Simultaneous spectrofluorimetric determination of scopoletin and ...

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Hari Singh Gour Vishwavidyalaya, Sagar (MP) 470003. India; Tel.: +91-7582-264582; ... district, Chattisgarh, India, from January to March,. 2007. The herb was ...
Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional Medicines, 2008, 3 ( 6 )

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Simultaneous spectrofluorimetric determination of scopoletin and mangiferin in a methanolic extract of Canscora decussata Schult. Neeraj K. Sethiya a, Alok Nahata a, V. K. Dixit a*

a. Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya Sagar (M.P.) 470003, India

Abstract Scopoletin and mangiferin are two phenolic compounds detected in Canscora decussata (Gentianaceae). A simple, accurate and sensitive method for their determination in crude drug was developed using spectrofluorimetry. Scopoletin exhibited a strong bright blue fluorescence at excitation and emission wavelengths of 430 nm and 460 nm, respectively, and mangiferin exhibited a strong apricot yellow green fluorescence at excitation and emission wavelengths of 248 nm and 520 nm, respectively. The method was statistically validated and found suitable for quantitation of mangiferin and scopoletin in the methanolic extract of C. decussata. The proposed spectrofluorimetric method provides a faster and low cost quality control by simultaneous routine analysis of scopoletin and mangiferin in C. decussata and its formulations. Key words: Canscora decussata; gentianaceae; spectrofluorimetry; coumarin; xanthone; scopoletin; mangiferin; shankhpushpi

Ayurvedic Pharmacopoeia of India, Shankhpushpi consists of the whole plant of Convolvulus pluricaulis Choisy (Convulvulaceae). The pharmacopoeial monograph also mentions in its note that Clitorea ternatea Linn. (Papilionaceae) and Evolvulus alsinoides Linn. (Convulvulaceae) are used as Shankhpushpi in certain parts of India [1]. In the eastern parts of India, Canscora decussata Schult. (CD) (Gentianaceae) is popularly known as “Shankhpushpi” and found up to an altitude of 1300 meters [2]. It is also grown in Sri Lanka and Myanmar. The entire plant, as well as its fresh juice, is used in medicine. It is used in the treatment of insanity, epilepsy and nervous debility. This plant contains bitter substances, oleoresin, triterpenes, alkaloids [3-5] and xanthones such as mangiferin [6, 7]. The presence of scopoletin in CD has been detected for the first time

Introduction I n t h e Ay u r v e d i c s y s t e m o f m e d i c i n e , ‘Shankhpushpi’ is considered as ‘Medhya Rasayana’ –meaning a drug, which rejuvenates, maintains, and potentiates intellect and memory. Formulations containing ‘shankhpushpi’ as a principal constituent are widely advertised in Indian print and electronic media as memory enhancers. According to the

* Author to whom correspondence should be addressed. Prof. V.K. Dixit Address:Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar (MP) 470003. India; Tel.: +91-7582-264582; Fax: +91-7582-264163; E-mail: dixitvk2011@ rediffmail.com Neeraj K. Sethiya E-mail: [email protected] Alok Nahata E-mail: [email protected] Received: 2008-05-27

Accepted: 2008-10-20

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Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional Medicines, 2008, 3 ( 6 )

by a co-TLC method. Fluorescence under UV light is a characteristic of the majority of natural coumarins [8-10]. These compounds are very easily detected, since they give characteristic fluorescent colors under UV light, which are intensified by further treatment with ammonia vapors. Scopoletin is a coumarin, which exhibits a blue violet fluorescence under UV light [11, 12]. Mangiferin and Scopoletin both contain heterocyclic fused rings, which are responsible for their intense fluorescence under UV light. Mangiferin and scopoletin in methanolic solution exhibit a bright blue fluorescence and a light green fluorescence, respectively, under UV light. Therefore, it was thought worthwhile to develop a sensitive, specific, simple, precise and accurate spectrofluorimetric method for the estimation of mangiferin and scopoletin in the methanolic extract of CD.

Aerial parts of CD were collected from Raipur district, Chattisgarh, India, from January to March, 2007. The herb was identified by Prof. S.C. Agarwal, Head, Department of Botany, Central Drug Research Institute, Lucknow and preserved in the herbarium of the institute (Voucher Specimen no. NS/ AN/ Cd2409).

Preparation of extracts Aerial parts of CD were shade dried at room temperature. Extraction was performed according to the method described by Nahata et al. [15]. The shade dried plant material (500 g) was coarsely powdered and subjected to extraction with petroleum ether (1500 ml) in a Soxhlet apparatus. The marc was then extracted with methanol (1500 ml) to obtain the methanolic extract. The yield of the extract was found to be 3.45 % (w/w). All chemicals used for the procedure were of analytical grade.

Isolation of scopoletin

Materials and methods

The presence of scopoletin in CD was detected for the first time by co-TLC method with a standard. It was then isolated by column chromatography with silica gel G (80-120 mesh) and its identity was confirmed by its melting point (204 °C), UV absorption maxima and superimposable FTIR spectral analysis. Additional NMR evidence further confirmed the identity of the isolate as scopoletin.

General experimental procedures The spectrofluorimetric study was carried out with a Shimadzu RF 5301 PC spectrofluorimeter, to determine the levels of fluorescence of the phenolic compounds in a stationary state. The light source used was a xenon 150 W lamp with an optical system composed of two automatic monochromators, one for excitation and the other for emission, of a mesh type to enable a suitable wide selection of excitation and emission wavelengths for the coumarins [13]. A quartz cell was used [14]. The detection system was an R 450-01 photomultiplier which transformed the fluorescent radiation emitted by the scopoletin solution in the cell into an electrical signal. Scopoletin (99.98 %) was obtained as a gift from Laila Impex Research Centre, Vijaywada (A.P.), India and pure mangiferin (99.96 %) was obtained from Natural Remedies Pvt. Ltd., Bangalore, India.

Interpretation of spectral data of the isolate The FTIR spectrum of the isolated compound is characteristic of 6-methoxy, 7-hydroxy coumarin i.e. scopoletin. The FTIR spectrum was identical to the reference standard which proved this. The H NMR spectrum showed a three proton singlet at 3.9δ corresponding to -OCH3, two pairs of doublets centered at 6.1 δ and 7.7 δ corresponding to C 3-H and C4-H with J=9 cps indicating that these protons were on adjacent carbon atoms of a double bond, two aromatic protons (singlets) C8-H and C5-H at 6.8 δ and 6.9 δ respectively, stretching at 9.9 δ indicative of a –

Plant material

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Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional Medicines, 2008, 3 ( 6 )

OH which shows one exchangeable proton with D2O. After taking into consideration the melting point and the analytical data obtained from UV, IR and NMR analysis, the isolated compound was considered to be scopoletin.

mangiferin. The volume of stock solution used was 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml, respectively. The samples were analyzed in the spectrofluorimeter against solvent blank (methanol). The wavelength and intensity of each sample was recorded and a standard curve was prepared for concentration versus the intensity of fluorescence.

Preliminary analysis A preliminary analysis was carried out to determine the wavelength at which maximum intensity is exhibited by pure scopoletin and mangiferin. For this purpose, 100 µg/ml samples of pure scopoletin and mangiferin were prepared in methanol. They were scanned spectrofluorimetrically to obtain the excitation and emission wavelengths. The λ max shown by scopoletin had an excitation at 430 nm and an emission at 460 nm while the λ max shown by mangiferin had an excitation at 248 nm and an emission at 520 nm.

Determination of scopoletin and mangiferin concentration in methanolic extract 10 mg of the methanolic extract was weighed accurately and dissolved in 10 ml of methanol with vigorous shaking. It was then filtered and the volume made up to 100 ml with methanol. This solution was analyzed in the spectrofluorimeter and intensity of fluorescence was recorded. The concentration of both scopoletin and mangiferin in the extract samples was determined from their standard curves.

Preliminary spectrofluorimetric screening of the methanolic extract for testing the wavelength range

Spectrofluorimetric method development for simultaneous estimation of scopoletin and mangiferin

The methanolic extract of CD was scanned in the spectrofluorimeter in concentrations of 0.1 µg/ml and 0.01 µg/ml. The scanning was performed at excitation and emission wavelengths of 200 nm to 700 nm and absorption maxima was found at 315 nm and 435 nm. This wavelength corresponded to λmax of mangiferin and scopoletin, respectively. No other absorption peaks were detected in the range screened. Scanning between 248 nm and 520 nm did not exhibit any other peak, thus the range selected for analysis was expected to screen only mangiferin and scopoletin in the test sample.

After the success of the spectrofluorimetric analysis in determining the concentration of scopoletin and mangiferin individually in the methanolic extract of CD, it was thought worthwhile to develop a method for the simultaneous estimation of scopoletin and mangiferin concentrations in crude drug samples. For this purpose, 1 g shade dried powdered drug was taken and subjected to methanolic extraction. The methanolic extract was transferred to a volumetric flask and the volume made up to 100 ml with methanol. The fluorescence intensity of this diluted sample was determined by spectrofluorimetry. The whole procedure was repeated three times to obtain triplicate readings. The concentration of scopoletin and mangiferin in all the samples was obtained by extrapolating from the standard curves. The mean concentration of scopoletin and mangiferin present in 1 g crude drug was determined. After determining the concentration of scopoletin and mangiferin per ml of the methanolic extract, the mean concentration

Preparation of standard curve Standard curves of scopoletin and mangiferin were prepared in methanol. First of all, stock solution containing 100 µg/ml of scopoletin and mangiferin was prepared in methanol. Then, this stock solution was used to prepare required dilutions containing 5, 10, 15, 20, 25 and 30 µg/ml of scopoletin and

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Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional Medicines, 2008, 3 ( 6 )

the observed concentrations corresponded to the theoretical concentrations from the standard curve. The % recoveries were calculated on the basis of determination of the analyte added to a sample containing a known amount of scopoletin and mangiferin (Table 1).

per gram of crude drug (mg/g) was calculated. The scopoletin and mangiferin content in crude drug powder of CD was found to be 4 mg/g and 7 mg/g, respectively. Thus, a simple spectrofluorimetric method for analysis of scopoletin and mangiferin in CD was developed. Further recovery studies were performed to validate this novel analytical method.

Results and discussion Standard curves for scopoletin and mangiferin were prepared at excitation and emission wavelengths of 430 nm and 460 nm and 248 nm and 520 nm, respectively, using a spectrofluorimeter. In both cases, the plots of concentration versus intensity exhibited a linear relationship. The equation of the straight line for scopoletin was y=34.642x and that for mangiferin was y=5.157x-114.06. A methanolic extract of CD was also analyzed at the same excitation and emission wavelengths. The scopoletin and mangiferin content calculated from the standard curve was found to be 4 mg/g and 7 mg/g, respectively. Thus, a simple analytical method was developed for determining concentrations of scopoletin and mangiferin simultaneously in CD. The developed method was validated for linearity, reproducibility and accuracy. The linearity was found to be in the range of 5-30 µg/ml. The correlation coefficients (r) were 0.9919 and 0.9937 for scopoletin and mangiferin, respectively, indicating good linearity between the fluorescence intensity and concentration. Scanning of the samples three times allowed the precision of the method to be checked. The reproducibility and accuracy of the method was checked by carrying out recovery studies. A known concentration of scopoletin and mangiferin was added to different concentrations of the methanolic extract i.e. 0.1, 0.2, 0.5 and 1.0 µg/ml. A sample of known concentration was added in equal volume to the various dilutions of the extract and analyzed spectrofluorimetrically to see whether the observed concentration obtained corresponded to the theoretical concentration obtained from the standard curve. The percentage

Analytical method validation - Linearity Standard solutions (5 µg/ml to 30 µg/ml) were prepared in methanol and the intensity of fluorescence was recorded in the spectrofluorimeter. The standard curve was prepared by plotting the concentration as the abscissa versus the intensity of fluorescence as the ordinate. A linear dependence of intensity on concentration was observed over the entire concentration range tested. - Precision and accuracy The precision of the method was checked using standard solutions of the methanolic extract at a concentration of 0.1, 0.2, 0.5 and 1.0 µg/ml, prepared by appropriate dilutions with methanol. The solutions were analyzed in a spectrofluorimeter at 430 nm and 460 nm (excitation and emission wavelengths) for scopoletin and 248 nm and 520 nm (excitation and emission wavelengths) for mangiferin and the intensities were recorded. The corresponding concentrations were extrapolated from the standard curve. The entire procedure was repeated three times for each dilution and the readings were expressed as Mean ± S.D. (n=3). Then, 0.1 µg/ml solutions of scopoletin and mangiferin were prepared by appropriate dilutions and analyzed spectrofluorimetrically. The concentrations of scopoletin and mangiferin were calculated for the sample. This sample of known concentration was added in an equal volume (1 ml) to all the previous dilutions, and analyzed to see whether

227

228

0.2

0.5

1.0

2

3

4

0.206±0.011

0.186±0.011

0.130±0.028

0.090±0.031

scopoletin

0.233±0.009

0.201±0.005

0.149±0.025

0.110±0.015

mangiferin

(mg/ml)

Analyte * content in extract

0.097

0.097

0.097

0.097

scopoletin

0.094

0.094

0.094

0.094

mangiferin

(mg/ml)

Analyte added

0.303±0.011

0.283±0.011

0.227±0.028

0.187±0.031

scopoletin

0.327±0.009

0.295±0.005

0.243±0.025

0.205±0.015

mangiferin

expected (mg/ml)

Total amount of analyte

0.304±0.001

0.288±0.031

0.226±0.009

0.189±0.009

scopoletin

0.328±0.004

0.292±0.039

0.247±0.039

0.204±0.045

mangiferin

(mg/ml)

Amount obtained

100.58±0.335

101.60±0.793

100.69±1.237

100.69±1.237

scopoletin

100.30±0.639

98.98±0.096

101.64±0.238

99.51±0.330

mangiferin

(%)

Percentage recovery

* As calculated from the standard curve. All values are Mean ± SD (n = 3). * Excitation (λmax: 430 nm and 248 nm for scopoletin and mangiferin respectively) and Emission (λ max: 460 nm and 520 nm for scopoletin and mangiferin respectively)

0.1

(µg/ml)

extract used

methanolic

1

No.

S.

ation of

Concentr-

Table 1. Validation of the spectrofluorimetric method * (percentage recovery of scopoletin and mangiferin)

Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional Medicines, 2008, 3 ( 6 )

Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional Medicines, 2008, 3 ( 6 )

recovery of scopoletin and mangiferin was found to be in the range of (98-102) % (Table 1). Hence, the developed spectrofluorimetric procedure is a quick and reliable method for the simultaneous quantitative monitoring of scopoletin and mangiferin in raw material, processed powder and in herbal preparations containing CD.

decussata. J Pharm Sci, 1973, 62: 137-9. [5] Chintalwar GJ, Chattopadhyay S. Structural confirmation of decussatin; a Swertia decussata xanthone. Nat Pro Res, 2006, 20: 53-6. [6] Ghosal S, Singh AK, Chaudhary RK. Chemical Constituents of Gentianaceae XX; natural occurrance of (-) – loliolide in Canscora decussata. J Pharm Sci, 1976, 65: 1549-51. [7] Ghosal S, Biswas K, Chaudhary RK. Chemical Constituents of Gentianaceae Part 22. Structures of new 1, 3, 5-tri and 1, 3, 5, 6, 7-pentaoxygenated xanthone of Canscora decussata Schult. J Pharm Sci, 1977, 14: 1597-605. [8] Murray RDH, Mendez J, Brown SA. The natural coumarins. Occurance, Chemistry and Biochemistry. John Wiley & Sons Ltd., London, 1982. [9] Evans WC. Farmacognosia. Nueva Editorial Interamericana, S.A.& Mc Graw-Hill, Mexico, 1991. [10] Gupta AK, Tandon N, Sharma M. Quality Standards of Indian Medicinal Plants. Indian Council of Medical Research, New Delhi, 2005. [11] Harborne JB. Phytochemical Methods- A Guide to modern techniques of plant analysis. Chapman and Hall, London, 1973. [12] Fernandez IME, Quesada RJ, Villalon MM, Lopez MMC. Comparison of methods for determining coumarins in distilled beverages. Food Chemistry, 2000, 70: 251-258. [13] Otsuka K, Zenibayashi Y. On the determination of scopoletin in aged liquors. Agricultural Biological Chemistry, 1974, 38: 1079-80. [14] Ira N. Fisicoquimica: Interamericana de Espana, S.A. & Mc Graw-Hill, Madrid, 1993. [15] Nahata A, Patil UK, Dixit VK. Anxiolytic activity of Evolvulus alsinoides and Convulvulus pluricaulis in rodents. Pharmaceutical Biology, In Press..

Acknowledgements The authors are grateful to Laila Impex Research Centre, Vijaywada (A.P.), India for generously providing a gift of scopoletin and would also like to thank Natural remedies Pvt. Ltd., Bangalore, India for providing a gift of mangiferin. Two of the authors, Neeraj Kumar Sethiya and Alok Nahata, would like to thank the University Grants Commission, New Delhi for providing junior research fellowships.

References [1] Anonymous. The Ayurvedic Pharmacopoeia of India. 1st ed., Ministry of Health and Family Welfare. Department of Indian Systems of Medicine and Homeopathy. Controller of Publications, Delhi, 2001. [2] Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, 1956. [3] Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. Nirali Prakashan, Pune, 2002. [4] Ghosal S, Chaudhary RK, Nath A. Chemical Constituents of Gentianaceae IV. New xanthone of Canscora

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