INTRODUCTION:

Triphala churna is most common among the formulations used in ayurvedic medicine, comprised of the fruits of three medicinally important plants namely Indian gooseberry (Amalaki, Emblica officinalis), Belleric myrobalan (Vibhitaka, Bahera, Terminalia belerica) and Chebulic myrobalan (Haritaki, Harda, Terminalia chebula). Triphala Churna is mentioned throughout the ancient literature of ayurvedic medicine as a tonifying blood cleanser and gentle laxative. It is highly prized for its ability to regulate the process of digestion and elimination. Triphala Churna plays an essential role in the treatment of a wide variety of conditions when used alone or in combination with other formulation¹.

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^2,3.

In this connection an effort has been made to develop the quality control parameter of Triphala Churna 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, HPLC⁴, capillary chromatography⁵ and electrophoresis⁶. We reported a HPTLC fingerprinting method for gallic acid in Triphala churna. 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 Triphala Churna.

MATERIAALS AND METHODS:

Plants:

Dried fruits of Emblica officinalis, Terminalia belerica, and Terminalia chebula were purchased from local market of Raipur (C.G.) 492010, INDIA and identified morphologically and microscopically by comparing with standard Pharmacopoeial Monograph ^[7-11].

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 Triphala churna 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 Triphala Churna:

Triphala churna, three batch name TP-I, TP-II, TP-III, were prepared in laboratory using method described in Ayurvedic Formulary of India¹².

Preparation of Gallic Acid Extract of Triphala Churna:

The gallic acid extract of Triphala churna was obtained by refluxing the powdered Triphala churna (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 Triphala churna, marketed formulation M1 and M2 and powdered crude drugs Emblica officinalis, Terminalia belerica, and Terminalia chebula 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 Triphala Churna, marketed formulations M1 and M2 and powdered drugs Emblica officinalis, Terminalia belerica, and Terminalia chebula 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 Triphala churna 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.

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

The limit of detection for gallic acid and the limit of quantification was 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=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.

Table: 1 Validation Parameters

Parameter	Value
Rf	0.38±0.04
Linearity (ng/spot)	150-750
Correlation coefficients r²	0.9997
LOD (ng /spot)	150
LOQ (μg /spot)	0.339
Precision( %RSD) a) Inter Day b) Intraday	0.64 1.3
Recovery Studies a) Accuracy( %RSD) b) SE c) Recovery%	0.35 0.40 99.72
Repeatability of sample application	0.642%
Repeatability of measurements	0.479%
No. of theoretical plates	4895

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

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). The concentration of gallic acid present in raw material of Emblica officinalis, Terminalia belerica, Terminalia chebula, was found to be 3.10±0.41% w/w, 8.47±0.82% w/w and 4.63±0.49% w/w respectively. In three identical laboratory batches of Triphala churna named TP-I, TP-II and TP-III, the concentration of gallic acid was found to be 5.39±0.48%, 5.42±0.46%, 5.41±0.52% w/w respectively with mean value 5.41±0.49%. Two marketed formulation of Triphala churna M₁ and M₂ showed gallic acid concentration to be 5.21±0.98% and 5.07±1.12%.

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, % RSD and standard error (SE) of 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.

Table 2: Estimation of gallic acid Content in Triphala Churna

Name		Gallic acid content %w/w	Confidence level (95%)
Emblica officinalis		3.10% ± 0.41	±0.44
Terminalia chebula		4.63%± 0.49	±0.47
Terminalia belerica		8.47%± 0.82	±0.88
Triphala churna	TP-I	5.39%±0.48	±0.46
	TP-II	5.42±0.46	±0.45
	TP-III	5.41%±0.52	±0.58
	M₁	5.21%±0.98	±0.92
	M₂	5.07%±1.12	±1.26

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

Name