Research Article Preparative Purification of Liriodendrin

Research ArticlePreparative Purification of Liriodendrin fromSargentodoxa cuneata by Macroporous Resin

Di-Hua Li1 Yan Wang2 Yuan-Shan Lv1 Jun-Hong Liu1 Lei Yang1

Shu-Kun Zhang1 and Yu-Zhen Zhuo1

1Tianjin Institute of Acute Abdominal Diseases of Integrated Traditional Chinese and Western Medicine Tianjin 300100 China2College of Pharmacy Tianjin University of Traditional Chinese Medicine Tianjin 300193 China

Correspondence should be addressed to Di-Hua Li dhli2013163com

Received 4 May 2015 Revised 23 June 2015 Accepted 24 June 2015

Academic Editor Gail B Mahady

Copyright copy 2015 Di-Hua Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The preparative purification of liriodendrin from Sargentodoxa cuneata using macroporous resin combined with crystallizationprocess was evaluated The properties of adsorptiondesorption of liriodendrin on eight macroporous resins were investigatedsystematically X-5 resin was selected as the most suitable medium for liriodendrin purification The adsorption of liriodendrin onX-5 resin fitted well with the pseudo-second-order kinetic model and Langmuir isotherm model Dynamic adsorptiondesorptiontests were performed using a glass column packed with X-5 resin to optimize the separation process of liriodendrin After onetreatment with X-5 resin the content of liriodendrin in the product was increased 4873-fold from 085 to 4142 with arecovery yield of 889 9748 liriodendrin was obtained by further crystallization and determined by HPLC The purifiedproduct possessed strong antioxidant activity In conclusion purification of liriodendrin might expend its further pharmacologicalresearches and further applications in pharmacy

1 Introduction

Sargentodoxa cuneata (Lardizabalaceae) is a well-known herbof traditional Chinesemedicine in China for treating abdom-inal pain due to acute appendicitis sore and ulcer dys-menorrheal amenorrhea traumatic swelling and rheumaticarthritis Modern studies have demonstrated that it pos-sesses various pharmacological effects such as antiviral [12] antibacterial [3] antioxidant [4 5] antitumor [6] anti-inflammatory [7] resistance myocardial ischemia [8] andantithrombotic [9] Among various active ingredients lirio-dendrin (Figure 1) has been proved to be one of the bioactivecompounds due to possessing of anti-inflammatory antinoci-ceptive [10] antiarrhythmic [11] antimyocardial ischemia [8]calcium channel antagonistic [12] inhibitory activity onHepG-2 cells [13] resisting glutamate-induced PC12 celldamage [14] increasing of heat shock factor 1 expression [15]and protecting SH-SY5Y cell effects [16]

Because of these beneficial pharmacological effects ofliriodendrin large quantities of pure compound are neededas chemical reference standard for further pharmacological

researches and further applications in pharmacy Thereforemany preparative purification methods of liriodendrin aredeveloped such as high-speed counter-current chromatog-raphy [17] HPLC [18] and silica gel column chromatography[10] But these methods have several disadvantages such ashaving large amount of organic solvents wastage being timeconsuming having poor yield and recovery or needing spe-cial instruments and are not suitable for large-scale industrialproduction Macroporous resins are one kind of durablehydrophilic polymer with advantages of good stability highadsorption capacity and selectivity fast adsorption and des-orption of mild conditions easy regeneration and saving ofthe cost The principle of adsorption is based on electrostaticforce hydrogen bonding interaction and size sieving actionbetween macroporous resins and different molecular fromthe solution [19] In recent years macroporous resins arewidely used as medium to enrich and separate the secondarymetabolites of traditional Chinesemedicine and nature plantincluding flavonoids [20 21] coumarins [22] terpenoids [23]lignans [24] and phenolics [25 26]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 861256 9 pageshttpdxdoiorg1011552015861256

2 BioMed Research International

O

O

OO

HOOH

OH

OH

O

O

OHHO

HO

OH

H3CO

OCH3

OCH3

OCH3

Figure 1 Chemical structure of liriodendrin

Liriodendrin after oral administration was in vivo trans-formation to syringaresinol which possesses strong antioxi-dant activity [10] However the antioxidant capacity of lirio-dendrin in vitro has not been investigated recently As a partof continuing research of expanding its further applicationsthe antioxidant activity of liriodendrin in vitrowas examined

In this study a simplemethod is developed for preparativepurification of liriodendrin from the stem of S cuneata usingmacroporous resin chromatography combined with crystal-lization process By static and dynamic adsorptiondesorp-tion tests on resins some process parameters such as feedconcentration and volume of sample solution flow rateethanol concentration and volume of desorption solutionare optimized for purification of liriodendrin After furthercrystallization liriodendrin is an obtained pure form andits structure is identified by NMR Moreover antioxidantactivity of liriodendrin is also evaluated in vitro for the firsttime

2 Materials and Methods

21 Materials and Reagents Liriodendrin standard was iso-lated from the stem of S cuneata in our previous study Thepurity was determined to be over 97 by normalization ofthe peak areas by HPLC Its structure was elucidated bycomparison of the NMR data with the literature [27] HPLC-grade acetonitrile and formic acidwere bought fromConcordTechnology Co Ltd (Tianjin China) 11-Diphenyl-2-picryl-hydrazyl (DPPH) and 221015840-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) were obtainedfrom sigmaChemical Co (Sigma-AldrichGmbH SternheimGermany) Other chemicals and reagents were analyticalgrade Deionized water was purified by a Milli-Q waterpurification system (Millipore Boston USA)

22 Adsorbents Macroporous resins including D101 AB-8X-5ADS-17 andNKAwere purchased fromNankaiHechengSampT Co Ltd (Tianjin China) and HPD100 HPD400 andHPD600 fromCangzhou BonAdsorber Technology Co Ltd(Hebei China) respectively Their physical properties weresummarized in Table 1 Before use these resins were soakedin 95 vv ethanol aqueous solutions for 24 h then washedwith 4 HCl aqueous solution deionized water and 4

Table 1 Physical properties of macroporous resins in test

Resin Average porediameter (nm)

Surface area(m2g) Polarity

AB-8 13-14 450ndash530 Weakly polarD101 10ndash12 600ndash700 NonpolarNKA 20ndash22 570ndash590 NonpolarX-5 29-30 500ndash600 NonpolarADS-17 25ndash30 90ndash150 Middle-polarHPD100 85ndash9 650ndash700 NonpolarHPD400 75ndash8 500ndash550 Middle-polarHPD600 8 550ndash600 Strong-polar

NaOHaqueous solution respectively andfinallywashedwithdeionized water

23 Preparation of Sample The dried stem of S cuneatawas powdered and sieved through a number 40 mesh Thepowder of sample (10 kg) was extracted with 8000mL of 95vv ethanol aqueous solutions under reflux for 90min andrepeated twice The extracted solutions were combined andfiltered and the filtrate was evaporated to dryness by a rotaryevaporator under reduced pressure at 60∘C The residue wassuspended in deionized water and suspended solution wascentrifuged at 3000 rpm for 30minThe supernatantwas usedas stock solution for the subsequent test

24 HPLC Analysis of Liriodendrin The HPLC system con-sisted of a SSI PC2000 chromatograph (SSI Scientific Sys-tems Inc USA) and a 2000ES evaporative light scatteringdetector (ELSD) (Alltech Alltech Associates Inc USA)An ODS-2 Hypersil C-18 column (250mm times 46mm id5 120583mThermo ScientificThermo Fisher Scientific Inc USA)was operated at 35∘C Solvents that constituted the mobilephase were acetonitrile (A) and 02 formic acid aqueoussolution (B) The separation was performed using a stepwisegradient elution of 0ndash14min 95ndash87 B 14ndash25min 87ndash87B and 25ndash45min 87ndash81 B then keeping 95 B to balancefor 10min The flow rate was 09mLmin ELSD was usedas the detection method with nebulizing gas flow rate of25 Lmin and drift tube temperature of 105∘C The impactor

BioMed Research International 3

position of ELSD was set off The stock standard solutionof liriodendrin was prepared and diluted six concentrationsof 37ndash370120583gmL It showed a good linearity over range of074ndash74120583g for liriodendrinThe regression equation for lirio-dendrin was 119884 = 163681119883 + 540736 (1198772 = 09992 119899 = 6)where 119884 is the logarithm peak area and 119883 is the logarithmmass of liriodendrin

25 Static Adsorption and Desorption Tests

251 Adsorption Resins Screening All macroporous resinswere screened through static adsorption test Pretreated resin(equivalent to 1 g dry resin) was put into a 50mL Erlenmeyerflask with stopper and 10mL aqueous solution of S cuneataextracts in Section 23 was added The flasks were shaken(180 rpm) for 8 h at 25∘C After adsorption equilibrium wasreached resins were washed by deionized water Then theresins were desorbed with 10mL 95 vv ethanol aqueoussolutions and the flasks were shaken (180 rpm) for 8 h at25∘C The solutions after adsorption and desorption weredetermined by HPLC The experiments were repeated threetimes for each resinThe following equationswere used to cal-culated adsorption capacity adsorption ratio and desorptionratio

Adsorption capacity is

119876119890=

119881119894(1198620 minus 119862119890)

119882

(1)

Adsorption ratio is

119864 =

1198620 minus 1198621198901198620times 100 (2)

where119876119890is the adsorption capacity at the adsorption equilib-

rium (mgg dry resin) 119864 is the adsorption ratio () 119881119894is the

initial volume of feed solution (mL) 1198620 and 119862119890 are the initialand equilibrium concentration of liriodendrin in solutionsrespectively (mgmL) and119882 is theweight of the dry resin (g)

Desorption ratio is

119863 =

119862119889119881119889

119881119894(1198620 minus 119862119890)

times 100 (3)

where 119863 is the desorption ratio () 119862119889is the concentration

of liriodendrin in the desorption solution (mgmL)119881119889is the

volume of the desorption solution (mL)1198811198941198620 and119862119890 are the

same as defined above

252 Adsorption Kinetics The adsorption kinetics of D101and X-5 resins were studied by mixing 10mL aqueous solu-tion of S cuneata extracts (the initial concentration of lirio-dendrin 2131mgmL) with pretreated resin (1 g dry resin)in each 50mL Erlenmeyer flask with stopper and shaken(180 rpm) at 25∘C Liriodendrin concentration in the solutionphase was monitored by HPLC at different time intervalsuntil equilibrium To examine the underlying mechanismof the adsorption kinetic process equations of pseudo-first-order model pseudo-second-order model and intraparticle

diffusion model were used to describe adsorption process[23]

Equation of pseudo-first-order kinetic model is

ln (119876119890minus119876119905) = ln119876

119890minus 1198961119905 (4)

Equation of pseudo-second-order kinetic model is

119905

119876119905

=

119905

119876119890

+

1119876119890

21198962 (5)

Equation of intraparticle diffusion kinetic model is

119876119905= 11989611989411990505+119862 (6)

where 119876119890and 119876

119905are the adsorption capacity at equilibrium

and at any time 119905 (mgg dry resin) respectively 1198961 (minminus1)1198962 (gmgsdotmin) and 119896

119894(mggsdotmin05) are the rate constant

for the adsorption process 119862 is the constant indicating theboundary layer thickness (mgg)

253 Adsorption Isotherms The equilibrium adsorptionisotherms of liriodendrin onD101 andX-5 resinswere studiedby the following experiment D101 or X-5 pretreated resin(1 g dry resin) and 10mL aqueous solution of S cuneataextracts (the initial concentration of liriodendrin at 02130426 0852 1279 1705 and 2131mgmL) were added intoa 50mL Erlenmeyer flask with stopper respectively andshaken (180 rpm) for 8 h at 25∘CThe equilibrium concentra-tion of liriodendrin was analyzed For further explanation ofhow molecules or ions of adsorbate interact with adsorbentsurface site the equilibrium adsorption isotherms of D101and X-5 resins were described by Freundlich and Langmuirequations respectively Freundlich equation described theadsorption behavior of the monomolecular layer as well asmultimolecular layer and Langmuir equation only describedthe monolayer adsorption [28 29]

Freundlich equation is

119876119890= 1198701198651198621119899119890 (7)

Langmuir equation is

119876119890=

11987601198701198711198621198901 + 119870119871119862119890

(8)

where 119870119865is the Freundlich constant an indicator of adsorp-

tion capacity 1119899 is an empirical constant related to mag-nitude of the adsorption driving force 1198760 is the theoreticalmaximum adsorption capacity (mgg dry resin) 119870

119871is the

Langmuir adsorption equilibrium constant119876119890and119862

119890are the

same parameters as in (1)

26 Dynamic Adsorption and Desorption Tests Dynamicadsorptiondesorption tests were performed with a glasscolumn (20mm times 300mm) wet-packed of X-5 resin (6 g dryresin) The bed volume (BV) was 30mL For the dynamicbreakthrough experiment aqueous solution of S cuneataextracts was loaded into the glass column at a prescribedflow rate (1 2 and 4 BVh) respectively The liriodendrin


concentrations in the effluents that were collected at 10mLintervals were determined by HPLC

For dynamic desorption experiment aqueous solutionof S cuneata extracts was loaded into the resin columnmentioned above After adsorption equilibrium the resincolumnwas washed first with 2 BV deionized water and thendesorption was conducted with different concentrations ofethanol aqueous solutions (10 20 30 40 50 60 70 80 and95 vv) at a flow rate of 1 BVh successively The volumeof each ethanol aqueous solution was 3 BV and each ethanoldesorption solution was analyzed by HPLC For the quantityof ethanol elution the test was performed as follows whenthe adsorption equilibrium was reached the resin columnwas washed first with 2 BV deionized water and then waseluted with 6 BV 40 vv ethanol aqueous solutions at a flowrate of 1 BVh The elution was collected at 1 BV intervalsand determined by HPLC All the 40 vv ethanol elutionwas collected and concentrated to dryness under reducedpressure at 60∘C to give refined product

27 Crystallization of Liriodendrin The refined product wasextracted with methyl alcohol-chloroform (1 4 vv) underreflux for 40min and repeated twice The extracted solutionswere combined and filtered and the filtrate was concentratedto dryness under reduced pressure at 60∘C The residue wasdissolved in methyl alcohol (1 10 wv) After being kept at20ndash30∘C for several days the solution was centrifuged toobtain crystals of liriodendrin Then crystals were washed bymethyl alcohol

28 Antioxidant Activity

281 DPPH Radical Scavenging Assay The DPPH radicalscavenging activity of samples was evaluated by the methodreported [30] Serial dilutions of the liriodendrin inmethanol(1mL) were added to 4mL methanol solution of DPPH(01mML) After 30min the absorbance was measured at517 nm against a control at room temperature Vitamin C wasused as a positive The DPPH radical scavenging activity wascalculated using the following equation

DPPH∙ scavenging effect ()

=

119860control minus 119860 sample

119860controltimes 100

(9)

282 ABTS∙+ Scavenging Assay ABTS∙+ scavenging assaywas performed by the literature method [30] The ABTS∙+solution was generated by the interaction of ABTS (7mML)and potassium persulfate (245mML) stored in the dark atroom temperature for 12 h The ABTS∙+ solution was diluteduntil absorbance was 0700 plusmn 0025 at 734 nm with ethanolThen serial dilutions of the liriodendrin in ethanol (1mL)were added to the ABTS∙+ solution (4mL) After 30minthe absorbance was measured at 734 nm against a control atroom temperature Vitamin C was used as a positive The

Table 2 Adsorption capacity and adsorption and desorption ratiosof liriodendrin on different resins at 25∘C

Resin Adsorption capacitya(mgg)

Adsorptionratiosa ()

Desorptionratioa ()

AB-8 14206 plusmn 0167 8333 plusmn 098 9245 plusmn 049D101 14535 plusmn 0282 8526 plusmn 166 9165 plusmn 039NKA 12299 plusmn 0176 7214 plusmn 103 8327 plusmn 083X-5 15870 plusmn 0135 9309 plusmn 079 9288 plusmn 037ADS-17 10024 plusmn 0267 5896 plusmn 121 7541 plusmn 088HPD100 14038 plusmn 0102 8234 plusmn 060 8798 plusmn 041HPD400 11157 plusmn 0187 6545 plusmn 166 8230 plusmn 191HPD600 9219 plusmn 0187 5408 plusmn 110 8953 plusmn 081aValues are means plusmn SD (119899 = 3)

scavenging capability of ABTS∙+ was calculated using the fol-lowing equation

ABTS∙+ scavenging effect ()

=



(10)

3 Results and Discussion

31 Resins Screening The selection of proper macroporousresins was performed in accordance with the structures andpolarities of adsorbates and resins [23] In the paper eightmacroporous resins of different properties were studied at25∘C As shown in Table 2 macroporous resins of nonpolarand weakly polar exhibited better adsorption capacity anddesorption ratios than others This may be caused for specialstructure of liriodendrin Although liriodendrin moleculecontained polar glucuronyl group the symmetrical structureof liriodendrin make it possess feature of nonpolar or weaklypolar Therefore nonpolar and weakly polar resins wereapplicable to adsorption of liriodendrin Adsorption capacityand adsorption ratios of D101 andX-5 resins were higher thanother resins and desorption ratios of AB-8 D101 and X-5resins were similar and higher than other resins ThereforeD101 and X-5 resins were selected to further evaluate for theirproperties in the following experiment

32 Adsorption Kinetics Adsorption kinetics on D101 and X-5 resins were evaluated at 25∘C Adsorption kinetic curveswere shown in Figure 2(a) For the two resins the adsorp-tion capacities toward liriodendrin increased rapidly in the30min and an asymptotic curve was reached at about120min The fast initial adsorption was likely due to rapidattachment of liriodendrin to the surface of resin and anasymptotic period due to diffusion of liriodendrin into themicropores of resin with high intraparticle mass transferresistance which indicated that the behaviors of two resinsbelonged to the fast adsorption resin type [31] The param-eters of three kinetic models were shown in Table 3(a)According to the calculated correlation coefficient (1198772 ge099) the pseudo-second-order kinetic model performed


Table 3 Kinetic (a) and isotherm (b) parameters of liriodendrin on D101 and X-5 resins at 25∘C

(a) Kinetic parameters of liriodendrin on D101 and X-5 resins at 25∘C

Resin D101 X-5Pseudo-first-order

Equation ln(136468 minus 119876119905) = 26135 minus 00596119905 ln(145831 minus 119876

119905) = 26799 minus 00634119905

1198772 09883 099001198961 (minminus1) 00596 00634119876119890(mgg) 136468 145831

Pseudo-second-orderEquation 119905119876

119905= 00694119905 + 04759 119905119876

119905= 00650119905 + 04177

1198772 09988 099911198962 (gmgsdotmin) 00101 00101119876119890(mgg) 144092 153846

Intraparticle diffusionEquation 119876

119905= 07261119905

05+ 49887 119876

119905= 07662119905

05+ 54919

1198772 07080 06936119896119894(mggsdotmin05) 07261 07662119862 (mgg) 49887 54919

(b) Isotherm parameters of liriodendrin on D101 and X-5 resins at 25∘C

Resin D101 X-5Freundlich

Linear equation ln119876119890= 04731 ln119862

119890+ 29920 ln119876

119890= 04069 ln119862

119890+ 33704

119870119865

199255 2909021119899 04731 040691198772 09701 08236

LangmuirLinear equation 119862

119890119876119890= 00648119862

119890+ 00054 119862

119890119876119890= 00597119862

119890+ 00014

1198760 (mgg) 154321 167504119870119871

120000 4264291198772 09948 09988

0

2

4

6

8

10

12

14

16

18

0 30 60 90 120 150 180 210 240 270Time (min)

D101X-5

Qe

(mg

g re

sin)

(a)

0

2

4

6

8

10

12

14

16

18

00 02 04 06 08

D101X-5

Ce (mgmL)

Qe

(mg

g re

sin)

(b)

Figure 2 Adsorption kinetics curves (a) and adsorption isotherms curves (b) for liriodendrin on D101 and X-5 resins at 25∘C


0

02

04

06

08

1

12

14

16

18

0 10 20 30 40 50 60 70 80 90 100Feed volume (mL)

Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

1BVh2BVh4BVh

(a)

0

5

10

15

20

25

30

35

40

45

0 10 20 30 40 50 60 70 80 90 100Ethanol concentration ()

Mas

s of l

iriod

endr

in (m

g)

(b)

0

05

1

15

2

25

3

0 2 4 6 8Elution volume (BV)

Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

(c)

Figure 3 Dynamic adsorption and desorption test curves on X-5 resin (a) Dynamic leakage curve (b) gradient elution curve and (c)isocratic desorption curve of liriodendrin on X-5 resin column at 25∘C

better to describe the adsorption process on X-5 and D101resins which suggested that the adsorption rate was con-trolled by chemical adsorption mechanism through sharingor exchange of electrons between adsorbate and adsorbent[32] In terms of the rate constant 119896

2 the adsorption rate of

X-5 and D101 resin was the same

33 Adsorption Isotherms Adsorption isotherms curveswereobtained for liriodendrin on D101 and X-5 resins at 25∘C Asshown in Figure 2(b) adsorption capacities increased withincreasing initial concentration and a turning point wasobserved when the initial liriodendrin concentration was1705mgmLTherefore this concentration of liriodendrin insample solution was selected in the following test Table 3(b)listed the two isotherms equations and relative parametersThe calculated correlation coefficients were shown that theLangmuir model fitted the test data more suitable than theFreundlich model in the studied concentration range whichsuggested themonolayer coverage of liriodendrin on the resin[22 28] The theoretical maximum adsorption capacities 1198760

on D101 and X-5 resins determined from the Langmuirequation were 154321mgg and 167504mggTherefore X-5resin was selected for dynamic adsorption and desorptiontests

34 Dynamic Adsorption-Desorption Tests

341 Dynamic Breakthrough Curves on X-5 Resin Dynamicbreakthrough tests were studies based on the feed con-centration the volume of effluent flow rate and temper-ature The initial feed concentration of liriodendrin was1705mgmL (from Section 33) and the temperature at 25∘Cand then the effect of flow rate was investigated As shownin Figure 3(a) the different flow rates showed noticeabledifferent breakthrough volume When the flow rate was1 BVh the best adsorption performance could be obtainedThe breakthrough volume of liriodendrin on X-5 resin was55mL (about 2 BV) at a flow rate of 1 BVh when the lirio-dendrin concentration in effluents reached 1 of the initialconcentration [18] The adsorption capacity of liriodendrinon X-5 resin was 15630mgg dry resin


Table 4 The purities and recoveries of liriodendrin in the two-steppurification

Step Purity () Recovery () Yield (mg)Crude extract 085 mdash mdashX-5 resin 4142 889 2013a

Crystallization 9748 637 2707baThe amount of refined sample was obtained from 55 g raw material in theX-5 resin treatmentbThe amount of liriodendrin was obtained from 1000mg refined sample bycrystallization

342 Dynamic Desorption on X-5 Resin In the process ofdynamic desorption the concentration and the volume ofdesorption solution were investigated Dynamic desorptionwas performed with gradient and isocratic elution modes atthe flow rate of 1 BVh respectively As shown in Figure 3(b)the desorption ability increased by the increase of ethanolconcentration When the concentration of ethanol reached40 vv the liriodendrin absorbed by X-5 resin could befully elutedTherefore 40 vv ethanol aqueous solution wasselected as the eluent in isocratic elutionThe isocratic elutionresult showed that the concentration of liriodendrin reachedmaximum values when eluting with 2 BV of 40 vv ethanolaqueous solutions When the volume of elution reached5 BV liriodendrin could be completely desorbed from X-5 resin (Figure 3(c)) Therefore the volume of desorptionsolution was selected to be 5 BV Under the above optimizedcondition all the 40 vv ethanol elution was collected andconcentrated to dryness under reduced pressure at 60∘C togive refined product

35 Purity Analysis and Identification of Liriodendrin Afterpurification on X-5 resin the purity of liriodendrin increasedfrom 085 of S cuneata extracts to 4142 and the liri-odendrin of recovery rate was 889 Such crystallizationresulted in a product with 9748 purity and the recovery ratewas 637 (Table 4) As shown in Figures 4(a) and 4(b) theHPLC chromatograms of S cuneata extracts before and aftertreatment on X-5 resin were compared Figure 4(c) showedthat the purity of liriodendrin crystals was determined to be9748 by HPLC For further chemical structure identifica-tion of liriodendrin NMR analysis was performed and thedata was shown as follows 1H-NMR (DMSO-d

6 400MHz)

120575 665 (4H s H-2 21015840 6 61015840) 493 (2H d 119869 = 68Hz H-110158401015840) 466 (2H d 119869 = 36Hz H-7 71015840) 431 (2H m Hb-9 91015840)419 (2H m Ha-9 91015840) 375 (12H s 4 times OCH

3) 13C-NMR

(DMSO-d6 100MHz) 120575c 1526 (C-3 31015840 5 51015840) 1371 (C-4 41015840)

1338 (C-1 11015840) 1043 (C-2 21015840 6 61015840) 1027 (C-110158401015840) 850 (C-7 71015840)772 (C-510158401015840) 765 (C-310158401015840) 741 (C-210158401015840) 713 (C-9 91015840) 700 (C-410158401015840) 609 (C-610158401015840) 564 (-OCH

3) 536 (C-8 81015840) The data were

identical with those reported in the literature [27]

36 Antioxidant Activity The antioxidant effect of plant sam-ples can be evaluated by several in vitro tests Since the assayresults of the antioxidant effect depend on the method useda combined assay of several methods is required In this

(vol

ts)

0

100

200

(min)5 10 15 20 25 30 35 40 45 50

(a)

(vol

ts)

(min)5 10 15 20 25 30 35 40 45 50

0

100

200

300

(b)

(min)5 10 15 20 25 30 35 40 45 50

(vol

ts)0

100

200

300

(c)

Figure 4 HPLC chromatograms of samples before treatment (a)and after treatment (b) on X-5 resin liriodendrin purified by crys-tallization (c)

study the antioxidant activity of liriodendrin isolated fromS cuneata was analysed using DPPH and ABTS

DPPH radical scavenging activity test aims to measurethe capacity of sample to scavenge the stable radical DPPHformed in solution by donation of hydrogen atom or anelectron [33] As shown in (Figure 5(a)) the IC

50value of

liriodendrin is 1361 120583gmL which is less important than thatof vitamin C (IC

50= 698 120583gmL)

ABTS∙+ scavenging assay is a widely accepted modelto determine the total antioxidant activity As shown in(Figure 5(b)) the IC

50values of liriodendrin and vitamin C

are 16842 and 2329 120583gmL respectivelyThe ABTS∙+ scavenging assay revealed that liriodendrin

possessed weak ABTS∙+ scavenging capacities However ithas been suggested that liriodendrin plays an important rolein DPPH inhibitory activities and oxygen radical absorbancecapacities On the other hand liriodendrin after oral admin-istration was in vivo transformation to syringaresinol whichpossesses strong antioxidant activity [10] The antioxidantactivity of liriodendrin in vitro and vivo needed to be furtherresearched

4 Conclusions

In the study highly concentrated liriodendrin was success-fully prepared After S cuneata extracts were purified on


0

20

40

60

80

100

0 30 60 90 120 150

LiriodendrinVitamin C

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

Concentration (120583gmL)

(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)

Figure 5 DPPH radical scavenging activity (a) and ABTS∙+ scavenging assay (b)

X-5 resin purity of liriodendrin was 4142 Further crys-tallization resulted in a product with 9748 purity andthe recovery rate was 637 The results indicated that theestablished method was simple and effective thus showingpotential for industrial scale isolation of liriodendrin fromSargentodoxa cuneata in the future Also such productionof highly concentrated liriodendrin as an antioxidant agentmight expand its further applications both in industrialproduction and in pharmacy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors thank Dr Lili Wang (Tianjin Institute of Phar-maceutical Research) for NMR experiments

References

[1] J-P Guo J Pang X-W Wang Z-Q Shen M Jin and J-WLi ldquoIn vitro screening of traditionally used medicinal plants inChina against enterovirusesrdquoWorld Journal of Gastroenterologyvol 12 no 25 pp 4078ndash4081 2006

[2] G Rucker R Mayer and J S Shin-Kim ldquoTriterpene saponinsfrom the Chinese drug lsquoDaxuetengrsquo (Caulis sargentodoxae)rdquoPlanta Medica vol 57 no 5 pp 468ndash470 1991

[3] J Chang andRCase ldquoPhenolic glycosides and ionone glycosidefrom the stemof Sargentodoxa cuneatardquoPhytochemistry vol 66no 23 pp 2752ndash2758 2005

[4] H-B Li C-C Wong K-W Cheng and F Chen ldquoAntioxidantproperties in vitro and total phenolic contents in methanolextracts from medicinal plantsrdquo LWTmdashFood Science and Tech-nology vol 41 no 3 pp 385ndash390 2008

[5] J Tang Y Qian Q-Q Li X-Q Xu and Z Ouyang ldquoCinnamicacid derivatives from the ethyl acetate fraction of Sargentodoxacuneatardquo Chemistry of Natural Compounds vol 48 no 1 pp118ndash119 2012

[6] S C Mao Q Q Gu C B Cui B Han B Cai and H B LiuldquoPhenolic compounds from Sargentodoxa cuneat (Oliv) RehdEt Wils and their antitumor activitiesrdquo Chinese Journal ofMedicinal Chemistry vol 14 no 6 pp 326ndash330 2004

[7] I Sakakibara M Yoshida K Hayashi and M Maruno ldquoAnti-inflammatory activities of glycosides from sargentodoxa cune-ata stemsrdquo Chemical Abstracts vol 122 pp 282ndash230 1997

[8] P Zhang S Q Yan Y D Shao and Z L Li ldquoStudy of an aqueousextract of sargentodoxa cuneata on anti-myocardial ischemiardquoActa Academiae Medicinae Primae Shanghai vol 15 no 3 pp191ndash194 1988

[9] L Zhu D L Lin C L Gu et al ldquoEffects of an aqueous extractof sargentodoxa cuneata on platelet aggregation coronary flowthrombus formation and plasma and platelet cAMP levelsrdquoActa Academiae Medicinae Primae Shanghai vol 13 no 5 pp346ndash350 1986

[10] H-J Jung H-J Park R-G Kim et al ldquoIn vivo anti-inflam-matory and antinociceptive effects of liriodendrin isolated fromthe stem bark of Acanthopanax senticosusrdquo Planta Medica vol69 no 7 pp 610ndash616 2003

[11] C Feng B-G Li X-P Gao H-Y Qi and G-L ZhangldquoA new triterpene and an antiarrhythmic liriodendrin fromPittosporum brevicalyxrdquoArchives of Pharmacal Research vol 33no 12 pp 1927ndash1932 2010

[12] N Lami S Kadota T Kikuchi and Y Momose ldquoConstituentsof the roots of Boerhaavia diffusa L III Identification of Ca2+channel antagonistic compound from the methanol extractrdquo


Chemical and Pharmaceutical Bulletin vol 39 no 6 pp 1551ndash1555 1991

[13] X-K Ran X-T Wang P-P Liu et al ldquoCytotoxic constituentsfrom the leaves of Broussonetia papyriferardquo Chinese Journal ofNatural Medicines vol 11 no 3 pp 269ndash273 2013

[14] MGan Y Zhang S Lin et al ldquoGlycosides from the root of Iodescirrhosardquo Journal of Natural Products vol 71 no 4 pp 647ndash6542008

[15] J-W Nam S-Y Kim T Yoon et al ldquoHeat shock factor 1inducers from the bark of Eucommia ulmoides as cytoprotectiveagentsrdquo Chemistry amp Biodiversity vol 10 no 7 pp 1322ndash13272013

[16] D L Zhao DW Shen Y T Chi F Liu L B Zou andH B ZhuldquoLiriodendrin protects SH-SY5Y cells from dopamine-inducedcytotoxicityrdquo Journal of Chinese Pharmaceutical Sciences vol 16no 4 pp 294ndash299 2007

[17] S Feng S Ni and W Sun ldquoPreparative isolation and purifi-cation of the lignan pinoresinol diglucoside and liriodendrinfrom the bark of EucommiaUlmoidesOliv by high speed coun-tercurrent chromatographyrdquo Journal of Liquid Chromatographyand Related Technologies vol 30 no 1 pp 135ndash145 2007

[18] M S Kamel K M Mohamed H A Hassanean K Ohtani RKasai and K Yamasaki ldquoIridoid and megastigmane glycosidesfrom Phlomis aureardquo Phytochemistry vol 55 no 4 pp 353ndash3572000

[19] J K Zhang X Y Zhu F L Luo et al ldquoSeparation and purifi-cation of neohesperidin from the albedo of Citrus reticulatacv Suavissima by combination of macroporous resin and high-speed counter-current chromatographyrdquo Journal of SeparationScience vol 35 no 1 pp 128ndash136 2012

[20] Y Dong M Zhao D Sun-Waterhouse et al ldquoAbsorption anddesorption behaviour of the flavonoids from Glycyrrhiza glabraL leaf on macroporous adsorption resinsrdquo Food Chemistry vol168 pp 538ndash545 2015

[21] Z Q Du K Wang Y Tao L Chen and F Qiu ldquoPurificationof baicalin and wogonoside from Scutellaria baicalensis extractsby macroporous resin adsorption chromatographyrdquo Journal ofChromatography B Analytical Technologies in the Biomedicaland Life Sciences vol 908 no 1 pp 143ndash149 2012

[22] Y Liu H M Bi J H Hu M Shang and H Y Zhou ldquoPuri-fication process of daphnetin from Zushima herb by macrop-orous adsorbent resinrdquo Asian Journal of Chemistry vol 25 no7 pp 4047ndash4050 2013

[23] RWang X G Peng L L Wang et al ldquoPreparative purificationof peoniflorin and albiflorin from peony rhizome using macro-porous resin and medium-pressure liquid chromatographyrdquoJournal of Separation Science vol 35 no 15 pp 1985ndash1992 2012

[24] S Liu Y Chen L Gu et al ldquoPurification of eleutherosidesby macroporous resin and the active fractions of anti-inflam-matory and antioxidant activity from Acanthopanax senticosusextractrdquo Analytical Methods vol 5 no 15 pp 3732ndash3740 2013

[25] L Lin H Zhao Y Dong B Yang and M Zhao ldquoMacroporousresin purification behavior of phenolics and rosmarinic acidfrom Rabdosia serra (MAXIM) HARA leafrdquo Food Chemistryvol 130 no 2 pp 417ndash424 2012

[26] P-C Sun Y Liu Y-T Yi H-J Li P Fan and C-H Xia ldquoPre-liminary enrichment and separation of chlorogenic acid fromHelianthus tuberosus L leaves extract by macroporous resinsrdquoFood Chemistry vol 168 pp 55ndash62 2015

[27] W B Huang D Y Kong and P M Yang ldquoStudies on lignanconstituents of Clematis armandii Franchrdquo Chinese Journal ofNatural Medicines vol 1 no 4 pp 199ndash202 2003

[28] C Li Y Zheng X Wang S Feng and D Di ldquoSimultaneousseparation and purification of flavonoids and oleuropein fromOlea europaea L (olive) leaves using macroporous resinrdquo Jour-nal of the Science of Food and Agriculture vol 91 no 15 pp2826ndash2834 2011

[29] B Zhang R Yang Y Zhao andC-Z Liu ldquoSeparation of chloro-genic acid from honeysuckle crude extracts by macroporousresinsrdquo Journal of Chromatography B Analytical Technologies inthe Biomedical and Life Sciences vol 867 no 2 pp 253ndash2582008

[30] M Ozturk U Kolak G Topcud S Oksuz and M I Choud-hary ldquoAntioxidant and anticholinesterase active constituentsfrom Micromeria cilicica by radical-scavenging activity-guidedfractionationrdquo Food Chemistry vol 126 no 1 pp 31ndash38 2011

[31] C Ma J Tang HWang G Tao X Gu and L Hu ldquoPreparativepurification of salidroside from Rhodiola rosea by two-stepadsorption chromatography on resinsrdquo Journal of SeparationScience vol 32 no 2 pp 185ndash191 2009

[32] Y Sun C-X Lin M-H Liu and Y-F Liu ldquoEquilibriumadsorption behaviors and kinetic characteristics of oxymatrineon a spherical cellulose adsorbentrdquo BioResources vol 6 no 1pp 631ndash640 2011

[33] N Khaled-Khodja L Boulekbache-Makhlouf and K MadanildquoPhytochemical screening of antioxidant and antibacterialactivities of methanolic extracts of some Lamiaceaerdquo IndustrialCrops and Products vol 61 pp 41ndash48 2014

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


O

O

OO

HOOH

OH

OH

O

O

OHHO

HO

OH

H3CO

OCH3

OCH3

OCH3

Figure 1 Chemical structure of liriodendrin

Liriodendrin after oral administration was in vivo trans-formation to syringaresinol which possesses strong antioxi-dant activity [10] However the antioxidant capacity of lirio-dendrin in vitro has not been investigated recently As a partof continuing research of expanding its further applicationsthe antioxidant activity of liriodendrin in vitrowas examined

In this study a simplemethod is developed for preparativepurification of liriodendrin from the stem of S cuneata usingmacroporous resin chromatography combined with crystal-lization process By static and dynamic adsorptiondesorp-tion tests on resins some process parameters such as feedconcentration and volume of sample solution flow rateethanol concentration and volume of desorption solutionare optimized for purification of liriodendrin After furthercrystallization liriodendrin is an obtained pure form andits structure is identified by NMR Moreover antioxidantactivity of liriodendrin is also evaluated in vitro for the firsttime

2 Materials and Methods

21 Materials and Reagents Liriodendrin standard was iso-lated from the stem of S cuneata in our previous study Thepurity was determined to be over 97 by normalization ofthe peak areas by HPLC Its structure was elucidated bycomparison of the NMR data with the literature [27] HPLC-grade acetonitrile and formic acidwere bought fromConcordTechnology Co Ltd (Tianjin China) 11-Diphenyl-2-picryl-hydrazyl (DPPH) and 221015840-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) were obtainedfrom sigmaChemical Co (Sigma-AldrichGmbH SternheimGermany) Other chemicals and reagents were analyticalgrade Deionized water was purified by a Milli-Q waterpurification system (Millipore Boston USA)

22 Adsorbents Macroporous resins including D101 AB-8X-5ADS-17 andNKAwere purchased fromNankaiHechengSampT Co Ltd (Tianjin China) and HPD100 HPD400 andHPD600 fromCangzhou BonAdsorber Technology Co Ltd(Hebei China) respectively Their physical properties weresummarized in Table 1 Before use these resins were soakedin 95 vv ethanol aqueous solutions for 24 h then washedwith 4 HCl aqueous solution deionized water and 4

Table 1 Physical properties of macroporous resins in test

Resin Average porediameter (nm)

Surface area(m2g) Polarity

AB-8 13-14 450ndash530 Weakly polarD101 10ndash12 600ndash700 NonpolarNKA 20ndash22 570ndash590 NonpolarX-5 29-30 500ndash600 NonpolarADS-17 25ndash30 90ndash150 Middle-polarHPD100 85ndash9 650ndash700 NonpolarHPD400 75ndash8 500ndash550 Middle-polarHPD600 8 550ndash600 Strong-polar

NaOHaqueous solution respectively andfinallywashedwithdeionized water

23 Preparation of Sample The dried stem of S cuneatawas powdered and sieved through a number 40 mesh Thepowder of sample (10 kg) was extracted with 8000mL of 95vv ethanol aqueous solutions under reflux for 90min andrepeated twice The extracted solutions were combined andfiltered and the filtrate was evaporated to dryness by a rotaryevaporator under reduced pressure at 60∘C The residue wassuspended in deionized water and suspended solution wascentrifuged at 3000 rpm for 30minThe supernatantwas usedas stock solution for the subsequent test

24 HPLC Analysis of Liriodendrin The HPLC system con-sisted of a SSI PC2000 chromatograph (SSI Scientific Sys-tems Inc USA) and a 2000ES evaporative light scatteringdetector (ELSD) (Alltech Alltech Associates Inc USA)An ODS-2 Hypersil C-18 column (250mm times 46mm id5 120583mThermo ScientificThermo Fisher Scientific Inc USA)was operated at 35∘C Solvents that constituted the mobilephase were acetonitrile (A) and 02 formic acid aqueoussolution (B) The separation was performed using a stepwisegradient elution of 0ndash14min 95ndash87 B 14ndash25min 87ndash87B and 25ndash45min 87ndash81 B then keeping 95 B to balancefor 10min The flow rate was 09mLmin ELSD was usedas the detection method with nebulizing gas flow rate of25 Lmin and drift tube temperature of 105∘C The impactor






119876119890=

119881119894(1198620 minus 119862119890)

119882

(1)

Adsorption ratio is

119864 =

1198620 minus 1198621198901198620times 100 (2)




Desorption ratio is

119863 =

119862119889119881119889

119881119894(1198620 minus 119862119890)

times 100 (3)








ln (119876119890minus119876119905) = ln119876

119890minus 1198961119905 (4)


119905

119876119905

=

119905

119876119890

+

1119876119890

21198962 (5)


119876119905= 11989611989411990505+119862 (6)

where 119876119890and 119876







119876119890= 1198701198651198621119899119890 (7)


119876119890=

11987601198701198711198621198901 + 119870119871119862119890

(8)



119871is the


119890are the










=



(9)





Desorptionratioa ()




=



(10)









119905) = 26799 minus 00634119905

1198772 09883 099001198961 (minminus1) 00596 00634119876119890(mgg) 136468 145831


119905= 00694119905 + 04759 119905119876

119905= 00650119905 + 04177

1198772 09988 099911198962 (gmgsdotmin) 00101 00101119876119890(mgg) 144092 153846


119905= 07261119905

05+ 49887 119876

119905= 07662119905

05+ 54919

1198772 07080 06936119896119894(mggsdotmin05) 07261 07662119862 (mgg) 49887 54919




119890+ 29920 ln119876

119890= 04069 ln119862

119890+ 33704

119870119865

199255 2909021119899 04731 040691198772 09701 08236


119890119876119890= 00648119862

119890+ 00054 119862

119890119876119890= 00597119862

119890+ 00014

1198760 (mgg) 154321 167504119870119871

120000 4264291198772 09948 09988

0

2

4

6

8

10

12

14

16

18

0 30 60 90 120 150 180 210 240 270Time (min)

D101X-5

Qe

(mg

g re

sin)

(a)

0

2

4

6

8

10

12

14

16

18

00 02 04 06 08

D101X-5

Ce (mgmL)

Qe

(mg

g re

sin)

(b)



0

02

04

06

08

1

12

14

16

18

0 10 20 30 40 50 60 70 80 90 100Feed volume (mL)

Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

1BVh2BVh4BVh

(a)

0

5

10

15

20

25

30

35

40

45


Mas

s of l

iriod

endr

in (m

g)

(b)

0

05

1

15

2

25

3


Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

(c)















6 400MHz)


3) 13C-NMR

(DMSO-d6 100MHz) 120575c 1526 (C-3 31015840 5 51015840) 1371 (C-4 41015840)

1338 (C-1 11015840) 1043 (C-2 21015840 6 61015840) 1027 (C-110158401015840) 850 (C-7 71015840)772 (C-510158401015840) 765 (C-310158401015840) 741 (C-210158401015840) 713 (C-9 91015840) 700 (C-410158401015840) 609 (C-610158401015840) 564 (-OCH

3) 536 (C-8 81015840) The data were



(vol

ts)

0

100

200

(min)5 10 15 20 25 30 35 40 45 50

(a)

(vol

ts)

(min)5 10 15 20 25 30 35 40 45 50

0

100

200

300

(b)

(min)5 10 15 20 25 30 35 40 45 50

(vol

ts)0

100

200

300

(c)




50value of


50= 698 120583gmL)





4 Conclusions



0

20

40

60

80

100

0 30 60 90 120 150


DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)





Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






119876119890=

119881119894(1198620 minus 119862119890)

119882

(1)

Adsorption ratio is

119864 =

1198620 minus 1198621198901198620times 100 (2)




Desorption ratio is

119863 =

119862119889119881119889

119881119894(1198620 minus 119862119890)

times 100 (3)








ln (119876119890minus119876119905) = ln119876

119890minus 1198961119905 (4)


119905

119876119905

=

119905

119876119890

+

1119876119890

21198962 (5)


119876119905= 11989611989411990505+119862 (6)

where 119876119890and 119876







119876119890= 1198701198651198621119899119890 (7)


119876119890=

11987601198701198711198621198901 + 119870119871119862119890

(8)



119871is the


119890are the










=



(9)





Desorptionratioa ()




=



(10)









119905) = 26799 minus 00634119905

1198772 09883 099001198961 (minminus1) 00596 00634119876119890(mgg) 136468 145831


119905= 00694119905 + 04759 119905119876

119905= 00650119905 + 04177

1198772 09988 099911198962 (gmgsdotmin) 00101 00101119876119890(mgg) 144092 153846


119905= 07261119905

05+ 49887 119876

119905= 07662119905

05+ 54919

1198772 07080 06936119896119894(mggsdotmin05) 07261 07662119862 (mgg) 49887 54919




119890+ 29920 ln119876

119890= 04069 ln119862

119890+ 33704

119870119865

199255 2909021119899 04731 040691198772 09701 08236


119890119876119890= 00648119862

119890+ 00054 119862

119890119876119890= 00597119862

119890+ 00014

1198760 (mgg) 154321 167504119870119871

120000 4264291198772 09948 09988

0

2

4

6

8

10

12

14

16

18

0 30 60 90 120 150 180 210 240 270Time (min)

D101X-5

Qe

(mg

g re

sin)

(a)

0

2

4

6

8

10

12

14

16

18

00 02 04 06 08

D101X-5

Ce (mgmL)

Qe

(mg

g re

sin)

(b)



0

02

04

06

08

1

12

14

16

18

0 10 20 30 40 50 60 70 80 90 100Feed volume (mL)

Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

1BVh2BVh4BVh

(a)

0

5

10

15

20

25

30

35

40

45


Mas

s of l

iriod

endr

in (m

g)

(b)

0

05

1

15

2

25

3


Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

(c)















6 400MHz)


3) 13C-NMR

(DMSO-d6 100MHz) 120575c 1526 (C-3 31015840 5 51015840) 1371 (C-4 41015840)

1338 (C-1 11015840) 1043 (C-2 21015840 6 61015840) 1027 (C-110158401015840) 850 (C-7 71015840)772 (C-510158401015840) 765 (C-310158401015840) 741 (C-210158401015840) 713 (C-9 91015840) 700 (C-410158401015840) 609 (C-610158401015840) 564 (-OCH

3) 536 (C-8 81015840) The data were



(vol

ts)

0

100

200

(min)5 10 15 20 25 30 35 40 45 50

(a)

(vol

ts)

(min)5 10 15 20 25 30 35 40 45 50

0

100

200

300

(b)

(min)5 10 15 20 25 30 35 40 45 50

(vol

ts)0

100

200

300

(c)




50value of


50= 698 120583gmL)





4 Conclusions



0

20

40

60

80

100

0 30 60 90 120 150


DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)





Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








=



(9)





Desorptionratioa ()




=



(10)









119905) = 26799 minus 00634119905

1198772 09883 099001198961 (minminus1) 00596 00634119876119890(mgg) 136468 145831


119905= 00694119905 + 04759 119905119876

119905= 00650119905 + 04177

1198772 09988 099911198962 (gmgsdotmin) 00101 00101119876119890(mgg) 144092 153846


119905= 07261119905

05+ 49887 119876

119905= 07662119905

05+ 54919

1198772 07080 06936119896119894(mggsdotmin05) 07261 07662119862 (mgg) 49887 54919




119890+ 29920 ln119876

119890= 04069 ln119862

119890+ 33704

119870119865

199255 2909021119899 04731 040691198772 09701 08236


119890119876119890= 00648119862

119890+ 00054 119862

119890119876119890= 00597119862

119890+ 00014

1198760 (mgg) 154321 167504119870119871

120000 4264291198772 09948 09988

0

2

4

6

8

10

12

14

16

18

0 30 60 90 120 150 180 210 240 270Time (min)

D101X-5

Qe

(mg

g re

sin)

(a)

0

2

4

6

8

10

12

14

16

18

00 02 04 06 08

D101X-5

Ce (mgmL)

Qe

(mg

g re

sin)

(b)



0

02

04

06

08

1

12

14

16

18

0 10 20 30 40 50 60 70 80 90 100Feed volume (mL)

Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

1BVh2BVh4BVh

(a)

0

5

10

15

20

25

30

35

40

45


Mas

s of l

iriod

endr

in (m

g)

(b)

0

05

1

15

2

25

3


Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

(c)















6 400MHz)


3) 13C-NMR

(DMSO-d6 100MHz) 120575c 1526 (C-3 31015840 5 51015840) 1371 (C-4 41015840)

1338 (C-1 11015840) 1043 (C-2 21015840 6 61015840) 1027 (C-110158401015840) 850 (C-7 71015840)772 (C-510158401015840) 765 (C-310158401015840) 741 (C-210158401015840) 713 (C-9 91015840) 700 (C-410158401015840) 609 (C-610158401015840) 564 (-OCH

3) 536 (C-8 81015840) The data were



(vol

ts)

0

100

200

(min)5 10 15 20 25 30 35 40 45 50

(a)

(vol

ts)

(min)5 10 15 20 25 30 35 40 45 50

0

100

200

300

(b)

(min)5 10 15 20 25 30 35 40 45 50

(vol

ts)0

100

200

300

(c)




50value of


50= 698 120583gmL)





4 Conclusions



0

20

40

60

80

100

0 30 60 90 120 150


DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)





Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






119905) = 26799 minus 00634119905

1198772 09883 099001198961 (minminus1) 00596 00634119876119890(mgg) 136468 145831


119905= 00694119905 + 04759 119905119876

119905= 00650119905 + 04177

1198772 09988 099911198962 (gmgsdotmin) 00101 00101119876119890(mgg) 144092 153846


119905= 07261119905

05+ 49887 119876

119905= 07662119905

05+ 54919

1198772 07080 06936119896119894(mggsdotmin05) 07261 07662119862 (mgg) 49887 54919




119890+ 29920 ln119876

119890= 04069 ln119862

119890+ 33704

119870119865

199255 2909021119899 04731 040691198772 09701 08236


119890119876119890= 00648119862

119890+ 00054 119862

119890119876119890= 00597119862

119890+ 00014

1198760 (mgg) 154321 167504119870119871

120000 4264291198772 09948 09988

0

2

4

6

8

10

12

14

16

18

0 30 60 90 120 150 180 210 240 270Time (min)

D101X-5

Qe

(mg

g re

sin)

(a)

0

2

4

6

8

10

12

14

16

18

00 02 04 06 08

D101X-5

Ce (mgmL)

Qe

(mg

g re

sin)

(b)



0

02

04

06

08

1

12

14

16

18

0 10 20 30 40 50 60 70 80 90 100Feed volume (mL)

Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

1BVh2BVh4BVh

(a)

0

5

10

15

20

25

30

35

40

45


Mas

s of l

iriod

endr

in (m

g)

(b)

0

05

1

15

2

25

3


Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

(c)















6 400MHz)


3) 13C-NMR

(DMSO-d6 100MHz) 120575c 1526 (C-3 31015840 5 51015840) 1371 (C-4 41015840)

1338 (C-1 11015840) 1043 (C-2 21015840 6 61015840) 1027 (C-110158401015840) 850 (C-7 71015840)772 (C-510158401015840) 765 (C-310158401015840) 741 (C-210158401015840) 713 (C-9 91015840) 700 (C-410158401015840) 609 (C-610158401015840) 564 (-OCH

3) 536 (C-8 81015840) The data were



(vol

ts)

0

100

200

(min)5 10 15 20 25 30 35 40 45 50

(a)

(vol

ts)

(min)5 10 15 20 25 30 35 40 45 50

0

100

200

300

(b)

(min)5 10 15 20 25 30 35 40 45 50

(vol

ts)0

100

200

300

(c)




50value of


50= 698 120583gmL)





4 Conclusions



0

20

40

60

80

100

0 30 60 90 120 150


DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)





Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

02

04

06

08

1

12

14

16

18

0 10 20 30 40 50 60 70 80 90 100Feed volume (mL)

Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

1BVh2BVh4BVh

(a)

0

5

10

15

20

25

30

35

40

45


Mas

s of l

iriod

endr

in (m

g)

(b)

0

05

1

15

2

25

3


Con

cent

ratio

n of

lirio

dend

rin (m

gm

L)

(c)















6 400MHz)


3) 13C-NMR

(DMSO-d6 100MHz) 120575c 1526 (C-3 31015840 5 51015840) 1371 (C-4 41015840)

1338 (C-1 11015840) 1043 (C-2 21015840 6 61015840) 1027 (C-110158401015840) 850 (C-7 71015840)772 (C-510158401015840) 765 (C-310158401015840) 741 (C-210158401015840) 713 (C-9 91015840) 700 (C-410158401015840) 609 (C-610158401015840) 564 (-OCH

3) 536 (C-8 81015840) The data were



(vol

ts)

0

100

200

(min)5 10 15 20 25 30 35 40 45 50

(a)

(vol

ts)

(min)5 10 15 20 25 30 35 40 45 50

0

100

200

300

(b)

(min)5 10 15 20 25 30 35 40 45 50

(vol

ts)0

100

200

300

(c)




50value of


50= 698 120583gmL)





4 Conclusions



0

20

40

60

80

100

0 30 60 90 120 150


DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)





Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







6 400MHz)


3) 13C-NMR

(DMSO-d6 100MHz) 120575c 1526 (C-3 31015840 5 51015840) 1371 (C-4 41015840)

1338 (C-1 11015840) 1043 (C-2 21015840 6 61015840) 1027 (C-110158401015840) 850 (C-7 71015840)772 (C-510158401015840) 765 (C-310158401015840) 741 (C-210158401015840) 713 (C-9 91015840) 700 (C-410158401015840) 609 (C-610158401015840) 564 (-OCH

3) 536 (C-8 81015840) The data were



(vol

ts)

0

100

200

(min)5 10 15 20 25 30 35 40 45 50

(a)

(vol

ts)

(min)5 10 15 20 25 30 35 40 45 50

0

100

200

300

(b)

(min)5 10 15 20 25 30 35 40 45 50

(vol

ts)0

100

200

300

(c)




50value of


50= 698 120583gmL)





4 Conclusions



0

20

40

60

80

100

0 30 60 90 120 150


DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)





Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

20

40

60

80

100

0 30 60 90 120 150


DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


(a)

0

10

20

30

40

50

60

70

80

90

100

0 300 600 900 1200 1500


ABT

S ra

dica

l sca

veng

ing

activ

ity (

)


(b)





Acknowledgment


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Preparative Purification of Liriodendrin