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Eriodictyol 7-O-β-D glucopyranoside from Coreopsis tinctoria Nutt. ameliorates lipid disorders via protecting mitochondrial function and suppressing lipogenesis

Eriodictyol 7-O-β-D glucopyranoside from Coreopsis tinctoria Nutt. ameliorates lipid disorders... MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 Eriodictyol 7‑O‑β‑D glucopyranoside from Coreopsis tinctoria Nutt. ameliorates lipid disorders via protecting mitochondrial function and suppressing lipogenesis 1* 1,2* 1* 1* 1 1 1 YUYAN LIANG , HAI NIU , LIMEI MA , DAN DU , LI WEN , QING XIA and WEN HUANG Laboratory of Ethnopharmacology, Department of Integrated Traditional Chinese and Western Medicine, Regenerative Medicine Research Center, West China Hospital, West China Medical School; College of Mathematics, Sichuan University, Chengdu, Sichuan 610041, P.R. China Received May 17, 2016; Accepted April 4, 2017 DOI: 10.3892/mmr.2017.6743 Abstract. Coreopsis tinctoria (snow chrysanthemum) has been protein expression levels of disuld fi e‑isomerase A3 precursor reported to exert antihyperlipidemic effects. The present study and fatty acid synthase, thus suppressing FFA-induced aimed to identify the active compounds of Coreopsis tinctoria lipogenesis in HepG2 cells. In conclusion, the present study and to investigate the molecular mechanisms underlying its identie fi d compound 2 as one of the main active compounds in effects on lipid dysregulation by measuring lipid levels, reac- Coreopsis tinctoria responsible for its lipid-lowering effects. tive oxygen species, lipid peroxidation and fatty acid synthesis. Compound 2 was revealed to possess antihyperlipidemic The present results demonstrated that snow chrysanthemum properties, exerted via reducing oxidative stress, protecting aqueous extracts signic fi antly reduced serum lipid levels and mitochondrial function and suppressing lipogenesis. oxidative stress in vivo. The main compounds that were isolated were identie fi d as flavanomarein (co mpound 1) and eriodictyol Introduction 7-O-β-D glucopyranoside (compound 2). Compounds 1 and 2 demonstrated potent antioxidative properties, including free Lipid dysregulation serves a critical role in the progres- radical scavenging activity, inhibition of lipid peroxidation, as sion of cardiovascular diseases (1), metabolic syndrome (2) well as lipid-lowering effects in human HepG2 hepatocellular and non-alcoholic fatty liver disease (3). These disorders carcinoma cells treated with free fatty acids (FFAs). Compound pose major public health concerns, and are associated with 2 was revealed to suppress the elevation of triglyceride levels family burden and a high socioeconomic cost (2). Currently and inhibit lipid peroxidation following FFA treatment. In addi- available lipid-lowering agents used in the treatment of hyper- tion, it was demonstrated to signic fi antly reduce intracellular lipidemia include statins and br fi ates; however, these agents levels of reactive oxygen species and improve the mitochon- have been associated with serious adverse effects, including drial membrane potential and adenosine triphosphate levels, gastrointestinal disturbances, severe muscle damage and thus protecting mitochondrial function in FFA-treated HepG2 hepatotoxicity (4). Therefore, natural products and herbal cells. Furthermore, compound 2 markedly suppressed the medicines with improved safety profiles have garnered atten - tion for the treatment of lipid disorders. The capitula of Coreopsis tinctoria, also known as snow chrysanthemum, have been used in the form of a tea-like beverage for the prevention of cardiovascular disorders, Correspondence to: Professor Wen Huang or Professor Qing Xia, diarrhea and diabetes in traditional Chinese medicine (5). Laboratory of Ethnopharmacology, Department of Integrated Coreopsis tinctoria has been revealed to contain high Traditional Chinese and Western Medicine, Regenerative Medicine concentrations of flavonoids (6), and it has been reported to Research Center, West China Hospital, West China Medical School, exert anti-inflammatory effects (5), to promote pancreatic Sichuan University, 1 Keyuan 4 Road, Gaopeng Avenue, Chengdu, cell recovery (7,8) and to regulate lipid metabolism in hyper- Sichuan 610041, P.R. China lipidemic mice (9). However, the main active compounds of E-mail: huangwen@scu.edu.cn Coreopsis tinctoria, as well as their exact pharmacologic E-mail: qing.xia.sppc@gmail.com effects on hyperlipidemia, have yet to be elucidated. An increasing body of evidence has demonstrated that Contributed equally oxidative stress is a key trigger in the progression of hyperlip- Key words: eriodictyol 7-O-β-D glucopyranoside, snow idemia (1,10). Lipids are thought to be among the most sensitive chrysanthemum aqueous extracts, flavanomarein, hyperlipidemia, biological molecules in terms of reactive oxygen species (ROS) oxidative stress, mitochondrial function, lipogenesis susceptibility (11). In addition, lipid peroxidation is known to disturb the integrity of cellular membranes, leading to leakage of cytoplasmic enzymes, which in turn causes cell death LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER and cell death ultimately drives disease progression (11,12). subjected to polyamide resin (Chongqing Change Chemical A previous study has demonstrated that flavonoids have the Co., Ltd., Chongqing, China) column chromatography eluted capacity for anti-oxidative activities by reducing the produc- with water, 30% ethyl alcohol and 70% ethyl alcohol to give tion of ROS and preventing lipid peroxidation, which may be three fractions (A-C, respectively). Fraction B was chroma- associated with alleviated hyperlipidemia (13). tographed over a Sephadex LH-20 column (GE Healthcare The aim of the present study was to identify the main Bio-Sciences, Uppsala, Sweden) eluted with 50% methanol active compounds of Coreopsis tinctoria, to evaluate their to give six fractions 1 to 6. Compound 1 was obtained and antihyperlipidemic properties in vivo, and to investigate the further purified by ecrystallization with 100% methanol molecular mechanisms underlying their effects on lipid regu- from fraction 6. Compound 2 was obtained by Prep. HPLC lation in vitro. (Sh i mad a z u, Y MC‑Pack ODS‑A; 5 µm, 250x 2 0 m m; Shimadzu Corporation) from fraction 3. The mobile phase was Materials and methods acetonitrile (18%; solvent B): water (82%; solvent A), and the o fl w rate was 6 ml/min. Compounds 1 and 2 were identie fi d by 1 13 Materials. Commercially available analytical reagents were H NMR (600 MHz) and C NMR (150 MHz) run on AV II purchased from Shanghai Aladdin Bio-Chem Technology Co., spectrometer (Bruker Corporation, Ettlingen, Germany). Ltd. (Shanghai, China). Dulbecco's modie fi d Eagle's medium HPLC profiles of SCAE, compound 1 and 2, were (DMEM), fetal bovine serum (FBS), trypsin and peni- analyzed using a reverse column (LC-20A, Inertsil ODS‑SP; cillin-streptomycin-glutamine were obtained from Beyotime 4.6x150 nm; 3.5 µm; Shimadzu Corporation). Equal quantities Institute of Biotechnology (Haimen, China). Dimethylsulfoxide (20 µl) of SCAE, compounds 1 and 2, were used for analysis. (for MTT assay), 2,2-diphenyl-picrylhydrazyl (DPPH), They were eluted at a 1 ml/min fl ow rate with solvent A, water thiobarbituric acid (TBA), bovine serum albumin (BSA), with 0.1% formic acid, and solvent B (acetonitrile with 0.1% MTT, 2',7'-dichlorofluorescein diacetate (DCFH-DA) and formic acid) at 280 nm. The gradient started from 15% B for mouse monoclonal anti‑GAPDH antibody (1:10,000; cat the fi rst 5 min, then to 65% by 15 min, and finally to 100% by no. G8795) were purchased from Sigma‑Aldrich; Merck 20 min at 22˚C. KGaA (Darmstadt, Germany). Rabbit monoclonal antifatty acid synthase (FAS; 1:1,000; cat no. 3180S) and rabbit mono- Animals. The animal experiments were approved by the Ethics clonal anti‑protein disuld fi e‑isomerase A3 precursor (ERp57; Committee of the Institutional Animal Care and Treatment 1:1,000; cat no. 2881S) antibodies were purchased from Cell Committee of Sichuan University (permit no. 2014002B; Signaling Technology, Inc. (Danvers, MA, USA). Horseradish Chengdu, China). Male Kunming mice (weight, 18‑22 g; peroxide-conjugated goat antimouse immunoglobulin (Ig)G age, 4-6 weeks) were provided by the Chengdu Dashuo (1:5,000; cat no. sc‑2005) and goat anti‑rabbit IgG (1:5,000; cat Experimental Animal Co, Ltd. (Chengdu, China). The mice no. sc-2004) were purchased from Santa Cruz Biotechnology, were housed in controlled temperature (22±1˚C) and humidity Inc. (Dallas, TX, USA). (55±5%) conditions, under a 12/12 h light/dark cycle with free access to food and water. Preparation and analysis of snow chrysanthemum aqueous extract and its main compounds. Snow chrysanthemum, the Animal experiments. The mice were divided into the following capitulum of Coreopsis tinctoria, was collected in the Uighur 3 groups (n=10 mice/group): Groups I, II and III. Mice in Autonomous Region of Xinjiang Province in September group I were maintained on a normal pellet diet, whereas mice 2012, and was identified by Professor Yu-Hai Guo (China in groups II and III were maintained on a high-fat diet for the Agricultural University, Beijing, China). A voucher specimen induction of hyperlipidemia, which consisted of the following: (cat no. 201209) was preserved in the herbarium of the Normal diet supplemented with 10% cholesterol, 10% lard, 2% Laboratory of Ethnopharmacology of West China Hospital, sodium deoxycholic acid and 0.1% propylthiouracil. Following West China Medical School of Sichuan University (Sichuan, 21 days, group II were treated with 0.5% sodium carboxymethyl China). Air-dried snow chrysanthemum (100 g) was ground cellulose (vehicle). Group III received SCAE (60 mg/kg; into a powder and decocted with distilled water (0.8 l) by compounds 1 and 2). Treatments were given orally twice a day heating reu fl x extraction at 98˚C for 3 h. Subsequently, the for 42 days. At the end of the study, the mice were sacric fi ed, snow chrysanthemum aqueous extract (SCAE) was dried until and blood, liver and kidney tissue samples were collected for water content was <10%. The total flavonoid content in SCAE biochemical analysis. Serum was separated by centrifugation was assessed using a colorimetric method, as previously at 1,000 x g for 15 min at 4˚C, then assays of total cholesterol described (14). (TC), triglyceride (TG), low-density lipoprotein-cholesterol Its main compounds flavanomarein (compound 1) and (LDL-C), glutathione peroxidase (GSH-Px) and nitric oxide eriodictyol 7-O-β-D glucopyranoside (compound 2) were synthase (NOS) levels were performed. Liver samples were isolated and purified using preparative high-performance homogenized (10%, w/v) in cold saline, then centrifuged at liquid chromatography (HPLC; Shimadzu Corporation, Kyoto, 1,000 x g for 15 min at 4˚C. The supernatant was used for Japan). Preparative HPLC was carried out on a SHIMADZU assaying the superoxide dismutase (SOD) and malondialde- LC-6AD instrument with an SPD-20A detector, using a hyde (MDA) levels. Kidney samples were homogenized (10%, YMC‑Pack ODS‑A column (250x20 mm; 5 µm; YMC Co., w/v) in cold saline and centrifuged at 1,000 x g for 10 min at Ltd., Kyoto, Japan). The dried powders (5 kg) of Coreopsis tinc‑ 4˚C for the lipid peroxidation assay. Protein concentration was toria were extracted three times successively with water and determined using a bicinchoninic acid (BCA) protein assay 70% ethyl alcohol to obtain the crude extract. The extract was kit (Beyotime Institute of Biotechnology). The commercially MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 available kits used for these measurements included: TC assay 1 mmol/l palmitate (cat no. P9767) (both from Sigma‑Aldrich; kit (cat no. A111-1), TG assay kit (cat no. A110-1), LDL-C assay Merck KGaA) at a ratio of 2:1, and was diluted in the culture kit (cat no. A113‑1), GSH‑Px assay kit (Colorimetric method; medium to obtain the desired final concentration (1 mmol/l). cat no. A005), Total NOS assay kit (cat no. A014-2), Total (T-) In addition, the FFAs mixture contained BSA (10% w/v; SOD assay kit (Hydroxylamine method; cat no. A001‑1) and Sigma‑Aldrich; Merck KGaA), as previously described (18). MDA assay kit (TBA method; cat no. A003‑1; (all from Nanjing HepG2 cells, cultured to 75% conu fl ence, were treated with Jiancheng Bioengineering Institute, Nanjing, China) kits, either DMEM containing BSA (10% w/v; Sigma‑Aldrich; according to the manufacturers' protocol. High-density lipo- Merck KGaA) as a control, or HepG2 cells, cultured to 75% protein cholesterol (HDL-C) levels were calculated according conu fl ence, were treated with 1 mmol/l FFAs alone or together to the following formula: HDL-C = TC-[(1/5xTG) + LDL-C]. with compounds 1 or 2 (25 µmol/l). A total of 24 h following treatment at 37˚C, cells were stained using Oil Red O to assess Antioxidant assays. The putative free radical-scavenging prop- intracellular lipid droplet accumulation, as previously described erties of SCAE were investigated using DPPH, as previously by Cui et al (19). described (15,16). Various concentrations of compounds 1 To further investigate the effects of compounds 1 and 2 on and 2 (0, 10, 20, 40, 80 and 160 µmol/l), were added to intracellular lipid levels, HepG2 cells at 75% conu fl ence were 500 µmol/l alcoholic DPPH solution. A total of 500 µmol/l treated for 24 h as aforementioned. FFA-containing medium was alcoholic DPPH solution, without compounds 1 and 2, was removed and the cells were washed twice with PBS. The cells used as the control. Following incubation for 30 min in the from the various treatment groups were lysed in 1% Triton-X-100 dark at room temperature, the absorbance of each sample was (cat no. T8787; Sigma‑Aldrich; Merck KGaA) for 30 min on ice. measured at 517 nm. The cell lysates were determined using a BCA protein assay kit Lipid peroxidation was assessed using the TBA method, (Beyotime Institute of Biotechnology) and were diluted in 1% as previously described (17). Briey fl , mouse liver and kidney Triton‑X‑100 to obtain the final concentration of 5 mgprot/ml, samples were homogenized (10%, w/v) in cold saline and then prepared for TG level assessments using the Triglyceride centrifuged at 1,000 x g for 15 min at 4˚C. Then, the liver Quantic fi ation Colorimetric/Fluorometric kit (BioVision, Inc., and kidney tissue homogenates (100 µl, 10%) were mixed with Milpitas, CA, USA), according to the manufacturer's protocol. 100 µl compounds 1 or 2 (10, 20, 40, 80 and 160 µmol/l), and ferrous sulfate (8 µl, 70 mmol/l) was added to each mixture. Cell lipid peroxidation assay. To further evaluate the effects of The mixtures were incubated for 30 min at 37˚C. Subsequently, compounds 1 and 2 on intracellular lipid peroxidation, HepG2 300 µl 20% acetic acid and 300 µl 0.8% TBA in 1.1% sodium cells at 75% conu fl ence were treated with compounds 1 or 2 dodecyl sulfate was added, and the final mixtures were incu - (25 µmol/l), together with 1 mmol/l FFAs for 24 h. Cell lysates bated at 95˚C for 60 min. Following cooling, the mixtures were were obtained as aforementioned using 1% Triton-X-100 to centrifuged at 5,000 x g for 10 min at 4˚C and their absorbance assess lipid peroxidation via measuring MDA levels, using was measured at 532 nm (17). a commercially available MDA kit (cat no. A003‑4; Nanjing The IC value denotes the effective concentration of Jiancheng Bioengineering Institute), according to the manufac- compounds 1 or 2 used to reduce 50% of available DPPH turer's protocol. radicals or inhibit 50% of liver and kidney lipid peroxidation. The IC value of compounds 1 and 2 was calculated using Intracellular ROS production. HepG2 cells (1x10 cells/well) SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). were incubated in a 24‑well plate (Costar; Corning Incorporated) for 24 h at 37˚C. HepG2 cells at 75% conu fl ence were plated in Cell culture and viability assay. The human HepG2 hepatocel- 24-well plates and were treated with 1 mmol/l FFAs alone or lular carcinoma cell line was obtained from the Cell Bank of the together with 25 µmol/l compound 2 for 24 h. Subsequently, Shanghai Institute of Biochemistry and Cell Biology, Chinese cells were incubated with 10 µmol/l membrane-permeable Academy of Sciences (cat no. TCHu72; Shanghai, China). oxidation‑sensitive uo fl rescent dye DCFH‑DA (cat no. D6883; Cells were cultured at 37˚C in DMEM supplemented with Sigma‑Aldrich; Merck KGaA) for 20 min at 37˚C. Stained cells 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, were observed under an Eclipse Ti laser scanning confocal as previously described (18). HepG2 cells (5x10 cells/well) microscope (Nikon Corporation, Tokyo, Japan) and photo- were seeded in each well of 96‑well plates (Costar; Corning micrographs were captured. In addition, HepG2 cells were Incorporated, Corning, NY, USA) and cultured for 24 h at treated with 1 mmol/l FFAs alone or together with compound 37˚C. Cells were then incubated with compounds 1 or 2 (0, 2 (25 µmol/l) for 24 h in a black opaque 96-well microplate 1, 5, 25, 125 or 625 µmol/l) at 37˚C for 24 h. Cells without (Corning Incorporated). Subsequently, cells were incubated treatment with compounds 1 and 2 were used as the controls. with 10 µmol/l DCFH‑DA for 20 min at 37˚C. During this Cell viability was assessed using an MTT assay, as previously process, DCFH‑DA is cleaved and oxidized to green fluorescent described (18). 2'‑7‑'‑dichlorof luorescein via ROS mediation (DCF; excita - tion/emission, 488/530 nm), the level of which was measured Cell lipid accumulation assays. HepG2 cells (4x10 cells/well) using the Synergy™ Mx microplate reader (BioTek Instruments, were incubated in a 6‑well plate (Costar; Corning Incorporated) Inc., Winooski, VT, USA). for 24 h at 37˚C. HepG2 cells cultured to 75% confluence were exposed to 1 mmol/l free fatty acids (FFAs) for 24 h to Mitochondrial membrane potential (∆Ψm) analysis. HepG2 assess hepatic lipid accumulation and lipid peroxidation. The cells (1x10 cells/well) were incubated in a 24-well plate FFA mixture contained 1 mmol/l oleate (cat no. O7501) and (Costar; Corning Incor porated) for 24 h at 37˚C. HepG2 LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER Table I. Lipid-lowering effects of SCAE on high-fat diet-induced hyperlipidemic mice. Group TC (mmol/l) TG (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) I 2.01±0.51 0.73±0.37 0.46±0.11 1.5±0.13 a a a a II 3.98±0.78 1.66±0.61 0.99±0.13 0.41±0.18 b b c c III 2.96±0.61 1.11±0.42 0.71±0.12 0.82±0.18 a b c P<0.001 vs. group I; P<0.05, P<0.001 vs. group II. Data are presented as the mean ± standard deviation (n=10 mice/group). SCAE, snow chrysanthemum aqueous extract; TC, total cholesterol; TG, triglyceride; LDL‑C, low‑density lipoprotein cholesterol; HDL‑C, high‑density lipoprotein cholesterol; Group I, normal group; Group II, high‑fat diet group; Group III, SCAE group. cells at 75% confluence were treated with 1 mmol/l FFAs USA). The membrane was blocked with 5% non-fat milk for alone or together with 25 µmol/l compound 2 for 24 h. Cells 1 h at room temperature (~22˚C), and then incubated with were stained with 5 µg/ml JC-1 dye, as a ∆Ψm indicator, for anti-GAPDH, anti-ERp57 and anti-FAS primary antibodies at 15 min (20), and then observed under an Eclipse Ti laser 4˚C over night. Following washing three times with TBST (TBS scanning confocal microscope (Nikon Corporation). In addi- containing 0.1% Tween‑20; cat no. P0231; Beyotime Institute tion, HepG2 cells (4x10 cells/well) were plated in 6-well of Biotechnology), the membranes were incubated with horse- plates for 24 h at 37˚C, then treated with 1 mmol/l FFAs radish peroxidase-conjugated goat anti-mouse and anti-rabbit alone or together with compound 2 (25 µmol/l) for 24 h at IgG secondary antibodies at room temperature for 2 h. Protein 37˚C. Cells were harvested by trypsinization, stained with bands were visualized by enhanced chemiluminescence using 5 µg/ml JC‑1 dye (cat no. M34152; Thermo Fisher Scientic fi , SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Inc., Waltham, MA, USA) without cell fixation for 15 min at Fisher Scientic fi , Inc.). 37˚C, then washed twice with ice‑cold PBS and resuspended in 0.5 ml ice‑cold FBS‑free DMEM. The intensity of fluores - Statistical analysis. The statistical signic fi ance of the differ - cence was determined using a MoFlo Cytomation, Modular ences between groups was assessed using one-way analysis f low cytometer (Dako; Agilent Technologies, Inc., Santa of variance followed by a post hoc Scheffé's test for multiple Clara, CA, USA) and the data were analyzed with Summit comparisons. Data are expressed as the mean ± standard devia- software version 4.3 (Cytomation, Inc., Fort Collins, CO, tion of three repeated experiments. P<0.05 was considered to USA). indicate a statistically signica fi nt difference. Statistical analysis was performed using SPSS software version 19.0 (IBM Corp.). Intracellular adenosine triphosphate (ATP) levels. HepG2 cells (4x10 cells/well) were incubated in a 6-well plate for 24 h at Results 37˚C. HepG2 cells were then treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. Subsequently, Antihyperlipidemic effects of SCAE. The present results demon- cells were lysed using an ATP assay kit (cat no. A22026; strated that SCAE (60 mg/kg) signic fi antly decreased the serum Invitrogen; Thermo Fisher Scientific, Inc.) according to the levels of TC, TG and LDL-C by ~26, 33 and 28%, respectively, manufacturer's instructions, centrifuged at 12,000 x g for 5 min whereas it increased the serum levels of HDL-C by >2-fold, at 4˚C, and the supernatants were collected. Protein concentra- compared with the high‑fat diet group (P<0.05; Table I). In addi- tion was determined using a BCA protein assay kit (Beyotime tion, treatment with SCAE (60 mg/kg) resulted in a signic fi ant Institute of Biotechnology) and cells were transferred to a increase in hepatic SOD and serum GSH-Px concentrations black opaque 96-well microplate (Corning Incorporated). (P<0.05), as well as a significant decrease in hepatic MDA Cellular ATP levels were also assessed using the ATP assay kit levels (P<0.05) in hyperlipidemic mice maintained on a high-fat (Invitrogen; Thermo Fisher Scientic fi , Inc.) with the Synergy™ diet (Table II). Mx microplate reader (BioTek Instruments, Inc.), according to The main compounds of SCAE were isolated using HPLC the manufacturer's protocol (21). and were identified as compound 1 and compound 2 by comparing the NMR results with previous reports (22,23) ( Western blot analysis. HepG2 cells were treated with 1 mmol/l Figs. 1 and 2). The antioxidative properties of compounds 1 FFAs alone or together with compound 2 (25 µmol/l) for and 2 were assessed using free radical-scavenging DPPH and 24 h. Cells were lysed using radioimmunoprecipitation assay lipid peroxidation TBA assays. Compound 2 was revealed lysis buffer (Beyotime Institute of Biotechnology) containing to exert more potent antioxidative effects compared with 1 mmol/l phenylmethane sulfonylu fl oride for 20 min on ice. compound 1 (Table III). Subsequently, cell lysates were centrifuged at 12,000 x g for 10 min at 4˚C. The protein concentration was determined using Effects of compounds 1 and 2 on lipid accumulation in HepG2 a BCA protein assay kit (Beyotime Institute of Biotechnology). cells. Following treatment of HepG2 cells with compounds 1 Equal amounts (40 µg) of extracted protein samples were and 2, no detectable morphological changes and toxicity were separated by 15% SDS-PAGE and transferred onto a polyvi- observed (data not shown). Treatment with compounds 1 and 2 nylidene u fl oride membrane (EMD Millipore, Billerica, MA, (25 µmol/l) was demonstrated to significantly reduce lipid MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 Table II. Antioxidative effects of SCAE on high-fat diet-induced hyperlipidemic mice. Group Serum GSH-Px (U/ml) Serum NOS (U/ml) Liver SOD (U/mgprot) Liver MDA (nmol/mgprot) I 1,162.76±81.33 22.54±2.21 61.31±2.85 2.18±0.42 a b a a II 776.74±42.10 19.33±2.03 43.22±2.35 4.59±0.61 c c c III 991.22±22.53 21.05±1.43 55.9±2.89 1.94±0.37 a b c P<0.001, P<0.01 vs. group I; P<0.001 vs. group II. Data are presented as the mean ± standard deviation (n=10 mice/group). SCAE, snow chrysanthemum aqueous extract; GSH‑Px, glutathione peroxidase; NOS, nitric oxide synthase; SOD, superoxide dismutase; MDA, malondial- dehyde; Group I, normal group; Group II, high‑fat diet group; Group III, SCAE group. Figure 1. HPLC profiles of SCAE and its main compounds were analyzed using gradient HPLC and 20 µl of the samples. HPLC profiles of (A) SCAE, (B) compound 1 and (C) compound 2. HPLC, high‑performance liquid chromatography; SCAE, snow chrysanthemum aqueous extract; compound 1, a fl vano - marein; compound 2, eriodictyol 7‑O‑β‑D glucopyranoside; AU, absorbance unit; PDA, photodiode array. Figure 2. Chemical structures of the main compounds of snow chrysanthemum aqueous extract, (A) a fl vanomarein and (B) eriodictyol 7‑O‑β-D glucopyrano- side. accumulation in FFA-treated HepG2 cells (Fig. 3A and B). Effects of compound 2 on ROS production in HepG2 cells. In addition, compounds 1 and 2 signic fi antly suppressed the HepG2 cells exposed to FFAs exhibited increased intracel- FFA-induced elevation in hepatocellular TG levels to 81 and lular ROS production, as demonstrated by the increased 62%, respectively (P<0.001; Fig. 3C). ROS-mediated oxidation of the acetate moieties of DCFH-DA to green DCF. DCF u fl orescence intensity was revealed to be Effects of compounds 1 and 2 on lipid peroxidation in increased by 4-fold in HepG2 cells exposed to FFAs compared HepG2 cells. As presented in Fig. 3D, lipid peroxidation was with control cells. Notably, compound 2 was demonstrated to signic fi antly enhanced in HepG2 cells exposed to 1 mmol/l significantly suppress the FFA‑induced increase in hepatic ROS FFAs compared with control cells. However, treatment with generation (Fig. 4). compounds 1 and 2 was revealed to significantly inhibit hepatic lipid peroxidation (P<0.001). Notably, compound 2 Effects of compound 2 on ∆Ψm. As presented in Fig. 5A, appeared to exert more potent effects on hepatic lipid accu- HepG2 cells exposed to FFAs demonstrated decreased ∆Ψm, mulation and peroxidation compared with compound 1, thus whereas treatment with compound 2 was revealed to reverse suggesting that compound 2 may be characterized by higher the FFA-induced ∆Ψm decrease. Flow cytometric analysis also biological activity. demonstrated that HepG2 cells exposed to FFAs exhibited a LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER Figure 3. Compounds 1 and 2 inhibited lipid accumulation, and reduced TG synthesis and lipid peroxidation induced by treatment with FFAs in HepG2 cells. Human HepG2 hepatocellular carcinoma cells were treated with 1 mmol/l FFAs alone or together with compounds 1 and 2 (25 µmol/l) for 24 h. (A) Compounds 1 and 2 inhibited lipid accumulation in HepG2 cells, as demonstrated following staining with Oil Red O. Photomicrographs were captured under x400 magnifi - cation. (a) Control cells; (b) FFA‑treated cells; (c) FFA‑ and compound 1‑treated cells; (d) FFA‑ and compound 2‑treated cells. (B) Treatment with compounds 1 and 2 abolished the FFA‑induced increase in cell lipid content. (C) Treatment with compounds 1 and 2 signic fi antly reduced TG levels. (D) Lipid peroxidation, assessed using cellular MDA content, was signic fi antly inhibited following treatment with compounds 1 and 2. Data are presented as the mean ± standard ### *** deviation. P<0.001 vs. control cells; P<0.001 vs. FFA‑treated cells. Compound 1, flavanomarein; Compound 2, eriodictyol 7‑O‑ β‑D glucopyranoside; TG, triglyceride; FFA, free fatty acid; MDA, malondialdehyde. Figure 4. Compound 2 inhibited FFA-induced ROS production in HepG2 cells. Human HepG2 hepatocellular carcinoma cells were treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. (A) DCF green u fl orescence was visualized in (a) Control, (b) FFA‑treated and (c) FFA‑ and compound 2‑treated cells. Photomicrographs were captured under x400 magnic fi ation. (B) DCF fluorescence intensity was quantie fi d in control, FFA‑treated ## * and FFA- and compound 2-treated cells. Data are presented as the mean ± standard deviation. P<0.01 vs. control cells; P<0.05 vs. FFA-treated cells. Compound 2, eriodictyol 7-O-β‑D glucopyranoside; FFA, free fatty acid; ROS, reactive oxygen species; DCF, 2'‑7'‑dichlorou fl orescein. Table III. IC of the antioxidative capabilities of SCAE signic fi ant decrease (35%) in ∆Ψm, which was signic fi antly atten- compounds 1 and 2 in vitro, and in liver and kidney samples uated following treatment with compound 2 (Fig. 5B and C). isolated from mice. Effects of compound 2 on intracellular ATP levels. Following IC exposure of HepG2 cells to FFAs, intracellular ATP levels were --------------------------------------------------------------------------------------- signic fi antly decreased, whereas treatment with compound 2 Assay Compound 1 (µmol/l) Compound 2 (µmol/l) was revealed to counter act the FFA-induced decrease in ATP levels (Fig. 5D). These findings suggested that compound 2 DPPH 44.12±1.18 27.02±1.40 may ameliorate hepatic lipid accumulation due to its protective TBA (liver) 61.61±1.68 43.22±2.92 effects on mitochondrial function, exerted through the reduc- TBA (kidney) 140.97±9.11 59.97±3.30 tion in ROS production and the regulation of ∆Ψm and ATP production. Data are presented as the mean ± standard deviation of three samples and of three independent experiments. IC , half maximal inhibitory concen- Effects of compound 2 on the expression of lipogenesis‑ tration; SCAE, snow chrysanthemum aqueous extract; Compound 1, associated proteins. As presented in Fig. 6, following exposure flavanomarein; Compound 2, eriodictyol 7‑O‑ β‑D glucopyranoside; to FFAs for 24 h, the expression levels of the lipogenesis-asso- DPPH, 2, 2‑diphenyl‑picrylhydrazyl; TBA, thiobarbituric acid. ciated proteins ERp57 and FAS appeared to be upregulated. MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 Figure 5. Compound 2 prevented the ∆Ψm collapse and maintained cellular ATP levels in FFA-treated HepG2 cells. Human HepG2 hepatocellular carcinoma cells were treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. (A) JC‑1 staining was used to assess ∆Ψm. Red fluores - cence indicates high ∆Ψm, whereas green u fl orescence indicates low ∆Ψm. (a) Control cells; (b) FFA‑treated cells; (c) FFA‑ and compound 2‑treated cells. Photomicrographs were captured under x400 magnic fi ation. (B) Flow cytometric analysis of ∆Ψm, assessed using JC‑1 u fl orescence. (a) Control cells; (b) FFA‑treated cells; (c) FFA‑ and compound 2‑treated cells. (C) Relative ∆Ψm levels were quantie fi d in control, FFA‑ and FFA‑ and compound 2‑treated cells. (D) Intracellular ATP levels were assessed in control, FFA- and FFA- and compound 2-treated cells. Data are presented as the mean ± standard deviation. ### *** P<0.001 vs. control cells; P<0.001 vs. FFA-treated cells. Compound 2, eriodictyol 7-O-β‑D glucopyranoside; ∆Ψm, mitochondrial membrane potential; ATP, adenosine triphosphate; FFA, free fatty acid. LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER with FFAs leads to increased lipid accumulation, TG synthesis and lipid peroxidation, and has been used to evaluate the effects of putative lipid-lowering agents on lipid accumulation and lipid peroxidation in vitro (18,28). The present results suggested that compound 2 may be characterized by more potent lipid-lowering capabilities compared with compound 1. In addition, compound 2 appeared to exert stronger anti- oxidative effects, as demonstrated by its greater capabilities for scavenging free radicals and inhibiting lipid peroxidation compared with compound 1. These results suggested that compound 2 may be the main bioactive compound of SCAE Figure 6. Compound 2 downregulated the protein expression of ERp57 and FAS in HepG2 cells treated with FFAs. Human HepG2 hepatocellular responsible for its lipid-lowering effects, due to its potent anti- carcinoma cells were treated with 1 mmol/l FFAs alone or together with oxidative capabilities. compound 2 (25 µmol/l) for 24 h. ERp57 and FAS protein expression levels Mitochondria have been identie fi d as the center of cellular were evaluated using western blot analysis. The results are representative of lipid metabolism and one of the main sources of intracellular three independent experiments. Compound 2, eriodictyol 7-O-β-D glucopy- ranoside; ERp57, disuld fi e‑isomerase A3 precursor; FAS, fatty acid synthase; ROS generation (29,30). Excessive fatty acid metabolism FFA, free fatty acid. has been associated with increased ROS generation, as well as decreased activity of antioxidant enzymes, ultimately resulting in mitochondrial damage (3,10,25). Malfunctioning Compound 2 was demonstrated to markedly attenuate the mitochondria release higher quantities of ROS (30), thus FFA-induced upregulation in ERp57 and FAS expression. resulting in a vicious cycle of mitochondrial dysfunction, These results suggested that compound 2 may suppress hepatic decreased mitochondrial fatty acid β-oxidation and increased lipid accumulation through the suppression of lipogenesis, via TG synthesis. The present results demonstrated that compound downregulating the expression of proteins involved in lipogen- 2 counteracted the FFA-induced increase in intracellular esis, including ERp57 and FAS. ROS production. Furthermore, it was revealed to prevent the FFA-induced collapse of the ∆Ψm and the decrease in cellular Discussion ATP levels, thus suggesting that compound 2 may protect mitochondrial function. Hyperlipidemia has been identie fi d as an important risk factor The endoplasmic reticulum (ER) is known to serve a for the development of atherosclerosis (1) and acute necrotic central role in de novo lipogenesis. Oxidative and ER stress pancreatitis (24). Coreopsis tinctoria is a herbal medicine have been reported to occur simultaneously or successively, used to regulate lipid metabolism in traditional Chinese and ER stress has been associated with hepatic lipid accu- medicine (9). However, the exact pharmacological effects of mulation (31). The ER-associated protein ERp57 has been Coreopsis tinctoria, as well as the main active compounds and revealed to be upregulated during FFA-induced cellular the molecular mechanisms responsible for these effects, have steatosis, whereas its knockdown signic fi antly reduced lipid yet to be elucidated. In the present study, SCAE was demon- accumulation in steatotic cells (31). FAS has been identie fi d as strated to decrease serum lipid levels in a mouse model of a key enzyme during lipogenesis, as it catalyzes the terminal hyperlipidemia, possibly due to its antioxidative properties. Its steps in de novo fatty acid synthesis (32). The present results main active compounds, compounds 1 and 2, were revealed to demonstrated that compound 2 downregulated the protein decrease lipid accumulation in HepG2 cells, possibly through expression levels of ERp57 and FAS in FFA-treated HepG2 the reduction of oxidative stress, the protection of mitochon- cells. These results suggested that compound 2 may prevent drial function and the suppression of lipogenesis. de novo lipogenesis, via suppressing the expression of ERp57 Administration of a high-fat diet has been reported to and FAS in hepatocytes. increase fat and cholesterol intake, decrease the β-oxidation In conclusion, the present study suggested that compound of fatty acids and accelerate TG synthesis in rats, resulting 2 may be the main active compound of Coreopsis tinctoria, in increased TC and TG levels in the bloodstream (25). The responsible for its lipid-regulating effects. Furthermore, present results suggested that the flavonoid‑rich SCAE may compound 2 was demonstrated to enhance the endogenous attenuate lipid disorders and regulate TG levels. The liver antioxidative defense mechanisms of hepatocytes and to is primarily responsible for lipid synthesis, metabolism and protect mitochondria against oxidative damage. In addition, transportation (13,26), and hyperlipidemia has been reported its effects on ER stress reduction and the inhibition of de novo to increase hepatic lipid content, thus enhancing ROS genera- lipogenesis may be involved in the molecular mechanisms tion and lipid peroxidation (27). A previous study revealed that underlying the lipid-lowering effects of SCAE. The present a fl vonoids may attenuate hyperlipidemia, possibly due to their results suggested that compound 2 may have potential for the potent antioxidative effects (25). The present study suggested development of novel therapeutic strategies for the treatment that SCAE may enhance the endogenous antioxidative defense of patients with hyperlipidemia. mechanisms of hepatocytes, thus ameliorating hyperlipidemia, due to its high flavonoid content and potent antioxidative Acknowledgements properties. In the present study, compounds 1 and 2 were the main The present study was supported by the National Natural compounds isolated from SCAE. Treatment of HepG2 cells Science Foundation of China (grant no. 81673710). MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 17. Grespan R, Aguiar RP, Giubilei FN, Fuso RR, Damião MJ, References Silva EL, Mikcha JG, Hernandes L, Bersani Amado C and Cuman RK: Hepatoprotective effect of pretreatment with 1. Abliz A, Aji Q, Abdusalam E, Sun X, Abdurahman A, Zhou W, Thymus vulgar is Essential Oil in experimental model of acet- Moore N and Umar A: Effect of Cydonia oblonga Mill. leaf aminophen-induced injury. Evid Based Complement Alternat extract on serum lipids and liver function in a rat model of hyper- Med 2014: 954136, 2014. lipidaemia. J Ethnopharmacol 151: 970-974, 2014. 18. Seo MS, Hong SW, Yeon SH, Kim YM, Um KA, Kim JH, 2. Cignarella A, Bellosta S, Corsini A and Bolego C: Hypolipidemic Kim HJ, Chang KC and Park SW: Magnolia ofc fi inalis attenuates therapy for the metabolic syndrome. Pharmacol Res 53: 492-500, free fatty acid-induced lipogenesis via AMPK phosphorylation in hepatocytes. J Ethnopharmacol 157: 140-148, 2014. 3. Luedde T, Kaplowitz N and Schwabe RF: Cell death and cell 19. Cui W, Chen SL and Hu KQ: Quantic fi ation and mechanisms of death responses in liver disease: Mechanisms and clinical oleic acid-induced steatosis in HepG2 cells, Am J Transl Res 2: relevance. Gastroenterology 147: 765-783, 2014. 95-104, 2010. 4. Irudayaraj SS, Sunil C, Duraipandiyan V and Ignacimuthu S: 20. Sung DK, Chang YS, Kang S, Song HY, Park WS and Lee BH: In vitro antioxidant and antihyperlipidemic activities of Comparative evaluation of hypoxic‑ischemic brain injury by o fl w Toddaliaasiatica (L) Lam. Leaves in Triton WR-1339 and high cytometric analysis of mitochondrial membrane potential with fat diet induced hyperlipidemic rats. Food Chem Toxicol 60: JC-1 in neonatal rats. J Neurosci Methods 193: 232-238, 2010. 135-140, 2013. 21. Gyamfi D, Everitt HE, Tewfik I, Clemens DL and Patel VB: 5. Zhang Y, Shi S, Zhao M, Chai X and Tu P: Coreosides A-D, Hepatic mitochondrial dysfunction induced by fatty acids and C14-polyacetylene glycosides from the capitula of Coreopsis ethanol. Free Radic Biol Med 53: 2131-2145, 2012. tinctoria and its anti-inflammatory activity against COX-2. 22. Pan J, Zhang S, Yan L, Tai J, Xiao Q, Zou K, Zhou Y and Wu J: Fitoterapia 87: 93-97, 2013. Separation of flavanone enantiomers and flavanone glucoside 6. Zhang Y, Mourboul A and Li ZY: Research advance in medicinal diastereomers from Balanophora involucrata Hook. F. by plants from genus Coreopsis. 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Eur J Pharmacol 572: 74-81, noids on tBHP and cytokine-induced cell injury in pancreatic MIN6 cells. J Ethnopharmacol 139: 485-492, 2012. 25. Feng LJ, Yu CH, Ying KJ, Hua J and Dai XY: Hypolipidemic and 9. Li YL, Chen X, Xue J, Liu J, Chen X and Wulasihan M: Flavonoids antioxidant effects of total a fl vonoids of Perilla frutescens leaves furom Coreopsis tinctoria adjust lipid metabolism in hyperlipid- in hyperlipidemia rats induced by high-fat diet. Food Res Int 44: emia animals by down-regulating adipose differentiation-related 404-409, 2011. protein. Lipids Health Dis 13: 193, 2014. 26. Neuschwander-Tetri BA: Hepatic lipotoxicity and the patho- 10. Gong G, Qin Y, Huang W, Zhou S, Wu X, Yang X, Zhao Y genesis of nonalcoholic steatohepatitis: The central role of and Li D: Protective effects of diosgenin in the hyperlipidemic nontriglyceride fatty acid metabolites. Hepatology 52: 774-788, rat model and in human vascular endothelial cells against hydrogen peroxide-induced apoptosis. 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Gerhäuser C, Klimo K, Heiss E, Neumann I, Gamal-Eldeen A, Tsui WM and Yu ZL: ERp57 is up-regulated in free fatty Knauft J, Liu GY, Sitthimonchai S and Frank N: Mechanism-based acids-induced steatotic L-02 cells and human nonalcoholic fatty in vitro screening of potential cancer chemopreventive agents. livers. J Cell Biochem 110: 1447-1456, 2010. Mutat Res 523-524: 163-172, 2003. 32. Menendez JA and Lupu R: Fatty acid synthase and the lipo- 16. Hajiaghaalipour F, Kanthimathi MS, Sanusi J and genic phenotype in cancer pathogenesis. Nat Rev Cancer 7: Rajarajeswaran J: White tea (Camellia sinensis) inhibits prolif- 763-777, 2007. eration of the colon cancer cell line, HT-29, activates caspases and protects DNA of normal cells against oxidative damage. Food Chem 169: 401-410, 2015. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Medicine Reports Pubmed Central

Eriodictyol 7-O-β-D glucopyranoside from Coreopsis tinctoria Nutt. ameliorates lipid disorders via protecting mitochondrial function and suppressing lipogenesis

Molecular Medicine Reports , Volume 16 (2) – Jun 9, 2017

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Copyright: © Liang et al.
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1791-2997
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1791-3004
DOI
10.3892/mmr.2017.6743
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Abstract

MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 Eriodictyol 7‑O‑β‑D glucopyranoside from Coreopsis tinctoria Nutt. ameliorates lipid disorders via protecting mitochondrial function and suppressing lipogenesis 1* 1,2* 1* 1* 1 1 1 YUYAN LIANG , HAI NIU , LIMEI MA , DAN DU , LI WEN , QING XIA and WEN HUANG Laboratory of Ethnopharmacology, Department of Integrated Traditional Chinese and Western Medicine, Regenerative Medicine Research Center, West China Hospital, West China Medical School; College of Mathematics, Sichuan University, Chengdu, Sichuan 610041, P.R. China Received May 17, 2016; Accepted April 4, 2017 DOI: 10.3892/mmr.2017.6743 Abstract. Coreopsis tinctoria (snow chrysanthemum) has been protein expression levels of disuld fi e‑isomerase A3 precursor reported to exert antihyperlipidemic effects. The present study and fatty acid synthase, thus suppressing FFA-induced aimed to identify the active compounds of Coreopsis tinctoria lipogenesis in HepG2 cells. In conclusion, the present study and to investigate the molecular mechanisms underlying its identie fi d compound 2 as one of the main active compounds in effects on lipid dysregulation by measuring lipid levels, reac- Coreopsis tinctoria responsible for its lipid-lowering effects. tive oxygen species, lipid peroxidation and fatty acid synthesis. Compound 2 was revealed to possess antihyperlipidemic The present results demonstrated that snow chrysanthemum properties, exerted via reducing oxidative stress, protecting aqueous extracts signic fi antly reduced serum lipid levels and mitochondrial function and suppressing lipogenesis. oxidative stress in vivo. The main compounds that were isolated were identie fi d as flavanomarein (co mpound 1) and eriodictyol Introduction 7-O-β-D glucopyranoside (compound 2). Compounds 1 and 2 demonstrated potent antioxidative properties, including free Lipid dysregulation serves a critical role in the progres- radical scavenging activity, inhibition of lipid peroxidation, as sion of cardiovascular diseases (1), metabolic syndrome (2) well as lipid-lowering effects in human HepG2 hepatocellular and non-alcoholic fatty liver disease (3). These disorders carcinoma cells treated with free fatty acids (FFAs). Compound pose major public health concerns, and are associated with 2 was revealed to suppress the elevation of triglyceride levels family burden and a high socioeconomic cost (2). Currently and inhibit lipid peroxidation following FFA treatment. In addi- available lipid-lowering agents used in the treatment of hyper- tion, it was demonstrated to signic fi antly reduce intracellular lipidemia include statins and br fi ates; however, these agents levels of reactive oxygen species and improve the mitochon- have been associated with serious adverse effects, including drial membrane potential and adenosine triphosphate levels, gastrointestinal disturbances, severe muscle damage and thus protecting mitochondrial function in FFA-treated HepG2 hepatotoxicity (4). Therefore, natural products and herbal cells. Furthermore, compound 2 markedly suppressed the medicines with improved safety profiles have garnered atten - tion for the treatment of lipid disorders. The capitula of Coreopsis tinctoria, also known as snow chrysanthemum, have been used in the form of a tea-like beverage for the prevention of cardiovascular disorders, Correspondence to: Professor Wen Huang or Professor Qing Xia, diarrhea and diabetes in traditional Chinese medicine (5). Laboratory of Ethnopharmacology, Department of Integrated Coreopsis tinctoria has been revealed to contain high Traditional Chinese and Western Medicine, Regenerative Medicine concentrations of flavonoids (6), and it has been reported to Research Center, West China Hospital, West China Medical School, exert anti-inflammatory effects (5), to promote pancreatic Sichuan University, 1 Keyuan 4 Road, Gaopeng Avenue, Chengdu, cell recovery (7,8) and to regulate lipid metabolism in hyper- Sichuan 610041, P.R. China lipidemic mice (9). However, the main active compounds of E-mail: huangwen@scu.edu.cn Coreopsis tinctoria, as well as their exact pharmacologic E-mail: qing.xia.sppc@gmail.com effects on hyperlipidemia, have yet to be elucidated. An increasing body of evidence has demonstrated that Contributed equally oxidative stress is a key trigger in the progression of hyperlip- Key words: eriodictyol 7-O-β-D glucopyranoside, snow idemia (1,10). Lipids are thought to be among the most sensitive chrysanthemum aqueous extracts, flavanomarein, hyperlipidemia, biological molecules in terms of reactive oxygen species (ROS) oxidative stress, mitochondrial function, lipogenesis susceptibility (11). In addition, lipid peroxidation is known to disturb the integrity of cellular membranes, leading to leakage of cytoplasmic enzymes, which in turn causes cell death LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER and cell death ultimately drives disease progression (11,12). subjected to polyamide resin (Chongqing Change Chemical A previous study has demonstrated that flavonoids have the Co., Ltd., Chongqing, China) column chromatography eluted capacity for anti-oxidative activities by reducing the produc- with water, 30% ethyl alcohol and 70% ethyl alcohol to give tion of ROS and preventing lipid peroxidation, which may be three fractions (A-C, respectively). Fraction B was chroma- associated with alleviated hyperlipidemia (13). tographed over a Sephadex LH-20 column (GE Healthcare The aim of the present study was to identify the main Bio-Sciences, Uppsala, Sweden) eluted with 50% methanol active compounds of Coreopsis tinctoria, to evaluate their to give six fractions 1 to 6. Compound 1 was obtained and antihyperlipidemic properties in vivo, and to investigate the further purified by ecrystallization with 100% methanol molecular mechanisms underlying their effects on lipid regu- from fraction 6. Compound 2 was obtained by Prep. HPLC lation in vitro. (Sh i mad a z u, Y MC‑Pack ODS‑A; 5 µm, 250x 2 0 m m; Shimadzu Corporation) from fraction 3. The mobile phase was Materials and methods acetonitrile (18%; solvent B): water (82%; solvent A), and the o fl w rate was 6 ml/min. Compounds 1 and 2 were identie fi d by 1 13 Materials. Commercially available analytical reagents were H NMR (600 MHz) and C NMR (150 MHz) run on AV II purchased from Shanghai Aladdin Bio-Chem Technology Co., spectrometer (Bruker Corporation, Ettlingen, Germany). Ltd. (Shanghai, China). Dulbecco's modie fi d Eagle's medium HPLC profiles of SCAE, compound 1 and 2, were (DMEM), fetal bovine serum (FBS), trypsin and peni- analyzed using a reverse column (LC-20A, Inertsil ODS‑SP; cillin-streptomycin-glutamine were obtained from Beyotime 4.6x150 nm; 3.5 µm; Shimadzu Corporation). Equal quantities Institute of Biotechnology (Haimen, China). Dimethylsulfoxide (20 µl) of SCAE, compounds 1 and 2, were used for analysis. (for MTT assay), 2,2-diphenyl-picrylhydrazyl (DPPH), They were eluted at a 1 ml/min fl ow rate with solvent A, water thiobarbituric acid (TBA), bovine serum albumin (BSA), with 0.1% formic acid, and solvent B (acetonitrile with 0.1% MTT, 2',7'-dichlorofluorescein diacetate (DCFH-DA) and formic acid) at 280 nm. The gradient started from 15% B for mouse monoclonal anti‑GAPDH antibody (1:10,000; cat the fi rst 5 min, then to 65% by 15 min, and finally to 100% by no. G8795) were purchased from Sigma‑Aldrich; Merck 20 min at 22˚C. KGaA (Darmstadt, Germany). Rabbit monoclonal antifatty acid synthase (FAS; 1:1,000; cat no. 3180S) and rabbit mono- Animals. The animal experiments were approved by the Ethics clonal anti‑protein disuld fi e‑isomerase A3 precursor (ERp57; Committee of the Institutional Animal Care and Treatment 1:1,000; cat no. 2881S) antibodies were purchased from Cell Committee of Sichuan University (permit no. 2014002B; Signaling Technology, Inc. (Danvers, MA, USA). Horseradish Chengdu, China). Male Kunming mice (weight, 18‑22 g; peroxide-conjugated goat antimouse immunoglobulin (Ig)G age, 4-6 weeks) were provided by the Chengdu Dashuo (1:5,000; cat no. sc‑2005) and goat anti‑rabbit IgG (1:5,000; cat Experimental Animal Co, Ltd. (Chengdu, China). The mice no. sc-2004) were purchased from Santa Cruz Biotechnology, were housed in controlled temperature (22±1˚C) and humidity Inc. (Dallas, TX, USA). (55±5%) conditions, under a 12/12 h light/dark cycle with free access to food and water. Preparation and analysis of snow chrysanthemum aqueous extract and its main compounds. Snow chrysanthemum, the Animal experiments. The mice were divided into the following capitulum of Coreopsis tinctoria, was collected in the Uighur 3 groups (n=10 mice/group): Groups I, II and III. Mice in Autonomous Region of Xinjiang Province in September group I were maintained on a normal pellet diet, whereas mice 2012, and was identified by Professor Yu-Hai Guo (China in groups II and III were maintained on a high-fat diet for the Agricultural University, Beijing, China). A voucher specimen induction of hyperlipidemia, which consisted of the following: (cat no. 201209) was preserved in the herbarium of the Normal diet supplemented with 10% cholesterol, 10% lard, 2% Laboratory of Ethnopharmacology of West China Hospital, sodium deoxycholic acid and 0.1% propylthiouracil. Following West China Medical School of Sichuan University (Sichuan, 21 days, group II were treated with 0.5% sodium carboxymethyl China). Air-dried snow chrysanthemum (100 g) was ground cellulose (vehicle). Group III received SCAE (60 mg/kg; into a powder and decocted with distilled water (0.8 l) by compounds 1 and 2). Treatments were given orally twice a day heating reu fl x extraction at 98˚C for 3 h. Subsequently, the for 42 days. At the end of the study, the mice were sacric fi ed, snow chrysanthemum aqueous extract (SCAE) was dried until and blood, liver and kidney tissue samples were collected for water content was <10%. The total flavonoid content in SCAE biochemical analysis. Serum was separated by centrifugation was assessed using a colorimetric method, as previously at 1,000 x g for 15 min at 4˚C, then assays of total cholesterol described (14). (TC), triglyceride (TG), low-density lipoprotein-cholesterol Its main compounds flavanomarein (compound 1) and (LDL-C), glutathione peroxidase (GSH-Px) and nitric oxide eriodictyol 7-O-β-D glucopyranoside (compound 2) were synthase (NOS) levels were performed. Liver samples were isolated and purified using preparative high-performance homogenized (10%, w/v) in cold saline, then centrifuged at liquid chromatography (HPLC; Shimadzu Corporation, Kyoto, 1,000 x g for 15 min at 4˚C. The supernatant was used for Japan). Preparative HPLC was carried out on a SHIMADZU assaying the superoxide dismutase (SOD) and malondialde- LC-6AD instrument with an SPD-20A detector, using a hyde (MDA) levels. Kidney samples were homogenized (10%, YMC‑Pack ODS‑A column (250x20 mm; 5 µm; YMC Co., w/v) in cold saline and centrifuged at 1,000 x g for 10 min at Ltd., Kyoto, Japan). The dried powders (5 kg) of Coreopsis tinc‑ 4˚C for the lipid peroxidation assay. Protein concentration was toria were extracted three times successively with water and determined using a bicinchoninic acid (BCA) protein assay 70% ethyl alcohol to obtain the crude extract. The extract was kit (Beyotime Institute of Biotechnology). The commercially MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 available kits used for these measurements included: TC assay 1 mmol/l palmitate (cat no. P9767) (both from Sigma‑Aldrich; kit (cat no. A111-1), TG assay kit (cat no. A110-1), LDL-C assay Merck KGaA) at a ratio of 2:1, and was diluted in the culture kit (cat no. A113‑1), GSH‑Px assay kit (Colorimetric method; medium to obtain the desired final concentration (1 mmol/l). cat no. A005), Total NOS assay kit (cat no. A014-2), Total (T-) In addition, the FFAs mixture contained BSA (10% w/v; SOD assay kit (Hydroxylamine method; cat no. A001‑1) and Sigma‑Aldrich; Merck KGaA), as previously described (18). MDA assay kit (TBA method; cat no. A003‑1; (all from Nanjing HepG2 cells, cultured to 75% conu fl ence, were treated with Jiancheng Bioengineering Institute, Nanjing, China) kits, either DMEM containing BSA (10% w/v; Sigma‑Aldrich; according to the manufacturers' protocol. High-density lipo- Merck KGaA) as a control, or HepG2 cells, cultured to 75% protein cholesterol (HDL-C) levels were calculated according conu fl ence, were treated with 1 mmol/l FFAs alone or together to the following formula: HDL-C = TC-[(1/5xTG) + LDL-C]. with compounds 1 or 2 (25 µmol/l). A total of 24 h following treatment at 37˚C, cells were stained using Oil Red O to assess Antioxidant assays. The putative free radical-scavenging prop- intracellular lipid droplet accumulation, as previously described erties of SCAE were investigated using DPPH, as previously by Cui et al (19). described (15,16). Various concentrations of compounds 1 To further investigate the effects of compounds 1 and 2 on and 2 (0, 10, 20, 40, 80 and 160 µmol/l), were added to intracellular lipid levels, HepG2 cells at 75% conu fl ence were 500 µmol/l alcoholic DPPH solution. A total of 500 µmol/l treated for 24 h as aforementioned. FFA-containing medium was alcoholic DPPH solution, without compounds 1 and 2, was removed and the cells were washed twice with PBS. The cells used as the control. Following incubation for 30 min in the from the various treatment groups were lysed in 1% Triton-X-100 dark at room temperature, the absorbance of each sample was (cat no. T8787; Sigma‑Aldrich; Merck KGaA) for 30 min on ice. measured at 517 nm. The cell lysates were determined using a BCA protein assay kit Lipid peroxidation was assessed using the TBA method, (Beyotime Institute of Biotechnology) and were diluted in 1% as previously described (17). Briey fl , mouse liver and kidney Triton‑X‑100 to obtain the final concentration of 5 mgprot/ml, samples were homogenized (10%, w/v) in cold saline and then prepared for TG level assessments using the Triglyceride centrifuged at 1,000 x g for 15 min at 4˚C. Then, the liver Quantic fi ation Colorimetric/Fluorometric kit (BioVision, Inc., and kidney tissue homogenates (100 µl, 10%) were mixed with Milpitas, CA, USA), according to the manufacturer's protocol. 100 µl compounds 1 or 2 (10, 20, 40, 80 and 160 µmol/l), and ferrous sulfate (8 µl, 70 mmol/l) was added to each mixture. Cell lipid peroxidation assay. To further evaluate the effects of The mixtures were incubated for 30 min at 37˚C. Subsequently, compounds 1 and 2 on intracellular lipid peroxidation, HepG2 300 µl 20% acetic acid and 300 µl 0.8% TBA in 1.1% sodium cells at 75% conu fl ence were treated with compounds 1 or 2 dodecyl sulfate was added, and the final mixtures were incu - (25 µmol/l), together with 1 mmol/l FFAs for 24 h. Cell lysates bated at 95˚C for 60 min. Following cooling, the mixtures were were obtained as aforementioned using 1% Triton-X-100 to centrifuged at 5,000 x g for 10 min at 4˚C and their absorbance assess lipid peroxidation via measuring MDA levels, using was measured at 532 nm (17). a commercially available MDA kit (cat no. A003‑4; Nanjing The IC value denotes the effective concentration of Jiancheng Bioengineering Institute), according to the manufac- compounds 1 or 2 used to reduce 50% of available DPPH turer's protocol. radicals or inhibit 50% of liver and kidney lipid peroxidation. The IC value of compounds 1 and 2 was calculated using Intracellular ROS production. HepG2 cells (1x10 cells/well) SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). were incubated in a 24‑well plate (Costar; Corning Incorporated) for 24 h at 37˚C. HepG2 cells at 75% conu fl ence were plated in Cell culture and viability assay. The human HepG2 hepatocel- 24-well plates and were treated with 1 mmol/l FFAs alone or lular carcinoma cell line was obtained from the Cell Bank of the together with 25 µmol/l compound 2 for 24 h. Subsequently, Shanghai Institute of Biochemistry and Cell Biology, Chinese cells were incubated with 10 µmol/l membrane-permeable Academy of Sciences (cat no. TCHu72; Shanghai, China). oxidation‑sensitive uo fl rescent dye DCFH‑DA (cat no. D6883; Cells were cultured at 37˚C in DMEM supplemented with Sigma‑Aldrich; Merck KGaA) for 20 min at 37˚C. Stained cells 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, were observed under an Eclipse Ti laser scanning confocal as previously described (18). HepG2 cells (5x10 cells/well) microscope (Nikon Corporation, Tokyo, Japan) and photo- were seeded in each well of 96‑well plates (Costar; Corning micrographs were captured. In addition, HepG2 cells were Incorporated, Corning, NY, USA) and cultured for 24 h at treated with 1 mmol/l FFAs alone or together with compound 37˚C. Cells were then incubated with compounds 1 or 2 (0, 2 (25 µmol/l) for 24 h in a black opaque 96-well microplate 1, 5, 25, 125 or 625 µmol/l) at 37˚C for 24 h. Cells without (Corning Incorporated). Subsequently, cells were incubated treatment with compounds 1 and 2 were used as the controls. with 10 µmol/l DCFH‑DA for 20 min at 37˚C. During this Cell viability was assessed using an MTT assay, as previously process, DCFH‑DA is cleaved and oxidized to green fluorescent described (18). 2'‑7‑'‑dichlorof luorescein via ROS mediation (DCF; excita - tion/emission, 488/530 nm), the level of which was measured Cell lipid accumulation assays. HepG2 cells (4x10 cells/well) using the Synergy™ Mx microplate reader (BioTek Instruments, were incubated in a 6‑well plate (Costar; Corning Incorporated) Inc., Winooski, VT, USA). for 24 h at 37˚C. HepG2 cells cultured to 75% confluence were exposed to 1 mmol/l free fatty acids (FFAs) for 24 h to Mitochondrial membrane potential (∆Ψm) analysis. HepG2 assess hepatic lipid accumulation and lipid peroxidation. The cells (1x10 cells/well) were incubated in a 24-well plate FFA mixture contained 1 mmol/l oleate (cat no. O7501) and (Costar; Corning Incor porated) for 24 h at 37˚C. HepG2 LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER Table I. Lipid-lowering effects of SCAE on high-fat diet-induced hyperlipidemic mice. Group TC (mmol/l) TG (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) I 2.01±0.51 0.73±0.37 0.46±0.11 1.5±0.13 a a a a II 3.98±0.78 1.66±0.61 0.99±0.13 0.41±0.18 b b c c III 2.96±0.61 1.11±0.42 0.71±0.12 0.82±0.18 a b c P<0.001 vs. group I; P<0.05, P<0.001 vs. group II. Data are presented as the mean ± standard deviation (n=10 mice/group). SCAE, snow chrysanthemum aqueous extract; TC, total cholesterol; TG, triglyceride; LDL‑C, low‑density lipoprotein cholesterol; HDL‑C, high‑density lipoprotein cholesterol; Group I, normal group; Group II, high‑fat diet group; Group III, SCAE group. cells at 75% confluence were treated with 1 mmol/l FFAs USA). The membrane was blocked with 5% non-fat milk for alone or together with 25 µmol/l compound 2 for 24 h. Cells 1 h at room temperature (~22˚C), and then incubated with were stained with 5 µg/ml JC-1 dye, as a ∆Ψm indicator, for anti-GAPDH, anti-ERp57 and anti-FAS primary antibodies at 15 min (20), and then observed under an Eclipse Ti laser 4˚C over night. Following washing three times with TBST (TBS scanning confocal microscope (Nikon Corporation). In addi- containing 0.1% Tween‑20; cat no. P0231; Beyotime Institute tion, HepG2 cells (4x10 cells/well) were plated in 6-well of Biotechnology), the membranes were incubated with horse- plates for 24 h at 37˚C, then treated with 1 mmol/l FFAs radish peroxidase-conjugated goat anti-mouse and anti-rabbit alone or together with compound 2 (25 µmol/l) for 24 h at IgG secondary antibodies at room temperature for 2 h. Protein 37˚C. Cells were harvested by trypsinization, stained with bands were visualized by enhanced chemiluminescence using 5 µg/ml JC‑1 dye (cat no. M34152; Thermo Fisher Scientic fi , SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Inc., Waltham, MA, USA) without cell fixation for 15 min at Fisher Scientic fi , Inc.). 37˚C, then washed twice with ice‑cold PBS and resuspended in 0.5 ml ice‑cold FBS‑free DMEM. The intensity of fluores - Statistical analysis. The statistical signic fi ance of the differ - cence was determined using a MoFlo Cytomation, Modular ences between groups was assessed using one-way analysis f low cytometer (Dako; Agilent Technologies, Inc., Santa of variance followed by a post hoc Scheffé's test for multiple Clara, CA, USA) and the data were analyzed with Summit comparisons. Data are expressed as the mean ± standard devia- software version 4.3 (Cytomation, Inc., Fort Collins, CO, tion of three repeated experiments. P<0.05 was considered to USA). indicate a statistically signica fi nt difference. Statistical analysis was performed using SPSS software version 19.0 (IBM Corp.). Intracellular adenosine triphosphate (ATP) levels. HepG2 cells (4x10 cells/well) were incubated in a 6-well plate for 24 h at Results 37˚C. HepG2 cells were then treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. Subsequently, Antihyperlipidemic effects of SCAE. The present results demon- cells were lysed using an ATP assay kit (cat no. A22026; strated that SCAE (60 mg/kg) signic fi antly decreased the serum Invitrogen; Thermo Fisher Scientific, Inc.) according to the levels of TC, TG and LDL-C by ~26, 33 and 28%, respectively, manufacturer's instructions, centrifuged at 12,000 x g for 5 min whereas it increased the serum levels of HDL-C by >2-fold, at 4˚C, and the supernatants were collected. Protein concentra- compared with the high‑fat diet group (P<0.05; Table I). In addi- tion was determined using a BCA protein assay kit (Beyotime tion, treatment with SCAE (60 mg/kg) resulted in a signic fi ant Institute of Biotechnology) and cells were transferred to a increase in hepatic SOD and serum GSH-Px concentrations black opaque 96-well microplate (Corning Incorporated). (P<0.05), as well as a significant decrease in hepatic MDA Cellular ATP levels were also assessed using the ATP assay kit levels (P<0.05) in hyperlipidemic mice maintained on a high-fat (Invitrogen; Thermo Fisher Scientic fi , Inc.) with the Synergy™ diet (Table II). Mx microplate reader (BioTek Instruments, Inc.), according to The main compounds of SCAE were isolated using HPLC the manufacturer's protocol (21). and were identified as compound 1 and compound 2 by comparing the NMR results with previous reports (22,23) ( Western blot analysis. HepG2 cells were treated with 1 mmol/l Figs. 1 and 2). The antioxidative properties of compounds 1 FFAs alone or together with compound 2 (25 µmol/l) for and 2 were assessed using free radical-scavenging DPPH and 24 h. Cells were lysed using radioimmunoprecipitation assay lipid peroxidation TBA assays. Compound 2 was revealed lysis buffer (Beyotime Institute of Biotechnology) containing to exert more potent antioxidative effects compared with 1 mmol/l phenylmethane sulfonylu fl oride for 20 min on ice. compound 1 (Table III). Subsequently, cell lysates were centrifuged at 12,000 x g for 10 min at 4˚C. The protein concentration was determined using Effects of compounds 1 and 2 on lipid accumulation in HepG2 a BCA protein assay kit (Beyotime Institute of Biotechnology). cells. Following treatment of HepG2 cells with compounds 1 Equal amounts (40 µg) of extracted protein samples were and 2, no detectable morphological changes and toxicity were separated by 15% SDS-PAGE and transferred onto a polyvi- observed (data not shown). Treatment with compounds 1 and 2 nylidene u fl oride membrane (EMD Millipore, Billerica, MA, (25 µmol/l) was demonstrated to significantly reduce lipid MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 Table II. Antioxidative effects of SCAE on high-fat diet-induced hyperlipidemic mice. Group Serum GSH-Px (U/ml) Serum NOS (U/ml) Liver SOD (U/mgprot) Liver MDA (nmol/mgprot) I 1,162.76±81.33 22.54±2.21 61.31±2.85 2.18±0.42 a b a a II 776.74±42.10 19.33±2.03 43.22±2.35 4.59±0.61 c c c III 991.22±22.53 21.05±1.43 55.9±2.89 1.94±0.37 a b c P<0.001, P<0.01 vs. group I; P<0.001 vs. group II. Data are presented as the mean ± standard deviation (n=10 mice/group). SCAE, snow chrysanthemum aqueous extract; GSH‑Px, glutathione peroxidase; NOS, nitric oxide synthase; SOD, superoxide dismutase; MDA, malondial- dehyde; Group I, normal group; Group II, high‑fat diet group; Group III, SCAE group. Figure 1. HPLC profiles of SCAE and its main compounds were analyzed using gradient HPLC and 20 µl of the samples. HPLC profiles of (A) SCAE, (B) compound 1 and (C) compound 2. HPLC, high‑performance liquid chromatography; SCAE, snow chrysanthemum aqueous extract; compound 1, a fl vano - marein; compound 2, eriodictyol 7‑O‑β‑D glucopyranoside; AU, absorbance unit; PDA, photodiode array. Figure 2. Chemical structures of the main compounds of snow chrysanthemum aqueous extract, (A) a fl vanomarein and (B) eriodictyol 7‑O‑β-D glucopyrano- side. accumulation in FFA-treated HepG2 cells (Fig. 3A and B). Effects of compound 2 on ROS production in HepG2 cells. In addition, compounds 1 and 2 signic fi antly suppressed the HepG2 cells exposed to FFAs exhibited increased intracel- FFA-induced elevation in hepatocellular TG levels to 81 and lular ROS production, as demonstrated by the increased 62%, respectively (P<0.001; Fig. 3C). ROS-mediated oxidation of the acetate moieties of DCFH-DA to green DCF. DCF u fl orescence intensity was revealed to be Effects of compounds 1 and 2 on lipid peroxidation in increased by 4-fold in HepG2 cells exposed to FFAs compared HepG2 cells. As presented in Fig. 3D, lipid peroxidation was with control cells. Notably, compound 2 was demonstrated to signic fi antly enhanced in HepG2 cells exposed to 1 mmol/l significantly suppress the FFA‑induced increase in hepatic ROS FFAs compared with control cells. However, treatment with generation (Fig. 4). compounds 1 and 2 was revealed to significantly inhibit hepatic lipid peroxidation (P<0.001). Notably, compound 2 Effects of compound 2 on ∆Ψm. As presented in Fig. 5A, appeared to exert more potent effects on hepatic lipid accu- HepG2 cells exposed to FFAs demonstrated decreased ∆Ψm, mulation and peroxidation compared with compound 1, thus whereas treatment with compound 2 was revealed to reverse suggesting that compound 2 may be characterized by higher the FFA-induced ∆Ψm decrease. Flow cytometric analysis also biological activity. demonstrated that HepG2 cells exposed to FFAs exhibited a LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER Figure 3. Compounds 1 and 2 inhibited lipid accumulation, and reduced TG synthesis and lipid peroxidation induced by treatment with FFAs in HepG2 cells. Human HepG2 hepatocellular carcinoma cells were treated with 1 mmol/l FFAs alone or together with compounds 1 and 2 (25 µmol/l) for 24 h. (A) Compounds 1 and 2 inhibited lipid accumulation in HepG2 cells, as demonstrated following staining with Oil Red O. Photomicrographs were captured under x400 magnifi - cation. (a) Control cells; (b) FFA‑treated cells; (c) FFA‑ and compound 1‑treated cells; (d) FFA‑ and compound 2‑treated cells. (B) Treatment with compounds 1 and 2 abolished the FFA‑induced increase in cell lipid content. (C) Treatment with compounds 1 and 2 signic fi antly reduced TG levels. (D) Lipid peroxidation, assessed using cellular MDA content, was signic fi antly inhibited following treatment with compounds 1 and 2. Data are presented as the mean ± standard ### *** deviation. P<0.001 vs. control cells; P<0.001 vs. FFA‑treated cells. Compound 1, flavanomarein; Compound 2, eriodictyol 7‑O‑ β‑D glucopyranoside; TG, triglyceride; FFA, free fatty acid; MDA, malondialdehyde. Figure 4. Compound 2 inhibited FFA-induced ROS production in HepG2 cells. Human HepG2 hepatocellular carcinoma cells were treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. (A) DCF green u fl orescence was visualized in (a) Control, (b) FFA‑treated and (c) FFA‑ and compound 2‑treated cells. Photomicrographs were captured under x400 magnic fi ation. (B) DCF fluorescence intensity was quantie fi d in control, FFA‑treated ## * and FFA- and compound 2-treated cells. Data are presented as the mean ± standard deviation. P<0.01 vs. control cells; P<0.05 vs. FFA-treated cells. Compound 2, eriodictyol 7-O-β‑D glucopyranoside; FFA, free fatty acid; ROS, reactive oxygen species; DCF, 2'‑7'‑dichlorou fl orescein. Table III. IC of the antioxidative capabilities of SCAE signic fi ant decrease (35%) in ∆Ψm, which was signic fi antly atten- compounds 1 and 2 in vitro, and in liver and kidney samples uated following treatment with compound 2 (Fig. 5B and C). isolated from mice. Effects of compound 2 on intracellular ATP levels. Following IC exposure of HepG2 cells to FFAs, intracellular ATP levels were --------------------------------------------------------------------------------------- signic fi antly decreased, whereas treatment with compound 2 Assay Compound 1 (µmol/l) Compound 2 (µmol/l) was revealed to counter act the FFA-induced decrease in ATP levels (Fig. 5D). These findings suggested that compound 2 DPPH 44.12±1.18 27.02±1.40 may ameliorate hepatic lipid accumulation due to its protective TBA (liver) 61.61±1.68 43.22±2.92 effects on mitochondrial function, exerted through the reduc- TBA (kidney) 140.97±9.11 59.97±3.30 tion in ROS production and the regulation of ∆Ψm and ATP production. Data are presented as the mean ± standard deviation of three samples and of three independent experiments. IC , half maximal inhibitory concen- Effects of compound 2 on the expression of lipogenesis‑ tration; SCAE, snow chrysanthemum aqueous extract; Compound 1, associated proteins. As presented in Fig. 6, following exposure flavanomarein; Compound 2, eriodictyol 7‑O‑ β‑D glucopyranoside; to FFAs for 24 h, the expression levels of the lipogenesis-asso- DPPH, 2, 2‑diphenyl‑picrylhydrazyl; TBA, thiobarbituric acid. ciated proteins ERp57 and FAS appeared to be upregulated. MOLECULAR MEDICINE REPORTS 16: 1298-1306, 2017 Figure 5. Compound 2 prevented the ∆Ψm collapse and maintained cellular ATP levels in FFA-treated HepG2 cells. Human HepG2 hepatocellular carcinoma cells were treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. (A) JC‑1 staining was used to assess ∆Ψm. Red fluores - cence indicates high ∆Ψm, whereas green u fl orescence indicates low ∆Ψm. (a) Control cells; (b) FFA‑treated cells; (c) FFA‑ and compound 2‑treated cells. Photomicrographs were captured under x400 magnic fi ation. (B) Flow cytometric analysis of ∆Ψm, assessed using JC‑1 u fl orescence. (a) Control cells; (b) FFA‑treated cells; (c) FFA‑ and compound 2‑treated cells. (C) Relative ∆Ψm levels were quantie fi d in control, FFA‑ and FFA‑ and compound 2‑treated cells. (D) Intracellular ATP levels were assessed in control, FFA- and FFA- and compound 2-treated cells. Data are presented as the mean ± standard deviation. ### *** P<0.001 vs. control cells; P<0.001 vs. FFA-treated cells. Compound 2, eriodictyol 7-O-β‑D glucopyranoside; ∆Ψm, mitochondrial membrane potential; ATP, adenosine triphosphate; FFA, free fatty acid. LIANG et al: ERIODICTYOL 7-O-β-D GLUCOPYRANOSIDE AMELIORATES LIPID DISORDER with FFAs leads to increased lipid accumulation, TG synthesis and lipid peroxidation, and has been used to evaluate the effects of putative lipid-lowering agents on lipid accumulation and lipid peroxidation in vitro (18,28). The present results suggested that compound 2 may be characterized by more potent lipid-lowering capabilities compared with compound 1. In addition, compound 2 appeared to exert stronger anti- oxidative effects, as demonstrated by its greater capabilities for scavenging free radicals and inhibiting lipid peroxidation compared with compound 1. These results suggested that compound 2 may be the main bioactive compound of SCAE Figure 6. Compound 2 downregulated the protein expression of ERp57 and FAS in HepG2 cells treated with FFAs. Human HepG2 hepatocellular responsible for its lipid-lowering effects, due to its potent anti- carcinoma cells were treated with 1 mmol/l FFAs alone or together with oxidative capabilities. compound 2 (25 µmol/l) for 24 h. ERp57 and FAS protein expression levels Mitochondria have been identie fi d as the center of cellular were evaluated using western blot analysis. The results are representative of lipid metabolism and one of the main sources of intracellular three independent experiments. Compound 2, eriodictyol 7-O-β-D glucopy- ranoside; ERp57, disuld fi e‑isomerase A3 precursor; FAS, fatty acid synthase; ROS generation (29,30). Excessive fatty acid metabolism FFA, free fatty acid. has been associated with increased ROS generation, as well as decreased activity of antioxidant enzymes, ultimately resulting in mitochondrial damage (3,10,25). Malfunctioning Compound 2 was demonstrated to markedly attenuate the mitochondria release higher quantities of ROS (30), thus FFA-induced upregulation in ERp57 and FAS expression. resulting in a vicious cycle of mitochondrial dysfunction, These results suggested that compound 2 may suppress hepatic decreased mitochondrial fatty acid β-oxidation and increased lipid accumulation through the suppression of lipogenesis, via TG synthesis. The present results demonstrated that compound downregulating the expression of proteins involved in lipogen- 2 counteracted the FFA-induced increase in intracellular esis, including ERp57 and FAS. ROS production. Furthermore, it was revealed to prevent the FFA-induced collapse of the ∆Ψm and the decrease in cellular Discussion ATP levels, thus suggesting that compound 2 may protect mitochondrial function. Hyperlipidemia has been identie fi d as an important risk factor The endoplasmic reticulum (ER) is known to serve a for the development of atherosclerosis (1) and acute necrotic central role in de novo lipogenesis. Oxidative and ER stress pancreatitis (24). Coreopsis tinctoria is a herbal medicine have been reported to occur simultaneously or successively, used to regulate lipid metabolism in traditional Chinese and ER stress has been associated with hepatic lipid accu- medicine (9). However, the exact pharmacological effects of mulation (31). The ER-associated protein ERp57 has been Coreopsis tinctoria, as well as the main active compounds and revealed to be upregulated during FFA-induced cellular the molecular mechanisms responsible for these effects, have steatosis, whereas its knockdown signic fi antly reduced lipid yet to be elucidated. In the present study, SCAE was demon- accumulation in steatotic cells (31). FAS has been identie fi d as strated to decrease serum lipid levels in a mouse model of a key enzyme during lipogenesis, as it catalyzes the terminal hyperlipidemia, possibly due to its antioxidative properties. Its steps in de novo fatty acid synthesis (32). The present results main active compounds, compounds 1 and 2, were revealed to demonstrated that compound 2 downregulated the protein decrease lipid accumulation in HepG2 cells, possibly through expression levels of ERp57 and FAS in FFA-treated HepG2 the reduction of oxidative stress, the protection of mitochon- cells. These results suggested that compound 2 may prevent drial function and the suppression of lipogenesis. de novo lipogenesis, via suppressing the expression of ERp57 Administration of a high-fat diet has been reported to and FAS in hepatocytes. increase fat and cholesterol intake, decrease the β-oxidation In conclusion, the present study suggested that compound of fatty acids and accelerate TG synthesis in rats, resulting 2 may be the main active compound of Coreopsis tinctoria, in increased TC and TG levels in the bloodstream (25). The responsible for its lipid-regulating effects. Furthermore, present results suggested that the flavonoid‑rich SCAE may compound 2 was demonstrated to enhance the endogenous attenuate lipid disorders and regulate TG levels. The liver antioxidative defense mechanisms of hepatocytes and to is primarily responsible for lipid synthesis, metabolism and protect mitochondria against oxidative damage. In addition, transportation (13,26), and hyperlipidemia has been reported its effects on ER stress reduction and the inhibition of de novo to increase hepatic lipid content, thus enhancing ROS genera- lipogenesis may be involved in the molecular mechanisms tion and lipid peroxidation (27). A previous study revealed that underlying the lipid-lowering effects of SCAE. The present a fl vonoids may attenuate hyperlipidemia, possibly due to their results suggested that compound 2 may have potential for the potent antioxidative effects (25). The present study suggested development of novel therapeutic strategies for the treatment that SCAE may enhance the endogenous antioxidative defense of patients with hyperlipidemia. mechanisms of hepatocytes, thus ameliorating hyperlipidemia, due to its high flavonoid content and potent antioxidative Acknowledgements properties. 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Molecular Medicine ReportsPubmed Central

Published: Jun 9, 2017

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