Chemical composition and hepatoprotective effect of free phenolic extract from barley during malting process

In this study, No.Ganpi4 of barley was steeped and malted to investigate the changes of phenolic compounds during malting process. The free phenolic extract from raw barley (FPEB) was analyzed by HPLC and predominant compounds were (+)-catechin, protocatechuate and quercetin. The FPEB was evaluated for hepatoprotective effect in vivo and in vitro. Intragastric administration of FPEB (100, 200 and 400 mg/kg/bw) to mice significantly weakened the effects of hepatic damage induced by CCl4 toxicity on serum markers, including serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total-bilirubin, total cholesterol and total triglycerides. FPEB administration also increased the hepatic levels of antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. Histopathological examinations further confirmed that FPEB could protect the liver from CCl4-induced damage. In vitro, the experimental results demonstrated that FPEB could reduce BRL hepatocyte apoptosis and damage induced by CCl4. These results suggest that FPEB exerts an effective protection for hepatic injury, and barley has the potential as a functional food to prevent hepatic injury.

HPLC analysis of free phenolic extract. In order to intuitively compare phenolic changes, HPLC chromatogram of extracts for raw grains and sample after 48 h of germination are shown in Fig. 2. It can be seen that nine phenolic compounds such as gallic acid, protocatechuate, (+)-catechin, vanillic acid, caffeic acid, (−)-epicatechin, p-coumaric acid, ferulic acid, and quercetin were determined. Meanwhile, although some peaks are strongly response on the UV absorbance at 280 nm, they were not characterized in the present study. For unambiguous identification further studies are required by using authentic compounds. We found there were some new arisen substances while some intrinsic phenolic compounds increased or decreased, and even disappeared after germination. The RP-HPLC quantitative analytical result of the phenolic compounds are shown in Table 1. The data showed that (+)-catechin, protocatechuate, quercetin, ferulic acid and gallic acid were the main phenolic compounds. The (−)-epicatechin, (+)-catechin and p-coumaric acid decreased significantly after the steeping stage (p < 0.05). Xu et al. 29 showed a significant decrease of different phenolic acids on oat during steeping; Lu et al. 18 also reported a significant decrease on Gan 4 barley during steeping. Enzymes at germination could stimulate the materials conversion or biosynthesis; meanwhile germination softened the tissue of cell walls and led to easier extraction and better releases of phenolic compounds 30 . In terms of raw grains, almost all monomer phenolic acids were the highest. Steeping caused significant decline of most phenolic acids, especially, p-coumaric acid and caffeic acid contents even declined to lower limit of detection. After steeping, most of the phenolic acids contents reached the highest at G48, and the protocatechuate content at G48 was 2.18 times in comparison with raw grains. Table 1 showed (+)-catechin was the leading phenolic acid; the second was protocatechuate in barley. Lu et al. 18 also concluded (+)-catechin was the most abundant phenolic compound, followed by ferulic acid in the raw barley and corresponding malts.

Hepatoprotective effects of FPEB in vivo.
Effect of FPEB on liver indices. The effects of the FPEB on the liver indices are shown in Table 2. Compared with the control group, the mice in the CCl 4 -treated group had a significant increase in liver index (p < 0.01), indicating that their hepatic tissue have been severely damaged, and that the livers were swollen as a result of exposure to CCl 4 toxicity, which was supported by previous study 24 . However, the group treated with three doses of FPEB for 3 weeks showed a significantly lower (p < 0.05) liver index compared with that of the CCl 4 -treated group, which revealed that the administering of the tested FPEB had a preventive effect against CCl 4 -induced hepatic damage in the mice.
Effects of the FPEB on serum AST, ALT, TC, TG, AKP and albumin activity levels. Hepatic function is often measured by serum ALT and AST enzymatic activities, and there are still other important indexes, such as TC, TG, AKP and albumin level in serum to assess hepatic function. As depicted in Table 3, the serum AST and ALT enzymatic activities of the CCl 4 -treated group sharply increased from 28 to 109.0 U/L (p < 0.05) and from 28.0 to 114.3 U/L (p < 0.05) compared with the control group, respectively. Treatments with FPEB at different dosage reduced the AST and ALT activities (p < 0.05). The results exhibited dose-effect relationship, and high-dosage   (400 mg/kg/bw) showed particularly better effect, which is even close to the BP group compared with those of the CCl 4 -treated group. Results of serum TC and TG levels are also shown in Table 3. The serum TC and TG level was significantly increased in the CCl 4 -treated group compared with the normal control group (p < 0.05). FPEB pretreatment at a dose of 100, 200 and 400 mg/kg prior to CCl 4 injection markedly prevented the elevation (p < 0.05) of TG. However, improvement effect of FPEB at low-dosage (100 mg/kg/bw) on TC level was not obvious, only slightly lower compared with the CCl 4 -treated group. Nonetheless, FPEB pretreatment at 200 and 400 mg/kg/bw relatively had a good protective effect on the serum TC and TG levels, similar to that of the reference compound BP. As shown in Table 3, in the control group, the serum AKP and albumin levels were 11.8 U/L and 36.7 g/L, respectively. After CCl 4 exposure, serum AKP markedly increased by 43.3% while albumin levels decreased by 35.5% (p < 0.05). However, the CCl 4 -induced elevation of these values was significantly weakened by pretreatment with FPEB at 200 and 400 mg/kg/bw. Pretreatment with FPEB at a low dose of 100 mg/kg/bw led to a slight change in AKP and albumin levels, yet, it was insignificantly different from that of the CCl 4 -treated group (p > 0.05).
Effects of FPEB on SOD, CAT, GPx and lipid peroxidation. Antioxidant enzymes, such as GPx (glutathione peroxidase), CAT (catalase) and SOD (superoxidedismutase) play an important role in effectively suppressing free radical oxidation damage in human metabolism. Furthermore, they are also the major elements in human bodies that increase immunity, prevent diseases and balance the metabolism process of oxygen free radical and lipid peroxidation reaction. Therefore, the activities of these antioxidant enzymes are closely related to liver detoxification function 24 . As shown in Table 4, the levels of hepatic SOD, CAT, and GPx activities in the CCl 4 -treated group were significantly lower than those of the control group (p < 0.01). In the CCl 4 -treated group, the activity levels of the hepatic antioxidant enzymes SOD, CAT and GPx decreased respectively by 46.7%, 35.4% and 35.4% as compared to the control group. On the contrary, the groups treated with FPEB at three different doses significantly increased (p < 0.05) in levels of SOD, CAT and GPx activities, and the preventive effect of FPEB treatment at 400 mg/kg/bw was similar to that of BP treatment. In this study, lipid peroxidation was measured and expressed as nmol of malondialdehyde equivalents (MDAeq) per milligram of proteins according to kit instruction. The effects of FPEB pretreatment on the CCl 4 -induced alteration of MDA level are presented in Table 4. The liver lipid peroxidation level or MDA content was significantly increased in the CCl 4 -treated group compared with that of the control group (p < 0.05). However, the FPEB treatment at 400 mg/kg/bw markedly relieved the induced elevation in the lipid peroxidation level (p < 0.05), and exhibited a positive protective effect which similar to that of BP treatment.
Histopathological examination of mouse livers. Histopathological examination can provide firsthand evidence of the effects of the investigated components against acute CCl 4 -induced hepatic injury 7 . As shown in Fig. 3, hepatic tissue of the control group contained typical, regular and intact hepatic cellular structure, along with a well-preserved cytoplasm, a prominent nucleus and nucleolus and visible central veins (Fig. 3A). In contrast, hepatic tissue of CCl 4 treatment exhibited severe histological changes and damage with vague cellular boundary, extensively deformed hepatocyte and collapse of parenchyma (Fig. 3B). However, the hepatic histopathological    changes induced by CCl 4 exposure were gradually ameliorated and recovered by FPEB treatment with the increase of dose (Fig. 3D,E). The FPEB administered at 400 mg/kg/bw was obviously effective and even close to the positive control BP drug in hepatic protection effect. This is because the hepatic cells of high-dose and positive group had a comparatively regular appearance with a well-preserved cytoplasm and prominent nuclei (Fig. 3E,F), which showed that FPEB exerted a good hepatoprotective effect. The histopathological result also supported our above biochemical results regarding the serum hepatotoxic markers and hepatic oxidative stress systems.
Hepatoprotective effects of FPEB in vitro. The results of CCl 4 -induced injury and cell viability of BRL hepatocytes in vitro are presented in Table 5 according to Gao et al. 24 . Cell viability significantly decreased by 74.0% in the CCl 4 -treated group compared with the control group. There was a significant increase of cell viability in the FPEB-treated groups compared with that of the CCl 4 -treated group (p < 0.01), and also there were obvious dose-response way between cell viability and the tested concentration of FPEB. However, cell viability of FPEB-treated groups was far lower than that of control group. The LDH, ALT and AST activities in CCl 4 -treated BRL hepatocytes supernatants with the administration of FPEB were also evaluated. Similarly, ALT and AST are important indicators of liver injury as previously mentioned 31 . As shown in Table 5, the AST, ALT and LDH activities were significantly higher in the supernatants of the CCl 4 -treated group compared with those of the control group (p < 0.01) and the LDH, AST and ALT levels was increased by 168.8%, 330.7% and 230.6% respectively. However, pretreatment with FPEB at 25, 50 and 100 μg/mL reduced the elevation of the AST, ALT and LDH activities caused by CCl 4 treatment (p < 0.01) in a dose-dependent manner. No difference in cell viability, LDH, ALT and AST activities was found between BP and treatment group with 100 μg/mL FPEB. This strongly proved  23,24,33 . Accordingly in this study, a relatively higher phenolic compound existed in FPEB could explains the positive or protective ability of FPEB against the hepatic injury caused by CCl 4 exposure in mice and BRL hepatocytes. However, after being ingested, the metabolism of polyphenol compounds resulted in the formation of new and often completely different compounds compared to the ingested molecules in the body 34 . Therefore, the hepatoprotective effect of free phenolic extract from barley still needs to be further studied by in vivo experiments.

Conclusion
In conclusion, the data obtained from this study indicates that steeping and malting resulted in significant decreases in FPC compared to raw grains. The (+)-catechin, protocatechuate, quercetin, ferulic acid and gallic acid were the main phenolic compounds in barley. Additionally, the result of the animals experiment indicates that pretreatment with FPEB was effective in the prevention of oxidative stress and liver damage induced by CCl 4 in rats, as revealed by the marked decrease in the hepatic lipid peroxidation content, the reduced serum AST, ALT, TC, TG and AKP levels, the enhanced hepatic SOD, CAT and GPx activities. Furthermore, histopathological morphologic improvement also demonstrates the beneficial effects of FPEB pretreatment. The results in vitro experiment also indicates that FPEB could decrease BRL hepatocyte apoptosis and damage induced by CCl 4 . Therefore, the phenolic extract from barley could be used as a hepatoprotective agent. This is the first report on the hepatoprotective effect of phenolics from barley. These results are expected to provide a scientific basis for the further development and exploitation of barley resource. Preparation of barley samples. Barley cultivar (Hordeum uulgure) Ganpi4, a naked variety, was obtained from Lanzhou in Gansu province during the crop year. Barley grains were selected in whole grain, sterilized using a 0.05% solution of sodium hypochlorite for 30 s, and then washed three times with deionized water before steeping. The grains were spread out on clean and humid gauze in thermostat incubator (HWS-280, Hangzhou Huier Instruments, Zhejiang, China) at 16 °C for 12 hours. The grains were marked as steeping sample. Other barley grains was continued to sprout for an established time, and the samples were respectively taken out from incubator at 24 h, 48 h, 72 h and 96 h, which were successively marked as G24, G48, G72 and G96. After the treatment mentioned above, samples were oven-dried at 35 °C temperature, and then milled with a micro plant grinding machine (HuaChen1000; Zhejiang, China) to a powder about 0.5 mm. Samples with different treatment were stored at −40 °C prior to analysis. Raw grains were also freeze-dried and used as reference samples in all performed analyses.

Materials and methods.
Extraction of free phenolic compounds. Free phenolic compounds were extracted according to the method with minor modifications 35 . Briefly, 5 g milled samples was defatted with 50 mL of hexane at room temperature by a rotary shaker (HY-5, Jintan billion electronics co., Ltd., Jiangsu, China.) and then treated with 25 mL of methanol for 20 min in the rotary shaker. The homogenates were centrifuged (Eppendorf5810R, Germany) at 4000 g for 10 min. The supernatant was removed following centrifugation while extraction of the precipitate was repeated twice. The three supernatants were collected, combined, and evaporated to dryness at 45 °C under   Table 5. Effects of pretreatment with FPEB on cell viability and LDH, AST and ALT activities of BRL hepatocyte . Values represent means of three independent replicates ± SD. Different letters within a column indicate statistically significant differences between the means (p < 0.01).

Analytical methods. Determination of phenolic content. Phenolic content was determined in terms of
Folin-Ciocalteu colorimetric method described by Payet et al. 36 with minor modifications. The 0.5 mL diluted extracts or standard solutions was mixed with 2.5 mL deionized water and 0.5 mL 1.0 M Folin-Ciocalteu reagent in volumetric flask. After 10 minuets, 1.5 mL 7.5% sodium carbonate was added and mixed thoroughly. After two hours of incubation at room temperature, the absorbance at 765 nm was detected with a UV-2000 spectrometer (Hitachi). Methanol was used as the blank and gallic acid (GA) was used for calibration of standard curve (0-10 μg/mL). The total phenolic content was expressed as micrograms of gallic acid equivalents (GAE) per gram of dry weight (µg of GAE/g of dw). After three days of environmental adaptation, the mice were randomly divided into the normal control, CCl 4 -intoxicated, the positive bifendate (BP) group and three experimental groups. The three test groups were respectively given 100, 200, or 400 mg/kg/bw of FPEB (0.3 mL, ig) once daily for 21 consecutive days. The animals from the normal and CCl4-intoxicated groups were also given the same volume of physiological saline, and the positive bifendate (BP) group received 200 mg/kg/bw reference drug BP (0.3 mL, ig) once daily for 21 consecutive days. On the 22nd day, all the groups except control group were given a 1% CCl 4 /peanut oil dissolved in peanut oil (v/v) by intraperitoneal injection (ip, 0.3 mL), while the control group mice merely received peanut oil. After two hours, all the groups were fasted, but allowed water and libitum as usual, for 24 h. The mice were then sacrificed for obtaining blood and livers. Serum was separated by centrifugation at 3000 rpm for 10 min, and then stored at −20 °C until analysis. A liver was dissected out from each mouse and washed immediately with ice cold saline to remove as much blood as possible, and then stored at −40 °C until further analysis. The liver indexes were calculated based on records of body weight, and the corresponding liver weights of every mouse (liver index = liver weight/body weight × 100%).

HPLC
Determination of Biochemical Parameters in Serum-AST, ALT, TC, TG, AKP and albumin. Liver damage was assessed by estimating the enzymatic activities of serum AST, ALT and AKP, as well as serum TC, TG and albumin level, using the corresponding commercial kits (Nanjing Jiancheng Institute of Biotechnology, China), respectively. The results were expressed according to the kit instruction.
Determination of antioxidant enzyme activities. Liver homogenate 10.0% (w/v) was prepared with frozen normal saline and centrifuged at 3000 rpm for 15 min. The homogenate supernatant was used for the measurement of SOD, CAT and GPx. All of these enzymes were determined according to the instructions on the commercial kits (Nanjing Jiancheng Institute of Biotechnology, China). SOD and CAT were expressed as units/mg protein (U/ mgprot) and GPx was expressed as μmol/gprot.

Determination of lipid peroxidation and parameters of hepatic function in Liver.
Lipid peroxidation was measured according to MDA diagnostic kits (Nanjing Jiancheng Institute of Biotechnology, China). The results were expressed as nmol/mg protein. The Parameters (TC and TG) of hepatic function was measured using commercially available diagnostic kits, and the results were expressed as mmol/gprot.
Histopathological assessment of liver damage. Samples of liver tissues were fixed in 10% neutral buffered formalin for 24 h. Specimens were dehydrated with ethanol solution of different concentration, and embedded in paraffin; these specimens were also sectioned at 5 μm and stained with hematoxylin-eosin (H&E) for histopathological examination. The histopathological changes in the sections were observed by a light photomicroscope. Finally, the images were examined and evaluated for pathological change analysis Determination of the hepatoprotective effect of FPEB in vitro. Determination of cell viability. BRL hepatocytes were seeded at 2.5 × 10 4 /mL, and 100 μL was added in each 96-well plate. After 12 h of incubation, 100 μL of different concentrations of FPEB (100, 50, 25 μg/mL, in DMEM containing 0.1% dimethyl sulphoxide) was added. The CC1 4 -treated group and the control group were given 100 μL of DMEM. Then, after 12 h of incubation, 50 μL PBS was added to the control group, while the other groups were given 50 μL CC1 4 (100 mM) to induce cell injury. 6 h later, cell viability was determined. Cell viability was assessed by microculture tetrazolium assay and the results were expressed as percent cell viability.
Determination of the LDH, AST and ALT activities in supernatants. BRL hepatocytes were seeded at 2.5 × 10 4 / mL, and 500 μL was added in each 24-well plate. Following 12 h of incubation, 500 μL of different concentrations of FPEB (100, 50, 25 μg/mL, in DMEM containing 0.1% dimethyl sulphoxide) was added. The CC1 4 -treated group and the control group were given 500 μL of DMEM. After 12 h of incubation, 250 μL PBS was added to the control group, while the other groups were given 250 μL CC1 4 (100 mM) to induce cell injury. The LDH, AST and ALT activities in supernatants were determined after 6 h.