Research Article Simultaneous Quantification of Ten Active Components in Traditional Chinese Formula Sijunzi Decoction Using a UPLC-PDA Method

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1 Journl of Anlyticl Methods in Chemistry, Article ID , 8 pges Reserch Article Simultneous Quntifiction of Ten Active Components in Trditionl Chinese Formul Sijunzi Decoction Using UPLC-PDA Method Kng An, 1,2,3 Guo Jin-rui, 1 Zhng Zhen, 2 nd Wng Xio-long 1 1 School of Phrmcy, Nnjing University of Chinese Medicine, 138 Xinlin Rod, Nnjing , Chin 2 Discipline of Chinese nd Western Integrtive Medicine, Nnjing University of Chinese Medicine, 138 Xinlin Rod, Nnjing , Chin 3 Stte Key Lbortory of Nturl Medicines, Chin Phrmceuticl University, Nnjing , Chin Correspondence should be ddressed to Wng Xio-long; x.greg.wng@gmil.com Received 25 September 2013; Accepted 17 April 2014; Published 20 My 2014 Acdemic Editor: Xiu-Ping Yn Copyright 2014 Kng An et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Sijunzi decoction (SJZT), trditionl Chinese formul (TCMF) consisting of four herbs, hs been widely used for the tretment of vrious gstrointestinl symptoms. owever, its moderniztion process is hindered by the lck of powerful qulity control method tht covers the mjor ctive components in the formul. The im of this study ws to estblish UPLC method for the quntittive determintion of ten ctive components in Sijunzi decoction including ginsenoside Rg 1,Re,Rb 1, liquiritin, liquiritigenin, glycyrrhizic cid, trctylenolide I, trctylenolide II, trctylenolide III, nd pchymic cid. Seprtion ws chieved using n ACQUITY UPLC BEC 18 column (2.1 mm 100 mm, 1.7 μm) with grdient elution progrm consisting of cetonitrile nd 0.1% phosphoric cid solution. The detection wvelengths were set t 203, 254, 222, nd 267 nm. The method ws vlidted for linerity, ccurcy, precision, limit of detection, nd limit of quntifiction. The vlidted method ws successfully pplied to the simultneous quntifiction of ten ctive compounds from severl finished btches of SJZT. This vlidted tht UPLC method is expected to provide new bsis for the qulity control of SJZT. 1. Introduction Trditionl Chinese herbl formultion (TCMF) hs been widely used in the clinic for its well-proven efficcy with few side effects. Sijunzi decoction (SJZT) is one of the most fmous TCMFs consisting of four herbs: Rdix Ginseng, Pori cocos, Rhizom Atrctylodis Mcrocephle, nd Rdix Glycyrrhize. In Chin, SJZT hs long been used for the tretment of gstrointestinl disorders such s chronic gstritis nd gstric nd duodenl ulcer, nd it could effectively ttenute nuse, vomiting, nd dirrhe [1]. Clinicl studies show tht SJZT could effectively restore the homeostsis of the digestive trct in ptients [2]. More recently, SJZT hs been shown to meliorte the intestinl flor disturbnce in rt models of spleen deficiency syndrome [3]. Moreover, emerging evidences re showing tht SJZT nd modified SJZT could ply good supporting role in suppressing tumors nd confer protective effect on gstrointestinl mucos dmge induced by chemotherpy [4]. Supporting these well-confirmed phrmcologicl efficcies, recent yers hve seen n incresing knowledge of the chemicl components from SJZT. PLC-MS ws exploited to nlyze the mjor components of SJZT, nd eight ginsenosides (ginsenosides Rg 1,Re,Rf,Ro,Rb 1,Rc,Rb 2,nd Rd) nd glycyrrhizic cid were identified through structurl elucidtion [1]. Recently, by employing the UPLC-Q-TF- MS technique, 66 phytochemicl compounds were detected in Sijunzi decoction formul nd 58 of them including ginsenosides, flvonoids, triterpenoid, nd coumrins were tenttively identified by compring the ccurte mss nd frgment informtion with the correltive references dt [5]. It should be noted tht the constituents nd contents of the

2 2 Journl of Anlyticl Methods in Chemistry min ctive components existing in SJZT my be influenced by hrvest time, plnt origin, nd mnufcturing procedures, which could significntly ffect the phrmcologicl effects nd necessitte the qulity ssessment of SJZT. Undoubtedly, it is not esy to simultneously determine ll the components existing in the formul. owever, simultneous nlysis of the min ctive components my be one possible solution. Previous studies hve quntittively determined the min components existing in the four individul herbs of SJZT, nmely, Rdix Ginseng [6, 7], Pori cocos [8], Rhizom Atrctylodis Mcrocephle[9, 10], nd Glycyrrhiz urlensis [11 13], by using PLC or UPLC methods. owever, stisfctory quntittive method of the mjor ctive components in SJZT for qulity control purposes is not vilble. Simultneous nlysis for the min ctive compounds in ech herb of SJZT hs been suggested s one possible solution. The im of this reserch ws to develop convenient, relible, nd sensitive nlyticl method to determine the quntity of mjor compounds in SJZT by using ultrperformnce liquid chromtogrphy (UPLC). Specificlly, ginsenoside Rg 1, Re, Rb 1, liquiritin, liquiritigenin, glycyrrhizic cid, trctylenolide I, trctylenolide II, trctylenolide III, nd pchymic cid were selected s the mrker constituents for the reltively high contents in the individul herbs nd their vlidted phrmcologicl effects, such s ntiinflmmtion, brin protection effects, ntioxidtion effect, nd hypoglycemic effect [1, 5]. The potentil ppliction of thisstudycouldnotonlysupportqulitycontrolofsjztbut lso provide theoreticl bsis for further in-depth reserch of SJZT in clinicl reserch. 2. Experimentl 2.1. Regents nd Chemicls. The four crude herbs, Rdix Ginseng, Pori cocos, Rhizom Atrctylodis Mcrocephle, nd Glycyrrhize urlensis, were purchsed from Nnjing Trffic ospitl (Nnjing, Chin). All smples were identified by one of the uthors (Professor Wng Xio-Long) s uthentic herbl medicine. Ginsenoside Rg 1,Re,ndRb 1 were purchsed from Jilin University (Chngchun, Chin); liquiritin, liquiritigenin, nd glycyrrhizic cid were purchsed from ChineseFoodndDrugInspectionInstitute;Atrctylenolide III, Atrctylenolide I, Atrctylenolide II, nd pchymic cid were purchsed from Sichun Weikeqi Biologicl Co., Ltd (Chengdu, Chin). The ten compounds used in the nlysis were of nlyticl grde nd their purity ws more thn 98%. Their chemicl structures re shown in Figure1.Acetonitrile nd methnol (PLC grde) were purchsed from Fisher Scientific (Wlthm, MA, USA); phosphoric cid (nlyticl grde) ws purchsed from Nnjing Chemicl Regents Compny (Nnjing, Chin); wter ws purified by Millipore Milli-Q system (Millipore, MA, USA); other regents nd chemicls were ll obtined from vrious commercil sources nd were of nlyticl grde Chromtogrphic Anlysis. The nlytes were seprted on n ACQUITY UPLC -clss system with PDA detector using 2.1 mm 100 mm 1.7 μm ACQUITY BE C18 column with flow rte of 0.3 ml min 1.Seprtionws chieved using grdient method. Acetonitrile (A) nd 0.1% phosphoric cid (B) were selected nd the grdient solvent system ws s follows: 0 5 min, (A) 19%; 5 13 min, 19% 30%; min, 30% 50%; min, 50% 60%; min, (A) 60% 68%; min, (A) 68% 19%; nd min, (A) 19%. The injection volume of smples ws 5 μl nd temperture of the column oven ws mintined t 30 C. The UV wvelength ws set t 203 nm (for ginsenoside Rg 1, Re, nd Rb 1 ), 254 nm (for liquiritin, glycyrrhizic cid, nd liquiritigenin), 222 nm (for trctylenolide I nd III), 276 nm (for trctylenolide II), nd 242 nm (for pchymic cid) Preprtion of SJZT Smples. According to the originl composition of SJZT, the four constituting herbs including Rdix Ginseng (100 g), Pori cocos (100 g), Rhizom Atrctylodis Mcrocephle(100 g), nd Glycyrrhize urlensis (50 g) werecrushedintosmllpiecesndthenmixednddecocted twicein3500mlwterfor1hinglssflsk.thedecoction ws filtered through 8 lyers of guze; the filtrte ws concentrted in vcuum t 60 C t finl concentrtion of 2 g/ml. Ethnolwsddedtonliquotof5mLSJZTovernight in order to remove the polyscchrides. The superntnt ws trnsferred into test tube nd evported to dryness with vcuum t room temperture. Finlly, the residue ws reconstituted in 5 ml methnol by vortex mixing for 5 min nd centrifuged t 16,000 rpm for 10 minutes. 5 μl superntnt ws injected into chromtogrphic systems for nlysis Preprtion of Stndrd Solutions. The stndrd stock solutions of ginsenoside Rg 1 (30 μg/ml), ginsenoside Re ( μg/ml), ginsenoside Rb 1 (30 μg/ml), liquiritin (9 μg/ml), liquiritigenin (87.00 μg/ml), glycyrrhizic cid (11 μg/ml), trctylenolide III (59.00 μg/ml), trctylenolide I (71.40 μg/ml), trctylenolide II (50.60 μg/ml), nd pchymic cid (20 μg/ml) were prepred in methnol. These solutions were stored t 4 C ndwerestblefortlest1month Preprtion of Negtive Control Smples of SJZT. The negtive control smples of SJZT were prepred by deriving one herb from the prescriptions. The herbs were ccurtely weighed ccording to the prescription of SJZT nd prepred with the sme procedure s for the smple preprtion Method Vlidtion. Specificity, linerity, limit of detection (LD), limit of quntifiction (LQ), precision (repetbility nd intr- nd interssy), nd ccurcy (recovery) of this UPLC method were evluted in ccordnce with Interntionl Conference on rmoniztion (IC) [14]. The stndrd clibrtion curve for the linerity ssy ws prepred with seven different concentrtions of diluted stndrd solutions (ginsenoside Rg 1, ginsenoside Re, ginsenoside Rb 1, liquiritin, glycyrrhizic cid, liquiritigenin, trctylenolide I, trctylenolide II, trctylenolide III, nd

3 Journl of Anlyticl Methods in Chemistry 3 glc- -glc Liquiritigenin glc Liquiritin Ginsenoside Rg 1 glc- -glc-rh Ginsenoside Re glc-glc- Glycyrrhizic cid glc-glc- Ginsenoside Rb 1 Pchymic cid Atrctylenolide I Atrctylenolide III Atrctylenolide II Figure 1: The chemicl structure of ginsenoside Rg 1,Re,Rb 1, liquiritin, liquiritigenin, glycyrrhizic cid, trctylenolide I, trctylenolide II, trctylenolide III, nd pchymic cid. pchymic cid). The lower limit of quntifiction (LLQ) ws determined s the lowest concentrtion point of the stndrd curve nd the signl-to-noise rtio ws higher thn 10. The lower limit of detection (LLD) ws defined s the mount tht could be detected with signl-to-noise rtio of 3. The precision of the nlyticl method ws evluted by intrbtch nd interbtch vribility. Three different concentrtions of stndrds (low, medium, nd high) were prepred. The quntity of ech component ws determined bytherespectiveclibrtioncurve.rsdwsusedtomesure precision. The interbtch reproducibility test ws crried out on three different btches. Recovery studies were crried out by spiking three concentrtions of mixed stndrds t low (50% of the known

4 4 Journl of Anlyticl Methods in Chemistry A 254 nm b c b c C 222 nm b c D 276 nm 9 b c B 203 nm E 242 nm 10 b c Figure 2: Ultrperformnce liquid chromtogrphy (UPLC) chromtogrms t different wvelengths of stndrd mixture (), SJZT extrction (b), nd negtive control smples (c). Peks: (1) liquiritin, (2) liquiritigenin, (3) ginsenoside Rg 1, (4) ginsenoside Re, (5) ginsenoside Rb 1,(6) glycyrrhizic cid, (7) trctylenolide III, (8) trctylenolide I, (9) trctylenolide II, nd (10) pchymic cid. mounts), medium (100% of the known mounts), nd high (200%oftheknownmounts)inthe5mLofSJZT.Then, the spiked smples were then extrcted, processed, nd quntified in ccordnce with the methods mentioned bove. 3. Results nd Discussion 3.1. ptimiztion of the UPLC Conditions. ptimiztion of the seprtion conditions for PLC nlysis ws performed including the mobile phse composition, grdient elution progrm, nd wvelength. To obtin chromtogrms with better resolution of djcent peks within shorter time, the chromtogrphic conditions were optimized. Methnol nd cetonitrile were compred in the experiment. The result showed tht cetonitrile ws much better s it could result in better resolution nd shorter time for nlysis. In ddition, wter nd 0.1% phosphoric cid/wter were investigted nd the result showed tht 0.1% phosphoric cid/wter ws better thn wter. As result ACQUITY UPLC BE C 18 column (2.1 mm 100 mm, 1.7 μm) with cetonitrile nd

5 Journl of Anlyticl Methods in Chemistry 5 Tble 1: The liner regression dt, LDs, nd LQs of ten compounds. 2 Components Regression equtions R Liner rnge (μg/ml) LDs (μg/ml) LQs (μg/ml) Ginsenoside Rg 1 y = x Ginsenoside Re y = x Ginsenoside Rb 1 y = 1465x Liquiritin y = x Liquiritigenin y = x Glycyrrhizic cid y = x Atrctylenolide III y = 34419x Atrctylenolide I y = 18610x Atrctylenolide II y = 40016x Pchymic cid y = x y=ax+b; y is pek re; x is concentrtion of the nlytes (μg/ml); r is the correltion coefficient of the eqution. Tble 2: Precision, repetbility, nd stbility of ten compounds in SJZT (n =6). Anlytes Precision Levels (ug/ml) Intrdy RSD (%) Interdy RSD (%) Ginsenoside Rg % % % 0.29% % 1.04% % 0.22% Ginsenoside Re % 0.42% % 0.93% Ginsenoside Rb % 0.16% % 0.22% % 0.98% % 0.70% Liquiritin % % % 1.03% % 0.70% Liquiritigenin % 0.49% % 0.31% % 1.04% Glycyrrhizic cid % 1.13% % 0.72% % 0.46% Atrctylenolide III % 0.50% % 0.83% % 0.58% Atrctylenolide I % 0.97% % 0.72% % 0.64% Atrctylenolide II % 0.62% % 0.72% % 0.48% Pchymic cid % 1.10% % 0.37% RSD: reltive stndrd devition. Repetbility RSD (%) Stbility RSD (%) 0.98% 1.01% 0.87% 1.04% 1.20% 0.92% 0.48% 0.57% 0.27% 0.92% 0.28% 0.73% 0.63% 0.84% 0.81% 0.92%

6 6 Journl of Anlyticl Methods in Chemistry Components Contents (ug/ml) Tble 3: Recovery of eight components in Sijunzi (n =3). Quntity dded (ug/ml) Theoreticl mount (ug/ml) Recorded mount (ug/ml) Recovery (%) RSD (%) Ginsenoside Rg Ginsenoside Rb Ginsenoside Re Liquiritin Liquiritigenin Glycyrrhizic cid Atrctylenolide III Atrctylenolide I Recovery (%) = (recorded mount originl mount)/spiked mount 100%. 0.1% phosphoric cid/wter ws selected s the preferred chromtogrphic conditions. Moreover, different grdient profiles were lso optimized. Actully, we tried to simplify the grdient elution system nd shorten the nlysis time, but peks for trctylenolide III nd trctylenolide I hve not been completely seprted except for the current condition to in the grdient progrm mentioned bove. In this experiment, the specificity of UV bsorption ws lso investigted, using the present chromtogrphic conditions nd compring SJZT smple with stndrd mixture. The UV bsorbnce nd the best UV detection wvelength of ech compound in SJZT were confirmed s follows: ginsenoside Rg 1,Re,ndRb 1 (203 nm), liquiritin, glycyrrhizic cid, nd liquiritigenin (254 nm), trctylenolide I nd trctylenolide III (222 nm), trctylenolide II (276 nm), nd pchymic cid (242 nm) Vlidtion of the UPLC Method. The vlidtion study llowed the evlution of the method for its suitbility for routine nlysis Specificity. Representtive chromtogrms of the stndrd solution, smple solution, nd negtive control smples t different UV wvelength were shown in Figure 2. The chromtogrphic peks were identified by compring their retention time with tht of ech reference compound. In ddition, chromtogrms of the negtive control smples further confirmed the specificity of this method Clibrtion Curves, LDs, nd LQs. The linerity of the developed method ws ssessed using seven different concentrtions ech of ginsenoside Rg 1, ginsenoside Re, ginsenoside Rb 1, liquiritin, glycyrrhizic cid, liquiritigenin, trctylenolide I, trctylenolide II, trctylenolide III, nd pchymic cid nd the observed concentrted rnges were s follows: , , , , , , , , , nd μg/mL, respectively. The correltion coefficient (R 2 ) of ll biomrkers hs good linerity of over > in the forementioned rnges. The LDs nd LQs of the ten nlytes were nd μg/ml, respectively (Tble 1).

7 Journl of Anlyticl Methods in Chemistry 7 Tble 4: Contents of eight components in three btches of SJZT. Contents (mg/g) Smple Source Ginsenoside Ginsenoside Ginsenoside Liquiritin Liquiritigenin Glycyrrhizic Atrctylenolide Rg 1 Re Rb 1 cid III Atrctylenolide I 1 Btch Btch Btch Precision, Repetbility, nd Stbility. Intrdy nd interdy vritions were chosen to determine the precision of the developed ssy. The nlyzed dt showed tht reltive stndrd devition (RSD) of intr- nd interdy ws in the rnge of % nd 1.10% (n =6), the RSD of the SJZTrepetbility,ndstbilitytestwsintherngeof 0.89% nd 0.57% 1.04%, respectively (Tble 2). None of the precision, repetbility, nd stbility dt exceeded 5%, except trctylenolide II nd pchymic cid (the content of those two components were lower thn the LDs) Accurcy. The ccurcy of the method ws ssessed by recovery ssy. The spiked smples were then extrcted, processed, nd quntified in ccordnce with the methods mentioned bove. The mesured dt showed tht the recovery of the investigted components rnged from 95.07% to %, nd their RSD vlues were ll less thn 3.0% (Tble 3). Recovery dt represented the ccurcy of the method nd is sufficient for usul nlysis Applictions. The estblished nlyticl method ws subsequently pplied for the simultneous determintion of the ten mrkers in 3 btches of SJZT. The results re presented in Tble 4. The results showed tht there re remrkble differences mong the contents of the ten components in SJZT from the sme or different btches. 4. Conclusion In this study, UPLC method for the simultneous determintion of ten ctive ingredients in SJZT hs been developed nd the results showed tht it could be used for the qulity controlofthesjzt.thus,thisvlidtedthtuplcmethod could be expected to provide new bsis for the qulity control of SJZT. Conflict of Interests The uthors declre tht there is no conflict of interests regrding publiction of this pper. Authors Contribution Kng An nd Guo Jin-rui contributed eqully to this work. Acknowledgments This study ws finncilly supported by the Youth Nturl Science Fund of Nnjing University of Chinese Medicine (no. 11XZR09), Ntionl Nturl Science Fund of Chin (no ), pen Project Progrm of Stte Key Lbortory of Nturl Medicines, Chin Phrmceuticl University (no. SKLNMKF201209), nd the Priority Acdemic Progrm Development of Jingsu igher Eduction Institutions (PAPD). References [1] Y. Liu, J. Yng, nd Z. Ci, Chemicl investigtion on Sijunzi decoction nd its two mjor herbs Pnx ginseng nd Glycyrrhiz urlensis by LC/MS/MS, Journl of Phrmceuticl nd Biomedicl Anlysis,vol.41,no.5,pp ,2006. [2]J..Guo,G.Chen,S.Q.Yng,M..Wei,ndX.Chen, Clinicl observtion of the role of Chenxi Sijunzi decoction in promoting the recovery of gstrointestinl function in criticlly ill ptients, Zhongguo Wei Zhong Bing Ji Jiu Yi Xue,vol.24,no. 11, pp , [3] Z.Wng,Y.Peng,ndX.B.Li, Effectofsijunzidecoctiononthe intestinl flor disturbnce in two rt models of Pi-deficiency syndrome, Zhongguo Zhong Xi Yi Jie e Z Zhi, vol. 29, no. 9, pp , [4] C.Ling,S..Zhng,ndZ.D.Ci, Effectsoferlyintestinl ppliction of sijunzi decoction on immune function in postopertionl ptients of gstrointestinl tumor, Zhongguo Zhong XiYiJieeZZhi,vol.25,no.12,pp ,2005. [5] Y.Wng,S.e,X.Cheng,Y.Lu,Y.Zou,ndQ.Zhng, UPLC- Q-TF-MS/MS fingerprinting of Trditionl Chinese Formul SiJunZiTng, JournlofPhrmceuticlndBiomediclAnlysis,vol.80,pp.24 33,2013. [6] P. N. Brown, R. Yu, T. Cin et l., Determintion of ginsenoside content in Pnx ginseng C.A. Meyer nd Pnx quinquefolius L. root mterils nd finished products by high-performnce liquid chromtogrphy with ultrviolet bsorbnce detection: interlbortory study, Journl of AAC Interntionl, vol. 96, no. 1, pp , [7] X. Jin, L. Y. Zhu,. Shen et l., Influence of sulphur-fumigtion on the qulity of white ginseng: quntittive evlution of mjor ginsenosides by high performnce liquid chromtogrphy, Food Chemistry,vol.135,no.3, pp ,2012. [8]S.Che,Q.Li,Y.-S.uo,X..Chen,ndK.S.Bi, RP- PLC simultneous determintion of five triterpenoid cids in different prts of Pori cocos by UV wvelengths switch, Yoxue Xuebo,vol.45,no.4,pp ,2010.

8 8 Journl of Anlyticl Methods in Chemistry [9] Q. Chen,. e, P. Li, J. Zhu, nd M. Xiong, Identifiction nd quntifiction of trctylenolide I nd trctylenolide III in Rhizom Atrctylodes Mcrocephl by liquid chromtogrphyion trp mss spectrometry, Biomedicl Chromtogrphy, vol. 27,no.6,pp ,2013. [10].P.Chen,J..Zhng,X.Y.Wng,ndL.Chen, Contents comprision of trctylode I, II, III in rhizom trctylodis mcrocephle nd the processed with soils, Zhong Yo Ci, vol. 34, no. 3, pp , [11] Y. P. Wu, X. S. Meng, Y. R. Bo, S. Wng, nd T. G. Kng, Simultneous quntittive determintion of nine ctive chemicl compositions in trditionl Chinese medicine Glycyrrhiz by RP-PLC with full-time five-wvelength fusion method, The Americn Journl of Chinese Medicine,vol.41,no.1,pp , [12] Y. T. Kml, M. Singh, E. T. Tmboli, R. Prveen, S. M. Zidi, nd S. Ahmd, Rpid RP-PLC method for the quntifiction of glbridin in crude drug nd in polyherbl formultion, Journl of Chromtogrphic Science,vol.50,no.9,pp ,2012. [13]S.Gupt,R.Shrm,P.Pndotr,S.Jgln,ndA.P.Gupt, Chromolithic method development, vlidtion nd system suitbility nlysis of ultr-sound ssisted extrction of glycyrrhizic cid nd glycyrrhetinic cid from Glycyrrhiz glbr, Nturl Product Communictions, vol.7,no.8,pp , [14] Interntionl conference of hrmoniztion Q2B: Vlidtion of nlyticl procedures methodology. US FDA Federl Register, pp , 1997.

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