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Journal of Chromatography A, 1180 (2008) 53–58

pH-gradient counter-current chromatography isolation of natural antioxidantchlorogenic acid from Lonicera japonica Thumb. using an upright coil

planet centrifuge with three multi-layer coils connected in series

Tingting Wang, Xinhang Jiang, Lu Yang, Shihua Wu ∗Zhejiang University, Research Center of Siyuan Natural Pharmacy and Biotoxicology,

College of Life Sciences, Zijinggang Campus, Hangzhou 310058, China

Received 2 September 2007; received in revised form 29 November 2007; accepted 30 November 2007Available online 8 December 2007

bstract

A new pH-gradient counter-current chromatography method for the isolation of chlorogenic acid from flowers and buds of Lonicera japonicahumb. has been successfully established using a novel upright coil planet centrifuge with three multi-layer coils connected in series with 600 mLapacity. The crude extracts were first prepared by direct extraction with hot water and following concentration to remove the solution. Then thewo-phase solvent system composed of ethyl acetate–n-butanol–water (2:1:3, v/v) was applied to the separation. Its neutral upper phase was used astationary phase, whereas both its neutral lower phase and base lower phase with 10 mM NH3 were employed as mobile phase with gradient elution

n the head to tail mode. As a result, 330 mg quantity of crude extract was purified in one-step separation for 180 min, yielding 20.5 mg chlorogeniccid with over 98% purity. Structure of the compound is further identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) anduclear magnetic resonance (NMR). 2007 Elsevier B.V. All rights reserved.

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eywords: Counter-current chromatography; Chlorogenic acid; Lonicera japonaterials

. Introduction

Chlorogenic acid (5-O-caffeoyl-quinic acid), an ester of caf-eic acid with quinic acid, has been received considerablettentions for their wide distribution and potential biologicalffects [1]. It is one of the most abundant polyphenols inhe human diet with coffee, fruits and vegetables as its majorources. It is also an important bioactive compound and rich inome traditional Chinese medicine, such as Flowers and budsf Lonicera japonica Thumb. (L. japonica), and the leaves ofucommia lmodies, both which have been used for the treat-ent of affection by exopathogenic wind-heat or epidemic

ebrile disease at the early stage, carbuncles, furuncles and

wellings for centuries [2]. A large number of studies revealedhat chlorogenic acid has potential anti-inflammatory, analgesic,ntipyretic [3], antimutagenic [4,5], and anticarcinogenic activ-

∗ Corresponding author. Tel.: +86 571 88206287; fax: +86 571 88206287.E-mail address: drwushihua@zju.edu.cn (S. Wu).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.11.112

pH-gradient; Flos lonicerae; Polyphenol; Antioxidant; Natural products; Plant

ties [6,7]. It can inhibit Bc-Abl tyrosine kinase and triggers38 mitogen-activated protein kinase-dependent apoptosis inhronic myelogenous leukemic cells [8]. Like other dietaryolyphenols, chlorogenic acid is a potential antioxidant [9,10].n vitro, it can limit low-density lipid (LDL) oxidation, theajor determinant of the initial events in atherosclerosis [11].

t removes particularly toxic reactive species by scavenginglkylperoxyl radicals and may prevent carcinogenesis by reduc-ng the DNA damage they use [12–15]. In vivo, when addedo the diet, it inhibits chemically induced carcinogenesis of thearge intestine, liver and tongue in rats and hamsters [16–19].hlorogenic acid inhibits activator protein-1, NF-kB, anditogen-activated protein kinase (MAPKs) and induces phase 2

etoxifying enzyme activity [20]. Furthermore, it also acts as anntioxidant in plant and protects against lipid peroxidation [21].

The important bioactive activities both in plants and animals

ncourage the development of strategies to increase chloro-enic acid levels in plants [22] and obtain more productionf chlorogenic acid with higher purity [23,24]. Extraction ofhlorogenic acid from plants is an efficient approach to obtain

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his compound. However, due to the existence of some com-ounds with similar functional groups to chlorogenic acid, likeaffeic acid, gallic acid, protocatechuic acid and vanillic acid25], it is difficult to purify chlorogenic acid from plant. Eveno, some researches make arduous efforts to get chlorogeniccid of high purity. The conventional used chemical separationechnique was time- and solvent-consuming and was a compli-ated process [26,27]. Thin-layer chromatography method cannly obtain a tiny amount of target product and the preparativeiquid chromatography separation technique is a good but expen-ive approach to purify chlorogenic acid with need of expensivetationary material [28,29].

To overcome the issue, several new separation methods wereeveloped to obtain chlorogenic acid of the high purity. Li etl. [23] developed a molecular imprinting technique to separatehlorogenic acid from the extract if E. ulmodies leaves. However,he preparation of molecularly imprinted polymer monolithictationary phase is complex and some time-consuming for morehan 12 h. Further, it suffers a risk of irreversible adsorptive lossf samples onto the solid stationary phase matrices. Lu et al. [24]eveloped a high-speed counter-current chromatography (CCC)ethod for separation of chlorogenic acid from Flos lonicera.CC is a useful method for rapid chromatography purificationmploying highly efficient fraction by a hybrid technique ofiquid–liquid counter-current distribution and liquid chromatog-aphy, in conjunction with the use of centrifugal force to provideigorous mixing between stationary and mobile phase, as wells retention of a very large fraction of the stationary phase [30].he method uses no solid support matrix thus eliminates theomplications resulting from the solid support matrix, such asrreversible adsorptive sample loss and deactivation, tailing ofolute peaks, and contamination. Therefore, it has been success-ully applied to the analysis and separation of various naturalnd synthetic products [31–33]. However, it is difficult to obtainsatisfactory two-phase solvent system for separation of chloro-enic acid using common CCC method because of its littleolubility in non-polar solvents. Thus the previous CCC methodas to use the solvent system of n-butanol–acetic–water (4:1:5,/v), a hydrophilic solvent system with high ratio of organiccid, which is characterized by low interfacial and high viscos-ty and shows the opposite hydrodynamic behavior that alwaysistribute the lower phase on the head side and the upper phasen the tail side. As a result, the CCC separation using this sys-em require that lower phase was used as the stationary phasehile the upper phase was used as the mobile phase in the head

o tail elution mode [31,34]. Due to the reasons, the retention oftationary phase on the coiled column is rather low, only 38%24]. Therefore, development of a new technique for large-scaleeparation and purification of chlorogenic acid is very importantor the potential application of chlorogenic acid.

In this paper, a new pH-gradient CCC method for sepa-ation of chlorogenic acid has been successfully establishedor the first time. As well known, pH-related CCC techniques

uch as pH-peak-focusing and pH-zone-refining CCC, offerarious advantages over conventional CCC methods, such asarge sample capacity, high concentration of eluted fractions,nd enrichment and detection of minor components present in

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. A 1180 (2008) 53–58

large quantity of the crude sample [31]. The methods haseen successfully applied to the separation of natural and syn-hetic products including acidic and basic derivatives of aminocids, oligopeptides, hydroxyxanthene dye, alkaloids, indoleuxin, and structural, geometrical and optical isomers, and son [35]. Recently, some new developments of the pH-relatedCC, such as pH-modulated stepwise elution CCC [36,37], andH-gradient CCC [38] were established to separate hydrox-anthraquinones and cinnamic acid, which display the higherartition efficiency resulting in that complex target compoundsave to be clearly separated. So far, no report has been publishedn the use of pH-related CCC for the isolation and purification ofhlorogenic acid. Therefore, the present paper aims to develop aH-gradient CCC method to isolate and purify chlorogenic acidrom flowers and buds of L. japonica.

. Experimental

.1. Apparatus

The CCC isolation and purification of chlorogenic acid from. japonica was performed by a new upright CCC device with

hree multi-layer coil connected in series with total capacity of00 mL. Upright CCC is different from traditional horizontalCC. It not only utilizes a centrifugal force generated fromoth rotational and synchronous planetary motion of coiledolumns containing two immiscible liquid phases, but also useshe orthogonal gravitational force, which will benefit to improvehe efficiency of CCC separation [39]. Up to now, several prac-icable separation and purification of natural products have beenuccessfully performed by using the upright CCC [40–42]. Inhe present work, the upright CCC device with three multi-layeroiled columns was designed and fabricated by Research Centerf Siyuan Natural Pharmacy and Biotoxicology, Zhejiang Uni-ersity, Hangzhou, China. Its general design principle is shownn Fig. 1A where three upright cylindrical column holders areymmetrically arranged around the centrifuge axis as similar torevious upright device with four columns [40] and the type-HSCCC with three horizontal multi-layer coils connected in

eries [43]. Each holder undergoes an identical synchronouslanetary motion: revolution around the centrifuge axis and rota-ion about its own axis at the same angular velocity in the sameirection as indicated by arrows. These holders are connected ineries with flow tubes. All constructs ensure that the system per-its the effluent flow in and out the system through the rotating

olumns without the use of a rotary seal which would become aource of leakage and contamination.

The fabricated CCC apparatus holds three identical multi-ayer coils in the symmetrical positions around the rotary framet distance of 10 cm from the central axis of the centrifuge toaintain perfect balance of centrifuge system without the use ofcounterweight. Each separation column was made by windingsingle piece of 4 mm i.d. and 1 mm wall thickness polytetraflu-

roethylene (PTFE) tubing directly onto the holder hub of 10 cmiameter, forming three layers of right-handed and left-handedoils alternating in each layer between a pair of flanges spaced0 cm apart. The β value of the multi-layer coil varies from 0.5

T. Wang et al. / J. Chromatogr. A 1180 (2008) 53–58 55

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ig. 1. (A) The design principle and (B) the photograph of the connection ofentrifuge.

t the internal terminal to 0.8 at the external terminal (β = r/Rhere r is the distance from the coil to the holder shaft, and R

he revolution radius or the distance between the holder axis andentral axis of the centrifuge). As shown in Fig. 1B, these multi-ayer coils are connected in series on the rotary frame using aow tube (PTFE, 1.6 mm i.d. and 0.7 mm wall thickness) to givetotal capacity of 600 mL while the unique gear arrangement on

he rotary frame establishes a twist-free mechanism of the flowubes so that continuous elution can be performed without these of a rotary seal.

The apparatus is equipped with a Microwolf PA-05 speedontroller (Microwolf Electric Co., Shanghai, China) to regulatehe revolution speed ranging from 0 to 1000 rpm, and a temper-ture control unit to control the separation temperature rangingorm 20 to 60 ◦C. In addition, this CCC system is equipped withP270 metering pump, a UV 230+ spectrometer (Elite Analyti-al Instrument Co., Dalian, China), BSZ-100 fraction collector, aheodyne 3725i-038 injection valve and Sepu3000 data analysis

ystem (Puhui Scientific Instrument Co., Hangzhou, China).The high-performance liquid chromatography (HPLC) used

as an Agilent 1100 system including a G1379A degasser, a1311A QuatPump, a G1367A Wpals, a G1316A column oven,G1315B diode assay detector (DAD), and an Agilent ChemSta-

ion for LC. The column used was a reversed-phase C18 columnZorbax Eclipse XDB-C18 narrow-bore, 150 mm × 2.1 mm i.d.,.5 �m).

.2. Reagents

All organic solvents used for CCC were of analytical gradend purchased from Huadong Chemicals, Hangzhou, China. The

ater was purified by means of a Milli-Q system (Millipore,edford, MA, USA) and used for all solutions and dilutions.ethanol used for HPLC analysis was of chromatographic grade

nd purchased from Merck, Darmstadt, Germany. The standard

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multi-layer coiled columns of present CCC apparatus with upright coil planet

hlorogenic acid was purchased from National Institute for theontrol of Pharmaceutical and Biological Products, Beijing,hina.

The flowers and buds of L. japonica were purchased fromhe Huqingyutang Museum of Traditional Chinese Medicines,angzhou, China. A voucher specimen with reference number2006-1068 is kept in the Research Centre of Siyuan Naturalharmacy and Biotoxicology, Zhejiang University, China.

.3. Preparation of crude extracts

Dried and powdered flowers of L. japonica (150 g) wasxtracted directly three times with pure water (3× 1 L) in aoiling water bath for 1 h per time. Then, the extract solu-ions were combined and evaporated under reduced pressure and0 ◦C to about 200 mL. The concentrated solution was furthero lyophilize, yielding a 50 g of crude extracts.

.4. Preparation of two-phase solvent system and sampleolutions

The two-phase solvent system used was composed of ethylcetate–n-butanol–water with the volume ratio of 2:1:3. Theolvent mixture was thoroughly equilibrated in a separatory fun-el at room temperature and the two phases were separatedhortly before use. For the present preparative CCC separa-ion, the total volume of the prepared two phases each time isL.

The sample solutions were prepared as follows: the crude

qual volumes of both upper and lower phases at suitable con-entration according to the preparative scale of CCC separation.fter the centrifugation to remove the precipitate, the super-atant liquid was collected as sample solution.

5 atogr. A 1180 (2008) 53–58

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Fig. 2. HPLC-UV profiles (330 nm) of crude extracts from the flowers and budsof Lonicera japonica Thumb. as well as the structure of chlorogenic acid (1).Conditions: column, Zorbax Eclipse XDB-C18 narrow-bore (150 mm × 2.1 mmi.d., 3.5 �m); column temperature, 35 ◦C; mobile phase, C (methanol) and D(a2

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.5. Separation procedure

Preparative pH-gradient CCC was performed as follows: thehree upright multi-layer coil columns connected in series wererst entirely filled with upper phase as stationary phase, and then

he sample solution was injected through the sample port and theower while both neutral lower phase (A) and base lower phaseith 10 mM NH3 (B) as a mobile phase was pumped in head-to-

ail elution mode at a linear gradient elution with the flow rate ofmL/min while the column was rotated at 500 rpm. The volume

atio of A and B started at 100:0 and then continuously changedrom 100:0 to 0:100 for 130 min. The separation temperatureas controlled at 30 ◦C. The effluent was monitored on-line at40 nm and automatically collected in 20 mL test tube per 3 minsing a BSZ-100 fraction collector. Peak fractions were collectedccording to the elution profile and HPLC detection.

.6. HPLC analysis and identification of CCC peakractions

HPLC analyses of the crude extracts and CCC peak fractionsere performed with a reversed-phase C18 column (Zor-ax Eclipse XDB-C18 narrow-bore, 150 mm × 2.1 mm i.d.,.5 �m). The mobile phase was C (methanol) and D (0.03%rifluoroacetic acid aqueous solution) at the gradient: C from% to 80% and D from 95% to 20% for 20 min. The flow-rateas 0.3 mL min−1, and the effluent was monitored at 330 nm.Identification of the CCC peak fraction was carried out

y electrospray ionization tandem mass spectrometry (ESI-S/MS), and nuclear magnetic resonance (NMR) spectra asell as reference standard. Positive ESI-MSn analyses wereerformed using Thermo Finnigan LCQ Daca XP Plus spec-rometer with an ESI interface. MS/MS analyses of selected ionsere performed in the ion trap by collision-induced dissociation

CID) with helium. NMR experiments were carried out using aruker Advanced DMX 500 NMR spectrometer with dimethyl

ulfoxide (DMSO-d6) as solvent and tetramethylsilane (TMS)s internal standard.

. Results and discussion

.1. HPLC analyses of crude extracts

Flowers and buds of L. japonica, commonly known as ‘Jiny-nhua’ in traditional Chinese medicine, has been reported toossess the constituents of iridoid glucoside [44] and polyphe-olic compounds, i.e., hyperoside, chlorogenic acid, luteolin andaffeic acid [45,46]. HPLC analysis (Fig. 2) revealed that crudeater extracts of L. japonica contained several constituents with

imilar UV absorbance to chlorogenic acid.

.2. Preparative CCC separation

Successful CCC separation depends on the correct two-phaseolvent system. However, it is difficult to obtain a satisfactorywo-phase solvent system using common CCC method due tonusual solubility of the chlorogenic acid. Chlorogenic acid is

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0.03% trifluoroacetic acid aqueous solution) at the gradient: C from 5% to 80%nd D from 95% to 20% for 20 min; flow rate, 0.3 mL min−1; injection volume,�L.

ot soluble in non-polar solvents, and has a littile solubitlty inthyl acetate, but it has some solubility in methanol and ethanol,nd is freely soluble in water. According to these properties,he previous CCC method has to use the solvent system of n-utanol–acetic–water (4:1:5, v/v), a hydrophilic solvent systemith high ratio of organic acid, which is characterized by low

nterfacial and high viscosity and shows the opposite hydrody-amic behavior that always distribute the lower phase on theead side and the upper phase on the tail side. As a result, theCC separation using this system require that lower phase wassed as the stationary phase while the upper phase was used ashe mobile phase in the head-to-tail elution mode. Due to theeasons, the retention of stationary phase on the coiled columns rather low, only 38% [24].

Partition coefficient experiments as reported [24] revealedhat chlorogenic acid can be easily soluble in some polar two-hase solvents systems, i.e. ethyl acetate–n-butanol–water witharious volume ratios, i.e. 1:1:2, 2:1:3, 4:1:5, 1:4:5, and 3:2:5nd so on. Clearly, the contents of chlorogenic acid in upperf these systems are commonly more than one in their lowerhase. This is the reason for the retention of chlorogenic acidn the column for long time and even not eluted using classicalCC method. However, it can be efficiently used for separationf chlorogenic acid from other constituent with more polar prop-rties. Furthermore, the system of ethyl acetate–n-butanol–waters different from the hydrophilic system of n-butanol–waterharacterized by low interfacial and high viscosity and showedhe opposite hydrodynamic behavior which always distributeshe lower phase on the head side and upper phase on the tailide. Due to the existence of ethyl acetate, the systems ofthyl acetate–n-butanol–water may be classified as moderateydrophobic solvent systems with shorter settling time and lowernterfacial tension, which distributes the upper phase on the headide and the lower phase on the tail side. The system has been

sed for long to isolate several compounds including phenoiclycosides [47], isoflavones [48,49], salidroside [50], gallic acid51], saponins [52], and p-nitrophenyl glucose [53] using upperhase as stationary phase and lower phase as mobile phase in

matogr. A 1180 (2008) 53–58 57

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Fig. 3. (A and B) Preparative CCC profiles of the crude extracts from the flow-ers and buds of L. japonica and (C) HPLC analysis of chlorogenic acid productpurified by pH-gradient CCC separation; (1) chlorogenic acid. CCC separa-tion conditions: column, multi-layer coil of 4.0 mm i.d. PTFE tube with a totalcapacity of 600 mL; rotary speed, 500 rpm; column temperature, 30 ◦C; solventsystem for (A), basic solvent system of ethyl acetate–n-butanol–water (2:1:3,v/v) containing 10 mM NH3 and its upper phase as stationary phase as well asits lower phase as mobile phase, and for (B and C), neutral upper phase of thesystem of ethyl acetate–n-butanol–water (2:1:3, v/v) as stationary phase whileboth neutral lower and base lower phase with 10 mM NH3 as mobile phase witha linear gradient elution exchanged from 100:0 for 70 min and then to 0:100 for130 min; elution mode, head-to-tail; flow rate, 5 mL min−1; detection, 340 nm;sample size, 330 mg dissolved in 4 mL upper phase and 4 mL lower phase; sam-pats

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he head to tail elution. These experiments indicated it is veryracticable and easy to achieve higher retention of stationaryhase more than 50%.

pH-related CCC is very useful for isolation of severalompounds including organic acids, alkaloids, bases, andeptides. As above experiments, the chlorogenic acid alwaysetained in the column for long time and even not elutedsing the upper phase of acidic or neutral systems of ethylcetate–n-butanol–water as stationary phase and its lower phases mobile phase in head-to-tail elution. However, it could note efficiently separated and always appeared at front of mobilehase elution with other polar constituents using the basicolvent system of ethyl acetate–n-butanol–water containing0 mM NH3 (Fig. 3A). Clearly, chlorogenic acid is moreoluble in lower phase of the basic system as mobile phase thants upper phase as stationary phase resulting in rapid elutionithout any separation. Thus, a pH-gradient CCC methods waseveloped as follows.

Fig. 3B shows the preparative CCC separation of 330 mgf the crude extracts using the solvent system composed ofthyl acetate–n-butanol–water (2:1:3, v/v). The neutral upperhase was used as stationary phase while both neutral lowerhase and base lower phase were used as mobile phase at ainear gradient from 100:0 for 70 min and then to 0:100 for30 min. The retention of stationary phase was more than0% when sample solution injected was supernatant liquidfter centrifugation for removing precipitate. In order to saveolvents and time, the other eluting compounds after the targetubstances were removed by pumping out the stationary phasenstead of eluting them with the mobile phase because of thetationary phase was used only once. After the separation theCC peak fractions were collected respectively according toCC profile and further HPLC analysis.

The structure of product chlorogenic acid corresponding tohe CCC peak 1 fractions was identified by ESI-MS/MS andMR spectra. The compound indicates characteristic ion at m/z55.3 due to [M + H]+ in positive ESI-MS and ion at m/z 353.3ue to [M − H]− in negative mode, corresponding to the molec-lar weight of 354.3 of chlorogenic acid. The positive precursoron at m/z 355.3 produced prominent fragment ion at m/z 163.1hile negative precursor ion at m/z 353.3 produced prominent

ragments at m/z 191.3 and 179.1, which is in very close agree-ent with its caffeoyl and quinic groups. 1H NMR spectral data

500 MHz, DMSO-d6), δ 7.42 (1H, d, J = 15.9 Hz, H-7′), 7.031H, d, J = 2.0 Hz, H-2′), 6.98 (1H, dd, J = 2.0, 8.2 Hz, H-6′),.76 (1H, d, J = 8.2 Hz, H-5′), 6.15 (1H, d, J = 15.9 Hz, H-8′),.06 (1H, br d, J = 6.8 Hz, H-5), 3.93 (1H, br s, H-3), 3.57 (1H,r d, J = 6.8 Hz, H-4), 1.99–2.04 (2H, m, H-6), 1.94 (1H, m, H-ax), 1.78 (1H, m, H-2eq); 13C NMR spectral data (125 MHz,MSO-d6), δ 175.0 (C-7), 165.7 (C-9′), 148.5 (C-4′), 145.4 (C-′), 144.8 (C-3′), 125.4 (C-1′), 121.2 (C-6′), 115.7 (C-5′), 114.7C-2′), 114.3 (C-8′), 73.6 (C-1), 70.9 (C-4), 70.6 (C-5), 68.3 (C-), 37.2 (C-6), 36.5 (C-2). These data obtained were identical to

he authentic chlorogenic acid and previously published studies54,55].

As a result, 20.5 mg chlorogenic acid with over 98% purityFig. 3C) were obtained in one-step separation for 180 min,

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les, crude sample solution without further centrifugation for (A), and withdditional centrifugation to remove the emulsifiable precipitates for (B); reten-ion of the stationary phase, (A) 51.5% and (B) 62.5%. For HPLC conditions,ee Section 2.

hich clearly indicated the pH-gradient elution was very effi-ient for the fast CCC separation of the chlorogenic acid.

. Conclusions

In conclusion, a new pH-gradient CCC isolation of chloro-enic acid from flowers and buds of L. japonica was successfully

erformed using a novel upright coil planet centrifuge with threeulti-layer coils connected in series. Differing from classicalCC separation, pH-gradient CCC method permits use of sim-le solvent system to retain much stationary phase for higher

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artition efficiency, which is more favorable for organic acid,ases, polyphenols and other constituents. The present resultslearly demonstrated the pH-gradient CCC method is powerfulor the separation of chlorogenic acid from crude extracts ofowers and buds of L. japonica.

cknowledgements

This work was supported by National Natural Science Foun-ation of China (20602031) and Zhejiang Provincial Naturalcience Foundation of China (Y304118). The authors alsocknowledge the fabrication of the present CCC instrument byenior engineer Yucheng Wu.

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