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Separation and Purification Technology 66 (2009) 329–339

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Separation and Purification Technology

journa l homepage: www.e lsev ier .com/ locate /seppur

ystematic purification of polydatin, resveratrol and anthraglycoside B fromolygonum cuspidatum Sieb. et Zucc.

alei Zhanga,b,c, Xiunan Lia, Dongxia Haoa, Guisheng Lid, Benming Xud, Guanghui Maa, Zhiguo Sua,∗

National Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, ChinaGraduate School of the Chinese Academy of Sciences, Beijing 100039, ChinaShandong Engineering Research Centre for Natural Drugs, Yantai, Shandong 264003, ChinaSchool of Pharmacy Yantai University, Yantai, Shandong 264005, China

r t i c l e i n f o

rticle history:eceived 29 September 2008eceived in revised form 10 December 2008ccepted 17 December 2008

eywords:olygonum cuspidatum Sieb. et Zucc.olydatinesveratrolnthraglycoside B

a b s t r a c t

For comprehensive utilization of Polygonum cuspidatum Sieb. et Zucc., a systematic, environmentalfriendly preparative process was developed for purification of polydatin, resveratrol and anthraglyco-side B simultaneously from the herb. The process was simple, consisting of macroporous resin adsorptionand reversed-phase liquid chromatography. Macroporous resin adsorption separated the solvent extractinto fraction I containing polydatin and fraction II containing resveratrol as well as anthraglycoside B. Thefractions I and II were further purified by reversed-phase chromatography to get the fractions correspond-ing to polydatin, resveratrol and anthraglycoside B, respectively. Two polydivinylbenzene microspheres(PDVB-SPG and PDVB-Swelling) were prepared by SPG membrane emulsification and by two-step acti-vated swelling polymerization, respectively. These new media were compared with a commercial C18

urification bonded silica medium in the reversed-phase liquid chromatography step. The PDVB-SPG column demon-strated a better separation compared with C18 and PDVB-Swelling column. The pH value of the mobilephase showed little effect on the separation of polydatin and resveratrol, and large effect on the separationof anthraglycoside B for both HPLC analysis and preparative reversed-phase liquid chromatography. Afterthe chromatographic process and a further crystallization, polydatin, resveratrol and anthraglycoside B

1.3%

were purified from 7.5%,81.3%, 77.4% and 80.2%.

. Introduction

Bioactive molecules are an integral and natural part of plantnd animal products, normally present in very low concentrations.or economical production of these compounds, it is desirable if asany as possible bioactive components are simultaneously recov-

red from one plant and animal source with adequate purity andield. If the separation steps are integrated properly, it would beossible to obtain more than one products from one medical herb.oreover, simultaneous separation of multiple products may elim-

nate waste disposal and reduce the demand of natural resources.he effect would benefit the environment.

The root of Polygonum cuspidatum Sieb. et Zucc., known as

huzhang” in Chinese, is one of the most widely used traditionalerbal medicine for treatment of atherosclerosis, cough, asthma,ypertension, cancer, etc. [1]. The known constituents mostly asso-iated with these properties are stilbenes and anthraquinones

∗ Corresponding author. Tel.: +86 10 62561817; fax: +86 10 62561813.E-mail address: zgsu@home.ipe.ac.cn (Z. Su).

383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.seppur.2008.12.013

and 2.4% to 98.8%, 98.2% and 98.6%, respectively, with total recoveries of

© 2009 Elsevier B.V. All rights reserved.

[1,2]. The major stilbenes are polydatin and resveratrol [2]. It hasbeen proposed that resveratrol and polydatin possess biologicalfunctions including anti-oxidative [2,3], anti-cancer [4,5], and anti-inflammatory [6]. The major anthraquinones are anthraglycoside B(emodin-8-O-�-d-glucoside), emodin, physcion and chrysophanol,in which the content of anthraglycoside B is the highest [7]. In ourprevious study, anthraglycoside B was found to provide neuropro-tection against cerebral ischemia-reperfused injury and glutamateinduced neuronal damage through exerting antioxidative effectsand inhibiting glutamate neurotoxicity [8]. In this paper, an inte-grated process was developed for separation and purification ofpolydatin, resveratrol and anthraglycoside B simultaneously fromP. cuspidatum to ensure an economical process for large scaleproduction. The structures of polydatin (1), resveratrol (2) andanthraglycoside (3) B are shown in Fig. 1.

The conventional methods of purifying polydatin and resvera-trol from P. cuspidatum Sieb.et Zucc. utilized liquid–liquid partition

and silica gel column chromatography [9], which required largeamount of organic solvents such as chloroform. If scaled up, theprocess would be highly toxic and environmentally unfriendly.Recently, resveratrol and polydatin were purified by high-speedcounter-current chromatography (HSCCC) [10,11]. Compared with

330 D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339

datin,

tfu

taeTraplcrfcassas

apcwbp

TdirazadoHp

Fig. 1. Chemical structures of poly

raditional column liquid chromatography, HSCCC has an advantageor non-use of solid media. However, HSCCC would be difficult orneconomical on scale-up due to its process nature.

Preparative chromatography offers advantages of high produc-ivity, high selectivity and maturity for operation, both in laboratorynd in production. It has been used successfully for selective recov-ry of target metabolites from various crude plant extracts [12].he key issue is how to select a solid medium and a process toeduce the cost and increase the selectivity. Macroporous resindsorption was used to separate polydatin and resveratrol in therevious literature [13,14], but the purity was poor because of the

ow resolution. It is only suitable for initial separation of targetompound from the crude extract. High performance preparativeeversed-phase chromatography (HPP-RPC) is a powerful techniqueor separation and purification of natural products, but the pro-ess could be very expensive due mainly to the solid media thatre mostly spherical silica beads with C18 bonded. Preparation ofuch high quality silica beads with spherical shape and controlledize is very expensive. Furthermore, the beads cannot be used inlkaline pH, thus it is impossible to do depyrogenation with alkaliolution.

On the other hand, synthetic polymeric beads have been testeds an alternative to silica ones for recovery and separation of naturalroducts. These polymer microspheres possess much better chemi-al stability and functionalization capacity than the silica, yet couldithstand reasonably high pressure. They are, however, may also

e expensive if they are made through conventional seed swellingolymerization.

In this paper, a two-stage purification scheme was designed.he first step was macroporous resin adsorption to separate poly-atin, resveratrol and anthraglycoside B from majority of other

mpurities. The second step was reversed-phase liquid chromatog-aphy. In this chromatographic step, we compared both silicand polymer beads. The polymer beads were polydivinylben-ene microspheres prepared by SPG membrane emulsification

nd two-step activated swelling polymerization. The beads wereirectly used without any surface modification. The influencesf pH of the mobile phase were also investigated in analyticalPLC and the preparative reversed-phase liquid chromatogra-hy.

resveratrol and anthraglycoside B.

2. Experimental

2.1. Materials and chemicals

The root of P. cuspidatum Sieb.et Zucc. was collected in Shanxiprovince of China. It was dried and pulverized to 100 meshes. Thereference compounds of polydatin and resveratrol with the purityof >99% were purchased from Shanxi Scidoor Hi-Tech Biology Co.,Ltd. (Xian, China). Anthraglycoside B was purified with the purity of>98% in our laboratory. The chemical structure of anthraglycoside Bwas identified by ESI mass spectrometry (ESI-MS) and nuclear mag-netic resonance (NMR). Methanol and acetonitrile were of HPLCgrade and purchased from Tianjin Shield Company, Tianjin, China.Water used was reverse osmosis water. All other chemicals were ofanalytical grade.

The HPLC column used for analytical purpose was a Discov-ery C18 column (250 mm × 4.6 mm ID, 5 �m, Supelco, USA). TheHPD-600 macroporous resin was purchased from Cangzhou BaoenChemical Co. (Cangzhou, China). The C18 medium, named YWGC18 with average particle diameter of 40 �m, was purchased fromTianjin No. 2 Chemical Plant (Tianjin, China). Two polydivinyl-benzene media, PDVB-SPG and PDVB-Swelling microsphere withaverage diameter of 30 �m were respectively prepared by SPGmembrane emulsification and suspension polymerization, and bythe two-step activated swelling method [15]. The former PDVB-SPG microspheres, developed by our laboratory, were fabricatedwith divinylbenzene as monomer, heptane as porogen and benzoylperoxide as initiator respectively. The later PDVB-Swelling micro-spheres were prepared with polystyrene microspheres of 5 �mas initial seeds, divinylbenzen, heptane and dioctanoyl peroxiderespectively as monomer, porogen, and initiator.

2.2. Apparatus

The apparatus used for HPLC and preparative liquid chromatog-

raphy was Agilent 1100 series system and Agilent HPLC workstation(Agilent Technologies, USA). Preparative liquid chromatographyfor scale-up was carried out on Waters chromatography systemcomposed of a model 515 pump, a model 2487 Dual � AbsorbanceDetector (Waters, USA). Full UV spectra were recorded using an UV-

ificati

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D. Zhang et al. / Separation and Pur

401 spectrophotometer (Shimadzu, Japan), equipped with 1 cmuartz cuvettes. Nuclear magnetic resonance (NMR) spectrometersed here was Bruker AV-400 (Bruker, Switzerland). ESI Masspectra (ESI-MS) were recorded on a Mariner mass spectrometerPerSeptive, Biosystems, USA).

.3. HPLC analysis

The analyses were performed with a Discovery C18 at 35 ◦C. Theow rate was kept at 1.0 ml/min constantly. The mobile phase wascetonitrile–water (26:74, v/v) of varying pH values (separately 3.5,.5, 5.5 and 6.5). Quantitative determination of polydatin, resver-trol and anthraglycoside B were performed by using an externaltandard based on the area of peak under the optimal HPLC analyt-cal conditions.

.4. Preparation of the solvent extract of P. cuspidatum Sieb.etucc

To optimize the extraction conditions for the best recovery ofolydatin, resveratrol and anthraglycoside B, different ethanol pro-ortions as the solvent (0, 25, 50, 75 and 95%, v/v, in distilled water)ere compared. The results showed that the best delivery of these

hree compounds were reached using 75% ethanol, ratio of sol-ent/solid of 8 ml/g, at 40 min ultrasonic bath and three extractionsn dark. The extract was combined and evaporated under reducedressure at about 60 ◦C until the content of ethanol reached 10%.he solution was filtered and the filtrate was evaporated to drynesso get the solvent extract.

.5. Column chromatography separation

.5.1. Macroporous resin column separationHPD-600 was suspended in 95% (v/v) ethanol–water overnight,

nd slurry packed in a 200 mm × 25 mm ID column and equilibratedith water at a flow rate of 5 ml/min. About 4 g of the solvent extractas dissolved in 25 ml of 10% ethanol and loaded onto the column.

he column was then washed with 3CVs of 10% ethanol to removeon-adsorbed impurities, and eluted with 6CVs of 30% ethanol toet fraction I and then with 4CVs of 65% ethanol to get fraction II.inally the column was regenerated with 95% ethanol. The collectedractions corresponding to fractions I and II were evaporated undereduced pressure with a rotary vacuum evaporator at about 60 ◦Cept from light. The residues were used as the fractions for furtherurification using RP chromatography.

.5.2. Comparison of preparative liquid chromatography of C18,DVB-SPG and PDVB-Swelling media purification of resveratrolnd anthraglycoside B from the fraction II

The preparative liquid chromatography was carried out on threeeversed-phase columns (250 mm × 4.6 mm ID) that were slurry-acked with three RP media (C18, PDVB-SPG and PDVB-Swelling),espectively. Three media were washed with ethanol extensivelyefore use. The columns were then washed with the washing sol-ents. The washing solvents used 20% ethanol for the C18 columnnd 30% ethanol for the PDVB-SPG and PDVB-Swelling column.bout 1.5 mg of the fraction II was dissolved in 200 �l of theashing solvents and was applied to the columns that were pre-

quilibrated with the washing solvents. The columns were thenashed with the washing solvents, respectively. After washing, the

18 column was eluted with 25% ethanol to get resveratrol, 30%

lution to get anthraglycoside B and finally regenerated with 100%thanol, whereas the PDVB-SPG and PDVB-Swelling columns wereluted with 35% ethanol and finally regenerated with 100% ethanol.ll the chromatographic experiments were conducted at a flowate of 2 ml/min and monitored at a UV wavelength of 290 nm at

on Technology 66 (2009) 329–339 331

ambient temperature. The peaks corresponding to resveratrol andanthraglycoside B were collected and subjected to analytical HPLCto determine the content and the product recovery.

2.5.3. Comparison of preparative liquid chromatography of C18,PDVB-SPG and PDVB-Swelling media purification of polydatinfrom the fraction I

The preparative liquid chromatography was carried out on thethree reversed-phase columns (250 mm × 4.6 mm ID). For the C18column, the mobile phase was 15% ethanol for washing and elut-ing polydatin, and finally regenerated with 100% ethanol. For thePDVB-SPG and PDVB-Swelling column, the mobile phase was 25%ethanol for washing and eluting polydatin, and finally regeneratedwith 100% ethanol. Sample loading was 10 mg for C18 column and16 mg for PDVB-SPG and PDVB-Swelling column. The flow rate was2 ml/min and the detection wavelength was 290 nm. The peaks cor-responding to polydatin were collected and subjected to analyticalHPLC to determine the content and the product recovery.

2.5.4. Scale-up of PDVB-SPG column for purification of polydatinfrom the fraction I and purification of resveratrol andanthraglycoside B from the fraction II

A 300 mm × 7.8 mm ID column was slurry packed with PDVB-SPG medium and was washed with a washing solution. About17.3 mg of the fraction II or 28.5 mg of the fraction I was dissolved inthe washing solvents and was applied to the columns, respectively.The washing solutions, eluting solutions and regenerating solutionswere the same as ones listed in Section 2.5.2 for the fraction II, andthe same as ones listed in Section 2.5.3 for the fraction I. All thechromatographic experiments were conducted at a mobile-phaseflow rate of 5 ml/min and monitored at a UV wavelength of 290 nmat ambient temperature. The peaks corresponding to resveratrol,anthraglycoside B and polydatin were collected and subjected toanalytical HPLC to determine the content and the product recovery.

2.6. Crystallization of polydatin, resveratrol and anthraglycosideB and structure determination

The collected fractions of polydatin, resveratrol and anthragly-coside B from PDVB-SPG reversed-phase chromatography wereevaporated under reduced pressure with a rotary vacuum evap-orator at about 60 ◦C kept from light until a few crystals appeared,respectively. The crystals of polydatin, resveratrol and anthraglyco-side B were collected and dried and then submitted to ESI-MS andMNR structure determination. 1H NMR and 13C NMR were recordedat 400 MHz in deuterated DMSO.

3. Results and discussion

3.1. HPLC analysis

The solvent extract and each fraction from column chromatog-raphy were analyzed by HPLC. Full UV spectra (200–600 nm) ofthe methanol solutions of polydatin, resveratrol, anthraglycoside Band the solvent extract were recorded and compared to choose thedetection wavelength. The �max of polydatin and resveratrol were219 nm, 306 nm and 320 nm, whereas the �max of anthraglycoside Bwere 221 nm, 284 nm, 421 nm and 502 nm. UV absorbance of thesethree compounds was different, whereas all could be detected wellat 290 nm. So a wavelength at 290 nm was used for simultaneousdetermination of these three compounds.

In order to select an appropriate condition for the HPLC anal-ysis of components of P. cuspidatum Sieb.et Zucc., several elutionsystems were tested with methanol, acetonitrile and water in dif-ferent proportions. When acetonitrile–water (26:74, v/v) was usedas the mobile phase, major peaks could be obtained and each peak

332 D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339

of Poly

gpFaIovaIbtrww

wAhaa

The pKa values of polydatin and resveratrol are higher than 9.39,while the pKa values of anthraglycoside B are 6.27 and 6.71. Whenthe pH of the mobile phase was less than 6.5, the polydatin andresveratrol would exist in the neutral forms, and the behavior in

Table 1pKa of resveratrol, polydatin and anthraglycoside B.

Fig. 2. HPLC chromatograms of the solvent extract

ot baseline separation. While the pH value of the mobile phaselays an important role in HPLC analysis of the extract of the herb.ig. 2 compares the retention behaviors of polydatin, resveratrolnd anthraglycoside B at 26% acetonitrile with varying pH values.t could be easily observed that the elution time and peak shapef resveratrol and polydatin showed little difference with the pHarying from 3.5 to 6.5, whereas the elution time and peak shape ofnthraglycoside B showed significant difference when pH changed.nterestingly, when the pH was less than 5.5, resveratrol was elutedefore anthraglycoside B. However, when the pH was 6.5, the elu-ion order was inverted so that anthraglycoside B was eluted beforeesveratrol. Moreover, anthraglycoside B showed good peak shapehen the pH was less than 4.5, and poor peak shape when the pHas over 5.5.

The reversal of the elution order and the change of peak shape

ere due to the pH of the mobile phase, which was changing.s Horváth et al., Pietrzyk et al. and other investigators [16–20]ave demonstrated, the chromatographic behavior of the solutesre basically controlled by two major effects, a hydrophobic effectnd a reversible ionization of the solute in the mobile phase. The

gonum cuspidatum Sieb.et Zucc. at different eluent.

behavior of iongenic solutes in the columns of non-polar station-ary phases is controlled by two main events, the dissociation ofthe solutes in the mobile phase and the interaction between thesolutes species and the stationary phase [20]. The dissociation ofthe solutes is determined mainly by the pKa’s of the solute and thepH of the mobile phase.

The pKa values of polydatin, resveratrol and anthraglycoside Bpredicted from ACDLabs 6.0 pKa calculator are shown in Table 1.

Compounds pKa

Resveratrol 11.00(13-OH) 10.02(1-OH) 9.40(11-OH)Polydatin 10.01(1-OH) 9.39(11-OH)Anthraglycoside B 6.71(1-OH) 6.27(6-OH)

D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339 333

d frac

tHcpmwcrotvss

3Pa

Prpt2ate

pB

Fig. 3. HPLC chromatograms of the solvent extract an

he columns was not affected by the pH value of the mobile phase.owever, at a pH of 6.5, closer to anthraglycoside B’s pKa, little

hange in pH showed significant effect on the elution time andeak shape. Anthraglycoside B, existed in the neutral forms, showedore hydrophobic, longer retention time and good peak shapehen the pH was less than 5.5. When the pH was 6.5, anthragly-

oside B became ionized and showed more hydrophilic, shorteretention time and poor peak shape. In order to get good separationf anthraglycoside B, the pH of the mobile phase should be adjustedo be less than 5.5. Thus, acetonitrile–water–acetic acid (26:74:0.1,/v) was used as the mobile phase. Fig. 3 shows HPLC analysis of theolvent extract, fractions I and II separated by macroporous resineparation.

.2. Comparison of preparative liquid chromatography of C18,DVB-SPG and PDVB-Swelling media purification of resveratrolnd anthraglycoside B from the fraction II

Three reversed-phase chromatography media including C18,DVB-SPG and PDVB-Swelling were compared in the purification ofesveratrol and anthraglycoside B from the fraction II. The mobilehases for these three media were optimized. For the C18 column,he best mobile phase was 20% ethanol for washing impurities,5% ethanol for eluting resveratrol and 30% ethanol for elutingnthraglycoside B. For the PDVB-SPG and PDVB-Swelling column,

he best mobile phase was 30% ethanol for washing impurities andluting resveratrol, 35% ethanol for eluting anthraglycoside B.

As the result shown in the HPLC analysis, the pH of the mobilehase played an important role in the separation of anthraglycoside. So the pH of the mobile phase was also compared in preparative

tions I and II purified by macroporous resin column.

purification. The pH values of washing and elution solvent were thesame and adjusted by acetic acid to 4.5, 5.5 and 6.5, respectively.Figs. 4–6 show the separation results of C18, PDVB-SPG and PDVB-Swelling column, which were in accordance with the result of HPLCanalysis.

The pH value showed little effect on the separation of resvera-trol and different degree effect on the separation of anthraglycosideB. The elution time of anthraglycoside B was getting shorter withthe pH rising from 4.5 to 6.5. When the pH was 6.5, resveratrol andanthraglycoside B were eluted in one peak for C18 and PDVB-SPGcolumn, anthraglycoside B were coeluted together with some impu-rities for PDVB-Swelling. When the pH was less than 5.5, resveratroland anthraglycoside B were separated in two peaks. So the pH of themobile phase should be less than 5.5. The better pH of the mobilephase may be in the range of 4.5–5.5 for the stability of two targetcompounds.

Table 2 showed the quantitative comparisons of the C18,PDVB-SPG and PDVB-Swelling columns while performing liquidchromatography separation of resveratrol and anthraglycosideB under the optimal conditions. Due to the serious peak tailingand the overlap of the peaks for resveratrol and anthraglycosideB with impurities, the recoveries and purities for both resveratroland anthraglycoside B were low on the C18 column. On the otherhand, resveratrol and anthraglycoside B were well resolved onPDVB-SPG and PDVB-Swelling columns. For the separation time,

C18 needed about 45 min, PDVB-SPG needed about 80 min andPDVB-Swelling needed about 150 min. The higher selectivity andlonger elution time of the PDVB-SPG and PDVB-Swelling columnsmay be result from two aspects. Firstly, the selectivity was dueto the homogeneity structure of these two kinds of media, which

334 D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339

Fig. 4. Profiles of liquid chromatography purification of resveratrol and anthraglycoside B from the fraction II by C18 column.

Table 2Comparison of the separation and recovery of polydatin, resveratrol and anthraglycoside B on C18, PDVB-SPG, PDVB-Swelling columns under the optimal conditions.

Column Resveratrol Anthraglycoside B Polydatin

Purity, % Recovery, % Purity, % Recovery, % Purity, % Recovery, %

C 83.6P 93.8P 93.1

dtttactPPihsSt

TC

M

CPP

18 87.6 75.2DVB-SPG 93.1 92.5DVB-Swelling 94.6 90.5

id not have irreversible adsorption of the target compounds andhe impurities. Secondly, the long elution time could be due tohe presence of interactions between the aromatic-rich surface ofhese two polystyrene resins and the benzene ring of resveratrolnd anthraglycoside B, but this interaction is absent for the targetompounds on bonded C18 packing. Comparatively, the elutionime for PDVB-Swelling was almost twice longer than that forDVB-SPG. Moreover, obvious peak tailing were observed for theDVB-Swelling medium. The specific surface area of PDVB-Swelling

s 1.4 times larger than that of PDVB-SPG. Because PDVB-Swellingas the same particle diameter and almost the same average poreize (Table 3), it can be speculated that large surface area of PDVB-welling might come from the contribution of its more microporeshan PDVB-SPG. SEM photos of PDVB-SPG and PDVB-Swelling

able 3haracteristics of C18, PDVB-SPG and PDVB-Swelling.

edia Specific surface area (m2/g) Particle dia

18 300 10–40DVB-SPG 500.7 30DVB-Swelling 693.3 30

82.1 86.2 75.492.2 92.8 92.191.7 92.6 93.7

microspheres were shown in Fig. 7. No obvious difference wasfound on the outer layer of the beads. Specific surface area andpore size distribution of dry PDVB-SPG and PDVB-Swelling micro-spheres were characterized by BET nitrogen adsorption/desorptionmeasurement with Quantasorb apparatus (Quantachrome Corp.,USA) at 60 ◦C. The pore size distribution of a diameter less than2 nm for the two microspheres was shown in Fig. 8. The microporesin PDVB-Swelling are significantly more than that in PDVB-SPG.

As we know, the micropores present in porous chromatographic

particles are undesirable because of their slow mass transfer fea-ture. Such a feature may come from the slow diffusion of targetmolecules in these pores. The consequence would be a broad elu-tion fraction with peak tailing and longer retention time as observedin Fig. 6 with PDVB-Swelling medium. Compared with the purity,

meter (�m) Average pore size (nm) pH stability

≤10 2–813.8 1–1214.2 1–12

D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339 335

Fig. 5. Profiles of liquid chromatography purification of resveratrol and anthraglycoside B from the fraction II by PDVB-SPG column.

Fig. 6. Profiles of liquid chromatography purification of resveratrol and anthraglycoside B from the fraction II by PDVB-Swelling column.

336 D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339

G and

rSmas

3Pf

mcet

FS

Fig. 7. SEM photos of PDVB-SP

ecovery and separation time, PDVB-SPG was better than PDVB-welling. The medium was therefore chosen as reversed-phaseedium for the separation and purification of resveratrol and

nthraglycoside B. The chromatography condition was kept theame with pH value of the mobile phase in the range of 4.5–5.5.

.3. Comparison of preparative liquid chromatography of C18,DVB-SPG and PDVB-Swelling media purification of polydatinrom the fraction I

The mobile phase was optimized with respect to these threeedia, including C18, PDVB-SPG and PDVB-Swelling. For the C18

olumn, the best mobile phase was 15% ethanol for washing andluting polydatin. For the PDVB-SPG and PDVB-Swelling column,he best mobile phase was 25% ethanol for washing and eluting

ig. 8. Specific surface area and pore size distribution of dry PDVB-SPG and PDVB-welling microspheres.

PDVB-Swelling microspheres.

polydatin. Quantitative comparison of the results of three reversed-phase media column separation of polydatin is shown in Table 2. Asin Fig. 9 and Table 2, the recoveries and purities for polydatin werelower on the C18 column than that on the PDVB-SPG and PDVB-Swelling columns. Similar to the result of purification of resveratroland anthraglycoside B from fraction II by reversed-phase chro-matography, longer elution time and peak tailing were found forPDVB-Swelling column. Therefore, PDVB-SPG column was selectedas the best medium for the separation and purification of polydatin.

3.4. Scale-up of PDVB-SPG column for purification of polydatin,resveratrol and anthraglycoside B

Fig. 10 shows the profiles of the PDVB-SPG column scaled-up forpurification of polydatin from fraction I, and purification of resver-atrol and anthraglycoside B from fraction II. Fig. 11 shows the HPLCanalysis of the fractions corresponding to polydatin, resveratrol andanthraglycoside B of the PDVB-SPG column liquid chromatography,respectively. As shown in Figs. 10 and 11, preparative separation ofthree target compounds on PDVB-SPG gave satisfactory results. Itdemonstrates that this reversed-phase chromatography process isreproducible, effective and feasible to scale-up.

3.5. Total purification results of polydatin, resveratrol andanthraglycoside B

Fig. 12 shows a brief diagram of the process for simultaneousseparation and purification of polydatin, resveratrol and anthragly-coside B from P. cuspidatum Sieb.et Zucc. Table 4 illustrates the

recovery and purity of each step. After extracted from the herb, thecrude extract was divided into two fractions by macroporous resincolumn separation, one containing polydatin, the other containingfraction II which were resveratrol and anthraglycoside B. FractionsI and II were purified by PDVB-SPG column liquid chromatography.

D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339 337

Fig. 9. Profiles of liquid chromatography purification of polydatin from the fraction I by C18, PDVB-SPG and PDVB-Swelling column.

Fig. 10. Profiles of scale-up of liquid chromatography purification of polydatin from the fraction I by PDVB-SPG column (A) and liquid chromatography purification ofresveratrol and anthraglycoside B from the fraction II by PDVB-SPG column (B).

Table 4The total result of purification of polydatin, resveratrol and anthraglycoside B.

Steps Polydatin Resveratrol Anthraglycoside B

Purity, % Recovery, % Purity, % Recovery, % Purity, % Recovery, %

Solvent extract 7.5 – 1.3 – 2.4 –Macroporous resin adsorption 32.6 92.3 8.2 91.7 15.8 92.8PDVB-SPG 92.8 92.1 93.1 92.5 93.8 92.2Crystallization 98.8 95.6 98.2 91.2 98.6 93.7Total recovery 81.3 77.4 80.2

338 D. Zhang et al. / Separation and Purification Technology 66 (2009) 329–339

Fig. 11. HPLC chromatograms of the peak fra

Fc

Iwlpiatrai

4

p

ig. 12. Diagram of the integrated process for simultaneous separation and purifi-ation of polydatin, resveratrol and anthraglycoside B.

ndividual fractions of polydatin, resveratrol and anthraglycoside B,ere obtained respectively. The fractions were then directly crystal-

ized and dried. It must be pointed out that the fractions containingolydatin and resveratrol should be kept from light because of their

nstability under light [4]. The purities of polydatin, resveratrolnd anthraglycoside B can reach more than 98% after final crys-allization, respectively. The structures of the obtained polydatin,esveratrol and anthraglycoside B were confirmed by IR, ESI-MS,nd NMR spectroscopy. These data were identical to those reportedn literature [4,21].

. Conclusion

A systematic purification process was set up for simultaneousurification of polydatin, resveratrol and anthraglycoside B from P.

ctions purified by PDVB-SPG column.

cuspidatum Sieb.et Zucc. using liquid chromatography. Initial effortto separate and purify polydatin, resveratrol and anthraglycosideB from the solvent extract by one-step reversed-phase liquid chro-matography was also tried. However the result was not satisfactory.Therefore, macroporous resin column separation was used as an ini-tial step to remove impurities and separate the solvent extract intotwo fractions. The obtained fractions were then purified by subse-quent reversed-phase chromatography. Comparatively, two kindsof polymeric media PDVB-SPG and PDVB-Swelling showed betterseparation results in the selectivity, recovery and purity than theC18 bonded silica medium. Moreover, PDVB-SPG column with fewermicropores, showed better peak shape and shorter run time thanPDVB-Swelling column. PDVB-SPG was then chosen as the chro-matography medium in the polishing purification of these threecompounds. Because of the difference between three target com-pounds’ pKa, the pH value of the mobile phase showed little effecton the separation of polydatin and resveratrol, but large effect onanthraglycoside B in both HPLC analysis and preparative liquidchromatography. A good peak shape of anthraglycoside B could beobtained when the mobile phase pH value was below 5.5. Consider-ing the stability of target compounds in the subsequent evaporationstep, the pH value was chosen in the range of 4.5–5.5. Based onthe optimal condition, highly purified polydatin, resveratrol andanthraglycoside B were obtained with the total recoveries of 81.3%,77.4% and 80.2% respectively.

Acknowledgements

This work was supported by National Basic Research Program ofChina (Grant No. 2007CB714305) and Natural Science Foundationof China (Grant Nos. 20536050 and 20636010).

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