot^-1) and ellagic acid (200 ng spot ^-1) and expressed as percentage relative standard deviation (% RSD) as shown in Table 1.

3.2.2 Repeatability:

The repeatability of method was affirmed by analysing 150 ng spot^-1 of gallic acid and 200 ng spot^-1of ellagic acid 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 gallic acid were 50 and 150 ng, respectively while for ellagic acid detection and quantification limit were 60 and 200 ng respectively as shown in Table 1 which indicates that the sensitivity of the method is adequate.

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

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery[%]	RSD[%]
Ashwagandharishta-T	50	288	100.35	0.93
	100	382	99.91	0.80
	150	478	100.07	0.64
Ashwagandharishta-M	50	284	99.99	0.93
	100	378	100.09	0.80
	150	472	99.86	0.68
Marketed Ashwagandharishta	50	282	99.88	0.74
	100	376	100.08	0.82
	150	471	100.14	0.54

Figure 1: Overlay HPTLC densitogram of gallic acid standard

Figure 2: Overlay HPTLC densitogram of gallic acid from samples of Ashwagandharishta

a, Ashwagandharishta-T; b, Ashwagandharishta-M; c, marketed Ashwagandharishta, (the mobile phase was toluene-ethyl acetate-formic acid-methanol, 6+6+1.2+0.25)

3.2.5 Specificity:

The specificity of the method was ascertained by analyzing reference standard and samples. The bands for gallic acid and ellagic acid from Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulations were confirmed by comparing the R_F and UV spectra of the separated bands with those from the standard.

3.2.6 Recovery:

The pre-analyzed samples of Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation were spiked with an additional 50, 100 and 150% of gallic acid standard and the mixtures were analysed again, in triplicate, by the proposed method, to check recovery of different amounts of gallic acid from the Ashwagandharishta-T, Ashwagandharishta-M and its marketed formulation. Recovery for gallic acid was found in between 99.81-100.10% in Ashwagandharishta-T, 100.05-100.24% in Ashwagandharishta-M and 99.92-100.30% 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 an additional 50, 100 and 150% of ellagic acid standard and the mixtures were analysed again, in triplicate, by the proposed method, to check the recovery of different amounts of ellagic acid from Ashwagandharishta-T, M and its marketed formulation and it was found in between 99.91-100.35% in Ashwagandharishta-T, 99.86-100.09% in Ashwagandharishta-M and 99.88-100.14% in marketed Ashwagandharishta as shown in Table 4.

3.3 Estimation of gallic acid and ellagic acid in Ashwagandharishta-T, Ashwagandharishta-M and in its marketed formulation:

The suitability of the method was examined by estimation of gallic acid in Ashwagandharishta-T, M and its marketed formulation. Bands of R_F0.49 ± 0.02 were observed in the densitogram for gallic acid standard (Figure 1) while the bands of same R_F were observed in the densitogram obtained from the gallic acid isolated from Ashwagandharishta-T, M and its marketed formulation (Figure 2). Similarly, ellagic acid was also estimated in Ashwagandharishta-T, M and its marketed formulation. Bands of R_F0.46 ± 0.02 were observed in the densitogram for ellagic acid standard (Figure 3) while the bands of same R_Fwere observed in the densitogram obtained from ellagic acid isolated from Ashwagandharishta-T, M and its marketed formulation (Figure 4).

Gallic acid was found to be 0.0281, 0.0279 and 0.0280 %w/w in Ashwagandharishta-T, M and its marketed formulation respectively while ellagic acid was found to be 0.0191, 0.0189 and 0.0188 %w/w in Ashwagandharishta-T, Ashwagandharishta-M and in marketed Ashwagandharishta respectively as shown in Table 5.

4. CONCLUSION:

This HPTLC technique was found to be simple, 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.

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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.

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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.

Received on 12.05.2012 Modified on 15.06.2012

Asian J. Research Chem. 5(6): June, 2012; Page 759-764

Sample	Gallic acid (% w/w)^a)	Ellagic acid (% w/w)^a)
Ashwagandharishta-T	0.0281±0.0004	0.0191±0.0003
Ashwagandharishta-M	0.0279±0.0003	0.0189±0.0003
Marketed Ashwagandharishta	0.0280±0.0004	0.0188±0.0002