INTRODUCTION:

Bhuvneshavara Vati among the most common formulas used for diarrhea (Atisara) in Ayurvedic medicine. Comprised of the fruits of five medicinal important plants, Indian gooseberry (Amalaki, Emblica officinalis), Belleric myrobalan (Vibhitaka, Terminalia belerica), Chebulic myrobalan (Haritaki, Terminalia chebula), Yamani (Ajowan, Trichyspermum ammi ), Bilvapesika( Bael, Aegle marmelous) and two mineral ingredients saindhava lavan ( rock salt),grahadhoom (Soot)( The Ayurvedic Formulary of India 2000, Jain et al 2007c).

The Ayurvedic formulations are lacking in their define quality control parameters and method hence, they are not being accepted globally. The World Health Organization (WHO) Assembly, 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 (World Health Organization1998; Jain et.al.2007b).

In this connection an effort has been made to develop the quality control parameter of Bhuvneshvara vati 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, HPLC (Mizukami et. al. 2007), capillary chromatography (Aucamp et. al. 2000) and electrophoresis (Prasongsidh et. al.1998). We reported a HPTLC fingerprinting method for gallic acid in Bhuvneshvara vati . 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 Bhuvneshvara vati .

MATERIAALS AND METHODS:

Plants

Dried fruits of Emblica officinalis, Terminalia belerica, and Terminalia chebula, Aegle marmelous, Trichyspermum ammi were purchased from local market of Raipur (C.G.) 492010, INDIA and identified morphologically and microscopically by comparing with standard Pharmacopoeial Monograph (The Ayurvedic Pharmacopoeia of India1986, Indian Herbal Pharmacopoeia1999, Quality Standards of Indian Medicinal Plants2003, Mukherjee 2002).

Chemicals

All the chemicals and solvents used were of A.R. Grade. Standard gallic acid was procured from Himedia laboratories Pvt. Limited Bombay, INDIA. Marketed formulation of Bhuvneshvara vati was purchased from local pharmacy store.

The study was performed at herbal drug laboratory of Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G), India.

Preparation of Bhuvneshvara vati

Bhuvneshvara vati , three batch name BV-I, BV-II, BV-III, were prepared in laboratory using method described in Ayurvedic Formulary of India (2000).

Preparation of gallic acid extract of Bhuvneshvara vati

The gallic acid extract of Bhuvneshvara vati was obtained by refluxing the powdered Bhuvneshvara vati (1gm) with 60 ml methanol for 1 hour. Resulting extract filtered and the marc was re-refluxed with 40 ml methanol for another one hour. The extract filtered and both the filtrates were combined. The methanolic extract was concentrated under vacuum till the semisolid mass obtained. The residue was dissolved in 100 ml methanol and filtered through sintered glass funnel (G-2) by vacuum filtration assembly. The same procedure was followed for each batch of Bhuvneshvara vati , marketed formulation M1 and M2 and powdered crude drugs Emblica officinalis, Terminalia belerica, Terminalia chebula, Aegle marmelous and Trichyspermum ammi and their gallic acid extract (100 ml) prepared.

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 Bhuvneshvara vati , marketed formulations M1 and M2 and powdered drugs Emblica officinalis, Terminalia belerica, Terminalia chebula, Aegle marmelous and Trichyspermum ammi 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 Bhuvneshvara vati of which the gallic acid content have been already estimated. The observations recorded and recovery was calculated

RESULTS AND DISCUSSION:

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 [Fig. 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.

Table: 1 Validation Parameters

S. No.	Parameter	Value
1	Rf	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%	0.91 99.84
8	Repeatability of sample application	0.642%
9	Repeatability of measurements	0.479%
10	No. of theoretical plates	4895

Rf : 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 Bhuvneshvara vati

S.no.	Name		Gallic acid content %w/w	Confidence level (95%)
1	Emblica officinalis		3.10% ± 0.41	±0.44
2	Terminalia chebula		4.63%± 0.49	±0.47
3	Terminalia belerica		8.47%± 0.82	±0.88
4	Aegle marmelous		1.82±0.73	±0.68
5	Trichyspermum ammi		0.26±0.653	±0.64
4	Bhuvneshvara vati	BV-I	2.61±0.468	±0.46
		BV-II	2.59±0.567	±0.45
		BV-III	2.63±0.392	±0.58
		M₁	2.01±0.721	±0.92
		M₂	2.22±0.973	±1.26

Mean ± SD of three determinations, TP-I: Bhuvneshvara vati Batch I, TP- II: Bhuvneshvara vati Batch II, TP-III: Bhuvneshvara vati Batch III, M₁ : Marketed formulation 1, M₂ : Marketed formulation 2

Table 3: Compilation of Data of Recovery Study

S.No	Amount of gallic acid (±g/ml)			RSD%	SE	Recovery%
	In sample	Added	Estimated
1 2 3	100 100 100	50 100 150	149.02±0.62 200.69±0.37 249.63±0.68	0.416 0.184 0.272	0.253 0.151 0.277	99.34±0.51 100.35±0.39 99.85.35±0.65
Mean				0.291	0.277	99.84

Mean ± SD of six determinations, RSD =Relative Standard Deviation, SE = Standard Error

The limit of detection for gallic acid and the limit of quantification was found to be 150 ng and 0.339μg/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=Rf value 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.

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(Fig. 2).The amount of gallic acid present in the raw materials and formulations was calculated using the respective calibration graph (Table-2

Fig. 1 TLC Densitometric chromatogram of gallic acid (Rf = 0.38)

Fig.2 Overlay absorption spectra of gallic acid in sample track with their respective standards

S T

Fig.2 Overlay absorption spectra of gallic acid in sample track with their respective standards

The concentration of gallic acid present in raw material was found to be 3.10 ±0.41% w/w in emblica officinalis, 8.47±0.82% w/w in terminalia belerica, 4.63±0.49% w/w in Terminalia chebula, 1.82±0.73% w/w in Aegle marmelous and 0.26±0.653% w/w in Trichyspermum ammi. Gallic acid content in three identical laboratory batch of Bhuvneshvara vati BV-I, BV-II and BV-III, is found to be 2.61±0.468%, 2.59±0.567% and 2.63±0.392% w/w respectively with mean value 2.61±0.476%. Two marketed formulation of Bhuvneshvara Vati M1 and M₂ shows gallic acid concentration 2.01±0.721% and 3.22±0.973%. 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 preanalysed 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, % RSD at three level were calculated, and found to be within the limit, as listed in [Table.1].This shows significant Precision of methods with 95% confidence level.

CONCLUSION:

The developed HPTLC method is simple, rapid, precise and accurate for routine estimation of gallic acid in Bhuvneshvara vati . The statistical analysis proved that the method is reproducible and efficient for the analysis of gallic acid, in Ayurvedic/ herbal formulations. As Bhuvneshvara vati is a good 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 Bhuvneshvara vati .

ACKNOWLEDGEMENT:

The authors are grateful to DST under FIST and UGC New Delhi for providing financial assistance under MRP(39-169/2010(SR)0 and SAP Scheme.

REFERENCES:

Aucamp, J.P., Hara, Y., Apostolides, Z., 2000, Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography, J Chromatogr A., 76(1-2):235-42.

Indian Herbal Pharmacopoeia, 1999, Volume II, Regional Research Laboratory Jammu, Indian drug Manufacturing Association Mumbai,PP: 50-57.

Jain, V., Saraf, S., and Saraf, S., 2007 b, Spectrophotometric Determination of Piperine in Trikatu Churna: An Ayurvedic Formulation, Asian Journal of Chemistry, 19(7): 5331-5335.

Jain,V., Saraf, S.,and Saraf, S., 2007a, Standardization of Triphala churna : Spectrophotometric Approach, Asian Journal of Chemistry, 19(2): 1406-1410

Jain V., Saraf S., and Saraf S.,2007c, Quantification of Tannic Acid In Ayurvedic Formulation Bhuvneshavara Vati: Spectrophotometric Approach For Routine Quality Control , Natural Product: An Indian Journal, 3, (2): 107-110.

Lazarowych, N. J. and Pekos, P. ,1998, Use of fingerprinting and marker compounds for identification and Standardization of botanical drugs: strategies for applying Pharmaceutical hplc analysis to Herbal products, Drug Information Journal, 32: 497–512,

Mizukami, Y., Sawai, Y., Yamaguchi, Y., 2007, Simultaneous analysis of catechins, gallic acid, strictinin, and purine alkaloids in green tea by using catechol as an internal standard, J Agric Food Chem., 55(13): 4957-64.

Mukherjee, P., 2002, Pharmacological Screening of Herbal Drug, Quality Control of Herbal Drug: An Approach to Evaluation of Botanicals; Eastern Publishers (Business Horizontal Ltd.), New Delhi,PP: 539-541.

Prasongsidh, B. C., and. Skurray, G.R., 1998, Capillary electrophoresis analysis of trans- and cis-resveratrol, quercetin, catechin and gallic acid in wine, , Food Chemistry, 62 (3) :355-358

Quality Standards of Indian Medicinal Plants, 2003, Volume I, Indian Council of Medicinal Research, New Delhi, PP: 198-212.

The Ayurvedic Formulary of India, 2000, Part I, 1^st english edition, Govt. of India, Ministry of Health and Family Planning, Dept. of Indian system of medicine and Homeopathic, Delhi, ,PP: 178.

The Ayurvedic Pharmacopoeia of India, 1986, Part I, Volume I, First edition, Ministry Of Health And Family Welfare, Government Of India, Department of Health, New Delhi, PP: 4,5,26, 47, 48.

World Health Organization, 1998, Quality Control Methods For Medicinal Plants Materials, Geneva, PP:1-15,

Received on 23.11.2011 Modified on 15.12.2011

Asian J. Research Chem. 5(2): February 2012; Page 193-196

INTRODUCTION:

Name

Table 3: Compilation of Data of Recovery Study

Mean