Jadhao M.P*, Bhusari KP, Shrikhande BK, Ghormade JM and Shrikhande VN
1Vidyabharati College of Pharmacy, Camp, Amravati – 444601.(M.S.)
2.Sharad Pawar College of Pharmacy, Wanadongari, Nagpur – 441110.(M.S.)
*Corresponding Author E-mail: monikajadhao2006@yahoo.co.in
ABSTRACT
A simple and reproducible high performance thin layer chromatography method was developed and validated for the estimation of kutkin in Picrorrhiza kurroa . The stationary phase used was precoated silica gel 60F254. The solvent system of chloroform: methanol (8.5:1.8, v/v) was used as mobile phase. the detection of spot was carried out at 258 nm. the method was validated in terms of linearity, accuracy, precision and specificity. The linearity was observed in the range of 480-1440 ng. The Kutkin content of 246.90 µg per 100mg was observed in the sample. The average percentage recovery value of 98.55% was obtained. The proposed method being precise and sensitive can be used for detection, monitoring and quantification of Kutkin in Picrorrhiza kurroa.
KEYWORDS: Validation., Picrorrhiza kurroa, Kutkin, HPTLC
INTRODUCTION:
Standardization of ayurvedic drugs and plant material is the need of the day. The pharmacopoeias containing monographs on plant materials describe only the physicochemical parameters. Hence, modern methods describing the identification and quantification of active compound may be useful for proper standardization of the herb.1 Picrorrhiza kurroa (Scrophulariaceae), an iridoid bitter glycoside which contain C9 monoterpine glycosides. Dried rhizome have been used for centuries, in ayurvedic preparations for the treatment of jaundice, antiperiodic, stomachic, antioxidant, antidiabetic, antiasthmatic, antihealing treatment. Among the complex mixture of biologically active compounds in the plant, kutkin can be used as an analytical marker to determine the quality of plant material of different sources.2,3
During crop improvement and drug analysis ,a sensitive and accurate analytical method is required for the quantitation of important compound like Kutkin, which is present at plant. Although few methods such as gravimetric, colorimetric, spectrophotometric methods have been reported for the quantitative estimation of Kutkin, many of these procedures are time consuming, not very precise and required multiple step extraction and purification.4 The present HPTLC method offers a sensitive, accurate, and reliable method for routine detection and quantification of Kutkin present in Picrorrhiza kurroa extract.
MATERIALS AND METHOD:
The std. kutkin (1 mg) was accurately weighed. It was dissolved in methanol (2-3 ml) and sonificated for 5 min. The volume was made with methanol (5 ml) to produce a final conc. (500 ng/µl).4 From this soln, the soln (3 ml) was further diluted to 5 ml (final conc. of 120 ng/µl).This reference standard was used as stock solution.
Standardized kutki powder was obtained from an herbal powder manufacturer. The kutki powder (0.5 g) was weighed accurately and dissolved in methanol (7 ml). The soln was sonicated for 5 min and filtered though Whatman filter paper (No.01). The volume was made up with methanol (10 ml). The std. soln (120 ng/µl) and the sample soln (50 µg/µl) were applied. Precoated HPTLC silica gel plates were prewashed with methanol in order to minimize noise ratio.6
The HPTLC plates were developed using a CAMAG twin through glass tank. A solvent combination of chloroform: methanol (8.5:1.8) was found to give best TLC resolution. The HPTLC runs were made in the laboratory conditions at 25± 50% relative humidity. After development the plates were withdrawn and dried and spots were visualized in UV light at absorbance of 258 nm.6,7
The methanol extract of powder and standard solution were applied in concentration of 6µl and 1µl,2µl,3µl,4µl. on precoated TLC plates with the use of Linomat IV applicator as a narrow band of 80 mm of length. Likewise three such plates were developed under chamber saturation condition. After air drying of the solvent,the plates were scanned at 258 nm. The amount of Kutkin present was determined using the calibration curve plotted between concentration and area of standard.8
TABLE 1: Statistical Data of Recovery Study of Kutkin from Picrorrhiza Kurroa
|
Track No. |
Sample applied (µg) [A] |
kutkin present in A (ng) [B] |
Std added to A (ng) [C] |
Total Kutkin applied (ng) B+C [D] |
Total kutkin recovered (ng) [E] |
% Recovery E/D x 100 [F] |
|
1 |
150 |
485.12 |
200 |
685.12 |
671.51 |
98.05 |
|
2 |
150 |
485.12 |
250 |
735.12 |
722.12 |
98.23 |
|
3 |
150 |
485.12 |
300 |
785.12 |
775.95 |
98.83 |
|
Mean |
98.13 |
|||||
|
S.D. |
0.9447 |
|||||
|
R.S.D. |
1.080 |
|||||
Fig 1- Calibration curves of std Kutkin
Fig 2:A typical HPTLC Chromatogram of kutkin .
HPTLC chromatogram represents peak of kutkin at 0.76
The method was validated and recovery studies were performed by standard addition method. Accurately measured amount of std. kutkin was added on the sample tracks on TLC plate in subsequently increasing conc. of 50 ng/spot. The chromatogram was developed and evaluated in scanner at 258 nm. The samples were refluxed and analysed separately as per procedure mentioned above.9
TABLE 2: Evaluated parameters
|
Parameters |
Values |
|
Linearity range (ng/ spot) standard deviation recovery (%) Rf Precision Reapetability of application (n=5) |
480-1440 1.6 98.05 to 98.83 0.76
2.645 |
The contents of kutkin were quantified and percentage recoveries were calculated (Table-1) using the proposed HPTLC method. The Rf of kutkin was found about 0.76.The content of Kutkin was found to be 246.90 µg per 100mg in test sample. The chromatograms of standard and test sample are shown in figs.2 and 3, respectively.
RESULTS AND DISCUSSION:
The calibration curve was linear in the range of 480-1440 ng/spot. for kutkin. Further the standard deviation 1.6 indicates good linearity between concentration and area. The method allows reliable quantification of kutkin and previous good resolution from other constituents of Picrorrhiza kurroa to ascertain the purity of peak.
Figure 3: Chromatogram of kutki powder containing kutkin
To ascertain the purity of peak in test sample, its in situ reflectance spectrum was compared with that of standard. The recovery values 98.05 to 98.83%,which were obtained indicate excellent reliability and reproducibility of the proposed method.
CONCLUSION:
It can be concluded that the proposed HPTLC method is rapid, simple and accurate for quantitative monitoring of kutkin in Picrorrhiza kurroa.
ACKNOWLEDGEMENTS:
Authors express their sincere gratitude to Mr. B. K. Shrikhande, Director, Baidyanath Research Foundation Ltd., Nagpur, for valuable guidance and providing valuable support to carry out research work.
REFERENCES:
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Received on 16.05.2008 Modified on 19.07.2009
Accepted on 12.08.2009 © RJPT All right reserved
Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 768-770