Food Chemistry 158 (2014) 433–437

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Preparative isolation and purification of phlorotannins from Ecklonia cava using centrifugal partition chromatography by one-step 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 Korea Natural Product Research Center, Hanyang University, Daejeon-dong, Ahnsan, Gyeongi-do, Republic of Korea c Natural Product Research Center, KIST Gangneung Institute, Daejeon-dong, Gangneung, Gangwon-do, Republic of Korea d Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 7 January 2013 Received in revised form 5 February 2014 Accepted 19 February 2014 Available online 28 February 2014 Keywords: Centrifugal partition chromatography (CPC) Phlorotannin Ecklonia cava

a b s t r a c t Various bioactive phlorotannins of Ecklonia cava (e.g., dieckol, eckol, 6,6-bieckol, phloroglucinol, phloroeckol, 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 bioactivecompounds 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), and fraction II (34.1 mg) with 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are isolated from the crude extract (500 mg) by a one-step CPC system. The purities of the isolated dieckol and phlorofucofuroeckol-A are P90% according to high performance liquid chromatography (HPLC) and electrospray ionization multi stage tandem mass spectrometry analyses. The purified 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are collected from fraction II by recycleHPLC. Thus, the CPC system is useful for easy and simple isolation of phlorotannins from E. cava. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction An edible brown seaweed Ecklonia cava, is known to show various biological activities such as anticancer, antioxidant, antiallergic 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 phlorotannins, including dieckol, pyrogallol-phloroglucinol-6,6-bieckol, 2,7-phloroglucinol-6,6-bieckol, and phlorofucofuroeckol-A (see structures in Fig. 1). Their chemical structures are shown in Fig. 1. In particular, dieckol is reported to possess inhibitory activity against a-glucosidase and a-amylase in vitro and alleviates postprandial hyperglycaemia in streptozotocin-induced diabetic mice (Lee et al., 2010). Pyrogallol-phloroglucinol-6,6-bieckol and 2,7-phloroglucinol-6,6-bieckol from E. cava have been evaluated for their antioxidant properties (Kang, Heo, Kim, Lee, Jeon, 2011; Kang, Lee, et al., 2011). Phlorofucofuroeckol-A from Eckolina stolonifera is reported to show inhibitory effects on FcRI expression ⇑ 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: [email protected] (J. Kim), [email protected] (Y.-J. Jeon). http://dx.doi.org/10.1016/j.foodchem.2014.02.112 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

which is known to be involved in the regulation of IgE-mediated allergic reactions (Shim, Choi, Byun, 2009). However, traditional methods for their purifications demand repetitive chromatography processes using Sephadex LH-20 column chromatography and reversed-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 time consuming, have problems with adsorption in the stationary phase, and can only be worked with limited amounts of the compounds. To solve these problems, a preparative centrifugal partition chromatography (CPC) system is used. A preparative CPC system, part of counter-current chromatography (CCC), is a non-solid support, preparative, liquid–liquid separation process based on the difference in the distribution of components in two immiscible liquid phases. This enables the isolation and purification of large quantities of compounds with purities 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 advantages such as versatile products, faster and inexpensive product development, retention of bioactivity integrity, higher throughput, higher yields, and reduced operating costs. The solutes are separated

434

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

HO

OH

OH

O O

OH

OH

O

O

OH

OH

OH

O O

O

O HO

HO

HO

OH

O OH

OH

OH

Dieck ol

HO OH

HO

Phlorof ucof uroeckol A HO HO

OH

O

O

HO

O

O

O

OH

OH

OH

O OH

O O

O

HO

HO OH

OH

OH

OH

O OH

O O

HO

OH

OH HO O

OH

O

2,7-phloroglucinol-6,6-bieckol HO

O OH OH

O

HO

OH

OH

O

LLB-M high performance CPC (Sanki Engineering, Kyoto, Japan) was used in preparative CPC. The total cell volume was 240 mL. A four-way switching valve incorporated in the CPC apparatus allowed its operation in either the descending or ascending mode. This CPC system was equipped with a Hitachi 6000 pump, an L-4000 UV detector (Hitachi, Japan), and Gilson FC 203B fraction collector (Gilson, France). The samples were manually injected through a Rheodyne valve (Rheodyne, CA, USA) with a 2 mL sample loop. The 1H-NMR spectra were measured with a JEOL JNM-LA 300 spectrometer (JEOL Ltd., Tokyo, Japan) and 13C-NMR spectra with a Bruker AVANCE 400 spectrometer (BRUKER, Germany). The mass spectra (FAB-MS and EIMS) were recorded on a JEOL JMS 700 spectrometer. The HPLC system consisted of a binary Gilson 321 pump, Gilson UV–Vis 151 detector, Gilson 234 auto-injector, and 506C interface module (Gilson). The recycle HPLC system was equipped with a liquid feed pump type L-7100 (JAI, Japan), and JAI UV detector 3702 (JAI). 2.3. Preparation of crude sample from E. cava

OH O

HO

2.2. Apparatus

OH

OH

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

O OH

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 and partitioned with ethyl acetate (1:1, v/v of sample). Then, the dried ethyl acetate fraction was stored in a refrigerator for CPC separation.

OH

Pyrogallol-phloroglucinol-6,6-bieckol Fig. 1. Chemical structures of phlorotannins from Ecklonia cava.

according to their partition coefficient (K), which is expressed as the ratio of their concentration in the stationary phase to that in the mobile phase (Berthod & Armstrong, 1988). The CPC system has been widely used for the separation of bioactive compounds from land plants (Marston, Borel, Hostettmann, 1988; BourdatDeschamps et al., 2004; Kim, Ahn, et al., 2006; Kim, Heo, et al., 2006). However, in the case of seaweeds, only a few algae such as 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 of phlorotannin compounds from E. cava.

2. Materials and methods

The CPC experiments were performed using a two-phase solvent system comprising n-hexane/ethyl acetate/methanol/water (2:7:3:7, v/v/v/v) solvent. The two phases were separated after thoroughly equilibrating the mixture in a separating funnel at room temperature. The upper organic phase was used as the stationary phase, and the lower aqueous phase was employed as the mobile phase. 2.5. CPC separation procedure The CPC column was initially filled with the organic stationary phase and rotated at 1000 rpm; the mobile phase was pumped into the column in the descending mode at the same flow rate used for separation (2 mL/min). When the mobile phase emerged from the column, it indicated that hydrodynamic equilibrium had been achieved (back pressure: 420.5 psi). The concentrated EtOAc fraction (500 mg) obtained from the 70% EtOH extract of E. cava was dissolved in 6 mL of a 1:1 (v/v) mixture of the two CPC solvent system phases and injected through the Rheodyne injection valve. The effluent from the CPC was monitored by UV at 290 nm, and 6 mL fractions were collected in 8 mL tubes by a fraction collector.

2.1. Materials 2.6. HPLC analysis of crude extracts from E. cava E. cava, collected from the coast of Jeju Island, South Korea, in June 2009, was ground and shifted through a 50 mesh standard testing sieve after drying in a freeze dryer SFDSMO6 (Samwon freezing engineering co., South Korea). The dried E. cava was stored in a refrigerator until use. All solvents used for the preparation of crude samples and CPC separation were analytical grade (Daejung Chemicals & Metals Co., Seoul, South Korea). HPLC grade solvents were purchased from Burdick & Jackson (MI, USA).

Here, 5 lL of a 5 mg/mL sample solution was directly injected on an Atlantis T3 column (3 lm 3.0  150 mm column) (Waters, USA) using a gradient acetonitrile–water solvent system at room temperature. The mobile phase comprised acetonitrile–water in gradient mode as follows: acetonitrile with 0.1% formic acid–water with 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 was 0.2 mL/min, and the UV absorbance was detected at 290 nm.

435

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

2.7. Recycle HPLC separation

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

The samples were manually injected through a Rheodyne 7725i valve (Rheodyne, CA, USA) with a 2 mL sample loop. Then, 1 mL of a 10 mg/mL sample solution was directly injected into the JAIGELODS-BP-L, SP-120–15 column (JAI, Japan) using an isocratic 30% acetonitrile–water solvent system. The flow rate was 1 mL/min with 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, a column oven, a binary pump, a DAD detector, and a degasser (Hewlett–Packard, Waldbronn, Germany) coupled to a Finnigan MAT LCQ ion-trap mass spectrometer (Finnigan MAT, San Jose, CA, USA). The MS was equipped with a Finnigan electrospray source and was capable of analysing ions up to m/z 2000. Xcalibur software (Finnigan MAT) was used for the MS operations. The chromatographic conditions are identical to those described in Section 2.6 and the flow cell outlet was connected to a splitting valve, from which a flow of 0.2 mL/min was diverted to the electrospray ion source via a short fused silica tubing. Negative ion mass spectra 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 temperature to 250 °C. The other conditions were as follows: capillary voltage, –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 fraction is known to contain various bioactive phlorotannins such as dieckol, phloroeckol, and 6,6-bieckol (Kang, Lee, Chae, Koh, et al., 2005; Kang, Lee, Chae, Zhang, et al., 2005). Therefore, the EtOAc fraction was selected for further experiments and expressed that its yield was 28% of EtOH extract (4 g). It was analysed using the described HPLC condition; its chromatogram is depicted in Fig. 2. The peaks 1 and 2 on the HPLC chromatogram have been confirmed 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 data showed that the phlorofucofuroeckol-A and pyrogallol-phloroglucinol-6,6-bieckol share the same HPLC peak. The partition coefficient (K) in a suitable two phase solvent systems is the most

Solvents

K-value

⁄

Dieckol

2,7phloroglucinol6,6-bieckol

Phlorofucofuroeckol A & pyrogallol-phloroglucinol6,6-bieckol

0.49 0.25 0.29 2.11

0.52 0.44 0.51 6.89

0.55 1.65 0.89 7.30

H:E:M:W

4:6:4:6 3:7:3:7 2:7:3:7 2:8:2:8 ⁄

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

important variable for successful separations of the target samples by preparative CPC. To select the most efficient separation system, several two-phase solvent systems with different compositions and volume ratios of two immiscible solvents such as n-hexane/ EtOAc/methanol/water were examined, and their K values were calculated (Table 1). A K value between 0.2 and 5 can be used without any excessive elution time associated with band broadening (Foucault, 1994; Sutherland & Fisher, 2009). The solvent n-hexane/EtOAc/methanol/water (2:7:3:7) exhibited the most efficient separation for each of the phlorotannins such as dieckol and phlorofucofuroeckol-A. The K values of dieckol, 2,7-phloroglucinol-6,6-bieckol and phlorofucofuroeckol A & pyrogallol-phloroglucinol-6,6-bieckol were 0.29, 0.51, and 0.89, respectively. Therefore, preparative CPC operated in the descending mode, with the upper phase acting as the stationary phase and the lower phase as the mobile phase. During the process, 500 mg crude of EtOAc extract was fractionated in a single run of 190 min, the retention of the stationary phase in the coil was 69.5%, and the pressure was 464 psi. The preparative CPC chromatogram is described in Fig. 3. The analysis of the HPLC peak area showed that the fractions I and III had purified compounds (dieckol and phlorofucofuroeckol-A, respectively) of up to 90% (Fig. 4A and C). The figure shows that 40.2 and 31.1 mg of dieckol and phlorofucofuroeckol-A, respectively (8.4% and 6.22% of the EtOAc fraction, respectively), were isolated and collected from 500 mg of the E. cava EtOAc fraction. In addition, both 2,7-phloroglucinol-6,6bieckol and pyrogallol-phloroglucinol-6,6-bieckol were present in fraction II (Fig. 4B). Previous reports related to the isolation of phlorotannins from E. cava have shown that dieckol and phlorofucofuroeckol A were generally isolated within 1% of EtOAc fraction (Ahn et al., 2007 and Lee et al., 2009). The isolation of the phlorotannins in previous reports resulted in significant losses of the sample because of the complicated processes and adsorption on Sephadex LH-20 and silica columns via hydroxyl branches.

Fig. 2. HPLC chromatogram of the EtOAc fraction from E. cava. Peak 1: dieckol; peak 2: 2,7-phloroglucinol-6,6-bieckol; peak 3: pyrolgallol-phloroglucino-6,6-bieckol and phlorofucofuroeckol A. (Chromatographic conditions, see Section 2).

436

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

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).

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

Therefore, preparative CPC can collect higher amounts and yields than previously reported for the phlorotannins. 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol could be obtained in 90% purity from fraction II using recycle HPLC. All the purified compounds were confirmed as dieckol, 2,7-phloroglucinol-6,6-bieckol, pyrogallol-phloroglucinol-6,6-bieckol, and phlorofucofuroeckol-A comparing the results with the previously reported 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 1H NMR, 13C NMR and HPLC–DAD–ESI/MS (negative ion mode) analyses. Peak A (dieckol): amorphous powder, 1H NMR (400 MHz, methanol-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: [MH] at m/z 741. Peak B-1 (2,700 -phloroglucinol-6,60 -bieckol): amorphous powder, 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, methanold6) 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) ; ESIMS: [MH] at m/z 973.37. Peak B-2 (pyrogallol-phloroglucinol-6,60 -bieckol): amorphous powder, 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: [MH] 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: [MH] at m/z 601.36. 4. Conclusions In this study, four phlorotannins dieckol, phlorofucofuroeckolA, 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol6,6-bieckol were isolated in high yields by a one-step CPC operation. It was demonstrated that the CPC system is a useful process for the isolation and purification of phlorotannins from E. cava.

437

References Ahn, G. N., Kim, K. N., Cha, S. H., Song, C. B., Lee, J., Heo, M. S., et al. (2007). Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage. European Food Research and Technology, 226, 71–79. Athukorala, Y., Kim, K. N., & Jeon, Y. J. (2006). Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava. Food and Chemical Toxicology, 44, 1065–1074. Berthod, A., & Armstrong, D. W. (1988). Centrifugal partition chromatography. I. General features. Journal of Liquid Chromatography, 11, 547–566. Bourdat-Deschamps, M., Herrenknecht, C., Akendengue, B., Laurens, A., & Hocquemiller, R. (2004). Separation of protoberberine quaternary alkaloids from a crude extract of Enantia chlorantha by centrifugal partition chromatography. Journal of Chromatography A, 1041, 143–152. Chevolot, L., Colliec-Jouault, S., Foucault, A., Ratiskol, J., & Sinquin, C. (1998). Preliminary report on fractionation of fucans by ion-exchange displacement centrifugal partition chromatography. Journal of Chromatography B, 706, 43–54. Chevolot, L., Foucault, A., Colliec-Jouault, S., Ratiskol, J., & Sinquin, C. (2000). Improvement purification of sulfated oligofucan by ion-exchange displacement centrifugal partition chromatography. Journal of Chromatography A, 869, 353–361. Delannay, E., Toribio, A., Boudesocque, L., Nuzillard, J. M., Zeches-Hanrot, M., Dardennes, E., et al. (2006). Renault multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratoryscale purifications. Journal of Chromatography A, 1127, 45. Foucault, A. P. (1994). Centrifugal partition chromatography. New York: Marcel Dekker. Heo, S. J., Ko, S. C., Cha, S. H., Kang, D. H., Park, H. S., Choi, Y. U., et al. (2009). Effect of phlorotannins isolated from Ecklonia cava on melanogenesis and their protective effect against photo-oxidative stress induced by UV-B radiation. Toxicology in Vitro, 23, 1123–1130. Kang, S. M., Heo, S. J., Kim, K. N., Lee, S. H., & Jeon, Y. J. (2011). Isolation and identification of new compound, 2,700 -phloroglucinol-6,60 -bieckol from brown algae, Ecklonia cava and its antioxidant effect. The Journal of Functional Foods, 4, 158–166. Kang, I. J., Jeon, Y. E., Yin, X. F., Nam, J. S., You, S. G., Hong, M. S., et al. (2011). Butanol extract of Ecklonia cava prevents production and aggregation of beta-amyloid, and reduces beta-amyloid mediated neuronal death. Food and Chemical Toxicology, 49, 2252–2259. Kang, K. A., Lee, K. H., Chae, S. W., Koh, Y. S., Yoo, B. S., Kim, J. H., et al. (2005). Triphlorethol-A from Ecklonia cava protects V79-4 lung fibroblast against hydrogen peroxide induced cell damage. Free Radical Research, 39, 883–892. Kang, K. A., Lee, K. H., Chae, S. W., Zhang, R., Jung, M. S., Lee, Y., et al. (2005). Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells. FEBS Letters, 579, 6295–6304. Kang, S. M., Lee, S. H., Heo, S. J., Kim, K. N., & Jeon, Y. J. (2011). Evaluation of antioxidant properties of a new compound, pyrogallol-phloroglucinol-6,60 bieckol isolated from brown algae, Ecklonia cava. Nutrition Research and Practice, 5, 495–502. Kim, C. Y., Ahn, M. J., & Kim, J. (2006). A preparative isolation and purification of arctigenin and matairesinol from Forsythia koreana by centrifugal partition chromatography. Journal of Separation Science, 29, 656–659. Kim, K. N., Heo, S. J., Song, C. B., Lee, J. H., Heo, M. S., Yeo, I. K., et al. (2006). Protective effect of Ecklonia cava enzymatic extracts on hydrogen peroxide-induced cell damage. Process Biochemistry, 41, 2393–2401. Kim, A. R., Lee, M. S., Shin, T. S., Hua, H., Jang, B. C., Choi, J. S., et al. (2011). Phlorofucofuroeckol A inhibits the LPS-stimulated iNOS and COX-2 expressions in macrophages via inhibition of NF-jB, Akt, and p38 MAPK. Toxicology in Vitro, 25, 1789–1795. Le, Q. T., Li, Y., Qian, Z. J., Kim, M. M., & Kim, S. K. (2009). Inhibitory effects of polyphenols isolated from marine alga Ecklonia cava on histamine release. Process Biochemistry, 44, 168–176. Lee, S. H., Park, M. H., Heo, S. J., Kang, S. M., Ko, S. C., Han, J. S., et al. (2010). Dieckol isolated from Ecklonia cava inhibits a-glucosidase and a-amylase in vitro and alleviates postprandial hyperglycemia in streptozotocin-induced diabetic mice. Food and Chemical Toxicology, 48, 2633–2637. Lee, Y., Qian, Z. J., Ryu, B. M., Lee, S. H., Kim, M. M., & Kim, S. K. (2009). Chemical components and its antioxidant properties in vitro: An edible marine brown alga, Ecklonia cava. Bioorganic & Medicinal Chemistry, 17, 1963–1973. Marston, A., Borel, C., & Hostettmann, K. (1988). Separation of natural products by centrifugal partition chromatography. Journal of Chromatography A, 450, 91–99. Michel, J. van Buel, Luuk, A. M. van der Wielen, & Karel, Ch. A. M. (1997). Luyben modelling gradient elution in centrifugal partition chromatography. Journal of Chromatography A, 773, 13–22. Shim, S. Y., Choi, J. S., & Byun, D. S. (2009). Inhibitory effects of phloroglucinol derivatives isolated from Ecklonia stolonifera on FceRI expression. Bioorganic & Medicinal Chemistry, 17, 4734–4739. Shim, S. Y., Le, Q. T., Lee, S. H., & Kim, S. K. (2009). Ecklonia cava extract suppresses the high-affinity IgE receptor, FceRI expression. Food and Chemical Toxicology, 47, 555–560. Sutherland, I. A., & Fisher, D. (2009). Role of counter-current chromatography in the modernisation of Chinese herbal medicines. Journal of Chromatography A, 1216, 740–753.