1. INTRODUCTION:

In recent years, there is a spurt in the interest regarding survival of Ayurvedic forms of medication. In the global perspective, there is a shift towards the use of medicine of herbal origin, as the dangers and the shortcomings of modern medicine have started getting more apparent. Majority of Ayurvedic formulations are prepared from the plants. Unfortunately, the Ayurvedic Pharmacopoeia and the formulary have been exempted from the standards attained by present day modern medicine which is continuously undergoing metamorphic changes and improvements in the standards of purity, safety and efficacy. Thus, maintaining the quality of Ayurvedic medications becomes the sole responsibility of the manufacturer⁷.

The Food and Drug Administration in India does not have the same stringent rules for quality of Ayurvedic preparations as they have for the quality of raw materials and finished products of allopathic drugs.

Though it is difficult to give a precise method of standardization, with the use of the modern analytical techniques available, it should be still possible to evolve some simple methods of quality control and identification of crude drugs. Traditional pharmacognostic work on indigenous medical plants is available but generally it is out of reach of Ayurvedic drug manufacturers. Certain efforts in this direction will assure the consumers the quality of the product and thus its efficacy⁸.

There is a great demand for herbal medicines in the developed as well as developing countries because of their wide biological activities, higher safety margin than the synthetic drugs and lesser costs. Since herbal medicines are prepared from materials of plant origin they are prone to contamination, deterioration and variation in composition. This gives rise to inferior quality of herbal products with little or no therapeutic efficacy. Most often the desired biological response is due to not one but a mixture of bioactive constituents and the relative proportion of active constituents can vary from plant to plant of the same species and also in different plant parts.

The World Heath Organization has recognized the importance of traditional medicines and has created strategies, guidelines and standards for botanical medicines. Proven agro-industrial technologies need to be applied to the cultivation and processing of medicinal plants and the manufacture of herbal medicines.

It is necessary to develop methods for rapid precise and accurate identification and estimation of active constituents in order to bring out consistency of important constituents in the formulations. This is especially true for export oriented products as the western countries have stringent quality control parameters. Most of the tests described in ancient texts however are based on observation and are subjective. Hence, it is essential to find out modern correlates of Ayurvedic quality control tests.

With the use of modern methods of analysis such as TLC, HPTLC, HPLC etc. it is possible to set up certain standards for analysis of a particular constituent from the Ayurvedic formulations.

One of the important methods of standardization of herbal drugs is isolation of Marker compounds from plants and standardization of herbal formulations with isolated markers.

2. Experimental:

2.1 Materials:

Standard catechin was supplied as a gift sample by UICT, Mumbai. All the solvents used for the analytical work were of Analytical grade (AR). The TLC and HPTLC studies were done using precoated TLC plates of Silica gel 60 GF₂₅₄(Merck). HPTLC studies were carried out using CAMAG LINOMAT IV applicator and CAMAG SCANNER III with WINCATS III software. All chemicals and reagents used are of HPLC and AR grade supplied by Merk Ltd.

3. Methodology:

3.1 Preparation of Acacia nilotica gel:

Bark of Acacia nilotica was procured frm local market Mumbai and authenticated by Piramal Life Sciences, Mumbai. Bark was dried and powdered for further processings. Dried, powdered bark of Acacia nilotica Linn. was extracted with acetone for 18 hours using Soxlet extraction apparatus. The extract was concentrated under vacuum and the color, nature and percent extractive values (w/w) with respect to dry powder were determined.

Acetone extract at a concentration of 10% w/w was triturated to form a fine non- gritty powder and uniformly dispersed in glycerine. This was then incorporated into an aqueous gel base using Carbopol as gelling agent at 0.5, 1 and 1.5% w/w concentrations. 10% NaOH was used as neutralizing agent.

The gel was evaluated for parameters like organoleptic properties, pH, spreadability, Stability studies were conducted at refrigeration and room temperatures. Accelerated stability studies were done as per ICH guidelines. Samples of the gel were evaluated for parameters like color, pH, extract content, spreadability and antimicrobial activity

3.2 HPTLC method development:

HPTLC method was developed for catechin. Standard catechin was dissolved in Methanol .Sttandard solution of Concentration (0.5 µg/μl) was prepared and spotted on TLC plate using LINOMAT IV applicator and CAMAG SCANNER III. Samples were spotted on precoated plates and the plates were the scanned at 278 nm which was the reported λmax for Catechin.The mobile phase used was toluene: ethyl acetate: methanol: formic acid (6: 6:1:0.1) as amobile phase.

Densitometric conditions:

Stationary phase : Silica gel 60 GF254 (Merck)

Saturation time : 30 min.

Development time :15 min.

Band width : 7 mm

Wavelength : 278 nm

Lamp : Deuterium

3.3 HPTLC method validation:

The developed method was validated as per the ICH guidelines. The following parameters are validated.

3.3.1 Accuracy:

Accuracy may often be expressed as percent recovery by the assay of known, added amounts of analyte. It is a measure of the exactness of the analytical method.

The accuracy of developed HPTLC method was determined by applying that method to samples to which known amounts of analyte have been added. The accuracy is then calculated from the test results as the percentage of analyte recovered by the assay. The known amount of standard catechin was added to the preanalyzed samples and was subjected to HPTLC study. At each level of recovery three determination were carried out

3.3.2 Precision:

The method was validated for precision by repeatability: Repeatability is obtained when the analysis is carried out in one laboratory by one operator using one piece of equipment over a relatively short time span. At least

· 5 or 6 determinations of

· three different matrices at

· two or three different concentrations

Should be done and the relative standard deviation calculated. The acceptance criteria for precision depend very much on the type of analysis. While for compound analysis in pharmaceutical quality control precision of better than 1 % RSD is easily achieved

The concentration selected for repeatability studies were 0.1 μg, 0.3 μg and 0.5 μg of Catechin.

3.3.3 Linearity and Range:

The linearity of an analytical method is its ability to elicit test results that are directly, or by a well defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. Linearity is usually expressed in terms of the variance around the slope of the regression line calculated according to an established mathematical relationship from test results obtained by the analysis of samples with varying concentrations of analyte.

Standards: The coefficient of correlation should be more than 0.995

3.3.4 Limit of detection :

The limit of detection is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. The limit of detection is usually expressed as the concentration of analyte in the sample.

In chromatography the detection limit is the injected amount that results in a peak with a height at least twice or three times as high as the baseline noise level. The limit of detection and limit of quantitation were determined based on calibration curves.

3.3.5 Limit of Quantitation:

Limit of Quantitation is a parameter of quantitative assay for low levels of compounds in ample matrices, such as impurities in bulk drug substances and degradation products in finished pharmaceuticals. It is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. The limit of Quantitation is expressed as the concentration of analyte in the sample.

The limit of Quantitation is the minimum injected amount that gives precise measurements, in chromatography typically requiring peak heights 20 times higher than baseline noise or 3 times the detection limit.

3.3.6 Robustmess:

The robustness of the method was studied and during this small changes were made in composition of mobile phase and effects of this were observed on the chromatographic behavior of the sample. The effects of following parameters were observed, Rf value, peak area, the various mobile phases used were,

1. Toluene: ethyl acetate: methanol: formic acid (6: 6:0.5:1)

2. Toluene: ethyl acetate: methanol: formic acid (5: 7:1:1)

3. Toluene: ethyl acetate: methanol: formic acid (7: 5:0.5:1)

3.4 Standardization of Acacia nilotica gel:

The methanol extract of Acacia nilotica gel was prepared with concentration of 100 mg/ml.Samples were spotted on precoated plates and the plates were the scanned at 278 nm which was the reported λmax for Catechin. The mobile phase used was toluene: ethyl acetate: methanol: formic acid (6: 6:1:0.1) as a mobile phase.

Densitometric conditions:

Stationary phase : Silica gel 60 GF₂₅₄(Merck)

Mobile phase : Toluene: ethyl acetate: methanol: formic acid (6: 6:1:0.1)

Saturation time : 30 min

Development time : 15 min

Band width : 7 mm

Wavelength : 278 nm

Lamp : Deuterium

4. Result and Discussion:

4.1 HPTLC method development:

The HPTLC method was successfully developed for Catechin. Catechin was found to be linear in the range 500-3000ng. The Coefficeint of Correlation was found to be 0.998

Figure No. 1 Calibration curve for Catechin

Figure 2: HPTLC fingerprint for standard Catechin at 278 nm

Figure 3: HPTLC fingerprint of methanolic extract of 100 μg of Acacia nilotica gel.

4.2 Standardization of Acacia nilotica gel :

With the developed HPTLC method Acacia nilotica gel was analysed. It contains mainly Catechin as major constituent. The % content of catechin in Acacia nilotica gel was determined from the standard plot of Catechin. The % content of catechin in Acacia nilotica gel was calculated from equation.

Table No: 1 Linearity studies of Catechin

Sr. No.	Concentration of catechin	Area Under Curve
1.	0.5 μg	1407.1
2.	1 μg	2079.2
3.	1.5 μg	2617.1
4.	2 μg	3350.5
5.	2.5 μg	4047.1
6.	3 μg	4644.5
7.	100μg of Methanolic extract of gel	1733.7

4.3 Method Validation:

1.From linearity studies it was found that Catechin showed good coefficient of correlation in the range 50- 300 ng/ band (r²= 0.998). The developed method was validated as per the ICH guidelines.

2. The accuracy of method was defined by the % recovery studies. Table No. 2 showed the result of % recovery studies. From this the method was found to be accurate.

Table No: 2 Results of Accuracy

Sample Injected	Obtained AUC	Expected AUC	Percent recovery (%)
300 μg of methanol extract of gel	1402.26 (A)
0.5 μg of Catechin	1201.2 (B)
300 μg of methanol extract of gel + 0.5 μg of Catechin	2578.1	2603.46 (A+B)	99.02 %
1 μg of Catechin	2104.6 (C)
300 μg of methanol extract of gel + 1 μg of Catechin	3528.1	3506.86 (A+C)	100.60%
1.5 μg of Caffeine	3152.8 (D)
300 μg of methanol extract of gel +1.5 μg of Catechin	4600.2	4555.06 (A+D)	100.99%

3.The low % RSD value indicate the suitability of the method.% RSD was found to be less than 2 % from repeatability studies, which proves the method was precise.

Table 3: Repeatability studies of Catechin

Sr. No.	Peak area for 0.5 μg	Peak area 1.5 μg	Peak area for 3 μg
1.	1343.3	2426.7	4463.8
2.	1340.6	2430.7	4492.3
3.	1330.5	2467.9	4497.9
4.	1342.6	2430.0	4507.2
5.	1350.5	2456.2	4536.5
Mean	1337.8	2442.3	4499.54
S.D.	7.198	18.559	26.254
% RSD	0.536	0.759	0.583

4. The limit of detection for Catechin was found to be 20 ngm and limit of quantitaion for catechin was found to be 50 ngm

5. The method was found to be robust and the results were given in the table no.4

Table No: 4 Result of Robustness

% Change in mobile phase	Rf	Peak area
60:60:5:10	0.28	3010.5
58:62:6:9	0.27	3125.1
59.5:60.1:9:10.1	0.28	3097.2

6. The developed method was used to determine the concentration of Catechin in Gel. In the % content of catechin in the Gel was found to be 7.6%

Table No:5 Results of Method validation

Sr. No.	Parameters For Method Validation	Result Values
1.	Precision (Repeatability)	0.0564
2.	Accuracy (% Recovery)	100.3 %
3.	Coefficient Of Correlation	0.998
4.	Limit of Detection	20 ngm
5.	Limit of Quantitation	50ngm
6.	Robustness	No Significant change in peak area and Rf

5. CONCLUSION:

The new HPTLC method was developed for Catechin and proposed method was validated as per ICH guidelines. The % RSD and standard deviation calculated for the method were low, indicating the high degree of precision of the method. The result of the recovery studies indicating the high degree of accuracy of the developed method. Hence, it can be concluded that the developed method can be used for the estimation of Catechin from herbal formulations.

6. REFERENCES:

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Received on 10.05.2011 Accepted on 22.05.2011

Res. J. Topical and Cosmetic Sci. 2(1): Jan. –June 2011 page 25-29