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Characterizations and hepatoprotective effect of polysaccharides from Mori Fructus in rats with alcoholic-induced liver injury
Accepted Manuscript Title: Characterizations and hepatoprotective effect of polysaccharides from Mori Fructus in rats with alcoholic-induced liver injury Authors: Xin Zhou, Qingfang Deng, Huaguo Chen, Enming Hu, Chao Zhao, Xiaojian Gong PII: DOI: Reference:
S0141-8130(16)32736-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.083 BIOMAC 7249
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
3-12-2016 27-2-2017 15-3-2017
Please cite this article as: Xin Zhou, Qingfang Deng, Huaguo Chen, Enming Hu, Chao Zhao, Xiaojian Gong, Characterizations and hepatoprotective effect of polysaccharides from Mori Fructus in rats with alcoholic-induced liver injury, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterizations and hepatoprotective effect of polysaccharides from Mori Fructus in rats with alcoholic-induced liver injury
Xin Zhou#*a,b,c, Qingfang Deng#a,b,c, Huaguo Chen*a,b,c, Enming Hua,b,c, Chao Zhaoa,b,c, Xiaojian Gonga,b,c
a
Key laboratory for Information System of Mountainous Areas and Protection of
Ecological Environment, Guizhou Normal University, 116 Baoshan North Rd., Guiyang, Guizhou, 550001, P. R. China b
Guizhou Engineering Laboratory for Quality Control & Evaluation Technology of
Medicine, 116 Baoshan North Rd., Guiyang, Guizhou, 550001, P. R. China c
The Research Center for Quality Control of Natural Medicine, Guizhou Normal
University, 116 Baoshan North Rd., Guiyang, Guizhou, 550001, P. R. China *Corresponding author Email address: [email protected]; [email protected] These authors contribute equally to this work.
#
1
Highlights
Preliminary structural characterization of Mori Fructus polysaccharides (MFPs) is investigated by UV spectrum, IR, high performance liquid chromatography (HPLC - DAD) and chemical analysis method, which mainly contained glucose, galacturonic acid, rhamnose and galactose.
The hepatoprotective effects and the acting mechanism of MFPs are validated in acute and chronic alcohol-induced liver injury model in mice, which indicating FMPS might be developed as a potential ingredient of functional food or natural agent for alcoholic liver injury, and the acting mechanism might be relate to its function of the recovery of antioxidase activities, elimination of free radicals, and inhibition of lipid peroxidation.
Abstract Crude polysaccharides of Mori Fructus (MFPs) are found to have anti-inflammatory antioxidant, and immuno-enhancing activities. However, the structure of the polysaccharides was ambiguous and its holistic hepatic protection evaluation was defective. This study was conducted to illustrate the characterization of MFPs, and evaluate its hepatoprotective activities. The results found that MFPs contained 67.93 ± 1.18% carbohydrates, 31.03 ± 0.54% uronic acid, and little protein and sulfate. The average molecular weight was ranging from 112.2 kDa to 181.9 kDa. Monosaccharide component analysis indicated that MFPs was mainly composed of glucose, galacturonic acid, rhamnose and galactose. Both the acute and subacute alcoholic-induced liver injury animal models were adopted to evaluate the MFPs’s 2
hepatoprotective activity. After administration of MFPs, both serological indexes (aspartate aminotransferase and alanine aminotransferase) and hepatic indicators (glutathione, superoxide dismutase, glutathione peroxidase and malondialdehyde) were improved by comparing with the non-MFPs group. The hepatic histopathology results also showed a prominent lipid degeneration and microvesicular steatosis attenuation in the MFPs groups. This outstanding hepatic protecting activity of MFPs might be related to its activation of ethanol dehydrogenase, elimination of free radicals and/or inhibition of lipid peroxidation capacities. MFPs might be a important active substances for preventing and remedying liver injury. Keywords: Mori Fructus polysaccharides; Hepatoprotective activity; Characterizations 1. Introduction Liver diseases have become one of the main concerns threatening human health around the world [1], particularly, in China and some other Asian countries. Liver diseases arise from many factors, such as excessive consumption of alcohol, bad customs of livelihood, toxic chemicals [2], environmental pollution [3], and long-term medication [4-6], etc. One of the most common liver diseases in the worldwide scope termed as alcoholic liver disease (ALD), has become a very important risk factor for morbidity and mortality [7], which is mainly caused by excessive consumption of alcohol [8]. So many attentions have been spent in finding effective and efficient agents to cure the ALD patients, and promote recovery of the ALD patients. Also some natural products, such as anthocyanins and polysaccharides with strong 3
antioxidant activities, have been expected as potential functional ingredients to treat ALD [9-13]. Mori Fructus, the fruit of Morus alba L., which contains a series of bioactive constituents, such as carbohydrates, vitamin, flavonoids, alkaloid, etc. [14, 15], has been used in traditional Chinese medicine for several thousand years. Studies on crude Mori Fructus polysaccharides (MFPs) have found that MFPs exhibit anti-inflammatory [16], enhance immunity [17], hypoglycemic action and hypolipidemic effect [18]. However, little information about MFPs in protecting liver injury, especially ALD, was reported. Our previous research has found that MFPs can enhance the activities of alcohol dehydrogenase in vitro [19], and exhibit a positive effect on protecting liver injury. So, in the present study a systematic investigation of MFPs, about the preparation and preliminary structure characterization was constructed. And the hepatoprotective activity of MFPs and its underlying mechanisms in alcohol-induced liver injury were also evaluated basing on acute and subacute model animals. 2. Materials and methods 2.1. Materials and reagents The fresh fruits of Morus alba L., collected in May of 2016 in the Guiyang medicinal botanical garden (Guiyang, China), was treated with a homogenizer machine and kept at -20 °C until use for extraction. The male mice of specific pathogen free (SPF) level were purchased from Changsha Tianqin Bio-Tech Co., Ltd. (Changsha, China) (number of animal license SCXK 4
(Xiang) 2014-0011). All experiments on animal in this study were approved by the Animal Ethics Committee of Guizhou Normal University. Galactose (Gal), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), mannose (Man), rhamnose (Rha), arabinose (Ara), xylose (Xyl), m-hydroxybiphenyl and 1-phenyl-3-methyl-5-pyrazolonde (PMP) were obtained from Guizhou science and technology Co., Ltd. (Guiyang, China). Elisa Kits of Alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were purchased from Shanghai MLBIO Biotechology Co., Ltd. (Shanghai, China). The hematoxylin solution and eosin solution were obtained from the Solarbio technology Ltd. (Beijing, China). All other chemicals and reagents were of chromatographic grade or analytical grade. 2.2. Preparation of MFPs MFPs was prepared by the enzyme assisted-ultrasonic extraction approach according to our previous reported method [19]. Briefly, the pulp of Mori Fructus (2.0 kg) underwent ethanol refluxing extraction and aether Saxhlet extraction to remove small molecule compounds and colored materials. Then the pretreated samples were suspended with water (22 L) and glucose oxidase (8 g) at 58 °C for 38 min, after that the sample was ultrasonically extracted for 30 min followed by centrifuged at 5000 rpm for 15 min. The collected supernatants were concentrated by a rotary evaporator to a proper volume. Protein was eliminated by the Sevag’s method followed by mixing with three volumes of absolute ethanol under vigorous stirring conditions and 5
kept at 4 °C for 36 h. After centrifugation at 5000 rpm for 15 min, the resulting precipitate was collected. The precipitate was reconstructed in distilled water and dialyzed with a molecular weight cut-off membrane (5 kDa) against distilled water for 24 h. The residue was freeze-dried to afford the MFPs. 2.3. Analysis of the Composition of MFPs The concentrations of total polysaccharide, protein, sulfate and uronic acid, and the molecular weight were determined. The total polysaccharides content in MFPs was analyzed by the phenol-sulfuric acid method [20] using glucose as the standard. The content of protein was analyzed by the Bradford assay using bovine serum albumin as a standard [21]. The sulfate content was determined according to the barium chloride-gelatin method using K2SO4 as control [22]. Uronic acid content was measured by the method of m-hydroxydiphenyl colorimetric by using GalA for the standard [23]. The molecular weight of MFPs were evaluated by HPLC equipped by Ultrahydrogel 250 (7.8 × 300 mm) and refractive index detector, eluted with NaNO3 (100 mM) at a flow rate of 0.6 mL/min, and using a series of known dextrans as standard. 2.4. Analysis of Monosaccharide Composition of MFPs The monosaccharide composition of MFPs was determined according to the existing method [24, 25] with a little modification. Briefly, hydrolyze the MFPs sample (10 mg) by 4 mL 2 M trifluoroacetic acid at 110 °C for 2 h to produce monosaccharides, repeatedly co-distill the resulting hydrolyzate with methanol to dryness and derivatized with 200 µL PMP (500 mM) under alkaline condition (100 µL 100 mM 6
NaOH) at 70 °C for 40 min. Then the solution was neutralized by 100 µL HCl (100 mM), excessive PMP was extracted repeatedly by chloroform. The residue was filtered through a 0.22 µm membrane for analysis. The standard monosaccharides were processed in the same way to obtain their PMP derivatives. All the PMP-labeled samples were analyzed on an Agilent 1260 HPLC system (Agilent Technologies, USA) equipped with Welch UItimate○R PAH column (4.6 × 250 mm, 5 µm, Welch Company, USA) and Diode-Array Detector. The mobile phase consisted of two solutions: A, phosphate buffered saline (PBS, pH 6.7); B, acetonitrile. The elution was programmed as following: 0~25 min, 13~19% B; 25~38 min, 19% B. Other operation chromatographic conditions were as follows: column temperature, 25 °C; detection wavelength, 250 nm; injection volume, 10 µL; constant flow rate, 1.0 mL/min. 2.5. FT-IR spectra of the MFPs The FT-IR spectrum of sample was collected with TENSOR27 spectrometer (Bruker， Germany). The samples were grinded with KBr and pressed into pellet for spectrometric measurement in the frequency range of 4000~400 cm−1. 2.6. Animals Grouping and Experimental Design The hepatoprotective effects of MFPs on ethanol-induced liver injury in mice were evaluated according to the reported method with some modifications. Briefly, the male mice of SPF level, 8-weeks-old, weighing 20 ± 2 g, were maintained under a standard environmental conditions with a controlled temperature of 22 ± 0.5 °C, a humidity of 55 ± 5% and a 12 h light-dark cycle and maintained with free access to 7
standard laboratory diet and water. All the experimental procedures involving animals were conducted in strict accordance with the international guidelines on the care and use of laboratory animals. 2.7. Ethanol-Induced Acute Liver Injury in Mice After an acclimatization period of 7 days, the mice were randomly divided into six groups (ten mice for each). Group I served as the normal control group; Group II served as alcohol model control group; Group III served as the bifendate-positive control group (150 mg/kg BW per day); Group IV-VI served as low-dose, moderate-dose and high-dose MFPs groups (50, 100 and 150 mg/kg BW per day, respectively). All groups were performed once a day for 30 consecutive days. Then, the mice expect Group I was administered orally with 14 mg/kg BW of 50% alcohol solution after the final treatment. After 16 h fasting following the last administration of ethanol, the blood samples were collected from the eyeballs and centrifuged at 3000 rpm for 10 min at 4 °C to afford the serums and stored at -80 °C until the future assay. The liver samples were dissected out immediately after the mice were killed, wash instantly with ice-cold PBS (pH 7.4), check the liver weight, and one part of the hepatic tissue was added to appropriate PBS (pH 7.4) and stored at -80 °C for the liver biochemical assays, whereas other part was rapidly divided and fixed in 10% formalin for pathological examination. 2.8. Ethanol-Induced Subacute Liver Injury in Mice These mice, acclimatizing for one week, were randomly divided into six groups of ten animals, including normal control group (Group 1), model control group (Group 2), 8
bifendate-positive group (Group 3) and MFPs groups (Group 4-6). MFPs groups mice were fed with MFPs in three different dose (50, 100 and 150 mg/kg BW per day for the low, medium and high dose, respectively) by gastric gavage. Mice in bifendate-positive group were given bifendate (150 mg/kg BW) in the same way, while mice in Group 1 and Group 2 were fed with equal volume distilled water by gastric gavage. And mice in group 2-6 also were administered orally with 12 mg/kg BW of 35% alcohol solution once a day, after 4 h from been bifendate or MFPs. All groups were carried out 30 consecutive days. Then, after 16 h fasting following the last administration, the mice were sacrificed via cervical dislocation. The blood samples were collected from the eyeballs immediately before sacrifice and centrifuged at 3000 rpm for 10 min at 4 °C to afford the serums. The serums were stored at -80 °C until the assay of the blood index (ALT and AST). Liver samples were dissected out and washed instantly with ice-cold PBS (pH 7.4), checked the liver weight, and one part of the hepatic tissue was added to appropriate PBS (pH 7.4) and stored at -80 °C for the liver biochemical assays, whereas another part was rapidly divided and fixed in 10% formalin for pathological examination. 2.9. Calculation of the Liver Index This paper obtained the liver index according to the follow calculation formula: The liver index =mice liver weight / mice weight × 100% 2.10. Serum Biochemical Assays The blood index (ALT and AST) were measured by a Spectra Max Plus 384 Enzyme mark analyzer (Molecular Devices Company, USA) with diagnostic reagent kits 9
(Shanghai MLBIO Biotechology Co., Ltd, China) according to the manufacturer’s instructions. 2.11. Liver Biochemical Assays The hepatic indicators (protein, GSH and MDA, SOD and GSH-Px) were determined by the commercial detection kits according to the manufacturer’s instructions. In brief, Liver samples were immediately homogenized in appropriate ice-cold PBS (50 mM, pH 7.4). The homogenate was centrifuged at 4 °C and 3000 rpm for 10 min, and then the supernatant was collected for the determinations of the content of protein, GSH and MDA, and the activities of GSH-Px and SOD. 2.12. Histopathologic Analysis Formalin-fixed liver tissue was processed by routine histology procedures and embedded in paraffin with the KH-TS automatic tissue spine-drier (Kuohai Medical Technology Co., Ltd, Xiaogan, China) and the KH-BQ automatic tissue embedded machines (Kuohai Medical Technology Co., Ltd, Xiaogan, China), use the KH-Q 2016 tissue slicer (Kuohai Medical Technology Co., Ltd, Xiaogan, China) to cut into 4 μm thick tissue sections, and then stained with hematoxylin and eosin (HE) by the KH-S automatic tissue staining apparatus (Kuohai Medical Technology Co., Ltd, Xiaogan, China), and subsequently examined under a MI12 invert light microscope (Minmei photoelectric technology Co., LTD, Guangzhou, China) of 40 × magnification for histopathological inspection. The results were apparently to photomicrographs. 2.13. Statistical Analysis 10
Data were expressed as means ± standard deviation (SD). SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analysis. The means among different groups were compared by one-way analysis of variance (ANOVA), while the Duncan’s multiple range tests was used in the post hoc multiple comparisons. Differences were regarded as statistically significant at the probability (P) less than 0.05. 3. Results 3.1. The Polysaccharides Yield of Mori Fructus The MFPs was prepared from the pretreatment Mori Fructus, by enzyme assisted extraction, ethanol precipitation, vacuum freeze-drying. The fresh Mori Fructus (2 kg) produced approximately 64.92 g of MFPs powder (the overall yield of MFPs was 3.25%). 3.2. The Concentrations of Total Polysaccharide, Protein, Sulfate, Uronic acid, Molecular weight On the basis of chemical analysis results, the nutrient compositions of MFPs contained 67.93 ± 1.18% total polysaccharide, 31.03 ± 0.54% uronic acid, and a small amount of sulfate and protein (Table 1). In addition, the weight-average molecular weight ranged from 112.2 kDa to 181.9 kDa (Fig. 1A). 3.3. Analysis of Monosaccharide Composition of MFPs For determination of the monosaccharide composition of MFPs, an Agilent 1260 HPLC system was used. As shown in Fig. 1B, the monosaccharides in MFPs hydrolyte were identified by comparing the retention times with standards. As 11
presented in Fig. 1C, MFPs was mainly composed of Glc (39.79), GalA (17.15), Rha (15.16) and Gal (14.48), along with a small quantity of GlcA, Xyl, Ara and Man. 3.4. FT-IR spectra of MFPs The FT-IR spectrum of MFPs was shown in Fig. 1D. The strong and broad characteristic absorption peaks around 3385.95 cm−1 for the stretching vibration of O-H, and a weak absorption peak at 2939.74 cm-1 indicated the C-H bonding. The absorption at 1607.92 cm-1 was related to the symmetric and asymmetric stretching vibration of C=O of the carboxylate groups. The relatively strong absorption peaks at 1416.10 cm-1 was due to the C-H bond deforming vibration. The relatively strong absorption peaks at 1075.90 cm-1 was caused by a pyranose form of sugar. An absorbance at nearly 826.58 cm-1 indicates the linkage of α-glycosides. 3.4. Effect of MFPs on Ethanol-induced Acute Liver Injury in Mice As shown in Table 2A, the liver index increased in model control groups compared to those in normal controls by 16.82%. However, this test indicated the administration of bifendate equaled to the middle dose of MFPs group on the protective effect on the alcohol-induced liver injury (P > 0.05). The highest dose of MFPs has significantly decreased the liver index by 29.14% (P < 0.05), compared with the administration of alcohol group. In Table 2A, we can see that compared with the normal control group, the serum ALT and AST levels in alcohol group elevated greatly, by 66.14% and 62.90% (P < 0.01), respectively, indicating that the alcohol-induced acute liver injury model in mice was well-established. The administration of MFPs showed a significant protective effect 12
on the alcohol-induced liver injury by remarkably preventing the elevation of serum levels of ALT and AST in a dose-dependent manner. The minimum dose of MFPs has similar potency to bifendate (P > 0.05). The highest dose of MFPs has significantly decreased the ALT level and the AST level by 34.79% and 31.71% (P < 0.05), respectively, compared with the administration of alcohol group. Table 2A shows that the administration of alcohol group had obviously increase in the MDA level and decrease in the GSH level, the activities levels of SOD and GSH-Px by 19.36%, 17.92%, 32.24% and 26.65% (P < 0.05), compared with the normal control group, respectively. The results indicated that the liver functions of the model control group were impaired in the present study. As for the activities of SOD and GSH-Px, and the GSH level we observed that the administration of MFPs significantly enhanced these activities (P < 0.05, P < 0.05, P < 0.01, for SOD, GSH-Px, and GSH, respectively), whereas decreased the MDA level compared with those of model control group. The highest dose group of MFPs has significantly increased the SOD, GSH and GSH-Px level by 20.23%, 73.22% and 40.90% (P < 0.05), respectively, and has significantly decreased the level of MDA by 30.10%, comparing with the administration of alcohol group. The histoarchitecture of hematoxylin and eosin-stained liver sections from each group mice was inspected by light microscopy, and the typical pathological sections of each group mice are shown in Fig. 2A1-A6. In control group (group I, Fig. 2A1), the histologic characteristics included normal morphology of hepatic lobules which was observed with well designated hepatic cells, and no necrotic and apoptotic hepatic 13
cells were found and hepatic sinusoids looked clear. Also, there were a lot of free ribosomes and rough surfaced endoplasmic reticulum. In general, this photomicrographs of pathology section from a healthy liver. In model group (group II, Fig. 2A2), however, animals exposed to ethanol exhibit hepatocytes with steatosis, lipid degeneration, diffuse ballooning degeneration, and cytoplasm loosening, indicating disruption of the normal liver architecture. Conversely, bifendate and MFPs treatment significantly reversed the ethanol-induced liver damage. Bifendate-treated and MFPs-treated mice showed an integrated liver cell architecture, and only seldom cells had ballooning and steatosis, and hepatocyte regeneration and no necrotic cells were observed, which expressed the cells were well preserved (Fig. 2A6). 3.5. Effect of MFPs on Subacute Ethanol-induced Liver Injury in Mice In Table 2B, we can see that compared with the normal control group, the liver index, caused a significant elevation compared with the administration of alcohol group by 19.77% (P < 0.05), respectively. The administration of MFPs showed a significant protective effect on the alcohol-induced liver injury by remarkably preventing the elevation of the liver index in a dose-dependent manner. The minimum dose of MFPs has similar potency to bifendate (P > 0.05). The highest dose of MFPs has significantly decreased the liver index by 14.93% (P < 0.05) compared with the administration of alcohol group. The activities of plasma ALT and AST, are the significant elevation of indicating liver injury. As shown in Table 2B, levels of ALT and AST increased in model control groups (14.37 and 31.91 U/L, respectively) compared to those in normal controls, 14
indicating that the subacute alcohol-induced liver injury model in mice was well-established in this study. However, these two plasmas test indicators the administration of bifendate/MFPs groups were a significant protective effect on the alcohol-induced liver injury. The highest dose of MFPs has significantly decreased the ALT level and the AST level by 45.23% and 54.38% (P < 0.05), respectively, compared with the administration of alcohol group. As shown in Table 2B, the MDA level obviously increased and the GSH level, and the activities levels of SOD and GSH-Px were dramatically decreased in the administration of alcohol group (P < 0.05, P < 0.05, P < 0.05 for GSH, SOD and GSH-Px, respectively) compared with those in normal group. The results confirmed again that the model was established successfully in this study. However, pretreatment with MFPs could improve markedly the GSH level by 52.45% and the activities were of SOD and GSH-Px by 19.65% and 20.63%, whereas could decrease the MDA level by 22.22%, in livers of alcohol-treated mice. Moreover, significantly high levels of activities were observed in the livers of mice that had been treated with bifendate (246.69 and 385.20 U/mg protein for SOD and GSH-Px, respectively, P < 0.01). The highest/middle dose of MFPs has similar potency to bifendate (P > 0.05). The results suggest that MFPs had the protective action for the ethanol-induced subacute liver injury. The representative photomicrographs of liver sections from the experimental groups were shown in Fig. 2B1-B6. The pathology section examination from the normal control group (group 1, Fig. 2B1) showed that the liver tissue organization is regular 15
and the hepatic lobules of them was clear without liquid droplets, liver sinusoid express normal, and the hepatic cord was well-arranged. However, the photomicrographs of liver sections from the model groups (group 2, Fig. 2B2) which treated by ethanol demonstrated that cytoplasm loosening of hepatic cells, diffuse ballooning degeneration and microvesicular steatosis, lipid degeneration, diffuse ballooning degeneration led to disrupted liver lobule structure. Moreover, there were acidophilic and necrotic liver cells in the photomicrograph. Granular degeneration could be found in some liver cells. And the inflammatory cells infiltrated were observed in the necrotic lesion. Conversely, the groups dealt with bifendate and MFPs significantly reversed the ethanol-induced liver injury. The liver tissue displayed an intact architecture, and only a few liver cells had ballooning and steatosis, and hepatocyte regeneration and no necrotic cells were observed (Fig. 2B6). 4. Discussion Alcohol-induced liver injury, one of the most common reasons of liver diseases, has been an increasingly important public-health problem worldwide. Oxidative stress, induced the damage of tissue by the imbalance of prooxidant and antioxidant, is thought to play a key role in the pathogenesis of alcohol-induced liver injury [26, 27]. Alcohol mediates oxidative stress in multiple ways involving the release of liver enzymes such as AST, ALT and ALP in serum, lipid peroxidation, depletion of cytoprotective antioxidant functions, and the generation of reactive oxygen species (ROS) [28]. ROS, one kind of pro-oxidants including superoxide radical, hydrogen peroxide, nitric oxide and hydroxyl radical, are generated naturally by cells in the 16
biological systems [29]. It is very important for keeping the biological systems from damage to rapidly clear the normally produced ROS by antioxidants. SOD and GSH-Px, can scavenge ROS and terminate the free-radical chain reaction, are critical antioxidants, and play important roles in hepatoprotection. Lack of SOD and GSH-Px is concerned with the redundantly accumulation of ROS, which could cause lipid peroxidation, tissues and organs injury, diseases and aging [30]. MDA, the stable metabolite of lipid peroxidation products, is associated with free radical effect in biological system. GSH can protect thiol group (-SH) of enzymes, adjust the synthesis of ribonucleotide and neutralize free radicals. In China, many herbal medicines been used to prevent and remedy the ALD, and some literatures also suggest that the mulberry water extracts can prevent alcohol-induced liver injury and CCl4-induced liver damage and fibrosis [11, 31]. However, the effect of MFPs on ethanol-induced liver injury is not clear. Therefore, the current study explored the hepatoprotective effect of MFPs. Alcohol played an inducement role in liver injury. Therefore, the alcohol-induced liver injury model is widely used to test the liver-protection effects of many bioactive substances, such as foods, drugs and herbs [32-34]. Bifendate, synthesized from schisandrin C with liver protective effect in rodents, is usually used as the drug to treat hepatitis, and also is regarded as a positive control for researching other hepatic protectors [35, 36]. So this study used bifendate as a positive control to establish acute and subacute alcohol-induced liver injury in mice model to evaluate the liver-protection effect of MFPs by histopathologic examination, and determining the 17
antioxidant indicators (SOD, GSH, and GSH-Px) and the lipid peroxidation product (MDA) in liver, as well as liver function index (AST and ALT) in serum. Compared normal group and positive group with alcohol model group showed that an apparent decline in the ALT, AST, DMA levels, and the level of SOD and GSH-Px have been obviously enhanced, which indicated the acute and subacute alcohol-induced liver injury model in mice were set up successfully. The histopathologic examination, which expressed cellular correlates of injury after ethanol administration (Fig. 2). This hepatocytes damage resulted in an increase in liver index (Table 2A and Table 2B). Cellular membranes breakage caused intracellular substances leaks, and accompanied by the elevation of serum liver markers. Therefore, the levels of AST and ALT in serum, which were established counters of hepatic damage, were improved. MFPs administration promoted biochemical and histologic modulation demonstrating liver repair to normality. The determination of the antioxidant indicators (SOD, GSH, and GSH-Px) and the lipid peroxidation product (MDA) in liver, as well as liver function index (AST and ALT) in serum, demonstrated that the MDA level obviously lower, while the a levels of GSH, SOD and GSH-Px were dramatically higher in the normal group, compared with those of the administration of alcohol group. It indicated that alcohol administration was likely significantly reduced the biosystems of anti-oxidations, thus caused the accumulation of free radicals, and caused lipid peroxidation, subsequently. In addition, the results showed that the MDA level significantly reduced and the SOD activities, GSH-Px activities, the GSH level were significantly increased in MFPs 18
group compared with the alcohol-induced liver injury group. Furthermore, MFPs exhibited significantly inhibition function to superoxide radical and lipid peroxidation, as well as bifendate. In conclusion, MFPs has a fine liver-protection effect on acute and subacute alcohol-induced liver injury, which might relate to its function of antioxidase activities, elimination of free radicals, and inhibition of lipid peroxidation. The results agree with the previous researches that some polysaccharides have protective function on ALD [12, 37]. Literature sources available showed that numerous complicated factors, included molecular weight, monosaccharide composition and conformation, affect the bioactivity of polysaccharide [38]. The monosaccharide composition analysis indicated that MFPs was mainly composed of Glc, GalA, Rha and Gal. In addition, MFPs contented lots of uronic acid. Therefore, experimental results indicated that structural properties and composition of MFPs might have greatest impact on its anti-oxidative stress, and then be related to its hepatoprotective effect. 5. Conclusions In this study, the preliminary structure characterization of and the hepatoprotective activity of MFPs were carried out. The assay of the preliminary structure characterization demonstrated that MFPs mainly contained 67.93 ± 1.18% total polysaccharide, 31.03 ± 0.54% uronic acid and little protein and sulfate. And MFPs was mainly composed of Glc, GalA, Rha and Gal, along with a small quantity of GlcA, Xyl, Ara and Man. Meanwhile, the assay of hepatoprotective activity in vivo demonstrated that the ALT level and AST level in serum, the liver levels of MDA are 19
remarkably lower, the liver levels of GSH, the liver activities of SOD and GSH-Px are significantly higher in MFPs group compared with the administration of alcohol group. Moreover, the histopathologic examination showed that the administration of MFPs satisfactorily repaired the alcohol-related liver damage. In conclusion, MFPs has a significant liver-protection effect on acute and subacute alcohol-induced liver injury, which might be related to its function of the recovery of antioxidase activities, elimination of free radicals, and inhibition of lipid peroxidation. This finding showed that MFPs might be one of important active substances for preventing and remedying ALD. Acknowledgements This project was supported by the National Natural Science Foundation of China [grant numbers 31570358), Guizhou Province High Level Creative Talents Cultivation [grant numbers [2015]4033], Guizhou Province Special Found for Modern Science and Technology Industrial of Traditional Chinese medicine research and development [grant numbers [2013]5005].
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dimethyl-dicarboxylate, Acta pharmaceutica Sinica 18(9) (1983) 714. [37] J. Xiao, Y. Zhu, Y. Liu, G.L. Tipoe, F. Xing, K.-F. So, Lycium barbarum polysaccharide attenuates alcoholic cellular injury through TXNIP-NLRP3 inflammasome pathway, Int J Biol Macromol 69(0) (2014) 73-78. [38] É.C. Francisco, T.T. Franco, R. Wagner, E. Jacob-Lopes, Assessment of different carbohydrates as exogenous carbon source in cultivation of cyanobacteria, Bioproc Biosyst Eng (2014) 1-9.
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Figure Captions Fig. 1 The preliminary characterizations of MFPs, including molecular sizes (A), HPLC chromatograms of PMP derivatives of monosaccharide standard samples and hydrolyzate (B), and the monosaccharide composition (C), and infrared spectrums (D).. Fig. 2 Effect of MFPs on alcohol-induced acute and subacute liver injury mice liver tissue histopathology. The livers of mice were removed and stained with HE staining and observed under a MI12 invert light microscope (× 40 magnification). A1-A6 showed the effects of MFPs on alcohol-induced acute liver injury mice liver tissue histopathology (A1, normal control group; A2, alcohol model control group; A3, administrated with bifendate at a dose of 150 mg/kg; A4, administrated with MFPs at a dose of 50 mg/kg; A5, administrated with MFPs at a dose of 100 mg/kg; A6, administrated with MFPs at a dose of mg/kg). B1-B6 showed the effects of MFPs on alcohol-induced subacute liver injury mice liver tissue histopathology (B1, normal control group; B2, alcohol model control group; B3, administrated with bifendate at a dose of 150 mg/kg; B4, administrated with MFPs at a dose of 50 mg/kg; B5, administrated with MFPs at a dose of 100 mg/kg; B6, administrated with MFPs at a dose of 150 mg/kg). Fig. 3 A schematic diagram of the proposed mechanism by which MFPs prevents alcohol-induced liver damage. MFPs prevents alcohol-induced liver damage might relate to its function of the recovery of antioxidase activities, elimination of free radicals, inhibition of lipid peroxidation and anti-oxidative stress.
25
Fig. 1
26
Fig. 2
27
Fig. 3
28
Table 1 The chemical composition of MFPs. Content (%) Component MFPs
Polysaccharide
Sulfate
Uronic acid
Protein
67.93 ± 1.18
6.39 ± 0.04
31.03 ± 0.54
3.52 ± 0.02
29
Table 2A Effects of MFPs on the GSH level and MDA level, the activities of SOD and GSH-Px in liver, and the ALT and AST levels in serum of alcohol-induced acute liver injury mice. Groupa Normal Model Positive MFPs-H MFPs-M MFPs-L SOD (U/mg protein) 249.67 ± 15.21# 204.91 ± 10.01 243.71 ± 7.98# 246.37 ± 11.02# 231.12 ± 8.71 214.58 ± 9.23 GSH-Px (U/mg protein) 440.20 ± 17.51# 322.90 ± 8.13 443.42 ± 10.65# 435.06 ± 6.98# 385.08 ± 14.73 346.87 ± 16.46 GSH (nmol/mg protein) 265.98 ± 10.78# 180.22 ± 7.12 258.42 ± 14.57# 256.20 ± 13.15# 232.08 ± 15.01 205.44 ± 9.69 MAD (nmol/mg protein) 0.54 ± 0.14# 0.67 ± 0.36 0.57 ± 0.29# 0.59 ± 0.11# 0.61 ± 0.21 0.64 ± 0.17 AST (U/L) 39.30 ±2.97# 64.02 ± 7.82 44.94 ± 3.21# 43.72 ± 2.57# 50.79 ± 5.18 58.82 ± 6.54 ALT (U/L) 29.71 ±4.11# 49.09 ± 4.59 30.86 ± 3.42# 32.01 ± 3.79# 38.57 ± 4.14 42..46 ± 3.33 Liver index (%) 3.51 ± 0.45# 4.22 ±0.31 3.86 ±0.23 3.59 ±0.16# 3.81 ±0.31 4.07 ±0.44 a Normal, normal control group; Model, alcohol model control group; Positive, administrated with bifendate at a dose of 150 mg/kg; MFPs-H, administrated with MFPs at a dose of 150 mg/kg; MFPs-M, administrated with MFPs at a dose of 100 mg/kg; MFPs-L, administrated with MFPs at a dose of 50 mg/kg. #
Data were presented as mean ± SD (n = 10), values with alcohol model control group are significantly different (P < 0.05).
Table 2B Effects of MFPs on the GSH level and MDA level, the activities of SOD and GSH-Px in liver, and the ALT and AST levels in serum of alcohol-induced subacute liver injury mice. Groupa Normal Model Positive MFPs-H MFPs-M MFPs-L SOD (U/mg protein) 246.69 ± 3.45# 198.21 ± 2.78 238.09 ± 21.56# 248.29 ± 5.61# 236.31 ± 4.14# 229.68 ± 2.97 GSH-Px (U/mg protein) 385.20 ± 4.51# 305.74 ± 8.12 372.12 ± 7.56# 380.94 ± 5.41# 357.35 ± 6.71 322.69 ± 6.12 GSH(nmol/mg protein) 232.47 ± 4.17# 110.55 ± 1.03 212.22 ± 5.28# 206.36 ± 2.11# 164.85 ± 3.01 136.12 ± 4.87 MAD (nmol/mg protein) 0.55 ± 0.02# 0.71 ± 0.72 0.55 ± 1.01# 0.59 ± 0.42# 0.61 ± 0.13 0.65 ± 0.57 AST (U/L) 45.85 ± 10.42# 77.76 ± 13.11 53.74 ± 7.97 50.51 ±7.5259# 58.09 ± 4.51 69 ± 12.13 ALT (U/L) 28.71 ± 5.61# 43.08 ± 11.08 30.34 ± 9.45 29.60 ±7.18# 32.63 ± 2.55 38.80 ± 5.82 Liver index (%) 2.84 ± 0.65# 3.54 ± 0.32 3.01 ± 0.34 2.88 ± 0.21# 3.27 ± 0.39 3.37± 0.32 a Normal, normal control group; Model, alcohol model control group; Positive, administrated with bifendate at a dose of 150 mg/kg; MFPs-H, administrated with MFPs at a dose of 150 mg/kg; MFPs-M, administrated with MFPs at a dose of 100 mg/kg; MFPs-L, administrated with MFPs at a dose of 50 mg/kg. #
Data were presented as mean ± SD (n = 10), values with alcohol model control group are significantly different (P < 0.05).
30
S0141-8130(16)32736-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.083 BIOMAC 7249
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
3-12-2016 27-2-2017 15-3-2017
Please cite this article as: Xin Zhou, Qingfang Deng, Huaguo Chen, Enming Hu, Chao Zhao, Xiaojian Gong, Characterizations and hepatoprotective effect of polysaccharides from Mori Fructus in rats with alcoholic-induced liver injury, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterizations and hepatoprotective effect of polysaccharides from Mori Fructus in rats with alcoholic-induced liver injury
Xin Zhou#*a,b,c, Qingfang Deng#a,b,c, Huaguo Chen*a,b,c, Enming Hua,b,c, Chao Zhaoa,b,c, Xiaojian Gonga,b,c
a
Key laboratory for Information System of Mountainous Areas and Protection of
Ecological Environment, Guizhou Normal University, 116 Baoshan North Rd., Guiyang, Guizhou, 550001, P. R. China b
Guizhou Engineering Laboratory for Quality Control & Evaluation Technology of
Medicine, 116 Baoshan North Rd., Guiyang, Guizhou, 550001, P. R. China c
The Research Center for Quality Control of Natural Medicine, Guizhou Normal
University, 116 Baoshan North Rd., Guiyang, Guizhou, 550001, P. R. China *Corresponding author Email address: [email protected]; [email protected] These authors contribute equally to this work.
#
1
Highlights
Preliminary structural characterization of Mori Fructus polysaccharides (MFPs) is investigated by UV spectrum, IR, high performance liquid chromatography (HPLC - DAD) and chemical analysis method, which mainly contained glucose, galacturonic acid, rhamnose and galactose.
The hepatoprotective effects and the acting mechanism of MFPs are validated in acute and chronic alcohol-induced liver injury model in mice, which indicating FMPS might be developed as a potential ingredient of functional food or natural agent for alcoholic liver injury, and the acting mechanism might be relate to its function of the recovery of antioxidase activities, elimination of free radicals, and inhibition of lipid peroxidation.
Abstract Crude polysaccharides of Mori Fructus (MFPs) are found to have anti-inflammatory antioxidant, and immuno-enhancing activities. However, the structure of the polysaccharides was ambiguous and its holistic hepatic protection evaluation was defective. This study was conducted to illustrate the characterization of MFPs, and evaluate its hepatoprotective activities. The results found that MFPs contained 67.93 ± 1.18% carbohydrates, 31.03 ± 0.54% uronic acid, and little protein and sulfate. The average molecular weight was ranging from 112.2 kDa to 181.9 kDa. Monosaccharide component analysis indicated that MFPs was mainly composed of glucose, galacturonic acid, rhamnose and galactose. Both the acute and subacute alcoholic-induced liver injury animal models were adopted to evaluate the MFPs’s 2
hepatoprotective activity. After administration of MFPs, both serological indexes (aspartate aminotransferase and alanine aminotransferase) and hepatic indicators (glutathione, superoxide dismutase, glutathione peroxidase and malondialdehyde) were improved by comparing with the non-MFPs group. The hepatic histopathology results also showed a prominent lipid degeneration and microvesicular steatosis attenuation in the MFPs groups. This outstanding hepatic protecting activity of MFPs might be related to its activation of ethanol dehydrogenase, elimination of free radicals and/or inhibition of lipid peroxidation capacities. MFPs might be a important active substances for preventing and remedying liver injury. Keywords: Mori Fructus polysaccharides; Hepatoprotective activity; Characterizations 1. Introduction Liver diseases have become one of the main concerns threatening human health around the world [1], particularly, in China and some other Asian countries. Liver diseases arise from many factors, such as excessive consumption of alcohol, bad customs of livelihood, toxic chemicals [2], environmental pollution [3], and long-term medication [4-6], etc. One of the most common liver diseases in the worldwide scope termed as alcoholic liver disease (ALD), has become a very important risk factor for morbidity and mortality [7], which is mainly caused by excessive consumption of alcohol [8]. So many attentions have been spent in finding effective and efficient agents to cure the ALD patients, and promote recovery of the ALD patients. Also some natural products, such as anthocyanins and polysaccharides with strong 3
antioxidant activities, have been expected as potential functional ingredients to treat ALD [9-13]. Mori Fructus, the fruit of Morus alba L., which contains a series of bioactive constituents, such as carbohydrates, vitamin, flavonoids, alkaloid, etc. [14, 15], has been used in traditional Chinese medicine for several thousand years. Studies on crude Mori Fructus polysaccharides (MFPs) have found that MFPs exhibit anti-inflammatory [16], enhance immunity [17], hypoglycemic action and hypolipidemic effect [18]. However, little information about MFPs in protecting liver injury, especially ALD, was reported. Our previous research has found that MFPs can enhance the activities of alcohol dehydrogenase in vitro [19], and exhibit a positive effect on protecting liver injury. So, in the present study a systematic investigation of MFPs, about the preparation and preliminary structure characterization was constructed. And the hepatoprotective activity of MFPs and its underlying mechanisms in alcohol-induced liver injury were also evaluated basing on acute and subacute model animals. 2. Materials and methods 2.1. Materials and reagents The fresh fruits of Morus alba L., collected in May of 2016 in the Guiyang medicinal botanical garden (Guiyang, China), was treated with a homogenizer machine and kept at -20 °C until use for extraction. The male mice of specific pathogen free (SPF) level were purchased from Changsha Tianqin Bio-Tech Co., Ltd. (Changsha, China) (number of animal license SCXK 4
(Xiang) 2014-0011). All experiments on animal in this study were approved by the Animal Ethics Committee of Guizhou Normal University. Galactose (Gal), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), mannose (Man), rhamnose (Rha), arabinose (Ara), xylose (Xyl), m-hydroxybiphenyl and 1-phenyl-3-methyl-5-pyrazolonde (PMP) were obtained from Guizhou science and technology Co., Ltd. (Guiyang, China). Elisa Kits of Alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were purchased from Shanghai MLBIO Biotechology Co., Ltd. (Shanghai, China). The hematoxylin solution and eosin solution were obtained from the Solarbio technology Ltd. (Beijing, China). All other chemicals and reagents were of chromatographic grade or analytical grade. 2.2. Preparation of MFPs MFPs was prepared by the enzyme assisted-ultrasonic extraction approach according to our previous reported method [19]. Briefly, the pulp of Mori Fructus (2.0 kg) underwent ethanol refluxing extraction and aether Saxhlet extraction to remove small molecule compounds and colored materials. Then the pretreated samples were suspended with water (22 L) and glucose oxidase (8 g) at 58 °C for 38 min, after that the sample was ultrasonically extracted for 30 min followed by centrifuged at 5000 rpm for 15 min. The collected supernatants were concentrated by a rotary evaporator to a proper volume. Protein was eliminated by the Sevag’s method followed by mixing with three volumes of absolute ethanol under vigorous stirring conditions and 5
kept at 4 °C for 36 h. After centrifugation at 5000 rpm for 15 min, the resulting precipitate was collected. The precipitate was reconstructed in distilled water and dialyzed with a molecular weight cut-off membrane (5 kDa) against distilled water for 24 h. The residue was freeze-dried to afford the MFPs. 2.3. Analysis of the Composition of MFPs The concentrations of total polysaccharide, protein, sulfate and uronic acid, and the molecular weight were determined. The total polysaccharides content in MFPs was analyzed by the phenol-sulfuric acid method [20] using glucose as the standard. The content of protein was analyzed by the Bradford assay using bovine serum albumin as a standard [21]. The sulfate content was determined according to the barium chloride-gelatin method using K2SO4 as control [22]. Uronic acid content was measured by the method of m-hydroxydiphenyl colorimetric by using GalA for the standard [23]. The molecular weight of MFPs were evaluated by HPLC equipped by Ultrahydrogel 250 (7.8 × 300 mm) and refractive index detector, eluted with NaNO3 (100 mM) at a flow rate of 0.6 mL/min, and using a series of known dextrans as standard. 2.4. Analysis of Monosaccharide Composition of MFPs The monosaccharide composition of MFPs was determined according to the existing method [24, 25] with a little modification. Briefly, hydrolyze the MFPs sample (10 mg) by 4 mL 2 M trifluoroacetic acid at 110 °C for 2 h to produce monosaccharides, repeatedly co-distill the resulting hydrolyzate with methanol to dryness and derivatized with 200 µL PMP (500 mM) under alkaline condition (100 µL 100 mM 6
NaOH) at 70 °C for 40 min. Then the solution was neutralized by 100 µL HCl (100 mM), excessive PMP was extracted repeatedly by chloroform. The residue was filtered through a 0.22 µm membrane for analysis. The standard monosaccharides were processed in the same way to obtain their PMP derivatives. All the PMP-labeled samples were analyzed on an Agilent 1260 HPLC system (Agilent Technologies, USA) equipped with Welch UItimate○R PAH column (4.6 × 250 mm, 5 µm, Welch Company, USA) and Diode-Array Detector. The mobile phase consisted of two solutions: A, phosphate buffered saline (PBS, pH 6.7); B, acetonitrile. The elution was programmed as following: 0~25 min, 13~19% B; 25~38 min, 19% B. Other operation chromatographic conditions were as follows: column temperature, 25 °C; detection wavelength, 250 nm; injection volume, 10 µL; constant flow rate, 1.0 mL/min. 2.5. FT-IR spectra of the MFPs The FT-IR spectrum of sample was collected with TENSOR27 spectrometer (Bruker， Germany). The samples were grinded with KBr and pressed into pellet for spectrometric measurement in the frequency range of 4000~400 cm−1. 2.6. Animals Grouping and Experimental Design The hepatoprotective effects of MFPs on ethanol-induced liver injury in mice were evaluated according to the reported method with some modifications. Briefly, the male mice of SPF level, 8-weeks-old, weighing 20 ± 2 g, were maintained under a standard environmental conditions with a controlled temperature of 22 ± 0.5 °C, a humidity of 55 ± 5% and a 12 h light-dark cycle and maintained with free access to 7
standard laboratory diet and water. All the experimental procedures involving animals were conducted in strict accordance with the international guidelines on the care and use of laboratory animals. 2.7. Ethanol-Induced Acute Liver Injury in Mice After an acclimatization period of 7 days, the mice were randomly divided into six groups (ten mice for each). Group I served as the normal control group; Group II served as alcohol model control group; Group III served as the bifendate-positive control group (150 mg/kg BW per day); Group IV-VI served as low-dose, moderate-dose and high-dose MFPs groups (50, 100 and 150 mg/kg BW per day, respectively). All groups were performed once a day for 30 consecutive days. Then, the mice expect Group I was administered orally with 14 mg/kg BW of 50% alcohol solution after the final treatment. After 16 h fasting following the last administration of ethanol, the blood samples were collected from the eyeballs and centrifuged at 3000 rpm for 10 min at 4 °C to afford the serums and stored at -80 °C until the future assay. The liver samples were dissected out immediately after the mice were killed, wash instantly with ice-cold PBS (pH 7.4), check the liver weight, and one part of the hepatic tissue was added to appropriate PBS (pH 7.4) and stored at -80 °C for the liver biochemical assays, whereas other part was rapidly divided and fixed in 10% formalin for pathological examination. 2.8. Ethanol-Induced Subacute Liver Injury in Mice These mice, acclimatizing for one week, were randomly divided into six groups of ten animals, including normal control group (Group 1), model control group (Group 2), 8
bifendate-positive group (Group 3) and MFPs groups (Group 4-6). MFPs groups mice were fed with MFPs in three different dose (50, 100 and 150 mg/kg BW per day for the low, medium and high dose, respectively) by gastric gavage. Mice in bifendate-positive group were given bifendate (150 mg/kg BW) in the same way, while mice in Group 1 and Group 2 were fed with equal volume distilled water by gastric gavage. And mice in group 2-6 also were administered orally with 12 mg/kg BW of 35% alcohol solution once a day, after 4 h from been bifendate or MFPs. All groups were carried out 30 consecutive days. Then, after 16 h fasting following the last administration, the mice were sacrificed via cervical dislocation. The blood samples were collected from the eyeballs immediately before sacrifice and centrifuged at 3000 rpm for 10 min at 4 °C to afford the serums. The serums were stored at -80 °C until the assay of the blood index (ALT and AST). Liver samples were dissected out and washed instantly with ice-cold PBS (pH 7.4), checked the liver weight, and one part of the hepatic tissue was added to appropriate PBS (pH 7.4) and stored at -80 °C for the liver biochemical assays, whereas another part was rapidly divided and fixed in 10% formalin for pathological examination. 2.9. Calculation of the Liver Index This paper obtained the liver index according to the follow calculation formula: The liver index =mice liver weight / mice weight × 100% 2.10. Serum Biochemical Assays The blood index (ALT and AST) were measured by a Spectra Max Plus 384 Enzyme mark analyzer (Molecular Devices Company, USA) with diagnostic reagent kits 9
(Shanghai MLBIO Biotechology Co., Ltd, China) according to the manufacturer’s instructions. 2.11. Liver Biochemical Assays The hepatic indicators (protein, GSH and MDA, SOD and GSH-Px) were determined by the commercial detection kits according to the manufacturer’s instructions. In brief, Liver samples were immediately homogenized in appropriate ice-cold PBS (50 mM, pH 7.4). The homogenate was centrifuged at 4 °C and 3000 rpm for 10 min, and then the supernatant was collected for the determinations of the content of protein, GSH and MDA, and the activities of GSH-Px and SOD. 2.12. Histopathologic Analysis Formalin-fixed liver tissue was processed by routine histology procedures and embedded in paraffin with the KH-TS automatic tissue spine-drier (Kuohai Medical Technology Co., Ltd, Xiaogan, China) and the KH-BQ automatic tissue embedded machines (Kuohai Medical Technology Co., Ltd, Xiaogan, China), use the KH-Q 2016 tissue slicer (Kuohai Medical Technology Co., Ltd, Xiaogan, China) to cut into 4 μm thick tissue sections, and then stained with hematoxylin and eosin (HE) by the KH-S automatic tissue staining apparatus (Kuohai Medical Technology Co., Ltd, Xiaogan, China), and subsequently examined under a MI12 invert light microscope (Minmei photoelectric technology Co., LTD, Guangzhou, China) of 40 × magnification for histopathological inspection. The results were apparently to photomicrographs. 2.13. Statistical Analysis 10
Data were expressed as means ± standard deviation (SD). SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analysis. The means among different groups were compared by one-way analysis of variance (ANOVA), while the Duncan’s multiple range tests was used in the post hoc multiple comparisons. Differences were regarded as statistically significant at the probability (P) less than 0.05. 3. Results 3.1. The Polysaccharides Yield of Mori Fructus The MFPs was prepared from the pretreatment Mori Fructus, by enzyme assisted extraction, ethanol precipitation, vacuum freeze-drying. The fresh Mori Fructus (2 kg) produced approximately 64.92 g of MFPs powder (the overall yield of MFPs was 3.25%). 3.2. The Concentrations of Total Polysaccharide, Protein, Sulfate, Uronic acid, Molecular weight On the basis of chemical analysis results, the nutrient compositions of MFPs contained 67.93 ± 1.18% total polysaccharide, 31.03 ± 0.54% uronic acid, and a small amount of sulfate and protein (Table 1). In addition, the weight-average molecular weight ranged from 112.2 kDa to 181.9 kDa (Fig. 1A). 3.3. Analysis of Monosaccharide Composition of MFPs For determination of the monosaccharide composition of MFPs, an Agilent 1260 HPLC system was used. As shown in Fig. 1B, the monosaccharides in MFPs hydrolyte were identified by comparing the retention times with standards. As 11
presented in Fig. 1C, MFPs was mainly composed of Glc (39.79), GalA (17.15), Rha (15.16) and Gal (14.48), along with a small quantity of GlcA, Xyl, Ara and Man. 3.4. FT-IR spectra of MFPs The FT-IR spectrum of MFPs was shown in Fig. 1D. The strong and broad characteristic absorption peaks around 3385.95 cm−1 for the stretching vibration of O-H, and a weak absorption peak at 2939.74 cm-1 indicated the C-H bonding. The absorption at 1607.92 cm-1 was related to the symmetric and asymmetric stretching vibration of C=O of the carboxylate groups. The relatively strong absorption peaks at 1416.10 cm-1 was due to the C-H bond deforming vibration. The relatively strong absorption peaks at 1075.90 cm-1 was caused by a pyranose form of sugar. An absorbance at nearly 826.58 cm-1 indicates the linkage of α-glycosides. 3.4. Effect of MFPs on Ethanol-induced Acute Liver Injury in Mice As shown in Table 2A, the liver index increased in model control groups compared to those in normal controls by 16.82%. However, this test indicated the administration of bifendate equaled to the middle dose of MFPs group on the protective effect on the alcohol-induced liver injury (P > 0.05). The highest dose of MFPs has significantly decreased the liver index by 29.14% (P < 0.05), compared with the administration of alcohol group. In Table 2A, we can see that compared with the normal control group, the serum ALT and AST levels in alcohol group elevated greatly, by 66.14% and 62.90% (P < 0.01), respectively, indicating that the alcohol-induced acute liver injury model in mice was well-established. The administration of MFPs showed a significant protective effect 12
on the alcohol-induced liver injury by remarkably preventing the elevation of serum levels of ALT and AST in a dose-dependent manner. The minimum dose of MFPs has similar potency to bifendate (P > 0.05). The highest dose of MFPs has significantly decreased the ALT level and the AST level by 34.79% and 31.71% (P < 0.05), respectively, compared with the administration of alcohol group. Table 2A shows that the administration of alcohol group had obviously increase in the MDA level and decrease in the GSH level, the activities levels of SOD and GSH-Px by 19.36%, 17.92%, 32.24% and 26.65% (P < 0.05), compared with the normal control group, respectively. The results indicated that the liver functions of the model control group were impaired in the present study. As for the activities of SOD and GSH-Px, and the GSH level we observed that the administration of MFPs significantly enhanced these activities (P < 0.05, P < 0.05, P < 0.01, for SOD, GSH-Px, and GSH, respectively), whereas decreased the MDA level compared with those of model control group. The highest dose group of MFPs has significantly increased the SOD, GSH and GSH-Px level by 20.23%, 73.22% and 40.90% (P < 0.05), respectively, and has significantly decreased the level of MDA by 30.10%, comparing with the administration of alcohol group. The histoarchitecture of hematoxylin and eosin-stained liver sections from each group mice was inspected by light microscopy, and the typical pathological sections of each group mice are shown in Fig. 2A1-A6. In control group (group I, Fig. 2A1), the histologic characteristics included normal morphology of hepatic lobules which was observed with well designated hepatic cells, and no necrotic and apoptotic hepatic 13
cells were found and hepatic sinusoids looked clear. Also, there were a lot of free ribosomes and rough surfaced endoplasmic reticulum. In general, this photomicrographs of pathology section from a healthy liver. In model group (group II, Fig. 2A2), however, animals exposed to ethanol exhibit hepatocytes with steatosis, lipid degeneration, diffuse ballooning degeneration, and cytoplasm loosening, indicating disruption of the normal liver architecture. Conversely, bifendate and MFPs treatment significantly reversed the ethanol-induced liver damage. Bifendate-treated and MFPs-treated mice showed an integrated liver cell architecture, and only seldom cells had ballooning and steatosis, and hepatocyte regeneration and no necrotic cells were observed, which expressed the cells were well preserved (Fig. 2A6). 3.5. Effect of MFPs on Subacute Ethanol-induced Liver Injury in Mice In Table 2B, we can see that compared with the normal control group, the liver index, caused a significant elevation compared with the administration of alcohol group by 19.77% (P < 0.05), respectively. The administration of MFPs showed a significant protective effect on the alcohol-induced liver injury by remarkably preventing the elevation of the liver index in a dose-dependent manner. The minimum dose of MFPs has similar potency to bifendate (P > 0.05). The highest dose of MFPs has significantly decreased the liver index by 14.93% (P < 0.05) compared with the administration of alcohol group. The activities of plasma ALT and AST, are the significant elevation of indicating liver injury. As shown in Table 2B, levels of ALT and AST increased in model control groups (14.37 and 31.91 U/L, respectively) compared to those in normal controls, 14
indicating that the subacute alcohol-induced liver injury model in mice was well-established in this study. However, these two plasmas test indicators the administration of bifendate/MFPs groups were a significant protective effect on the alcohol-induced liver injury. The highest dose of MFPs has significantly decreased the ALT level and the AST level by 45.23% and 54.38% (P < 0.05), respectively, compared with the administration of alcohol group. As shown in Table 2B, the MDA level obviously increased and the GSH level, and the activities levels of SOD and GSH-Px were dramatically decreased in the administration of alcohol group (P < 0.05, P < 0.05, P < 0.05 for GSH, SOD and GSH-Px, respectively) compared with those in normal group. The results confirmed again that the model was established successfully in this study. However, pretreatment with MFPs could improve markedly the GSH level by 52.45% and the activities were of SOD and GSH-Px by 19.65% and 20.63%, whereas could decrease the MDA level by 22.22%, in livers of alcohol-treated mice. Moreover, significantly high levels of activities were observed in the livers of mice that had been treated with bifendate (246.69 and 385.20 U/mg protein for SOD and GSH-Px, respectively, P < 0.01). The highest/middle dose of MFPs has similar potency to bifendate (P > 0.05). The results suggest that MFPs had the protective action for the ethanol-induced subacute liver injury. The representative photomicrographs of liver sections from the experimental groups were shown in Fig. 2B1-B6. The pathology section examination from the normal control group (group 1, Fig. 2B1) showed that the liver tissue organization is regular 15
and the hepatic lobules of them was clear without liquid droplets, liver sinusoid express normal, and the hepatic cord was well-arranged. However, the photomicrographs of liver sections from the model groups (group 2, Fig. 2B2) which treated by ethanol demonstrated that cytoplasm loosening of hepatic cells, diffuse ballooning degeneration and microvesicular steatosis, lipid degeneration, diffuse ballooning degeneration led to disrupted liver lobule structure. Moreover, there were acidophilic and necrotic liver cells in the photomicrograph. Granular degeneration could be found in some liver cells. And the inflammatory cells infiltrated were observed in the necrotic lesion. Conversely, the groups dealt with bifendate and MFPs significantly reversed the ethanol-induced liver injury. The liver tissue displayed an intact architecture, and only a few liver cells had ballooning and steatosis, and hepatocyte regeneration and no necrotic cells were observed (Fig. 2B6). 4. Discussion Alcohol-induced liver injury, one of the most common reasons of liver diseases, has been an increasingly important public-health problem worldwide. Oxidative stress, induced the damage of tissue by the imbalance of prooxidant and antioxidant, is thought to play a key role in the pathogenesis of alcohol-induced liver injury [26, 27]. Alcohol mediates oxidative stress in multiple ways involving the release of liver enzymes such as AST, ALT and ALP in serum, lipid peroxidation, depletion of cytoprotective antioxidant functions, and the generation of reactive oxygen species (ROS) [28]. ROS, one kind of pro-oxidants including superoxide radical, hydrogen peroxide, nitric oxide and hydroxyl radical, are generated naturally by cells in the 16
biological systems [29]. It is very important for keeping the biological systems from damage to rapidly clear the normally produced ROS by antioxidants. SOD and GSH-Px, can scavenge ROS and terminate the free-radical chain reaction, are critical antioxidants, and play important roles in hepatoprotection. Lack of SOD and GSH-Px is concerned with the redundantly accumulation of ROS, which could cause lipid peroxidation, tissues and organs injury, diseases and aging [30]. MDA, the stable metabolite of lipid peroxidation products, is associated with free radical effect in biological system. GSH can protect thiol group (-SH) of enzymes, adjust the synthesis of ribonucleotide and neutralize free radicals. In China, many herbal medicines been used to prevent and remedy the ALD, and some literatures also suggest that the mulberry water extracts can prevent alcohol-induced liver injury and CCl4-induced liver damage and fibrosis [11, 31]. However, the effect of MFPs on ethanol-induced liver injury is not clear. Therefore, the current study explored the hepatoprotective effect of MFPs. Alcohol played an inducement role in liver injury. Therefore, the alcohol-induced liver injury model is widely used to test the liver-protection effects of many bioactive substances, such as foods, drugs and herbs [32-34]. Bifendate, synthesized from schisandrin C with liver protective effect in rodents, is usually used as the drug to treat hepatitis, and also is regarded as a positive control for researching other hepatic protectors [35, 36]. So this study used bifendate as a positive control to establish acute and subacute alcohol-induced liver injury in mice model to evaluate the liver-protection effect of MFPs by histopathologic examination, and determining the 17
antioxidant indicators (SOD, GSH, and GSH-Px) and the lipid peroxidation product (MDA) in liver, as well as liver function index (AST and ALT) in serum. Compared normal group and positive group with alcohol model group showed that an apparent decline in the ALT, AST, DMA levels, and the level of SOD and GSH-Px have been obviously enhanced, which indicated the acute and subacute alcohol-induced liver injury model in mice were set up successfully. The histopathologic examination, which expressed cellular correlates of injury after ethanol administration (Fig. 2). This hepatocytes damage resulted in an increase in liver index (Table 2A and Table 2B). Cellular membranes breakage caused intracellular substances leaks, and accompanied by the elevation of serum liver markers. Therefore, the levels of AST and ALT in serum, which were established counters of hepatic damage, were improved. MFPs administration promoted biochemical and histologic modulation demonstrating liver repair to normality. The determination of the antioxidant indicators (SOD, GSH, and GSH-Px) and the lipid peroxidation product (MDA) in liver, as well as liver function index (AST and ALT) in serum, demonstrated that the MDA level obviously lower, while the a levels of GSH, SOD and GSH-Px were dramatically higher in the normal group, compared with those of the administration of alcohol group. It indicated that alcohol administration was likely significantly reduced the biosystems of anti-oxidations, thus caused the accumulation of free radicals, and caused lipid peroxidation, subsequently. In addition, the results showed that the MDA level significantly reduced and the SOD activities, GSH-Px activities, the GSH level were significantly increased in MFPs 18
group compared with the alcohol-induced liver injury group. Furthermore, MFPs exhibited significantly inhibition function to superoxide radical and lipid peroxidation, as well as bifendate. In conclusion, MFPs has a fine liver-protection effect on acute and subacute alcohol-induced liver injury, which might relate to its function of antioxidase activities, elimination of free radicals, and inhibition of lipid peroxidation. The results agree with the previous researches that some polysaccharides have protective function on ALD [12, 37]. Literature sources available showed that numerous complicated factors, included molecular weight, monosaccharide composition and conformation, affect the bioactivity of polysaccharide [38]. The monosaccharide composition analysis indicated that MFPs was mainly composed of Glc, GalA, Rha and Gal. In addition, MFPs contented lots of uronic acid. Therefore, experimental results indicated that structural properties and composition of MFPs might have greatest impact on its anti-oxidative stress, and then be related to its hepatoprotective effect. 5. Conclusions In this study, the preliminary structure characterization of and the hepatoprotective activity of MFPs were carried out. The assay of the preliminary structure characterization demonstrated that MFPs mainly contained 67.93 ± 1.18% total polysaccharide, 31.03 ± 0.54% uronic acid and little protein and sulfate. And MFPs was mainly composed of Glc, GalA, Rha and Gal, along with a small quantity of GlcA, Xyl, Ara and Man. Meanwhile, the assay of hepatoprotective activity in vivo demonstrated that the ALT level and AST level in serum, the liver levels of MDA are 19
remarkably lower, the liver levels of GSH, the liver activities of SOD and GSH-Px are significantly higher in MFPs group compared with the administration of alcohol group. Moreover, the histopathologic examination showed that the administration of MFPs satisfactorily repaired the alcohol-related liver damage. In conclusion, MFPs has a significant liver-protection effect on acute and subacute alcohol-induced liver injury, which might be related to its function of the recovery of antioxidase activities, elimination of free radicals, and inhibition of lipid peroxidation. This finding showed that MFPs might be one of important active substances for preventing and remedying ALD. Acknowledgements This project was supported by the National Natural Science Foundation of China [grant numbers 31570358), Guizhou Province High Level Creative Talents Cultivation [grant numbers [2015]4033], Guizhou Province Special Found for Modern Science and Technology Industrial of Traditional Chinese medicine research and development [grant numbers [2013]5005].
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Figure Captions Fig. 1 The preliminary characterizations of MFPs, including molecular sizes (A), HPLC chromatograms of PMP derivatives of monosaccharide standard samples and hydrolyzate (B), and the monosaccharide composition (C), and infrared spectrums (D).. Fig. 2 Effect of MFPs on alcohol-induced acute and subacute liver injury mice liver tissue histopathology. The livers of mice were removed and stained with HE staining and observed under a MI12 invert light microscope (× 40 magnification). A1-A6 showed the effects of MFPs on alcohol-induced acute liver injury mice liver tissue histopathology (A1, normal control group; A2, alcohol model control group; A3, administrated with bifendate at a dose of 150 mg/kg; A4, administrated with MFPs at a dose of 50 mg/kg; A5, administrated with MFPs at a dose of 100 mg/kg; A6, administrated with MFPs at a dose of mg/kg). B1-B6 showed the effects of MFPs on alcohol-induced subacute liver injury mice liver tissue histopathology (B1, normal control group; B2, alcohol model control group; B3, administrated with bifendate at a dose of 150 mg/kg; B4, administrated with MFPs at a dose of 50 mg/kg; B5, administrated with MFPs at a dose of 100 mg/kg; B6, administrated with MFPs at a dose of 150 mg/kg). Fig. 3 A schematic diagram of the proposed mechanism by which MFPs prevents alcohol-induced liver damage. MFPs prevents alcohol-induced liver damage might relate to its function of the recovery of antioxidase activities, elimination of free radicals, inhibition of lipid peroxidation and anti-oxidative stress.
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Fig. 1
26
Fig. 2
27
Fig. 3
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Table 1 The chemical composition of MFPs. Content (%) Component MFPs
Polysaccharide
Sulfate
Uronic acid
Protein
67.93 ± 1.18
6.39 ± 0.04
31.03 ± 0.54
3.52 ± 0.02
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Table 2A Effects of MFPs on the GSH level and MDA level, the activities of SOD and GSH-Px in liver, and the ALT and AST levels in serum of alcohol-induced acute liver injury mice. Groupa Normal Model Positive MFPs-H MFPs-M MFPs-L SOD (U/mg protein) 249.67 ± 15.21# 204.91 ± 10.01 243.71 ± 7.98# 246.37 ± 11.02# 231.12 ± 8.71 214.58 ± 9.23 GSH-Px (U/mg protein) 440.20 ± 17.51# 322.90 ± 8.13 443.42 ± 10.65# 435.06 ± 6.98# 385.08 ± 14.73 346.87 ± 16.46 GSH (nmol/mg protein) 265.98 ± 10.78# 180.22 ± 7.12 258.42 ± 14.57# 256.20 ± 13.15# 232.08 ± 15.01 205.44 ± 9.69 MAD (nmol/mg protein) 0.54 ± 0.14# 0.67 ± 0.36 0.57 ± 0.29# 0.59 ± 0.11# 0.61 ± 0.21 0.64 ± 0.17 AST (U/L) 39.30 ±2.97# 64.02 ± 7.82 44.94 ± 3.21# 43.72 ± 2.57# 50.79 ± 5.18 58.82 ± 6.54 ALT (U/L) 29.71 ±4.11# 49.09 ± 4.59 30.86 ± 3.42# 32.01 ± 3.79# 38.57 ± 4.14 42..46 ± 3.33 Liver index (%) 3.51 ± 0.45# 4.22 ±0.31 3.86 ±0.23 3.59 ±0.16# 3.81 ±0.31 4.07 ±0.44 a Normal, normal control group; Model, alcohol model control group; Positive, administrated with bifendate at a dose of 150 mg/kg; MFPs-H, administrated with MFPs at a dose of 150 mg/kg; MFPs-M, administrated with MFPs at a dose of 100 mg/kg; MFPs-L, administrated with MFPs at a dose of 50 mg/kg. #
Data were presented as mean ± SD (n = 10), values with alcohol model control group are significantly different (P < 0.05).
Table 2B Effects of MFPs on the GSH level and MDA level, the activities of SOD and GSH-Px in liver, and the ALT and AST levels in serum of alcohol-induced subacute liver injury mice. Groupa Normal Model Positive MFPs-H MFPs-M MFPs-L SOD (U/mg protein) 246.69 ± 3.45# 198.21 ± 2.78 238.09 ± 21.56# 248.29 ± 5.61# 236.31 ± 4.14# 229.68 ± 2.97 GSH-Px (U/mg protein) 385.20 ± 4.51# 305.74 ± 8.12 372.12 ± 7.56# 380.94 ± 5.41# 357.35 ± 6.71 322.69 ± 6.12 GSH(nmol/mg protein) 232.47 ± 4.17# 110.55 ± 1.03 212.22 ± 5.28# 206.36 ± 2.11# 164.85 ± 3.01 136.12 ± 4.87 MAD (nmol/mg protein) 0.55 ± 0.02# 0.71 ± 0.72 0.55 ± 1.01# 0.59 ± 0.42# 0.61 ± 0.13 0.65 ± 0.57 AST (U/L) 45.85 ± 10.42# 77.76 ± 13.11 53.74 ± 7.97 50.51 ±7.5259# 58.09 ± 4.51 69 ± 12.13 ALT (U/L) 28.71 ± 5.61# 43.08 ± 11.08 30.34 ± 9.45 29.60 ±7.18# 32.63 ± 2.55 38.80 ± 5.82 Liver index (%) 2.84 ± 0.65# 3.54 ± 0.32 3.01 ± 0.34 2.88 ± 0.21# 3.27 ± 0.39 3.37± 0.32 a Normal, normal control group; Model, alcohol model control group; Positive, administrated with bifendate at a dose of 150 mg/kg; MFPs-H, administrated with MFPs at a dose of 150 mg/kg; MFPs-M, administrated with MFPs at a dose of 100 mg/kg; MFPs-L, administrated with MFPs at a dose of 50 mg/kg. #
Data were presented as mean ± SD (n = 10), values with alcohol model control group are significantly different (P < 0.05).
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