1.0 INTRODUCTION:

Drakshasava is a polyherbal hydroalcoholic preparation and is used to improve digestion, as blood purifier, in the treatment of anaemia and advised as a choice of remedy in respiratory problems. The chief ingredient of Drakshasava is draksha, dried fruits of Vitis vinifera¹. The composition and properties of fruits of Vitis vinifera have been extensively investigated and it was reported that they contain large amount of phenolic compounds as catechins, epicatechin, quercetin and gallic acid, dimeric, trimeric and tetrameric procyanidins². These compounds have many favourable effects on human health such as lowering of human low density lipoproteins, reduction of heart disease and cancer because of their antioxidant property^3-6.

Furthermore, no validated HPTLC method has been reported for the quantification of gallic acid and catechin from Drakshasava. Since, standardization is an important aspect for establishing the quality and efficacy of Ayurvedic formulations or any polyherbal formulation.

Therefore, a proper scientific validation as chromatographic fingerprinting is required for quantification of marker compounds for quality control purposes.

2.0 MATERIALS AND METHOD:

2.1 Preparation of Drakshasava-T

This was prepared by the method as given in Ayurvedic Formulary of India, Part-II¹. The ingredients were procured from local market, Jamnagar. Identification of 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 CIMAP for future reference.

According to this method, dried fruits of Vitis vinifera were crushed and then placed in polished vessel of brass along with prescribed quantity of water (16.384 L), and allowed to steep overnight. After overnight steeping, this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity (4.096 L), then the heating was stopped and it was filtered through unstarched muslin cloth in cleaned and fumigated vessel and after that jaggery and honey were added and mixed properly. Then dhataki flowers (Woodfordia floribunda) and prescribed quantity of coarsely powdered prakshepa dravyas as Myristica fragrans (flowers), Eugenia caryophyllus (flower bud), Cubeba officinalis (fruits), Santalum album (heart wood), Piper nigrum (fruits), Cinnamomum zeylanicum (stem bark), Eletteria cardamomum (seeds) and Cinnamomum tamala (leaves) were added and this sweet filtered fluid was placed for fermentation in incubator for fifteen days at 33ºC± 1ºC. After fifteen days, completion of fermentation was confirmed by standard tests⁷. The fermented preparation was filtered with unstarched muslin cloth and kept in cleaned covered vessel for further next seven days. Then, it was poured in clean amber colored glass bottles previously rinsed with ethyl alcohol, packed and labelled properly.

2.2 Preparation of Drakshasava-M

Method of preparation was same as followed with Drakshasava-T, only dhataki flowers were replaced with yeast for inducing fermentation⁸.

2.3 Instrumentation

Chromatography was performed on 20 x 10 cm HPTLC plate coated with layers of silica gel 60 F₂₅₄ (Merck, Darmstadt, Germany) having thickness of 0.25 mm. 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. Densitometric scanning was performed with a Desaga TLC scanner CD 60 in reflectance absorbance mode controlled by ProQuant software (v1.06; Desaga) resident in the system. The slit dimensions were 4 x 0.02 mm and the scanning speed 100 nm s^-1. The radiation source was a deuterium lamp emitting continuous UV radiation between 190-360 nm.

2.4 Chemicals

All solvents used were of analytical grade and were purchased from Merck. Gallic acid (purity 98%) was purchased from SD fine, Mumbai while catechin (purity 98%) was purchased from Yucca Enterprises, Mumbai, India.

2.5 Preparation of stock solution and working 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.

2.6 Preparation of stock solution and working standard solution of catechin

Stock solution of 400 µg ml^-1 of catechin was prepared by dissolving 20 mg of accurately weighed catechin 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 catechin, respectively. 10µl each of the standard solutions of catechin (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.

2.7 Preparation of test sample

1 g (equivalent to 0.90 mL) of each of the test formulation of Drakshasava as Drakshasava-T, Drakshasava-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 Drakshasava 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.

2.8 Chromatographic condition

The mobile phase was selected as a mixture of toluene-ethyl acetate-formic acid-methanol, 6 + 6 + 1.2 + 0.25 (v/v) for gallic acid while mixture of toluene-ethyl acetate-formic acid-methanol, 9 + 9 + 1.5 + 0.6 (v/v) for catechin 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 vapour 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 in reflectance absorbance mode at λ = 290 nm for gallic acid and λ = 292 nm for catechin. The amount of compounds chromatographed was determined from the intensity of diffusely reflected light.

2.9 Method validation

The method was validated for linearity, sensitivity, specificity, precision and accuracy. ICH guidelines were followed for the validation of analytical method developed for sensitivity, precision, repeatability and accuracy⁹.

To determine the linearity, calibration curves were plotted over a concentration range of 150-750 ng spot^-1 for gallic acid and 200-1000 ng spot^-1 for catechin and the densitometric scanning of solutions of gallic acid and catechin was performed at λ = 290 nm and 292 nm respectively. Linear regression data for the calibration plot of gallic acid and catechin are listed in Table 1. A good linear relationship between response (peak area) and amount for gallic acid was obtained over the range of 150-750 ng/band while for catechin 200-1000 ng spot^-1.

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

Parameter	Gallic acid	Catechin
Instrumental Precision (% RSD, n = 6)	0.54	1.22
Repeatability (% RSD, n = 6)	0.58	1.26
LOD (ng)	50	70
LOQ (ng)	150	200
Linear range (n = 3)	150-750 ng/spot	200-1000 ng/spot
Correlation coefficient (r)	0.9996	0.9997
Linearity (Regression equation)	y = 6715.2x+1925.5	y = 2274.2x+483.67

The sensitivity of the method was measured in terms of limit of detection (LOD) and limit of quantification (LOQ) which were measured by visual evaluation while specificity of the method was ascertained by analyzing reference standard and test samples.

Six replicates of three samples of different concentration of gallic acid and catechin as low, medium and high were used for the determination of precision. Intra-day and inter-day precision were carried out. The accuracy of the method was determined by calculating the recoveries of gallic acid and catechin in mixture by standard addition method. To measure the accuracy, known amount of standard solutions of gallic acid and catechin were added to a pre-quantified sample solution and then their response (peak area) was measured and percentage recovery was calculated. Each response was taken as the average of three determinations. The suitability of the method was examined by estimation of gallic acid and catechin content in test formulations of Drakshasava.

3.0 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 + 1.5 + 0.6 (v/v) resulted in sharp catechin peaks of R_F0.43 ± 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 Instrumental precision

Instrumental precision was checked by repeated scanning (n = 6) of the same spot of gallic acid (150 ng spot^-1) and catechin (200 ng spot ^-1) and expressed as relative standard deviation (% RSD) as shown in Table 1.

3.3 Repeatability

The repeatability of method was affirmed by analysing 150 ng spot^-1 of gallic acid and 200 ng spot^-1 of catechin individually on TLC plate (n = 6) and expressed as % RSD as shown in Table 1.

3.4 LOD and LOQ

The limit of detection and quantification were determined by visual evaluation and were found 50 and 150 ng respectively for gallic acid while 70 and 200 ng for catechin respectively as shown in Table 1 which indicates that the sensitivity of the method is adequate.

3.5 Linearity

A good linear relationship between response (peak area) and amount was obtained over the range of 150-750 ng/band for gallic acid while 200-1000 ng/band for catechin. Linear regression data for the calibration plot as correlation coefficients were found to be 0.9996 and 0.9997 for gallic acid and catechin respectively. The lower limit of quantification was defined as the lowest concentration in the calibration curve.

3.6 Specificity

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

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

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery [%]	RSD [%]
Drakshasava-T	50	0.385±0.0040	100.35±1.04	1.04
	100	0.513±0.0032	100.13±0.63	0.63
	150	0.641±0.0025	100.11±0.39	0.39
Drakshasava-M	50	0.380±0.0031	99.83±0.85	0.85
	100	0.507±0.0026	99.80±0.52	0.52
	150	0.634±0.0035	99.89±0.55	0.55
Marketed Drakshasava	50	0.384±0.0036	100.26±0.94	0.94
	100	0.510±0.0021	100.06±0.41	0.41
	150	0.639±0.0032	100.10±0.50	0.50

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

Sample	Amount of drug added [%]	Theoretical content [ng]	Recovery [%]	RSD [%]
Drakshasava-T	50	0.293±0.0038	99.89±1.05	1.05
	100	0.390±0.0031	100.08±0.64	0.64
	150	0.487±0.0043	99.79±0.73	0.73
Drakshasava-M	50	0.291±0.0030	100.34±0.84	0.84
	100	0.386±0.0031	99.91±0.65	0.65
	150	0.482±0.0031	99.93±0.51	0.51
Marketed Drakshasava	50	0.292±0.0032	100.45±0.86	0.86
	100	0.390±0.0032	100.43±0.68	0.68
	150	0.485±0.0033	99.93±0.54	0.54

Table 5-Estimation of gallic acid and catechin from Drakshasava-T, Drakshasava-M and its marketed formulation by proposed HPTLC method.

Sample	Gallic acid (% w/w)^a)	Catechin (% w/w)^a)
Drakshasava-T	0.0256±0.0003	0.0195±0.0003
Drakshasava-M	0.0254±0.0002	0.0193±0.0003
Marketed Drakshasava	0.0255±0.0002	0.0194±0.0005

^a) Mean ± SD, n = 3

3.7 Intra-day and inter-day precision

The intra-day and inter-day precision of the method were estimated by analyzing six replicates of three samples of different concentration as low, medium and high containing 150, 450 and 750 ng spot^-1 of gallic acid and 200, 600 and 1000 ng spot ^-1 of catechin on the same day for intra- day precision while on different days for inter-day precision and the results are expressed as percentage relative standard deviation (% RSD) in Table 2^10-11.The percentage relative standard deviation (% RSD) for intra-day precision and inter-day precision was found within acceptable limit.

3.8 Accuracy

The accuracy of the method was determined by standard addition method. The pre-analyzed samples of Drakshasava-T, Drakshasava-M and its marketed formulation were spiked with 50, 100 and 150% of gallic acid standard and the mixtures were analysed again, in triplicate, by the proposed method, to check the recovery of different amounts of gallic acid from the Drakshasava-T, Drakshasava-M and its marketed formulation. Recovery for gallic acid was found in between 100.11-100.35 % in Drakshasava-T, 99.80-99.89 % in Drakshasava-M and 100.06-100.26 % in the marketed formulation of Drakshasava as depicted in Table 3. Similarly, the pre-analyzed samples of Drakshasava-T, M and its marketed formulation were spiked with 50, 100 and 150% of catechin standard and the mixtures were analysed again, in triplicate, by the proposed method, to check the recovery of different amounts of catechin from Drakshasava-T, M and its marketed formulation. Recovery for catechin was found to be 99.79-100.08 % in Drakshasava-T, 99.91-100.34 % in Drakshasava-M and 99.93-100..45 % in marketed Drakshasava as shown in Table 4.

3.9 Estimation of gallic acid and catechin in Drakshasava-T, Drakshasava-M and its marketed formulation

The suitability of the method was examined by estimation of gallic acid in Drakshasava-T, M and its marketed formulation. Bands of R_F0.49 ± 0.02 were observed in the densitogram of gallic acid standard (Figure 1) while the bands of same R_F were observed in the densitogram obtained from the gallic acid isolated from Drakshasava-T, M and its marketed formulation (Figure 2). Similarly, catechin was also estimated in Drakshasava-T, M and its marketed formulation. Bands of R_F0.43 ± 0.02 were observed in the densitogram of catechin standard (Figure 3) while the bands of same R_Fwere observed in the densitogram obtained from catechin isolated from Drakshasava-T, M and its marketed formulation (Figure 4). Gallic acid was found to be 0.0256, 0.0254 and 0.0255 %w/w in Drakshasava-T, M and its marketed formulation respectively while catechin was found to be 0.0195, 0.0193 and 0.0194 %w/w in Drakshasava-T, Drakshasava-M and in its marketed formulation respectively as shown in Table 5.

Figure 1: Overlay HPTLC densitogram of gallic acid standard

Figure 2: Overlay HPTLC densitogram of gallic acid from samples of Drakshasava.

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

(the mobile phase was toluene-ethyl acetate-formic acid-methanol, 6+6+1.2+0.25 v/v)

Figure 3: Overlay HPTLC densitogram of catechin standard

Figure 4: Overlay HPTLC densitogram of catechin from samples of Drakshasava.

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

(the mobile phase was toluene-ethyl acetate-formic acid-methanol, 9+9+1.5+0.6 v/v)

4.0 CONCLUSION:

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

5.0 REFERENCES:

1. The Ayurvedic Formulary of India Part-II, Controller of Publications, Delhi; 2000:35.

2. Baydar NG, Ozkan G, Sagdic O. Total phenolic contents and antibacterial activities of grape (Vitis vinifera L.) extracts. Food Control 2004; 15: 335-339.

3. Frankel EN, Kanner J, German JB, parks E, Kinsella JE. Inhibition of oxidation of human low density lipoprotein by phenolic substances in red wine. Lancet 1993; 341 (20): 454-457.

4. Mayer AS, Yi OS, Person DA, waterhouse DL, Frankel EN. Inhibition of human low density lipoprotein oxidation in relation to composition of phenolic antioxidants in grapes (Vitis vonofera). Journal of Agriculture and Food Chemistry 1997; 45: 1638-1643.

5. Teissure PL, Frankel EN, Waterhouse AL, Peleg H, German GB. Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and wines. Journal of Science Food and Agriculture 1996; 70:55-61.

6. Waterhouse AL. Wine antioxidants may reduce heart disease and cancer. Presentation of American Chemical Society, Washington; 1994.

7. Mishra S, Bhaisazya Kalpana Vigyan. Varanasi (India): Chaukambha Surbharati Prakashan; 2005:253-254.

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

9. ICH guidelines Q2 (R1). Validation of analytical procedures. Methodology, Geneva, 2006.

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

11. R.P.W. Scott, Encyclopedia of Chromatography, 10^th edn., Marcel Dekker, USA, 2001, p.p. 252-254.

Received on 25.07.2012 Modified on 09.08.2012

Asian J. Research Chem. 5(8): August, 2012; Page 1033-1037

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	2913.95	0.55	2912.10	0.64
	450	4910.59	0.41	4909.48	0.48
	750	6952.91	0.34	6951.22	0.42
Catechin	200	929.21	1.27	928.15	1.46
	600	1835.14	0.90	1834.64	1.17
	1000	2761.17	0.64	2760.54	0.85