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Research Paper
Journal of Pharmacy And Pharmacology
Protective effect of fucosterol isolated from the edible brown algae, Ecklonia stolonifera and Eisenia bicyclis, on tert-butyl hydroperoxide- and tacrine-induced HepG2 cell injury Jae Sue Choia*, Yu Ran Hana*, Jeong Su Byeona, Se-Young Choungb, Hee Sook Sohnc and Hyun Ah Jungc* a c
Department of Food Science and Nutrition, Pukyong National University, Busan, bCollege of Pharmacy, Kyeong Hee University, Seoul and Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, Korea
Keywords fucosterol; hepatoprotection; oxidative stress; tacrine; tert-butyl hydroperoxide Correspondence Hyun Ah Jung, Department of Food Science and Human Nutrition, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju 561-756, Korea. E-mail: [email protected] Jae Sue Choi, Department of Food Science and Nutrition, Pukyong National University, 45 Yongso-ro, Nam-gu, Busan 608-737, Korea. E-mail: [email protected] Received August 19, 2014 Accepted February 1, 2015 doi: 10.1111/jphp.12404 *These authors contributed equally to this work.
Abstract Objectives Fucosterol is the primary sterol found in brown algae. Recently, considerable interest has been generated regarding fucosterol due to its potential antioxidant, anti-inflammatory and antidiabetic effects. The aim of this study was to investigate the protective effects of fucosterol on tert-butyl hydroperoxide (t-BHP)- and tacrine-induced oxidative stress in HepG2 cells. Methods Fucosterol by itself exhibited no cytotoxicity at concentrations below 100 μm by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The increased intracellular reactive oxygen species (ROS) and decreased glutathione levels observed in t-BHP- and tacrine-treated HepG2 cells were ameliorated by fucosterol pretreatment, indicating that the protective effects of fucosterol are mediated by the induction of cellular defence mechanisms against oxidative stress. Moreover, elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in tacrine-treated mice were significantly reduced after oral administration of fucosterol. Key findings The hepatoprotective effects of fucosterol may occur via an increase in the hepatic level of glutathione and a decrease in ROS production, thereby preventing hepatic damage and the resultant increases in ALT and AST activity. Conclusion These results suggest that fucosterol may be an effective hepatoprotective agent that could be useful for preventive therapies against oxidative stress-related hepatotoxicity.
Introduction Oxidative stress, which can be induced by various environmental factors including xenobiotics, drugs and ionizing radiation, leads to the excessive production of reactive oxygen species (ROS). In this study, tert-butyl hydroperoxide (t-BHP) and tacrine were used to induce liver toxicity because the stressors have been reported to generate ROS, which subsequently react with DNA, proteins and lipids, thereby leading to hepatotoxicity. t-BHP triggers the generation of harmful free radical intermediates, such as peroxyl and alkoxyl radicals, which readily cross cellular membranes and lead to the production of highly reactive hydroxyl radicals.[1] Tacrine (1,2,3,4tetrahydro-9-aminoacridine), an acetylcholinesterase inhibitor, was originally developed for the treatment of
Alzheimer’s disease. However, tacrine has been shown to exert hepatotoxic side effects in 30–50% of all treated patients, which substantially limits its clinical use.[2,3] Therefore, in recent years, a great deal of research has been aimed at identifying natural products that can confer protective effects against t-BHP- and tacrine-induced cytotoxicity.[4–6] In these studies, the immortalized human hepatoma cell line HepG2 has frequently been employed for the screening of hepatoprotective agents against t-BHPand tacrine-induced cytotoxicity, because this cell line is known to retain many of the relevant cellular functions,[7] and is also known to be comparable with rat primary hepatocytes in terms of t-BHP- and tacrine-induced cytotoxicity.[8]
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Hepatoprotective effect of fucosterol
Jae Sue Choi et al.
Although several recent attempts have focused on discovering effective hepatoprotective agents derived from natural sources, research focusing on marine algae as sources of therapeutic agents against hepatotoxicity is still in its infancy. Only a few species of Gracilaria, Sargassum, Ulva and Ecklonia have been investigated so far.[9–15] A number of studies have investigated the hepatoprotective effects of the brown algae Ecklonia stolonifera and Eisenia bicyclis on t-BHP- and tacrine-treated HepG2 cells. In particular, researchers have been interested in the polar EtOAc and n-BuOH fractions derived from these algae, and have identified their individual polar phlorotannin components, including dioxynodehydroeckol, phlorofucofuroeckol A, eckol and 2-phloroeckol as effective hepatoprotective agents via an activity-guided fractionation strategy.[13,16,17] In addition to these various polar hepatoprotective components derived from seaweeds, fucosterol, which has been isolated from the nonpolar fractions of the marine alga Pelvetia siliquosa, was also found to be an intriguing nonpolar candidate that exerted hepatoprotective effects in CCl4intoxicated rats.[18] Although fucosterol is the most abundant sterol found in seaweeds, investigations of its ability to exert hepatoprotective effects have been limited. A great deal of research has focused on hepatoprotective effect of phlorotannins, whereas to the best of our knowledge, this is the first report demonstrating whether fucosterol isolated from the edible brown alga extract exerts a hepatoprotective effect on t-BHP- and tacrine-treated HepG2 cells. Therefore, the aim of this study was to ascertain whether fucosterol isolated from the edible brown algae E. stolonifera and E. bicyclis protects cells against oxidative stress in t-BHP- and tacrine-treated HepG2 cells, and also whether fucosterol protects from liver damage in vivo, as measured by the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in tacrine-treated mice.
Materials and Methods General
Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), t-BHP, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxyl acid (trolox), bovine serum albumin, silymarin and tacrine were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Minimum essential medium (MEM), penicillin– streptomycin, 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA), fetal bovine serum (FBS), sodium pyruvate and nonessential amino acids were purchased from GibcoBRL Life Technologies (Grand Island, NY, USA). The glutathione (GSH) assay kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). The serum ALT and AST enzymatic kit was purchased from Asan (Seoul, Korea). All chemicals and solvents used were of analytical grade.
Isolation of fucosterol The leafy thalli of E. stolonifera were collected at Tongyoung, in April 2011 and authenticated (Professor Y. J. Choi, the Marine Bioscience and Technology at Kyongsang National University). Voucher specimens (no. 20110428) have been deposited in the author’s laboratory (J. S. Choi). Leafy thalli of E. bicyclis were harvested at Chungsapo, Busan in February 2009, and were authenticated (C. H. Sohn, Pukyong National University, 2009). Voucher specimens (No. 20090228) were deposited in the author’s (JSC) laboratory. Fucosterol was isolated from E. stolonifera and E. bicyclis and according to the method described by Jung et al.[19,20] Fucosterol was identified by physical properties and spectroscopic methods, including 1H-NMR and 13C-NMR, as well as by comparison with published data and TLC analysis.[20] The structure of fucosterol is shown in Figure 1. Fucosterol was obtained as an amorphous white powder. The melting point (m.p.) was observed at 290–292°C; the optical rotation was −38° (c = 0.5, CHCl3).
Cell culture
All 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectra were measured in deuterated chloroform (CDCl3) using a JEOL JNM ECP-400 spectrometer (JEOL, Tokyo, Japan) at 400 MHz for 1H-NMR and 100 MHz for 13CNMR. Chemical shifts were referenced to the appropriate residual solvent peak (7.25 ppm for 1H-NMR and 77.0 ppm for 13C-NMR). Column chromatography was performed using silica (Si) gel 60 (70–230 mesh, Merck, Darmstadt, Germany). Thin layer chromatography (TLC) was conducted on precoated Merck Kieselgel 60 F254 plates (20 × 20 cm, 0.25 mm, Merck, Darmstadt, Germany) using 50% H2SO4 as a spray reagent. All solvents for column chromatography were of reagent grade and were acquired from commercial sources. 2
Chemicals
The HepG2 (human hepatocarcinoma) cell line was purchased from American Type Culture Collection (HB-8065,
HO Figure 1
Structure of fucosterol isolated from Ecklonia stolonifera.
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Jae Sue Choi et al.
Hepatoprotective effect of fucosterol
Cell viability was assessed using the MTT assay as previously described.[21] In brief, HepG2 cells were seeded into a 96-well plate at a density of 2.5 × 104 cells per well and incubated at 37°C for 24 h. The cells were then fed with fresh MEM containing various concentrations (25, 50 and 100 μm) of fucosterol, and then incubated for 24 h. For the cytoprotective assay, after 24 h incubation with fucosterol, the medium was replaced with medium containing either t-BHP (200 μm) or tacrine (300 μm). Cells were then incubated for 2 h or 24 h, respectively, before the addition of 100 μl of MTT solution (0.5 mg/ml in phosphate-buffered saline (PBS)) followed by a 2 h incubation. To measure the proportion of surviving cells, the medium was replaced with 100 μl of DMSO. Control cells were treated with 0.1% DMSO; this concentration exhibited no cytotoxicity as measured by this assay. The resultant absorbances at 570 nm were measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
2013; approval number: 2013-05). Imprinting control regions (ICR) mice (male, 25–30 g) were purchased from the Samtako Bio Korea Co. (Gyeonggi Province, Korea) and permitted free access to a standard chow diet and tap water. All mice were acclimatized for 1 week before the experiments and maintained at 22 ± 2°C with a relative humidity of 50 ± 5% and a 12 h light–dark cycle. Animals were randomly assigned to five groups, with eight to nine animals in each group. Treatments were administered orally with 20% propylene glycol in saline solution for mice in groups 1 (control) and 2 (tacrine), whereas mice in groups 3 to 5 were treated orally with 200 μl fucosterol (25, 50 and 100 mg/kg) for three consecutive days. On the final (third) day, 100 μl of tacrine was injected after the conclusion of the oral administration, except for mice in the control group. Tacrine was dissolved in distilled water and given in an intraperitoneal injection. Tacrine doses (15 mg/kg) were designed to mimic the human dose (up to 160 mg) on a body area basis. To assess the resultant levels of hepatotoxicity, blood was collected 6 h after tacrine was administrated and the serum was separated for liver function tests. In addition, mouse livers were isolated and stored at −75°C before analysis. The serum levels of ALT and AST were determined using enzymatic kits (Asan, Seoul, Korea). During the experimental period, there were no statistically significant differences between the control and the fucosterol-treated mice (up to 1000 mg/kg) with regard to changes in body weight. No case of diarrheal symptoms was also found.
Measurement of the level of intracellular reactive oxygen species
Measurement of glutathione
Manassas, VA, USA). Cells were maintained in MEM containing 2.0 mM l-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate and 10% FBS at 37°C in a humidified atmosphere with 5% CO2. Medium was changed every 48 h. Samples were dissolved in DMSO before being added to cells; the final concentration of DMSO did not exceed 0.1%.
Cell viability and cytoprotective assay in HepG2 cells
The level of intracellular ROS production was measured using the oxidant-sensitive fluorescence probe, DCFHDA.[22] To determine the extent of intracellular ROS scavenging activity, HepG2 cells were seeded in black 96-well plates at a density of 2.5 × 104 cells/well and incubated with various concentrations of fucosterol (25, 50 and 100 μm) for 24 h. Cells were then exposed to either t-BHP (200 μm) or tacrine (300 μm) for 2 h or 24 h to induce ROS production and subsequently incubated with DCFH-DA (20 μm) for 30 min. The resultant fluorescence intensities were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader (FL × 800; Bio-Tek Instruments Inc., Winooski, UT, USA).
Animals Animal experiments were conducted according to experimental protocols and procedures approved by the Animal Ethics Committee of Pukyong National University (review date: 22 May 2013 ∼ 4 June 2013; approval date: 12 June
Total intracellular glutathione (tGSH) (including the thiolreduced glutathione (GSH) and the disulfide-oxidized glutathione (GSSG); i.e. tGSH = GSH + GSSG) levels were measured by Tietze’s enzymatic recycling method using GSH reductase, according to the instructions in a commercial GSH assay kit (Cayman Chemical, Ann Arbor, MI, USA). In brief, confluent HepG2 cells grown in culture dishes were treated with fucosterol (25, 50 and 100 μm) in MEM for 24 h. After the incubation with fucosterol, cells were treated with either t-BHP (200 μm) or tacrine (300 μm) for 2 h or 24 h, respectively. Cells were then washed twice with cold PBS, harvested using a rubber policeman, resuspended in 250 μl of PBS containing 1 mM EDTA and homogenized by a freeze-thaw method to extract tGSH. Lysates were then cleared by centrifugation, and an aliquot of the resultant supernatant was reserved for the protein assay. For deproteinization, an equal amount of 5% w/v metaphosphoric acid was added to the residual supernatant. After centrifugation (10 000 × g for 15 min at 4°C), the resultant supernatant (400 μl) was neutralized with 20 μl of 50% v/v triethanolamine to measure the tGSH level
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Hepatoprotective effect of fucosterol
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in the sample. The tGSH concentration was then determined by the kinetic method according to the procedure described in the assay kit; concentrations are expressed as μmol/mg protein.
independent experiments unless otherwise indicated. Differences were considered significant at P < 0.05 and marked by different letters.
Results Statistical analysis
Cytotoxicity of fucosterol
The results were analysed by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. All data are expressed as means ± standard deviation from at least three 120 a
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Effect of fucosterol on tert-butyl hydroperoxide- and tacrine-induced hepatotoxicity
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Figure 2 Effect of fucosterol on cell viability in HepG2 cells. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) method. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. Data shown represent means ± standard deviation of triplicate experiments. Each group marked by a represented no significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
Relative cell viability (%)
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To determine whether fucosterol exerts a hepatoprotective effect on t-BHP- or tacrine-treated HepG2 cells, cells were pretreated with the indicated concentrations (0, 25, 50 and 100 μm) of fucosterol for 24 h and then treated with either 200 μm t-BHP (Figure 4a) or 300 μm tacrine (Figure 4b). Cells were then incubated for either 2 or 24 h, respectively. As shown in Figure 4a and 4b, treatment with fucosterol
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t-BHP (µM)
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Before determining whether fucosterol exerts any hepatoprotective activity, the cytotoxicity of fucosterol on HepG2 cells was first measured by the MTT assay. As shown in Figure 2, fucosterol did not show any cytotoxicity at concentrations up to 100 μm. The cytotoxicities of t-BHP and tacrine were also measured using the MTT assay (Figure 3). Compared with untreated cells, treatment of HepG2 cells with either t-BHP (0–200 μm) or tacrine (0–2000 μm) dramatically decreased cell viability in a dose-dependent manner (Figure 3a and 3b). The values of the 50% inhibitory concentration of t-BHP and tacrine for inducing cell death were determined to be approximately 200 μm and 300 μm, respectively. These concentrations were used in subsequent hepatoprotective assays with fucosterol.
a 100 80
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Figure 3 Cytotoxic effects of tert-butyl hydroperoxide (t-BHP) and tacrine in HepG2 cells. Cell viability was determined using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Cells were incubated for 2 h or 24 h with t-BHP (a) or tacrine (b) at the indicated concentrations. Data are represented as means ± standard deviation of triplicate experiment. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
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© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Jae Sue Choi et al.
Hepatoprotective effect of fucosterol
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Figure 4 Cytoprotective effect of fucosterol on tert-butyl hydroperoxide (t-BHP) and tacrine-treated HepG2 cells. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. After this time, cells were treated with either t-BHP (200 μM, a) or tacrine (300 μM, b) and incubated for either 2 h or 24 h, respectively. Control values were obtained in the absence of t-BHP, tacrine and fucosterol. Silymarin was used as a positive control. Data are represented as means ± standard deviation of triplicate experiments. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
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0 t-BHP (300 µM) Fucosterol (µM) Silymarin (µg/ml) Trolox (µM)
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Relative intracellular ROS level (%)
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Figure 5 Effect of fucosterol on the level of intracellular reactive oxygen species (ROS) in tert-butyl hydroperoxide (t-BHP)- and tacrine-treated HepG2 cells. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. After this time, cells were treated with either t-BHP (200 μM) (a) or tacrine (300 μM) (b) for either 2 h or 24 h and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (20 μM) and then incubated for 30 min to induce the generation of ROS. Data are represented as means ± standard deviation of triplicate experiments. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
increased the cell viability of both t-BHP- and tacrinetreated HepG2 cells in a dose-dependent manner. The respective cell viabilities achieved upon treatment with 100 μm fucosterol were found to be 71.5 ± 1.2% and 84.6 ± 2.7%, similar to those achieved upon treatment with 12.5 μg/ml silymarin (82.5 ± 3.4% and 78.9 ± 2.1%, respectively), a well-known hepatoprotective agent.[23] The values of the 50% effective concentration of fucosterol for rescuing the cell viability of t-BHP- or tacrine-treated HepG2 cells were determined to be 102.5 ± 7.8 μm and 78.6 ± 3.4 μm, respectively.
Effect of fucosterol on the levels of intracellular ROS in t-BHP- and tacrine-treated HepG2 cells Both t-BHP and tacrine stimulate the redox status of cells, thereby resulting in the generation of ROS.[24] Therefore, in
our study, cells were treated with either t-BHP or tacrine to induce oxidative stress. ROS generation was then assessed using the ROS-sensitive fluorescence indicator DCFHDA.[22] The effect of fucosterol on ROS generation in t-BHPand tacrine-treated HepG2 cells is shown in Figure 5a and 5b. Pretreatment of different concentrations of fucosterol significantly inhibited the generation of ROS in a dosedependent manner in both t-BHP- and tacrine-treated HepG2 cells. Moreover, at concentrations exceeding 50 μm, fucosterol almost completely inhibited the generation of ROS in t-BHP-treated HepG2 cells (Figure 5a), while fucosterol also showed the inhibitory potential against ROS generation in tacrine-treated HepG2 cells, reducing the level of ROS to less than about 70% that in control cells at the indicated concentrations (Figure 5b). The positive controls silymarin (50 μg/ml) and trolox (10 μm) inhibited the generation of ROS by about 60% in both t-BHP- and tacrinetreated HepG2 cells.
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Hepatoprotective effect of fucosterol
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Effect of fucosterol on the level of intracellular GSH in t-BHP- and tacrine-treated HepG2 cells GSH is a potent intracellular antioxidant that plays a primary role in the detoxification of toxic oxygen radicals such as H2O2 and O2–, thereby alleviating lipid peroxidation and cell injury. GSH exists in both a thiol-reduced (GSH) and a disulfide-oxidized (GSSG) form. In the redox cycle of GSH, it is oxidized to GSSG, which in turn is reduced back to GSH by GSSG reductase at the expense of nicotinamide adenine dinucleotide phosphate (NADPH).[25] As shown in Figure 6a and 6b, the reduced levels of intracellular GSH resulting from treatment with either t-BHP or tacrine were significantly rescued by treatment with fucosterol in a dose-dependent manner. Treatment with 100 μm fucosterol also restored GSH levels to the control group. GSH levels were also significantly increased in cells upon pretreatment with 25 μg/ml silymarin; silymarin- and t-BHP-treated cells exhibited a higher GSH level than silymarin- and tacrine-treated HepG2 cells.
Effect of fucosterol on tacrine-treated hepatotoxicity in mice Since our data indicated that fucosterol exerts hepatoprotective effect on t-BHP- and tacrine-treated HepG2 cells, we further evaluated whether fucosterol exhibits an in-vivo hepatoprotective effect against tacrineinduced hepatotoxicity in mice. To check the possible toxicity of fucosterol on animals, acute toxicity test was conducted. Various concentrations of fucosterol up to 1000 mg/kg were administered to animals and the treated mice did not present any behavioural alteration, convulsion
Table 1 mice
Effects of fucosterol on tacrine-induced hepatotoxicity in
Group Control Tacrine Fucosterol – –
Treatment dose (mg/kg)
Serum ALT (IU/L)
Serum AST (IU/L)
–
9.30 ± 3.75a 21.00 ± 4.54b 18.74 ± 9.80b 16.53 ± 7.09bc 14.78 ± 2.66c
122.50 ± 75.73a 359.03 ± 97.53b 356.75 ± 30.59b 320.04 ± 174.48bc 294.20 ± 131.84c
15 25 50 100
ICR (male, 25–30 g) mice received oral administration of fucosterol (25, 50 and 100 mg/kg) once daily for 3 consecutive days. Animals were randomly assigned to five groups, with eight animals in control and tacrine groups; nine animals in fucosterol-treated groups. Control mice were given saline. Tacrine was administered intraperitoneally at a dose of 15 mg/kg. At 6 h post-tacrine treatment, serum was collected from the blood of each mouse’s heart. To assess the resultant hepatotoxicity, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were measured. Values are represented as means ± standard deviation for each group. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
(b) 400
a
ad
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0 t-BHP (200 µM) Fucosterol (µM) Silymarin (µg/ml)
b
GSH level (µmol/mg/min)
GSH level (µmol/mg/min)
(a)
and death for 24 h (data not shown). Tacrine has been reported to cause leakages of ALT and AST in rodents.[26] A single dose (15 mg/kg body weight) of tacrine was given to mice by intraperitoneal injection; after 6 h, this dose resulted in elevated serum ALT and AST levels compared with the vehicle control group (Table 1). However, pretreatment with fucosterol at doses of 25, 50 and 100 mg/kg body weight markedly attenuated this cytotoxic effect of tacrine. Moreover, the hepatoprotective effect of fucosterol at its highest dose (100 mg/kg body weight) resulted in ALT level similar to those of the control group indicating that fucosterol has the potential to reduce tacrine-induced hepatotoxicity.
300
a ad
200
c
c
c
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25 50
100 25
Tacrine (300 µM) Fucosterol (µM) Silymarin (µg/ml)
25 50
100 25
Figure 6 Effect of fucosterol on the intracellular glutathione (GSH) level in tert-butyl hydroperoxide (t-BHP)- and tacrine-treated HepG2 cells. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. After this time, cells were treated with either t-BHP (200 μM) (a) or tacrine (300 μM) (b) for either 2 h or 24 h. Whole cell lysates were then made and used in the GSH assay. Data are presented as means ± standard deviation of triplicate experiments. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
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© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Discussion Seaweeds that belong to a group of marine plants known as algae are very popular foods as sea vegetables in China, Japan and South Korea. Moreover, a large number of seaweed cultivators, producers and consumers can be found in the world.[27] Recent studies that have focused on the biological and pharmacological activity of seaweeds have shown them to be potentially prolific sources of highly bioactive secondary metabolites that might represent useful leads in the development of new pharmaceutical agents.[28] Several varieties of bioactive novel compounds such as phlorotannins, diterpenes, polysaccharides, phytosterols and phytopigments from seaweeds have been isolated; moreover, many of these compounds have been demonstrated to possess numerous biological activity, including hepatoprotective activity.[13,16,29] Individual phlorotannins isolated from the active EtOAc fraction of the MeOH extract of the edible brown algae E. stolonifera and E. bicyclis have been reported to exert strong hepatoprotective activity in tacrine- and t-BHP-treated HepG2 cells, respectively.[13,16] Eckol and 2-phloroeckol have also been shown to inhibit the expression of Fas-mediated cell death proteins, including tBid, caspase-3 and poly (ADP-ribose) polymerase, and to suppress the release of cytochrome c from mitochondria to the cytosol in a dose-dependent manner in tacrinetreated HepG2 cells.[17] Bioassay-guided fractionation strategies are commonly used to isolate and identify individual active components that could represent potential biological and pharmacological targets. These strategies have become increasingly popular, and have been successfully applied to many plant sources.[30,31] However, it appears unlikely that functional foods such as nutraceuticals and pharmaceuticals will be successfully developed, since the activity of the seaweed extracts/fractions themselves sometimes do not correlate with the activity of their ingredients. Indeed, the magnitudes of the activity from different extracts/fractions might be significantly different from the activity of the individual compounds contained therein, due to compositional and content differences.[32] Thus, by screening active principles from extracts or fractions, it is often difficult to evaluate the exact biological activity of each component. Although the nonpolar n-hexane and dichloromethane fractions derived from MeOH extracts did not show any hepatoprotective effect on tacrine- or t-BHP-treated HepG2 cells,[13,16] fucosterol isolated from nonpolar fractions of the marine alga P. siliquosa did exhibit a hepatoprotective effect in CCl4-intoxicated rats. In this case, the hepatoprotective effect was manifested by an inhibition of serum transaminase activity, such as serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase, as well as an increase in hepatic antioxidant enzymes such as
Hepatoprotective effect of fucosterol
superoxide dismutase, catalase and GSH peroxidase.[18] Due to the discrepancies in the literature described above, we attempted to evaluate whether the fucosterol contained in E. stolonifera and E. bicyclis extracts exerted hepatoprotective activity on tacrine- or t-BHP-treated HepG2 cells. We found that fucosterol isolated from the nonpolar fraction with lower activity did exert a hepatoprotective effect, likely mediated through the induction of cellular defence mechanisms against oxidative stress, on both tacrine- and t-BHP-treated HepG2 cells. Enhanced ROS production and GSH depletion are major parts of the mechanism involved in tacrine- and t-BHPinduced cytotoxicity.[33,34] Both ROS and reactive nitrogen species (RNS) are known to play a central role in liver diseases such as hepatocellular carcinoma, viral and alcoholic hepatitis, and nonalcoholic steatosis.[35] GSH is an essential intracellular reducing substance for the maintenance of thiol groups on intracellular proteins and antioxidant molecules in living organisms. Perturbation of GSH status in a biological system has been reported to lead to serious consequences. Therefore, the use of antioxidants has been suggested to prevent or alleviate the pathology of liver diseases caused by oxidative stress.[36,37] Phenolic compounds, in particular polyphenols, are believed to be at least in part responsible for these effects by chelating metal ions, preventing radical formation, indirectly modulating enzyme activity and altering the expression levels of antioxidant and detoxifying enzymes.[38] Apart from polyphenols, the nonphenolic compound oleanolic acid belongs to a large group of structurally diverse natural products that also includes sterols, steroids and triterpenoid saponins.[39] The nonpolar compound oleanolic acid was reported to exert a hepatoprotective effect and to protect not only from acute liver injury induced by chemicals, but also from fibrosis and cirrhosis caused by chronic liver diseases.[40] Oleanolic acid has been shown to increase the nuclear accumulation of Nrf2, a key transcriptional regulator of antioxidant and detoxifying enzymes, thereby leading to the induction of Nrf2-dependent genes that play a key role in the protection of the liver. Oleanolic acid was also found to activate Nrf2independent cytoprotective mechanisms in Nrf2-null mice.[40] As stated above, both nonpolar compounds as well as polar compounds have been reported to exert hepatoprotective effects via various signalling pathways. Growing interest has focused on fucosterol, a nonpolar maritime sterol. Fucosterol has been reported to be the main and characteristic sterol found in brown algae, and its contents commonly range from 82.9 ± 1.8 to 97.3 ± 0.6% in the brown seaweeds analysed to date.[41] Although fucosterol is the most abundant sterol found in seaweeds, investigations of its ability to exert hepatoprotective effects have been limited. Although a great deal of research has
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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focused on cholinesterase inhibition,[42] the antioxidant,[18] antidiabetic,[43] anti-osteoporotic,[44] antifungal[45] and antiadipogenic effects[46] of fucosterol, to the best of our knowledge, this is the first report demonstrating that fucosterol exerts a hepatoprotective effect on both t-BHP- and tacrinetreated HepG2 cells. In this study, we demonstrated that fucosterol (25– 100 μm) significantly increased the levels of GSH and also inhibited ROS generation in a dose-dependent manner in both t-BHP- and tacrine-treated HepG2 cells. These results indicate that the hepatoprotective properties of fucosterol may be, at least in part, attributed to the direct antioxidative activity of fucosterol via the increase of hepatic antioxidant activity. Tacrine has been shown to cause leakage of ALT and AST in mice,[25] suggesting that liver injury induced by tacrine in animals can serve as an in-vivo model for screening the hepatoprotective activity of drugs. A single dose of tacrine given to mice by intraperitoneal injection resulted in elevated serum levels of ALT and AST compared with the vehicle control group after 24 h (Table 1). Consistent with the in-vitro assay data presented here, pretreatment of fucosterol significantly attenuated the cytotoxic effect of tacrine; moreover, fucosterol also exerted a hepatoprotective effect at a high dosage (100 mg/kg body weight). This effect resulted in ALT level comparable to those in control animals, indicating that fucosterol has the potential to reduce the hepatotoxicity induced by tacrine. Both ALT and AST are associated with hepatocellular liver injury, followed by leakage of AST and ALT into the blood stream.[47] In the
References 1. Grunberger D et al. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 1988; 44: 230– 232. 2. Watkins PB et al. Hepatotoxic effects of tacrine administration in patients with Alzheimer’s disease. JAMA 1994; 271: 992–998. 3. Galisteo M et al. Hepatotoxicity of tacrine: occurrence of membrane fluidity alterations without involvement of lipid peroxidation. J Pharmacol Exp Ther 2000; 294: 160–167. 4. Alía M et al. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J Biochem Mol Toxicol 2005; 19: 119–128. 8
present work, the levels of ALT and AST differently decreased/returned to that of control group, because AST is released into serum in proportion to cellular damage and is most elevated in the acute phase of cellular necrosis, whereas release of ALT occurs early in liver damage and remains elevated for a relative longer period.[48] The decrease in serum ALT and serum AST levels caused by fucosterol indicates that it can stabilize and reverse the plasma membrane damage caused by tacrine. These changes in clinically relevant liver biomarkers can be interpreted to indicate a functional improvement of hepatocytes induced by fucosterol.
Conclusion Fucosterol appears to exert its hepatoprotective effects by increasing the hepatic GSH level and decreasing ROS production, thereby preventing hepatic damage and increased ALT and AST activity. These results suggest that fucosterol may be an effective hepatoprotective agent, and could potentially be useful for preventive therapies against oxidative stress-related hepatotoxicity.
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).
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Research Paper
Journal of Pharmacy And Pharmacology
Protective effect of fucosterol isolated from the edible brown algae, Ecklonia stolonifera and Eisenia bicyclis, on tert-butyl hydroperoxide- and tacrine-induced HepG2 cell injury Jae Sue Choia*, Yu Ran Hana*, Jeong Su Byeona, Se-Young Choungb, Hee Sook Sohnc and Hyun Ah Jungc* a c
Department of Food Science and Nutrition, Pukyong National University, Busan, bCollege of Pharmacy, Kyeong Hee University, Seoul and Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, Korea
Keywords fucosterol; hepatoprotection; oxidative stress; tacrine; tert-butyl hydroperoxide Correspondence Hyun Ah Jung, Department of Food Science and Human Nutrition, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju 561-756, Korea. E-mail: [email protected] Jae Sue Choi, Department of Food Science and Nutrition, Pukyong National University, 45 Yongso-ro, Nam-gu, Busan 608-737, Korea. E-mail: [email protected] Received August 19, 2014 Accepted February 1, 2015 doi: 10.1111/jphp.12404 *These authors contributed equally to this work.
Abstract Objectives Fucosterol is the primary sterol found in brown algae. Recently, considerable interest has been generated regarding fucosterol due to its potential antioxidant, anti-inflammatory and antidiabetic effects. The aim of this study was to investigate the protective effects of fucosterol on tert-butyl hydroperoxide (t-BHP)- and tacrine-induced oxidative stress in HepG2 cells. Methods Fucosterol by itself exhibited no cytotoxicity at concentrations below 100 μm by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The increased intracellular reactive oxygen species (ROS) and decreased glutathione levels observed in t-BHP- and tacrine-treated HepG2 cells were ameliorated by fucosterol pretreatment, indicating that the protective effects of fucosterol are mediated by the induction of cellular defence mechanisms against oxidative stress. Moreover, elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in tacrine-treated mice were significantly reduced after oral administration of fucosterol. Key findings The hepatoprotective effects of fucosterol may occur via an increase in the hepatic level of glutathione and a decrease in ROS production, thereby preventing hepatic damage and the resultant increases in ALT and AST activity. Conclusion These results suggest that fucosterol may be an effective hepatoprotective agent that could be useful for preventive therapies against oxidative stress-related hepatotoxicity.
Introduction Oxidative stress, which can be induced by various environmental factors including xenobiotics, drugs and ionizing radiation, leads to the excessive production of reactive oxygen species (ROS). In this study, tert-butyl hydroperoxide (t-BHP) and tacrine were used to induce liver toxicity because the stressors have been reported to generate ROS, which subsequently react with DNA, proteins and lipids, thereby leading to hepatotoxicity. t-BHP triggers the generation of harmful free radical intermediates, such as peroxyl and alkoxyl radicals, which readily cross cellular membranes and lead to the production of highly reactive hydroxyl radicals.[1] Tacrine (1,2,3,4tetrahydro-9-aminoacridine), an acetylcholinesterase inhibitor, was originally developed for the treatment of
Alzheimer’s disease. However, tacrine has been shown to exert hepatotoxic side effects in 30–50% of all treated patients, which substantially limits its clinical use.[2,3] Therefore, in recent years, a great deal of research has been aimed at identifying natural products that can confer protective effects against t-BHP- and tacrine-induced cytotoxicity.[4–6] In these studies, the immortalized human hepatoma cell line HepG2 has frequently been employed for the screening of hepatoprotective agents against t-BHPand tacrine-induced cytotoxicity, because this cell line is known to retain many of the relevant cellular functions,[7] and is also known to be comparable with rat primary hepatocytes in terms of t-BHP- and tacrine-induced cytotoxicity.[8]
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Although several recent attempts have focused on discovering effective hepatoprotective agents derived from natural sources, research focusing on marine algae as sources of therapeutic agents against hepatotoxicity is still in its infancy. Only a few species of Gracilaria, Sargassum, Ulva and Ecklonia have been investigated so far.[9–15] A number of studies have investigated the hepatoprotective effects of the brown algae Ecklonia stolonifera and Eisenia bicyclis on t-BHP- and tacrine-treated HepG2 cells. In particular, researchers have been interested in the polar EtOAc and n-BuOH fractions derived from these algae, and have identified their individual polar phlorotannin components, including dioxynodehydroeckol, phlorofucofuroeckol A, eckol and 2-phloroeckol as effective hepatoprotective agents via an activity-guided fractionation strategy.[13,16,17] In addition to these various polar hepatoprotective components derived from seaweeds, fucosterol, which has been isolated from the nonpolar fractions of the marine alga Pelvetia siliquosa, was also found to be an intriguing nonpolar candidate that exerted hepatoprotective effects in CCl4intoxicated rats.[18] Although fucosterol is the most abundant sterol found in seaweeds, investigations of its ability to exert hepatoprotective effects have been limited. A great deal of research has focused on hepatoprotective effect of phlorotannins, whereas to the best of our knowledge, this is the first report demonstrating whether fucosterol isolated from the edible brown alga extract exerts a hepatoprotective effect on t-BHP- and tacrine-treated HepG2 cells. Therefore, the aim of this study was to ascertain whether fucosterol isolated from the edible brown algae E. stolonifera and E. bicyclis protects cells against oxidative stress in t-BHP- and tacrine-treated HepG2 cells, and also whether fucosterol protects from liver damage in vivo, as measured by the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in tacrine-treated mice.
Materials and Methods General
Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), t-BHP, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxyl acid (trolox), bovine serum albumin, silymarin and tacrine were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Minimum essential medium (MEM), penicillin– streptomycin, 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA), fetal bovine serum (FBS), sodium pyruvate and nonessential amino acids were purchased from GibcoBRL Life Technologies (Grand Island, NY, USA). The glutathione (GSH) assay kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). The serum ALT and AST enzymatic kit was purchased from Asan (Seoul, Korea). All chemicals and solvents used were of analytical grade.
Isolation of fucosterol The leafy thalli of E. stolonifera were collected at Tongyoung, in April 2011 and authenticated (Professor Y. J. Choi, the Marine Bioscience and Technology at Kyongsang National University). Voucher specimens (no. 20110428) have been deposited in the author’s laboratory (J. S. Choi). Leafy thalli of E. bicyclis were harvested at Chungsapo, Busan in February 2009, and were authenticated (C. H. Sohn, Pukyong National University, 2009). Voucher specimens (No. 20090228) were deposited in the author’s (JSC) laboratory. Fucosterol was isolated from E. stolonifera and E. bicyclis and according to the method described by Jung et al.[19,20] Fucosterol was identified by physical properties and spectroscopic methods, including 1H-NMR and 13C-NMR, as well as by comparison with published data and TLC analysis.[20] The structure of fucosterol is shown in Figure 1. Fucosterol was obtained as an amorphous white powder. The melting point (m.p.) was observed at 290–292°C; the optical rotation was −38° (c = 0.5, CHCl3).
Cell culture
All 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectra were measured in deuterated chloroform (CDCl3) using a JEOL JNM ECP-400 spectrometer (JEOL, Tokyo, Japan) at 400 MHz for 1H-NMR and 100 MHz for 13CNMR. Chemical shifts were referenced to the appropriate residual solvent peak (7.25 ppm for 1H-NMR and 77.0 ppm for 13C-NMR). Column chromatography was performed using silica (Si) gel 60 (70–230 mesh, Merck, Darmstadt, Germany). Thin layer chromatography (TLC) was conducted on precoated Merck Kieselgel 60 F254 plates (20 × 20 cm, 0.25 mm, Merck, Darmstadt, Germany) using 50% H2SO4 as a spray reagent. All solvents for column chromatography were of reagent grade and were acquired from commercial sources. 2
Chemicals
The HepG2 (human hepatocarcinoma) cell line was purchased from American Type Culture Collection (HB-8065,
HO Figure 1
Structure of fucosterol isolated from Ecklonia stolonifera.
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Hepatoprotective effect of fucosterol
Cell viability was assessed using the MTT assay as previously described.[21] In brief, HepG2 cells were seeded into a 96-well plate at a density of 2.5 × 104 cells per well and incubated at 37°C for 24 h. The cells were then fed with fresh MEM containing various concentrations (25, 50 and 100 μm) of fucosterol, and then incubated for 24 h. For the cytoprotective assay, after 24 h incubation with fucosterol, the medium was replaced with medium containing either t-BHP (200 μm) or tacrine (300 μm). Cells were then incubated for 2 h or 24 h, respectively, before the addition of 100 μl of MTT solution (0.5 mg/ml in phosphate-buffered saline (PBS)) followed by a 2 h incubation. To measure the proportion of surviving cells, the medium was replaced with 100 μl of DMSO. Control cells were treated with 0.1% DMSO; this concentration exhibited no cytotoxicity as measured by this assay. The resultant absorbances at 570 nm were measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
2013; approval number: 2013-05). Imprinting control regions (ICR) mice (male, 25–30 g) were purchased from the Samtako Bio Korea Co. (Gyeonggi Province, Korea) and permitted free access to a standard chow diet and tap water. All mice were acclimatized for 1 week before the experiments and maintained at 22 ± 2°C with a relative humidity of 50 ± 5% and a 12 h light–dark cycle. Animals were randomly assigned to five groups, with eight to nine animals in each group. Treatments were administered orally with 20% propylene glycol in saline solution for mice in groups 1 (control) and 2 (tacrine), whereas mice in groups 3 to 5 were treated orally with 200 μl fucosterol (25, 50 and 100 mg/kg) for three consecutive days. On the final (third) day, 100 μl of tacrine was injected after the conclusion of the oral administration, except for mice in the control group. Tacrine was dissolved in distilled water and given in an intraperitoneal injection. Tacrine doses (15 mg/kg) were designed to mimic the human dose (up to 160 mg) on a body area basis. To assess the resultant levels of hepatotoxicity, blood was collected 6 h after tacrine was administrated and the serum was separated for liver function tests. In addition, mouse livers were isolated and stored at −75°C before analysis. The serum levels of ALT and AST were determined using enzymatic kits (Asan, Seoul, Korea). During the experimental period, there were no statistically significant differences between the control and the fucosterol-treated mice (up to 1000 mg/kg) with regard to changes in body weight. No case of diarrheal symptoms was also found.
Measurement of the level of intracellular reactive oxygen species
Measurement of glutathione
Manassas, VA, USA). Cells were maintained in MEM containing 2.0 mM l-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate and 10% FBS at 37°C in a humidified atmosphere with 5% CO2. Medium was changed every 48 h. Samples were dissolved in DMSO before being added to cells; the final concentration of DMSO did not exceed 0.1%.
Cell viability and cytoprotective assay in HepG2 cells
The level of intracellular ROS production was measured using the oxidant-sensitive fluorescence probe, DCFHDA.[22] To determine the extent of intracellular ROS scavenging activity, HepG2 cells were seeded in black 96-well plates at a density of 2.5 × 104 cells/well and incubated with various concentrations of fucosterol (25, 50 and 100 μm) for 24 h. Cells were then exposed to either t-BHP (200 μm) or tacrine (300 μm) for 2 h or 24 h to induce ROS production and subsequently incubated with DCFH-DA (20 μm) for 30 min. The resultant fluorescence intensities were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader (FL × 800; Bio-Tek Instruments Inc., Winooski, UT, USA).
Animals Animal experiments were conducted according to experimental protocols and procedures approved by the Animal Ethics Committee of Pukyong National University (review date: 22 May 2013 ∼ 4 June 2013; approval date: 12 June
Total intracellular glutathione (tGSH) (including the thiolreduced glutathione (GSH) and the disulfide-oxidized glutathione (GSSG); i.e. tGSH = GSH + GSSG) levels were measured by Tietze’s enzymatic recycling method using GSH reductase, according to the instructions in a commercial GSH assay kit (Cayman Chemical, Ann Arbor, MI, USA). In brief, confluent HepG2 cells grown in culture dishes were treated with fucosterol (25, 50 and 100 μm) in MEM for 24 h. After the incubation with fucosterol, cells were treated with either t-BHP (200 μm) or tacrine (300 μm) for 2 h or 24 h, respectively. Cells were then washed twice with cold PBS, harvested using a rubber policeman, resuspended in 250 μl of PBS containing 1 mM EDTA and homogenized by a freeze-thaw method to extract tGSH. Lysates were then cleared by centrifugation, and an aliquot of the resultant supernatant was reserved for the protein assay. For deproteinization, an equal amount of 5% w/v metaphosphoric acid was added to the residual supernatant. After centrifugation (10 000 × g for 15 min at 4°C), the resultant supernatant (400 μl) was neutralized with 20 μl of 50% v/v triethanolamine to measure the tGSH level
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in the sample. The tGSH concentration was then determined by the kinetic method according to the procedure described in the assay kit; concentrations are expressed as μmol/mg protein.
independent experiments unless otherwise indicated. Differences were considered significant at P < 0.05 and marked by different letters.
Results Statistical analysis
Cytotoxicity of fucosterol
The results were analysed by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. All data are expressed as means ± standard deviation from at least three 120 a
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Figure 2 Effect of fucosterol on cell viability in HepG2 cells. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) method. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. Data shown represent means ± standard deviation of triplicate experiments. Each group marked by a represented no significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
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To determine whether fucosterol exerts a hepatoprotective effect on t-BHP- or tacrine-treated HepG2 cells, cells were pretreated with the indicated concentrations (0, 25, 50 and 100 μm) of fucosterol for 24 h and then treated with either 200 μm t-BHP (Figure 4a) or 300 μm tacrine (Figure 4b). Cells were then incubated for either 2 or 24 h, respectively. As shown in Figure 4a and 4b, treatment with fucosterol
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Before determining whether fucosterol exerts any hepatoprotective activity, the cytotoxicity of fucosterol on HepG2 cells was first measured by the MTT assay. As shown in Figure 2, fucosterol did not show any cytotoxicity at concentrations up to 100 μm. The cytotoxicities of t-BHP and tacrine were also measured using the MTT assay (Figure 3). Compared with untreated cells, treatment of HepG2 cells with either t-BHP (0–200 μm) or tacrine (0–2000 μm) dramatically decreased cell viability in a dose-dependent manner (Figure 3a and 3b). The values of the 50% inhibitory concentration of t-BHP and tacrine for inducing cell death were determined to be approximately 200 μm and 300 μm, respectively. These concentrations were used in subsequent hepatoprotective assays with fucosterol.
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Figure 3 Cytotoxic effects of tert-butyl hydroperoxide (t-BHP) and tacrine in HepG2 cells. Cell viability was determined using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Cells were incubated for 2 h or 24 h with t-BHP (a) or tacrine (b) at the indicated concentrations. Data are represented as means ± standard deviation of triplicate experiment. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
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Hepatoprotective effect of fucosterol
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bc
60 40 20 0
Tacrine (300 µM) Fucosterol (µM) 50 100 12.5 Silymarin (µg/ml)
25
50 100 12.5
Figure 4 Cytoprotective effect of fucosterol on tert-butyl hydroperoxide (t-BHP) and tacrine-treated HepG2 cells. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. After this time, cells were treated with either t-BHP (200 μM, a) or tacrine (300 μM, b) and incubated for either 2 h or 24 h, respectively. Control values were obtained in the absence of t-BHP, tacrine and fucosterol. Silymarin was used as a positive control. Data are represented as means ± standard deviation of triplicate experiments. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
(b) a
100 80 60 40
d b bc
20
0 t-BHP (300 µM) Fucosterol (µM) Silymarin (µg/ml) Trolox (µM)
e
c
Relative intracellular ROS level (%)
Relative intracellular ROS level (%)
(a) 120
120
a
100 80 60
b
b
bc
b
c
40 20 0
25 50 100 50 10
Tacrine (300 µM) Fucosterol (µM) Silymarin (µg/ml) Trolox (µM)
25 50 100 50 10
Figure 5 Effect of fucosterol on the level of intracellular reactive oxygen species (ROS) in tert-butyl hydroperoxide (t-BHP)- and tacrine-treated HepG2 cells. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. After this time, cells were treated with either t-BHP (200 μM) (a) or tacrine (300 μM) (b) for either 2 h or 24 h and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (20 μM) and then incubated for 30 min to induce the generation of ROS. Data are represented as means ± standard deviation of triplicate experiments. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
increased the cell viability of both t-BHP- and tacrinetreated HepG2 cells in a dose-dependent manner. The respective cell viabilities achieved upon treatment with 100 μm fucosterol were found to be 71.5 ± 1.2% and 84.6 ± 2.7%, similar to those achieved upon treatment with 12.5 μg/ml silymarin (82.5 ± 3.4% and 78.9 ± 2.1%, respectively), a well-known hepatoprotective agent.[23] The values of the 50% effective concentration of fucosterol for rescuing the cell viability of t-BHP- or tacrine-treated HepG2 cells were determined to be 102.5 ± 7.8 μm and 78.6 ± 3.4 μm, respectively.
Effect of fucosterol on the levels of intracellular ROS in t-BHP- and tacrine-treated HepG2 cells Both t-BHP and tacrine stimulate the redox status of cells, thereby resulting in the generation of ROS.[24] Therefore, in
our study, cells were treated with either t-BHP or tacrine to induce oxidative stress. ROS generation was then assessed using the ROS-sensitive fluorescence indicator DCFHDA.[22] The effect of fucosterol on ROS generation in t-BHPand tacrine-treated HepG2 cells is shown in Figure 5a and 5b. Pretreatment of different concentrations of fucosterol significantly inhibited the generation of ROS in a dosedependent manner in both t-BHP- and tacrine-treated HepG2 cells. Moreover, at concentrations exceeding 50 μm, fucosterol almost completely inhibited the generation of ROS in t-BHP-treated HepG2 cells (Figure 5a), while fucosterol also showed the inhibitory potential against ROS generation in tacrine-treated HepG2 cells, reducing the level of ROS to less than about 70% that in control cells at the indicated concentrations (Figure 5b). The positive controls silymarin (50 μg/ml) and trolox (10 μm) inhibited the generation of ROS by about 60% in both t-BHP- and tacrinetreated HepG2 cells.
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Effect of fucosterol on the level of intracellular GSH in t-BHP- and tacrine-treated HepG2 cells GSH is a potent intracellular antioxidant that plays a primary role in the detoxification of toxic oxygen radicals such as H2O2 and O2–, thereby alleviating lipid peroxidation and cell injury. GSH exists in both a thiol-reduced (GSH) and a disulfide-oxidized (GSSG) form. In the redox cycle of GSH, it is oxidized to GSSG, which in turn is reduced back to GSH by GSSG reductase at the expense of nicotinamide adenine dinucleotide phosphate (NADPH).[25] As shown in Figure 6a and 6b, the reduced levels of intracellular GSH resulting from treatment with either t-BHP or tacrine were significantly rescued by treatment with fucosterol in a dose-dependent manner. Treatment with 100 μm fucosterol also restored GSH levels to the control group. GSH levels were also significantly increased in cells upon pretreatment with 25 μg/ml silymarin; silymarin- and t-BHP-treated cells exhibited a higher GSH level than silymarin- and tacrine-treated HepG2 cells.
Effect of fucosterol on tacrine-treated hepatotoxicity in mice Since our data indicated that fucosterol exerts hepatoprotective effect on t-BHP- and tacrine-treated HepG2 cells, we further evaluated whether fucosterol exhibits an in-vivo hepatoprotective effect against tacrineinduced hepatotoxicity in mice. To check the possible toxicity of fucosterol on animals, acute toxicity test was conducted. Various concentrations of fucosterol up to 1000 mg/kg were administered to animals and the treated mice did not present any behavioural alteration, convulsion
Table 1 mice
Effects of fucosterol on tacrine-induced hepatotoxicity in
Group Control Tacrine Fucosterol – –
Treatment dose (mg/kg)
Serum ALT (IU/L)
Serum AST (IU/L)
–
9.30 ± 3.75a 21.00 ± 4.54b 18.74 ± 9.80b 16.53 ± 7.09bc 14.78 ± 2.66c
122.50 ± 75.73a 359.03 ± 97.53b 356.75 ± 30.59b 320.04 ± 174.48bc 294.20 ± 131.84c
15 25 50 100
ICR (male, 25–30 g) mice received oral administration of fucosterol (25, 50 and 100 mg/kg) once daily for 3 consecutive days. Animals were randomly assigned to five groups, with eight animals in control and tacrine groups; nine animals in fucosterol-treated groups. Control mice were given saline. Tacrine was administered intraperitoneally at a dose of 15 mg/kg. At 6 h post-tacrine treatment, serum was collected from the blood of each mouse’s heart. To assess the resultant hepatotoxicity, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were measured. Values are represented as means ± standard deviation for each group. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
(b) 400
a
ad
ad
cd
300 c 200 100
0 t-BHP (200 µM) Fucosterol (µM) Silymarin (µg/ml)
b
GSH level (µmol/mg/min)
GSH level (µmol/mg/min)
(a)
and death for 24 h (data not shown). Tacrine has been reported to cause leakages of ALT and AST in rodents.[26] A single dose (15 mg/kg body weight) of tacrine was given to mice by intraperitoneal injection; after 6 h, this dose resulted in elevated serum ALT and AST levels compared with the vehicle control group (Table 1). However, pretreatment with fucosterol at doses of 25, 50 and 100 mg/kg body weight markedly attenuated this cytotoxic effect of tacrine. Moreover, the hepatoprotective effect of fucosterol at its highest dose (100 mg/kg body weight) resulted in ALT level similar to those of the control group indicating that fucosterol has the potential to reduce tacrine-induced hepatotoxicity.
300
a ad
200
c
c
c
100 b 0
25 50
100 25
Tacrine (300 µM) Fucosterol (µM) Silymarin (µg/ml)
25 50
100 25
Figure 6 Effect of fucosterol on the intracellular glutathione (GSH) level in tert-butyl hydroperoxide (t-BHP)- and tacrine-treated HepG2 cells. Cells were pretreated with the indicated concentrations (25, 50 and 100 μM) of fucosterol for 24 h. After this time, cells were treated with either t-BHP (200 μM) (a) or tacrine (300 μM) (b) for either 2 h or 24 h. Whole cell lysates were then made and used in the GSH assay. Data are presented as means ± standard deviation of triplicate experiments. Each group marked by different letter represented significant differences between groups at P < 0.05 (Kruskal–Wallis test followed by Dunn’s test).
6
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Jae Sue Choi et al.
Discussion Seaweeds that belong to a group of marine plants known as algae are very popular foods as sea vegetables in China, Japan and South Korea. Moreover, a large number of seaweed cultivators, producers and consumers can be found in the world.[27] Recent studies that have focused on the biological and pharmacological activity of seaweeds have shown them to be potentially prolific sources of highly bioactive secondary metabolites that might represent useful leads in the development of new pharmaceutical agents.[28] Several varieties of bioactive novel compounds such as phlorotannins, diterpenes, polysaccharides, phytosterols and phytopigments from seaweeds have been isolated; moreover, many of these compounds have been demonstrated to possess numerous biological activity, including hepatoprotective activity.[13,16,29] Individual phlorotannins isolated from the active EtOAc fraction of the MeOH extract of the edible brown algae E. stolonifera and E. bicyclis have been reported to exert strong hepatoprotective activity in tacrine- and t-BHP-treated HepG2 cells, respectively.[13,16] Eckol and 2-phloroeckol have also been shown to inhibit the expression of Fas-mediated cell death proteins, including tBid, caspase-3 and poly (ADP-ribose) polymerase, and to suppress the release of cytochrome c from mitochondria to the cytosol in a dose-dependent manner in tacrinetreated HepG2 cells.[17] Bioassay-guided fractionation strategies are commonly used to isolate and identify individual active components that could represent potential biological and pharmacological targets. These strategies have become increasingly popular, and have been successfully applied to many plant sources.[30,31] However, it appears unlikely that functional foods such as nutraceuticals and pharmaceuticals will be successfully developed, since the activity of the seaweed extracts/fractions themselves sometimes do not correlate with the activity of their ingredients. Indeed, the magnitudes of the activity from different extracts/fractions might be significantly different from the activity of the individual compounds contained therein, due to compositional and content differences.[32] Thus, by screening active principles from extracts or fractions, it is often difficult to evaluate the exact biological activity of each component. Although the nonpolar n-hexane and dichloromethane fractions derived from MeOH extracts did not show any hepatoprotective effect on tacrine- or t-BHP-treated HepG2 cells,[13,16] fucosterol isolated from nonpolar fractions of the marine alga P. siliquosa did exhibit a hepatoprotective effect in CCl4-intoxicated rats. In this case, the hepatoprotective effect was manifested by an inhibition of serum transaminase activity, such as serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase, as well as an increase in hepatic antioxidant enzymes such as
Hepatoprotective effect of fucosterol
superoxide dismutase, catalase and GSH peroxidase.[18] Due to the discrepancies in the literature described above, we attempted to evaluate whether the fucosterol contained in E. stolonifera and E. bicyclis extracts exerted hepatoprotective activity on tacrine- or t-BHP-treated HepG2 cells. We found that fucosterol isolated from the nonpolar fraction with lower activity did exert a hepatoprotective effect, likely mediated through the induction of cellular defence mechanisms against oxidative stress, on both tacrine- and t-BHP-treated HepG2 cells. Enhanced ROS production and GSH depletion are major parts of the mechanism involved in tacrine- and t-BHPinduced cytotoxicity.[33,34] Both ROS and reactive nitrogen species (RNS) are known to play a central role in liver diseases such as hepatocellular carcinoma, viral and alcoholic hepatitis, and nonalcoholic steatosis.[35] GSH is an essential intracellular reducing substance for the maintenance of thiol groups on intracellular proteins and antioxidant molecules in living organisms. Perturbation of GSH status in a biological system has been reported to lead to serious consequences. Therefore, the use of antioxidants has been suggested to prevent or alleviate the pathology of liver diseases caused by oxidative stress.[36,37] Phenolic compounds, in particular polyphenols, are believed to be at least in part responsible for these effects by chelating metal ions, preventing radical formation, indirectly modulating enzyme activity and altering the expression levels of antioxidant and detoxifying enzymes.[38] Apart from polyphenols, the nonphenolic compound oleanolic acid belongs to a large group of structurally diverse natural products that also includes sterols, steroids and triterpenoid saponins.[39] The nonpolar compound oleanolic acid was reported to exert a hepatoprotective effect and to protect not only from acute liver injury induced by chemicals, but also from fibrosis and cirrhosis caused by chronic liver diseases.[40] Oleanolic acid has been shown to increase the nuclear accumulation of Nrf2, a key transcriptional regulator of antioxidant and detoxifying enzymes, thereby leading to the induction of Nrf2-dependent genes that play a key role in the protection of the liver. Oleanolic acid was also found to activate Nrf2independent cytoprotective mechanisms in Nrf2-null mice.[40] As stated above, both nonpolar compounds as well as polar compounds have been reported to exert hepatoprotective effects via various signalling pathways. Growing interest has focused on fucosterol, a nonpolar maritime sterol. Fucosterol has been reported to be the main and characteristic sterol found in brown algae, and its contents commonly range from 82.9 ± 1.8 to 97.3 ± 0.6% in the brown seaweeds analysed to date.[41] Although fucosterol is the most abundant sterol found in seaweeds, investigations of its ability to exert hepatoprotective effects have been limited. Although a great deal of research has
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Hepatoprotective effect of fucosterol
Jae Sue Choi et al.
focused on cholinesterase inhibition,[42] the antioxidant,[18] antidiabetic,[43] anti-osteoporotic,[44] antifungal[45] and antiadipogenic effects[46] of fucosterol, to the best of our knowledge, this is the first report demonstrating that fucosterol exerts a hepatoprotective effect on both t-BHP- and tacrinetreated HepG2 cells. In this study, we demonstrated that fucosterol (25– 100 μm) significantly increased the levels of GSH and also inhibited ROS generation in a dose-dependent manner in both t-BHP- and tacrine-treated HepG2 cells. These results indicate that the hepatoprotective properties of fucosterol may be, at least in part, attributed to the direct antioxidative activity of fucosterol via the increase of hepatic antioxidant activity. Tacrine has been shown to cause leakage of ALT and AST in mice,[25] suggesting that liver injury induced by tacrine in animals can serve as an in-vivo model for screening the hepatoprotective activity of drugs. A single dose of tacrine given to mice by intraperitoneal injection resulted in elevated serum levels of ALT and AST compared with the vehicle control group after 24 h (Table 1). Consistent with the in-vitro assay data presented here, pretreatment of fucosterol significantly attenuated the cytotoxic effect of tacrine; moreover, fucosterol also exerted a hepatoprotective effect at a high dosage (100 mg/kg body weight). This effect resulted in ALT level comparable to those in control animals, indicating that fucosterol has the potential to reduce the hepatotoxicity induced by tacrine. Both ALT and AST are associated with hepatocellular liver injury, followed by leakage of AST and ALT into the blood stream.[47] In the
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Conclusion Fucosterol appears to exert its hepatoprotective effects by increasing the hepatic GSH level and decreasing ROS production, thereby preventing hepatic damage and increased ALT and AST activity. These results suggest that fucosterol may be an effective hepatoprotective agent, and could potentially be useful for preventive therapies against oxidative stress-related hepatotoxicity.
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).
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