In vitro and in vivo antioxidant activity of polyphenols extracted from black highland barley

Accepted Manuscript In Vitro and In Vivo Antioxidant activity of Polyphenols Extracted from Black Highland Barley Yingbin Shen, Hui Zhang, Liling Cheng, Li Wang, Haifeng Qian, Xiguang Qi PII: DOI: Reference:

S0308-8146(15)01291-1 http://dx.doi.org/10.1016/j.foodchem.2015.08.083 FOCH 18031

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

22 February 2015 18 August 2015 20 August 2015

Please cite this article as: Shen, Y., Zhang, H., Cheng, L., Wang, L., Qian, H., Qi, X., In Vitro and In Vivo Antioxidant activity of Polyphenols Extracted from Black Highland Barley, Food Chemistry (2015), doi: http://dx.doi.org/ 10.1016/j.foodchem.2015.08.083

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In Vitro and In Vivo Antioxidant activity of Polyphenols Extracted from Black Highland Barley Yingbin Shen, Hui Zhang*, Liling Cheng, Li Wang, Haifeng Qian, Xiguang Qi State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122 Wuxi, China *Corresponding Author: Hui Zhang State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122 Wuxi, China Telephone: +86-139-211-77990. Fax: +86-510-853-29099 E-mail: [email protected] Abbreviations

AI, atherosclerosis index; BW, body weight; BHLPE, polyphenol extract from black highland barley; CAT, catalase; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; FRAP, ferric reducing antioxidant power; GPX, glutathione peroxidase; GAE, gallic acid equivalents; GSH-Px, glutathione peroxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL_C, high-density lipoprotein cholesterol; HFD, high fat diet; HO-1, heme oxygenase-1; H&E, hematoxylin eosin staining; HPLC, high performance liquid chromatography; KM, Kun Ming; LPL, lipoprotein lipase; LDL_C, low-density lipoprotein cholesterol; MDA, malondialdehytde; NADH, nicotinamide adenine dinucleotide; NQO1, quinone oxidoreductase-1; Nrf2, transcription factor NF-E2-related factor 2; NBT, nitrotetrazolium blue chloride; NFD, normal fat diet; RT-PCR, reverse transcriptase polymerase chain reaction; ROS, reactive oxygen species; SOD, superoxide dismutase; T-AOC, total antioxidant capacity; TG, triglyceride; TC, total cholesterol; TPTZ, 2,4,6-tripyridyl-s-triazine; TPC, total phenolic contents; TFC, total flavonoid contents; TAC, total anthocyanins content; PMS, N-methylphenazonium methyl sulphate

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ABSTRACT The objective of this study was to determine the antioxidant capacity of polyphenols extracted from black highland barley (BHLPE) in vitro and in vivo. BHLPE was found to have strong superoxide radical, hydroxyl radical and 2, 2diphenyl-1-picrylhydrazyl radical-scavenging activity, ferric reducing antioxidant power and moderate metal ion-chelating activity. Compared with a high fat diet (HFD) group, mice that were administered 600 mg BHLPE /kg body weight showed significant decreases in total cholesterol, low-density lipoprotein cholesterol and the atherosclerosis index, in addition to markedly increased high-density lipoprotein cholesterol levels. Furthermore, the antioxidant defense system and antioxidant gene expression were significantly improved in vivo in mice that were administered BHLPE compared with mice in the HFD group. These results suggest that BHLPE has significant potential as a natural antioxidant to promote health and to reduce the risk of disease.

KEYWORDS: black highland barley, in vitro, in vivo, antioxidant activity, oxidative stress

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1. Introduction Oxidative stress is manifested by the excessive production of reactive oxygen species (ROS) and an insufficient or defective antioxidant defense system. Meanwhile, oxidative stress causes profound alterations in various biological structures, including cellular membranes, lipids, proteins and nucleic acids (Zitka, Skalickova, Gumulec, Masarik, Adam, Hubalek, et al., 2012). Interest in identifying biomarkers for disease, in which oxidative stress is always involved, is increasingly growing. Oxidative stress is involved in aging and in various diseases, including diabetes mellitus(J. F. Liu, Liu, Chen, Chang, & Chen, 2013; Mensink, Hesselink, Russell, Schaart, Sels, & Schrauwen, 2007), atherosclerosis(Bonomini, Tengattini, Fabiano, Bianchi, & Rezzani, 2008; Nwose, Jelinek, Richards, Tinley, & Kerr, 2009; Pihl, Zilmer, Kullisaar, Kairane, Magi, & Zilmer, 2006), Alzheimer's disease(Shen, Callaghan, Juzwik, Xiong, Huang, & Zhang, 2010; Sinha, Saha, Basu, Pal, & Chakrabarti, 2010), Parkinson's disease(Kaur, Chauhan, & Sandhir, 2011; Nikam, Nikam, & Ahaley, 2009) and some cancers (Oueslati, Ksouri, Falleh, Pichette, Abdelly, & Legault, 2012). However, epidemiological studies have shown that increased consumption of whole grains and their products has been consistently associated with the reduction of these chronic diseases associated with oxidative stress (Borneo & Leon, 2012; Kristensen, Toubro, Jensen, Ross, Riboldi, Petronio, et al., 2012; Slavin, 2009; Steffen, Jacobs, Murtaugh, Moran, Steinberger, Hong, et al., 2003; Van der Kamp, 2012). Therefore, the antioxidant activity of various whole grains has been intensively studied during recent years. Highland barley, known in Chinese as Qing Ke, is a principal food crop in Tibet. The Highland barley crop occupies the most area and has the highest field production because it is the only crop that can be grown at high altitudes of 4200-4500 m above sea level (Z. F. Liu, Yao, Yu, & Zhong, 2013). It is very adaptable to low temperature and also tolerates high sunlight. A previous study focused on β-glucan, which is present at a higher concentration in the seeds of highland barley than in other cereals. The β-glucan in highland barley reduced serum glucose and serum lipids, as well as insulin resistance and the risk of arterial sclerosis in high-fat induced C57BL/6 mice (Tian, Song, Liu, Su, Sun, & Li, 2013). Highland barley contains various phytochemicals such as phenolic acids (Bonoli, Verardo, Marconi, & Caboni, 3

2004; Gong, Jin, Wu, Wu, & Zhang, 2012; Siebenhandl, Grausgruber, Pellegrini, Del Rio, Fogliano, Pernice, et al., 2007) and anthocyanins (Kohyama, Ono, & Yanagisawa, 2008). Furthermore, Highland barley showed a strong antioxidant activity in a previous study (Gong, Jin, Wu, Wu, & Zhang, 2012; M.-J. Kim, Hyun, Kim, Park, Kim, Kim, et al., 2007). Several studies have reported that crude extracts of grains or whole grains have potential in vivo antioxidant activity (J. Kim & Noh, 2013; Omwamba, Li, Sun, & Hu, 2013; L. F. Wang, Sun, Yi, Wang, & Ju, 2012). However, few studies reported the antioxidant activity of black highland barley systematically and explored the mechanism by which it regulates the antioxidant defense system in vivo. Thus, the objective of this study was (1) to determine the phenolic compounds in the polyphenol extract from black highland barley (BHLPE) by high performance liquid chromatography (HPLC), and (2) to evaluate the antioxidant activity of BHLPE in vitro and in vivo. 2. Materials and Methods 2.1. Chemicals A total antioxidant capacity (T-AOC) kit, superoxide dismutase (SOD) kit, catalase (CAT) kit, glutathione peroxidase (GSH-Px) kit, malondialdehytde (MDA) kit and bovine serum albumin (BSA) protein assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Folin-Ciocalteu reagent, p-coumaric acid, ferulic acid, gallic acid, catechin, 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) and rutin (RT) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Nitroblue tetrazolium (NBT) chloride, N-methylphenazonium methyl sulphate (PMS), nicotinamide adenine dinucleotide (NADH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ferrozine and 2,4,6-tripyridyl-s-triazine (TPTZ) were purchased from J&K Scientific Co., Ltd. (Beijing, China). All other chemicals and solvents were of analytical grade. 2.2. Sample preparation Black Highland barley (Hordcum vulgare L. var. nudum Hook. f., Zangqing 2000, Tibet, China) was obtained from Diqing Shangri-la Highland Barley Resource Development Co., Ltd. The samples were milled into whole grain flour using a 60-mesh size screen, thoroughly mixed and then stored at -20°C. 4

Total phenolic compounds were extracted as previously described with minor modifications (L. F. Wang, Chen, Xie, Ju, & Liu, 2013). Briefly, Black Highland Barley flour was digested with 2 M sodium hydroxide at room temperature for 2 hours. The mixture was neutralized with an appropriate amount of hydrochloric acid. Petroleum ether was used to extract the lipids in the mixture. The ethyl acetate fraction was evaporated to dryness. Phenolic compounds were reconstituted in 80% chilled methanol and stored at -20°C for analysis. 2.3. Determination of the Total Phenolic content, Total Flavonoid and total anthocyanin content The total phenolic content (TPC) of BHLPE was determined using the Folin-Ciocalteu colorimetric method described by Singleton et al. with some modifications(Singleton, 1965). Gallic acid was used as the standard, and the total phenolic content was expressed as mg GAE/g BHLPE. Briefly, appropriate dilutions of extracts were oxidized with Folin-Ciocalteu reagent, and the reaction was neutralized with sodium carbonate. The absorbance of the resulting blue color was measured at 760 nm after 90 min. The total flavonoid content (TFC) was determined by a colorimetric method previously described with some modifications (Zilic, Sukalovic, Dodig, Maksimovic, Maksimovic, & Basic, 2011). Appropriate dilutions of sample extracts were reacted with sodium nitrite, followed by the formation of a flavonoid-aluminum complex using aluminum chloride. The absorbance at 510 nm was immediately measured and compared with that of rutin standards. The flavonoid content is expressed as mg RT/g DW. The total anthocyanin content (TAC) of BLHPE was measured using a spectophotometric pH differential protocol reported by Lopez-Martinez (Lopez-Martinez, Oliart-Ros, Valerio-Alfaro, Lee, Parkin, & Garcia, 2009). Absorbance readings at 535 nm were taken and corrected for background absorbance at 700 nm in a photodiode array spectrophotometer. Anthocyanins are expressed as mg of cyanidin-3 glucoside equivalents/100 g, using a molar extinction coefficient of 25,965 M-1cm-1 and a molecular weight of 449.2 g/mol. The data are reported as the mean± standard deviation (SD) for three replicates. 2.4. HPLC Analysis 5

The phenolic ingredients in samples were analyzed by HPLC using the method reported by Guo (Guo, Ma, Parry, Gao, Yu, & Wang, 2011) with modifications. The HPLC system employed was a Waters (Milford, MA, USA) 1525 binary HPLC pump separation module with 20 µL injected by hand and a Waters 2996 photodiode array detector. Separation was performed with an Hanbon C18 column (250*4.6 mm, 5 µm) at 35°C with gradient elution employing solution A: methanol, and solution B: acetic acid–water solution (0.1% acetic acid), which were delivered at a flow rate of 0.8 mL/min as follows: 0 min, 25% (A); 2 min, 35% (A); 10 min, 40% (A); 15 min, 75% (A); 20 min, 25% (A); and 25 min, 25% (A). The UV spectrum between 254 and 400 nm was recorded for peak characterization. Phenolic ingredients were quantified by the peak area of their maximum absorption wavelength. 2.5. Antioxidant activity in vitro 2.5.1 DPPH radical scavenging ability The antioxidant activity of the obtained extracts was also tested using DPPH as previously described with some modifications (Zhang, Zhang, Wang, Guo, Qi, & Qian, 2011). Briefly, the DPPH free radical scavenging activity of grain extracts was determined using a 2×10-4 M DPPH solution. Each sample of BLHPE (0.5 ml) was mixed with 4 ml 2×10-4M DPPH in ethanol. The mixture was shaken, and then left to stand for 60 min in the dark. The absorbance was measured at 517 nm in a spectrophotometer. The absorbance of the control was obtained by replacing the sample with 80% methanol. The DPPH radical scavenging activity of the sample was calculated as follows: DPPH radical scavenging activity (%) = [1－absorbance of sample/absorbance of control] × 100. 2.5.2 Ferric reducing antioxidant power assay The Ferric reducing antioxidant power assay (FRAP) was conducted according to the procedures described by Muller with some modifications (Muller, Frohlich, & Bohm, 2011). In our procedure, freshly prepared FRAP reagent (2.5 ml of a 10 mM TPTZ solution in 40 mM HCl, 2.5 ml of 20 mM FeCl3 and 25 ml of 0.1 M acetate buffer, pH 3.6) was incubated at 37°C for 10 min. Then, 0.05 ml of rapeseed extract and 2 ml of FRAP reagent were transferred to a 10-ml volumetric flask and brought to volume with redistilled water. The blue solution obtained was incubated at room 6

temperature for 20 min. The absorbance was measured at 593 nm against a reagent blank (2 ml of FRAP reagent brought to 10 mL with redistilled water) using a UV spectrophotometer in a 1-cm quartz cell. Trolox was used as the positive control, and the results are expressed as A593nm. 2.5.3. Superoxide anion scavenging activity Measurement of the superoxide anion scavenging activity of BHLPE was based on the method described by Liu and Chang with slight modifications (F. Liu, Ooi, & Chang, 1997). Superoxide radicals are generated in PMS-NADH systems by the oxidation of NADH and assayed by the reduction of NBT. In this experiment, superoxide radicals were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 1 mL of NBT (50 µM) solution, 1 mL NADH (78 µM) solution and the sample solution. The reaction was initiated by adding 1 mL of PMS solution (10 µM) to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against a blank. A decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. The percentage inhibition of superoxide anion radical generation for three parallel measurements was calculated using the following formula: Inhibition (%) = [(A control –A sample)/A control] ×100 In this formula, A control is the absorbance of control and A sample is the absorbance in the presence of the extract or a standard. The IC50 value represents the concentration of the compounds that caused 50% inhibition of hydroxyl radical formation. 2.5.4. Assay of hydroxyl radical scavenging activity The hydroxyl radical scavenging activity of BHLPE was determined based on a method from a previous study with some modifications (Su, Wang, & Liu, 2009). The hydroxyl radical was generated through a Fenton reaction in a system of FeSO4 and H2O2. The reaction mixture consisted of 1.0 ml FeSO4 (9 mmol/L), 1.0 mL H2O2 (8.8 mmol/L), and 1.0 mL of various concentrations of BHLPE and 1.0 mL of salicylic acid (9 mmol/ L). The mixture (4.0 mL) was incubated at 37°C for 1 h, and the absorbance of the solution at 510 nm was recorded. Trolox was used as a positive 7

control. The scavenging activity was calculated using the following formula: Scavenging activity (%) = [1－(A1－ A2)/A0]×100. In this formula, A0 is the absorbance of the control (without extract), A1 is the absorbance of the sample, and A2 is the absorbance without hydrogen peroxide. The IC50 value represents the concentration of the compounds that caused 50% inhibition of hydroxyl radical formation. 2.5.5. Metal Chelating Activity The metal chelating activity of BHLPE was determined by the method of Liang (Liang, Chang, Liang, Hung, & Hsieh, 2014) with minor modifications. A methanol solution of the samples (0.5 ml) at various concentrations (0.05, 0.1, 0.15 0.2 mg/ml) was mixed with methanol (3 ml) FeCl2·4H2O (2 mM, 0.1 ml) and ferrozine (5 mM, 0.2 ml). The mixture was incubated in the dark for 10 min. The control contained all the reagents without the sample and was used as the blank. The metal chelating activity was determined by measuring the absorbance at 562 nm using a spectrophotometer. The metal chelating activity of ethylenediamine tetra-acetic acid (EDTA) was also determined for comparison. The metal chelating activity (%) = (1− absorbance of sample/absorbance of control) × 100%. 2.6. Antioxidant activity in vivo 2.6.1. Animals and experimental design The license number for using experimental animals was SCXK (SH) 2012-0002. The experimental protocol was developed according to the institution’s guideline for the care and use of laboratory animals by Jiangnan University. Animal maintenance and experimental procedures were approved by the Animal Ethics Committee of Jiangnan University. Forty male mice weighing 20±2 g (four weeks old) were used (Shanghai Laboratory Animal Center). Animals were maintained under standard conditions (23±2°C, relative humidity 55±0.5%, 12 h light-dark cycle). Prior to experiments, the animals were adapted by being fed a normal fat diet (NFD) for one week. Mice in the NFD and high fat diet (HFD) 8

groups were fed with physiological saline by gavage. Mice in the BHLPE treatment groups were fed BHLPE at two different doses (300 and 600 mg/kg BW per day) by gavage. The composition of the experimental diets was shown in Table 1. All groups were fed once daily for 42 consecutive days. 2.6.2. Biochemical assays Twenty-four hours after the last drug administration, the mice were euthanized. Blood samples were collected and centrifuged at 4000 x g at 4°C for 10 min to produce the sera. The livers were quickly removed, washed and homogenized in ice-cold physiological saline to prepare a 10% (w/v) homogenate. The homogenate was centrifuged at 4000 x g at 4°C for 10 min to remove cellular debris, and the supernatant was collected for analysis. The levels of T-AOC, MDA, CAT, SOD and GSH-Px levels were determined following the manufacturer’s instructions. Briefly, the CAT activity was determined by measuring the absorbance of the yellow H2O2–ammonium molybdate complex at 405 nm. SOD activity was measured via the inhibition of hydroxylamine oxidation by the superoxide radicals generated in the xanthine–xanthine oxidase system. GSH-Px activity was measured on the basis of the reaction of GSH and 5, 5-dithiobis-(2-nitrobenzoic acid). All activities are expressed as units per milliliter (U/ml) in serum or units per milligram of protein (U/mg protein) in liver. The protein content in the liver supernatants was determined by the Lowry method using bovine serum albumin as the standard (Lowry, Rosebrough, Farr, & Randall, 1951). Triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL_C) and low-density lipoprotein cholesterol (LDL_C) levels of serum were analyzed on a Mairui Automatic Analyzer (Shen Zhen, China) using commercial enzymatic kits following the manufacturer’s instructions. Atherosclerotic index (Al) was calculated using the following formula: (TC-HDL_C)/HDL_C. 2.7. Gene expression in liver For determining mRNA expression, total RNA was first extracted from frozen tissue with Trizol reagent. The quantity and quality of the RNA were verified by measuring the A260/A280 ratio and by gel electrophoresis. Total RNA 9

was reverse transcribed to cDNA according to the manufacturer’s instructions (AMV First Strand cDNA Synthesis Kit). The mRNA expression was quantified using real-time polymerase chain reaction (RT-PCR). The primer sequences are listed in Figure 4-A. RT-PCR was carried out using ABI StepOne plus under the following conditions: 40 cycles of denaturation at 95°C for 10 s, annealing and extension at 60°C for 40 s. Porcine primers were designed using Primer Premier 5.0 and synthesized by the GeneRay Biotechnology Co. (Shanghai, China). Three independent RT-PCR reactions were carried out for each sample. The relative expression level of the target genes was calculated as a ratio to the housekeeping gene GAPDH. A house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as internal control to normalize the RT-PCR data. The relative expression level of a gene in a given sample was represented as 2-(∆∆Ct) as compared with the normal fat diet (NFD) group. ∆∆Ct=∆Ct (experimental gene in each group)-∆Ct (NFD group), ∆Ct=Ct (experimental)-Ct (GAPDH). 2.8. Statistical Analysis The data are expressed as the mean ±SD (standard deviation). Analysis of variance (ANOVA) was carried out to determine significant differences by the SPSS (Statistical Package for the Social Science) statistical software (version 17.0, SPSS Inc., USA). The significance of the difference was checked by the Duncan test, and differences were considered significant at p<0.05 or very significant at p<0.01. 3. Results and Discussion 3.1. TPC, TFC, TAC and phenolic compounds in BHLPE TPC, TFC and TAC of BHLPE were 171.77 mg GAE/g, 16.22 mgRT /g and 3.76 mg Cy-3-O-G/g, respectively (data not shown). Figure 1 indicates that BHLPE was rich in phenolic compounds. Ferulic acid, p-coumaric acid and catechin were identified via HPLC by comparison with standard phenolics. The concentrations of each phenolic compound were 19.14, 14.59 and 4.78 mg/g, respectively, which indicated that ferulic acid and p-coumaric acid were the main phenolic acids in BHLPE. These results were partly consistent with a previous study that showed that ferulic 10

acid and p-coumaric acid were the major phenolic acids in barley (Andersson, Lampi, Nystrom, Piironen, Li, Ward, et al., 2008; Hernanz, Nunez, Sancho, Faulds, Williamson, Bartolome, et al., 2001). It has also been reported that phenolic compounds are known to be powerful chain-breaking antioxidants and have scavenging ability due to their hydroxyl groups. These results indicated that the pronounced in vitro and in vivo antioxidant activity of BHLPE was possibly due to its high phenolic content. Phenolic esters can be hydrolyzed to release phenolic acids by different methods. Alkaline, acidic or enzymatic hydrolysis methods are used to release bound phenolics from the cereal matrix. Generally, alkaline hydrolysis is mostly used for extracting bound phenolics from grains at room temperature (Adom & Liu, 2002), and therefore, alkaline hydrolysis was used in this study to extract the total phenolics including the free and bound forms. Thus, TPC and TAC from BHLPE were higher than previously reported because a different extraction method and highland barley type was used in our study (Bellido & Beta, 2009; Gong, Jin, Wu, Wu, & Zhang, 2012). 3.2. Antioxidant activity in vitro 3.2.1. Scavenging activity on DPPH radical of BHLPE Scavenging of DPPH radicals is a widely used model to evaluate the free radical scavenging activity of mixed and pure antioxidants from plants. In the DPPH assay, antioxidants are able to reduce the stable DPPH radical (purple) to the non-radical form, DPPH-H (yellow). The DPPH scavenging activity of an antioxidant is attributed to its hydrogen donating ability. As shown in Figure 2-A, the scavenging activities of DPPH radical by BHLPE and Trolox increased with increasing concentration. At a concentration of 0.25 mg/mL, the DPPH scavenging activity of BHLPE and Trolox were 67.32% and 82.04%, respectively. Furthermore, it should be noted that the scavenging activity of Trolox was higher than that of BHLPE (p<0.05). The IC50 values of BHLPE and Trolox were 196.61 and 170.54 µg/ml, respectively. Therefore, the results indicated that BHLPE had strong DPPH radical scavenging activity. 3.2.2. Ferric reducing antioxidant power of BHLPE The FRAP assay treats the antioxidants contained in the samples as reductants in a redox-linked colorimetric assay, 11

and the value reflects the reducing power of the antioxidants. This method has been frequently used for a rapid evaluation of the total antioxidant capacity of various antioxidants from grains and vegetables. The ferric reducing antioxidant power of BHLPE and Trolox is shown in Figure 2-B. The FRAP results were well correlated with concentration, and value for Trolox was higher than that for BHLPE (P < 0.05). At a concentration of 0.5 mg/ml, the scavenging activities of BHLPE and Trolox were 1.02 and 1.73, respectively. These values suggested that the BHLPE had a higher ferric reducing antioxidant power.

3.2.3. Superoxide anion radical-scavenging activity of BHLPE

The superoxide anion radical is the most common free radical generated in vivo. Superoxide anion, derived from dissolved oxygen by a PMS-NADH coupling reaction, reduces NBT. The decrease in absorbance at 560 nm in the presence of antioxidants indicates the consumption of superoxide anions. Figure 2-C shows percentage inhibition of superoxide anion radical generation for different amounts of BHLPE, compared with the same concentration of Trolox. The scavenging activity of all samples was well correlated with concentration, and the scavenging activity of Trolox was higher than that of BHLPE (P < 0.05). At a concentration of 0.2 mg/ml, the scavenging activity of BHLPE and Trolox were 63.11% and 75.49%, respectively, and the IC50 values were 143.88 and 98.19µg/ml, respectively. These values suggested that the BHLPE is a good scavenger of the superoxide radical.

3.2.4. Scavenging of hydroxyl radical by BHLPE

The hydroxyl radical is the most reactive radical known and can attack and damage almost every macromolecule in a living cell. The most highly characterized biological damage caused by hydroxyl radical is its capacity to stimulate lipid peroxidation, which occurs when hydroxyl radical is generated near a membrane and attacks the fatty acid side chains of membrane phospholipids. As shown in Figure 2-D, the scavenging activities of BHLPE and Trolox increased with increasing concentration. At a concentration of 0.2 mg/ml, the scavenging activity for BHLPE and Trolox was 25.31% and 69.77%, respectively, and Trolox showed a higher hydroxyl radical scavenging activity than BHLPE (p < 12

0.05). The IC50 values of BHLPE and Trolox were 415.27 and 146.31 µg/ml, respectively, indicating that BHLPE is a good scavenger of hydroxyl radical.

3.2.5. Metal ion chelating activity of BHLPE

The ability of BHLPE to chelate ferrous ions is shown in Figure 2-E, which shows that the scavenging activities of BHLPE and EDTA increased with increasing concentration. At a concentration of 0.2 mg/ml, the metal ion chelating capacity of BHLPE and EDTA was 49.45% and 85.21%, respectively. Furthermore, EDTA showed a higher metal ion chelating capacity than BHLPE (p<0.05). The IC50 values of BHLPE and EDTA were 187.82 and 43.95 µg/ml, respectively. The metal chelating activity of BHLPE was significantly lower than that of EDTA at the same concentration. This result indicates that BHLPE has moderate metal chelating activity that increases with an increase in the concentration of BHLPE.

3.3. Antioxidant activity in vivo

3.3.1. Effect of BHLPE on the growth of mice fed a high fat diet

A high-fat diet has been shown to produce a more rapid weight gain in rodents (J. Wang, Cao, Wang, & Sun, 2011). In the present study, daily weight gain (DWG) in the HFD group increased by 9.04% (p>0.05) compared with the NFD group (Table 2). Furthermore, compared with mice in the NFD group, food intake (FI), feed efficiency (FE), liver weight (FW) and the liver index (LI) of mice in the HFD group were not significantly changed (p>0.05) (Table 2). However, compared with mice in the HFD group, the daily weight gain of mice in the HFDL group and HFDH group decreased by 20.68% and 24.97%, respectively (p<0.05) (Table 2). Furthermore, the FI, FE, LW and LI of mice in the HFDL and HFDH groups were not significantly changed compared with mice in the HFD group (p>0.05) (Table 2). These results indicated that a high-fat diet had no significant effect on the growth and biomarkers of mice, whereas the administration of BHLPE affected the DWG of mice during the dietary treatment.

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3.3.2. Effect of BHLPE on lipid status and atherosclerosis index in serum

Compared with the NFD group, the serum levels of TG, TC, LDL_C and AI in the HFD group were increased by 102.33%, 67.15%, 76.77% and 334%, respectively (Table 2). Compared with the HFD group, the serum levels of TC, LDL_C and AI in the HFDH group were decreased by 23.33%, 26.29% and 38.70%, respectively (p<0.05) (Table 2), whereas HDL_C levels were increased by 17.80% (p<0.05). Furthermore, there was no difference between the HFD group and the HFDL group in the serum levels of TG, TC, HDL_C, LDL_C and AI (p>0.05) (Table 2). These results suggested that the administration of BHLPE could improve the lipid status of mice during the dietary treatment. Many researchers have proved that the increased serum levels of TC and LDL_C raise the risk of atherosclerosis and CVD(Berliner & Heinecke, 1996). On the contrary, a raised serum level of HDL_C was associated with a reduced risk of atherosclerosis because high density lipoprotein in serum is thought to facilitate the translocation of excess cholesterol from the peripheral tissue to the liver for further catabolism(Assmann, 2004). In the present study, mice fed a high-cholesterol diet for 42 days had significantly higher levels of serum TG, TC, LDL_C and AI, as well as lower HDL_C levels, compared with mice maintained on a normal diet. These results are in agreement with those reported in several studies (Tang, Yang, Shi, & Le, 2011; R. L. Yang, Le, Li, Zheng, & Shi, 2006). However, oral gavage of BHLPE dramatically hindered the increase in the serum levels of TC, LDL_C and AI, and the decrease in the serum level of HDL_C. This effect partly agrees with a previous investigation on rats supplemented with sorghum (J. Kim & Noh, 2013), showing that the concentrations of serum triglycerides, TC, HDL_C, and non-HDL_C were significantly lower in rats given sorghum extract (7.5 g/kg diet). Similarly, Wang (L. F. Wang, Sun, Yi, Wang, & Ju, 2012) reported that the administration of extracted polyphenols from Adlay was effective for significantly decreasing the serum levels of TC, LDL_C in rats. Furthermore, according to the references studied the antiatherogenic property of phenolic compounds from other sources, we suspected the reduction of TC induced by BHLPE might be due to a decrease in cholesterol absorption and biosynthesis and an increase in fecal bile acid and cholesterol excretion(Raederstorff,

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Schlachter, Elste, & Weber, 2003).

3.3.3. Effect of BHLPE on the antioxidant activity of serum and liver

Compared with the NFD group, the serum levels of T-AOC, CAT, SOD and GSH-Px in the HFD group were decreased by 27.82%, 45.13%, 25.58% and 16.21%, respectively (p<0.05) (Table 2), whereas the MDA level was increased by 43.52% in mice treated with BHLPE. However, compared with the HFD group, T-AOC, CAT, SOD and GSH-Px serum levels in the HFDH group were increased by 17.97%, 44.62%, 32.06% and 19.57%, respectively (p<0.05), whereas the MDA level decreased by 31.83% (p<0.05) (Table 2). In contrast, no changes of T-AOC, MDA, CAT, SOD and GSH-Px levels in the serum of the HFDL group were found (p>0.05) (Table 2). These results suggested that the administration of 600 mg/Kg BW BHLPE could alleviate the oxidative stress in the serum of mice caused by a high fat diet. Compared with the NFD group, the T-AOC, CAT, SOD and GSH-Px levels in the liver in the HFD group were decreased by 53.08%, 59.24%, 21.12% and 46.98%, respectively (p<0.05) (Table 2), whereas the MDA level was increased by 91.03%. However, compared with the HFD group, the T-AOC, CAT, SOD and GSH-Px levels in the liver in the HFDH group were increased by 26.67%, 49.61%, 32.06% and 14.90%, respectively, whereas the MDA level was decreased by 72.15% (p<0.05) (Table 2). In contrast, the T-AOC, MDA, CAT, SOD and GSH-Px levels in the livers from the HFDL group were not changed compared with those from the HFD group (p>0.05) (Table 2). Thus, these results suggested that the administration of 600 mg/Kg BW BHLPE can improve the antioxidant activity in the livers of mice fed a high fat diet. The free radical damage hypothesis is an ageing theory that states that the excessive generation of ROS or free radicals can lead to cell and tissue damage or death (Ramalingam & Kim, 2014; Sayin, Ibrahim, Larsson, Nilsson, Lindahl, & Bergo, 2014). MDA is a principal product of lipid oxidation and has been found to be elevated in various diseases related to free radical damage, so it has been widely used as an index of lipid peroxidation (Liao, Ning, Chen,

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Wei, Yuan, Yang, et al., 2014). SOD, GSH-Px and CAT are the most important antioxidant enzymes that inhibit free radical formation and are usually used as biomarkers to indicate ROS production (Queguineur, Goya, Ramos, Angeles Martin, Mateos, & Bravo, 2012; X. Yang, Yang, Guo, Jiao, & Zhao, 2013). SOD is an extremely effective defense enzyme that converts superoxide anions into hydrogen peroxide (H2O2). CAT is a common enzyme found in nearly all living organisms that catalyzes the decomposition of hydrogen peroxide into water and oxygen. CAT can also oxidize other toxins, such as formaldehyde, formic acid, phenols, and alcohols. GSH-Px is the general name of an enzyme family with peroxidase activity, whose main biological role is to protect the host organism from oxidative damage. The biochemical function of GSH-Px is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. In the present study, changes in the activities of antioxidant enzymes, MDA and T-AOC of mice were investigated (Liu, Jia, Kan, & Jin, 2013). These results suggested that the administration of BHLPE at a dose of 600 mg/kg BW could improve the lipid oxidation in the serum and liver of mice induced by a high fat diet. As reported, the enhanced activities of antioxidant enzymes were partially due to an increase in mRNA expression for these enzymes (Ha, Na, & Kim, 2013). The activities of the antioxidant enzymes SOD and GSH-Px increased while the MDA level decreased in the liver of aged mice treated with the barley extract in a previous study (Omwamba, Li, Sun, & Hu, 2013).

3.3.4. Effect of BHLPE on hepatic steatosis

To investigate the inhibitory effect of dietary BHLPE administration on hepatic fat accumulation, we analyzed the liver tissue histology using H&E staining. The cell architecture of the liver in normal mice showed normal hepatocytes with a central vein (Figure 3-A). Significant microvesicular steatosis accompanied by partial mild inflammation was observed in the HDF group, as shown in Figure 3-B. However, the degree of hepatic fat accumulation was substantially alleviated by dietary intake of BHLPE at the dose of 600 mg/kg BW, as indicated by the reduced surface area of steatosis observed in livers of mice in the HFDH group (Figure 3-D). These results suggested that the administration of

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600 mg/Kg BW BHLPE could improve the liver steatosis caused by a high fat diet. This effect was the same as that of other antioxidant or phenolic compounds previously reported (Jang, Park, Kim, Lee, Hwang, Park, et al., 2012; Park, Jung, Lee, Cho, Jung, Hong, et al., 2013). 3.4. Gene expression in liver As shown in Figure 4, a high-fat diet significantly decreased the relative gene expression levels of the transcription factor NF-E2-related factor 2 (Nrf2), glutathione peroxidase (Gpx), SOD, heme oxygenase 1 (HO-1) and lipoprotein lipase (LPL) in the HFD group compared with those in the NFD group. However, no significant differences in NAD(P)H quinone oxidoreductase1 (NQO-1) expression were noted between the NFD and the HFD group. The relative gene expression levels of Nrf2, Gpx, SOD, HO-1, NQO-1 and LPL in the HFDH group increased by 78.65%, 113.69%, 38.06%, 127.96%, 20.08% and 56.35% compared with those of the HFD group, respectively (p<0.05) (Figure 4). The relative gene expression of Nrf2, Gpx, SOD, and HO-1 in the HFDL group increased by 59.64%, 96.76%, 21.43%, and 83.31% compared with the HFD group, respectively (p<0.05) (Figure 4). However, the relative gene expression levels of NQO-1 and LPL in the HFDL group were not significantly altered compared with the levels in the HFD group (Figure 4). Nrf2 plays an important role in the transcription of antioxidant enzymes, and its increased level certainly collaborated to raise the expression of SOD, CAT, and Gpx, resulting in higher activities of these enzymes (Vicente, Ishimoto, & Torres, 2014). Increased levels of cytosolic Nrf2 promote its translocation to the nucleus, activating the transcription of ARE-responsive genes (Volz, Boettler, Winkler, Teller, Schwarz, Bakuradze, et al., 2012). A study showed that a high fat diet decreased the expression of Nrf2 and its target genes and that the reduced expression was related to increased oxidative stress in the liver of rats.(Tanaka, Aleksunes, Yeager, Gyamfi, Esterly, Guo, et al., 2008) Some natural compounds, such as quercetin, curcumin, or blueberry, were shown to increase the mRNA expression of antioxidant enzymes such as HO-1 and NQO1 via the activation of Nrf2 (Y. P. Wang, Cheng, Zhang, Mu, & Wu, 2010). In our study, the results showed that BHLPE increased the expression of antioxidant genes, including Nrf2, Gpx, SOD, 17

HO-1, NQO-1and LPL. 4. Conclusions In summary, BHLPE was found to have strong superoxide radical, hydroxyl radical and DPPH scavenging activity, a high ferric reducing antioxidant power and a moderate metal ion chelating activity. Our data clearly demonstrated that mice that were administered 600 mg/kg BW BHLPE showed significantly decreased serum levels of TC, LDL_C and AI, and markedly increased HDL_C levels, compared with mice in the HFD group. The antioxidant defense system and gene expression were improved after the oral gavage of BHLPE. These results suggest that BHLPE has potent antioxidant activity and might be utilized as a novel natural antioxidant in food and therapeutics.

Conflicts of interest

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Time (min) Figure 1. Phenolic compounds in BHLPE analyzed by HPLC (280nm). A: Standard catechin；B: Standard p-coumaric acid; C: Standard freulic acid; D, BHLPE.1, catechin (8.670min); 2, p-coumaric acid (17.311min); 3, freulic acid (17.663min)

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Figure 2. In vitro antioxidant activities of BHLPE in different concentrations A, DPPH radical scavenging ability ; B, Ferric reducing antioxidant power assay; C, super-oxide radical scavenging activity; D, hydroxyl radical scavenging activity; E, metal-ion chelating activity.

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Figure 3. Effect of BHLPE administration on liver histology in mice fed with a high-fat diet (HE,100×). A:NFD; B:HFD; C: HFD+ BHLPE（300mg/kgBW）; D: HFD+ BHLPE（600mg/kgBW）

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Figure 4. Sequences of primers used in quantitative real-time reverse transcription PCR and effects of BHLPE on antioxidant gene expression in liver (n=10)

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Highlights


Ferulic acid and p-coumaric acid were the major phenolic acids in BHLPE




BHLPE was found to have strong superoxide radical, hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, ferric reducing antioxidant power and moderate metal ion chelating activity.




Mice that were administered 600 mg BHLPE/kg body weight showed significantly improved lipid status, antioxidant defense system and antioxidant gene expression compared with mice in the HFD group.

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