This article was downloaded by: [Sanmuga Priya Ekambaram] On: 10 June 2014, At: 04:39 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Liquid Chromatography & Related Technologies Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljlc20 HPTLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ANALYSIS OF EMODIN AND CHRYSOPHANOL IN Cassia tora LINN METHANOLIC EXTRACT E. Sanmuga Priya a , P. Senthamil Selvan a & P. P. Prakash a a Department of Pharmaceutical Technology , Anna University of Technology , Tiruchirappalli , Tamilnadu , India Accepted author version posted online: 12 Dec 2012.Published online: 01 Aug 2013. To cite this article: E. Sanmuga Priya , P. Senthamil Selvan & P. P. Prakash (2013) HPTLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ANALYSIS OF EMODIN AND CHRYSOPHANOL IN Cassia tora LINN METHANOLIC EXTRACT, Journal of Liquid Chromatography & Related Technologies, 36:18, 2525-2533 To link to this article: http://dx.doi.org/10.1080/10826076.2012.723087 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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This article was downloaded by: [Sanmuga Priya Ekambaram]On: 10 June 2014, At: 04:39Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Liquid Chromatography &Related TechnologiesPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/ljlc20
HPTLC METHOD DEVELOPMENT ANDVALIDATION FOR SIMULTANEOUSANALYSIS OF EMODIN ANDCHRYSOPHANOL IN Cassia tora LINNMETHANOLIC EXTRACTE. Sanmuga Priya a , P. Senthamil Selvan a & P. P. Prakash aa Department of Pharmaceutical Technology , Anna University ofTechnology , Tiruchirappalli , Tamilnadu , IndiaAccepted author version posted online: 12 Dec 2012.Publishedonline: 01 Aug 2013.
To cite this article: E. Sanmuga Priya , P. Senthamil Selvan & P. P. Prakash (2013) HPTLC METHODDEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ANALYSIS OF EMODIN AND CHRYSOPHANOL INCassia tora LINN METHANOLIC EXTRACT, Journal of Liquid Chromatography & Related Technologies,36:18, 2525-2533
To link to this article: http://dx.doi.org/10.1080/10826076.2012.723087
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.
This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
HPTLC METHOD DEVELOPMENT AND VALIDATION FORSIMULTANEOUS ANALYSIS OF EMODIN AND CHRYSOPHANOLIN Cassia tora LINN METHANOLIC EXTRACT
E. Sanmuga Priya, P. Senthamil Selvan, and P. P. Prakash
Department of Pharmaceutical Technology, Anna University of Technology,Tiruchirappalli, Tamilnadu, India
& Anthraquinone glycosides are the major constituents of Cassia tora Linn (Caesulpinaceae)among which emodin and chrysophanol were found to be the active glycosides present as majorconstituents. The objective of the present article is to develop a simple, precise, and rapid thin-layerchromatographic method for the simultaneous estimation of emodin and chrysophanol in Cassiatora extract and to validate the method as per ICH guidelines. TLC analysis was performed onprecoated silica gel 60 F254 plates with Toluene: ethyl acetate (9:1) as mobile phase. On scanningat 435 nm emodin and chrysophanol had shown good resolution with Rf values ‘‘0.31� 0.02and 0.85� 0.03,’’ respectively. The method was validated as per ICH guidelines. The percentagecontent of emodin and chrysophanol was found to be 0.037 and 0.067%, respectively. The linearityrange was found to be 10–300 lg=band and average recovery values were found to be 94 and 95%for emodin and chrysophanol, respectively. The developed method did not involve any derivatisationprocedure and thus was found to be simple, precise, and reproducible for the routine analysis ofemodin and chrysophanol.
Cassia tora Linn (Caesulpinaceae) is an annual foetid herb, with a heightof 30–90 cm, mainly found in the states of Uttar Pradesh and MadhyaPradesh in India. The seeds of Cassia tora are rhombohedral and brown incolor, about 30–50 in number. According to Ayurveda, the leaves and seedsare acrid, laxative, antiperiodic, anthelmintic, ophthalmic, liver tonic,cardiotonic, and expectorant. The leaves and seeds are useful in leprosy,ringworm, flatulence, colic, dyspepsia, constipation, cough, bronchitis,and cardiac disorders. Roasted seeds are substituted for coffee, like tephrosia
Address correspondence to E. Sanmuga Priya, Department of Pharmaceutical Technology, AnnaUniversity of Technology, Tiruchirappalli - 620 024, Tamilnadu, India. E-mail: [email protected]
Journal of Liquid Chromatography & Related Technologies, 36:2525–2533, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2012.723087
Journal of Liquid Chromatography & Related Technologies, 36:2525–2533, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2012.723087
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seeds. The major constituent of various parts of Cassia tora Linn werefound to be anthraquinone glycosides and flavonoids. The phytochemicalinvestigation on the seeds have shown Chrysophanol, Physcion, Emodin,Chryso obtusin, Obtusin, Chryso obtusin 2 O-b-D glucoside, Obtusin 2O-b-D glucoside,[1] Rubrafusarin, Chrysophonic 9-anthrone, Naptho apyrone toralactune.[2,3] The seeds found to possess various pharmacologicalactivities like hypolipidemic,[4] antigenotoxic,[5] antihepatotoxic,[6] antitu-mor, antifungal,[7] antibacterial,[8] antimutagenic,[9] and hypotensive.[10]
Cassia tora is one of the most important sources of medicinally impor-tant phytochemicals and widely used in Ayurvedic and Chinese system ofmedicine. Most of the works have been reported on the seeds and theirmain pharmacological actions are based on the anthraquinone glycosidesderivatives. Molecular mechanism of emodin action shows transition fromlaxative ingredient to an anti-tumor agent and mutagenic and genotoxiceffects.[11] Chrysophanol is the marker constituent in Cassia tora Linn.[12]
Various derivatives of chrysophanol and emodin were reported to have anti-cancer activities.[13]
The anthraquinone glycosides like emodin and chrysophanol (Figure 1)in Cassia tora Linn seeds were reported to have various pharmacologicalactivities and various HPLC methods of analysis were reported in theliterature.[14–16] HPTLC method of analysis has several advantages overHPLC-like simultaneous processing of standard and samples, less mobilephase consumption; no interference from previous analysis, multiple sam-pling, simple, inexpensive and easy handling, etc. Also, HPTLC has theadvantages of many-fold possibilities of detection in analyzing herbal med-icines. Thus, HPTLC is rapidly gaining importance in pharmaceuticalapplications and to the best of our knowledge there is no report on quan-titative determination of anthraquinone glycosides in Cassia tora seeds byHPTLC. Hence, the aim of the present article is to develop a novel, rapid,sensitive, simple, and accurate HPTLC method of analysis for the simul-taneous estimation of anthraquinone glycosides like emodin and chryso-phanol and to study some validation parameters for the method as perICH guidelines.
FIGURE 1 Structure of emodin and chrysophanol.
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EXPERIMENTAL
Plant Materials
Cassia tora Linn seeds were collected from Kolli hills, Namakal Districtin Tamil Nadu from November–December. It was identified by the Directorof the Raninat Herbarium and center for Molecular Systematics, St.Joseph’s College (campus), Tiruchirappalli, Tamil Nadu, India and thevoucher specimen number is PP 001.
Analytical-grade solvents and chemicals were procured from Qualigensfine chemicals, Mumbai. TLC aluminium plates precoated with silica gel60 F254 were obtained from Merck Ltd (Mumbai, India). The standardsemodin (93%) and chrysophanol (95%) was obtained as complementarysamples from Institute of Jammu tawi, India.
Preparation of Extract
The shade dried and coarsely powdered seeds of Cassia tora Linn(500 g) were exhaustively extracted with methanol (3� 3 l) in a glass perco-lator at room temperature (25� 2�C). The pooled methanolic extract wasfiltered using whatman filter paper and concentrated under vacuum at45�C until dryness. The extract was stored in a vacuum desiccator.
TLC Analysis
Chromatographic ConditionsPrecoated silica gel aluminium TLC sheets 60 F254 were used for spot-
ting the samples as bands with 6mm width using Camag microlitre syringe.Camag Linomat 5 applicator with 100 mL syringe was used for spotting thesamples. Ascending development was carried out in a toluene: ethyl acetate(9:1) in a twin trough chamber (20� 10 cm) previously saturated with sol-vent for 30min at room temperature. The development of the chromato-gram was approximately 80mm from the point of application. The plateswere removed and the solvent was dried. Camag TLC scanner 3 was usedfor densitometric scanning of the bands at 435 nm. The operating softwarewas Camag win CATS software, Switzerland.
Preparation of Standard Solution
The standard solution of emodin and chrysophanol was prepared byindividually weighing the standards and dissolving in methanol to obtaina concentration of 100 mg=mL as stock solution, respectively. These solutionswere further diluted to obtain solution of 10mg=mL which was used for theanalysis.
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Preparation of Sample Solution
500mg of Cassia tora methanolic extract was transferred to a 25mLvolumetric flask and about 15mL of methanol was added. The flask contentwas sonicated for 15min in a sonicator and then diluted upto the mark(25mL) with methanol. The solution was then filtered through Whatmanfilter and used for further analysis.
Validation Parameters
The method was validated for various parameters like specificity,linearity, accuracy, precision, and sensitivity as per ICH guidelines.[17]
Specificity
The method specificity was confirmed by simultaneously applying stan-dard, blank, and sample on the same TLC plate. Emodin and chrysophanolin Cassia tora extract were confirmed by comparing the Rf values with theirrespective standards.
Linearity
Different volumes (1, 2, 5, 10, 20, and 30mL) were taken separatelyfrom emodin and chrysophanol working standard solutions (10mg=mL)and applied separately to TLC plates, then the procedure under chromato-graphic conditions was followed. Calibration graphs for each glycoside wereconstructed by plotting concentration of standards vs. average peak areaafter scanning at 435 nm.
Accuracy
The method accuracy was done by performing recovery study at 3different concentrations (80, 100, and 120%) in triplicates. A known andvarying amount of the standards emodin and chrysophanol was added tothe extract and analyzed according to the previously mentioned procedure.The total content as well as the recovery was determined.
Precision
i. As per ICH guideline, the repeatability study was done in a maximum ofsix determinations at 100% of test concentration. Sample from the samevial but different tracks on the same concentration of chrysophanol andemodin standard were developed, dried and scanned at 435 nm. Thearea of all the bands was measured and % RSD was calculated.
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ii. For intermediate precision, the standard stock solutions of emodin andchrysophanol (10mg=mL) were applied on TLC plate, developed, driedand scanned at 435 nm 7 times in 4 different days without changing theplate position. The percentage RSD was calculated for the peak area.
Limit of Detection and Limit of Quantitation
ICH recommendations[17] using visual non instrumental method werefollowed to calculate the values of LOD and LOQ. The limit of detection(LOD) was considered as 3:1 and limit of quantitation (LOQ) as 10:1.
RESULTS AND DISCUSSION
Various HPLC methods were reported for the estimation of anthraqui-none glycosides in the genus Cassia.[14–16] There is no reported HPTLCmethod for the simultaneous determination of anthraquinones in Cassiagenus. Considering these facts, the present article was designed to developa simple and precise HPTLC method for the simultaneous estimation ofemodin and chrysophanol, the major and significant anthraquinone glyco-side of Cassia tora Linn.
Development of Optimum Mobile Phase
The TLC mobile phase was optimized by testing different solvent systemwith varying composition of different polarity. Some of the mobile phaseswere toluene: ethyl acetate (9.5:0.5), toluene: ethylacetate (9:1) and chloro-form: methanol (9:1). Among which chloroform: methanol (9:1) was foundto be highly polar for the glycosides as they eluted the metabolites to the sol-vent front. Toluene: ethyl acetate (9.5:0.5) has eluted only emodin and notchrysophanol, whereas the toluene: ethylacetate (9:1) found to show elutionof both emodin and chrysophanol with clear separation and good differencein the Rf values. The optimum mobile phase for analysis of emodin andchrysophanol was found to be Toluene: Ethyl acetate (9:1, v=v). As the com-pounds were colored, the scanning was carried out at 435nm (visible range),the absorption maximum for emodin and chrysophanol. The optimizedmobile phase has shown good separation and resolution with Rf values0.31� 0.02 for emodin and 0.85� 0.03 for chrysophanol (Figure 2).
Method Validation
The method validation parameters were studied and their values wereshown in Table 1.
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Specificity
The specificity of the phytochemicals emodin and chrysophanolpresent in the extract was confirmed by comparing with the Rf values ofthe extract with those of the standard bands. (Figure 2). The method wasfound to be specific with good separation between emodin and chrysopha-nol in Cassia tora methanolic extract.
Linearity
The linearity was determined by applying different concentrations ofworking standard solutions of the two glycosides. The data was processedby linear least square regression and the correlation coefficient (r2) wasdetermined. The r values for chrysophanol and emodin was found to be‘‘0.9867 and 0.9918,’’ respectively. The linearity range was found to be
FIGURE 2 HPTLC chromatogram showing standard mixture of emodin and chrysophanol in Cassiatora methanolic extract. (Color figure available online.)
TABLE 1 Method Validation Parameters in Quantitation of Emodin and Chrysophanol
10–300 mg=band. The calibration graph and TLC plate showing linearityrange of emodin and chrysophanol is shown in Figures 3 and 4.
Precision
The precision or repeatability study was done in six determinations of thesame concentration of chrysophanol and emodin but on the different tracks.In total, six tracks were scanned in repeated procedure and the chromato-grams were recorded. The repeatability of measurement (n¼ 6) for peak areaof the standards has been expressed in terms of percent coefficient of vari-ation (RSD%). The results are shown in Table 1 which depict that themethodis precise for the analysis of anthraquinone glycosides in Cassia tora seeds.
Limit of Detection and Limit of Quantitation
The LOD was found to be 0.24 mg=band for chrysophanol and 0.29 mg=band for emodin. The LOQ was found to be 0.75 mg=band for chrysophanoland 0.99 mg=band for emodin.
Accuracy
The accuracy of the method was assessed by recovery studies by usingthree different (80, 100, and 120%) concentrations of standard chrysopha-nol and emodin with the Cassia tora methanolic extract and spiked sample.The percentage recovery for ‘‘emodin was 94% and for chrysophanol 95%,’’respectively.
Estimation of Emodin and Chrysophanol in Cassia toraMethanolic Extract
The amount of chrysophanol and emodin estimated by the mentionedmethod is reported in Table 2. The method proposed is simple, accurate,
FIGURE 3 Precoated silica gel 60 F254 TLC plates showing linearity range of emodin and chrysophanolunder UV lamp. (Color figure available online.)
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and precise and the assay can be performed without any special pretreat-ment of the sample. The compounds chrysophanol and emodin werecolored and thus they can be determined in the visible region wavelengthwithout the need of derivatization and spraying reagent. Furthermore, themethod validation parameter proves the repeatability of the method. Thus,the developed HPTLC method can be used for routine analysis of theextracts in pharmaceutical Industries.
FIGURE 4 Calibration curves of emodin and chrysophanol. (Color figure available online.)
TABLE 2 Amount of Marker Compounds Present in Cassia tora Linn Methanolic Extract
Sample Name of the sample Content (%) %RSD (n¼ 3)
1. Emodin 0.037 0.922. Chrysophanol 0.067 0.78
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ACKNOWLEDGMENT
The authors thank Dr. A. V. Anoop, Director, Cholayil Products andServices, Koyambedu, Chennai, India for their help in providing HPTLCinstrument facility.
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