Protective Mechanisms of Acacetin against d-Galactosamine and

Nov 10, 2014 - Hong-Ik Cho†, Jin-Hyun Park†, Hyo-Sun Choi†, Jong Hwan Kwak†, ... Pengran Cao , Pingyao Xie , Xuebiao Wang , Jinmei Wang , Jinf...
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Protective Mechanisms of Acacetin against D‑Galactosamine and Lipopolysaccharide-Induced Fulminant Hepatic Failure in Mice Hong-Ik Cho,† Jin-Hyun Park,† Hyo-Sun Choi,† Jong Hwan Kwak,† Dong-Ung Lee,‡ Sang Kook Lee,§ and Sun-Mee Lee*,† †

School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea § College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea ‡

ABSTRACT: This study examined the hepatoprotective effects of acacetin (1), a flavonoid isolated from Agastache rugosa, against Dgalactosamine (GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were given an intraperitoneal injection of 1 (25, 50, and 100 mg/kg), or the vehicle alone (5% dimethyl sulfoxide−saline), 1 h before GalN (800 mg/kg)/LPS (40 μg/kg) treatment and sacrificed at 6 h after GalN/LPS injection. GalN/LPS markedly increased mortality and serum aminotransferase activity, and these increases were attenuated by 1. GalN/LPS increased serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, while 1 attenuated TNF-α levels and further increased IL-6 levels. GalN/LPS increased protein expression of toll-like receptor 4, phosphorylation of extracellular signal-related kinase, and p38 and c-Jun N-terminal kinase and increased nuclear protein expression of nuclear factor κB; these increases were attenuated by 1. GalN/LPS increased Atg5 and Atg7 protein expressions, and these increases were augmented by 1. GalN/LPS activated autophagic flux as indicated by decreased microtubule-associated protein 1 light chain 3-II and sequestosome1/p62 protein expression. This activation was enhanced by 1. These findings suggest that 1 protects against GalN/LPS-induced liver injury by suppressing TLR4 signaling and enhancing autophagic flux.

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damage.5 Several studies have demonstrated critical links between autophagy and inflammatory diseases such as viral infection and hepatitis. Hepatitis B virus (HBV) infection inhibits autophagic degradation by impairing lysosomal maturation, which contributes to the development of HBVassociated hepatocellular carcinoma.6 Recently, Amir et al. demonstrated that overexpression of the autophagy-related gene Beclin-1 reduces GalN/LPS-induced liver injury.7 Moreover, several studies have demonstrated the interrelationship between TLR and autophagy in inflammatory diseases. Knockdown, as well as pharmacological inhibition, of TLR4 decreases LPS-induced autophagy, which attenuates the intracellular bactericidal activity in peritoneal mesothelial cells.8 In addition, Xu et al. showed that LPS induces autophagy in human and murine macrophages through TLR4 signaling.9 In oriental folk medicine, Agastache rugosa (Fisch. & C.A. Mey.) Kuntze (Lamiaceae) has been widely used for treatment of fungal diseases, gastrointestinal disorders, and inflammatory diseases. Recently, we screened the water and 70% ethanol extract of A. rugosa and its active components for hepatoprotective agents. Among them, acacetin (5,7-dihydroxy-4′-methoxyflavone, 1), a major active flavonoid compound of A. rugosa, showed a hepatoprotective effect in primary

ulminant hepatic failure (FHF) is a life-threatening clinical syndrome that includes the development of hepatic encephalopathy, severe coagulopathy, hydroperitoneum, and jaundice in patients. The prognosis of FHF is quite poor, and there is no effective therapy for the disease, other than liver transplantation. Administration of lipopolysaccharide (LPS) in D-galactosamine (GalN)-sensitized mice has been recognized as a promising experimental model that is closely similar to clinical FHF.1 GalN/LPS can stimulate numerous inflammatory responses including the production of proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and other cellular mediators that activate improper inflammation.2 Activation of the toll-like receptor (TLR) system and associated downstream signaling plays an important role in infectious and inflammatory disease states. Ben Ari et al. reported that GalN/LPS injection upregulates hepatic TLR4 mRNA expression in mice, and gene silencing of TLR4 attenuates the inflammatory response and liver injury.3 Autophagy is a self-digestion-recycling process in which autophagic substrates are sequestered by a double-membrane autophagosome and are delivered to lysosomes for proteolytic degradation.4 Autophagy mostly functions as a pro-survival mechanism by removing misfolded molecules and dysfunctional organelles and regulating energy balance in diverse situations. However, a large amount of evidence indicates that an unbalanced autophagic response directly results in cell © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 17, 2014

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attenuated by pretreatment with 1 at 25, 50, and 100 mg/kg (Table 1). Compound 1 alone at 100 mg/kg did not affect the

hepatocytes (data not shown). A plethora of studies showed that 1 has various pharmacological effects including antibacterial, antitumor, and anti-inflammatory effects.10,11 Compound 1 inhibits cell growth and induces apoptosis in Hep G2 cells12 and human non-small-cell lung cancer A549 cells by inactivating the mitogen-activated protein kinase (MAPK) signaling pathway.13 In addition, compound 1 protects dopaminergic neuron cells by inhibiting production of proinflammatory factors including nitric oxide and TNF-α in vitro. In animal models of Parkinson’s disease, 1 suppresses degeneration of dopaminergic neurons in vivo.11 However, there is limited information available on the hepatoprotective effects of 1 in vivo. Therefore, we investigated the hepatoprotective effects of acacetin (1) in FHF and its specific molecular mechanisms, especially focusing on TLR4 signaling and autophagic flux.

Table 1. Effect of Acacetin (1) on Serum ALT Activity in Mice after GalN/LPS Injectiona group control 1 + control GalN/LPS 1 + GalN/LPS

dose (mg/kg) 100 25 50 100

ALT (U/L) 49.6 64.4 618.4 520.1 395.6 361.3

± ± ± ± ± ±

9.9 13.9 43.7c 46.6b 52.1b 48.1b,d

a

Liver damage was assessed 6 h after GalN/LPS injection by measurement of circulating serum ALT activity. Mice were intraperitoneally administered acacetin (1) 1 h before GalN/LPS injection (n = 8−10). The values are represented as mean ± SEM. b,cDenote significant differences (p < 0.05, p < 0.01) versus control group. d Denotes significant difference (p < 0.01) versus GalN/LPS group.

mortality and serum ALT level (data not shown). On the basis of these results, 1 at 100 mg/kg was selected as the optimal effective dose for evaluating the molecular mechanisms of 1 against GalN/LPS-induced hepatic injury. Histological analyses of the liver samples with hematoxylin and eosin (H&E) staining strongly supported the protective effect of compound 1. As shown in Figure 2, the histological features of the control group showed normal lobular architecture and cell structure. Compound 1 alone did not affect the histological features. However, liver sections isolated at 6 h after GalN/LPS administration showed multiple areas of portal inflammation and hepatocellular necrosis, as well as a moderate increase in inflammatory cell infiltration; this histologic damage was ameliorated by pretreatment with 1. Collectively, these results suggest that 1 suppresses GalN/LPSinduced hepatocellular damage, which may be useful in potential clinical applications for treating liver diseases. Acacetin (1) Modulates TNF-α and IL-6 Production in GalN/LPS-Induced Fulminant Hepatic Failure. The liver is a major organ of the innate immune system, and the inflammatory process contributes to diverse pathological events. Kupffer cells, resident macrophages in the liver, are activated by various bacterial stimuli, including LPS and superantigens, and produce a number of signaling molecules causing inflammatory responses. TNF-α is the first-released inflammatory cytokine produced by Kupffer cells in response to tissue damage.14 A previous study demonstrated that TNF-α was increased in livers of GalN/LPS-treated mice, and blockage of TNF-α afforded these mice a significant survival advantage and prevented hepatic injury.15 IL-6 is a typical pleiotropic cytokine produced by Kupffer cells in response to stimulation, such as LPS, and acts as both a pro-inflammatory and an antiinflammatory cytokine in the acute phase response for maintaining homeostasis in the liver.16 Previous studies showed that IL-6 is essential for liver regeneration after partial hepatectomy and confers resistance to liver injury by xenobiotics and ischemia/reperfusion injury.17,18 In this study, at 6 h after GalN/LPS administration, the level of serum TNFα and IL-6 dramatically increased to 5.6-fold and 6.3-fold compared with those in the control group, respectively. Compound 1 attenuated the increase in serum TNF-α level, while it augmented the increase in IL-6 level (Table 2).

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RESULTS AND DISCUSSION Acacetin (1) Ameliorates GalN/LPS-Induced Fulminant Hepatic Failure. Administration of LPS in combination with GalN in mice causes acute liver injury that is clinically similar to viral hepatitis in both functional and morphological features.1 For this reason, this animal model has been widely used for investigating the molecular mechanisms underlying the pathophysiology of FHF and evaluating the biological activities of hepatoprotective agents. In the GalN/LPS group, mice began to die 7 h after GalN/ LPS administration and the survival rate stabilized at 20% at 24 h. However, pretreatment with 1 (25, 50, and 100 mg/kg) before GalN/LPS administration reduced the mortality in a dose-dependent manner. With 100 mg/kg of 1, the survival rate was 80% 24 h after GalN/LPS injection (Figure 1). In the control group, the level of serum alanine aminotransferase (ALT), a marker of hepatocellular damage, was 49.6 ± 9.9 U/L. At 6 h after GalN/LPS injection, the level of serum ALT significantly increased to 618.4 ± 43.7 U/L. This increase was

Figure 1. Effect of acacetin (1) on lethality induced by GalN/LPS. All groups consisted of 10 mice. Mice were intraperitoneally administered vehicle or 1 (25, 50, or 100 mg/kg) 1 h before GalN (800 mg/kg)/ LPS (40 μg/kg) treatment. B

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Figure 2. Effect of acacetin (1) on the histological changes in the liver at 6 h after GalN/LPS injection. Mice were intraperitoneally administered 100 mg/kg acacetin (1) 1 h before GalN/LPS injection (n = 8−10). Typical images were chosen from each experimental group (original magnification 200×): (A) control; (B) 1 + control, normal hepatic architecture; (C) GalN/LPS, extensive areas of inflammation, necrosis, and lymphocyte infiltration around the overall area; (D) 1 + GalN/LPS, mild hepatocellular damage and inflammatory infiltration.

compared with that of the control group, and this was attenuated by 1 (Figure 3A). Activation of TLR4 initiates the recruitment of its downstream adaptor proteins and subsequently results in the activation of nuclear factor kappa B (NF-κB) and MAPK to induce expression of proinflammatory cytokine genes leading to liver failure.23,24 Previous studies have demonstrated involvement of NF-κB activation in TLR4-mediated FHF by GalN/ LPS injection.3 In this study, the nuclear level of NF-κB increased to 1.9-fold compared with that in the control group, and this was attenuated by pretreatment with 1 (Figure 3B). MAPKs are serine/threonine/tyrosine-specific protein kinases that are involved in directing cellular responses to a diverse array of stimuli.25 Extracellular signal-related kinases (ERKs), p38 MAPK, and c-Jun N-terminal kinases (JNKs) are members of the MAPK family. Previous study showed scoparone, a natural organic compound of Artemisia capillaris (Compositae), exhibited hepatoprotective effects through down-regulation of MAPK activation in GalN/LPS-injected mice.26 At 6 h after GalN/LPS injection, phosphorylation of p38, ERK, and JNK increased to 3.7-fold, 4.3-fold, and 1.8-fold compared with those in the control group, respectively, and these increases were attenuated by 1 (Figure 3C). Collectively, these results indicate that 1 may suppress the activation of the TLR4mediated inflammatory signaling pathway in FHF. Acacetin (1) Activates Autophagic Flux in GalN/LPSInduced Fulminant Hepatic Failure. Autophagy is a dynamic and continuous process by which cytoplasmic substrates are sequestered in autophagosomes and degraded in autolysosomes upon fusion of the autophagosomes with lysosomes.4 The formation of autophagosomes is a three-step process characterized by nucleation, elongation, and completion of the phagophore. Class III PI3K complex containing Beclin-1, ultraviolet irradiation resistance-associated gene protein, and Bif-1 initiate the nucleation of a phagophore,

Table 2. Effect of Acacetin (1) on Serum TNF-α and IL-6 Levels in Mice after GalN/LPS Injectiona group control 1 + control GalN/LPS 1 + GalN/LPS

TNF-α (pg/mL)

IL-6 (pg/mL)

± 1.1 + 1.0 ± 14.9b ± 9.3d

96.7 ± 4.9 131.0 ± 24.9 605.8 ± 96.8b 1011 ± 97.0b,c

14.1 13.1 78.9 27.4

a

The levels of serum TNF-α and IL-6 were determined at 6 h after GalN/LPS injection. Mice were intraperitoneally administered 100 mg/kg acacetin (1) 1 h before GalN/LPS injection (n = 8−10). The values are represented as mean ± SEM. bDenotes significant difference (p < 0.01) versus control group. c,dDenote significant differences (p < 0.05, p < 0.01) versus GalN/LPS group.

Acacetin (1) Suppresses TLR4 Signaling in GalN/LPSInduced Fulminant Hepatic Failure. TLRs are a family of pattern recognition receptors that detect specific patterns of microbial components, particularly those derived from pathogens, and control innate immune responses.19 There is considerable evidence that hepatic TLR4 significantly contributes to the pathogenesis of GalN/LPS-induced FHF.3 Indeed, TLR4 antagonist E5564, as well as the anti-inflammatory agent bicyclol, exhibit hepatoprotective effects through downregulation of TLR4 expression in GalN/LPS-induced liver injury.20,21 Due to their lower toxicity and multiple pharmacological properties, medicinal plants and their bioactive components have been actively investigated as pharmacological strategies to treat inflamed-liver conditions. In our previous study, anethole, a major component of Foeniculum vulgare Mill. (Apiaceae), showed a hepatoprotective effect against liver ischemia/ reperfusion injury via inhibition of the TLR4 signaling pathway.22 In this study, at 6 h after GalN/LPS injection, the level of TLR4 protein expression increased to 1.7-fold C

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Figure 3. Effect of acacetin (1) on TLR4 (A) and nuclear NF-κB (B) protein expression and MAPK phosphorylation (C) in the liver at 6 h after GalN/LPS injection. Mice were intraperitoneally administered 100 mg/kg acacetin (1) 1 h before GalN/LPS injection (n = 8−10). TLR4 levels and total and phospho-p38, ERK, and JNK levels in the liver were measured by Western blot analysis at 6 h after GalN/LPS injection. NF-κB was measured on the nuclear extracts from liver by Western blot analysis at 6 h after GalN/LPS injection. The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus control group; +, ++ denote significant differences (p < 0.05, p < 0.01) versus GalN/LPS group.

Figure 4. Effect of acacetin (1) on Atg5 and Atg7 (A) and LC3-II, p62, and Beclin-1 (B) protein expression in the liver at 6 h after GalN/LPS injection. Mice were intraperitoneally administered 100 mg/kg acacetin (1) 1 h before GalN/LPS injection (n = 8−10). The protein expression was measured by Western blot analysis at 6 h after GalN/LPS injection. The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus control group; +, ++ denote significant differences (p < 0.05, p < 0.01) versus GalN/LPS group.

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Figure 5. Effect of acacetin (1) on autophagic vacoules in the liver at 6 h after GalN/LPS injection. Mice were intraperitoneally administered 100 mg/kg acacetin (1) 1 h before GalN/LPS injection (n = 8−10). Autophagic vacuoles (arrows indicated) were observed from transmission electron microscopy images: (A) control; (B) 1 + control; (C) GalN/LPS; (D) 1 + GalN/LPS. The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus control group; ++ denotes significant difference (p < 0.01) versus GalN/LPS group.

which recruits different Atg proteins.27 The Atg5−Atg12− Atg16L complex plays an essential role in the elongation of the phagophore,28 and microtubule-associated protein 1 light chain 3 (LC3)-II, which is a lipidation form of LC3-I by Atg7, acts as a structural component of the autophagosomes.29 Autophagy is considered a cellular housekeeper against intrinsic and extrinsic stress, while it also causes autophagic cell death, which is called type II programmed cell death, by rapidly eliminating the cytoplasmic components.5 A plethora of studies have shown that autophagy plays an important role in the innate immune system. During Yersinia enterocolitica infection, activated autophagy mediates phagocytosis of the pathogens, which inhibits improper inflammation.30 Yuan et al. also reported that stimulation of autophagy by LPS in rat cardiomyocytes protects against programmed cell death.31 In diethylnitrosamineinduced liver injury, TLR2-activated autophagy in liver cells and its genetic inhibition facilitated development of hepatocellular carcinoma.32 A more recent study demonstrated that gene silencing of Atg7 in mice accelerated the mitochondrial death pathway in the liver and subsequently increased mortality in GalN/LPS-induced FHF.7 However, the pattern change of autophagy and its process have not been elucidated in the GalN/LPS-induced FHF model. In this study, at 6 h after GalN/LPS injection, the levels of Atg5 and Atg7 protein expression significantly increased to 1.3-fold and 1.6-fold, respectively, compared to those of the control group; these increases were augmented by pretreatment with 1 (Figure 4A). In addition, the protein expression level of LC3-II, a reliable

marker of the autophagosome, and sequestosome1/p62 (p62), a selective substrate for autophagy, significantly decreased to about 70.1% and 36.9% at 6 h after GalN/LPS injection, respectively, compared to those of the control group. The decrease of LC3-II was attenuated, and the decrease of p62 was further decreased by pretreatment with 1 (Figure 4B). However, there were no significant changes in Beclin-1 protein expression among any of the experimental groups (Figure 4B). The lysosomal-dependent turnover of LC3-II and p62 has emerged as a measure of bona f ide autophagic proteolysis, which is commonly termed autophagic flux, because these proteins are both degraded in the autolysosome.33 In particular, the amount of LC3-II, which increases transiently upon induction of autophagy, could be decreased after longer periods of autophagy activation.34 Our data of LC3-II and p62 suggest that 1 reinforces GalN/LPS-induced autophagic flux. These observations were confirmed by transmission electron microscopy (TEM). Compared with the basal level of autophagic vacuoles in the control group (1.1 ± 0.3), the number of autophagic vacuoles was increased in the GalN/LPS group (2.7 ± 0.4). Pretreatment with 1 augmented the number of autophagic vacuoles in the GalN/LPS group (4.9 ± 0.4) (Figure 5). Taken together, these results indicate that 1 may enhance GalN/LPS-induced autophagic flux. In conclusion, these findings suggest that acacetin (1) protects the liver against GalN/LPS-induced FHF by suppressing TLR4 signaling and activating autophagic flux. In addition, the results of the acute toxicity test (LD50 = 933 mg/ E

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kg) suggest that acacetin (1) is a safe agent for clinical use.35 Thus, we propose that acacetin (1) might be useful as a potential therapeutic medication for the treatment of hepatic failure.

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spectrophotometric procedure using a ChemiLab ALT assay kit (IVDLab Co., Ltd., Uiwang, Korea). Serum TNF-α and IL-6 Levels. Circulating levels of TNF-α and IL-6 were quantified at 6 h after GalN/LPS injection using commercial mouse enzyme-linked immunosorbent assay kits (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions. Histological Analysis. Liver sections were stained as described previously.39 Liver specimens for histopathological analysis were obtained 6 h after GalN/LPS injection. Samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, sliced into 5 μm sections, and stained with H&E, followed by blinded histological assessment. Histological changes were evaluated in randomly chosen histological fields at 200× magnification. Transmission Electron Microscopy. Liver tissues were fixed in 2.5% glutaraldehyde, 1% formaldehyde, and 100 mM sodium phosphate (pH 7.2). Samples were rinsed in 0.1 M Na cocadylate (pH 7.4), postfixed in 2% osmium tetroxide in 0.1 M Na cacodylate, and washed in 0.1 M Na cacodylate. The samples were then dehydrated in a graded series of ethanol and propylene oxide and embedded in epoxy resin (Taab 812 resin; Marivac Industries, Montreal, QC, Canada). Ultrathin (60−70 nm) sections were counterstained with uranyl acetate and lead citrate and viewed using a Hitachi 7600 transmission electron microscope (Hitachi HighTechnologies America, Inc., Schaumburg, IL, USA) equipped with a Macrofire monochrome progressive scan CCD camera (Optronics, Inc., Muskogee, OK, USA) and AMTv image capture software (Advanced Microscopy Techniques, Corp., Danvers, MA, USA). Isolation of Cytosolic and Nuclear Proteins. Fresh liver tissue was isolated and homogenized in PRO-PREP (iNtRON Biotechnology Inc., Seongnam, Korea) for whole protein samples and in NE-PER (Pierce Biotechnology Inc., Rockford, IL, USA) for nuclear and cytosolic protein samples, according to the manufacturer’s instructions. Protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology Inc.). Western Blot Immunoassay. To determine the content of TLR4, phospho-p38, p38, phospho-ERK, ERK, phospho-JNK, JNK, NF-κB, sequestosome1/p62, LC3, Beclin-1, Atg5, and Atg7 protein expression, 20 μg of soluble protein from whole liver homogenate was used. Protein samples were loaded per lane on 10−17% polyacrylamide gels, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes using the Semi-Dry Trans-Blot Cell (Bio-Rad Laboratories, Hercules, CA, USA). Transferred membranes were washed with 0.1% Tween 20 in 1× Tris-buffered saline (TBS/T) and blocked for 1 h at room temperature with 5% (w/v) skim milk powder or 5% (w/v) bovine serum albumin powder in TBS/T. Blots were then incubated overnight at 4 °C with primary antibodies. After washing five times for 8 min each in TBS/T, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature, followed by detection using an ECL detection system (iNtRON Biotechnology Inc.), according to the manufacturer’s instructions. The intensity of the immunoreactive bands was determined using TotalLab TL 120 software (Nonlinear Dynamics Ltd., Newcastle, UK). The following primary antibodies were used: phospho-p38, p38, phosphoERK, ERK, phospho-JNK, JNK, Beclin-1, Atg5, and Atg7 (Cell Signaling Technology, Beverly, MA, USA); NF-κB and p62 (Abcam, Cambridge, MA, USA); LC3 (Novus Biologicals, Littleton, CO, USA); TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and β-actin (Sigma-Aldrich). Statistical Analysis. All results are presented as mean ± standard error of the mean (SEM). The overall significance of results was analyzed by one-way ANOVA. Differences between compared groups were considered statistically significant at p < 0.05 with the appropriate Bonferroni correction for multiple comparisons. Survival data were analyzed by Kaplan−Meier curves and the log-rank test.

EXPERIMENTAL SECTION

General Experimental Procedures. NMR experiments were performed on a Bruker AVANCE III 700 spectrometer, working at 700 MHz for proton and 176 MHz for carbon with the usual pulse sequences. ESIMS data were obtained on an Agilent 1100 LC/MSD trap classic (Agilent Technologies, Santa Clara, CA, USA). Column chromatography was carried out on a Lipophilic Sephadex column (25−100 μm; Sigma-Aldrich, St. Louis, MO, USA) and silica gel 60 (230−400 mesh; Merck, Darmstadt, Germany). Plant Material. The aerial parts of Agastache rugosa were collected in September 2012 at Gyeongju, Gyeongsangbuk-do, Korea, and identified by Prof. Je-Hyun Lee, College of Oriental Medicine, Dongguk University, Gyeongju, Korea. A voucher specimen (SKKUPh-12-001) was deposited in the Laboratory of Pharmacognosy, School of Pharmacy, Sungkyunkwan University. Extraction and Isolation. The dried aerial parts of A. rugosa (3.25 kg) were cut into small pieces and extracted with MeOH (30 L) three times at room temperature. After maceration, total filtrate was concentrated to dryness under reduced pressure, and the MeOH extract (228.3 g) was suspended in distilled water (2.0 L). The resulting solution was partitioned consecutively with organic solvents to yield hexane (34.1 g), CH2Cl2 (20.0 g), EtOAc (17.9 g), n-BuOH (25.8 g), and H2O (127.2 g) fractions. The EtOAc fraction was fractionated on a Lipophilic Sephadex column using MeOH as eluent to give seven subfractions (E-1 to E-7). Subfraction E-5 was rechromatographed over a silica gel column using a stepwise elution with EtOAc−MeOH−H2O (100:3:2, 100:10:7, and 100:30:20) to afford seven further subfractions (E-5-1 to E-5-7). Compound 1 (438.5 mg) was obtained by silica gel column chromatography (hexane−EtOAc−MeOH, 10:10:1 and 10:10:2) from the subfraction E-5-3. The compound obtained was a yellowish, amorphous powder and exhibited spectroscopic data (1H NMR, 13C NMR, and ESIMS) consistent with literature values.36 The purity of 1 was determined to be 98.8% by HPLC analysis. Animal Treatment. Male 6-week-old ICR mice (24−26 g, Orient Bio, Inc., Seongnam, Korea) were kept in a temperature- and humidity-controlled room (25 ± 1 °C and 55 ± 5%, respectively) under a 12 h light−dark cycle. All experiments were approved by the Animal Care Committee of Sungkyunkwan University School of Pharmacy (SUSP14-01) and performed in accordance with the guidelines of the National Institutes of Health (NIH publication No. 86-23, revised 1985). Mice were fasted for 18 h before the experiments, but were allowed free access to tap water. Mice received an intraperitoneal injection with GalN (800 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) and LPS (40 μg/kg Escherichia coli O111:B4; SigmaAldrich) dissolved in phosphate-buffered saline, except for the normal control. Compound 1 (25, 50, and 100 mg/kg) was suspended in 5% dimethyl sulfoxide (DMSO)−saline and intraperitoneally administered 1 h before the GalN/LPS treatment, while other groups received an equivalent volume of 5% DMSO−saline as the vehicle. The dose and timing of 1 treatments were selected based on previous reports37,38 and on preliminary studies. Animals were randomly divided into six groups (each group, n = 8−10): (1) vehicle-treated control (control), (2) 1 at 100 mg/kg-treated control (1 + control), (3) vehicle-treated GalN/LPS (GalN/LPS), (4) 1 at 25 mg/kg-treated GalN/LPS (1 25 mg/kg + GalN/LPS), (5) 1 at 50 mg/kg-treated GalN/LPS (1 50 mg/ kg + GalN/LPS), (6) 1 at 100 mg/kg-treated GalN/LPS (1 100 mg/ kg + GalN/LPS). Mice were anesthetized with ketamine (55 mg/kg) and xylazine (7 mg/kg), then sacrificed at 6 h after GalN/LPS injection, and blood and liver samples were collected. Mortality and Serum ALT Activity. The survival rate of animals was monitored during the 24 h after GalN/LPS treatment. Serum ALT activity at 6 h after GalN/LPS injection was measured by the standard F

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-31-290-7712. Fax: +82-31-292-8800. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant (12172MFDS989) from Ministry of Food and Drug Safety in 2013 (“Studies on the Identification of Efficacy of Biologically Active Components from Oriental Herbal Medicines”). H.-I.C. (NRF2012H1A2A1016419) received “Global Ph.D. Fellowship Program” support from the NRF funded by the Ministry of Education, Science, and Technology (MEST).

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dx.doi.org/10.1021/np500537x | J. Nat. Prod. XXXX, XXX, XXX−XXX