INTRODUCTION:

The most of the Ayurvedic formulation are lacking in their defined quality control parameters and method of its evaluation.¹ The World Health Organization (WHO) in its resolution WHA 31.33 (1978), WHA 40.33 (1987), WHA 42.43 (1989) has emphasized the need to ensure the quality of medicinal plant products by using modern controlled technique and applying suitable standards^2,. The present paper is an effort to develop the quality control parameter of Drakshadi gutika by spectrophotometric determination using tannic acid as an internal standard.

Drakshadi gutika is effective for amlapitta (hyperacidity), hrddaha (heart disease), kanthadaha (itching of throat), trsna (thirst), murccha (syncope), agnimandaya (digestive impairment), bhrama (vertigo) and amavata (rheumatism) is official in Ayurveduc formulary in India. Main ingredients include Terminalia belerica (Vibhitaka), vitis vinifera (draksha), and Jaggery (Khanda) from Soccharum officinarum ^{3, 4}.

In this connection an effort has been made to develop the quality control parameter of Drakshadi gutika by HPTLC method for determining gallic acid as an internal standard which is as a important and major content in the formulation. Gallic acid is hydrolysable tannin found in many plants and chemically it is 3,4,5- trihydroxy benzoic acid. Estimation of gallic acid in formulations can be done in order to develop fingerprint of the formulation. Some of the methods reported so far, for the estimation of gallic acid are based on visible spectroscopy, HPLC⁵, capillary chromatography⁶ and electrophoresis⁷. We reported a HPTLC fingerprinting method for gallic acid in Drakshadi gutika. The TLC densitometric analysis of gallic acid is a simple, precise, and accurate method which can be considered as one of the quality control method for routine analysis of Drakshadi gutika.

EXPERIMENTAL:

Plants:

All the crude drugs were purchased from local market Raipur (C.G.), India and identified on the basis of morphological and microscopical characters and compared with standard Pharmacopoeial Monograph.^8-11

Chemicals:

All the chemicals and solvents were used of A.R. Grade.

Preparation of Drakshadi gutika:

Drakshadi gutika, three batches name DG-I, DG-II, DG-III, were prepared in laboratory using method described in Ayurvedic Formulary¹².

Preparation of extract of Drakshadi gutika:

The gallic acid extract for each batch of Drakshadi gutika and separately powdered amla, and draksha were prepared as per the method described by Jain et.al^13,14.

Chromatographic conditions

The Camag HPTLC instrument was used for estimation consist of Linomat V semi automatic sample applicator, TLC scanner 3, CATS V.4.06 software for interpretation of the data, Hamilton syringe and Camag twin trough chamber. The pre-coated silica gel G60 F₂₅₄ was used as stationary phase, purchased from E. Merck. India. The TLC plates were pre-washed with methanol, and activated at 115^o C for about 30 min. The gallic acid was well resolved on the precoated silica gel G60 F₂₅₄ on aluminum sheets, the mobile phase was toluene: ethyl acetate: formic acid (3: 2: 0.4v/v), chamber saturation time 20 min, migration distance 70 mm, wavelength scanning at 280 nm, band width 8 mm, slit dimension 5 * 0.45 mm, scanning speed 20 nm/sec, and the source of radiation was a deuterium lamp. Sample was prepared by using methanol as solvent.

Preparation of standard solution of gallic acid

Gallic acid stock solution (75 ng/ml) was prepared by dissolving 7.5 mg of accurately weighed gallic acid in methanol and made up the volume up to 100 ml with methanol.

Method Validation

The method was validated for linearity, accuracy, limit of detection, limit of quantification, inter-day and intra-day assay precision, repeatability of measurement, and repeatability of sample application.

Estimation of gallic acid

The appropriate aliquots from gallic acid extract of each batch of Drakshadi gutika and powdered drugs Terminalia belerica and Vitis vinifera were withdrawn in 10 ml volumetric flask separately. The corresponding concentrations of gallic acid against respective peak area were determined using calibration curve of gallic acid.

Recovery Studies

The recovery studies performed at three levels were done by adding known amount of gallic acid to extract of Drakshadi gutika of which the gallic acid content have been already estimated. The observations recorded and recovery was calculated

RESULTS AND DISCUSSION:

Fingerprint method for Drakshadi gutika with TLC Densitometric Methods (HPTLC) using gallic acid as a standard developed. The gallic acid is a major content in all the three ingredients of the formulation.

The TLC procedure was optimized with a view to develop a stability indicating assay method. The standard and the sample were run in different solvent systems. Better results were obtained with mobile phase consisting of toluene: ethyl acetate: formic acid (3: 2: 0.4v/v) with R_f values of 0.38±0.06 for gallic acid (Figure 1). The spot was resolved on the chromatogram that showed the good resolution. To a pre-washed activated TLC plate, 2-10 µl of standard stock solution of gallic acid was spotted with Linomat V semi sample applicator. The plate was developed and scanned. The peak areas of each standard were obtained from the software, and a calibration graph of concentration against peak area was plotted. A good linear relationship was obtained over a concentration range of 150-750 ng/spot of gallic acid. The correlation coefficient (r²) value was 0.9997, indicates the good linearity between the concentration and peak area.

The limit of detection for gallic acid and the limit of quantification were found to be 150 ng and 0.378μg/ml respectively. These values are considered to be good enough for a reasonable accuracy in most of the laboratories worldwide.

Intra-day assay precision was found by analysis of standard drug three times on the same day. Inter-day assay precision was carried out using the standard drug on three different days, and % relative standard deviation (RSD) was calculated. The RSD was found to be less than 2 for both inter-day and intra-day assay precision. The low values indicate robustness of the method. Repeatability of sample application was assessed by spotting 10 μl of drug solution for 6 times. From the peak areas, the % RSD (0.642) was determined. Repeatability of measurement was determined by spotting 10 µl of standard drug solution on TLC plate. After development, spot was scanned six times without changing position. The % RSD calculated for gallic acid is 0.479.

The efficiency of the method is determined by means of number of theoretical plates. It was calculated using the formula, n=16x²/y², where x=R_fvalue of drugs and y=width of peaks. The number of theoretical plates was found to be 4895. The complete validation parameters are shown in table 1.

Table 1: Validation Parameter of gallic acid

S. No.	Parameter	Value
1	R_f	0.38±0.04
2	Linearity (ng/spot)	150-750
3	Correlation coefficients r²	0.9997
4	LOD(ng /spot)	150
5	LOQ(μg /spot)	0.339
6	Precision( %RSD) a) Inter Day b) Intraday	0.64 1.3
7	Recovery Studies a)Accuracy( %RSD) b)Recovery%	0. 435 99.32
8	Repeatability of sample application	0.642%
9	Repeatability of measurements	0.479%
10	No. of theoretical plates	4895

R_f : Retention factor, RSD : Relative standard deviation, LOD : Limit of detection, LOQ: Limit of quantification, SE: Standard error

Table 2: Estimation of gallic acid content in Drakshadi gutika

S.no.	Name		Gallic acid content %w/w*	Confidence level (95%)
1	Terminalia belerica		8.912 ± 0.41	±0.44
2	Vitis vinifera		0.73±0.63	±0.62
3	Drakshadi gutika	DG-I	3.307±0. 52	±0.54
		DG-II	3.301±0.63	±0.67
		DG-III	3.314±0.35	±0.36
		M-I	2.289±0.74	±0.76
		M-II	1.033±0.65	±0.64

*Mean ± SD of six determinations

Fig. 1 TLC Densitometric chromatogram of gallic acid

The sample aliquot of raw materials along with laboratory and marketed formulation was applied, and the plate developed with the mobile phase. The band of gallic acid in sample extract was confirmed by overlaying their UV absorption spectra with those of standard gallic acid using a Camag TLC scanner 3.The amount of gallic acid present in the raw materials and formulations was calculated using the respective calibration graph (Table 2). The concentration of gallic acid present in raw material was found to be 8.912 ± 0.41% w/w in Terminalia belerica, and 0.73±0.63% w/w in Vitis vinifera. Gallic acid content in three identical laboratory batch DG-I, DG-II and DG-III, was found to be 3.307%±0.52%, 3.301%±0.63% and 3.314%±0.35% w/w respectively. The gallic acid content in all the three different batches is found to be in close proximities with each other.

The recovery studies were carried out for the accuracy parameter. The study was carried out at three levels. To the powdered formulation, the standard drugs of gallic acid were added at 50% 100% and 150% levels; dilutions were made, and analyzed by the method. The mean of % recovery, and % RSD of three levels were calculated, and found to be within the limit, as listed in table.1.This shows significant Precision of methods with 95% confidence level.

The developed HPTLC method is simple, rapid, precise and accurate for routine estimation of gallic acid in Drakshadi gutika. The statistical analysis proved that the method is reproducible and efficient for the analysis of gallic acid, in ayurvedic/ herbal formulations. As Drakshadi gutika is a important source of gallic acid, these findings can be used as routine chromatographic fingerprinting method for the standardization of the raw materials as well as the finished formulation of Drakshadi gutika.

Acknowledgement:

The authors are grateful to DST under FIST and UGC New Delhi for providing financial assistance under MRP (39-169/2010(SR) and SAP Scheme.

REFERENCES:

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Received on 25.10.2013 Accepted on 15.11.2013

Asian J. Pharm. Ana. 3(4): Oct. - Dec. 2013; Page 111-114