Hepatoprotective Effects of Pleurotus Ostreatus Protein Hydrolysates Yielded by Pepsin Hydrolysis

: Pleurotus ostreatus protein extract (POPE) was prepared by the alkali precipitation method with 0.3% (w/v) NaOH. POPEP-III with a MW of 3000–5000 Da was acquired by pepsin enenzymatic hydrolysis. POPEP-III displayed noteworthy effects of 1,1-diphenyl-2-picrylhydrazyl DPPH and H 2 O 2 scavenging activities, Fe 2+ chelating ability, lipid peroxidation inhibition capacity, and metal reducing power. The administration of POPEP-III in mice significantly prevented prior CCl 4 -induced strengthen serum ALT and AST activities, changing from 365.44 ± 36.87 IU/L to 220.23 ± 22.27 IU/L and 352.52 IU/L to 206.75 ± 17.26 IU/L, respectively (p < 0.001), and suppressed hepatic malondialdehyde (MDA) formation from 15.28 ± 3.47 nmol/mg prot to 10.04 ± 2.06 nmol/mg prot (p < 0.001). Mice treated with POPEP-III demonstrated augmented activities of superoxide dismutase (SOD) in the liver, from 187.49 ± 19.81 U/mg prot to 233.35 ± 34.23 U/mg prot, and of glutathione peroxidase (GSH-Px), from 84.01 ± 14.54 U/mg prot to 115.9 ± 16.57 U/mg prot (p < 0.05). POPEP-III also prevented CCl 4 -induced oxidative liver histological alteration. The results suggest that POPEP-III can protect the liver from CCl 4 -induced oxidative damage.


Introduction
As one of the most important organs, the liver plays crucial roles in protein, lipid, saccharide, and drug metabolism in the human body [1,2]. When the liver is exposed to excessive dietary fats, sugars and proteins, alcohols, or drugs, the heavy burden applied on the liver creates difficulty and might lead to liver diseases. In liver metabolism, some of these substances are transformed into free radical-mediated ROS by cytochrome P450 [3]. Liver diseases are common, inducing death at a high ratio of approximately 2% of all death [4]. Liver diseases, such as subclinical icteric hepatitis, hepatic fibrosis, cirrhosis and hepatocellular carcinoma, are related to free radical-mediated ROS.
ROS can be generated in the liver mitochondria [5] and participate in the process of liver disease induction and development [6][7][8].
Free radical-mediated reactive oxygen species (ROS) have attracted appreciable attention based on their role in liver diseases and current methods for scavenging of ROS. CCl4, a classical hepatotoxin, is reductively de-halogenated by cytochrome P450 to trichloromethyl free radicals (CCl3OO· or ·CCl3 ) and depletion of antioxidant enzymes in hepatic parenchyma cells and initiates the process of lipid peroxidation [8]. To protect the liver from damaging oxidation, numerous antioxidants have been explored. Aqueous, alcoholic, or supercritical CO2 extracts with antioxidant activity from selected medicinal herbs demonstrated hepatoprotective effects, although the substances in these extracted mixtures are structurally unclear [9][10][11][12]. One antioxidant chemical, silymarin from the Silybum marianum seeds, is commonly applied for hepatoprotection [13] and has side effects that include gastrointestinal complaints. Currently, mushrooms are increasingly under examination for their hepatoprotective values. Among the mushrooms explored for hepatoprotective substances, polysaccharides were ranked as the predominant component, and certain mixtures extracted from mushrooms by alcohol, water or other solvents have become topics of research [14][15][16]. Notably little research has focused on the hepatoprotective peptides from mushrooms [17], especially the hydrolysates from mushroom proteins.
Pleurotus ostreatus is widely cultivated around the world and is reported to contain bioactive compounds that can protect health due to their antioxidant properties. Among these compounds, P. ostreatus polysaccharides have attracted increasing attention [18,19]. Selected compounds extracted by ethanol from P. ostreatus showed high antioxidant activities, but the constituent was unclear [20,21]. An extraction with water from P. ostreatus solid culture presented immune-nutritional recovery [22]. Polysaccharides from P. ostreatus are of interest due to their multiple bioactivities, including antitumor, anti-inflammatory and cytotoxic effects [23]. Proteins in P. ostreatus have many bioactivities, including anti-tumor effects and use as an adjuvant for HBV DNA vaccine [24,25]. However, most of the previous works were relative to the nutritional evaluation for mushroom proteins, and few current studies focus on the bioactive natural component of low molecular weight peptides with multi-action bioactivity hydrolyzed by enzymes from the P. ostreatus proteins or other edible mushrooms. As the output of P. ostreatus is tremendous, it is very important to develop the procedure of health-keeping to extend the industry chain, and improve people's health with the natural products respectively. In our study, the antioxidant activity of P. ostreatus protein hydrolysates obtained with five proteases was investigated in vitro using biochemical assays. The highest antioxidant activity fractionated hydrolysates were refined through ultrafiltration. Moreover, the fraction with the highest antioxidant activity was investigated via an in vivo model of CCl4 intoxication. Physical characteristics, such as the degree of hydrolysis and amino acid composition of the hydrolysates fractions, were explored.

Degree of Hydrolysis
Different efficiency was acquired in hydrolyzing POPE for the peptide bonds, as the five enzymes recognize different bonds and function differently. Among pepsin, trypsin, dispase, papain, and bromelin, pepsin had the highest efficiency in POPE hydrolysis ( Table 1). The statistics indicated a mild DH of 16.01 ± 0.12% after POPE incubation with pepsin. Hydrolysis proceeded for 1 h at 37 °C and with pH 2.0. DH is defined as the percentage of peptide bonds cleaved. It was well established that DH can affect the functionality of hydrolysates, including antioxidant activity or reducing power. Generally, these effects become more evident when DH is modified more extensively.

Amino Acid Composition
The amino acid compositions of the hydrolysates suggested that they are rich in Leu, Phe, Glu, Met, Ala, Val, and Asp (Table 2). In addition, the THAA content in POPEP-III was higher than the other three fractions. For protein hydrolysate and peptides, the higher content in hydrophobicity the higher solubility in lipid they have. Moreover, high solubility enhances their antioxidant activity [26]. These highest antioxidant activities were presumably because of the highest content of THAA.

Preparation of Protein Hydrolysatess and Antioxidant Activity
Five different proteases, i.e., pepsin, trypsin, dispase, papain and bromelin, originating from different sources were used in the enzymatic hydrolysis of POPE. The antioxidant activities of the hydrolysate hydrolyzed by each protease are shown in Figure 1 and Table 3. Among the hydrolysates, POPEP derived from POPE hydrolyzed by pepsin showed the strongest antioxidant activity. This suggested that when disrupted by proteases, the active amino acids of protein might be exposed, and the antioxidant activity increases [27].  POPEP was further separated using three kinds of UF membranes (10,000, 5000, and 3000 Da MWCO) according to molecular size, and four kinds of fractions, POPEP-I (>10,000 Da), POPEP-II (5000-10,000 Da), POPEP-III (3000-5000 Da) and POPEP-IV (<3000 Da) were obtained. POPEP-III had the highest antioxidant activity among the four fractions ( Figure 2, Table 4). The scavenging activity of POPEP-III for DPPH and H2O2 was the highest among the samples. The reducing power and Fe 2+ chelating activity of POPEP-III exhibited the highest antioxidant activity among these samples. However, all the samples had no significant differences in inhibitory effects on lipid peroxidation. POPEP-III has certain similarities with peptides with alcalase-derived hydrolysate from alfalfa leaf protein [28]. Considering that POPEP-III was found to possess the highest antioxidant activity, this fraction was analyzed for molecular weight distribution. The chromatographic data suggested that POPEP-III was composed of molecular weights of 4076 Da and 3287 Da two major fractions.

Effects of POPEP-III on Liver Weight, HI and Body Weight in Mice
The changes in liver weight, HI and body weight, in different experimental groups of mice are shown in Table 5. The liver weight (p < 0.05) and HI (p < 0.05) increased significantly compared with the normal control after CCl4 treatment. However, it could be mitigated by pretreatment with POPEP-III, similar to the results of pretreatment with bifendate. Additionally, from the data, we note that the HI was partially dose-dependent on POPEP-III. A decrease in body weight was observed in the CCl4-treated groups (p < 0.05), but to a certain extent, POPEP-III could alleviate the weight decrease.

Effects of POPEP-III on AST, ALT, MDA, GSH-Px, and SOD Activities
Some acquainted enzymes in serum, such as ALT and AST, were used as biochemical criteria for early acute hepatic damage. As displayed in Figure 3 and Table 6, CCl4 treatment significantly increased the activities of ALT and AST in serum. The activity of ALT is 24.68 ± 5.64 IU/L and that of AST is 44.62 ± 5.25 IU/L in group I (the normal group). However, the activities of ALT and AST are 365.44 ± 36.87 IU/L and 352.52 ± 13.44 IU/L respectively, in group II treated with CCl4 only (p < 0.001). The pretreatment of POPEP-III reduced the activities of ALT and AST in a dose-dependent manner as caused by CCl4 (p < 0.05 vs CCl4-intoxicated group II). At a dose of 200 mg/kg.bw POPEP-III reduced the ALT and AST activities to 220. 23    CCl4 affected the antioxidant parameters of the liver tissue of mice. As shown in Figure 4a and Table 7, MDA, the product of membrane lipid peroxidation, its levels increased from 5.28 ± 1.55 nmol/mg prot in group I to 15.28 ± 3.47 nmol/mg prot (p < 0.001) in group II. When treated with POPEP-III (100 mg/kg, 200 mg/kg), levels of MDA decreased to 12.07 ± 2.16 nmol/mg prot in group IV and to 10.04 ± 2.06 nmol/mg prot in group V, but all are higher than the level of group III treated with bifendate. More interestingly, the level of group VI treated with POPE was the same as that of group II and did not demonstrate positive effects. These results indicate that hydrolysatess are more effective in membrane protection than unhydrolysated POPE but less effective than bifendate.
SOD and GSH-Px protect liver cells from oxidative damage by scavenging superoxide anions. In this work ( Figure 4B,C and Table 7), an apparent decrease was observed in the SOD level from 237.65 ± 36.06 U/mg prot (p < 0.01) (Group I) to 187.49 ± 19.81 U/mg prot (Group II) and in GSH-Px level from 152.11 ± 12.58 U/mg prot (p < 0.01) (Group I) to 84.01 ± 14.54 U/mg prot (Group II). When treated with POPEP-III at a dosage of 100 mg/kg, the contant of SOD and GSH-Px in Group IV are similar to the level of Group III treated with bifendate, but the contant of SOD and GSH-Px in Group V are lower compared with Group III. Similar to the effect of MDA adjustment, POPE did not demonstrate the positive effect.   Values are presented as the mean ± SD: (###) p < 0.001 compared with normal group; (***) p < 0.001, (**) p < 0.01, and (*) p < 0.05 compared with the CCl4-treated control group.

GSH-Px (U/mg prot)
As expected, a significant decrease of the MDA and increase of the GSH-Px and SOD enzyme activities occurred with pretreatment of POPEP-III. MDA content, SOD and GSH-Px activity reached 10.04 ± 2.06 nmol/mg prot, 233.35 ± 34.23 U/mg prot and 115.9 ± 16.57 U/mg prot (p < 0.05), respectively, and 100 mg/kg.bw of POPEP-III had a relatively slighter influence.

Histopathology Examination
In this seaction, histopathological observation of the liver was performed to supply further support for the biochemical analysis. Compared with the hepatic cellular architecture of mice tissue from the normal control group (Figure 5A), administration of CCl4 to mice resulted in extensive liver damage characterized by formation of large vacuoles, infiltration of inflammatory cells, severe cellular degeneration, necrosis and nuclear fragmentation ( Figure 5B). However, bifendate pretreatment effectively protected the liver against CCl4-induced damage ( Figure 5C), and pathological observation showed only hepatocyte swelling around portal areas. In addition, POPEP-III pretreatment showed slighter hepatic injury and ameliorated liver damage by diminution of large vacuole formation and attenuation of cellular degeneration ( Figure 5D,E). For the undigested protein POPE group ( Figure 5F), POPE pretreatment did not markedly improve CCl4-induced acute liver injury.

Chemicals and Reagents
The diagnostic kits for activity assays of ALT, AST, MDA, SOD, and GSH-Px were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Linoleic acid, DPPH, BSA, and ferrozine were purchased from Sigma-Aldrich (Steinheim, Germany). The UF membrane reactor system for fractionation of hydrolysate was sourced from Sartoyius AG (Goettingen, Germany). For the proteases used in enzymatic hydrolysis, pepsin, trypsin, and dispase were acquired from Sigma-Aldrich (Steinheim, Germany), while papain and bromelin were purchased from Solarbio (Beijing, China). All of the other chemicals used in this study were of analytical grade.

Preparation of Crude P. ostreatus Protein
The procedures used for isolating POPE are those from Souza [29] with some modifications. The fresh fruiting bodies of P. ostreatus were purchased from a local farm in Beijing. The preparation of P. ostreatus protein extract (POPE) involved a multistep procedure that entailed soaking treatment with deionized water, centrifugation, and extraction with NaOH solution. Fresh P. ostreatus fruiting bodies were homogenized in deionized water (1:1, w/v) whith a Waring blender. The homogenate was centrifuged (10,000× g, 4 °C, 30 min) after overnight extraction at 4 °C. The supernatant was added with 20% (w/v) NaOH solution until the concentration of NaOH reached 0.3% (w/v) at 25 °C. The dispersion was centrifuged at 10,000× g for 20 min at 4 °C after a 5-h stand at room temperature. The obtained precipitate was dialyzed in deionized water for 72 h to remove free NaOH and was subsequently lyophilized. This product was POPE.

Enzymatic Hydrolysis of POPE
The lyophilized POPE was dissolved in distilled water to reach a 0.05 g/mL concentration. The pH of the POPE solution was adjusted to the optimal condition for the enzymes used (Table 8). Each enzyme (pepsin, trypsin, dispase, papain, and bromelin) was added proportionally to the POPE solution according to the enzyme/substrate ratio based on the original form of specific enzyme. At the end of the incubation, the solution was heated at 100 °C for 15 min to deactivate the enzyme. Hydrolysate was subsequently obtained by centrifugation (10,000× g, 4 °C, and 10 min). hydrolysate was lyophilized for 96 h at −70 °C and 0.04 Mbar. All hydrolysates powders were stored at −20 °C for further analysis.

Ultrafiltration
POPEP was sequentially filtrated using three different MWCO ranges UF membranes of 10,000, 5000, and 3000 Da, respectively. The fractions were designed as follows: POPEP-I with MW distribution >10,000 Da, POPEP-II with MW distribution of 5000-10,000 Da, POPEP-III with MW distribution of 3000-5000 Da and POPEP-IV with MW distribution <3000 Da. All four fractions of POPEP were lyophilized. After that, the four fractions were stored at −20 °C for further analysis.

Degree of Hydrolysis (DH)
Protein sample solubility was evaluated according to the method of Saeed [30]. Aqueous POPEP solution (1% w/v) was prepared at pH 7 (adjusted with either 2 M HCl or NaOH). Solutions were stirred at room temperature for 30 min and centrifuged at 1200× g for 10 min at 4 °C. The protein concentration of the supernatant was determined according to the Lowry method [31]. Solubility was expressed as the percent of total protein of the original sample.

Analysis of Amino Acid Composition and Molecular Weight Distribution of POPEs
The lyophilized fractions POPE, POPEP-I, POPEP-II, POPEP-III and POPEP-IV were digested in HCl (6.0 M) solution at 110 °C for 24 h under nitrogen atmosphere. An automated amino acid analyzer (L-8800; Hitachi High Technologies Corp., Tokyo, Japan) equipped with a visible analytical detector was used before post-column derivatization with ninhydrin. Each sample (1 µL) was injected into an analytical column (sulfonic cation-exchange resin, 4.6 mm i.d., 60 mm length, 3 µm particle size; Tokyo, Japan) at 135 °C with detection at 570 nm. An amino acid standard solution containing 16 amino acids was used as the internal standard.
The POPEP fraction which has the highest antioxidant activity was further analyzed in terms of molecular weight distribution by gel filtration chromatography. The peptides were loaded onto a Sephadex G-25 column (1.0 cm × 10 cm, GE-Healthcare, Boston, MA, USA) and eluted with deionized water at a flow rate of 0.2 mL/min. The fractions were collected and monitored at 220 nm. A molecular weight calibration curve was obtained from the following standards: vitamin B12 (1,355 Da), L-glutathione (oxidized) (612 Da), L-tyrosine (181 Da), and BSA (6,700 Da).

Free Radical Scavenging Activity
The capacity of all protein hydrolysate fractions tracking free radicals was determined using DPPH [32,33]. The scavenging activity of hydrolysate samples is showed as the percentage disappearance of DPPH relative to a control mixture with deionized water instead of the sample. The mathematical calculations followed Equation (1): Scavenging of DPPH radical (%) = ((Acontrol − Asample)/Acontrol) × 100% (1) where Acontrol is the absorbance of the system using deionized water instead of the sample, and Asample is the absorbance of the system in the presence of the tested sample.

Hydrogen Peroxide Scavenging Activity
The ability to scavenge hydrogen peroxide (H2O2) was measured following Ganie [34] with some medications. The detection system included 200 µL of phosphate buffer (0.1 M, pH 7.4), 40 µL of H2O2 solution (0.3%), and 100 µL of sample solution (5 mg/mL), and the absorbance was measured at 230 nm after 10 min. The scavenging of H2O2 was calculated as similar to Equation (1).

Reducing Power
The reducing power of the peptide samples was measured following our previous work [35]. An amount of 1 mL of 5 mg/mL sample solution was mixed with 1 mL of 0.2 M phosphate buffer saline (pH 6.6) and 1 mL of 0.1% K3Fe(CN)6 solution and incubated at 50 °C for 20 min. Then, 1 mL of 10% trichloroacetic acid solution was subsequently added. The mixture was centrifuged at 3000× g for 10 min. Next, 1 mL of the upper layer was mixed with 1 mL of distilled water and 0.2 mL of 0.3% FeCl3 solution, and the absorbance was measured spectrophotometrically at 700 nm.

Inhibitory Effect on Lipid Peroxidation
The inhibitory activity on lipid peroxidation was determined using a slightly modified method of conjugated diene [36]. First, 25 µL of sample (5 mg/mL) was incubated with 500 µL of 10 mM linoleic acid emulsion in 0.2 M sodium phosphate buffer (pH 6.6) in the dark at 37 °C for 24 h. Second, 1.5 mL of 60% methanol was added. Finally, the mixture absorbance was measured at 234 nm. The inhibitory activity on lipid peroxidation was measured as similar to Equation (1).

Ferrous Ion Chelating Activity
The chelating activity on Fe 2+ was detected following the procedure described by our earlier study [37] with some modifications. First, 200 µL of samples (5 mg/mL) was mixed with 550 µL water and 10 µL ferrous chloride (2 mM). The reaction was initiated by adding 40 µL of ferrozine (5 mM). The reactant was shaken to evenly mix and held at 25 °C for 10 min. Finally, the mixture absorbance was measured at 562 nm against a blank. The Fe 2+ chelating activity was expressed as similar to Equation (1).

Animal Experiments
Male Kunming mice (22-25 g) were obtained from Beijing HFK Bioscience Company. The animals were housed for 7 days to acclimatize them to the environment and temperature (20-25 °C) with a 12 h light/dark cycle. The animals were fed with a standard diet and given water ad libitum.
The in vivo hepatoprotective activity of the POPEP-III was examined against CCl4-induced hepatotoxicity in Kunming mice. Forty-two mice were randomly allocated into six groups (I-VI), with seven mice in each group as follows: All groups accepted allocated intragastric administration treatment for 10 successive days. In the normal group and control group, animals received formal saline at a dosage of (12 mL/kg, i.g.) once daily, and the mice in bifendate group were treated with bifendate (200 mg/kg in distilled water, i.g.) once daily. In the three remaining experimental groups, the mice were pretreated with POPEP-III (100 and 200 mg/kg, i.g.) and POPE (200 mg/kg, i.g.) once daily. On the 11th day, all mice except those in group I received an intraperitoneal gastric perfusion of a CCl4/olive oil mixture (2%, 12 mL/kg, i.g.), and the normal control group received an equal amount of olive oil alone [38].
All mice were treated with anesthetic via intraperitoneal injection of pentobarbital sodium (30 mg/kg). Approximately 1 mL of blood was drawn from the mouse abdominal artery. The blood was allowed to coagulate for 30 min at room temperature. The serum was isolated by centrifugation at 978× g for 10 min at 4 °C and used in biochemical index detection. The removed livers were washed repeatedly with saline until clean. A small portion of the liver was conserved in 10% (v/v) formaldehyde for further histopathological analysis, and the remaining liver was homogenized with pre-cooled saline (1:9, w/v) for biochemical index detection.

Biochemical Assays
The activities of the serum ALT and AST enzymes were assayed according to the guidelines of the diagnostic kits, and the results are showed in IU/L. The liver homogenate in saline solution (1:9, w/v) was centrifuged at 8000× g for 10 min at 4 °C, and the supernatant was collecte. The supernatant was stored at −20 °C. The activities of SOD, GSH-Px, and MDA content and hepatic protein concentrations in the liver were measured according to the diagnostic kits instructions. The activities of SOD, GSH-Px, and MDA content were normalized with reference to protein, and the results are showed in units per milligram of protein, units per milligram of protein, and nanomoles per milligram of protein. The HI was calculated as the Equation (2): HI = (Liver weight/Body weight) × 100% (2)

Histopathological Study on the Liver
The fresh liver was removed from each mouse after dissection, and liver tissue was fixed whith 10% formalin solution. Then the fixed liver tissues were dehydrated in alcohol and embedded in paraffin using the standard microtechnique. Sections (1.5 cm × 1.5 cm × 5 µm) stained with hematoxylin and eosin (H&E) were observed under light microscopy (×20 magnification) for histopathological studies [39].

Statistical Analysis
Experimental results are expressed as the mean ± SD (standard deviation). The p-values < 0.05 were treated as statistically significant (SPSS, version 20.0).

Conclusions
In this study, we obtained a peptide named POPEP-III from P. ostreatus by pepsin hydrolysis. POPEP-III exhibit better antioxidant activity in vivo and in vitro. It can significant increase the activity of SOD and GSH-Px, and decrease the content of MAD in serum in mice prior to CCl4. Serum ALT and AST are index of liver, many liver diseases could lead the serum ALT and AST level increase. In our study, POPEP-III can significance decrease the activity of AlT and AST induced by CCl4. The results demonstrate that the POPEP-III could be a liver protect reagent to protect the liver from oxidative damage.
In summary, we hydrolyzed proteins from P. ostreatus fruiting bodies using pepsin and tested the hepatoprotective effects of POPE and POPEP hydrolysate for potential application in health support. The experiments showed that as an antioxidant ingredient, POPEP-III plays a role by halting free radical chain reactions. Additionally, POPEP-III promoted the activities of SOD and GSH-Px, decreased the level of MDA, and protected the liver from the attacks of free radical-mediated ROS.