Isoorientin Prevents Hyperlipidemia and Liver Injury by Regulating

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Isoorientin prevents hyperlipidaemia and liver injury by regulating lipid metabolism, antioxidant capability and inflammatory cytokine release in high-fructose-fed mice Li Yuan, Xiao Han, Wenfeng Li, Daoyuan Ren, and Xing-Bin Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00290 • Publication Date (Web): 09 Mar 2016 Downloaded from http://pubs.acs.org on March 13, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Isoorientin prevents hyperlipidaemia and liver injury by regulating

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lipid metabolism, antioxidant capability and inflammatory cytokine release in

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high-fructose-fed mice

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Li Yuan*, Xiao Han, Wenfeng Li, Daoyuan Ren, Xingbin Yang*

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College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710119, China

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*Corresponding author: Li Yuan, Ph. D., Xian, Shaanxi, 710119, China.

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Tel./fax: 029-85310517; email: [email protected]

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*Corresponding author: Xingbin Yang, Ph. D., Xian, Shaanxi, 710119, China. Tel./fax: 029-85310517; email: [email protected]

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Abbreviations: ISO, isoorientin; HF, high fructose; TG, total triglyceride; TC, total

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cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density

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lipoprotein-cholesterol; VLDL, very low-density lipoprotein-cholesterol; ALT,

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alanine

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alkaline phosphatase; T-SOD, total superoxide dismutase; GSH-Px, glutathione

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peroxidase; MDA, malondialdehyde; apoA-Iapolipoprotein A1; apoB, apolipoprotein

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B; IL-6, interleukin 6; IL-1,interleukin 1; FAS, fatty acid synthase.

aminotransferase;

AST,

aspartate

aminotransferase;

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ALP,

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ABSTRACT

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Isoorientin (ISO), a natural flavonoid, has been found to have multiple biological

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properties. In the present study, obese mice with high-fructose (HF)-induced liver

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injury were used to investigate the hepatoprotective effects of ISO. The results

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showed that ISO significantly reduced the serum lipid parameters in the mice fed 20%

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HF water. Meanwhile, ISO appeared to alleviate HF-induced lipid metabolic disorders

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by increasing the serum levels of apoA-I and decreasing the serum apoB levels,

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apoB/apoA-I ratio and FAS activity in the liver. ISO also remarkably ameliorated

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HF-induced hepatic oxidative injury and inflammation by decreasing ALT, AST and

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ALP levels; enhancing antioxidant enzyme activities; and inhibiting inflammatory

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cytokine (TNF-α, IL-1, IL-6) release. Histopathology of liver stained by H&E and Oil

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Red O showed the liver steatosis and oxidative injury after HF treatment and the

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protective effect of ISO. Furthermore, aortic pathology observation found that ISO

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had a protective effect on the vascular endothelium. This is the first report that ISO

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efficiently inhibited HF-induced hyperlipidaemia and liver injury by ameliorating

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lipid metabolism, enhancing the antioxidant defensedefence system and regulating the

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secretion of inflammatory cytokines.

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isoorientin,

hyperglycaemia,

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KEYWORDS:

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lipid metabolism, hepatic inflammation

liver

damage,

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oxidative

stress,

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INTRODUCTION

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Fructose is widely used in the food industry as high-fructose syrup which has

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55-90% of fructose, and its consumption has been increasing significantly in recent

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years.1 However, many evidences show that a high-fructose intake can

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ultimately result in metabolic disorders, including obesity, insulin resistance,

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hypertension, hyperlipidaemia and hyperglycaemia.2,3 Moreover, the HF diet also

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results in inflammation, oxidative damage and hepatic steatosis.

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number of studies have suggested that the natural oligosaccharides, flavonoids and

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alkaloids from plants, such as (-)-epocatechin, 6 stachyose and tea polyphenols7 and

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betaine 8, play a promising role in preventing the metabolic syndromes induced by an

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HF diet.

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A large

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Isoorientin (3',4',5,7-tetrahydroxy-6-C-glucopyranosyl flavone; ISO; Fig. 1)

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exists in the human diet and can be isolated from several edible plants, including

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buckwheat, 9 Patrinia10 and corn silks11. It has been reported that ISO exhibits various

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physiological activities,12 for instance, antioxidant activity, anti-nociceptive and

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anti-inflammatory activities in mice.

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hepatoprotective effect against CCL4-induced oxidative damage in rats15 and

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t-BOOH-induced oxidative damage in HepG2 cells.16 Our previous studies also found

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that ISO inhibits the proliferation of human hepatoblastoma cancer cells by inducing

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apoptosis and autophagy.17,18 ISO is able to protect the liver cells against hydrogen

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peroxide-induced oxidation stress.19 Additionally, ISO reduces TNF-induced insulin

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resistance in murine 3T3-F442A cells and human sc adipocytes.20 Therefore, we

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speculate that ISO might play an important role in preventing liver injury and

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Furthermore, ISO exhibits a significant

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hyperlipidaemia. However, the hepatoprotective and hypolipidaemic effects of ISO

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have yet to be explored in vivo.

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The objectives of the present study are to test the protective effects of ISO on

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hyperglycaemia, oxidative injury and hepatic steatosis in mice fed an HF diet. In

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addition, its effects on lipid metabolism and vascular endothelium are also

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investigated. This study should provide a clue for substantiating dietary and

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preventing hyperglycaemia and hepatic injury.

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MATERIAL AND METHODS

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Chemicals and reagents

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Isoorientin (purity ≥ 98 %, extracted from bamboo) was purchased from Forever

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Biotechnology, Ltd. (Shanghai, China). Food-grade fructose was from Senbo Biology

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Co., Ltd. (Xi’an, China). Haematoxylin and eosin (H&E) and Oil red O were obtained

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from Shanghai Lanji Technological Development Co., Ltd. (Shanghai, China). Assay

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kits of glucose, total triglyceride (TG), total cholesterol (TC), high-density

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lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), very

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low-density lipoprotein-cholesterol (VLDL), alanine aminotransferase (ALT),

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aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were from

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Changchun Huili Biotechnology Co., Ltd. (Changchun, China). The total superoxide

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dismutase (T-SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)

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assay kits were from the Nanjing Jiancheng Bioengineering Institute (Nanjing,

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Jiangsu, China). ELISA kits of institute, apolipoprotein A1 (apo-A1), apolipoprotein

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B (apo-B), TNF-α, interleukin 6 (IL-6), interleukin 1 (IL-1) and fatty acid synthase

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(FAS) were also purchased from the Nanjing Jiancheng Bioengineering Institute

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(Nanjing, Jiangsu, China). The BCA protein assay kit was from Thermo Scientific

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(Rockford, IL, USA). All other chemicals were analytical grade and were produced in

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China.

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Animals, diets and and experimental design

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Healthy male Kunming mice (weight 18-22 g) were from the Experimental

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Animal Center of Fourth Military Medical University (Xi’an, China), and were

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housed in standard temperature (22 ± 2℃) and humidity (60 ± 5 %) room with 12/12h

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light-dark cycle. All of the animals were allowed free access to tap water and the

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rodent chow (Qianmin Feed Factory, Experimental Animal Center of Fourth Military

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Medical University, Xi’an, China), which contained 40 % corn flour, 26 % wheat

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flour, 10 % bran, 10 % fish meal, 10 % bean cake, 2 % mineral, 1 % coarse meal, and

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1 % vitamin mix.

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After a week for adjustable feeding, mice were randomly divided into four

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groups (n = 10). In the normal control (ND) group, the mice were allowed free access

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to tap water and were given 0.4 mL physiological saline by gavage once daily. In the

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high-fructose (HF) group, mice both received 20 % high-fructose water and 0.4mL

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physiological saline. In the ISO-treated groups, the mice were administered with ISO

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at 20 and 40 mg/kg·bw (ig, 0.4 mL) as low- (HF+LISO) and high-dose (HF+HISO)

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treatments, respectively, except 20 % high-fructose water. The mice both in HF and

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ISO group were allowed free access to 20 % high-fructose water, and the 20 % HF

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water was renewed every other day. During 56 consecutive days of treatment, the

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body weights of all of the mince were measured once a week. After two hours of the

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last administration, mice were allowed free access to water for 8 h except food. Then,

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all of the mice were anesthetized by ether, and weighed sacrificed to obtain blood and

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livers. The blood samples were centrifuged at 1200×g for 20 min and stored at 4 ℃,

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and the liver samples were frozen at -80 ℃. All experimental protocols were adhered

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the Guidelines of Experimental Animal Administration.

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Biochemical and enzymatic activities evaluation

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The serum levels of TG, TC, VLDL-C, LDL-C, HDL-C, insulin, glucose, ALT,

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AST and ALP were determined using commercial kits with a Multiskan Go automatic

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analyser (Thermo Electron, America) according to the manufacturer's protocol.

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Lipid metabolism analysis

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The levels of apoA-I and apoB in serum and FAS levels in the liver were tested

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using a commercial RIA kit or an ELISA kit according with the manufacturer's

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instructions.

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Estimation of Biochemical properties and cytokines of the liver

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The liver samples were homogenized using an automatic homogenizer

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(F6/10-10G, FLUKO Equipment Shanghai Co., Ltd., Shanghai, China). After

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centrifuging at 5000 rpm for 10 min at 4 °C the supernatants were collected and

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measured protein concentrations using BCA assay kit. The levels of GSH-Px, T-SOD,

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MDA, TNF-α, IL-1 and IL-6 in the liver supernatant were determined using

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commercial kits or an ELISA kit according to the manufacturer's instructions.

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Histopathological and Thoracic Aortas observation

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Histopathology of the liver was measured using H&E and Oil Red O staining. The

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liver tissue samples were fixed with 10% neutral formalin solution, and then

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embedded in paraffin, cut into slices (5-6µm thickness) and stained only with H&E

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dye. Next, the stained sample tissues were processed using cryostat (CM1950, Leika,

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Germany) and stained by Oil Red O. The slides were examined under a light

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microscope (Olympus Optical Co., Ltd., Tokyo, Japan) for observations and

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photography.

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The thoracic aortas were fixed with 4% paraformaldehyde buffer solutionand

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were processed with the method of paraffin embedding and H&E staining, followed

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by observation under a light microscope.

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Statistical analysis

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All results were presented as the means ± standard errors (SE), and were

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analysed using the one-way factorial analysis of variance (ANOVA) and Duncan’s

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post-hoc test (SPSS 16.0).

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RESULTS

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Effects on food intake, body weight, BMI and liver weight

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Table 1 shows the effects of ISO on the food intake, body weights, BMI, liver and

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fat weights of the experimental mice. As shown in Table 1, food intake of HF-fed

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mice was lower than that of ND group mice (p < 0.05), and compared with HF group,

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there were no significant change of food intake in ISO-treated group. Water intake of

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ND group mice was slight lower than that of other groups, even though there was no

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significant difference (p < 0.05), and all groups had no differences (p < 0.05) at

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fructose intake. Additionally, there was no significant difference in the initial body

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weight among the all groups. However, by the end of 8 weeks trial, the final body

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weights of HF-fed mice were significantly higher than that of ND group mice.

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Comparing with the HF group, 20 and 40 mg/kg·bw of ISO supplementation

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effectively reduced body weight by 11.90 % and 11.07 % (p