Research Article Analysis of Flavone C-Glycosides in the Leaves of Clinacanthus nutans (Burm. f.) Lindau by HPTLC and HPLC-UV/DAD June Lee Chelyn, Maizatul Hasyima Omar, Nor Syaidatul Akmal Mohd Yousof, Ramesh Ranggasamy, Mohd Isa Wasiman, and Zakiah Ismail Phytochemistry Unit, Herbal Medicine Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia Correspondence should be addressed to June Lee Chelyn; [email protected] Received 3 July 2014; Accepted 7 September 2014; Published 22 October 2014 Academic Editor: Wanchai De-Eknamkul Copyright © 2014 June Lee Chelyn et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Clinacanthus nutans (family Acanthaceae) has been used for the treatment of inflammation and herpes viral infection. Currently, there has not been any report on the qualitative and quantitative determination of the chemical markers in the leaves of C. nutans. e C-glycosidic flavones such as shaſtoside, isoorientin, orientin, isovitexin, and vitexin have been found to be major flavonoids in the leaves of this plant. erefore, we had developed a two-step method using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) for the rapid identification and quantification of the flavones C-glycosides in C. nutans leaves. e TLC separation of the chemical markers was achieved on silica gel 60 plate using ethyl acetate : formic acid : acetic acid : water (100 : 11 : 11 : 27 v/v/v/v) as the mobile phase. HPLC method was optimized and validated for the quantification of shaſtoside, orientin, isovitexin, and vitexin and was shown to be linear in concentration range tested (0.4–200 g/mL, r 2 ≥ 0.996), precise (RSD ≤ 4.54%), and accurate (95–105%). e concentration of shaſtoside, orientin, vitexin, and isovitexin in C. nutans leave samples was 2.55–17.43, 0.00–0.86, 0.00–2.01, and 0.00–0.91 mmol/g, respectively. 1. Introduction Clinacanthus nutans (Burm. f.) Lindau (family Acanthaceae) is a medicinal herb that is native to many tropical Asia countries including Malaysia, ailand, Indonesia, and China [1]. is herb is highly reputed in ailand where it is known locally as Phaya Yo or Phaya Plong ong [2]. is herb is also gaining popularity in Malaysia whereby it is more commonly known as Belalai Gajah or Sabah Snake Grass [3]. e fresh leaves of this herb have been traditionally used to treat skin rashes and bites caused by insects and snakes [2, 4]. In addition, the ethanolic extracts from the leaves of C. nutans have also been used clinically in the form of topical cream to treat Herpes genitalis and Herpes zoster lesions [5]. Some of the bioactive components that have been isolated from C. nutans include the chlorophyll phaeophytin deriva- tives isolated from the chloroform extract of C. nutans leaves and a mixture of cerebrosides and monoacylmonogalactosyl- glycerol isolated from the ethanolic extract of the fresh leaves of C. nutans in which they were shown to inhibit Herpes simplex virus-1 (HSV-1) activity [2, 4]. Other studies have also reported anti-inflammatory and immune-modulatory activities in the ethanolic extracts of C. nutans leaves [6, 7]. However, the exact compound or phytochemicals which are responsible for the anti-inflammatory and immune- modulatory activities observed are unknown. From the known phytochemicals, the six C-glycosyl flavones (shown in Figure 1), shaſtoside (1), isomollupentin 7-O--glucopyranoside, orientin (2), isoorientin (3), vitexin (4), and isovitexin (5) isolated from the methanolic extract of C. nutans leaves, were of interest due to their rare occurrence in plants [8]. e flavones C-glycosides are an important subclass of the flavonoids group of compounds and have been reported in plant from families Verbenaceae, Passifloraceae, Caryophyllaceae, and Polygonaceae [8]. ey possess impor- tant biological activities including antimicrobial activity (isoorientin, vitexin), hepatoprotective activity (isoorientin), and antioxidant activity (isovitexin) [9–11]. Despite that, there Hindawi Publishing Corporation e Scientific World Journal Volume 2014, Article ID 724267, 6 pages http://dx.doi.org/10.1155/2014/724267
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Research ArticleAnalysis of Flavone C-Glycosides in the Leaves ofClinacanthus nutans (Burm f) Lindau by HPTLCand HPLC-UVDAD
June Lee Chelyn Maizatul Hasyima Omar Nor Syaidatul Akmal Mohd YousofRamesh Ranggasamy Mohd Isa Wasiman and Zakiah Ismail
Phytochemistry Unit Herbal Medicine Research Centre Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia
Correspondence should be addressed to June Lee Chelyn jchels87yahoocom
Received 3 July 2014 Accepted 7 September 2014 Published 22 October 2014
Academic Editor Wanchai De-Eknamkul
Copyright copy 2014 June Lee Chelyn et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Clinacanthus nutans (family Acanthaceae) has been used for the treatment of inflammation and herpes viral infection Currentlythere has not been any report on the qualitative and quantitative determination of the chemical markers in the leaves of C nutansTheC-glycosidic flavones such as shaftoside isoorientin orientin isovitexin and vitexin have been found to bemajor flavonoids inthe leaves of this plant Therefore we had developed a two-step method using thin-layer chromatography (TLC) and high pressureliquid chromatography (HPLC) for the rapid identification and quantification of the flavones C-glycosides in C nutans leavesThe TLC separation of the chemical markers was achieved on silica gel 60 plate using ethyl acetate formic acid acetic acid water(100 11 11 27 vvvv) as themobile phaseHPLCmethodwas optimized and validated for the quantification of shaftoside orientinisovitexin and vitexin andwas shown to be linear in concentration range tested (04ndash200 120583gmL r2 ge 0996) precise (RSDle 454)and accurate (95ndash105)The concentration of shaftoside orientin vitexin and isovitexin in C nutans leave samples was 255ndash1743000ndash086 000ndash201 and 000ndash091mmolg respectively
1 Introduction
Clinacanthus nutans (Burm f) Lindau (family Acanthaceae)is a medicinal herb that is native to many tropical Asiacountries includingMalaysiaThailand Indonesia andChina[1] This herb is highly reputed inThailand where it is knownlocally as Phaya Yo or Phaya Plong Thong [2] This herbis also gaining popularity in Malaysia whereby it is morecommonly known as Belalai Gajah or Sabah Snake Grass [3]The fresh leaves of this herb have been traditionally used totreat skin rashes and bites caused by insects and snakes [2 4]In addition the ethanolic extracts from the leaves ofC nutanshave also been used clinically in the form of topical cream totreat Herpes genitalis and Herpes zoster lesions [5]
Some of the bioactive components that have been isolatedfrom C nutans include the chlorophyll phaeophytin deriva-tives isolated from the chloroform extract of C nutans leavesand amixture of cerebrosides andmonoacylmonogalactosyl-glycerol isolated from the ethanolic extract of the fresh leaves
of C nutans in which they were shown to inhibit Herpessimplex virus-1 (HSV-1) activity [2 4] Other studies havealso reported anti-inflammatory and immune-modulatoryactivities in the ethanolic extracts of C nutans leaves [67] However the exact compound or phytochemicals whichare responsible for the anti-inflammatory and immune-modulatory activities observed are unknown
From the known phytochemicals the six C-glycosylflavones (shown in Figure 1) shaftoside (1) isomollupentin7-O-120573-glucopyranoside orientin (2) isoorientin (3) vitexin(4) and isovitexin (5) isolated from the methanolic extract ofC nutans leaves were of interest due to their rare occurrencein plants [8] The flavones C-glycosides are an importantsubclass of the flavonoids group of compounds and have beenreported in plant from families Verbenaceae PassifloraceaeCaryophyllaceae and Polygonaceae [8] They possess impor-tant biological activities including antimicrobial activity(isoorientin vitexin) hepatoprotective activity (isoorientin)and antioxidant activity (isovitexin) [9ndash11]Despite that there
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 724267 6 pageshttpdxdoiorg1011552014724267
2 The Scientific World Journal
O
O
OHO
OH
OHOH
O
Shaftoside (1)
O
OOO
O
O
O
O
Orientin (2)
O
O
O
Isoorientin (3)
O
O
O
Vitexin (4)
O
O
O
Isovitexin (5)
HOH2C
HOH2C
HOH2C
HOH2C
HOH2C
HOH2C
HO
HO
HOHO
OH
OH
OHOH
OHHOHO
HOHO
OH
OH
HO
HOHO
OH
OH
OH
OHOH
HO
HOHO
OH
OH
OH
OH
HO
HOHO
OH
OH
OHHO
HOHO
Isomollupentin 7-O-120573-glucopyranoside
Figure 1 Chemical structures of known flavone C-glycosides shaftoside (1) isomollupentin 7-O-120573-glucopyranoside orientin (2) isoorientin(3) vitexin (4) and isovitexin (5)
is currently no report on the qualitative and quantitativedetermination of the chemical markers including the C-glycosyl flavones in the leaves of C nutans which hinder thedevelopment ofC nutansmedicinal productsThepurpose ofthis study is therefore to develop a separation technique usinghigh performance thin-layer chromatography (HPTLC) andhigh performance liquid chromatography coupled to a photo-diode array detector (HPLC-UVDAD) for the simultaneousdetermination and quantification of flavones C-glycosidesin C nutans that could accurately identify C nutans rawmaterials and products
2 Materials and Methods
21 Plant Materials Fresh leaves of C nutans werecollected from three different geographical locations inMalaysia The locations that were selected are TaipingPerak (L1) Kota Tinggi Johor (L2) and Sendayan NegeriSembilan (L3) Voucher specimens UPMIBSUBH39-12UPMIBSUBH46-12 and SBID 01912 were preparedand authenticated by botanists from the UniversityPutra Malaysia (UPM) and the Forest Research InstituteMalaysia (FRIM) The voucher specimens were depositedin the herbarium of FRIM and Institute for MedicalResearch
22 Chemicals and Standards Chemical standards shafto-side isoorientin orientin isovitexin and vitexin (all primarygrades) were purchased from Chromadex (Irvine CA USA)Solvents acetonitrile and ethanol (both HPLC grade) andethyl acetate acetic acid and formic acid (analytical grade)were purchased from Merck (Merck Co Darmstadt Ger-many) Water was purified with an ultrapure water system(Sartorius Germany)
23 Extract and Standard Preparation The fresh leaves wereoven dried at 40∘C then grind to fine powder The ethanolicextract was prepared by adding ethanol (5mL) to 05 g ofpowdered raw material sonicated for 30 minutes and thenfiltered using Whatman no 1 filter paper The supernatantis collected and used for HPTLC and HPLC analysis Thestandard stock solution (1mgmL) of shaftoside orientinisovitexin and vitexin was prepared in HPLC grade ethanoland stored at 4∘C Working solutions of lower concentration(shaftoside 20 40 60 80 100 150 175 and 200120583gmLorientin isovitexin vitexin 02 04 06 08 100 20 40 6080 and 100 ugmL) were prepared by appropriate dilution ofthe stock solutions in ethanol
24 HPTLC Analysis Thin-layer chromatography was per-formed on plates precoated with silica gel 60 F254 (Merck
The Scientific World Journal 3
Germany) Samples (5 120583L) were applied to the plate as 8mmwidth bands using Camag (Switzerland) Linomat V sampleapplicator The plate is then developed in an automaticdeveloping chamber (ADC2) (Camag Switzerland) usingethyl acetate formic acid acetic acid water (100 11 11 27vvvv) as themobile phaseDeveloping distancewas 70mmAfter development the plate was air-dried and derivatized byimmersing the plate in natural product reagent (2-aminoethyldiphenylborinate) (10 g) in methanol (200mL) and PEG 400(100 g) in methanol (200mL)
25 HPLC Analysis HPLC-UVDAD analysis was per-formed on a Waters Alliance 2695 (Millford MA USA)system connected to Waters 2996 photodiode array detector(DAD) The chromatographic separation was performedusing a Kinetex PFP column (250 times 46mm 5120583m Phe-nomenex USA) at 40∘C The solvent system consisted ofmixtures of water with 08 (vv) glacial acetic acid (solventA) and acetonitrile (solvent B) The solvents were degassedbefore delivering into the system Samples for HPLC analysiswere filtered through a 045 120583m membrane filter The opti-mized HPLC condition is as follows 5ndash19 B (30min) 19ndash95 B (3min) 95 B (5min) 95ndash5 B (1min) and 5 B(3min) The flow rate was 07mLmin The injection volumewas 10 120583L Signal was monitored at 330 nm
26 Validation of HPLC Method The optimized HPLCmethod was validated in terms of linearity limit of detection(LOD) limit of quantification (LOQ) precision and accuracyaccording to the International Conference onHarmonizationguidelines [12] The calibration curves were obtained by theexternal standard method on eight levels of concentration ofstandardmixtures with three injections per level Calibrationcurve was plotted using chromatogram peak areas on 330 nmagainst the known concentrations of standard solutions Alinear regression equation was calculated by least squaresmethod LOD and LOQ were estimated experimentally byinjecting a series of dilute solutions with known concen-tration until the signal-to-noise ratio reached 3 1 for LODand 10 1 for LOQThree different concentrations of standardmixtures (5 10 and 20 120583gmL) were used for intra- andinterday precision testing For intraday precision the peakarea and retention time of three different concentrationsof the standard solutions were determined in one day andexpressed as relative standard deviation (RSD) For interdayprecision the RSD of the standards were determined on threedifferent days Accuracy of the HPLC method was deter-mined by recovery tests analyzing sample extracts spikedwiththree different standard mixture concentrations (5 10 and20120583gmL)
3 Results and Discussion
31 HPTLC Screening Results TLC chromatographic condi-tions were optimized to obtain a system in which the flavonesC-glycosides would be separated in the best manner Themobile phase containing a mixture of ethylacetate formic
Figure 2 TLC profile of standards and extracts of C nutans leavesfrom different location S standard mixtures of shaftoside (119877
119891=
023) isoorientin (119877119891= 048) isovitexin (119877
119891= 056) orientin
(119877119891= 060) and vitexin (119877
119891= 066) C nutans samples L1 Taiping
Perak L2 Kota Tinggi Johor and L3 Sendayan Negeri Sembilan
acid acetic acid water (100 11 11 27 vvvv) gave goodseparation of the compounds of interest For visualiza-tion of the bands an additional step of derivatization wasnecessary using natural product reagent followed by PEG400 The marker compounds after derivatization observedunder UV 366 nm gave fluorescent bands on the TLC plateThe flavones C-glycosides with the apigenin backbone gavecharacteristic green coloured bands at retention factors (119877
119891)
of 023 (shaftoside) 056 (isovitexin) and 066 (vitexin) whileflavones C-glycosides with luteolin backbone gave distinctyellow bands for isoorientin and orientin 119877
119891048 and 060
respectively The retention time (119877119891) and colour of the
bands were compared between the standard mixture andsamples from three locations to confirm the presence ofthese markers in samples The preliminary TLC screeningresults showed that shaftoside was present in all three samplelocations Isoorientin isovitexin orientin and vitexin wereonly detected in two locations (L1 and L2) but were absent inthe third location (L3) as observed in Figure 2
32 Optimization of HPLC Method Several parameters wereoptimized to obtain the best HPLC chromatographic sep-aration The first parameter that was investigated was theuse of different mobile phase combination of either water-acetonitrile or water-methanol in which the combinationof water-acetonitrile gave better chromatographic resolu-tion compared to the later Addition of 08 glacial aceticacid to the mobile phase was found to improve peakresolution and peak shape and eliminate peak tailing ofthe marker compounds Besides that a gradient elutionmethod improved the resolution of compounds in samplecompared to isocratic mode For this study a shallow gra-dient profile from 5ndash19 B for 30 minutes was optimized
4 The Scientific World Journal
0255075
100
Mix standard solution(1) Shaftoside(2) Isoorientin
(3) Orientin(4) Isovitexin(5) Vitexin
12
3
45
(mAU
)
330nm
(min)2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
(a)
L3 Sendayan Negeri Sembilan
L2 Kota Tinggi Johor
L1 Taiping Perak
(mAU
)
(min)
8
6
4
0
minus236
27
18
9
040
30
20
10
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
a b
1
2 ce
a b
1
2 c 3
4
e 5
d
d
da b
1
2 c 3
4
e5
f
f
330nm
(b)
Figure 3 HPLC-UVDAD chromatogram of (a) standard solution mixture and (b) ethanolic extracts of C nutans leaves 120582 = 330 nm Cnutans samples L1 Taiping Perak L2 Kota Tinggi Johor and L3 Sendayan Negeri Sembilan (1) Shaftoside (2) isoorientin (3) orientin (4)vitexin and (5) isovitexin andashf Unknown
and used to quantitate markers of interest In additiondifferent chromatographic columns including Kinetex PFPcolumn (250 times 46mm 5 120583m Phenomenex USA) Sun-fire C18 (250 times 46mm 5120583m Waters USA) and SynergiPolar-RP (250 times 46mm 4120583m Phenomenex USA) weretested to improve chromatographic resolution between themarkers A satisfactory separation of marker compoundswas obtained using Kinetex PFP column (250 times 46mm5 120583mPhenomenexUSA)However investigation of the peakpurity of isoorientin in samples showed that the isoorientinpeak (2) was coeluting with another compound (Peak c) asshown in Figure 3(b)Therefore quantification of isoorientinwas not carried out in this study The HPLC separation wasmonitored at 330 nm according to the absorption maxima ofthemarker compoundsTheHPLC-UVDADchromatogramof the standard solutionmixture and the ethanolic extracts ofC nutans leaves at 330 nm are shown in Figures 3(a) and 3(b)respectively
33 HPLC Validation Data The linear range regressionequation correlation coefficient of each marker and LODand LOQ values are summarized in Table 1 The eight pointcalibration curve showed good linearity (1198772 = 0996) inthe given concentration ranges The LOD and LOQ valuesshowed the adequate sensitivity of the HPLC method devel-oped (Table 1) The interday and intraday precision rangefrom 062 to 454 (Table 2) showed that the method wasrepeatable The recovery values of the marker compoundsare presented in Table 2 The average recoveries whichranged from 9533 to 10513 showed good accuracy of themethod However recoveries were above 100 for certainsamples possibly due to the inaccuracy of spiking at lowerranges The compounds that showed high recoveries wereorientin 105 (amount recovered 525 120583gmL) shaftoside102 (1021 120583gmL) isovitexin 101 (509120583gmL) and vitexin102 and 101 (51120583gmL and 1015 120583gmL)
The Scientific World Journal 5
Table 1 Linear range regression equations LOD and LOQ for quantitative analysis of HPLC (119899 = 3)
Compound 119879119877(min) 120582 (nm) Linear range (120583gmL) Regression equationa
1198772 LOD (120583gmL) LOQ (120583gmL)
Shaftoside 2279 271 337 200ndash200 119910 = 28370119909 minus 230484 1198772= 0999 02 06
Orientin 2558 255 349 08ndash100 119910 = 27655119909 minus 27061 1198772= 0996 04 08
Isovitexin 2716 269 337 08ndash100 119910 = 34067119909 + 47328 1198772= 0999 04 08
Vitexin 2861 268 337 08ndash100 119910 = 29217119909 + 33931 1198772= 0997 04 08
a119910 = 119886119909 + 119887 where 119909 is concentration in 120583gmL and 119910 is area under curve at UV 330 nm wavelength
Table 2 Intra- and interday precision recovery and accuracy data
Compound Precision (RSD RT AUC) RecoveryConcentration (120583gmL) Intraday (119899 = 3) Interday (119899 = 6) Amount added (120583gmL) Recovery () RSD ()
Shaftoside5 324 280 5 9533 21810 296 211 10 10210 34920 085 339 20 9550 074
Orientin5 324 294 5 10513 14310 296 192 10 9953 10320 085 308 20 9811 010
Isovitexin5 155 096 5 10197 29310 418 243 10 9767 35320 070 329 20 9649 053
Vitexin5 238 211 5 10261 12510 311 454 10 10150 21020 062 348 20 9552 143
AUC area under curve
Table 3 Content of shaftoside orientin isovitexin and vitexin in samples of C nutans from different locations L1 Taiping Perak L2 KotaTinggi Johor and L3 Sendayan Negeri Sembilan
Compound Content of marker compounds (mmolg)L1 L2 L3
Shaftoside 1743 plusmn 001 1633 plusmn 003 255 plusmn 004Orientin 086 plusmn 004 059 plusmn 0003 NDIsovitexin 201 plusmn 002 103 plusmn 009 NDVitexin 091 plusmn 003 043 plusmn 0006 NDND not detectedValues are means plusmn SD 119899 = 3
34 Sample Analysis The optimized and validated methodwas applied to determine the concentration of the markercompounds in the samples from the three geographicallocations The content of shaftoside orientin isovitexin andvitexin in samples is presented in Table 3 The results showedthat shaftoside was the major flavone present in samplesfrom all three locations in which concentration ranges from255mmolg to 1743mmolg The other known flavones C-glycosides were present in lower amounts ranging fromthe highest for isovitexin (000ndash201mmolg) followed byorientin (000ndash086mmolg) to the lowest amount whichwas vitexin (000ndash091mmolg) Flavones C-glycosides arefavourable chemical markers due to their large structuraldiversity that could help to distinguish rapidly between otherplant species [13]The results also allowed us to conclude thatshaftoside would be best suited as chemical markers for Cnutans rawmaterial as it is found in all locations in the highest
quantity The lower amount of flavones detected includingshaftoside in L3 compared to L1 and L2 could be caused bydifferences in climate soil regime of the different region anddifferences in processing such as harvest time drying andstorage all of which may affect the composition of flavonoids
4 Conclusion
Chemical markers are essential as part of identification andalso quality control of herbal material In the present studyHPTLC technique was used for the rapid identification offive known flavones C-glycosides in C nutans raw materialsIn HPTLC the unique characteristic fluorescent bands afterderivatization provided important clues for the identificationof the major flavone present in the samples A secondstep using HPLC-UVDAD technique was employed for thesimultaneous detection and quantification of these known
6 The Scientific World Journal
flavonoids The HPLC-UVDAD method developed in thispresent study was effective accurate precise and sensitivefor the quantification of four known flavones C-glycosides(shaftoside orientin isovitexin and vitexin) of interestThe method was also applied to identify and quantify thechemical markers of interest in crude powdered leaves of Cnutans from three geographical regions in Malaysia Fromthe present study the compound shaftoside could be recom-mended as a marker for C nutans rawmaterial as it is presentin all locations and is also available as a commercial standardfor method validationThe current method developed in thisstudy is also useful for the evaluation of quality of C nutansraw materials and its commercial products
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and Director of Insti-tute for Medical Research for giving permission to publishthis paper Special record of appreciation for the supportand funding given by the Ministry of Agriculture (MoA)under the Malaysian Herbal Monograph (MHM) Develop-ment project that aims to assist the industries in not onlyauthenticating and confirming the rawmaterial used but alsothe quality of such raw material supplied
References
[1] I BurkillADictionary of Economic Products of theMalay Penin-sula vol 1 Ministry of Agriculture amp Cooperative MalaysiaKuala Lumpur Malaysia 1966
[2] S Sakdarat A ShuypromC Pientong T Ekalaksananan and SThongchai ldquoBioactive constituents from the leaves of Clinacan-thus nutans Lindaurdquo Bioorganic amp Medicinal Chemistry vol 17no 5 pp 1857ndash1860 2009
[3] Y K Yong J J Tan S S Teh et al ldquoClinacanthus nutansextracts are antioxidant with antiproliferative effect on culturedhuman cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 462751 8 pages 2013
[4] P Tuntiwachwuttikul Y Pootaeng-On P Phansa and W CTaylor ldquoCerebrosides and a monoacylmonogalactosylglycerolfrom Clinacanthus nutansrdquo Chemical amp Pharmaceutical Bul-letin vol 52 no 1 pp 27ndash32 2004
[5] C Kongkaew and N Chaiyakunapruk ldquoEfficacy of Clinacan-thus nutans extracts in patients with herpes infection system-atic review and meta-analysis of randomised clinical trialsrdquoComplementary Therapies in Medicine vol 19 no 1 pp 47ndash532011
[6] B Sriwanthana P Chavalittumrong andLChompuk ldquoEffect ofClinacanthus nutans onhuman cell-mediated immune responsein vitrordquoThai Journal of Pharmaceutical Sciences vol 20 no 4pp 261ndash267 1996
[7] VThongrakard and T Tencomnao ldquoModulatory effects ofThaimedicinal plant extract on proinflammatory cytokines-induced
apoptosis in human keratinocyte HaCat cellsrdquo African Journalof Biotechnology vol 9 no 31 pp 4999ndash5003 2010
[8] K-I Teshima T Kaneko K Ohtani et al ldquoC-glycosyl flavonesfrom Clinacanthus nutansrdquo Natural Medicines vol 51 no 6article 557 1997
[9] Y Zhang J Jiao C Liu X Wu and Y Zhang ldquoIsolation andpurification of four flavone C-glycosides from antioxidant ofbamboo leaves by macroporous resin column chromatogra-phy and preparative high-performance liquid chromatographyrdquoFood Chemistry vol 107 no 3 pp 1326ndash1336 2008
[10] F U Afifi E Khalil and S Abdalla ldquoEffect of isoorientinisolated from Arum palaestinum on uterine smooth muscle ofrats and guinea pigsrdquo Journal of Ethnopharmacology vol 65 no2 pp 173ndash177 1999
[11] Q F Huang S J Zhang L Zheng et al ldquoProtective effectof isoorientin-21015840-O-120572-l-arabinopyranosyl isolated from Gyp-sophila elegans on alcohol induced hepatic fibrosis in ratsrdquo Foodand Chemical Toxicology vol 50 no 6 pp 1992ndash2001 2012
[12] International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals forHuman Use Validation of Analytical Procedures Text andMethodology Q2(R1) 2005 httpwwwichorgfileadminPub-lic Web SiteICH ProductsGuidelinesQualityQ2 R1Step4Q2 R1 Guidelinepdf
[13] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of Separation Sciencevol 30 no 18 pp 3268ndash3295 2007
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2 The Scientific World Journal
O
O
OHO
OH
OHOH
O
Shaftoside (1)
O
OOO
O
O
O
O
Orientin (2)
O
O
O
Isoorientin (3)
O
O
O
Vitexin (4)
O
O
O
Isovitexin (5)
HOH2C
HOH2C
HOH2C
HOH2C
HOH2C
HOH2C
HO
HO
HOHO
OH
OH
OHOH
OHHOHO
HOHO
OH
OH
HO
HOHO
OH
OH
OH
OHOH
HO
HOHO
OH
OH
OH
OH
HO
HOHO
OH
OH
OHHO
HOHO
Isomollupentin 7-O-120573-glucopyranoside
Figure 1 Chemical structures of known flavone C-glycosides shaftoside (1) isomollupentin 7-O-120573-glucopyranoside orientin (2) isoorientin(3) vitexin (4) and isovitexin (5)
is currently no report on the qualitative and quantitativedetermination of the chemical markers including the C-glycosyl flavones in the leaves of C nutans which hinder thedevelopment ofC nutansmedicinal productsThepurpose ofthis study is therefore to develop a separation technique usinghigh performance thin-layer chromatography (HPTLC) andhigh performance liquid chromatography coupled to a photo-diode array detector (HPLC-UVDAD) for the simultaneousdetermination and quantification of flavones C-glycosidesin C nutans that could accurately identify C nutans rawmaterials and products
2 Materials and Methods
21 Plant Materials Fresh leaves of C nutans werecollected from three different geographical locations inMalaysia The locations that were selected are TaipingPerak (L1) Kota Tinggi Johor (L2) and Sendayan NegeriSembilan (L3) Voucher specimens UPMIBSUBH39-12UPMIBSUBH46-12 and SBID 01912 were preparedand authenticated by botanists from the UniversityPutra Malaysia (UPM) and the Forest Research InstituteMalaysia (FRIM) The voucher specimens were depositedin the herbarium of FRIM and Institute for MedicalResearch
22 Chemicals and Standards Chemical standards shafto-side isoorientin orientin isovitexin and vitexin (all primarygrades) were purchased from Chromadex (Irvine CA USA)Solvents acetonitrile and ethanol (both HPLC grade) andethyl acetate acetic acid and formic acid (analytical grade)were purchased from Merck (Merck Co Darmstadt Ger-many) Water was purified with an ultrapure water system(Sartorius Germany)
23 Extract and Standard Preparation The fresh leaves wereoven dried at 40∘C then grind to fine powder The ethanolicextract was prepared by adding ethanol (5mL) to 05 g ofpowdered raw material sonicated for 30 minutes and thenfiltered using Whatman no 1 filter paper The supernatantis collected and used for HPTLC and HPLC analysis Thestandard stock solution (1mgmL) of shaftoside orientinisovitexin and vitexin was prepared in HPLC grade ethanoland stored at 4∘C Working solutions of lower concentration(shaftoside 20 40 60 80 100 150 175 and 200120583gmLorientin isovitexin vitexin 02 04 06 08 100 20 40 6080 and 100 ugmL) were prepared by appropriate dilution ofthe stock solutions in ethanol
24 HPTLC Analysis Thin-layer chromatography was per-formed on plates precoated with silica gel 60 F254 (Merck
The Scientific World Journal 3
Germany) Samples (5 120583L) were applied to the plate as 8mmwidth bands using Camag (Switzerland) Linomat V sampleapplicator The plate is then developed in an automaticdeveloping chamber (ADC2) (Camag Switzerland) usingethyl acetate formic acid acetic acid water (100 11 11 27vvvv) as themobile phaseDeveloping distancewas 70mmAfter development the plate was air-dried and derivatized byimmersing the plate in natural product reagent (2-aminoethyldiphenylborinate) (10 g) in methanol (200mL) and PEG 400(100 g) in methanol (200mL)
25 HPLC Analysis HPLC-UVDAD analysis was per-formed on a Waters Alliance 2695 (Millford MA USA)system connected to Waters 2996 photodiode array detector(DAD) The chromatographic separation was performedusing a Kinetex PFP column (250 times 46mm 5120583m Phe-nomenex USA) at 40∘C The solvent system consisted ofmixtures of water with 08 (vv) glacial acetic acid (solventA) and acetonitrile (solvent B) The solvents were degassedbefore delivering into the system Samples for HPLC analysiswere filtered through a 045 120583m membrane filter The opti-mized HPLC condition is as follows 5ndash19 B (30min) 19ndash95 B (3min) 95 B (5min) 95ndash5 B (1min) and 5 B(3min) The flow rate was 07mLmin The injection volumewas 10 120583L Signal was monitored at 330 nm
26 Validation of HPLC Method The optimized HPLCmethod was validated in terms of linearity limit of detection(LOD) limit of quantification (LOQ) precision and accuracyaccording to the International Conference onHarmonizationguidelines [12] The calibration curves were obtained by theexternal standard method on eight levels of concentration ofstandardmixtures with three injections per level Calibrationcurve was plotted using chromatogram peak areas on 330 nmagainst the known concentrations of standard solutions Alinear regression equation was calculated by least squaresmethod LOD and LOQ were estimated experimentally byinjecting a series of dilute solutions with known concen-tration until the signal-to-noise ratio reached 3 1 for LODand 10 1 for LOQThree different concentrations of standardmixtures (5 10 and 20 120583gmL) were used for intra- andinterday precision testing For intraday precision the peakarea and retention time of three different concentrationsof the standard solutions were determined in one day andexpressed as relative standard deviation (RSD) For interdayprecision the RSD of the standards were determined on threedifferent days Accuracy of the HPLC method was deter-mined by recovery tests analyzing sample extracts spikedwiththree different standard mixture concentrations (5 10 and20120583gmL)
3 Results and Discussion
31 HPTLC Screening Results TLC chromatographic condi-tions were optimized to obtain a system in which the flavonesC-glycosides would be separated in the best manner Themobile phase containing a mixture of ethylacetate formic
Figure 2 TLC profile of standards and extracts of C nutans leavesfrom different location S standard mixtures of shaftoside (119877
119891=
023) isoorientin (119877119891= 048) isovitexin (119877
119891= 056) orientin
(119877119891= 060) and vitexin (119877
119891= 066) C nutans samples L1 Taiping
Perak L2 Kota Tinggi Johor and L3 Sendayan Negeri Sembilan
acid acetic acid water (100 11 11 27 vvvv) gave goodseparation of the compounds of interest For visualiza-tion of the bands an additional step of derivatization wasnecessary using natural product reagent followed by PEG400 The marker compounds after derivatization observedunder UV 366 nm gave fluorescent bands on the TLC plateThe flavones C-glycosides with the apigenin backbone gavecharacteristic green coloured bands at retention factors (119877
119891)
of 023 (shaftoside) 056 (isovitexin) and 066 (vitexin) whileflavones C-glycosides with luteolin backbone gave distinctyellow bands for isoorientin and orientin 119877
119891048 and 060
respectively The retention time (119877119891) and colour of the
bands were compared between the standard mixture andsamples from three locations to confirm the presence ofthese markers in samples The preliminary TLC screeningresults showed that shaftoside was present in all three samplelocations Isoorientin isovitexin orientin and vitexin wereonly detected in two locations (L1 and L2) but were absent inthe third location (L3) as observed in Figure 2
32 Optimization of HPLC Method Several parameters wereoptimized to obtain the best HPLC chromatographic sep-aration The first parameter that was investigated was theuse of different mobile phase combination of either water-acetonitrile or water-methanol in which the combinationof water-acetonitrile gave better chromatographic resolu-tion compared to the later Addition of 08 glacial aceticacid to the mobile phase was found to improve peakresolution and peak shape and eliminate peak tailing ofthe marker compounds Besides that a gradient elutionmethod improved the resolution of compounds in samplecompared to isocratic mode For this study a shallow gra-dient profile from 5ndash19 B for 30 minutes was optimized
4 The Scientific World Journal
0255075
100
Mix standard solution(1) Shaftoside(2) Isoorientin
(3) Orientin(4) Isovitexin(5) Vitexin
12
3
45
(mAU
)
330nm
(min)2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
(a)
L3 Sendayan Negeri Sembilan
L2 Kota Tinggi Johor
L1 Taiping Perak
(mAU
)
(min)
8
6
4
0
minus236
27
18
9
040
30
20
10
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
a b
1
2 ce
a b
1
2 c 3
4
e 5
d
d
da b
1
2 c 3
4
e5
f
f
330nm
(b)
Figure 3 HPLC-UVDAD chromatogram of (a) standard solution mixture and (b) ethanolic extracts of C nutans leaves 120582 = 330 nm Cnutans samples L1 Taiping Perak L2 Kota Tinggi Johor and L3 Sendayan Negeri Sembilan (1) Shaftoside (2) isoorientin (3) orientin (4)vitexin and (5) isovitexin andashf Unknown
and used to quantitate markers of interest In additiondifferent chromatographic columns including Kinetex PFPcolumn (250 times 46mm 5 120583m Phenomenex USA) Sun-fire C18 (250 times 46mm 5120583m Waters USA) and SynergiPolar-RP (250 times 46mm 4120583m Phenomenex USA) weretested to improve chromatographic resolution between themarkers A satisfactory separation of marker compoundswas obtained using Kinetex PFP column (250 times 46mm5 120583mPhenomenexUSA)However investigation of the peakpurity of isoorientin in samples showed that the isoorientinpeak (2) was coeluting with another compound (Peak c) asshown in Figure 3(b)Therefore quantification of isoorientinwas not carried out in this study The HPLC separation wasmonitored at 330 nm according to the absorption maxima ofthemarker compoundsTheHPLC-UVDADchromatogramof the standard solutionmixture and the ethanolic extracts ofC nutans leaves at 330 nm are shown in Figures 3(a) and 3(b)respectively
33 HPLC Validation Data The linear range regressionequation correlation coefficient of each marker and LODand LOQ values are summarized in Table 1 The eight pointcalibration curve showed good linearity (1198772 = 0996) inthe given concentration ranges The LOD and LOQ valuesshowed the adequate sensitivity of the HPLC method devel-oped (Table 1) The interday and intraday precision rangefrom 062 to 454 (Table 2) showed that the method wasrepeatable The recovery values of the marker compoundsare presented in Table 2 The average recoveries whichranged from 9533 to 10513 showed good accuracy of themethod However recoveries were above 100 for certainsamples possibly due to the inaccuracy of spiking at lowerranges The compounds that showed high recoveries wereorientin 105 (amount recovered 525 120583gmL) shaftoside102 (1021 120583gmL) isovitexin 101 (509120583gmL) and vitexin102 and 101 (51120583gmL and 1015 120583gmL)
The Scientific World Journal 5
Table 1 Linear range regression equations LOD and LOQ for quantitative analysis of HPLC (119899 = 3)
Compound 119879119877(min) 120582 (nm) Linear range (120583gmL) Regression equationa
1198772 LOD (120583gmL) LOQ (120583gmL)
Shaftoside 2279 271 337 200ndash200 119910 = 28370119909 minus 230484 1198772= 0999 02 06
Orientin 2558 255 349 08ndash100 119910 = 27655119909 minus 27061 1198772= 0996 04 08
Isovitexin 2716 269 337 08ndash100 119910 = 34067119909 + 47328 1198772= 0999 04 08
Vitexin 2861 268 337 08ndash100 119910 = 29217119909 + 33931 1198772= 0997 04 08
a119910 = 119886119909 + 119887 where 119909 is concentration in 120583gmL and 119910 is area under curve at UV 330 nm wavelength
Table 2 Intra- and interday precision recovery and accuracy data
Compound Precision (RSD RT AUC) RecoveryConcentration (120583gmL) Intraday (119899 = 3) Interday (119899 = 6) Amount added (120583gmL) Recovery () RSD ()
Shaftoside5 324 280 5 9533 21810 296 211 10 10210 34920 085 339 20 9550 074
Orientin5 324 294 5 10513 14310 296 192 10 9953 10320 085 308 20 9811 010
Isovitexin5 155 096 5 10197 29310 418 243 10 9767 35320 070 329 20 9649 053
Vitexin5 238 211 5 10261 12510 311 454 10 10150 21020 062 348 20 9552 143
AUC area under curve
Table 3 Content of shaftoside orientin isovitexin and vitexin in samples of C nutans from different locations L1 Taiping Perak L2 KotaTinggi Johor and L3 Sendayan Negeri Sembilan
Compound Content of marker compounds (mmolg)L1 L2 L3
Shaftoside 1743 plusmn 001 1633 plusmn 003 255 plusmn 004Orientin 086 plusmn 004 059 plusmn 0003 NDIsovitexin 201 plusmn 002 103 plusmn 009 NDVitexin 091 plusmn 003 043 plusmn 0006 NDND not detectedValues are means plusmn SD 119899 = 3
34 Sample Analysis The optimized and validated methodwas applied to determine the concentration of the markercompounds in the samples from the three geographicallocations The content of shaftoside orientin isovitexin andvitexin in samples is presented in Table 3 The results showedthat shaftoside was the major flavone present in samplesfrom all three locations in which concentration ranges from255mmolg to 1743mmolg The other known flavones C-glycosides were present in lower amounts ranging fromthe highest for isovitexin (000ndash201mmolg) followed byorientin (000ndash086mmolg) to the lowest amount whichwas vitexin (000ndash091mmolg) Flavones C-glycosides arefavourable chemical markers due to their large structuraldiversity that could help to distinguish rapidly between otherplant species [13]The results also allowed us to conclude thatshaftoside would be best suited as chemical markers for Cnutans rawmaterial as it is found in all locations in the highest
quantity The lower amount of flavones detected includingshaftoside in L3 compared to L1 and L2 could be caused bydifferences in climate soil regime of the different region anddifferences in processing such as harvest time drying andstorage all of which may affect the composition of flavonoids
4 Conclusion
Chemical markers are essential as part of identification andalso quality control of herbal material In the present studyHPTLC technique was used for the rapid identification offive known flavones C-glycosides in C nutans raw materialsIn HPTLC the unique characteristic fluorescent bands afterderivatization provided important clues for the identificationof the major flavone present in the samples A secondstep using HPLC-UVDAD technique was employed for thesimultaneous detection and quantification of these known
6 The Scientific World Journal
flavonoids The HPLC-UVDAD method developed in thispresent study was effective accurate precise and sensitivefor the quantification of four known flavones C-glycosides(shaftoside orientin isovitexin and vitexin) of interestThe method was also applied to identify and quantify thechemical markers of interest in crude powdered leaves of Cnutans from three geographical regions in Malaysia Fromthe present study the compound shaftoside could be recom-mended as a marker for C nutans rawmaterial as it is presentin all locations and is also available as a commercial standardfor method validationThe current method developed in thisstudy is also useful for the evaluation of quality of C nutansraw materials and its commercial products
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and Director of Insti-tute for Medical Research for giving permission to publishthis paper Special record of appreciation for the supportand funding given by the Ministry of Agriculture (MoA)under the Malaysian Herbal Monograph (MHM) Develop-ment project that aims to assist the industries in not onlyauthenticating and confirming the rawmaterial used but alsothe quality of such raw material supplied
References
[1] I BurkillADictionary of Economic Products of theMalay Penin-sula vol 1 Ministry of Agriculture amp Cooperative MalaysiaKuala Lumpur Malaysia 1966
[2] S Sakdarat A ShuypromC Pientong T Ekalaksananan and SThongchai ldquoBioactive constituents from the leaves of Clinacan-thus nutans Lindaurdquo Bioorganic amp Medicinal Chemistry vol 17no 5 pp 1857ndash1860 2009
[3] Y K Yong J J Tan S S Teh et al ldquoClinacanthus nutansextracts are antioxidant with antiproliferative effect on culturedhuman cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 462751 8 pages 2013
[4] P Tuntiwachwuttikul Y Pootaeng-On P Phansa and W CTaylor ldquoCerebrosides and a monoacylmonogalactosylglycerolfrom Clinacanthus nutansrdquo Chemical amp Pharmaceutical Bul-letin vol 52 no 1 pp 27ndash32 2004
[5] C Kongkaew and N Chaiyakunapruk ldquoEfficacy of Clinacan-thus nutans extracts in patients with herpes infection system-atic review and meta-analysis of randomised clinical trialsrdquoComplementary Therapies in Medicine vol 19 no 1 pp 47ndash532011
[6] B Sriwanthana P Chavalittumrong andLChompuk ldquoEffect ofClinacanthus nutans onhuman cell-mediated immune responsein vitrordquoThai Journal of Pharmaceutical Sciences vol 20 no 4pp 261ndash267 1996
[7] VThongrakard and T Tencomnao ldquoModulatory effects ofThaimedicinal plant extract on proinflammatory cytokines-induced
apoptosis in human keratinocyte HaCat cellsrdquo African Journalof Biotechnology vol 9 no 31 pp 4999ndash5003 2010
[8] K-I Teshima T Kaneko K Ohtani et al ldquoC-glycosyl flavonesfrom Clinacanthus nutansrdquo Natural Medicines vol 51 no 6article 557 1997
[9] Y Zhang J Jiao C Liu X Wu and Y Zhang ldquoIsolation andpurification of four flavone C-glycosides from antioxidant ofbamboo leaves by macroporous resin column chromatogra-phy and preparative high-performance liquid chromatographyrdquoFood Chemistry vol 107 no 3 pp 1326ndash1336 2008
[10] F U Afifi E Khalil and S Abdalla ldquoEffect of isoorientinisolated from Arum palaestinum on uterine smooth muscle ofrats and guinea pigsrdquo Journal of Ethnopharmacology vol 65 no2 pp 173ndash177 1999
[11] Q F Huang S J Zhang L Zheng et al ldquoProtective effectof isoorientin-21015840-O-120572-l-arabinopyranosyl isolated from Gyp-sophila elegans on alcohol induced hepatic fibrosis in ratsrdquo Foodand Chemical Toxicology vol 50 no 6 pp 1992ndash2001 2012
[12] International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals forHuman Use Validation of Analytical Procedures Text andMethodology Q2(R1) 2005 httpwwwichorgfileadminPub-lic Web SiteICH ProductsGuidelinesQualityQ2 R1Step4Q2 R1 Guidelinepdf
[13] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of Separation Sciencevol 30 no 18 pp 3268ndash3295 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
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International Journal ofPhotoenergy
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Carbohydrate Chemistry
International Journal of
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Journal of
Chemistry
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Advances in
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Analytical Methods in Chemistry
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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
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Chromatography Research International
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CatalystsJournal of
The Scientific World Journal 3
Germany) Samples (5 120583L) were applied to the plate as 8mmwidth bands using Camag (Switzerland) Linomat V sampleapplicator The plate is then developed in an automaticdeveloping chamber (ADC2) (Camag Switzerland) usingethyl acetate formic acid acetic acid water (100 11 11 27vvvv) as themobile phaseDeveloping distancewas 70mmAfter development the plate was air-dried and derivatized byimmersing the plate in natural product reagent (2-aminoethyldiphenylborinate) (10 g) in methanol (200mL) and PEG 400(100 g) in methanol (200mL)
25 HPLC Analysis HPLC-UVDAD analysis was per-formed on a Waters Alliance 2695 (Millford MA USA)system connected to Waters 2996 photodiode array detector(DAD) The chromatographic separation was performedusing a Kinetex PFP column (250 times 46mm 5120583m Phe-nomenex USA) at 40∘C The solvent system consisted ofmixtures of water with 08 (vv) glacial acetic acid (solventA) and acetonitrile (solvent B) The solvents were degassedbefore delivering into the system Samples for HPLC analysiswere filtered through a 045 120583m membrane filter The opti-mized HPLC condition is as follows 5ndash19 B (30min) 19ndash95 B (3min) 95 B (5min) 95ndash5 B (1min) and 5 B(3min) The flow rate was 07mLmin The injection volumewas 10 120583L Signal was monitored at 330 nm
26 Validation of HPLC Method The optimized HPLCmethod was validated in terms of linearity limit of detection(LOD) limit of quantification (LOQ) precision and accuracyaccording to the International Conference onHarmonizationguidelines [12] The calibration curves were obtained by theexternal standard method on eight levels of concentration ofstandardmixtures with three injections per level Calibrationcurve was plotted using chromatogram peak areas on 330 nmagainst the known concentrations of standard solutions Alinear regression equation was calculated by least squaresmethod LOD and LOQ were estimated experimentally byinjecting a series of dilute solutions with known concen-tration until the signal-to-noise ratio reached 3 1 for LODand 10 1 for LOQThree different concentrations of standardmixtures (5 10 and 20 120583gmL) were used for intra- andinterday precision testing For intraday precision the peakarea and retention time of three different concentrationsof the standard solutions were determined in one day andexpressed as relative standard deviation (RSD) For interdayprecision the RSD of the standards were determined on threedifferent days Accuracy of the HPLC method was deter-mined by recovery tests analyzing sample extracts spikedwiththree different standard mixture concentrations (5 10 and20120583gmL)
3 Results and Discussion
31 HPTLC Screening Results TLC chromatographic condi-tions were optimized to obtain a system in which the flavonesC-glycosides would be separated in the best manner Themobile phase containing a mixture of ethylacetate formic
Figure 2 TLC profile of standards and extracts of C nutans leavesfrom different location S standard mixtures of shaftoside (119877
119891=
023) isoorientin (119877119891= 048) isovitexin (119877
119891= 056) orientin
(119877119891= 060) and vitexin (119877
119891= 066) C nutans samples L1 Taiping
Perak L2 Kota Tinggi Johor and L3 Sendayan Negeri Sembilan
acid acetic acid water (100 11 11 27 vvvv) gave goodseparation of the compounds of interest For visualiza-tion of the bands an additional step of derivatization wasnecessary using natural product reagent followed by PEG400 The marker compounds after derivatization observedunder UV 366 nm gave fluorescent bands on the TLC plateThe flavones C-glycosides with the apigenin backbone gavecharacteristic green coloured bands at retention factors (119877
119891)
of 023 (shaftoside) 056 (isovitexin) and 066 (vitexin) whileflavones C-glycosides with luteolin backbone gave distinctyellow bands for isoorientin and orientin 119877
119891048 and 060
respectively The retention time (119877119891) and colour of the
bands were compared between the standard mixture andsamples from three locations to confirm the presence ofthese markers in samples The preliminary TLC screeningresults showed that shaftoside was present in all three samplelocations Isoorientin isovitexin orientin and vitexin wereonly detected in two locations (L1 and L2) but were absent inthe third location (L3) as observed in Figure 2
32 Optimization of HPLC Method Several parameters wereoptimized to obtain the best HPLC chromatographic sep-aration The first parameter that was investigated was theuse of different mobile phase combination of either water-acetonitrile or water-methanol in which the combinationof water-acetonitrile gave better chromatographic resolu-tion compared to the later Addition of 08 glacial aceticacid to the mobile phase was found to improve peakresolution and peak shape and eliminate peak tailing ofthe marker compounds Besides that a gradient elutionmethod improved the resolution of compounds in samplecompared to isocratic mode For this study a shallow gra-dient profile from 5ndash19 B for 30 minutes was optimized
4 The Scientific World Journal
0255075
100
Mix standard solution(1) Shaftoside(2) Isoorientin
(3) Orientin(4) Isovitexin(5) Vitexin
12
3
45
(mAU
)
330nm
(min)2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
(a)
L3 Sendayan Negeri Sembilan
L2 Kota Tinggi Johor
L1 Taiping Perak
(mAU
)
(min)
8
6
4
0
minus236
27
18
9
040
30
20
10
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
a b
1
2 ce
a b
1
2 c 3
4
e 5
d
d
da b
1
2 c 3
4
e5
f
f
330nm
(b)
Figure 3 HPLC-UVDAD chromatogram of (a) standard solution mixture and (b) ethanolic extracts of C nutans leaves 120582 = 330 nm Cnutans samples L1 Taiping Perak L2 Kota Tinggi Johor and L3 Sendayan Negeri Sembilan (1) Shaftoside (2) isoorientin (3) orientin (4)vitexin and (5) isovitexin andashf Unknown
and used to quantitate markers of interest In additiondifferent chromatographic columns including Kinetex PFPcolumn (250 times 46mm 5 120583m Phenomenex USA) Sun-fire C18 (250 times 46mm 5120583m Waters USA) and SynergiPolar-RP (250 times 46mm 4120583m Phenomenex USA) weretested to improve chromatographic resolution between themarkers A satisfactory separation of marker compoundswas obtained using Kinetex PFP column (250 times 46mm5 120583mPhenomenexUSA)However investigation of the peakpurity of isoorientin in samples showed that the isoorientinpeak (2) was coeluting with another compound (Peak c) asshown in Figure 3(b)Therefore quantification of isoorientinwas not carried out in this study The HPLC separation wasmonitored at 330 nm according to the absorption maxima ofthemarker compoundsTheHPLC-UVDADchromatogramof the standard solutionmixture and the ethanolic extracts ofC nutans leaves at 330 nm are shown in Figures 3(a) and 3(b)respectively
33 HPLC Validation Data The linear range regressionequation correlation coefficient of each marker and LODand LOQ values are summarized in Table 1 The eight pointcalibration curve showed good linearity (1198772 = 0996) inthe given concentration ranges The LOD and LOQ valuesshowed the adequate sensitivity of the HPLC method devel-oped (Table 1) The interday and intraday precision rangefrom 062 to 454 (Table 2) showed that the method wasrepeatable The recovery values of the marker compoundsare presented in Table 2 The average recoveries whichranged from 9533 to 10513 showed good accuracy of themethod However recoveries were above 100 for certainsamples possibly due to the inaccuracy of spiking at lowerranges The compounds that showed high recoveries wereorientin 105 (amount recovered 525 120583gmL) shaftoside102 (1021 120583gmL) isovitexin 101 (509120583gmL) and vitexin102 and 101 (51120583gmL and 1015 120583gmL)
The Scientific World Journal 5
Table 1 Linear range regression equations LOD and LOQ for quantitative analysis of HPLC (119899 = 3)
Compound 119879119877(min) 120582 (nm) Linear range (120583gmL) Regression equationa
1198772 LOD (120583gmL) LOQ (120583gmL)
Shaftoside 2279 271 337 200ndash200 119910 = 28370119909 minus 230484 1198772= 0999 02 06
Orientin 2558 255 349 08ndash100 119910 = 27655119909 minus 27061 1198772= 0996 04 08
Isovitexin 2716 269 337 08ndash100 119910 = 34067119909 + 47328 1198772= 0999 04 08
Vitexin 2861 268 337 08ndash100 119910 = 29217119909 + 33931 1198772= 0997 04 08
a119910 = 119886119909 + 119887 where 119909 is concentration in 120583gmL and 119910 is area under curve at UV 330 nm wavelength
Table 2 Intra- and interday precision recovery and accuracy data
Compound Precision (RSD RT AUC) RecoveryConcentration (120583gmL) Intraday (119899 = 3) Interday (119899 = 6) Amount added (120583gmL) Recovery () RSD ()
Shaftoside5 324 280 5 9533 21810 296 211 10 10210 34920 085 339 20 9550 074
Orientin5 324 294 5 10513 14310 296 192 10 9953 10320 085 308 20 9811 010
Isovitexin5 155 096 5 10197 29310 418 243 10 9767 35320 070 329 20 9649 053
Vitexin5 238 211 5 10261 12510 311 454 10 10150 21020 062 348 20 9552 143
AUC area under curve
Table 3 Content of shaftoside orientin isovitexin and vitexin in samples of C nutans from different locations L1 Taiping Perak L2 KotaTinggi Johor and L3 Sendayan Negeri Sembilan
Compound Content of marker compounds (mmolg)L1 L2 L3
Shaftoside 1743 plusmn 001 1633 plusmn 003 255 plusmn 004Orientin 086 plusmn 004 059 plusmn 0003 NDIsovitexin 201 plusmn 002 103 plusmn 009 NDVitexin 091 plusmn 003 043 plusmn 0006 NDND not detectedValues are means plusmn SD 119899 = 3
34 Sample Analysis The optimized and validated methodwas applied to determine the concentration of the markercompounds in the samples from the three geographicallocations The content of shaftoside orientin isovitexin andvitexin in samples is presented in Table 3 The results showedthat shaftoside was the major flavone present in samplesfrom all three locations in which concentration ranges from255mmolg to 1743mmolg The other known flavones C-glycosides were present in lower amounts ranging fromthe highest for isovitexin (000ndash201mmolg) followed byorientin (000ndash086mmolg) to the lowest amount whichwas vitexin (000ndash091mmolg) Flavones C-glycosides arefavourable chemical markers due to their large structuraldiversity that could help to distinguish rapidly between otherplant species [13]The results also allowed us to conclude thatshaftoside would be best suited as chemical markers for Cnutans rawmaterial as it is found in all locations in the highest
quantity The lower amount of flavones detected includingshaftoside in L3 compared to L1 and L2 could be caused bydifferences in climate soil regime of the different region anddifferences in processing such as harvest time drying andstorage all of which may affect the composition of flavonoids
4 Conclusion
Chemical markers are essential as part of identification andalso quality control of herbal material In the present studyHPTLC technique was used for the rapid identification offive known flavones C-glycosides in C nutans raw materialsIn HPTLC the unique characteristic fluorescent bands afterderivatization provided important clues for the identificationof the major flavone present in the samples A secondstep using HPLC-UVDAD technique was employed for thesimultaneous detection and quantification of these known
6 The Scientific World Journal
flavonoids The HPLC-UVDAD method developed in thispresent study was effective accurate precise and sensitivefor the quantification of four known flavones C-glycosides(shaftoside orientin isovitexin and vitexin) of interestThe method was also applied to identify and quantify thechemical markers of interest in crude powdered leaves of Cnutans from three geographical regions in Malaysia Fromthe present study the compound shaftoside could be recom-mended as a marker for C nutans rawmaterial as it is presentin all locations and is also available as a commercial standardfor method validationThe current method developed in thisstudy is also useful for the evaluation of quality of C nutansraw materials and its commercial products
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and Director of Insti-tute for Medical Research for giving permission to publishthis paper Special record of appreciation for the supportand funding given by the Ministry of Agriculture (MoA)under the Malaysian Herbal Monograph (MHM) Develop-ment project that aims to assist the industries in not onlyauthenticating and confirming the rawmaterial used but alsothe quality of such raw material supplied
References
[1] I BurkillADictionary of Economic Products of theMalay Penin-sula vol 1 Ministry of Agriculture amp Cooperative MalaysiaKuala Lumpur Malaysia 1966
[2] S Sakdarat A ShuypromC Pientong T Ekalaksananan and SThongchai ldquoBioactive constituents from the leaves of Clinacan-thus nutans Lindaurdquo Bioorganic amp Medicinal Chemistry vol 17no 5 pp 1857ndash1860 2009
[3] Y K Yong J J Tan S S Teh et al ldquoClinacanthus nutansextracts are antioxidant with antiproliferative effect on culturedhuman cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 462751 8 pages 2013
[4] P Tuntiwachwuttikul Y Pootaeng-On P Phansa and W CTaylor ldquoCerebrosides and a monoacylmonogalactosylglycerolfrom Clinacanthus nutansrdquo Chemical amp Pharmaceutical Bul-letin vol 52 no 1 pp 27ndash32 2004
[5] C Kongkaew and N Chaiyakunapruk ldquoEfficacy of Clinacan-thus nutans extracts in patients with herpes infection system-atic review and meta-analysis of randomised clinical trialsrdquoComplementary Therapies in Medicine vol 19 no 1 pp 47ndash532011
[6] B Sriwanthana P Chavalittumrong andLChompuk ldquoEffect ofClinacanthus nutans onhuman cell-mediated immune responsein vitrordquoThai Journal of Pharmaceutical Sciences vol 20 no 4pp 261ndash267 1996
[7] VThongrakard and T Tencomnao ldquoModulatory effects ofThaimedicinal plant extract on proinflammatory cytokines-induced
apoptosis in human keratinocyte HaCat cellsrdquo African Journalof Biotechnology vol 9 no 31 pp 4999ndash5003 2010
[8] K-I Teshima T Kaneko K Ohtani et al ldquoC-glycosyl flavonesfrom Clinacanthus nutansrdquo Natural Medicines vol 51 no 6article 557 1997
[9] Y Zhang J Jiao C Liu X Wu and Y Zhang ldquoIsolation andpurification of four flavone C-glycosides from antioxidant ofbamboo leaves by macroporous resin column chromatogra-phy and preparative high-performance liquid chromatographyrdquoFood Chemistry vol 107 no 3 pp 1326ndash1336 2008
[10] F U Afifi E Khalil and S Abdalla ldquoEffect of isoorientinisolated from Arum palaestinum on uterine smooth muscle ofrats and guinea pigsrdquo Journal of Ethnopharmacology vol 65 no2 pp 173ndash177 1999
[11] Q F Huang S J Zhang L Zheng et al ldquoProtective effectof isoorientin-21015840-O-120572-l-arabinopyranosyl isolated from Gyp-sophila elegans on alcohol induced hepatic fibrosis in ratsrdquo Foodand Chemical Toxicology vol 50 no 6 pp 1992ndash2001 2012
[12] International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals forHuman Use Validation of Analytical Procedures Text andMethodology Q2(R1) 2005 httpwwwichorgfileadminPub-lic Web SiteICH ProductsGuidelinesQualityQ2 R1Step4Q2 R1 Guidelinepdf
[13] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of Separation Sciencevol 30 no 18 pp 3268ndash3295 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
4 The Scientific World Journal
0255075
100
Mix standard solution(1) Shaftoside(2) Isoorientin
(3) Orientin(4) Isovitexin(5) Vitexin
12
3
45
(mAU
)
330nm
(min)2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
(a)
L3 Sendayan Negeri Sembilan
L2 Kota Tinggi Johor
L1 Taiping Perak
(mAU
)
(min)
8
6
4
0
minus236
27
18
9
040
30
20
10
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
a b
1
2 ce
a b
1
2 c 3
4
e 5
d
d
da b
1
2 c 3
4
e5
f
f
330nm
(b)
Figure 3 HPLC-UVDAD chromatogram of (a) standard solution mixture and (b) ethanolic extracts of C nutans leaves 120582 = 330 nm Cnutans samples L1 Taiping Perak L2 Kota Tinggi Johor and L3 Sendayan Negeri Sembilan (1) Shaftoside (2) isoorientin (3) orientin (4)vitexin and (5) isovitexin andashf Unknown
and used to quantitate markers of interest In additiondifferent chromatographic columns including Kinetex PFPcolumn (250 times 46mm 5 120583m Phenomenex USA) Sun-fire C18 (250 times 46mm 5120583m Waters USA) and SynergiPolar-RP (250 times 46mm 4120583m Phenomenex USA) weretested to improve chromatographic resolution between themarkers A satisfactory separation of marker compoundswas obtained using Kinetex PFP column (250 times 46mm5 120583mPhenomenexUSA)However investigation of the peakpurity of isoorientin in samples showed that the isoorientinpeak (2) was coeluting with another compound (Peak c) asshown in Figure 3(b)Therefore quantification of isoorientinwas not carried out in this study The HPLC separation wasmonitored at 330 nm according to the absorption maxima ofthemarker compoundsTheHPLC-UVDADchromatogramof the standard solutionmixture and the ethanolic extracts ofC nutans leaves at 330 nm are shown in Figures 3(a) and 3(b)respectively
33 HPLC Validation Data The linear range regressionequation correlation coefficient of each marker and LODand LOQ values are summarized in Table 1 The eight pointcalibration curve showed good linearity (1198772 = 0996) inthe given concentration ranges The LOD and LOQ valuesshowed the adequate sensitivity of the HPLC method devel-oped (Table 1) The interday and intraday precision rangefrom 062 to 454 (Table 2) showed that the method wasrepeatable The recovery values of the marker compoundsare presented in Table 2 The average recoveries whichranged from 9533 to 10513 showed good accuracy of themethod However recoveries were above 100 for certainsamples possibly due to the inaccuracy of spiking at lowerranges The compounds that showed high recoveries wereorientin 105 (amount recovered 525 120583gmL) shaftoside102 (1021 120583gmL) isovitexin 101 (509120583gmL) and vitexin102 and 101 (51120583gmL and 1015 120583gmL)
The Scientific World Journal 5
Table 1 Linear range regression equations LOD and LOQ for quantitative analysis of HPLC (119899 = 3)
Compound 119879119877(min) 120582 (nm) Linear range (120583gmL) Regression equationa
1198772 LOD (120583gmL) LOQ (120583gmL)
Shaftoside 2279 271 337 200ndash200 119910 = 28370119909 minus 230484 1198772= 0999 02 06
Orientin 2558 255 349 08ndash100 119910 = 27655119909 minus 27061 1198772= 0996 04 08
Isovitexin 2716 269 337 08ndash100 119910 = 34067119909 + 47328 1198772= 0999 04 08
Vitexin 2861 268 337 08ndash100 119910 = 29217119909 + 33931 1198772= 0997 04 08
a119910 = 119886119909 + 119887 where 119909 is concentration in 120583gmL and 119910 is area under curve at UV 330 nm wavelength
Table 2 Intra- and interday precision recovery and accuracy data
Compound Precision (RSD RT AUC) RecoveryConcentration (120583gmL) Intraday (119899 = 3) Interday (119899 = 6) Amount added (120583gmL) Recovery () RSD ()
Shaftoside5 324 280 5 9533 21810 296 211 10 10210 34920 085 339 20 9550 074
Orientin5 324 294 5 10513 14310 296 192 10 9953 10320 085 308 20 9811 010
Isovitexin5 155 096 5 10197 29310 418 243 10 9767 35320 070 329 20 9649 053
Vitexin5 238 211 5 10261 12510 311 454 10 10150 21020 062 348 20 9552 143
AUC area under curve
Table 3 Content of shaftoside orientin isovitexin and vitexin in samples of C nutans from different locations L1 Taiping Perak L2 KotaTinggi Johor and L3 Sendayan Negeri Sembilan
Compound Content of marker compounds (mmolg)L1 L2 L3
Shaftoside 1743 plusmn 001 1633 plusmn 003 255 plusmn 004Orientin 086 plusmn 004 059 plusmn 0003 NDIsovitexin 201 plusmn 002 103 plusmn 009 NDVitexin 091 plusmn 003 043 plusmn 0006 NDND not detectedValues are means plusmn SD 119899 = 3
34 Sample Analysis The optimized and validated methodwas applied to determine the concentration of the markercompounds in the samples from the three geographicallocations The content of shaftoside orientin isovitexin andvitexin in samples is presented in Table 3 The results showedthat shaftoside was the major flavone present in samplesfrom all three locations in which concentration ranges from255mmolg to 1743mmolg The other known flavones C-glycosides were present in lower amounts ranging fromthe highest for isovitexin (000ndash201mmolg) followed byorientin (000ndash086mmolg) to the lowest amount whichwas vitexin (000ndash091mmolg) Flavones C-glycosides arefavourable chemical markers due to their large structuraldiversity that could help to distinguish rapidly between otherplant species [13]The results also allowed us to conclude thatshaftoside would be best suited as chemical markers for Cnutans rawmaterial as it is found in all locations in the highest
quantity The lower amount of flavones detected includingshaftoside in L3 compared to L1 and L2 could be caused bydifferences in climate soil regime of the different region anddifferences in processing such as harvest time drying andstorage all of which may affect the composition of flavonoids
4 Conclusion
Chemical markers are essential as part of identification andalso quality control of herbal material In the present studyHPTLC technique was used for the rapid identification offive known flavones C-glycosides in C nutans raw materialsIn HPTLC the unique characteristic fluorescent bands afterderivatization provided important clues for the identificationof the major flavone present in the samples A secondstep using HPLC-UVDAD technique was employed for thesimultaneous detection and quantification of these known
6 The Scientific World Journal
flavonoids The HPLC-UVDAD method developed in thispresent study was effective accurate precise and sensitivefor the quantification of four known flavones C-glycosides(shaftoside orientin isovitexin and vitexin) of interestThe method was also applied to identify and quantify thechemical markers of interest in crude powdered leaves of Cnutans from three geographical regions in Malaysia Fromthe present study the compound shaftoside could be recom-mended as a marker for C nutans rawmaterial as it is presentin all locations and is also available as a commercial standardfor method validationThe current method developed in thisstudy is also useful for the evaluation of quality of C nutansraw materials and its commercial products
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and Director of Insti-tute for Medical Research for giving permission to publishthis paper Special record of appreciation for the supportand funding given by the Ministry of Agriculture (MoA)under the Malaysian Herbal Monograph (MHM) Develop-ment project that aims to assist the industries in not onlyauthenticating and confirming the rawmaterial used but alsothe quality of such raw material supplied
References
[1] I BurkillADictionary of Economic Products of theMalay Penin-sula vol 1 Ministry of Agriculture amp Cooperative MalaysiaKuala Lumpur Malaysia 1966
[2] S Sakdarat A ShuypromC Pientong T Ekalaksananan and SThongchai ldquoBioactive constituents from the leaves of Clinacan-thus nutans Lindaurdquo Bioorganic amp Medicinal Chemistry vol 17no 5 pp 1857ndash1860 2009
[3] Y K Yong J J Tan S S Teh et al ldquoClinacanthus nutansextracts are antioxidant with antiproliferative effect on culturedhuman cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 462751 8 pages 2013
[4] P Tuntiwachwuttikul Y Pootaeng-On P Phansa and W CTaylor ldquoCerebrosides and a monoacylmonogalactosylglycerolfrom Clinacanthus nutansrdquo Chemical amp Pharmaceutical Bul-letin vol 52 no 1 pp 27ndash32 2004
[5] C Kongkaew and N Chaiyakunapruk ldquoEfficacy of Clinacan-thus nutans extracts in patients with herpes infection system-atic review and meta-analysis of randomised clinical trialsrdquoComplementary Therapies in Medicine vol 19 no 1 pp 47ndash532011
[6] B Sriwanthana P Chavalittumrong andLChompuk ldquoEffect ofClinacanthus nutans onhuman cell-mediated immune responsein vitrordquoThai Journal of Pharmaceutical Sciences vol 20 no 4pp 261ndash267 1996
[7] VThongrakard and T Tencomnao ldquoModulatory effects ofThaimedicinal plant extract on proinflammatory cytokines-induced
apoptosis in human keratinocyte HaCat cellsrdquo African Journalof Biotechnology vol 9 no 31 pp 4999ndash5003 2010
[8] K-I Teshima T Kaneko K Ohtani et al ldquoC-glycosyl flavonesfrom Clinacanthus nutansrdquo Natural Medicines vol 51 no 6article 557 1997
[9] Y Zhang J Jiao C Liu X Wu and Y Zhang ldquoIsolation andpurification of four flavone C-glycosides from antioxidant ofbamboo leaves by macroporous resin column chromatogra-phy and preparative high-performance liquid chromatographyrdquoFood Chemistry vol 107 no 3 pp 1326ndash1336 2008
[10] F U Afifi E Khalil and S Abdalla ldquoEffect of isoorientinisolated from Arum palaestinum on uterine smooth muscle ofrats and guinea pigsrdquo Journal of Ethnopharmacology vol 65 no2 pp 173ndash177 1999
[11] Q F Huang S J Zhang L Zheng et al ldquoProtective effectof isoorientin-21015840-O-120572-l-arabinopyranosyl isolated from Gyp-sophila elegans on alcohol induced hepatic fibrosis in ratsrdquo Foodand Chemical Toxicology vol 50 no 6 pp 1992ndash2001 2012
[12] International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals forHuman Use Validation of Analytical Procedures Text andMethodology Q2(R1) 2005 httpwwwichorgfileadminPub-lic Web SiteICH ProductsGuidelinesQualityQ2 R1Step4Q2 R1 Guidelinepdf
[13] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of Separation Sciencevol 30 no 18 pp 3268ndash3295 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
The Scientific World Journal 5
Table 1 Linear range regression equations LOD and LOQ for quantitative analysis of HPLC (119899 = 3)
Compound 119879119877(min) 120582 (nm) Linear range (120583gmL) Regression equationa
1198772 LOD (120583gmL) LOQ (120583gmL)
Shaftoside 2279 271 337 200ndash200 119910 = 28370119909 minus 230484 1198772= 0999 02 06
Orientin 2558 255 349 08ndash100 119910 = 27655119909 minus 27061 1198772= 0996 04 08
Isovitexin 2716 269 337 08ndash100 119910 = 34067119909 + 47328 1198772= 0999 04 08
Vitexin 2861 268 337 08ndash100 119910 = 29217119909 + 33931 1198772= 0997 04 08
a119910 = 119886119909 + 119887 where 119909 is concentration in 120583gmL and 119910 is area under curve at UV 330 nm wavelength
Table 2 Intra- and interday precision recovery and accuracy data
Compound Precision (RSD RT AUC) RecoveryConcentration (120583gmL) Intraday (119899 = 3) Interday (119899 = 6) Amount added (120583gmL) Recovery () RSD ()
Shaftoside5 324 280 5 9533 21810 296 211 10 10210 34920 085 339 20 9550 074
Orientin5 324 294 5 10513 14310 296 192 10 9953 10320 085 308 20 9811 010
Isovitexin5 155 096 5 10197 29310 418 243 10 9767 35320 070 329 20 9649 053
Vitexin5 238 211 5 10261 12510 311 454 10 10150 21020 062 348 20 9552 143
AUC area under curve
Table 3 Content of shaftoside orientin isovitexin and vitexin in samples of C nutans from different locations L1 Taiping Perak L2 KotaTinggi Johor and L3 Sendayan Negeri Sembilan
Compound Content of marker compounds (mmolg)L1 L2 L3
Shaftoside 1743 plusmn 001 1633 plusmn 003 255 plusmn 004Orientin 086 plusmn 004 059 plusmn 0003 NDIsovitexin 201 plusmn 002 103 plusmn 009 NDVitexin 091 plusmn 003 043 plusmn 0006 NDND not detectedValues are means plusmn SD 119899 = 3
34 Sample Analysis The optimized and validated methodwas applied to determine the concentration of the markercompounds in the samples from the three geographicallocations The content of shaftoside orientin isovitexin andvitexin in samples is presented in Table 3 The results showedthat shaftoside was the major flavone present in samplesfrom all three locations in which concentration ranges from255mmolg to 1743mmolg The other known flavones C-glycosides were present in lower amounts ranging fromthe highest for isovitexin (000ndash201mmolg) followed byorientin (000ndash086mmolg) to the lowest amount whichwas vitexin (000ndash091mmolg) Flavones C-glycosides arefavourable chemical markers due to their large structuraldiversity that could help to distinguish rapidly between otherplant species [13]The results also allowed us to conclude thatshaftoside would be best suited as chemical markers for Cnutans rawmaterial as it is found in all locations in the highest
quantity The lower amount of flavones detected includingshaftoside in L3 compared to L1 and L2 could be caused bydifferences in climate soil regime of the different region anddifferences in processing such as harvest time drying andstorage all of which may affect the composition of flavonoids
4 Conclusion
Chemical markers are essential as part of identification andalso quality control of herbal material In the present studyHPTLC technique was used for the rapid identification offive known flavones C-glycosides in C nutans raw materialsIn HPTLC the unique characteristic fluorescent bands afterderivatization provided important clues for the identificationof the major flavone present in the samples A secondstep using HPLC-UVDAD technique was employed for thesimultaneous detection and quantification of these known
6 The Scientific World Journal
flavonoids The HPLC-UVDAD method developed in thispresent study was effective accurate precise and sensitivefor the quantification of four known flavones C-glycosides(shaftoside orientin isovitexin and vitexin) of interestThe method was also applied to identify and quantify thechemical markers of interest in crude powdered leaves of Cnutans from three geographical regions in Malaysia Fromthe present study the compound shaftoside could be recom-mended as a marker for C nutans rawmaterial as it is presentin all locations and is also available as a commercial standardfor method validationThe current method developed in thisstudy is also useful for the evaluation of quality of C nutansraw materials and its commercial products
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and Director of Insti-tute for Medical Research for giving permission to publishthis paper Special record of appreciation for the supportand funding given by the Ministry of Agriculture (MoA)under the Malaysian Herbal Monograph (MHM) Develop-ment project that aims to assist the industries in not onlyauthenticating and confirming the rawmaterial used but alsothe quality of such raw material supplied
References
[1] I BurkillADictionary of Economic Products of theMalay Penin-sula vol 1 Ministry of Agriculture amp Cooperative MalaysiaKuala Lumpur Malaysia 1966
[2] S Sakdarat A ShuypromC Pientong T Ekalaksananan and SThongchai ldquoBioactive constituents from the leaves of Clinacan-thus nutans Lindaurdquo Bioorganic amp Medicinal Chemistry vol 17no 5 pp 1857ndash1860 2009
[3] Y K Yong J J Tan S S Teh et al ldquoClinacanthus nutansextracts are antioxidant with antiproliferative effect on culturedhuman cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 462751 8 pages 2013
[4] P Tuntiwachwuttikul Y Pootaeng-On P Phansa and W CTaylor ldquoCerebrosides and a monoacylmonogalactosylglycerolfrom Clinacanthus nutansrdquo Chemical amp Pharmaceutical Bul-letin vol 52 no 1 pp 27ndash32 2004
[5] C Kongkaew and N Chaiyakunapruk ldquoEfficacy of Clinacan-thus nutans extracts in patients with herpes infection system-atic review and meta-analysis of randomised clinical trialsrdquoComplementary Therapies in Medicine vol 19 no 1 pp 47ndash532011
[6] B Sriwanthana P Chavalittumrong andLChompuk ldquoEffect ofClinacanthus nutans onhuman cell-mediated immune responsein vitrordquoThai Journal of Pharmaceutical Sciences vol 20 no 4pp 261ndash267 1996
[7] VThongrakard and T Tencomnao ldquoModulatory effects ofThaimedicinal plant extract on proinflammatory cytokines-induced
apoptosis in human keratinocyte HaCat cellsrdquo African Journalof Biotechnology vol 9 no 31 pp 4999ndash5003 2010
[8] K-I Teshima T Kaneko K Ohtani et al ldquoC-glycosyl flavonesfrom Clinacanthus nutansrdquo Natural Medicines vol 51 no 6article 557 1997
[9] Y Zhang J Jiao C Liu X Wu and Y Zhang ldquoIsolation andpurification of four flavone C-glycosides from antioxidant ofbamboo leaves by macroporous resin column chromatogra-phy and preparative high-performance liquid chromatographyrdquoFood Chemistry vol 107 no 3 pp 1326ndash1336 2008
[10] F U Afifi E Khalil and S Abdalla ldquoEffect of isoorientinisolated from Arum palaestinum on uterine smooth muscle ofrats and guinea pigsrdquo Journal of Ethnopharmacology vol 65 no2 pp 173ndash177 1999
[11] Q F Huang S J Zhang L Zheng et al ldquoProtective effectof isoorientin-21015840-O-120572-l-arabinopyranosyl isolated from Gyp-sophila elegans on alcohol induced hepatic fibrosis in ratsrdquo Foodand Chemical Toxicology vol 50 no 6 pp 1992ndash2001 2012
[12] International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals forHuman Use Validation of Analytical Procedures Text andMethodology Q2(R1) 2005 httpwwwichorgfileadminPub-lic Web SiteICH ProductsGuidelinesQualityQ2 R1Step4Q2 R1 Guidelinepdf
[13] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of Separation Sciencevol 30 no 18 pp 3268ndash3295 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
6 The Scientific World Journal
flavonoids The HPLC-UVDAD method developed in thispresent study was effective accurate precise and sensitivefor the quantification of four known flavones C-glycosides(shaftoside orientin isovitexin and vitexin) of interestThe method was also applied to identify and quantify thechemical markers of interest in crude powdered leaves of Cnutans from three geographical regions in Malaysia Fromthe present study the compound shaftoside could be recom-mended as a marker for C nutans rawmaterial as it is presentin all locations and is also available as a commercial standardfor method validationThe current method developed in thisstudy is also useful for the evaluation of quality of C nutansraw materials and its commercial products
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and Director of Insti-tute for Medical Research for giving permission to publishthis paper Special record of appreciation for the supportand funding given by the Ministry of Agriculture (MoA)under the Malaysian Herbal Monograph (MHM) Develop-ment project that aims to assist the industries in not onlyauthenticating and confirming the rawmaterial used but alsothe quality of such raw material supplied
References
[1] I BurkillADictionary of Economic Products of theMalay Penin-sula vol 1 Ministry of Agriculture amp Cooperative MalaysiaKuala Lumpur Malaysia 1966
[2] S Sakdarat A ShuypromC Pientong T Ekalaksananan and SThongchai ldquoBioactive constituents from the leaves of Clinacan-thus nutans Lindaurdquo Bioorganic amp Medicinal Chemistry vol 17no 5 pp 1857ndash1860 2009
[3] Y K Yong J J Tan S S Teh et al ldquoClinacanthus nutansextracts are antioxidant with antiproliferative effect on culturedhuman cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 462751 8 pages 2013
[4] P Tuntiwachwuttikul Y Pootaeng-On P Phansa and W CTaylor ldquoCerebrosides and a monoacylmonogalactosylglycerolfrom Clinacanthus nutansrdquo Chemical amp Pharmaceutical Bul-letin vol 52 no 1 pp 27ndash32 2004
[5] C Kongkaew and N Chaiyakunapruk ldquoEfficacy of Clinacan-thus nutans extracts in patients with herpes infection system-atic review and meta-analysis of randomised clinical trialsrdquoComplementary Therapies in Medicine vol 19 no 1 pp 47ndash532011
[6] B Sriwanthana P Chavalittumrong andLChompuk ldquoEffect ofClinacanthus nutans onhuman cell-mediated immune responsein vitrordquoThai Journal of Pharmaceutical Sciences vol 20 no 4pp 261ndash267 1996
[7] VThongrakard and T Tencomnao ldquoModulatory effects ofThaimedicinal plant extract on proinflammatory cytokines-induced
apoptosis in human keratinocyte HaCat cellsrdquo African Journalof Biotechnology vol 9 no 31 pp 4999ndash5003 2010
[8] K-I Teshima T Kaneko K Ohtani et al ldquoC-glycosyl flavonesfrom Clinacanthus nutansrdquo Natural Medicines vol 51 no 6article 557 1997
[9] Y Zhang J Jiao C Liu X Wu and Y Zhang ldquoIsolation andpurification of four flavone C-glycosides from antioxidant ofbamboo leaves by macroporous resin column chromatogra-phy and preparative high-performance liquid chromatographyrdquoFood Chemistry vol 107 no 3 pp 1326ndash1336 2008
[10] F U Afifi E Khalil and S Abdalla ldquoEffect of isoorientinisolated from Arum palaestinum on uterine smooth muscle ofrats and guinea pigsrdquo Journal of Ethnopharmacology vol 65 no2 pp 173ndash177 1999
[11] Q F Huang S J Zhang L Zheng et al ldquoProtective effectof isoorientin-21015840-O-120572-l-arabinopyranosyl isolated from Gyp-sophila elegans on alcohol induced hepatic fibrosis in ratsrdquo Foodand Chemical Toxicology vol 50 no 6 pp 1992ndash2001 2012
[12] International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals forHuman Use Validation of Analytical Procedures Text andMethodology Q2(R1) 2005 httpwwwichorgfileadminPub-lic Web SiteICH ProductsGuidelinesQualityQ2 R1Step4Q2 R1 Guidelinepdf
[13] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of Separation Sciencevol 30 no 18 pp 3268ndash3295 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
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