INTRODUCTION:

Traditional medicine ‘Ayurveda’ since antiquity it has been in serving the mankind in defending against chronic conditions. Nisha amalaki vatti is a polyherbal formulation containing Curcuma longa and Phyllanthus emblica used as anti-diabetic agents marketed by Ayush, India. Amla (Emblica officinalis) and turmeric (Curcuma longa) have long been known in India and many other countries as important dietary sources in addition to their use in traditional medicine for wound healing, inflammation and stomach acidity. The active phytoconstitution of Curcuma longa contains (Curcumin, demethoxycurcumin, bisdemethoxycurcumin) and Emblica officinalis (ascorbic acid and tannins)^1-2. Several investigators have determined the efficacy of both amla and turmeric for their medicinal activities. It has now been well established that oxidative stress plays an important role in these disorders³. In India, 19 species of Curcuma longa and 29 species of Emblica officinalis were found. Standardization of these compounds was reported for individual phytoconstituents by UV spectroscopy, HPLC, HPTLC, tandem mass spectroscopy, electrochemical and gravimetric analysis methods^4-17.

Nisha amalaki vatti is a polyherbal tablet marketed by Ayush India contains Curcuma longa and Embilica officinalis used for the treatment of diabetes.

Standardization by UV, HPLC and HPTLC method has not been reported for the simultaneous analysis of curcuminoids and ascorbic acid in combined polyherbal formulation. Thus the present study was designed to develop new analytical method to estimate the polyherbal drugs containing curcuminoids and ascorbic acid by simultaneous equation method¹⁸ using UV spectrophotometer.

MATERIALS AND METHOD:

Chemical and reagents:

Curcumin and ascorbic acid were purchased for S.D fine chemicals and Merck chemicals (Mumbai, India). Curcuma longa and Amla (Emblica officinalis) raw materials were obtained from local market of Nalgonda, A.P. India; standard extracts were obtained from Chemiloids Vijayawada. Methanol analytical grade solvent was obtained from Merck (Mumbai, India), Polyherbal formulation Nisha amalaki vatti (Ayurveda tablet) obtained from Ayush (Hyderabad A.P, India).

Instrumentation:

Spectroscopic analysis was carried out using Elico SL-197 UV/Vis-Double beam spectrophotometer with Spectra treaties software. Spectrophotometer with spectral width 2nm, wavelength accuracy of 0.5nm and a pair of 10mm matching quartz cells was used to measure absorbance of the resulting solutions.

Preparation of Standard stock solution:

Standard stock solution (A) of curcuminoids and ascorbic acid was prepared by dissolving 100mg of each drug in 100mL volumetric flask separately by using methanol. From the stock solution final concentration (100 µg/mL) of the individual working standards were prepared with methanol.

Procedure:

Simultaneous Equation Methods:

Working standards solution was scanned in the range of 200 to 600 nm to determine the λ _maxof both drugs using methanol as a blank. The λ_max of curcuminoids and ascorbic acid were found to be 447nm and 247nm respectively. From the stock solution (A) 10ml was taken and diluted to 100ml with methanol (B), form this solution (B) 1ml, 2ml, 3ml, 4ml, and 5ml were taken and volume is made up to 10ml in volumetric flask to get a concentration of 10, 20, 30, 40 and 50µg/ml. The absorbance of the resulting solution was measured at 447 nm and 247 nm respectively and a calibration curve were plotted at these wavelength. The absorptivity coefficients of these two drugs were determined using calibration curve equation¹⁸. Two simultaneous equations were formed using these adsorpitivity coefficient values. A1= 1058 × Cx + 177 × Cx + 179 × Cy and A2 = 738 × Cy, where Cx and Cy are the concentration of curcuminoids and ascorbic acid respectively in g/mL in the sample solution. A1 and A2 are the absorbance of the mixture at 247 nm and 447 nm, respectively. The concentration of Cx and Cy can be obtained as Cx = {(A2 ×177)-(A1×738)}/-78480 and Cy= {-(A2 × 1058)}/-78480.

Procedure for Analysis of Ayurvedic Polyherbal tablets (Nisha Amalaki Vatti):

S. No	Ascorbic acid		Curcuminoids
S. No	Conc. (µg/mL)	Absorbance ( at 247 nm)	Conc. (µg/mL)	Absorbance ( at 447 nm)
1	10	0.349	10	0.303
2	20	0.698	20	0.626
3	30	1.004	30	0.939
4	40	1.396	40	1.252
5	50	1.745	50	1.565

Twenty tablets (Nisha Amalaki vatti) were weighed accurately and the average weight was determined and then grounded to fine powder. Quantity equivalents to 0.1 g were transferred to 100 mL of volumetric flask and volume is adjusted with 100ml with methanol. The solution was centrifuged for five minutes at 3000 rpm. Centrifugation was found to be faster and more effective than filtration. Centrifugation forms a cake of excipients at the bottom of the test tube, which is not disturbed while drawing out the supernatant solution. This supernatant solution was pipette out and diluted appropriately with methanol to obtain the concentration of 10 µg/mL concentration of curcuminoids and ascorbic acid. For forming simultaneous equation, the solution was scanned from 200-600 nm. The absorptivity value at 247 nm and 447nm for both the drug were determined by checking the absorbance values of over a concentration range 10-50 µg/mL for working standards shown in table.2.

Validation of proposed method:

The proposed method was validated by studying several parameters such as accuracy, precision, and linearity.

Precision:

The repeatability of the sample application was calculated by repeating the assay six times for each concentration. Intraday precision were performed by analyzing sample solution on the same days on the different days at specific time intervals. The results of the same are show in table 3.

Accuracy:

To check the accuracy of the proposed method, recovery studies were carried out at 80,100 and 120% of the test concentration as per ICH guidelines. The recovery studies were performed three times at each level. Results of the formulation analysis recovery studies along with its statistical validation data are given in table 3.

Linearity:

The linearity of the measurement was evaluated by analyzing different concentration of the solution of curcuminoids and ascorbic acid. For the simultaneous equation method the Beer-Lambert’s concentration ranges was found to be from 10-50 µg/mL for curcuminoids and ascorbic acid respectively. The standard calibration curve and standard table for curcuminoids and ascorbic acid is given in fig.1 and table.1 respectively. Hence the proposed method can be used for the routinely employed for the estimation of curcuminoids and ascorbic acid in bulk and pharmaceutical Ayurvedic dosage forms.

RESULTS AND CONCLUSION:

The proposed method was found to be simple, sensitive, accurate, precise, economical and rapid for the routine simultaneous estimation of these two phytoconstituents in a combined dosage form. The value of the standard deviation and coefficient of variation were satisfactory shown in table 1, 2 and 3. In the simultaneous equation method tow wavelength of respective absorbance maxima i.e. 247 nm for ascorbic acid and 447 nm for curcuminoids were used for the analysis of the phytoconstitution in the standard and tablet.

Table: 1 Optical characteristics and Validation date of Curcuminoids and Ascorbic acid

Parameters	Ascorbic acid	Curcuminoids
Working λ_max (in Methanol)	247 nm	447 nm
Beer-Lamberts Law Range	10-50 µg/mL	10-50 µg/mL
Molar absorbitivity	1.94 × 10³	7.80 × 10³
Regression Values:
Slope	0.552152425	0.49806877
Regression	0.999393333	0.999979844
Mean	1.0384	0.937
Precision:
Interday (%CV)	0.1498	0.1267
Interday (%CV)	0.1078	0.1067

Table: 2 Regression Analysis of Calibration curves

Table 3: Analysis Date of formulation, Statistical validation and recovery studies

Drugs

Label claim (mg)

Amount

Found (mg)

Label claim (%)

Standard

Deviation

Coefficient of variation

Standard error

Amount Added

At (%) mg

Recovery

Curcuminoids

250

249.70

99.70

31.74

0.99939

0.2987

100

120

400

500

600

99.81

100.35

99.89

Ascorbic acid

250

247.56

98.62

28.618

0.99997

0.1895

100

120

400

500

600

98.98

99.92

100.45

Fig: 1 Showing the calibration curves and the linearity for curcuminoids and ascorbic acid

Fig: 2 Over lapping spectra showing curcuminoids and ascorbic acid

CONCLUSION:

The method described in this paper for the simultaneous estimation of curcuminoids and ascorbic acid are found to be simple, sensitive, accurate ,precise, rapid and economical. Hence the method could be used for the routine analysis of this phytoconstitution in their combined bulk and ayurvedic pharmaceutical dosage.

ACKNOWLEDGMENT:

The authors are grateful to the Director Ayush India; Chemiloids Vijayawada and Nalanda college of Pharmacy Management for providing need full supports.

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Received on 13.03.2010 Modified on 04.04.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 678-681