Journal of Food Composition and Analysis 23 (2010) 214–219

Original Article

Simultaneous determination of epicatechin, syringic acid, quercetin-3-O-galactoside and quercitrin in the leaves of Rhododendron species byusing a validated HPTLC method§

Nandini Sharma, Upendra Kumar Sharma, Ajai Prakash Gupta, Arun Kumar Sinha *

Natural Plant Products Division, Institute of Himalayan Bioresource Technology (CSIR), Palampur 176061, Himachal Pradesh, India

A R T I C L E I N F O

Article history:

Received 14 October 2008

Received in revised form 30 October 2009

Accepted 5 November 2009

Keywords:

Epicatechin

Syringic acid

Quercetin-3-O-galactoside

Quercitrin

HPTLC

Rhododendron

Biodiversity

Food analysis

Food composition

A B S T R A C T

A rapid and sensitive reverse phase high-performance thin-layer chromatographic (RP-HPTLC) method

has been developed for the simultaneous determination of four bioactive phenolics, viz. epicatechin (1),

syringic acid (2), quercetin-3-O-galactoside (3) and quercitrin (4) in the leaves of Rhododendron

arboreum, Rhododendron campanulatum and Rhododendron anthopogon. HPTLC was performed on pre-

coated RP-18 F254S TLC plates. For achieving good separation, the mobile phase of methanol–5% formic

acid in water, 50:50 (v/v) was used and densitometric determination of compounds was carried out at

290 nm in reflection/absorption mode. The method was validated in terms of linearity, precision,

accuracy, and sensitivity. The calibration curves were linear in the range of 200–1200 ng for compounds

1, 3 and 4 and 400–2400 ng for compound 2. Lower limits of detection obtained for compounds 1–4 were

20, 40, 25 and 25 ng respectively, while the limit of quantification obtained were 50, 115, 75 and 70 ng

respectively. The method was found to be reproducible and convenient for quantitative analysis of these

compounds in Rhododendron species.

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1. Introduction

The genus Rhododendron (family Ericaceae) consists of around250 species all over the world, ranging in size from few centimetresto giant trees. Fifty species are reported in India (Prakash et al.,2007), out of which three major species found in WesternHimalayas are Rhododendron arboreum Smith, Rhododendron

anthopogon D. Don and Rhododendron campanulatum D. Don.The flowers and leaves of these Rhododendron species possessnutritional, medicinal and toxicological properties, and also havecommercial importance.

R. arboreum is the national flower of Nepal. Its beautiful redflowers have a sweetish-sour taste and are consumed both raw andcooked in the form of salads, pickles, chutney or sour jelly(Manandhar, 2002; Facciola, 1990; Gupta, 1945), and some sourcesclaim that the tender leaves may be used as a cooked vegetable(Facciola, 1990; Tanaka, 1976). In addition, the leaves and flowersare used for treating many illnesses, from diabetes (Bhandary andKawabata, 2008) to rheumatism (Skidel, 1980). The fresh leaves

§ Institute of Himalayan Bioresource Technology (IHBT) Communication

No. 0818.

* Corresponding author. Tel.: +91 1894 230426; fax: +91 1894 230433.

E-mail address: aksinha08@rediffmail.com (A.K. Sinha).

0889-1575/$ – see front matter � 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.jfca.2009.11.003

and flowers of R. anthopogan are made into a health-promoting teaby Himalayan healers (Kunwar et al., 2006; Sharma et al., 2004)and the leaves are used for preparation of a non-alcoholic beverage(Gairola and Biswas, 2008). The leaves and flowers of R.

campanulatum are used to treat some diseases, but are largelyconsidered to be toxic (Chauhan, 1999).

Rhododendron genus is considered to be a rich source ofsecondary metabolites like simple phenols, flavonoids, flavanol-3-O-glycosides, phenolic acids, terpenoids, resins, etc. (Cao et al.,2004; Feng et al., 2005; Harborne and Williams, 1971), and severalcompounds have been isolated from many other species of genusRhododendron like Rhododendron dauricum, Rhododendron ponti-

cum, Rhododendron molle, Rhododendron ferrugineum (Cao et al.,2004; Kashiwada et al., 2001; Zhong et al., 2005; Chosson et al.,1998). However, few reports are available on the chemicalscreening of the above-mentioned three species found growingin the western Himalayan region (Swaroop et al., 2005; Kamil et al.,1995; Shafiullah et al., 1991). This plant deserves an investigationinto its chemical composition, particularly for the presence ofphenolic compounds.

Owing to the significant nutritional, medicinal and commercialvalue of the leaves of these three Rhododendron species, theobjective of the present work was to develop a simple, rapid andeffective quantitative method for the analysis of phenoliccompounds in them. For the separation and quantification of

Fig. 1. Chemical structure of quantified phenolic compounds (1–4).

N. Sharma et al. / Journal of Food Composition and Analysis 23 (2010) 214–219 215

secondary metabolites especially for the evaluation of botanicalmaterials, high performance thin-layer chromatography hasemerged as one of the most efficient tools in the last two decades.In comparison with HPLC, the greatest advantage of the HPTLC isthat it does not require extensive clean-up procedures of crudeplant extracts, even for quantitative analysis. Additionally,numerous samples can be run in a single analysis therebydramatically reducing analytical time. With HPTLC, the sameanalysis can be viewed using different wavelengths of light therebyproviding a more complete profile of the plant than is typicallyobserved with more specific types of analyses (Dhalwal et al.,2008; Wagner et al., 2008; Singh et al., 2005). Thus, in continuationof our studies on qualitative and quantitative analysis of plantsecondary metabolites (Singh et al., 2005; Sharma et al., 2008a,b,2007; Verma et al., 2005), we, herein report for the first time asimple and rapid RP-HPTLC method for the simultaneousdetermination and quantification of four phenolic compounds,viz. epicatechin (1), syringic acid (2), quercetin-3-O-galactoside (3)and quercitrin (4) (Fig. 1) in the leaves of R. arboreum, R.

campanulatum and R. anthopogon.

2. Materials and methods

2.1. Plant material and chemicals

The plant material was collected from western Himalayas, Indiaat an altitude ranging between 1500 and 4000 m above the meansea level, and was authenticated at the Biodiversity Department ofour institute. The collected plant material was dried at roomtemperature under shade. Epicatechin, syringic acid, quercetin-3-O-galactoside and quercitrin were purchased from Sigma (USA)and have purity more than 97%. The TLC plates RP-18 F254S

(20 cm � 10 cm) (E. Merck, Darmastadt, Germany) were usedwithout any pretreatment. Methanol and formic acid were of HPLCgrade (E. Merck). Deionised water was from J.T. Baker (USA).

2.2. Standard stock solution and sample preparation

Standard stock solutions containing 1 mg/10 mL of purecompounds 1–4 were prepared by dissolving 5 mg of accuratelyweighed compound in 50 mL of methanol and filtered through0.45 mm (Millipore) filter. Samples were prepared from air-driedpowdered leaves (1 g) each of R. arboreum, R. campanulatum, R.anthopogon collected from Western Himalayan region, India. Thepowder was defatted with petroleum ether prior to extraction withmethanol (3 � 50 mL) under ultra-sonication at 40 � 5 8C for30 min. The organic extracts were combined, filtered and concen-

trated in vacuo to obtain a crude extract. Known amount of extractswere taken and dissolved in HPLC grade methanol (final conc. 10 mg/mL) and filtered through 0.45 mm filter for HPTLC analysis.

2.3. HPTLC procedure

2.3.1. Instrumentation and operating conditions

A Camag HPTLC system equipped with an automatic TLCsampler (ATS 4), TLC scanner 3 integrated with software (WinCATSversion 1.4.2), UV cabinet and automatic developing chamberADC2 with humidity control facility was used for the analysis. Thesamples were applied using automated TLC sampler in 10 mmbands at 10 mm from the bottom, 15 mm from the sides and with8 mm space between the two bands. Plates were developed insoftware controlled Camag automatic developing chamber ADC2pre-saturated with the 10 mL of developing solvent phase for30 min at room temperature (25 � 2 8C) and relative humidity wasmaintained 45 � 1%. The plates were developed to a height of about8 cm from the base in methanol/5% formic acid in water, 50:50 (v/v).After development, the plate was removed, dried and spots werevisualized under UV (254 and 366 nm) light. Quantitative evaluationof the plate was performed in the reflectance/absorbance mode at290 nm with following conditions: slit width 6 mm � 0.3 mm,scanning speed 20 mm/s and data resolution 100 mm/step. To checkthe identity of the bands, UV absorption spectrum of each standardwas overlaid with the corresponding band in the sample track.

2.3.2. Calibration and quantification

For the preparation of calibration curve, appropriate dilutionswere made to get the desired concentrations in the quantificationrange. The obtained working standard solutions were then appliedon the RP-TLC plate for preparing six-point linear calibrationcurves. Compound 2 was applied as 4, 8, 12, 16, 20, 24 mL while 1, 3and 4 were spotted as 2, 4, 6, 8, 10, 12 mL. Sample solution (10 mL)was applied on RP-TLC plate in triplicate with similar band pattern.The experimental parameters were identical for all the aboveanalysis.

2.4. Method validation

2.4.1. Specificity

The specificity of the method was determined by analysing thebands of different standards and samples. The bands forcompounds 1–4 in sample solution were confirmed by comparingthe Rf and UV spectra with the reference standards. The peak purityof these compounds was assessed by comparing the spectra atthree different levels, i.e. peak start, peak apex and peak endpositions.

2.4.2. Accuracy

The accuracy of the method was determined by analysing thepercentage recovery of the compounds in samples. For it, three setswere prepared from one of the species, i.e. R. arboreum. Thesamples were spiked with three different concentrations: 1, 3 and4 (50, 100 and 200 ng) and 2 (100, 200 and 400 ng) beforeextraction. The spiked samples were extracted in triplicate andthen analyzed by proposed HPTLC method.

2.4.3. Precision

Instrumental precision was checked by repeated scanning ofthe same spot of epicatechin, syringic acid, quercetin-3-O-galacto-side and quercitrin (400 ng) six times each. The repeatability of thesample application and measurements of peak area was expressedin terms of percent relative standard deviation (%RSD). To studythe intra-day precision different concentration levels of 200, 400and 600 ng/spot of reference compounds (1–4) were spotted five

Fig. 2. CCD image of TLC plate, lanes 1–5: standard tracks, epicatechin (1), syringic

acid (2), quercetin-3-O-galactoside (3), quercitrin (4); 6–14: methanolic extract of

Rhododendron spp. (6–8 R. arboreum; 9–11 R. campanulatum; 12–14 R. anthopogon)

(A) at 254 nm and (2B) at 366 nm.

N. Sharma et al. / Journal of Food Composition and Analysis 23 (2010) 214–219216

times within 24 h and expressed in terms of percent relativestandard deviation (%RSD). For intermediate precision six deter-minations was repeated at concentration levels of 200, 400 and600 ng/spot of reference compounds over a period of 5 days andexpressed as %RSD.

2.4.4. Robustness

Robustness is a measure of the method to remain unaffected bysmall but deliberate variations in the method conditions, and is anindication of the reliability of the method. For the robustness,different parameters such as mobile phase composition, develop-ing TLC distance and different TLC plate lots were checked.

2.4.5. Sensitivity

The sensitivity of the method was determined with respect toLOD and LOQ. The stock solutions of the standards were seriallydiluted and applied on RP-TLC plates to plot the calibration curves.LOD was determined based on the lowest concentration detectedby the instrument from the standard while the LOQ wasdetermined based on the lowest concentration quantified in thesample.

3. Results and discussion

In order to develop an effective solvent system for theseparation of phenolic compounds, the analysis was first triedon normal phase HPTLC plates using various combinations likechloroform–ethyl acetate–acetic acid, chloroform–methanol,chloroform–ethyl acetate–methanol, chloroform–ethyl acetate–methanol–isopropyl alcohol and ethyl acetate–hexane in differentproportions but good separation of the compounds could not beachieved. This may be due to wide polarity and functionalitydifferences among these phenolic compounds. Therefore, ourattention shifted towards the use of RP-TLC plate. Here, for theoptimization of HPTLC system various solvent systems likemethanol-water-acetic acid, acetonitrile-water-acetic acid, aceto-nitrile-water-formic acid and methanol-water-formic acid indifferent ratios were tried, the one containing methanol–5%formic acid in water, 50:50 (v/v) gave the best resolution, withsymmetrical and reproducible peaks, of epicatechin (Rf = 0.63),syringic acid (Rf = 0.47), quercetin-3-O-galactoside (Rf = 0.28) andquercitrin (Rf = 0.21) from the other components of the sample

Fig. 3. HPTLC chromatogram of (A) standard track, (B) R. arboreum methanolic extract,

(scanned at 290 nm).

extracts and enabled their simultaneous quantification. The plateswere visualized at two different wavelengths 254 and 366 nm(Fig. 2A and B) as the compounds were found to absorb at variablespectrum range. In addition, this helped in the generating a betterfingerprint data whereby species could be well differentiated onenhanced visual identification of individual compounds. Themethod developed here was found to be quite selective with goodbaseline resolution of each compound (Fig. 3). The identity of thebands of compounds 1–4 in the sample extracts was confirmed byoverlaying their UV absorption spectra with those of the standards(Fig. 4).

The developed HPTLC method was validated for differentparameters like specificity, linearity, accuracy, precision, robust-ness, LOD and LOQ. The specificity of the method was ascertainedby analyzing the standard compounds and samples for the

(C) R. campanulatum methanolic extract and (D) R. anthopogon methanolic extract

Fig. 4. Overlay of UV absorption spectra of the compounds in the sample track with respective standards (A) epicatechin, (B) syringic acid, (C) quercetin-3-O-galactoside and

(D) quercitrin.

N. Sharma et al. / Journal of Food Composition and Analysis 23 (2010) 214–219 217

interference of other components. The bands for compounds 1–4were confirmed by comparing the Rf and spectra of the bands withthat of standards. Absence of any interfering peak indicated thatthe method was specific. The purity of bands was confirmed byoverlaying the absorption spectra at the start, middle, and endposition of the bands.

Linearity of compounds 1–4 was validated by the linearregression equation and correlation coefficient. The six-pointcalibration curves for compounds 1, 3 and 4 were found to belinear in the range of 200–1200 ng/spot and for compound 2 in therange of 400–2400 ng/spot. Regression equation and correlationcoefficient for the reference compound were: Y = 610.217 + 3.5183x

(0.9985) for 1, Y = 587.0926 + 6.9580x (0.9996) for 2,Y = 540.5656 + 5.0337x (0.9993) for 3, and Y = 568.0204 + 3.5209x

(0.9991) for 4 which revealed a good linearity response fordeveloped method and are presented in Table 1. RSD of the samplesvaried from 1.43% to 2.60% for all the compounds under analysis.Good recoveries were obtained by the fortification of the sample at

Table 1Rf and method validation parameters for the quantitative determination of epicatechin

Parameters 1

Rf 0.63

Linearity range (ng/spot) 200–1200

r2 0.9985

Repeatability of application (n = 6) 0.86

Repeatability of measurement (n = 6) 0.72

LOD (ng) 20

LOQ (ng) 50

Amount of compound present in plant material (ng/spot).

three concentration levels for compounds 1–4. It is evident from theresults that the percent recoveries for all the four compounds aftersample processing and applying were in the range of 95.45–98.50%as shown in Table 2.

The mobile phase with a slight differences in their composition,i.e. methanol-5% formic acid in water with three different ratios45:55, 50:50 and 55:45 (v/v) were used and developing distancewas checked varying between 7 and 9 cm and no considerableeffect on the analysis was recorded. Also different TLC plate lots ofthe same manufacturer had no influence on the chromatographicseparation.

The instrumental precision which was represented as repeat-ability of sample application and measurement of peak area wasfound to be 0.86 and 0.72 for epicatechin, 0.74 and 0.59 for syringicacid, 0.52 and 0.58 for quercetin-3-O-galactoside, 0.64 and 0.43 forquercitrin, respectively (Table 1). To study the variability ofmethod, the intra-day precision and inter-day precisions(expressed in terms of %RSD) were calculated for three different

(1), syringic acid (2), quercetin-3-O-galactoside (3) and quercitrin (4).

2 3 4

0.47 0.28 0.21

200–2400 200–1200 200–1200

0.9996 0.9993 0.9991

0.74 0.52 0.64

0.59 0.58 0.43

40 25 25

115 75 70

Table 2Recovery study of epicatechin (1), syringic acid (2), quercetin-3-O-galactoside (3) and quercitrin (4) by proposed HPTLC method.

Compounds Amount of compound present

In plant material (ng/spot)

Amount (ng/spot) of

standard added

Observed amount

(ng/spot)

% Recovery %RSD

1 735.85 50 771.85 98.22 1.38

100 821.16 98.24 1.22

200 919.76 98.28 1.17

2 765.57 100 852.59 98.50 1.67

200 949.06 98.29 1.08

400 1138.98 97.71 1.11

3 291.77 50 327.12 95.71 1.20

100 379.28 96.81 1.52

200 472.48 96.07 1.29

4 610.12 50 631.26 95.63 1.66

100 678.03 95.48 1.32

200 773.26 95.45 1.59

Table 3Content of epicatechin, syringic acid, quercetin-3-O-galactoside and quercitrin found in Rhododendron spp.

Species Epc SA Q-3-O-gal Qrc

Av (n = 3) ng/spot %RSD Av (n = 3) ng/spot %RSD Av (n = 3) ng/spot %RSD Av (n = 3) ng/spot %RSD

R. arboreum 735.85 765.57 291.77 610.12

2.65 2.77 2.36 0.84

R. campanulatum 211.56 535.93 446.71 239.48

2.36 1.89 2.43 1.14

R. anthopogon 220.44 1718.23 384.73 281.06

2.23 2.13 2.02 0.90

Epc = Epicatechin, SA = syringic acid, Q-3-O-gal = quercetin-3-O-galactoside, Qrc = quercitrin.

N. Sharma et al. / Journal of Food Composition and Analysis 23 (2010) 214–219218

concentration (200, 400, and 600 ng/spot) by measurement of peakarea for the compounds 1–4 and were observed in the range of0.41–1.37% and 0.67–2.04%, respectively, which demonstrated thegood precision of proposed method.

Lower limits of detection obtained for compounds 1–4 were 20,40, 25 and 25 ng respectively while the limit of quantificationobtained were 50, 115, 75 and 70 ng respectively (Table 1). Thisindicated that the proposed method exhibits a good sensitivity forthe quantification of above compounds.

The content of epicatechin, syringic acid, quercetin-3-O-galactoside and quercitrin was estimated in the methanolic extractof Rhododendron spp. by the proposed method and the resultsobtained are summarized in Table 3. The results showedinteresting differences in the amounts of these derivatives presentin the same genus. It is for the first time, a simple, accurate andrapid HPTLC method has been developed for the simultaneousquantification of four bioactive compounds (1–4) in leaves of R.

arboretum, R. campanulatum and R. anthopogon.

4. Conclusions

The presented study clearly gave evidence of the quantitativevariation of epicatechin, syringic acid, quercetin-3-O-galactoside,and quercitrin in different species of the same genus. Thedeveloped reverse phase HPTLC densitometric method for thesimultaneous quantification of above four phenolic compounds issimple, precise, specific, sensitive, and accurate. Further, thismethod can be effectively used for the quantification of thesecompounds in other species of the same genus also.

Acknowledgements

The authors are grateful to the Council of Scientific & IndustrialResearch, New Delhi, India for its financial support during the

course of this project. Authors are also thankful to the Director,IHBT for providing necessary facilities during the course of work.NS and UKS are thankful to CSIR, Delhi for the fellowship. Authorsgreatly acknowledge Dr. Brij Lal for his help in plant identificationand valuable suggestions.

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