INTRODUCTION:

The most of the Traditional formulation are lacking in their defined quality control parameters and method of its evaluation. The World Health Organization (WHO) has emphasized the need to ensure the quality of medicinal plant products by using modern controlled technique and applying suitable standards. [1, 2]

Chooranam are fine powder of crude drugs. Nilaavaarai Chooranam is an important Siddha formulation, is official in formulary of Siddha Medicine is combination of five reputed herbs, comprised of the Cassia angustifolia (Nilaavaarai) ,Zingiber officinale (Chukku), Trichyspermum ammi (Omum) and Embelia ribes (Vaividangam). The formulation is dispensed for the gaseous distension of stomach, hiccup, vomiting, constipation and biliousness. It is also useful as mild laxative [3].The present paper is an effort to develop the quality control parameter of Nilaavaarai Chooranam by spectrophotometric determination using 6- Gingerol as a internal standard. [6]-Gingerol (5-hydroxy-1-(4′hydroxy-3′-methoxyphenyl) - 3-decanone) is the biomarker constituent of Zingiber officinale.

The present study is an attempt to develop the fingerprint method for Nilaavaarai Chooranam by spectrophotometric determination using 6- Gingerol as a standard, which is as an important content in formulation. The UV spectrophotometric analysis which is a simple, precise, and accurate method that can be considered as one of the quality control method for routine analysis.

MATERIALS AND METHODS:

Procurement of Crude Drug

Crude drugs were procured from local market and identification was confirmed by macroscopic and microscopic characters and compared with standard Pharmacopoeial monograph. [4-7]

Preparation of the Formulation

Nilaavaarai Chooranam, three laboratory batches (named NC-I, NC-II and NC-III) were prepared in laboratory according to reported method of formulary of Siddha Medicine. The available one commercially brand M-1 of Nilaavaarai Chooranam was procured from local Pharmacy.[3]

Chemicals

Reference 6-Gingerol was purchased from Sigma–Aldrich (Germany). All solvents used were of analytical grade and procured from Merck (Mumbai, India).

Preparation of Standard Solution of 6-Gingerol

Accurately weighed 6-Gingerol (10 mg) was transferred in 100 ml volumetric flask and dissolved in and diluted to 100 ml with methanol. The final solution contained 100 mg of the Gingerol per ml of the solution.

Preparation of Calibration Curve for 6- Gingerol

Standard solutions of 6-Gingerol were pipetted into concentration range 10-60 mg/ml in a series of five 25 ml volumetric flask. The absorbance of the 6-Gingerol was measured at 282.5 nm against ethanol by UV-Vis spectrophotometer. The results are shown in Table 1, Figure 1.

Table 1: Calibration data of 6- Gingerol

Conc.(µg/ml)	Absorbance
10	0.247
20	0.509
30	0.869
40	1.132
50	1.501
60	1.858

Table 2: Validation parameters of 6-Gingerol

Parameters	Observations
Absorption maxima	282.5 nm
Beer’s law limit (µg/ml)	10-60
Correlation coefficient (r²)	0.997
Regression equation (y^*) Slope (a) Intercept (b)	Y = 0.32 X -0.110 0.032 0.110
Recovery Studies a)Accuracy( %RSD) b)SE c)Recovery%	0.096 0.108 99.83

Recovery

Standards of 6-Gingerol with the known amounts in solutions were spiked to the Nilaavaarai Chooranam solution of which the contents of 6-Gingerol had been determined before the addition of the standard chemical. Then, 6-Gingerol marker compound in Nilaavaarai Chooranam sample solutions were extracted, processed and quantified in accordance with the established procedures, and finally the recovery rates were calculated. Same procedures were applied for the three different marketed formulations.

Estimation of 6-Gingerol in the Formulations

6-Gingerol was isolated by the methods of Thresh and Garnett and Grier.1 gm of powdered ginger was exhausted with 95% alcohol and the percolate concentrated to a volume of one litre. An equal volume of water was added which caused the precipitation of a large part of the fats and resins, leaving most of the 6-Gingerol in solution. The solution was shaken out with petroleum ether and evaporated and the residue extracted by repeatedly boiling out with petroleum ether from which the 6-Gingerol was removed by shaking with 60% alcohol. On evaporating the alcohol 6-Gingerol was obtained as very pungent, yellow oil. The obtained extract was analyzed. The same procedure was applied for the estimation of 6-Gingerol in the laboratory and marketed formulations. The analysis was repeated six times. The possibility of interference from other components of extract in the analysis was studied. [8]

RESULT AND DISCUSSION:

The developed method was found to be reliable, accurate, precise and sensitive. The method involves absorbance measurement at 282.5 nm for 6-Gingerol corresponding to the absorption maxima of the herbal formulation Nilaavaarai Chooranam. The optical characteristic shows that 6-Gingerol obeys Beer Lambert’s law in concentration range 10-60 mg/ml at λ-max 282.5 nm. The correlation coefficient (r²), Regression equation, Precision and Accuracy were calculated for the spectrophometric method and results are summarized in table-2.

The r² value 0.997 indicates the good linearity between the concentration and absorbance. The estimation of 6-Gingerol content of Nilaavaarai Chooranam (three laboratory and one marketed samples) and powder of crude drug Ginger (Zingiber officinale) was carried out separately. The concentration of 6-Gingerol present in raw material was found to be 1.162 ± 0.0526 %w/w in Zingiber officinale. Content of 6-Gingerol in different batches of Nilaavaarai Chooranam (laboratories and marketed formulation) was found to be 0.1172 ± 0.0318%, 0.1153± 0.0692%, 0.1161± 0.0019 and 0.1016± 0.0234% w/w respectively for NC-I, NC-II, NC-III and M-1 respectively. Results are summarized in Table-3.

In order to obtain precision and accuracy the recovery study were performed by adding known amount of 6-Gingerol with pre-analyzed sample of 6-Gingerol in Nilaavaarai Chooranam. The result shows 99.83 % recovery of 6-Gingerol which shows reproducibility of the result. Results are summarized in Table-4.

Table 3: Estimation of 6-Gingerol content (% (w/w) in Nilaavaarai Chooranam

Formulations		6-Gingerol Content %(w/w)	Standard Deviation
Zingiber officinale		1.162	0.0526
Nilaavaarai Chooranam	NC-I	0.1172	0.0318
	NC –II	0.1153	0.0692
	NC-III	0.1161	0.0019
	M-1	0.1016	0.0234

Table 4: Compilation of data of recovery studies of 6-Gingerol in Nilaavaarai Chooranam

S No.	Conc. of sample (µg/ml)	Added Conc.(µg/ml)	Found Conc. (µg/ml)	Recovery %	% RSD	SE
1	10	5	14.987	99.91	0.06	0.05
2	15	5	19.93	99.65	0.196	0.23
3	20	15	24.982	99.93	0.032	0.05
Mean				99.83	0.096	0.108

Figure 1: Calibration curve of 6- Gingerol

CONCLUSION:

The present work was carried out for preparation, standardization and fingerprint development of Nilaavaarai Chooranam. The results obtained were equally compatible with that of marketed formulation. The proposed method of quantitative determination of 6-Gingerol in the raw plant material, extract, and marketed formulations and characterized by high sensitivity, relatively small error, and good reproducibility.

So far since no attempt has been made to evaluate the polyherbal formulation, the present work is a small step towards the development of quality control methods for polyherbal preparation. This will also help to produce uniform standard products, which will restore the faith in Siddha system.

REFERENCES:

1. World Health Organization, Quality Control Methods For Medicinal Plants Materials, Geneva, 1-15, (1998).

2. Jain T., and Dashora K., Spectrophotometric fingerprinting method for Unani formulation Hab-e-Azarakhi, Asian J. Pharm. Tech. 2012; 2(1),01-03

3. Formulary of Siddha Medicine. 4th ed., published by indian medical practitioners cooperative pharmacy and store ltd (IMPCOPS), Chennai, 1993, 45.

4. Indian Herbal Pharmacopoeia, Volume II, Regional Research Laboratory Jammu, Indian drug Manufacturing Association Mumbai, 93-101, (1999).

5. Quality Standards Of Indian Medicinal Plants, Volume I, Indian Council of Medicinal Research, New Delhi, 168-172,(2003).

6. Mukherjee, P., Pharmacological Screening of Herbal Drug, Quality Control of Herbal Drug: An Approach to Evaluation of Botanicals; Eastern Publishers (Business Horizontal Ltd.), New Delhi, 755-760, (2002).

7. The Ayurvedic Pharmacopoeia of India, Part I; vol. II, edn. I^st, Govt. of India, Ministry of health and Family Welfare, New Delhi, 133, (1999).

8. Tzucker R., Jordan CB. A study of the assay of ginger. J Am Pharm Assoc 1940;29(6):265-269.

Received on 23.12.2011 Modified on 25.01.2012

Asian J. Research Chem. 5(3): March 2012; Page 317-319

INTRODUCTION:

Absorbance

Nilaavaarai Chooranam

Table 4: Compilation of data of recovery studies of 6-Gingerol in Nilaavaarai Chooranam

% RSD

SE