Rapid validated high performance thin layer chromatography method for simultaneous estimation of mangiferin and scopoletin in Canscora decussata (South Indian Shankhpushpi) extract

Abstract

Mangiferin (polyphenolic xanthone) and scopoletin (phenolic coumarin) are well-studied biological markers present in Canscora decussata(Roxb.) Roem. & Schult., Gentianaceae. The objective set for the present studies is to establish and develop a new, simple, selective, sensitive, and precise high performance thin layer chromatography method for the simultaneous estimation of mangiferin and scopoletin in hydroalcoholic extract of C. decussata. The thin layer chromatographic separation of these biomarkers was carried out on aluminum plate pre-coated with silica gel 60F254, eluted with ethyl acetate:acetic acid:formic acid:water (10:0.5:0.5:1.5). The plate was then dried and densitometric scanning was performed at 254 nm using a Camag TLC scanner III. The system was found to give compact spots for mangiferin (RF 0.22) and scopoletin (RF 0.78). A good relationship of linear precision between the concentrations (100–600 ng/spot) and peak areas was obtained with correlation coefficient (r) of 0.9979 (mangiferin) and 0.9962 (scopoletin), respectively. The limits of detection and limit of quantification were determined to be 46 and 94 ng/spot for mangiferin and 31 and 78 ng/spot for scopoletin respectively. The percentage of recovery was found from 99.91 to 99.94% for mangiferin and 99.75 to 99.86% for scopoletin. Results obtained from recovery studies showed excellent reliability and reproducibility of the method. Present communication on validated high performance thin layer chromatography method may provide a new, selective, sensitive, and precise method to estimate mangiferin and scopoletin as phytomarkers in the hydroalcoholic extract of C. decussata used in Ayurvedic formulations.

Keywords
Mangiferin; Scopoletin; HPTLC; Validation

Introduction

Canscora decussata (Roxb.) Roem. & Schult., Gentianaceae, is popularly known as "Shankhpushpi" in southern India and is well known for its medicinal values. The fresh juice of the whole plant is used traditionally for the treatment of insanity, epilepsy and cognition debility (Sethiya et al., 2012Sethiya, N.K., Nahata, A., Dixit, V.K., Mishra, S.H., 2012. Cognition boosting effect of Canscora decussata (a South Indian Shankhpushpi). Eur. J. Integr. Med. 4, 113–121.). This plant is reported to contain several types of phenolics, xanthones, bitter substances, oleoresin, triterpenoids, loliolide, sterols and flavanoids. C. decussata has proven its therapeutic potential in acetylcholinesterase inhibition, CNS stimulation, memory enhancement, antioxidant, hypertension, convulsions, tuberculosis, immunomodulation, inflammation, hepatoprotection, spermatogenesis and postmenopausal osteoporosis (Sethiya et al., 2010aSethiya, N.K., Nahata, A., Dixit, V.K., 2010a. Anxiolytic activity of Canscora decussata in albino rats. J. Complement. Integr. Med. 7, 19.). Scopoletin (1) and mangiferin (2) are recognized as the major active principles largely responsible for the bio-potency of C. decussata (Sethiya et al., 2013;Sethiya, N.K., Raja, M.K.M.M., Mishra, S.H., 2013. Antioxidant markers based TLC-DPPH differentiation on four commercialized botanical sources of Shankhpushpi (medhya rasayana) – a preliminary assessment. J. Adv. Pharm. Technol. Res. 4, 25–30. Sethiya and Mishra, 2014Sethiya, N.K., Mishra, S.H., 2014. Investigation of mangiferin, as a promising natural polyphenol xanthone on multiple targets of Alzheimer's disease. J. Biol. Active Prod. Nat. 4, 111–119.). Mangiferin is a polyphenolic xanthone, whereas scopoletin is a coumarin, and they are responsible for the antioxidant and memory enhancing activities (Sethiya et al., 2009aSethiya, N.K., Nahata, A., Dixit, V.K., 2009a. Comparative thin layer chromatographic investigations on sources of Shankhpushpi. Pharmacogn. J. 1, 224–226.). There are some reports on the application of colorimetry (Jubert et al., 2012Jubert, E., Botha, M., Maicu, C., De Beer, D., Manley, M., 2012. Rapid screening methods for estimation of mangiferin and xanthone contents of Cyclopia subternata plant material. S. Afr. J. Bot. 82, 113–122.), spectrophotometry (Krivut et al., 1976Krivut, B.A., Fedyunina, N.A., Kocherga, S.I., Rusakoya, S.V., 1976. Spectrophotometric determination of mangiferin. Chem. Nat. Compd. 12, 36–38.), spectrofluorimetry (Nahata and Dixit, 2008Nahata, A., Dixit, V.K., 2008. Spectrofluorimetric estimation of scopoletin in Evolvulus alsinoides Linn. and Convulvulus pluricaulis Choisy. Indian J. Pharm. Sci. 70, 834–837.; Sethiya et al., 2008Sethiya, N.K., Nahata, A., Dixit, V.K., 2008. Simultaneous spectrofluorimetric determination of scopoletin and mangiferin in a methanol extract of Canscora decussata Schult. Asian J. Tradit. Med. 3, 224–229.), thin layer chromatography (TLC) fingerprinting (Sethiya et al., 2009bSethiya, N.K., Nahata, A., Mishra, S.H., Dixit, V.K., 2009b. An update on Shankhpushpi a cognition boosting Ayurvedic medicine. Zhong Xi Yi Jie He Xue Bao 7, 1001–1022.), high performance thin layer chromatography (HPTLC), liquid chromatography–mass spectrometry (LC–MS) and high performance liquid chromatography (HPLC) methods (Risner, 1994Risner, C.H., 1994. The determination of scopoletin in environmental tobacco smoke by high-performance liquid chromatography. J. Liq. Chromatogr. 17 (12), 2723–2736.; Kapadia et al., 2006Kapadia, N.S., Acharya, N.S., Acharya, S.A., Shah, M.B., 2006. Use of HPTLC to establish a distinct chemical profile for shankhpushpi and for quantification of scopoletin in Convolvulus pluricaulis Choisy and in commercial formulations of Shankhpushpi. J. Planar Chromatogr. Mod. TLC 19, 195–199.; Rastogi et al., 2007Rastogi, S., Pandey, M.M., Rawat, A.K.S., 2007. A new, convenient method for determination of mangiferin, an anti-diabetic compound, in Mangifera indica L. J. Planar Chromatogr. Mod. TLC 20, 317–320.; Suryawanshi et al., 2007Suryawanshi, S., Asthana, R.K., Gupta, R.C., 2007. Simultaneous estimation of mangiferin and four secoiridoid glycosides in rat plasma using liquid chromatography tandem mass spectrometry and its application to pharmacokinetic study of herbal preparation. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 858, 211–219.; Xia et al., 2007Xia, Y., Dai, Y., Wang, Q., Liang, H., 2007. Determination of scopoletin in rat plasma by high performance liquid chromatographic method with UV detection and its application to a pharmacokinetic study. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 857, 332–336.; Shastry et al., 2009Shastry, V., Haldankar, A., Kadam, N., 2009. HPLC estimation of mangiferin in Salacia chinensis Linn. Asian J. Chem. 21, 6679–6682.; Upadhyay et al., 2013Upadhyay, V., Sharma, N., Tiwari, A.K., Joshi, H.M., Malik, A., Singh, B., Kalakoti, B.S., 2013. Standardization of HPLC method of scopoletin in different extracts of Convolvulus pluricaulis. Int. J. Pharm. Sci. Drug Res. 5, 28–31.) for analysis of mangiferin and scopoletin. Among these analytical methods, mangiferin and scopoletin were analyzed either alone or in combination with one or two other secondary plant metabolites, but attempts for simultaneous quantitative analysis of both by HPTLC in a herbal drug and formulation to establish distinct chemical profiling of C. decussata from other related herbs are not available. In the last few decades, HPTLC has emerged as an efficient and powerful analytical technique for fingerprinting and quantification of marker compounds in herbal drugs due to its merits of reliability, simplicity, sensitivity, accuracy, suitability for high throughput screening and speed of estimation of the content of phytomarkers in herbs (Mukherjee et al., 2010Mukherjee, D., Kumar, N.S., Khatua, T., Mukherjee, P.K., 2010. Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract. Phytochem. Anal. 21, 556–560.). HPTLC is currently gaining momentum for marker-based standardization of herbal drugs and is commonly applied for the identification, assay and testing for purity, stability, dissolution or content uniformity of raw materials and formulated products (Bhatt et al., 2010Bhatt, M.K., Dholwani, K.K., Saluja, A.K., 2011. Isolation and structure elucidation of scopoletin from Ipomoea reniformis(Convolvulaceae). J. Appl. Pharm. Sci. 1, 138–144.; Trivedi et al., 2011Trivedi, A., Sethiya, N.K., Mishra, S.H., 2011. Preliminary pharmacognostic and phytochemical analysis of Granthika (Leonotis nepetaefolia). An Ayurvedic herb. Indian J. Tradit. Know. 10, 682–688.). Keeping in view the utility of C. decussata, and the lack of an appropriate simple TLC method for the simultaneous separation of mangiferin and scopoletin, it is proposed to develop a routine method of analysis for simultaneous qualitative and quantitative estimation of mangiferin and scopoletin in hydroalcoholic extract of C. decussata by HPTLC. The proposed method was validated by specificity, range, linearity, accuracy, precision, detection limit, quantitative limit, and robustness according to the ICH guidelines (ICH, 1996ICH, 1996. Q2B. Validation of Analytical Procedure: Methodology. International Conference on Harmonization, Geneva., 2005ICH, 2005. Q2A. Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization, Geneva.).

Material and methods

Plant material

Whole herb of Canscora decussata (Roxb.) Roem. & Schult., Gentianaceae, was collected (November, 2011) from the Ninai Ghat (Gujarat, India) and identified by Dr. S.C. Agrawal (Department of Botany, CDRI, Lucknow, India). Voucher specimens of plants (Pharmacy/CD/09-10/13/NS) have been deposited in Herbal Drug Technology Lab, Pharmacy Department, The M. S. University of Baroda, Gujarat (India).

Chemicals and reagents

Mangiferin reference standard (98%) was obtained from Sigma Aldrich (India), and scopoletin reference standard (98%) was obtained as gift sample from Laila Impex Laboratory, Vijayawada (India). Analytical-grade solvents were obtained from E-Merck, Mumbai, India. Pre-coated silica gel 60F254 TLC plates were purchased from Merck, Darmstadt (Germany).

Isolation and characterization of mangiferin

Extraction and isolation of mangiferin was done according to the method given in our previous studies (Sethiya et al., 2010bSethiya, N.K., Patel, M.B., Mishra, S.H., 2010b. Phytopharmacologic aspects of Canscora decussata Roem and Schult. Pharmacogn. Rev. 4, 49–57.). Powdered materials (120 g) were first defatted with petroleum ether (500 ml) and the marc remained after this was dried and extracted with methanol to obtain the methanol extract (yield 4.47%, w/w). The methanol extract of C. decussata was subjected to preparative thin layer chromatography using silica gel G TLC plates as adsorbent and n-butanol, acetic acid and water (4:1:2, v/v) as solvent mixture. A yellow amorphous powder was obtained after recrystallization of fraction obtained from preparative chromatography. The isolated compound was analyzed by IR, MS, 1H and 13C NMR and identified by comparison of their spectral data with that in the literature and for reference standard of mangiferin (Chaudhuri and Ghosal, 1971Chaudhuri, R.K., Ghosal, S., 1971. Xanthones of Canscora decussata Schult. Phytochemistry 10, 2425–2432.; Kim et al., 2006Kim, C.Y., Ahn, M., Kim, J., 2006. Preparative isolation of mangiferin from Anemarrhena asphodeloides rhizomes by centrifugal partition chromatography. J. Liq. Chromatogr. Relat. Technol. 29, 869–875.; Dineshkumar et al., 2010Dineshkumar, B., Mitra, A., Manjunatha, M., 2010. Studies on the antidiabetic and hypolipidemic potentials of mangiferin (xanthone glucoside) in streptozotocin-induced type 1 and type 2 diabetic model rats. Int. J. Adv. Pharm. Sci. 1, 75–85.; Luo et al., 2012Luo, F., Lv, Q., Zhao, Y., Hu, G., Huang, G., Zhang, J., Sun, C., Li, X., Chen, K., 2012. Quantification and purification of mangiferin from Chinese Mango (Mangifera indica L.) cultivars and its protective effect on human umbilical vein endothelial cells under H2O2-induced stress. Int. J. Mol. Sci. 13, 11260–11274.; Sellamuthu et al., 2012Sellamuthu, P.S., Arulselvan, P., Muniappan, B.P., Kandasamy, M., 2012. Effect of mangiferin isolated from Salacia chinensisregulates the kidney carbohydrate metabolism in streptozotocin-induced diabetic rats. Asian. Pac. J. Trop. Biomed. 2, S1583–S1587.; Bhuvaneswari, 2013Bhuvaneswari, K., 2013. Isolation of mangiferin from leaves of Mangifera indica Var Alphonso. Asian J. Pharm. Clin. Res. 6, 173–174.).

Isolation and characterization of scopoletin

Isolation of scopoletin was done by reported method of Nahata et al. (2010)Nahata, A., Patil, U.K., Dixit, V.K., 2010. Effect of Evolvulus alsinoides Linn. on learning behavior and memory enhancement activity in rodents. Phytother. Res. 24, 486–493.. The methanol extract of C. decussata was subjected to column chromatography using silica gel (80–120 mesh) as adsorbent and chloroform, methanol and toluene (8:1:1) as eluent. The fraction was further purified by preparative chromatography and recrystallized with acetone to get crystalline material. The isolated compound was analyzed by IR, MS, 1H and 13C NMR and identified by comparison of their spectral data with that in the literature and with reference standard of scopoletin (Goodwin and Kavanagh, 1949Goodwin, R.H., Kavanagh, F., 1949. The isolation of scopoletin, a blue-fluorescing compound from oat roots. Bull. Torrey Bot. Club 76, 255–265.; Silva et al., 2002Silva, W.P., Deraniyagala, S.A., Wijesundera, R.L., Karunanayake, E.H., Priyanka, U.M., 2002. Isolation of scopoletin from leaves of Hevea brasiliensis and the effect of scopoletin on pathogens of H. brasiliensis. Mycopathologia 153, 199–202.; Nahata et al., 2010Nahata, A., Patil, U.K., Dixit, V.K., 2010. Effect of Evolvulus alsinoides Linn. on learning behavior and memory enhancement activity in rodents. Phytother. Res. 24, 486–493.; Bhatt et al., 2011Bhatt, M.K., Dholwani, K.K., Saluja, A.K., 2011. Isolation and structure elucidation of scopoletin from Ipomoea reniformis(Convolvulaceae). J. Appl. Pharm. Sci. 1, 138–144.; Darmawan et al., 2012Darmawan, A., Kosela, S., Kardono, L.B.S., Syah, Y.M., 2012. Scopoletin, a coumarin derivative compound isolated from Macaranga gigantifolia Merr. J. Appl. Pharm. Sci. 2, 175–177.).

HPTLC

Instrumentation conditions

A Camag (Muttenz, Switzerland) HPTLC system including a Linomat V sample applicator, a Camag twin-trough plate development chamber, Camag TLC Scanner 3 and WinCATS integration software was used. Aluminum backed HPTLC plates 10 cm × 10 cm with 0.2 mm layers of silica gel 60F254 (E. Merck, Darmstadt, Germany), pre-washed with methanol, were used (Agrawal et al., 2013Agrawal, R., Sethiya, N.K., Mishra, S.H., 2013. Antidiabetic activity of alkaloids of Aerva lanata roots on streptozotocin-nicotinamide induced type-II diabetes in rats. Pharm. Biol. 51, 635–642.). The length of the chromatogram run was 8 cm. Subsequent to chromatographic development, TLC plates were dried in air with the help of a TLC plate dryer.

Preparation of standard solutions

A common stock solution (1 mg/ml) of mangiferin and scopoletin was prepared in methanol. It was further diluted (10 ml) with methanol to get stock solution of 100 μg/ml. Aliquots (1–6 ml) of this stock solution were transferred to 10 ml volumetric flasks and diluted to volume with methanol to furnish standard solutions containing 10, 20, 30, 40, 50, and 60 μg/ml.

Preparation of sample

Freshly collected whole herb of C. decussata was dried under shade and coarsely powdered. The 10 g of powder materials were extracted with methanol (70%, 50 ml) and after standing for 48 h at room temperature, the hydroalcoholic extract was drained off. This process of extraction at ambient temperature was repeated till exhaustive extraction of mangiferin and scopoletin was accomplished, which was thoroughly monitored by TLC analysis. The hydroalcoholic extract obtained was combined, filtered and evaporated to dryness under reduced pressure in a rotary evaporator at 45 °C and finally dried under high vacuum to furnish the final extract. A measured quantity of the dried hydroalcoholic extract of C. decussata was dissolved in methanol and filtered through Whatman qualitative filter paper no. 1, pore size 11 μm (Maidstone, UK), and the volume of the solution was adjusted with methanol in a volumetric flask to obtain a final concentration of 1 mg/ml. This solution was used for the HPTLC analysis.

Chromatographic studies and densitometric scanning

Thin layer chromatographic (TLC) studies were performed using different solvent systems, and finally, ethyl acetate, acetic acid, formic acid and water (10:0.5:0.5:1.5, v/v) were found to be the suitable mobile phase for the proper separation of mangiferin and scopoletin in a single track. These markers were further fingerprinted with hydroalcoholic extract of C. decussata to ascertain their presence. The plates were densitometrically scanned (254 nm) with slit dimension of 1 mm × 0.1 mm. After the development, bands in the extracts were identified by matching their RF values with those obtained as standards.

Calibration of mangiferin and scopoletin and their analysis in C. decussata extract

A 10 μl of each concentration (10–60 μg/ml) of 1:1 (w/v) mixture of mangiferin and scopoletin was applied by means of Linomat V sample applicator to get a final concentration of 100–600 ng/spot. This was plotted against peak area to obtain a calibration plot. Further, 10 μl of the extract solution (10 μg/spot) was applied. After applying the chromatography technique, the amounts of mangiferin and scopoletin present in the extract were determined by means of the calibration plot.

Method validation

Accuracy and precision

The repeatability of the sample application and measurement of peak area were carried out using six replicates of the same spot of mangiferin and scopoletin, which were expressed in terms of percentage relative standard deviation (% RSD) and standard error (SE). The intra-day precision was determined at three different concentration levels of different markers, 100, 300 and 500 ng/spot, six times on the same day, and the inter-day precision was determined at three different concentrations of markers, 100, 300 and 500 ng/spot, six times on five different interval days over a period of 1 week (Kumar et al., 2008Kumar, V., Mukherjee, K., Kumar, S., Mal, M., Mukherjee, P.K., 2008. Validation of HPTLC method for the analysis of taraxerol in Clitorea ternatea. Phytochem. Anal. 19, 244–250.).

Robustness of the method

By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different compositions such as ethyl acetate, acetic acid, formic acid and water (9:0.5:0.5:2, v/v) and ethyl acetate, acetic acid, formic acid and water (11:0.5:0.5:1, v/v) were tried and the chromatograms were run. The amount of mobile phase, temperature and relative humidity were varied in the range of +5%. The plates were pre-washed by methanol and activated at 60 ± 5 °C for 2, 5 and 7 min prior to chromatography. Time from spotting to chromatography and from chromatography to scanning was varied from 0, 20, 40 and 60 min. The robustness of the method was done at three different concentration levels, 100, 300 and 500 ng/spot (Agrawal et al., 2004Agrawal, H., Kaul, N., Paradkar, A.R., Mahadik, K.R., 2004. HPTLC method for guggulsterone. I. Quantitative determination of E- and Z-guggulsterone in herbal extract and pharmaceutical dosage form. J. Pharm. Biomed. Anal. 36, 33–34.).

Limits of detection and quantification

The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation (SD) of the response and the slope (S) of the calibration curve at levels approaching the LOD according to the formulas: LOD = 3.3 (SD/S) and LOQ = 10 (SD/S). The SD of the response was determined based on the SD of y-intercepts of regression lines (Tuzimski and Bartosiewicz, 2003Tuzimski, T., Bartosiewicz, E., 2003. Correlation of retention parameters of pesticides in normal and RP systems and their utilization for the separation of a mixture of ten urea herbicides and fungicides by two dimensional TLC on cyano propyl-bonded polar stationary phase and two-adsorbent-layer multi-K plate. Chromatographia 58, 781–788.). Three different levels (50, 100 and 200 ng/spot) of the mixed standard stock solution (25 μg/ml) of mangiferin and scopoletin were prepared and used accordingly. Blank methanol was spotted six times following the same method as explained in the instrument and chromatographic conditions and the signal-to-noise ratio was determined. The LOD and LOQ were experimentally verified by diluting the known concentration of mangiferin and scopoletin until the average responses were approximately 3 or 10 times the standard deviation of the response for six replicate determinations.

Specificity

The specificity of the method was ascertained by analyzing the standard drug and sample. The spots for mangiferin and scopoletin in sample were confirmed by comparing the RF values and spectra of the spot with that of standard. The peak purity of mangiferin and scopoletin were assessed by comparing the spectra at three different levels, i.e., peak start, peak apex and peak end positions of the spot (Abourashed and Mossa, 2004Abourashed, E.A., Mossa, J.S., 2004. HPTLC determination of caffeine in stimulant herbal products and power drinks. J. Pharm. Biomed. Anal. 36, 617–620.).

Recovery studies

To study the accuracy and precision of the method, recovery studies were performed by the method of standard addition. The recovery of added standard was studied at two different levels, each being analyzed in a manner similar to that described for the assay (Biringanine et al., 2006Biringanine, G., Chiarelli, M.T., Faes, M., Duez, P., 2006. A validation protocol for the HPTLC standardization of herbal products: application to the determination of acteoside in leaves of Plantago palmata Hook f.s. Talanta 69, 418–424.; Wagner et al., 2008Wagner, S., Urena, A., Reich, E., Merfort, I., 2008. Validated HPTLC methods for the determination of salicin in Salix sp. and of harpagoside in Harpogophytum procumbens. J. Pharm. Biomed. Anal. 48, 587–591.). The hydroalcoholic extract of C. decussata used for recovery studies was pre-analyzed by the developed method as mentioned in the experimental section and found to contain 44 mg of mangiferin and 4.5 mg of scopoletin per gram of extract. Thus, 25 μg of the same pre-analyzed extract contained 880 ng/spot of mangiferin and 90 ng/spot of scopoletin, which was used for the recovery studies. The pre-analyzed extract samples were reanalyzed by spiking with an extra 1:2 ratio i.e. 100 and 200 ng/spot of the respective standard of mangiferin and scopoletin. The experiment was conducted six times to check for the recovery of the mangiferin and scopoletin.

Results and discussion

Isolation and characterization of mangiferin

The isolated yellow amorphous compound showed a single spot and exactly matched with RF of reference standard of mangiferin in TLC plate. It melts between 273 and 276 °C (for reference standard 274 °C). It gave an apricot green yellow color with 1% ferric chloride reagent, a blue quench in UV at 254 nm and a light yellow fluorescence in UV at 366 nm. It was further characterized as mangiferin by UV with absorption maxima of 315 nm, mass spectroscopy [significant peaks at m/e 404 (M-18) (12); 368 (45); 326 (14); 300 (21); 285 (34); 273 (100)], mixed melting point (Superfit Melting Point Apparatus), UV absorption maxima (GBC-Cintra, Australia) and superimposable FTIR (Shimadzu FTIR 8400S) spectral analysis. The structure of mangiferin was confirmed by comparing physicochemical and spectral data with those in the published literature (Chaudhuri and Ghosal, 1971Chaudhuri, R.K., Ghosal, S., 1971. Xanthones of Canscora decussata Schult. Phytochemistry 10, 2425–2432.; Kim et al., 2006Kim, C.Y., Ahn, M., Kim, J., 2006. Preparative isolation of mangiferin from Anemarrhena asphodeloides rhizomes by centrifugal partition chromatography. J. Liq. Chromatogr. Relat. Technol. 29, 869–875.; Dineshkumar et al., 2010Dineshkumar, B., Mitra, A., Manjunatha, M., 2010. Studies on the antidiabetic and hypolipidemic potentials of mangiferin (xanthone glucoside) in streptozotocin-induced type 1 and type 2 diabetic model rats. Int. J. Adv. Pharm. Sci. 1, 75–85.; Luo et al., 2012Luo, F., Lv, Q., Zhao, Y., Hu, G., Huang, G., Zhang, J., Sun, C., Li, X., Chen, K., 2012. Quantification and purification of mangiferin from Chinese Mango (Mangifera indica L.) cultivars and its protective effect on human umbilical vein endothelial cells under H2O2-induced stress. Int. J. Mol. Sci. 13, 11260–11274.; Sellamuthu et al., 2012Sellamuthu, P.S., Arulselvan, P., Muniappan, B.P., Kandasamy, M., 2012. Effect of mangiferin isolated from Salacia chinensisregulates the kidney carbohydrate metabolism in streptozotocin-induced diabetic rats. Asian. Pac. J. Trop. Biomed. 2, S1583–S1587.; Bhuvaneswari, 2013Bhuvaneswari, K., 2013. Isolation of mangiferin from leaves of Mangifera indica Var Alphonso. Asian J. Pharm. Clin. Res. 6, 173–174.).

Isolation and characterization of scopoletin

The isolated crystalline white compound showed a single spot and exactly matched with RF of reference standard of scopoletin in TLC plate. It gave blue fluorescence in UV (254 and 366 nm) and melted at 204–206 °C (for reference standard 205 °C). It was further characterized as scopoletin by mixed melting point, UV absorption maxima and superimposable FTIR spectral analysis. The FTIR spectrum of the compound is characteristic of 6-methoxy, 7-hydroxy coumarin, i.e. scopoletin. A superimposable FTIR spectrum with the reference standard proved this fact. The structure of scopoletin was confirmed by comparing physicochemical and spectral data with those in the published literature (Goodwin and Kavanagh, 1949Goodwin, R.H., Kavanagh, F., 1949. The isolation of scopoletin, a blue-fluorescing compound from oat roots. Bull. Torrey Bot. Club 76, 255–265.; Silva et al., 2002Silva, W.P., Deraniyagala, S.A., Wijesundera, R.L., Karunanayake, E.H., Priyanka, U.M., 2002. Isolation of scopoletin from leaves of Hevea brasiliensis and the effect of scopoletin on pathogens of H. brasiliensis. Mycopathologia 153, 199–202.; Nahata et al., 2010Nahata, A., Patil, U.K., Dixit, V.K., 2010. Effect of Evolvulus alsinoides Linn. on learning behavior and memory enhancement activity in rodents. Phytother. Res. 24, 486–493.; Bhatt et al., 2011Bhatt, M.K., Dholwani, K.K., Saluja, A.K., 2011. Isolation and structure elucidation of scopoletin from Ipomoea reniformis(Convolvulaceae). J. Appl. Pharm. Sci. 1, 138–144.; Darmawan et al., 2012Darmawan, A., Kosela, S., Kardono, L.B.S., Syah, Y.M., 2012. Scopoletin, a coumarin derivative compound isolated from Macaranga gigantifolia Merr. J. Appl. Pharm. Sci. 2, 175–177.).

TLC fingerprinting of mangiferin and scopoletin in C. decussata extract

The selected mobile phase resolved mangiferin and scopoletin efficiently from other components of C. decussata extract. The RF of mangiferin and scopoletin was found to be 0.22 and 0.78, respectively. The results obtained by fingerprinting of mangiferin and scopoletin in hydroalcoholic extract of C. decussata are shown in Fig. 1.

Fig. 1.
(A) Chromatogram of combined standards mangiferin and scopoletin (100–600 ng/spot): peak 1 (RF = 0.22): peak 2 (RF = 0.78); mobile phase is ethyl acetate:acetic acid:formic acid:water (10:0.5:0.5:1.5, v/v). (B) Chromatogram of Canscora decussata extract (10 μg/spot); peaks 1, 2, 4, 6, 7, and 8 belong to other components present in the extract. Peak 3 is mangiferin (RF = 0.22) and peak 9 is scopoletin (RF = 0.78); mobile phase is ethyl acetate:acetic acid:formic acid:water (10:0.5:0.5:1.5, v/v).

Calibration of mangiferin and scopoletin and their analysis in C. decussata extract

The calibration plots (Fig. 2) were linear in the range 100–600 ng/spot and the correlation coefficient (r) of 0.9979 (mangiferin) and 0.9962 (scopoletin) were indicative of good linear dependence of peak area on concentration. The calibration curve was represented by the linear equation y = 486.2 + 0.9846x and y = 522.2 + 4.652x for mangiferin and scopoletin, respectively (where y is the response as peak area and x is the concentration). The estimated content of mangiferin was found to be 44 mg/g, whereas the content of scopoletin was found to be 4.5 mg/g of hydroalcoholic extract of C. decussata, respectively.

Fig. 2.
Calibration plot (A) mangiferin and (B) scopoletin.

Method validation

Accuracy and precision

The reproducibility of the method was determined by different analysis using the sample from the same homogeneous batch and repeatability was determined by intra-day and inter-day precision. To ascertain the effectiveness of the method, suitability tests were performed on a freshly prepared mixture of standard stock solutions of mangiferin and scopoletin spiked with pre-analyzed hydroalcoholic extract of C. decussata. The repeatability of sample application and measurement of peak area were expressed in terms of % RSD and the % RSDs for intra- and inter-day analysis are depicted in Table 1. Intra-day precision (% RSD) on the basis of content of mangiferin and scopoletin was found to be 0.015–0.023 and 0.078–0.15, whereas inter-day precision (% RSD) on the basis of the content was found to be 0.009–0.045 and 0.040–0.084, respectively. So, the TLC densitometric method was found to be precise with RSD for intra-day and for inter-day precision.

Table 1
Intra- and inter-day precision of HPTLC (n = 6).

Robustness of the method

The SD of peak areas was calculated for each parameter and % RSD was found to be <2%, which shows robustness of the method. This indicates that the proposed method was precise and reproducible.

Limit of detection and quantification

The LOD and LOQ were determined based on the standard deviation of the response of blank and slope estimated from the calibration curve of standard solution of mangiferin and scopoletin. The LOD and LOQ were calculated from the equations LOD = 3.3 (SD/S) and LOQ = 10 (SD/S). The LOD for mangiferin and scopoletin found were 46 and 31 ng/spot, respectively, whereas LOQ were found to be 94 and 78 ng/spot, respectively.

Specificity

The peak purity of mangiferin and scopoletin was assessed by comparing the spectra of standard at peak start, peak apex and peak end positions of the spots, i.e., r (start, middle) = 0.9973 and r (middle, end) = 0.9979. Good correlation (r = 0.9994) was also obtained between the standard and sample. It was also observed that the peak of standards of mangiferin and scopoletin did not interfere with the peak of mangiferin and scopoletin in the hydroalcoholic extract of C. decussata, therefore the method can be considered specific. The chromatogram of standards mangiferin and scopoletin from the extract was matched in a similar fashion.

Recovery studies

The recovery study was performed by the method of standard addition. The recoveries of added standards (mangiferin and scopoletin) were studied at two different levels. The results of content estimation and recovery studies of mangiferin and scopoletin from hydroalcoholic extract of C. decussata, after spiking it with 100 and 200 ng/spot of additional standards, are listed in Table 2. The average percent recoveries at two different levels were found in the range of 99.91–99.94% (mangiferin) and 99.75–99.86% (scopoletin) showing the reliability and reproducibility of the method respectively.

Table 2
Recovery study of mangiferin and scopoletin (n = 3).

Estimation of the content of marker in Ayurvedic botanicals is of the utmost importance in evaluating the phytochemical entity of the herb. The current study was performed to develop a HPTLC method for the simultaneous determination of mangiferin and scopoletin content in C. decussata; one of the botanicals claims used as a source of Shankhpushpi. The developed HPTLC method is precise, specific, and accurate for the determination of mangiferin and scopoletin. Statistical analysis proved that the method is inevitable for the analysis of mangiferin and scopoletin. Since the proposed mobile phase effectively resolves mangiferin and scopoletin, the method can be used for routine qualitative, as well as quantitative analysis, of these markers in C. decussata extracts used for Ayurvedic formulation. The method can be used to determine the purity of the drug available from various sources by detecting the related impurities which allows the determination of variations in the botanicals of Shankhpushpi. It may be extended to study the degradation of mangiferin and scopoletin under different stress conditions as per the recommendations of ICH guidelines.

Acknowledgements

The authors thank Laila Impex, Vijayawada, India for the gift sample of standard scopoletin. One of the authors Neeraj K. Sethiya is thankful to University Grants Commission, New Delhi (India) for providing Junior Research Fellowship for the project. Neeraj K. Sethiya and Ashish Trivedi are thankful to Anchrom HPTLC Technologists, Mumbai (India) for providing the facilities for technical advice regarding instrument handling.

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Publication Dates

  • Publication in this collection
    May-Jun 2015

History

  • Received
    11 Jan 2015
  • Accepted
    08 Apr 2015
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