INTRODUCTION:

Spectrophotometric is emerging as a versatile, high throughput & cost-effective technology that is uniquely suited to assessing the identity and quality of botanical materials. The World Health Organization (WHO) has emphasized the need to ensure the quality of herbal / Ayurvedic formulations by using suitable standards and technique. Astangavaleha is official in Ayurvedic formulary of India and it is the most common formulation used for respiratory disorders in Ayurvedic medicinal preparation. It Composition of the medicinal important plants Myrica esculenta, Inula racemosa, Pistacia integerrima, Trachyspermum ammi, Carum carvi, Zingiber officinale, Piper nigrum, Piper longum, madhu and Zingiber officinale (svarasa). Spectrophotometric determination tannic acid used as an internal standard. Polyphenols constitute one of the most numerous and ubiquitous groups of plant metabolites and are an integral part of both human and animal diets.Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. The main objective of the present study is to quantification of one of the active constituent of total polyphenols as tannic acid in Astangavaleha and crude drugs (Myrica esculenta, Inula racemosa and Pistacia integerrima).

Spectrophotometric analysis, which is a simple, precise and accurate method that can be considered as one of the quality control method for routine analysis. ^(1-7)

MATERIALS AND METHODS:

Plant material

Dried crude drugs of Myrica esculenta, Inula racemosa, Pistacia integerrima, Trachyspermum ammi, Carum carvi, Zingiber officinale, Piper nigrum, Piper longum were purchased from local market of Ujjain (M.P.) 456010, INDIA. The sample of crude drug was also authenticated by Dept. of Botany, Vikram University Ujjain (M.P.) 456010, India.

Preparation of the formulation

Astangavaleha was prepared in laboratory, as per the method described in Ayurvedic Formulary of India. The authenticated crude drugs of these formulations were crushed in to fine powder separately. Finally the prescribed weight of all the raw material was mixed together. Madhu was added when the preparation was cool & mixed well. The composition of Astangavaleha with their botanical identities and parts used were given in table 1. Three laboratory formulation batch of Astangavaleha were prepared using above mentioned methods and were named as AS-I, AS-II, AS-III. One Marketed formulations named AU-I was purchased from local pharmacy store Ujjain. These formulation samples were stored at identical conditions of temperature, light and moisture. ⁽²⁾

Table 1 Content of Astangavaleha

S. No.	Sanskrit Name	Hindi /common Name	Botanical Name	Family	Part Used	Quantity
1.	Katphala	Kaiphal	Myrica esculenta	Myricaceae	Fr.	1 part
2.	Pauskara	Pohakar	Inula racemosa	Asteraceae	Rt	1 part
3.	Srngi	Karkatasrangi	Pistacia integerrima	Anacardiaceae	Gi	1 part
4.	Yamani	Ajwain	Trachyspermum ammi	Umbelliferae	Fr.	1 part
5.	Karavi	Jira	Carum carvi	Umbelliferae	Fr.	1 part
6.	Sunthi	Adraka	Zingiber officinale	Zingiberaceae	Rz.	1 part
7.	Marica	Kalimirch	Piper nigrum	Piperaceae	Fr.	1 part
8.	Pippali	Lindi Pippal	Piper longum	Piperaceae	Fr.	1 part
9.	Madhu					Q. S.
10.	Ardrak Svarasa					Q. S.

Figure 1 Calibration curve of tannic acid

Method development of UV^{(3, 4, 9-12)}

Chemicals

All the chemicals and solvents were used of A.R. Grade. Standard tannic acid was procured from Himedia laboratories Pvt. Limited Bombay, INDIA

Instrument

Astangavaleha was estimated for their tannic acid contents against standard tannic acid solution on UV-Visible Spectrophotometer (Shimadzu, UV-1700, Pharmaspec).

Preparation of standard solution of tannic acid

As tannic acid has good solubility in 0.1N Hydrochloric acid, an accurately weighed tannic Acid (100 mg), from Himedia, A.R. Grade, was dissolved in 0.1N hydrochloric acid and volume was made up to 100 ml with 0.1N hydrochloric acid in volumetric flask. 2 ml of this solution was diluted with 0.1N hydrochloric acid up to 100 ml in volumetric flask to give 20 mg/ml tannic acid solution.

Preparation of tannic acid extract of Astangavaleha & crude drugs

Extract the powdered Astangavaleha (1gm) with 6 volume of denatured spirit on a shaker for 2 hours. Filter the extract and re extract the marc left with 4 volumes of denatured spirit for another 1hours. Filter and combine the filtrate. Concentrate the denatured spirit extract under vacuum till the semisolid mass is obtained. Dilute with distilled water (1:50) and keep it overnight at 5^oc. Now filter the extract and discard the flocculent precipitate. Extract the filtrate with equal volume of ethyl acetate thrice. Concentrate the ethyl acetate extract till the semisolid mass is obtained. Dissolve the residue in 75 ml 0.1N hydrochloric acid and filter through sintered glass funnel (G-2) by vacuum filtration assembly. The filtrate was centrifuged at 2000 rpm for 20 minutes, the supernatant was collected in 100 ml volumetric flask and volume was made with 0.1N hydrochloric acid. The same procedure was performed for each batch of Astangavaleha (AS-I, AS-II and AS-III), marketed formulations (AU-I) and separately powdered Myrica esculenta, Inula racemosa and Pistacia integerrima solution (100 ml) of their tannic acid extract were prepared.

Calibration curve of tannic acid

Standard solutions of tannic acid (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ml) were pipetted into concentration range 2-20 mg/ml in a series of five 25 ml volumetric flask. The absorbance of the tannic acid was measured at 277.2 nm against ethanol. The results were shown in Figure 1.

Limit of detection and limit of quantitation

In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ), blank methanol was spotted six times. The signal to noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting known concentrations of tannic acid until the average responses were approximately three or ten times the standard deviation of the responses for six replicate determinations.

Table 2 Recovery study

S. No.	Amount of tannic acid (mg/ml)			RSD%	SE	Recovery%
	Sample	Added	Estimated
1 2 3	100 100 100	50 100 150	148.97±0.572 199.02±0.781 248.97±0.524	0.383 0.392 0.210	0.233 0.318 0.213	99.31 99.51 99.58
Mean				0.328	0.254	99.46

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

Precision and accuracy

The method was validated for precision and accuracy, by performing the recovery studies at three levels by adding known amount of tannic acid extract of Astangavaleha of which the tannic acid content have been estimated previously. The data were obtained and recovery was calculated (Table 2).

RESULTS AND DISCUSSION:

In order to obtain precision and accuracy, the recovery study was performed at three levels by adding known amount of tannic acid with pre -analyzed sample of tannic acid in Astangavaleha. The experiment was repeated six times at both level (Table 3.18) and result shows 99.31 %, 99.51 % and 99.58 % recovery of tannic acid at all the level with mean value 99.46 % which prove reproducibility of the result. The %relative standard deviation (% RSD) value was found to be 0.383, 0.392 and 0.310 with mean 0.328 at all the level while the standard error was 0.233, 0.318 and 0.213 with mean 0.254 respectively. From the data’s it was observed that the present method of spectrophotometric determination of tannic acid was simple, precise, accurate and suitable for routine analysis of tannic acid in Astangavaleha (Table 3).

Table 3 Validation parameters of tannic acid

S. No.	Parameters	Observations
01	Absorption maxima	277.2 nm
02	Beer’s law limit (µg/ml)	2-20
03	Correlation coefficient (r²)	0.991
04	Regression equation (y) Slope (a) Intercept (b)	Y = 0.043 X -0.009 0.043 0.009
05	LOD(µg/ml)	0.478
06	LOQ (µg/ml)	1.580
07	Precision (% R.S.D.) (n = 6) Repeatability Intraday Precision Interday precision	0.472 0.522 1.217

Table 4 Tannic acid content (% w/w) in Astangavaleha (Mean ± SD of 6 determinations)

S. No.	Formulations and crude drugs		Tannic acid Content % (w/w)	Standard error
1	Myrica esculenta		5.37 ± 0.812	0.331
2	Inula racemosa		3.22± 0.389	0.158
3	Pistacia integerrima		7.92 ± 0.761	0.310
6	Astangavaleha	AS-I	0.2452±0.652	0.266
7		AS –II	0.2357±0.718	0.293
8		AS–III	0.2140±0.537	0.219
9		AU-I	0.2256±0.614	0.250

Estimation of tannic acid in Astangavaleha & crude drugs

The appropriate aliquots from tannic acid extract of each batch of Astangavaleha (AS-I, AS-II and AS-III), marketed formulations (AU-I) and separately powdered Myrica esculenta, Inula racemosa and Pistacia integerrima separately were withdrawn in 10 ml volumetric flask. Absorbance for aliquots of each was noted at 277.2 nm. The corresponding concentration of tannic acid against respective absorbance value was determined using the tannic acid calibration curve. The concentration of tannic acid present in raw material is found to be 5.37% ± 0.812 % w/w in Myrica esculenta, 13.22± 0.389 % w/w in Inula racemosa and 7.92 ± 0.761% w/w in Pistacia integerrima and in three identical laboratory batches of Astangavaleha (AS-I, AS-II, AS-III) and one marketed preparations (AU-1) 0.2452±0.652 % w/w, 0.2357±0.718 % w/w, 0.2140±0.537 % w/w and 0.2256±0.614 % w/w respectively (Table 4)

REFERENCES:

1. Ayurvedic Formulary of India: Ministry of Health and Family Welfare. Govt. of India, New Delhi: Controller of Publications; 1999.

2. Ayurvedic Pharmacopoeia of India, part II, first edition, Govt. of India, Ministry of Health and Family Welfare. Dept. of health.

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6. Laura Bravo, Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions, Nutrition Reviews, 56 (11), 317–333, November 1998.

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Received on 06.03.2014 Accepted on 22.03.2014

Asian J. Pharm. Ana. 4(1): Jan.-Mar. 2014; Page 42-44

Method development of UV (3, 4, 9-12)

Table 2 Recovery study

Precision and accuracy

The method was validated for precision and accuracy, by performing the recovery studies at three levels by adding known amount of tannic acid extract of Astangavaleha of which the tannic acid content have been estimated previously. The data were obtained and recovery was calculated (Table 2).

Table 4 Tannic acid content (% w/w) in Astangavaleha (Mean ± SD of 6 determinations)

Method development of UV^{(3, 4, 9-12)}