Food Chemistry 158 (2014) 433–437

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Food Chemistry

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Analytical Methods

Preparative isolation and purification of phlorotannins from Eckloniacava using centrifugal partition chromatography by one-step

http://dx.doi.org/10.1016/j.foodchem.2014.02.1120308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +82 51 629 5849 (J. Kim). Tel.: +82 64 754 3475;fax: +82 64 756 3493 (Y.-J. Jeon).

E-mail addresses: jikim@pknu.ac.kr (J. Kim), youjin@jejunu.ac.kr (Y.-J. Jeon).

Ji-Hyeok Lee a, Ju-Young Ko a, Jae-Young Oh a, Chul-Young Kim b, Hee-Ju Lee c, Jaeil Kim d,⇑, You-Jin Jeon a,⇑a Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Koreab Natural Product Research Center, Hanyang University, Daejeon-dong, Ahnsan, Gyeongi-do, Republic of Koreac Natural Product Research Center, KIST Gangneung Institute, Daejeon-dong, Gangneung, Gangwon-do, Republic of Koread Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 January 2013Received in revised form 5 February 2014Accepted 19 February 2014Available online 28 February 2014

Keywords:Centrifugal partition chromatography (CPC)PhlorotanninEcklonia cava

Various bioactive phlorotannins of Ecklonia cava (e.g., dieckol, eckol, 6,6-bieckol, phloroglucinol, phlo-roeckol, and phlorofucofuroeckol-A) are reported. However, their isolation and purification are not easy.Centrifugal partition chromatography (CPC) can be used to efficiently purify the various bioactive-compounds efficiently from E. cava. Phlorotannins are successfully isolated from the ethyl acetate (EtOAc)fraction of E. cava by CPC with a two-phase solvent system comprising n-hexane:EtOAc:methanol:water(2:7:3:7, v/v) solution. The dieckol (fraction I, 40.2 mg), phlorofucofuroeckol-A (fraction III, 31.1 mg), andfraction II (34.1 mg) with 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol areisolated from the crude extract (500 mg) by a one-step CPC system. The purities of the isolated dieckoland phlorofucofuroeckol-A are P90% according to high performance liquid chromatography (HPLC)and electrospray ionization multi stage tandem mass spectrometry analyses. The purified 2,7-phloroglu-cinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are collected from fraction II by recycle-HPLC. Thus, the CPC system is useful for easy and simple isolation of phlorotannins from E. cava.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

An edible brown seaweed Ecklonia cava, is known to showvarious biological activities such as anticancer, antioxidant, anti-allergic disease and anti-neurodegenerative disease activities(Athukorala, Kim, & Jeon, 2006; Kang et al., 2011; Kim, Ahn, &Kim, 2006; Kim, Heo, et al., 2006; Le, Li, Qian, Kim, & Kim, 2009;Shim, Le, Lee, Kim, 2009). E. cava has bioactive phlorotan-nins, including dieckol, pyrogallol-phloroglucinol-6,6-bieckol,2,7-phloroglucinol-6,6-bieckol, and phlorofucofuroeckol-A (seestructures in Fig. 1). Their chemical structures are shown inFig. 1. In particular, dieckol is reported to possess inhibitory activ-ity against a-glucosidase and a-amylase in vitro and alleviatespostprandial hyperglycaemia in streptozotocin-induced diabeticmice (Lee et al., 2010). Pyrogallol-phloroglucinol-6,6-bieckol and2,7-phloroglucinol-6,6-bieckol from E. cava have been evaluatedfor their antioxidant properties (Kang, Heo, Kim, Lee, Jeon, 2011;Kang, Lee, et al., 2011). Phlorofucofuroeckol-A from Eckolina stolo-nifera is reported to show inhibitory effects on FcRI expression

which is known to be involved in the regulation of IgE-mediatedallergic reactions (Shim, Choi, Byun, 2009). However, traditionalmethods for their purifications demand repetitive chromatographyprocesses using Sephadex LH-20 column chromatography and re-versed-phase high performance liquid chromatography (HPLC)(Heo et al., 2009; Kang, Heo, et al., 2011; Kang, Lee, et al., 2011;Kim et al., 2011). Additionally, the conventional methods are timeconsuming, have problems with adsorption in the stationaryphase, and can only be worked with limited amounts of the com-pounds. To solve these problems, a preparative centrifugal parti-tion chromatography (CPC) system is used.

A preparative CPC system, part of counter-current chromatogra-phy (CCC), is a non-solid support, preparative, liquid–liquid separa-tion process based on the difference in the distribution ofcomponents in two immiscible liquid phases. This enables the iso-lation and purification of large quantities of compounds with puri-ties of greater than 90% in a one-step process (Bourdat-Deschamps,Herrenknecht, Akendengue, Laurens, & Hocquemiller, 2004;Delannay et al., 2006; Michel, Luuk, & Karel, 1997). In addition,the CPC system also offers the following technological advantagessuch as versatile products, faster and inexpensive product develop-ment, retention of bioactivity integrity, higher throughput, higheryields, and reduced operating costs. The solutes are separated

O

OO

OH OOH

OH

OHHO

OO

O

OH

OH

OH

HO

HO OH

OO

OO

OHO

HOHO

OH

OH

OH

OH

OHHO

lokceiDAlokceorufocuforolhP

OO

O

OOH

HO

HO OH

O

OOH

OH

HOO

HO OH

O OH

OH

OH

OHHOOH

O

OHO

OH OOH

OH

OHHO

O

OHO

OHOH

OHO

OHOO

OH

OH OH

OH

2,7-phloroglucinol-6,6-bieckol

Pyrogallol-phloroglucinol-6,6-bieckol

Fig. 1. Chemical structures of phlorotannins from Ecklonia cava.

434 J.-H. Lee et al. / Food Chemistry 158 (2014) 433–437

according to their partition coefficient (K), which is expressed asthe ratio of their concentration in the stationary phase to that inthe mobile phase (Berthod & Armstrong, 1988). The CPC systemhas been widely used for the separation of bioactive compoundsfrom land plants (Marston, Borel, Hostettmann, 1988; Bourdat-Deschamps et al., 2004; Kim, Ahn, et al., 2006; Kim, Heo, et al.,2006). However, in the case of seaweeds, only a few algae suchas Ascophyllum nodosum have been subjected to CPC (Chevolot,Colliec-Jouault, Foucault, Ratiskol, & Sinquin, 1998; Chevolot,Foucault, Colliec-Jouault, Ratiskol, & Sinquin, 2000).

The focus of this study is on the simple and easy isolation ofphlorotannin compounds from E. cava.

2. Materials and methods

2.1. Materials

E. cava, collected from the coast of Jeju Island, South Korea, inJune 2009, was ground and shifted through a 50 mesh standardtesting sieve after drying in a freeze dryer SFDSMO6 (Samwonfreezing engineering co., South Korea). The dried E. cava was storedin a refrigerator until use.

All solvents used for the preparation of crude samples and CPCseparation were analytical grade (Daejung Chemicals & Metals Co.,Seoul, South Korea). HPLC grade solvents were purchased fromBurdick & Jackson (MI, USA).

2.2. Apparatus

LLB-M high performance CPC (Sanki Engineering, Kyoto, Japan)was used in preparative CPC. The total cell volume was 240 mL. Afour-way switching valve incorporated in the CPC apparatusallowed its operation in either the descending or ascending mode.This CPC system was equipped with a Hitachi 6000 pump, anL-4000 UV detector (Hitachi, Japan), and Gilson FC 203B fractioncollector (Gilson, France). The samples were manually injectedthrough a Rheodyne valve (Rheodyne, CA, USA) with a 2 mL sampleloop.

The 1H-NMR spectra were measured with a JEOL JNM-LA 300spectrometer (JEOL Ltd., Tokyo, Japan) and 13C-NMR spectra witha Bruker AVANCE 400 spectrometer (BRUKER, Germany). The massspectra (FAB-MS and EIMS) were recorded on a JEOL JMS 700 spec-trometer. The HPLC system consisted of a binary Gilson 321 pump,Gilson UV–Vis 151 detector, Gilson 234 auto-injector, and 506Cinterface module (Gilson). The recycle HPLC system was equippedwith a liquid feed pump type L-7100 (JAI, Japan), and JAI UV detec-tor 3702 (JAI).

2.3. Preparation of crude sample from E. cava

Dried E. cava (20 g) was extracted three times with 1 L of 70%ethanol (EtOH) for 3 h by sonication at room temperature (25 �C).The extract was concentrated in a rotary vacuum evaporator andpartitioned with ethyl acetate (1:1, v/v of sample). Then, the driedethyl acetate fraction was stored in a refrigerator for CPCseparation.

2.4. Preparation of two-phase solvent system and sample solution

The CPC experiments were performed using a two-phase sol-vent system comprising n-hexane/ethyl acetate/methanol/water(2:7:3:7, v/v/v/v) solvent. The two phases were separated afterthoroughly equilibrating the mixture in a separating funnel atroom temperature. The upper organic phase was used as the sta-tionary phase, and the lower aqueous phase was employed asthe mobile phase.

2.5. CPC separation procedure

The CPC column was initially filled with the organic stationaryphase and rotated at 1000 rpm; the mobile phase was pumped intothe column in the descending mode at the same flow rate used forseparation (2 mL/min). When the mobile phase emerged from thecolumn, it indicated that hydrodynamic equilibrium had beenachieved (back pressure: 420.5 psi). The concentrated EtOAc frac-tion (500 mg) obtained from the 70% EtOH extract of E. cava wasdissolved in 6 mL of a 1:1 (v/v) mixture of the two CPC solvent sys-tem phases and injected through the Rheodyne injection valve. Theeffluent from the CPC was monitored by UV at 290 nm, and 6 mLfractions were collected in 8 mL tubes by a fraction collector.

2.6. HPLC analysis of crude extracts from E. cava

Here, 5 lL of a 5 mg/mL sample solution was directly injectedon an Atlantis T3 column (3 lm 3.0 � 150 mm column) (Waters,USA) using a gradient acetonitrile–water solvent system at roomtemperature. The mobile phase comprised acetonitrile–water ingradient mode as follows: acetonitrile with 0.1% formic acid–waterwith 0.1% formic acid (0–40 min:10:90–40:60 v/v, 40–50 min:-�50:50 v/v, and 50–60 min:�100:0 v/v). The flow rate was0.2 mL/min, and the UV absorbance was detected at 290 nm.

Table 1K-values of phlorotannins from E. cava as solvents.

Solvents K-value

⁄H:E:M:W Dieckol 2,7phloroglucinol-6,6-bieckol

PhlorofucofuroeckolA & pyrogallol-phloroglucinol-6,6-bieckol

4:6:4:6 0.49 0.52 0.553:7:3:7 0.25 0.44 1.652:7:3:7 0.29 0.51 0.892:8:2:8 2.11 6.89 7.30

⁄ H:E:M:W = n-Hexan:Ethylacetate:Methanol:Water.

J.-H. Lee et al. / Food Chemistry 158 (2014) 433–437 435

2.7. Recycle HPLC separation

The samples were manually injected through a Rheodyne 7725ivalve (Rheodyne, CA, USA) with a 2 mL sample loop. Then, 1 mL ofa 10 mg/mL sample solution was directly injected into the JAIGEL-ODS-BP-L, SP-120–15 column (JAI, Japan) using an isocratic 30%acetonitrile–water solvent system. The flow rate was 1 mL/minwith UV absorbance detection at 290 nm.

2.8. HPLC–DAD–ESI/MS analysis of purified compounds

HPLC–DAD–ESI/MS analyses were carried out using a Hewlett–Packard 1100 series HPLC system equipped with an autosampler, acolumn oven, a binary pump, a DAD detector, and a degasser (Hew-lett–Packard, Waldbronn, Germany) coupled to a Finnigan MATLCQ ion-trap mass spectrometer (Finnigan MAT, San Jose, CA,USA). The MS was equipped with a Finnigan electrospray sourceand was capable of analysing ions up to m/z 2000. Xcalibur soft-ware (Finnigan MAT) was used for the MS operations. The chro-matographic conditions are identical to those described inSection 2.6 and the flow cell outlet was connected to a splittingvalve, from which a flow of 0.2 mL/min was diverted to the electro-spray ion source via a short fused silica tubing. Negative ion massspectra of the column eluate were recorded in the range m/z 100–2000. The source voltage was set to 4.5 kV and the capillary tem-perature to 250 �C. The other conditions were as follows: capillaryvoltage, –36.5 V; inter-octapole lens voltage, 10 V; sheath gas,80 psi (551.6 kPa); auxiliary gas, 20 psi (137.9 kPa).

3. Results and discussion

Previous studies show that among all the fractions from E. cava(n-hexane, chloroform, EtOAc, n-butanol fractions), the EtOAc frac-tion is known to contain various bioactive phlorotannins such asdieckol, phloroeckol, and 6,6-bieckol (Kang, Lee, Chae, Koh, et al.,2005; Kang, Lee, Chae, Zhang, et al., 2005). Therefore, the EtOAcfraction was selected for further experiments and expressed thatits yield was 28% of EtOH extract (4 g). It was analysed using thedescribed HPLC condition; its chromatogram is depicted in Fig. 2.The peaks 1 and 2 on the HPLC chromatogram have been con-firmed as dieckol and 2,7-phloroglucinol-6,6-bieckol, respectively,by both LC–DAD–ESI/MS and previous reports (Kang, Jeon, et al.,2011; Lee et al., 2009). In addition, the LC–DAD–ESI/MS datashowed that the phlorofucofuroeckol-A and pyrogallol-phloroglu-cinol-6,6-bieckol share the same HPLC peak. The partitioncoefficient (K) in a suitable two phase solvent systems is the most

Fig. 2. HPLC chromatogram of the EtOAc fraction from E. cava. Peak 1: dieckol; peak 2phlorofucofuroeckol A. (Chromatographic conditions, see Section 2).

important variable for successful separations of the target samplesby preparative CPC. To select the most efficient separation system,several two-phase solvent systems with different compositionsand volume ratios of two immiscible solvents such as n-hexane/EtOAc/methanol/water were examined, and their K values werecalculated (Table 1). A K value between 0.2 and 5 can be usedwithout any excessive elution time associated with bandbroadening (Foucault, 1994; Sutherland & Fisher, 2009). The sol-vent n-hexane/EtOAc/methanol/water (2:7:3:7) exhibited themost efficient separation for each of the phlorotannins such asdieckol and phlorofucofuroeckol-A. The K values of dieckol,2,7-phloroglucinol-6,6-bieckol and phlorofucofuroeckol A & pyro-gallol-phloroglucinol-6,6-bieckol were 0.29, 0.51, and 0.89, respec-tively. Therefore, preparative CPC operated in the descendingmode, with the upper phase acting as the stationary phase andthe lower phase as the mobile phase. During the process, 500 mgcrude of EtOAc extract was fractionated in a single run of190 min, the retention of the stationary phase in the coil was69.5%, and the pressure was 464 psi. The preparative CPC chro-matogram is described in Fig. 3. The analysis of the HPLC peak areashowed that the fractions I and III had purified compounds (dieckoland phlorofucofuroeckol-A, respectively) of up to 90% (Fig. 4A andC). The figure shows that 40.2 and 31.1 mg of dieckol and phlorofu-cofuroeckol-A, respectively (8.4% and 6.22% of the EtOAc fraction,respectively), were isolated and collected from 500 mg of theE. cava EtOAc fraction. In addition, both 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol were present infraction II (Fig. 4B). Previous reports related to the isolation ofphlorotannins from E. cava have shown that dieckol and phlorofu-cofuroeckol A were generally isolated within 1% of EtOAc fraction(Ahn et al., 2007 and Lee et al., 2009). The isolation of the phloro-tannins in previous reports resulted in significant losses of thesample because of the complicated processes and adsorption onSephadex LH-20 and silica columns via hydroxyl branches.

: 2,7-phloroglucinol-6,6-bieckol; peak 3: pyrolgallol-phloroglucino-6,6-bieckol and

Fig. 3. Preparative CPC seperation of the EtOAc fraction from E. cava. Solvents condition. (CPC conditions, see Section 2).

Fig. 4. HPLC chromatogram and ESI-MS spectra of CPC peak fractions (I, II and III). CPC peak fraction I (A); CPC peak fraction II (B); CPC peak fraction III (C) in Fig. 3.(Chromatographic and ESI-MS conditions, see Section 2).

436 J.-H. Lee et al. / Food Chemistry 158 (2014) 433–437

J.-H. Lee et al. / Food Chemistry 158 (2014) 433–437 437

Therefore, preparative CPC can collect higher amounts and yieldsthan previously reported for the phlorotannins. 2,7-phloroglu-cinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol couldbe obtained in 90% purity from fraction II using recycle HPLC. Allthe purified compounds were confirmed as dieckol, 2,7-phloroglu-cinol-6,6-bieckol, pyrogallol-phloroglucinol-6,6-bieckol, and phlo-rofucofuroeckol-A comparing the results with the previouslyreported 1H and 13C-NMR data (Kang, Heo et al., 2011; Kang, Lee,et al., 2011; Lee et al., 2009).

Identification of each CPC fraction was carried out using 1HNMR, 13C NMR and HPLC–DAD–ESI/MS (negative ion mode)analyses.

Peak A (dieckol): amorphous powder, 1H NMR (400 MHz, meth-anol-d4) d 6.15 (1H, s), 6.13 (1H, s), 6.09 (1H, d, 2.9 Hz), 6.06 (1H, d,2.9 Hz), 6.05 (1H, d, 2.9 Hz), 5.98 (1H, d, 2.8 Hz), 5.95 (1H, d,2.8 Hz), 5.92 (3H, m); 13C NMR (100 MHz, methanol-d4) d 161.8,160.1, 157.8, 155.9, 154.5, 152.4, 147.3, 147.2, 147.1, 146.9,144.3, 144.1, 143.4, 143.3, 138.6, 138.5, 126.5, 126.2, 125.6,125.5, 124.9, 124.6, 124.5, 99.9, 99.7, 99.5, 99.4, 97.6, 96.2, 95.8,95.7, 95.3; ESI-MS: [M�H]� at m/z 741.

Peak B-1 (2,700-phloroglucinol-6,60-bieckol): amorphous pow-der, 1H NMR (400 MHz, methanol-d4) d 5.57 (1H, s), 5.89 (1H, s),5.74 (1H, m), 5.84 (1H, m), 5.74 (1H, m), 6.25 (1H, s), 6.14 (1H,s), 5.84 (1H, m), 5.89 (1H, m), 5.84 (1H, m), 6.52 (1H, s), 6.14(1H, m), 6.44 (1H, m), 6.77 (1H, s), 6.72 (1H, s), 8.93 (1H, s), 8.93(1H, s), 9.19 (1H, s), 9.19 (1H, s), 9.19 (1H, s), 9.04 (1H, s), 8.26(1H, s), 9.94 (1H, s), 8.59 (1H, s), 9.88 (1H, s), 9.86 (1H, s), 9.25(1H, s), 9.75 (1H, s), 9.21 (1H, s); 13C NMR (100 MHz, methanol-d6) d 127.6, 143.0, 93.0, 137.1, 125.6, 147.2, 106.5, 152.2, 95.5,152.4, 127.6, 137.1, 162.0, 98.7, 160.3, 95.5, 160.3, 98.8, 124.3,147.2, 94.5, 144.1, 124.3, 147.2, 110.0, 144.1, 101.5, 151.8, 137.2,144.1, 159.7, 96.7, 157.1, 95.5, 157.1, 96.7, 159.8, 97.8, 159.3,95.2, 159.2, 97.9, 122.5, 153.9, 99.8, 156.8, 99.9, 152.8 (d) ; ESI-MS: [M�H]� at m/z 973.37.

Peak B-2 (pyrogallol-phloroglucinol-6,60-bieckol): amorphouspowder, 1H NMR (400 MHz, methanol-d4) d 6.10 (1H, s), 5.99(1H, s), 5.72 (1H, m), 5.75 (1H, m), 5.72 (1H, m), 6.25 (1H, s),6.14 (1H, s), 5.88 (1H, d, 2.21 Hz), 5.88 (1H, d, 2.21 Hz), 5.85 (1H,d, 2.21 Hz), 6.72 (1H, d, 2.2 Hz), 6.08 (1H, d, 2.2 Hz), 5.89 (1H, d,2.01 Hz), 5.54 (1H, d, 2.01 Hz), 5.89 (1H, d, 2.01 Hz), 9.116 (1H,s), 9.03 (1H, s), 8.92 (1H, s), 9.27 (1H, s), 9.20 (1H, s), 9.20 (1H, s),9.18 (1H, s), 9.03 (1H, s), 8.92 (1H, s), 8.25 (1H, s), 9.94 (1H, s),9.87 (1H, s), 9.87 (1H, s), 9.20 (1H, s), 9.20 (1H, s); 13C NMR(100 MHz, methanol-d4) d 125.2, 145.9, 95.5, 145.9, 125.1, 147.9,105.5, 148.4, 95.4, 151.9, 127.8, 135.3, 161.9, 97.9, 160.3, 95.5,160.3, 97.8, 124.4, 144.4, 94.4, 144.4, 124.5, 147.9, 105.4, 148.4,95.5, 151.4, 127.8, 138.3, 159.2, 100.2, 156.9, 99.5, 159.3, 96.3,154.0, 96.6, 152.1, 122.5, 153.5, 96.6, 159.9, 99.0, 159.8, 94.6,159.8, 98.6; ESI-MS: [M�H]� at m/z 973.03.

Peak C (phlorofucofuroeckol-A): amorphous powder, 1H NMR(400 MHz, methanol-d4) d: 6.63 (1H, s, H-7), 6.40 (1H, s, H-11),6.26 (1H, s, H-2), 5.97 (2H, d, J = 2.1 Hz, H-200, 600), 5.94 (1H, t,J = 1.9 Hz, H-40), 5.92 (1H, t, J = 2.0 Hz, H-400), 5.88 (2H, d,J = 2.1 Hz, H-20, 60). 13C NMR (100 MHz, CD3OD) d: 162.7, 162.6,161.0, 161.0, 154.0, 152.5, 152.0, 149.1, 149.0, 146.7, 144.7,139.2, 136.2, 128.9, 125.9, 125.6, 123.2, 106.2, 106.1, 100.8,100.2, 98.6, 98.5, 97.0, 96.2, 96.2; ESI-MS: [M�H]� at m/z 601.36.

4. Conclusions

In this study, four phlorotannins dieckol, phlorofucofuroeckol-A, 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol were isolated in high yields by a one-step CPCoperation. It was demonstrated that the CPC system is a useful pro-cess for the isolation and purification of phlorotannins from E. cava.

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