Research Article ISSN: 0974-6943

Nadagouda Smitha G. et al. / Journal of Pharmacy Research 2010, 3(5),1107-1109

Available online through www.jpronline.info Validated HPTLC method for mangiferin in Salacia chinensis
Nadagouda Smitha G. 1 *, Karigar Asif A2 ., Joshi V. G. 2, Sikarwar Mukesh S.3 , 1 Sonia College of Pharmacy, Dharwad, Karnataka, India. 2 Maratha Mandals College of Pharmacy, Belgaum, Karnataka 3 K.L.E. S College of Pharmacy Ankola, Karnataka-India.

Received on: 20-12-2009; Revised on: 16-03-2010; Accepted on:12-04-2010 ABSTRACT

A high performance thin layer chromatographic (HPTLC) method was developed and validated as per ICH (International conferences on harmonization) guidelines for simultaneous quantification of mangiferin in Salacia chinensis roots. For achieving good separation mobile phase of ethyl acetate methanol (40:60) on precoated silica gel 60 F254 HPTLC plates were used. The densitometric quantification of mangiferin was carried out at ?= 254 nm in reflection absorption mode after spraying with acetic anhydride: sulphuric acid: ethanol reagent. The calibration curve were linear with good correlation coefficient (0.998 0.999). The method was found to be reproducible for quantitative analysis of mangiferin in Salacia chinensis root collected from different locations and will serve as a quality control indicator to monitor the commercial production of mangiferin and its allied molecules.

Key words: Mangiferin, validation, (ICH) International Conference on Harmonization, Salacia chinensis, High performance thin layer chromatography. INTRODUCTION Today we find a renewed interest in traditional medicines. Human beings are exposed to these compounds through environmental exposure, consumption of contaminated food or during exposure to chemical substances in the polluted environment. In addition, human beings consume a lot of synthetic drugs during diseased conditions, which are essential to treat the diseases but have a variety of side effects and produce a variety of toxic manifestations [1]. Mangiferin is a xanthone glucoside and active phytochemical principle which is present in Salacia species. Salacia chinensis is a member of Hippocrateaceae family. The roots are used in indigenous system of medicine for the treatment of Diabetes, amenorrhea, dysmenorrhea, veneral diseases and shows astringent, abortificient properties. Mangeferin is called as Cglucosyl xanthone and 2--D- glucopyranosyl 1, 3, 6, 7- tetrahydroxy xanthone. Mangiferin is recommended to treat immunodeficiency diseases such as diabetes, hepatitis, arthritis, cardiac and mental disorders [2-3]. by the Botanist Prof. G. S. Naik, Department of Botany, G. C. Science and Art College, Ankola. A voucher herbarium specimen number GCSAC/ SC/03 was also preserved in the same college. The HPTLC plates (20 cm 10 cm) were used without any pretreatment. All chemicals and solvents were of analytical grade. METHODS Extraction and isolation of mangiferin: The roots of Salacia chinensis were washed thoroughly with water, shade dried, cut into the small pieces and were crushed to moderately coarse powder. It was extracted using 95% of the methanol in Soxhlet apparatus for 24 hours. The extract was concentrated by using rotatory evaporator at 45 50 0 C under reduced pressure. The methanolic root extract yield was about 23.5%.

A portion of the crude methanolic root extract was subjected HPTLC is a method of choice for quantification due to its high to column chromatography over silica gel with chloroform gradient elusample through output at low operating cost and short analysis time. Present study aims to develop validated HPTLC method for quantifica- tion using ethyl acetate in methanol 95:515:85 respectively for the total amount of 17 elutes collected in combine into bases of their thin layer tion of mangiferin in roots. chromatography similarities. The mangiferin yield was eluted on the fraction ratio of 40:60 (ethyl acetate: methanol) which was confirmed by EXPERIMENTAL TLC analysis. The concentrated fraction was subjected to recolumn chromatography and its purity was confirmed. The yield of the mangiferin Materials was found to be 7.8%. The authentic standard sample of mangiferin was purchased from sigma Aldrich Company and compared with the sample. Root bark of Salacia chinensis were collected in and around The isolated mangiferin closely resembled with the authentic purity which local forest area of Sirsi in Western Ghats, Karnataka and authenticated was found to be 99.4% w/w [4]. *Corresponding author.
Nadagouda Smitha G Asst. Professor, Sonia College of Pharmacy, S. R. Nagar, Dharwad-02, Karnataka, India Tel.: + 91-9341948625 E-mail:smithasanglad@ymail.com

HPTLC procedure HPTLC is a versatile separation technique and is official in most of the pharmacopoeias for determining content uniformity, pu1107-1109

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Nadagouda Smitha G. et al. / Journal of Pharmacy Research 2010, 3(5),1107-1109 rity, assay value, dissolution, drug-drug interaction, and bioavailability of the drugs. A camag HPTLC system equipped with an automatic TLC chamber, automated developing chamber, TLC scanner and integrated software win CATS version 1.4.2. was used for analysis. HPTLC was performed on precoated TLC layer Silica gel 60F254 (20x20cm) plates of 0.20mm layer thickness. Chromatography was carried in ADC which was pre-saturated with 20ml mobile phase ethyl acetate methanol (40:60) for 30 minutes at room temperature (2520C) and 505% relative humidity. The samples and standards were applied on the plates as 6mm wide bands with an automatic TLC sampler (ATS4) under the flow of N2 gas, 10mm from bottom, 10mm from the side, space between two bands was 6 mm of the plate and application speed 150nm/s. The flow of air in the laboratory was maintained unidirectional (laminar flow, towards exhaust). The length of the chromatograph run was 9cm from the base. Bands were visualized by spraying with the acetic anhydride: sulphuric acid: ethanol (01:01:10v/ v/v) followed by heating at 1100C for 2 min. quantitative evaluation of the plate was performed after 20 min in reflection absorption mode at 254nm[5]. Calibration curve of mangiferin The standard curve of mangiferin was prepared and over spotted through software. Standard compound calibration curve was established over six analyte level in duplicate by applying 2-20l of mangiferin on HPTLC plate of the working solution. The calibration curve was plotted between amount of analyte versus average response. Method of Validation The validation procedure considers issues of specificity, linearity, range, accuracy, precision, detection limit, quantitation limit, robustness and system suitability. The International Conference on Harmonization (ICH) has issued guidelines on validating analytical procedures, which are widely accepted and commonly used. Specificity Specificity indicates the extent to which an assay procedure specifically measures the analyte of interest. Spiking the sample with the analyte or related compounds and observing the effect on the estimations demonstrates assay specificity. If spiking with compounds related to the analyte produces no effect on the result, the procedure is considered specific. Specificity is particularly valuable when analyzing an analyte among several similar compounds present in the sample. In present study specificity of the method was ascertained by analyzing the standard and sample solutions. The bands of mangiferin were confirmed by comparing its RF value and overlaid spectra of the spotted bands with standards. Accuracy The accuracy of an analytical procedure is the closeness of agreement between the conventional true value (or an accepted reference value) and the value calculated. Accuracy should be specified across the range of analytical procedure and inferred from 9 measurements (triplicates of three concentrations in the range). Application of the procedure to test samples after spiking at three different levels of 50%, 100% and 150% of expected analyte concentration helps to determine the accuracy of the procedure. Here accuracy was evaluated by using the recovery test. Three concentration levels were tested (low, middle and high). At each level samples were prepare in triplicate and analyzed. Accuracy was expressed as percentage. Precision The precision of an analytical method is closeness of results for a series of measurements of multiple samples from the same homogeneous material. Precision is expressed at three levels of short, medium and long intervals, which are respectively referred to as repeatability, intermediate precision and reproducibility. Repeatability Repeatability is precision under the same operating conditions over a short interval of time (intra-day precision). Intermediate Precision Intermediate precision expresses intra-laboratory variations: different days, different analysts, different equipment and expressed as %RSD. Reproducibility Reproducibility is precision at the inter-laboratory level. It is especially important if the analytical procedure is to be used in different laboratories, for instance, a pharmacopoeial procedure. Robustness Robustness of an analytical method ensures that it performs well and has few variables affecting its performance. For robustness different parameters i.e. developing TLC distance, mobile phase composition, humidity, temperature, chamber dimensions and chamber saturation were studied [6-11]. Table 1. Results of the developed HPTLC method
S.No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Mangiferin (mg/ml) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Mean (RSD %) (n=5) 1.01 (1.41) 0.99 (1.32) 1.03 (1.34) 0.98 (1.02) 0.95 (1.43) 0.97 (1.39) 1.01 (1.42) 1.02 (1.48) 1.03 (1.31) 0.97 (1.33)

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Nadagouda Smitha G. et al. / Journal of Pharmacy Research 2010, 3(5),1107-1109 acetate methanol (40:60) afforded good resolution with RF 0.26 and 0.39. The proposed HPTLC method has been used for the quantification of mangiferin and it was found to be 1.54% (in average). CONCLUSION In the present study, an HPTLC method was developed for quantification of mangiferin in the root bark of the salacia chinensis traditionally used for the treatment of diabetes. The method is simple, cost effective and easily adaptable for simultaneous screening and quantitative determination of mangiferin as compared to other analytical techniques. This method was validated and found to be in linearity, accuracy, precision, specificity, selectivity, ruggedness and recovery. REFERENCES Fig1: HPTLC Chromatogram of Mangiferin RESULT AND DISCUSSION The active components were analyzed by HPTLC. Different parameters were determined like limit of quantification, linearity, accuracy, precision, specificity, selectivity, ruggedness, and recovery. The calibration curve was found to be linear with good correlation coefficient 0.998-0.999. These experiments were mainly aimed at checking the system stability for the quantitative analysis on a large throughput basis during quality control procedures. The inter day and intraday variation were compared at different concentration and was found to be within laboratory variations in different days. The results show that the average recovery at each level was more than 99%. For repeatability six samples of same concentration were prepared and analyzed by proposed method to determine variation arising from method and expressed as %RSD. The Related Standard Deviation (RSD) values were found to be below 2 %. The standard and test solutions were spotted on HPTLC plated and several slightly different combination of the three solvents were used to assess robustness. The modified mobile phase of ethyl
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Source of support: Nil, Conflict of interest: None Declared

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