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Research Paper

Journal of Pharmacy And Pharmacology

Protective effect of the edible brown alga Ecklonia stolonifera on doxorubicin-induced hepatotoxicity in primary rat hepatocytes Hyun Ah Junga, Jae-I Kimb, Se Young Choungc and Jae Sue Choid a Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, bNational of Food & Drug Safety Evaluation Scientific Food Investigation Team, Korea Food & Drug Administration, Chungcheongbuk-do, cDepartment of Preventive Pharmacy and Toxicology, College of Pharmacy, Kyung Hee University, Seoul, dDepartment of Food Science and Nutrition, Pukyong National University, Busan, Korea

Keywords doxorubicin; Ecklonia stolonifera; hepatotoxicity; phlorotannin; seaweed Correspondence Jae Sue Choi, Department of Food Science and Nutrition, Pukyong National University, 608–737, Korea. E-mail: [email protected] Received September 16, 2013 Accepted February 2, 2014 doi: 10.1111/jphp.12241

Abstract Objectives As part of our efforts to isolate anti-hepatotoxic agents from marine natural products, we screened the ability of 14 edible varieties of Korean seaweed to protect against doxorubicin-induced hepatotoxicity in primary rat hepatocytes. Methods Among the crude extracts of two Chlorophyta (Codium fragile and Capsosiphon fulvescens), seven Phaeophyta (Undaria pinnatifida, Sargassum thunbergii, Pelvetia siliquosa, Ishige okamurae, Ecklonia cava, Ecklonia stolonifera and Eisenia bicyclis), five Rhodophyta (Chondrus ocellatus, Gelidium amansii, Gracilaria verrucosa, Symphycladia latiuscula and Porphyra tenera), and the extracts of Ecklonia stolonifera, Ecklonia cava, Eisenia bicyclis and Pelvetia siliquosa exhibited significant protective effects on doxorubicin-induced hepatotoxicity, with half maximal effective concentration (EC50) values of 2.0, 2.5, 3.0 and 15.0 μg/ml, respectively. Key findings Since Ecklonia stolonifera exhibits a significant protective potential and is frequently used as foodstuff, we isolated six phlorotannins, including phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol A (4), dieckol (5) and triphloroethol-A (6). Phlorotannins 2 ∼ 6 exhibited potential protective effects on doxorubicin-induced hepatotoxicity, with corresponding EC50 values of 3.4, 8.3, 4.4, 5.5 and 11.5 μg/ml, respectively. Conclusion The results clearly demonstrated that the anti-hepatotoxic effects of Ecklonia stolonifera and its isolated phlorotannins are useful for further exploration and development of therapeutic modalities for treatment of hepatotoxicity.

Introduction Free radicals are generated via normal and pathological cell metabolism by external factors, including both foreign materials and UV radiation. During respiration and combustion, molecular oxygen reacts easily with free radicals to form reactive oxygen species (ROS).[1] Such reactive species are formed in the body as a consequence of aerobic metabolism and are capable of damaging any accessible intracellular components, including nucleic acids, proteins and lipids. ROS have also been implicated in hepatotoxicity. Several hepatotoxic model systems have been used to evaluate protective activity of natural products against hepatotoxicity. Previous studies have investigated cytotoxins, such as

carbon tetrachloride (CCl4), acetaminophen, thioacetamide, rubratoxin B,[2] tacrine,[3] doxorubicin,[4] H2O2[5] and lipopolysaccharide/D-galactosamine.[6] These hepatotoxins may be involved in the metabolism of cytochrome P450, followed by reactive toxic metabolites formation.[7,8] Doxorubicin (adriamycin) is used as an anticancer agent in a variety of neoplastic conditions, such as multiple myeloma,[9] osteogenic sarcoma,[10] lymphocytic leukaemia[11] and stomach cancer.[12] However, doxorubicin has also been found to induce hepatotoxicity and general organ toxicity in mammals.[4] The main toxic effects on hepatocytes include cell cycle arrest, oxidative stress and

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

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Hepatoprotective effect of Ecklonia on doxorubicin

Hyun Ah Jung et al.

disruption of the electron transport chain.[13] However, the exact mechanism of doxorubicin-induced hepatotoxicity is not fully understood. Therefore, a great deal of research to locate natural products that confer protective effects against doxorubicin-induced cytotoxicity has been conducted in recent years.[14–16] In these studies, primary rat hepatocytes were frequently employed to screen for hepatoprotective agents that protect against doxorubicin-induced cytotoxicity.[17] Many seaweed species, including Laminaria spp., Undaria spp., Hizikia spp., Ecklonia spp. and Porphyra spp., are consumed in the form of sushi wrappings, seasonings, condiments and vegetables in the diets of Pacific and East Asian countries. Ecklonia stolonifera Okamura, a perennial brown algae belonging to the family Laminariaceae, is commonly distributed in the middle Pacific coast around Korea and Japan. E. stolonifera usually grows well in subtidal zones at depths of 2–10 m and in the sublittoral zone. Species of Ecklonia have been used as phlorotannin-rich raw materials.[18] Phlorotannins are secondary metabolites of phloroglucinol (1,3,5-trihydroxybenzene) that have been polymerized through ether, phenyl or 1,4-dibenzodioxin linkages. These polyphenolic compounds show a variety of bioactivities, including antidiabetic complications,[18] hepatoprotective,[19,20] anti-plasmin inhibitory,[21] antiinflammatory,[22,23] antioxidant[24–27] and angiotensin converting enzyme-I inhibitory activity.[28] However, there have been no studies to date on the Korean seaweeds as well as their active components responsible for protecting against doxorubicin-induced hepatotoxicity. In this study, we investigate the effects of the ethanol (EtOH) extract and solventsoluble fractions of the brown alga E. stolonifera and six isolated phlorotannins against doxorubicin-induced hepatotoxicity in primary rat hepatocytes. Our results showed that the EtOH extracts of E. stolonifera and the associated phlorotannins significantly protect against doxorubicininduced hepatotoxicity.

Materials and Methods General experimental procedures 1

H- and 13C-NMR spectra were obtained using a JNM ECP400 spectrometer (Jeol, Tokyo, Japan) at 400 MHz and 100 MHz, respectively. 1H- and 13C -NMR chemical shifts were referenced with the residual solvent peaks (δH 3.31 and δC 49.0 for CD3OD; δH 2.50 and δC 39.5 for DMSO-d6). EI-MS data were collected with a GC-MS QP-5050A spectrometer (Shimadzu, Kyoto, Japan). LR FAB-MS data were assessed with a JMS-HX110/110A spectrometer (Jeol). IR spectra (KBr) were obtained using a Shimadzu FT-IR spectrometer (Tokyo, Japan). Column chromatography was conducted using silica gel 60 (70–230 mesh; Merck, Darmstadt, Germany), RP-18 Lichroprep (40–63 μm; Merck) and 2

Sephadex LH20 (20–100 μm; Sigma, St Louis, MO, USA). Thin-layer chromatography was conducted on precoated Kieselgel 60 F254 plates (20 × 20 cm, 0.25 mm) and RP-18 F254 plates (5 × 10 cm; Merck) using 50% H2SO4 as a spray reagent.

Chemicals and reagents The RPMI 1640 medium, trypsin-ethylene diaminetetraacetic acid (EDTA) and antibiotics used in this study were purchased from Gibco Laboratories (Grand Island, NY, USA). Fetal bovine serum was obtained from Hyclone Laboratories (Logan, UT, USA). Doxorubicin, silymarin and [2(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium] (WST-1) were purchased from Sigma Chemical Co. Tissue culture plates and all other tissue culture dishes were obtained from Nunc, Inc. (North Aurora, IL, USA). All other chemicals and solvents used in this study were purchased from E. Merck, Fluka or Sigma-Aldrich, unless otherwise stated.

Plant materials The leafy thalli of E. stolonifera (Lessoniaceae) were collected at Tongyoung City, South Korea, in April 2011 and authenticated by Prof Y. J. Choi of the Marine Bioscience and Technology at Kyongsang National University. The leafy thalli of Pelvetia siliquosa were collected from a seashore in Mokpo during February 2003, and were authenticated by Prof J. A. Shin of Cheonnam National University. All other leafy algal thalli were harvested at Chungsapo, Busan, during February 2009. Voucher specimens (no. 20011428) have been deposited in the author’s laboratory (J. S. Choi).

Extraction and isolation of phlorotannins 1–6 After washing with seawater and tap water, the samples were dried in the shade. All of the seaweeds (100 g) tested in this study were extracted with 95% ethanol. The weights of the ethanolic (EtOH) extracts are listed in Table 1. For further chemical investigation, a lyophilized powder (500 g) of leaf thalli of E. stolonifera was refluxed with EtOH (3 × 3 L) for 3 h, and each filtrate was concentrated to dryness in vacuo at 40°C to render the EtOH extract (116.6 g). The EtOH extract was then separately suspended in distilled H2O, and partitioned in sequence with n-hexane, CH2Cl2, EtOAc, n-BuOH and H2O to yield five fractions (Figure 1). The respective yields (%) of n-hexane (4.65 g), CH2Cl2 (4.27 g), EtOAc (4.17 g), n-BuOH (16.6 g) and H2O fractions (86.6 g) were 3.9, 3.7, 3.6, 17.3 and 74.3%. The active EtOAc fraction (4.17 g) obtained from E. stolonifera was subjected to chromatography on a silica gel column using

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Hyun Ah Jung et al.

Hepatoprotective effect of Ecklonia on doxorubicin

Table 1 Protective effects of ethanolic extracts of selected Korean seaweed species against doxorubicin-induced hepatotoxicity in primary rat hepatocytes

Phylum

Seaweed species

Chlorophyta

Capsosiphon fulvescens (C. Agardh) Setchell & N.L. Gardner Codium fragile (Suringar) Hariot Undaria pinnatifida (Harvey) Suringar Pelvetia siliquosa Tseng & Chang Sargassum thunbergii (Mertens ex Roth) Kuntze Ecklonia cava Kjellman Ishige okamurae Yendo Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellmann) Setchell Chondrus ocellatus Holmes Gelidium amansii (J.V. Lamouroux) J.V. Lamouroux Gracilaria verrucosa (Hudson) Papenfuss Symphyocladia latiuscula (Harvey) Yamada Porphyra tenera Kjellman Silymarin

Phaeophyta

Rhodophyta

Positive controlc a

Yield

EC50

Cytotoxicity

(g)b

(μg/ml)c

(μg/ml)c

4.95

>200

NDa

10.06 2.09 2.88 1.66 12.50 2.26 25.28 30.03 7.89 0.19

>200 >200 15.0 >200 2.50 >200 2.0 3.0 >200 >200

ND ND >500 ND 176 ND 200 >500 ND ND

8.37 3.17 2.28 –

>200 >200 >200 13.5

ND ND ND 240

ND, not determined. bYields of the ethanolic extracts of dried seaweed samples (100 g). cMean values of three assays.

Ecklonia stolonifera (500g) MeOH (95 °C reflux for 3h. 3L × 3 times) EtOH extract (116.6 g) Partitioned with n-hexane n-hexane layer (4.65 g)

Aqueous layer Partitioned with CH2CI2

CH2CI2 layer (4.27 g)

Aqueous layer Partitioned with EtOAc EtOAc layer (4.17 g)

Aqueous layer Partitioned with n-BuOH

n-BuOH layer (16.6 g) Figure 1

Aqueous layer (86.6 g)

Extraction and fractionation of Ecklonia stolonifera.

EtOAc–MeOH (50 : 1 to 5 : 1) as the eluent, which yielded 10 subfractions (EF01–EF10). Repeated column chromatography of EF01 (6.8 g) was conducted with a solvent mixture of n-hexane and EtOAc (50 : 1 to 0 : 1), yielding 11 subfractions (EF0101–EF0111). Compound 1 (2.2 mg) was purified from EF0104 (0.6 g) in an RP-18 column by eluting with aqueous MeOH (20% MeOH to 100% MeOH, gradient elution). Under identical solvent conditions, RP-18 column chromatography of EF0105 (3. g) led to the isola-

tion of 2 (0.9 mg) and 3 (25 mg). Compounds 4 (43.5 mg), 5 (42.6 mg) and 6 (2.8 mg) were purified from EF0106 (2.2 g) using RP-18 (20% MeOH to 100% MeOH, gradient elution) and Sephadex LH-20 columns (100% MeOH). Using spectroscopic methods, as well as by comparing with published data, isolated compounds 1–6 were identified and characterized as phloroglucinol, dioxinodehydroeckol, eckol, phlorofucofuroeckol-A, dieckol[24] and triphloroethol-A,[21] respectively.

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

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Phloroglucinol (1) IR νmax cm−1 3481, 1617, 1499, 1419; EI-MS m/z (rel. int., %): 126 [M]+ (100); 1H-NMR (400 MHz, CD3OD) δ: 5.78 (3H, s, H-2/H-4/H-6); 13CNMR (100 MHz, CD3OD) δ: 160.9 (C-1/C-3/C-5), 96.3 (C-2/C-4/C-6). Dioxinodehydroekol (2) IR νmax cm−1 3243, 1635, 1518, 1494, 1396, 1281, 1243, 1207, 1154, 1118, 1089, 1012, 810; Negative FAB-MS m/z: 369, Positive FAB-MS m/z: 370; 1 H-NMR (400 MHz, DMSO-d6) δ: 9.77 (1-OH), 9.64 (9-OH), 9.60 (6-OH), 9.27 (3-OH), 9.26 (11-OH), 6.10 (1H, s, H-7), 6.04 (1H, d, J = 2.7 Hz, H-2), 6.01 (1H, d, J = 2.7 Hz, H-10), 5.84 (1H, d, J = 2.7 Hz, H-4), 5.82 (1H, d, J = 2.7 Hz, H-12); 13C-NMR (100 MHz, DMSO-d6) δ: 153.3 (C-3), 153.0 (C-11), 146.1 (C-1), 146.0 (C-9), 142.1 (C-4a), 141.7 (C-12a), 140.1 (C-6), 137.2 (C-7a), 131.6 (C-13b), 125.9 (C-5a), 122.7 (C-8a), 122.5 (C-13a), 122.3 (C-14a), 98.8 (C-2/C-10), 97.6 (C-7), 93.9 (C-4/C-12). Eckol (3) Positive FAB-MS m/z: 372 [M]+; 1H-NMR (400 MHz, CD3OD) δ: 6.13 (1H, s, H-3), 5.94 (2H, s, H-6/ H-8), 5.93 (3H, s, H-2′/H-4′/H-6′); 13C-NMR (100 MHz, CD3OD) δ: 162.4 (C-1′), 160.7 (C-3′/C-5′), 150.0 (C-8), 147.7 (C-3), 147.6 (C-6), 144.7 (C-9a), 143.8 (C-1), 139.0 (C-4a), 126.1 (C-4), 125.3 (C-5a), 125.1 (C-10a), 100.3 (C-7), 99.9 (C-2), 98.2 (C-4′), 96.3 (C-9), 95.9 (C-2′/C-6′). Phlorofucofuroeckol A (4) Positive FAB-MS m/z: 602 [M]+; 1H-NMR (400 MHz, CD3OD) δ:) δ: 6.63 (1H, s, H-9), 6.40 (1H, s, H-13), 6.26 (1H, s, H-3), 5.97 (2H, d, J = 2.1 Hz, H-2″/H-6″), 5.94 (1H, t, J = 1.9 Hz, H-4′), 5.92 (1H, t, J = 2.0 Hz, H-4″), 5.88 (2H, d, J = 2.1 Hz, H-2′/H-6′); 13CNMR (100 MHz, CD3OD) δ: 162.7 (C-1″), 162.6 (C-1′), 161.0 (C-3′/C-5′), 161.0 (C-3″/C-5″), 154.0 (C-12a), 152.5 (C-10), 152.0 (C-11a), 149.1 (C-2), 149.0 (C-8), 146.7 (C-14), 144.7 (C-1), 139.2 (C-4), 136.2 (C-15a), 128.9 (C-5a), 125.9 (C-14a), 125.6 (C-4a), 123.2 (C-1/C-11), 106.2 (C-7), 106.1 (C-6), 100.8 (C-9), 100.2 (C-3), 98.6 (C-4′), 98.5 (C-4″), 97.0 (C-13), 96.2 (C-2″/C-6″), 96.2 (C-2′/C-6′). Dieckol (5) Positive FAB-MS m/z: 742 [M]+; 1H-NMR (400 MHz, CD3OD) δ: 6.15 (1H, s, H-3″), 6.13 (1H, s, H-3), 6.09 (2H, s, H-2″′/H-6″′), 6.06 (1H, d, J = 2.9 Hz, H-8), 6.05 (1H, d, J = 2.9 Hz, H-8″), 5.98 (1H, d, J = 2.8 Hz, H-6), 5.95 (1H, d, J = 2.8 Hz, H-6″), 5.92 (3H, s, H-2′/H-4′/H-6′); 13CNMR (100 MHz, CD3OD) δ: 162.7 (C-1′), 161.0 (C-3′/H5′), 158.6 (C-1″′), 156.8 (C-7), 155.3 (C-7″), 153.2 (C-3″′/ C-5″′), 148.1 (C-2″), 148.01 (C-2), 147.9 (C-9″), 147.7 (C-9), 145.1 (C-5a″), 145.0 (C-5a), 144.2 (C-4″), 144.1 (C-4), 139.4 (C-10a), 139.3 (C-10a″), 127.3 (C-4′′′), 127.0 (C-9a), 126.5 (C-1), 126.4 (C-1″), 125.7 (C-9a″), 125.5 (C-4a″), 125.4 (C-4a), 100.7 (C-8″), 100.6 (C-8), 100.3 (C-3), 100.2 (C-3″), 98.5 (C-4′), 97.0 (C-2″′/C-6″′), 96.7 (C-6″), 96.6 (C-6), 96.2 (C-2′/C-6′). Triphloroethol-A (6) 1H-NMR (400 MHz, DMSO-d6) δ: 5.94 (1H, d, J = 2.7 Hz, H-3), 5.84 (2H, s, H-3″, 5″), 5.82 4

(2H, d J = 1.9 Hz, H-2′, 6′), 5.80 (1H, d, J = 1.9 Hz, H-4′), 5.52 (1H, d, J = 2.7 Hz, H-5). 13C-NMR (100 MHz, DMSOd6) δ: 160.7 (C-1′), 158.7 (C-3′, 5′), 154.6 (C-4″), 154.3 (C-4), 153.1 (C-6), 151.1 (C-2, 2″, 6″), 123.3 (C-1), 122.3 (C-1″), 96.2 (C-3), 95.7 (C-4′), 94.8 (C-3″, 5″), 94.1 (C-2′, 6′), 92.8 (C-5).

Determination of the protective effects against doxorubicin-induced cytotoxicity in primary cultured rat hepatocytes The hepatoprotective activity of extracts and their isolated constituents was evaluated by a 2-(4-iodophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST1) colorimetric assay using cultured primary rat hepatocytes. In metabolically active cells, WST-1 is converted to a highly water-soluble formazan, allowing for simple and direct colorimetric measurement of cell viability and proliferation. Hepatocytes were isolated from male SD rats (250–300 g) by collagenase perfusion. Primary rat hepatocytes were cultured on gelled collagen type I coated 96-well plates at a density of 1.5 × 104 cells/100 μl/well in hepatozyme serum-free medium at 37°C in a humidified atmosphere of 95% air/5% CO2. Cells were pretreated with 10 μl of the desired compound (dissolved in DMSO and diluted to appropriate concentrations with hepatozyme, with the final concentration of DMSO adjusted to 0.1%). After preincubation for 30 min, 10 μl of 5 μm doxorubicin was added to each well, and the cells were incubated for 2 days (39–48 h). For the determination of cell viability, 10 μl of WST-1 was added to each well followed by incubation for 1.5 h. The optical density (OD) of the formazan solution was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 440 nm. The percent inhibition of tested agents was obtained according to the following formula:

inhibition (%) = [(OD (sample ) − OD (control )) (OD (normal ) − OD (control ))] ×100

Statistical analysis The Kruskal–Wallis test and the Mann–Whitney U-test were used to determine the statistical significance of the differences between values for various experimental and control groups. Data are expressed as the mean ± standard error of the mean, performed in triplicate graphically.

Results Protective effects of selected Korean seaweeds To identify hepatoprotective agents from marine plants, we screened the protective effect of the EtOH extracts of 14

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Hyun Ah Jung et al.

Hepatoprotective effect of Ecklonia on doxorubicin

different seaweed species against doxorubicin-induced hepatotoxicity in primary rat hepatocytes. The cytotoxic and protective effects of selected seaweeds are presented in Table 1. The cytotoxicity of the seaweed extracts used in this study was measured by WST-1 assay. As shown in Table 1, none of the EtOH extracts used in this study produced cytotoxic effects at concentrations of up to 100 μg/ml. Among the selected seaweeds, the EtOH extracts of brown algae E. stolonifera, E. cava, Eisenia bicyclis and Pelvetia siliquosa exhibited significant protective effects on doxorubicin-induced hepatotoxicity at effective concentration (EC50) values of 2.0, 2.5, 3.0 and 15 μg/ml, respectively. Since E. stolonifera is frequently used as a foodstuff in Korea, we further investigated its potential as a pharmaceuticals and nutraceuticals to treat hepatotoxicity.

Protective effect of EtOH extract and different fractions of E. stolonifera Because the crude EtOH extract of E. stolonifera had marked hepatoprotective effects, we successively partitioned the crude extract with several solvents using a bioassayguided fractionation strategy. As shown in Figure 1, the EtOH extract of E. stolonifera was dissolved in H2O, and successively partitioned with n-hexane, CH2Cl2, EtOAc and n-BuOH to obtain the respective solvent-soluble fractions and water residue. We next evaluated the cytotoxic and hepatoprotective effects of the different solvent-soluble fractions. With the exception of n-hexane and H2O fractions, the fractions exhibited a significant protective potential with respect to doxorubicin-induced hepatotoxicity (Table 2). Among the fractions tested, the EtOAc fraction was the most active (EC50 value = 3.0 μg/ml) compared with the positive control silymarin (EC50 value = 8.0 μg/ ml). In addition, the n-BuOH fraction also exhibited a potent protective effect, with a corresponding EC50 value of 8.4 μg/ml. However, the protective effects of the CH2Cl2 fraction (EC50 value = 40.0 μg/ml) on doxorubicin-

Table 2 Protective effects of the ethanolic extract and their subfractions against doxorubicin-induced hepatotoxicity in primary rat hepatocytes Test sample

EC50 value (μg/ml)b

Cytotoxicity (μg/ml)b

EtOH extract n-hexane fraction CH2Cl2 fraction EtOAc fraction n-BuOH fraction H2O fraction Silymarinc

2.5 ± 0.2 >200 40.0 ± 2.6 3.0 ± 0.3 8.4 ± 0.7 >200 8.0 ± 0.5

>200 NDa 43.9 ± 1.3 117.0 ± 2.6 >500 ND 215.0 ± 3.1

a ND, not determined. bEC50 are given as the means ± standard error of the mean of triplicate experiments. cUsed as a positive control.

induced hepatotoxicity were concurrent with cytotoxic concentrations, as determined by WST-1 assay, indicating that the CH2Cl2 fraction, on its own, was significantly cytotoxic. Neither the n-hexane nor H2O fractions exhibited significant protective effects up to 200 μg/ml, respectively.

Protective effects of phlorotannins isolated from E. stolonifera In this study, in which we sought to identify the bioactive components of E. stolonifera, the most active EtOAc fraction was subjected to repeated column chromatography to yield six phlorotannins, namely phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol A (4), dieckol (5) and triphloroethol-A (6). The structures of the phlorotannins are shown in Figure 2, and their cytotoxic or protective effects on doxorubicin-induced hepatotoxicity are presented in Table 3. Although phloroglucinol (1) exhibited no protective effects at concentrations of up to 200 μg/ml, other phlorotannins showed significant protective effects in the order of dioxinodehydroeckol (2) > phlorofucofuroeckol A (4) > dieckol (5) > eckol (3) > triphloroethol-A (6), with the following EC50 values: 3.4 ± 0.7, 4.4 ± 0.3, 5.5 ± 0.9, 8.3 ± 0.5 and 11.5 ± 1.1 μg/ml, respectively. The hepatoprotective effect exhibited by phlorotannins 2 ∼ 5 were comparable to the positive control silymarin (EC50 = 8.0 ± 0.5 μg/ml). However, while the concentration at which phlorofucofuroeckol A (4) produced cytotoxicity (69.0 ± 1.0 μg/ml) was higher than that of the isolated compounds, the viability of primary rat hepatocytes was not altered in the presence of phlorotannins at the indicated concentrations.

Discussion Plant-based therapies can have multiple health benefits with limited toxicity, and thus continue to be extensively researched as part of ongoing efforts to identify drugs from natural sources for the treatment of various ailments, including liver disease.[29,30] Although there have been several recent attempts at identifying effective hepatoprotective agents from natural sources, research focused on identifying effective hepatoprotective agents from marine algae is still in its infancy, and has been limited to only a few species of Gracilaria, Sargassum, Ulva and Ecklonia.[19,31–36] We previously reported that the brown alga E. stolonifera and its associated phlorotannins, including dioxinodehydroeckol, phlorofucofuroeckol A, eckol and 2-phloroeckol, exhibit protective effects against tacrineinduced hepatotoxicity in HepG2 cells.[19,37] However, the protective effects of the Korean seaweeds selected in this study, as well as the isolation and structural determination of components contained therein, against doxorubicininduced hepatotoxicity have not yet been reported, despite

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

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Hepatoprotective effect of Ecklonia on doxorubicin

Figure 2

Hyun Ah Jung et al.

Chemical structures of phlorotannins from Ecklonia stolonifera.

Table 3 Protective effects of phlorotannins isolated from Ecklonia stolonifera against doxorubicin-induced hepatotoxicity in primary rat hepatocyte Test sample

EC50 value (μg/ml)b

Cytotoxicity (μg/ml)b

Phloroglucinol (1) Dioxinodehydroeckol (2) Eckol (3) Phlorofucofuroeckol A (4) Dieckol (5) Triphloroethol A (6) Silymarinc

>200 3.4 ± 0.7 8.3 ± 0.5 4.4 ± 0.3 5.5 ± 0.9 11.5 ± 1.1 8.2 ± 0.5

NDa >250 >250 69.0 ± 1.6 >250 >250 235.0 ± 4.3

ND, not determined. bEC50 are given as the means ± standard error of the mean of triplicate experiments. cUsed as a positive control. a

their frequent use in foodstuffs. Thus, this study was designed to investigate the protective effect of 14 types of Korean seaweed extracts and their associated organic solvent-soluble fractions. To this end, we evaluated the effects of n-hexane, CH2Cl2, EtOAc, n-BuOH and H2O fractions from the most active extracts of E. stolonifera, as well as the most active phlorotannins, on doxorubicin-induced hepatotoxicity in primary rat hepatocytes using a bioassayguided fractionation strategy. 6

In this study, pretreatment with the EtOH extracts of E. stolonifera, E. cava, Eisenia bicyclis and Pelvetia siliquosa, the most active E. stolonifera solvent-soluble fractions, and the five phlorotannins (dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol A (4), dieckol (5) and triphloroethol-A (6)) from the most active EtOAc fraction exhibited significant protective effects against doxorubicin-induced hepatotoxicity. Furthermore, this is the first work demonstrating the hepatoprotective effect of dieckol (5) and triphloroethol-A (6). The hepatoprotective effects of these phlorotannins were comparable to that of the positive control silymarin (Table 3). Silymarin, a polyphenolic flavonoid derived from milk thistle, is an important hepatoprotective agent.[38] Silymarin is a very strong antioxidant, capable of scavenging both free radicals and ROS, resulting in the enhancement of cellular antioxidant defense machinery and increasing the antioxidant potential of cells, as well as protecting the liver from toxic substances.[39–41] The hepatoprotective results of individual phlorotannins suggest that the molecular weight of a phlorotannin is an important factor with respect to its hepatoprotective effects against doxorubicin-induced cytotoxicity. As shown in Figure 2, the lower molecular weight phloroglucinol (1) (MW = 126) exhibited no effect on doxorubicininduced cytotoxicity, while the higher molecular

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Hyun Ah Jung et al.

Hepatoprotective effect of Ecklonia on doxorubicin

weight phlorotannins, including dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol A (4), dieckol (5) and triphloroethol-A (6) did exert hepatoprotective effects. Although the mechanism underlying doxorubicininduced hepatotoxicity has not yet been fully elucidated, it has been established that doxorubicin induces liver injury mainly via the generation of free radicals and the activation of the NF-κB,[42] which suggests the involvement of ROS generation and lipid peroxidation in doxorubicin-induced cytotoxicity.[43] To this end, it has been demonstrated that both the scavenging and inhibitory activities on free radicals, including trichloromethyl free radicals (CCl3·) and trichloromethyl peroxy radicals (CCl3OO·) by means of cytochrome P450 systems-related CCl4 metabolism, are likely to be related to the hepatoprotective activity of cellular membranes and organelles by prevention of oxidation.[7,44] These results indicate that antioxidative compounds may exert protective effects against doxorubicininduced hepatotoxicity. We previously investigated the inhibition of total ROS generation by the isolated phlorotannins described in this study, and all were found to have inhibitory effects against ROS generation in the order of phlorofucofuroeckol A > eckol > dioxinodehydroeckol > dieckol.[25] Triphloroethol-A isolated from E. cava has been reported to protect cells against oxidative damage by reducing ROS or enhancing cellular antioxidant activity.[45] Antioxidative action is considered to be a rather complex process, and may include the prevention of free radical formation or free radical scavenging. Thus, the inhibition of ROS generation is considered to be one of the probable hepatoprotective mechanisms underlying the observed effects of the phlorotannins described above.

soluble fractions, including n-hexane, CH2Cl2, EtOAc, n-BuOH and H2O fractions from E. stolonifera, as well as the most active phlorotannins, was evaluated on doxorubicin-induced hepatotoxicity in primary rat hepatocytes. Among test samples, the EtOH extracts of E. stolonifera, E. cava, Eisenia bicyclis and Pelvetia siliquosa, the most active E. stolonifera solvent-soluble fractions, and the five phlorotannins (dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol A (4), dieckol (5) and triphloroethol-A (6)) from the most active EtOAc fraction exhibited significant protective effects against doxorubicin-induced hepatotoxicity. Together, the results of this study imply that E. stolonifera and its active phlorotannins protect against doxorubicin-induced hepatotoxicity, and thus hold great promise as novel therapeutic or preventive hepatoprotective agents. However, further studies are required to confirm this possibility by investigating hepatocyte morphology changes and induction of apoptosis, as well as to elucidate the mechanistic basis for the hepatoprotective effects associated with these phlorotannins. In addition, structure activity analysis of phlorotannins and evaluation of their hepatoprotective effects using animal models are needed.

Declarations Funding This work was financially supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries, Republic of Korea (811001-3).

Conclusion In this study, the protective effect of 14 types of Korean seaweed extracts and their associated organic solvent-

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Protective effect of the edible brown alga Ecklonia stolonifera on doxorubicin-induced hepatotoxicity in primary rat hepatocytes.

As part of our efforts to isolate anti-hepatotoxic agents from marine natural products, we screened the ability of 14 edible varieties of Korean seawe...
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