The structural characteristics, antioxidant and hepatoprotection activities of polysaccharides from Chimonanthus nitens Oliv. leaves

Journal Pre-proof The structural characteristics, antioxidant and hepatoprotection activities of polysaccharides from Chimonanthus nitens Oliv. leaves

Ximei Ye, Qi An, Si Chen, Xin Liu, Ning Wang, Xiang Li, Meng Zhao, Yi Han, Zitong Zhao, Kehui Ouyang, Wenjun Wang PII:

S0141-8130(19)36068-4

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.200

Reference:

BIOMAC 13981

To appear in:

International Journal of Biological Macromolecules

Received date:

1 August 2019

Revised date:

17 October 2019

Accepted date:

25 November 2019

Please cite this article as: X. Ye, Q. An, S. Chen, et al., The structural characteristics, antioxidant and hepatoprotection activities of polysaccharides from Chimonanthus nitens Oliv. leaves, International Journal of Biological Macromolecules(2018), https://doi.org/ 10.1016/j.ijbiomac.2019.11.200

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© 2018 Published by Elsevier.

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The structural characteristics, antioxidant and hepatoprotection activities of polysaccharides from Chimonanthus nitens Oliv. leaves Ximei Ye1, Qi An1, Si Chen2, Xin Liu1, Ning Wang1, Xiang Li1, Meng Zhao1, Yi Han1, Zitong Zhao1, Kehui Ouyang2, Wenjun Wang1,* 1

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Key Lab for Natural Products and Functional Foods of Jiangxi Province, College of Food Science and

Engineering, Jiangxi Agricultural University, Nanchang 330045, China 2

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College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China.

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*Corresponding Authors

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(Wenjun Wang) Tel: +86-791-83813655; Fax: +86-791-83813655;

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E-mail address: [email protected]

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Abstract This work aimed to investigate the structural characteristics, antioxidant activities and hepatoprotection effect of Chimonanthus nitens Oliv. leaves polysaccharides (COP) on alcohol-induced oxidative damage in mice. Physical and chemical analysis showed that COP contained four monosaccharides including arabinose (Ara), mannose (Man), glucose (Glu) and galactose (Gal), with mass percentages of 26.6%, 5.1%, 32.2% and 36.0%, respectively, which

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was a heteropolysaccharide with both α- and β- configurations. In vivo experiments indicated that oral administration COP significantly reduced the levels of ALT, AST and MDA in serum, and

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significantly increased the activity of SOD and GSH-Px. Mice pretreated with COP had a higher

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superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in liver and lower

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content of TNF-α, IL-6 and IL-1β in the liver and serum when compared with alcohol exposure. In

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addition, the liver histopathological changes induced by alcohol returned to normal in the COP pretreatment group. These results suggest that COP has a protective effect on acute liver injury

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induced by alcohol.

Keywords: polysaccharides; antioxidant; hepatoprotection; Chimonanthus nitens Oliv. leaves

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1． Introduction Alcohol consumption is spread worldwide, particularly, in China, its average alcohol rose 76% from 2005 to 2016. According to The World Health Organization Management published, 3 million deaths resulted from harmful use of alcohol, that also caused more than 200 diseases and injuries especially the alcohol liver damage [1]. Alcoholic liver disease (ALD) is a term that

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encompasses the liver manifestation of alcohol over consumption, including fatty liver, alcohol

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hepatitis, and chronic hepatitis with liver fibrosis or cirrhosis [2]. As a major organ in most vertebral species, liver has many important functions of detoxification metabolism on

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hepatotoxicants, thus many drinkers are seeking hepatoprotective medicines help [3]. However,

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the long-term use of synthetic hepatoprotective medicines causes side-effects and endanger

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people's health [4]. Therefore, it is imperative for alcohol consumers to explore effective natural

[5].

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compounds to protect against or slow down the progression of alcohol liver injury in the ear stage

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Chimonanthus nitens Oliv. leaves, is also called Gold tea, Xiang tea, Yan Ma San or Mao Shan tea which is deemed to be a very important Chinese traditional medicine [6, 7]. Chimonanthus nitens Oliv. is widely distributed in Sanqing Mountain, Jiangxi Province, Kaihua and Chun'an Counties in west Zhejiang Province, Baojing County, west Hunan Province [8]. According to Compendium of Materia Medica, Chimonanthus nitens Oliv. leaves show plenty biological activities such as heat-clearing, detoxifying, sobriety [9]. Chen et al reported that Chimonanthus nitens Oliv. leaves exhibited the function of reducing weight, antiappetite, and reducing TG and TC levels in obese model mice [10]. In addition, it was reported that Chimonanthus nitens Oliv. leaves had a good hepatoprotective effect [11]. Polysaccharides, which are the most abundant

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metabolite in Chimonanthus nitens Oliv. tea, possessed the potential of the hepatoprotective activity. In this study, we investigated the antioxidative, anti-inflammatory and hepatoprotective effects of polysaccharides form Chimonanthus nitens Oliv.

2. Materials and Methods

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2.1. Material and Chemicals

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The diagnostic kits used for investigating the activities of SOD, GSH-Px and MDA contents

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were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The kits for

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the TNF-α, IL-1β and IL-6 investigation were supplied by Wuhan Boster Biological Technology

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Co. Ltd. (Wuhan, China). All the other reagents and chemicals used in the present work were of

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analytical grade, and they were provided by local chemical suppliers in China.

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2.2. Extraction, isolation and purification of Chimonanthus nitens Oliv. polysaccharides

Chimonanthus nitens Oliv. polysaccharides (COP) were prepared by a previous method with minor modification [12]. In brief, the dried Chimonanthus nitens Oliv. leaves were ground into a powder and filtered through 60-mesh sieve. The powder was extracted twice with distilled water at 80 °C for 2 h after being removed lipids and soluble materials by treated with petroleum ether for 48 h. After filtering the solution, the filtrate is collected and concentrated by rotary evaporation at 55 °C, then mixed with three volumes of ethanol (95%, v/v). Stand for 24 h at room temperature, the precipitate was gathered after centrifugation at 3800 r/min for 15 min, then dissolved in distilled water and deproteinized via the Savage method (chloroform: n-butyl alcohol =4:1, repeat 6 to 9 times). The deproteinized solution was dialyzed in running water for 48 h then 24 h in

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distilled water and water was changed every few hours. The crude polysaccharides were collected after lyophilization. The crude product was dissolved in double-distilled water and further purified by DEAE-52 cellulose chromatography. Elution was conducted with distilled water, 0.1 m, 0.2 m, 0.3 m and 0.4 m NaCl solution (1 ml /min), respectively, then the distilled water components which is the eluent with the most abundant content was separated and collected. Then dialyzed,

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lyophilized and named COP for the further experiment.

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2.3 FT-IR analysis of COP

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The COP (1 mg) was mixed with KBr powder (100-200 mg) and then pressed into 1 mm

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pellets for infrared spectral analysis with a range of 400-4000 cm-1 [13].

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2.4 Scanning electron microscopy (SEM) of COP

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Take appropriate amount of dried COP powder and fix it on the sample table, then spray gold.

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Then observed with a scanning electron microscope and images were collected using XT microscope control software.

2.5 Thermogravimetric analysis of COP

Weigh COP 3-5 mg and place it in an alumina crucible, then conduct Thermogravimetric analysis (TGA) was recorded on a PerkinElmer TGA4000 at heating rate of 10 °C /min in N2.

2.6 Analysis of monosaccharides composition

The compositional monosaccharides of COP were analyzed by GC-MS (QP2010, Shimadzu, Japan) by previous procedure with proper modification [14]. Briefly, the identification and the

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quantification of monosaccharide compositions of COPs were estimated by GC-MS equipped with RXI-5 SIL MS chromatographic column (30 m*0.25 mm, coating thickness 0.25 μm). The analysis condition was as follows: the carrier gas was Helium, the flow rate of the carrier gas was 1 mL/min. The injector temperature was 250 °C, column temperature was 120 °C initial and column temperature increased to 250 °C by 3 °C/min and kept this temperature for 5 min. The

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monosaccharide identifications were processed by comparing them with the standard reagents

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rhamnose (Rha), fucose (Fuc), arabinose (Ara), xylose (Xyl), mannose (Man), glucose (Glu),

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2.7 Determination of molecular weight (MW)

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galactose (Gal).

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The relative molecular weight and homogeneity were evaluated by high performance gel permeation chromatography (HPGPC, LC-10A Shimadzu) according to the method reported

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previously [15]. The detailed operation conditions contained steaming water for the mobile phase,

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flow rate of 0.8 mL/min, the column temperature 40 °C. A series of Dextran T standards were used to establish the calibration curve of molecular weight determination.

2.8 Antioxidant activities of COP in vitro

The DPPH radical (DPPH˙), hydroxyl radical (˙OH), superoxide anion radical (O2˙−), scavenging activities and reducing power of COP were measured according to a reported method with proper modification [16].

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2.9 Animal experiments

2.9.1. Design of animal experiment

Male KM mice (20±2.1 g) were purchased from Hunan Silaike Laboratory Animal Co., Ltd., (SCXK (Xiang) 2016-0002, Changsha, China). All rats with a standard diet management regulations 20–22 °C, relative humidity 55 ± 10%, and 12 h/12 h light and dark cycle. After

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acclimation for 5 days, mice were randomly divided into the following six groups (n = 8): normal

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control (NC), alcohol acute liver injury (model group), bifendate pills group (PC, 150 mg/kg body

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weight), COP-L (150 mg/kg body weight) group, COP-M (300 mg/kg body weight) group,

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COP-H (600 mg/kg body weight) group. In the normal and model groups, mice were orally

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administered with a single dose of physiological saline once a day. In the positive control group, mice were orally administered with a single dose of bifendate pills (150 mg/kg body weight) once

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a day. Similarly, in the COP-treated groups, mice were orally administered with COP at 150, 300

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and 600 mg/kg body weight once a day. All the administrations were conducted from 3:00 pm to 4:00 pm and lasted 30 consecutive days. On the thirtieth day, after the last administration for 1 h, all the mice except those in the normal group were received 12 mL/kg Red Star Erguotou, and then all the mice were fasted strictly, but allowed free access to water as usual. 18 hours later, all mice were weighed, and then sacrificed to obtain blood and livers. All the experiments were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Association for Laboratory Animal Science and approved by the Animal Care and Use Committee of the Jiangxi Agricultural University (JXAUA01) and the related ethical regulations of our university.

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2.9.2 Biochemical assays

The sample of blood were collected from eyeballs and centrifuged at 1500×g for 15 min at 4 °C after stand for 1 h to afford the required serums and stored at −80 °C for further test. The liver was surgically removed, weighed, and frozen at −80 °C. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide

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dismutase (SOD), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) were tested

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using commercial kits according to the instructions. The livers were homogenized (1:9, w/v) in

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phosphate buffered solutions (0.2 mol/L, pH 7.4) centrifuged at 3800×g for 15 min to obtain liver

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homogenate for assessing ALT, AST, SOD, GSH-PX and MDA levels according to the kits

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instructions.

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2.9.3 Measurement of TNF-α and IL-6, IL-1β levels

(ELISA) kits.

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The TNF-α, IL-6 and IL-1β levels were measured using enzyme-linked immunosorbent assay

2.9.4 H&E staining

Liver tissue samples were collected from the right lobe of the liver, fixed with 10% formalin buffer, embedded in the liver, fragmented into 5-μm sections, and stained with hematoxylin and eosin (HE) [17].

2.10 Date analysis

All values are expressed as mean ± standard deviation (SD). SPSS 20.0 software was used for variance (ANOVN) analysis and Dunn’s test. *P < 0.05, **P < 0.01 was statistically significant.

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3. Results

3.1 FT-IR analysis

The FT-IR spectra of COP was show in Fig. 1. FT-IR showed a strong and broad absorption peak at 3404.54 cm−1, which suggests the hydroxyl groups of polysaccharide [13]. The weak band at 2931.33 cm−1 was due to C—H bonding [18]. In addition, the asymmetrical stretching peak of

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COP which appeared at 1629.16 cm-1, indicating the presence of C=O of the carboxylate groups

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[19, 20]. The relatively strong absorption peaks at 1384.27 cm−1 was due to the C—H bond

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deforming vibration [21]. The stretching peaks at 1153.68 cm−1 of asymmetric vibration of

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C—O—C glycosidic rings indicated that COP was pyran polysaccharide [22]. COP also showed

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strong absorption peaks at 1078.92 cm-1 and 1025.13 cm−1 respectively, corresponding to pyran

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ring highly consistent with the result above, which was possibly considered as the fingerprint of molecules [15, 23]. Moreover, the absorption peaks at 938.07 cm-1 and 859.17 cm-1 showed the

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presence of β- and α-configurations in COP [21, 24].

3.2 SEM and Thermogravimetric analysis of COP

The thermal stability of COP is shown in Fig. 2. It can be seen from the TGA curve that when the temperature is 101.1°C, the quality of polysaccharide tends to be stable after the first mass loss, which may be caused by water evaporation in polysaccharide molecules. When the temperature is 228.2°C, the second mass loss of polysaccharides occurs, because of the thermal decomposition of proteins and some polysaccharides in polysaccharides. When the temperature further increased to 350.9°C, the third loss occurred, which should be caused by the degradation of polysaccharide

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structure caused by too high temperature. When the temperature rose to 800°C, the residual mass of polysaccharide was 2.6%. This TGA graph indicates on the fact that at temperatures lower than 228.2°C the polysaccharide is chemically stable. The thermal behavior of polysaccharides is related to the differences in their structure and functional groups, and the physical and chemical changes of polysaccharides during heat treatment are unique to given polysaccharides[25].

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The electron micrographs of COP amplified 500 times, 1000 times, 5000 times and 10000

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times were shown in Fig. 3A, B, C and D, respectively. It can be seen from the figure that the COP

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3.3 GC-MS and HPGPC analysis of COP

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is hollow and loose, with rough protrusion on the surface.

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The monosaccharide compositions of COP were determined by comparing the retention time of standards and the results were shown in Fig. 4. Obviously, the COP primarily contained four

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monosaccharides including arabinose (Ara), mannose (Man), glucose (Glu) and galactose (Gal) in

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the mass percentages of 26.6%, 5.1%, 32.2% and 36.0%, respectively. The linearity of the method was determined using Dextran standards of different molecular weights to determine the homogeneity and molecular weight of COP. The calibration curve of Dextran standard was plotted as the molecular weights on a log scale versus the retention time. The regression equation was logMw= -0.2906tR+13.124 (Mw: weight-average molar mass (Da); tR: the retention time (min)) with a high correlation of R2 = 0.991 obtained by the standard curve. As show in Fig. 5, there were two peaks in the HPGPC profile of COP, indicating that COP had two fractions. Respectively, the molecular weights of COP were 33.94 kDa (tR = 29.571 min) and 1.63 kDa (tR = 34.096 min), based on calibration with standard dextran.

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3.4 Antioxidant activities of COP in vitro

As show in Fig. 6, antioxidant activities of COP were evaluated on the scavenging radicals (DPPH·, O2·− and OH·) and reducing power in vitro. It could be seen in Fig. 6A, COP had the ability to scavenge DPPH· at the selected concentration range from 0.5 mg/mL to 2.5 mg/mL, where DPPH· scavenging effects continually increased from 0% to 79.09%. The assay for

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scavenging O2·− showed that the antioxidant effect of COP had the same change trend with VC, in

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which COP displayed relatively high scavenging activity on O2·−. Hydroxyl radical scavenging

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activity of COP was shown in Fig. 6C, it could be observed that the scavenging activity

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against ·OH was in a concentration-dependent manner. Moreover, COP had a certain reducing power, which was also shown a dosage-dependent manner. Therefore, COP displayed potential

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antioxidant property, although it was inferior to the reference VC in antioxidant capacity.

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3.5 Effects of COP on the body weight, liver weight and liver index

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The effects of COP on the body weight and liver index in ALD mice are shown in Table 1 and Table 2. Clearly, the difference in the initial body weight of the mice was significant between NC group and the others except the PC group. However, the amounts of weight within the COP-L, COP-M and COP-H groups were higher than that in the MC group, but not significant (P < 0.05). Meanwhile, the liver weights of mice in the MC group were enhanced when compared with the weights in the NC group, indicating that liver damage occurred after acute alcohol injection. Clearly the COP showed potential effects on alleviating organ damage. Although compared with the model group, the prevention effect was significant, there was no significant difference between COP groups (P > 0.05). The liver indexes were decreased by 15.09% at the dose of 600 mg/kg

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body weight, by 16.98% at 300 mg/kg body weight, and by 13.21% at 150 mg/kg body weight in comparison with that in the MC group, respectively. The results indicated COP had certain hepatoprotective effects against the alcohol-induced liver injury. 3.6 Effects of COP on serum AST, ALT, MDA, SOD and GSH-Px The effects of COP on alcohol-induced elevation of serum AST and ALT were shown in Fig.

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7A and B. Compared with the normal group mice, the serum AST and ALT activities of the alcohol-induced group were significantly elevated (P < 0.01) after alcohol consumption. There

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was no significant difference between PC group and normal group (P > 0.05), suggesting that

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bifendate pills can reduce alcohol-induced acute oxidative liver injury. Compared with MC group,

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the activity of AST and ALT in serum of COP groups was significantly reduced (P < 0.01).

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In addition, compared with the NC group, the level of MDA in the serum induced by alcohol

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was significantly increased (P < 0.05), which was shown in Fig. 7C. After treatment with COP, abnormal growth was inhibited. Compared with MC group, MDA activity in serum of COP-M

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group was significantly reduced (P < 0.01), and there was no significant difference in therapeutic effect between COP-M group and PC group (P > 0.05), indicating that COP has a good therapeutic and preventive effect on liver injury caused by alcohol. Compared with MC group, the activities of SOD and GSH-Px in serum of alcohol-induced mice were significantly reduced (Fig. 7D and E, P < 0.05), indicating the presence of oxidative stress in alcohol-treated mice. However, COP treatment significantly improved the reduction of these parameters compared with the MC group. The activity of SOD and GSH-Px in serum of COP-M group was the highest, 57.7% and 106.2% higher than those in the MC group, respectively.

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3.7 Effects of COP on liver MDA, SOD and GSH-Px

Alcoholic liver injury can lead to an increase in reactive oxygen species/free radicals, leading to a significant decrease in the activity of non-enzymatic antioxidants and endogenous antioxidant enzymes [22, 26]. Liver GSH-Px and SOD levels in MC group were lower than those in NC group (Fig. 8B and C, P < 0.05).There was no significant change in liver GSH-Px level of COP low dose

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(150 mg/kg body weight) compared with MC group (P > 0.05). SOD and GSH-Px levels increased

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significantly with COP concentration (P < 0.05). In addition, malondialdehyde (MDA) can be used

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to determine lipid peroxidation in liver. Compared with NC group, MDA level in the liver of MC

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group was significantly increased (Fig. 8A). MDA levels significantly decreased after COP treatment (P < 0.05). The results showed that COP had significant therapeutic effect on

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alcohol-induced oxidative liver injury.

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3.8 Effects of COP on inflammatory cytokines

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IL-1β, TNF-α, and IL-6 are very important inflammatory factors, which may cause cell inflammation and death [27]. The effect of COP on serum and liver IL-1β, TNF-α, and IL-6 levels is shown in Fig. 9. Compared with the NC group, there was a significant elevation in the levels of IL-1β, TNF-α, and IL-6 in the MC group (P < 0.05). However, COP treatment significantly reduced the level of these parameters in both liver and serum, compared with MC group (P < 0.01). Compared with MC group, after pre-treating with COP at 300 mg/kg body weight, serum concentrations of IL-1β, TNF-α, and IL-6 decreased by 32.11%, 29.56% and 51.11%, respectively. The levels of IL-1β, TNF-α, and IL-6 in the liver were significantly lower than those in the MC group after the treatment with COP of 300 mg/kg body weight (P < 0.05). When compared with

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the results in other COP dose groups, similar conclusions can be obtained. The result indicated that COP had potential anti-inflammatory effects against ALD, which was reflected by suppressing the expression of these cytokines.

3.9 Histopathological observations of mouse liver

The histopathological observations of H&E staining of the livers were performed to

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fundamentally support the results of the biochemical analysis. Compared with the hepatic cellular

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architecture of mice tissues from the normal group (NC), liver sections from alcohol-treated mice

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(MC) showed severe cellular degeneration, hepatocyte necrosis, loss of cell boundaries, and fat

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droplet formation. However, the liver histopathological changes induced by alcohol were

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alleviated by COP therapy, and COP treated mice at 300 mg/kg body weight showed significant protective effects, where the liver displayed normal appearance with well-preserved cytoplasm,

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the cells were arranged tightly and neatly, the nuclei were prominent, and the nucleoli were visible,

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indicating that COP had an excellent protective effect on liver protection. This is consistent with the biochemical results of serum hepatic toxicity markers and hepatic oxidative stress system.

4. Discussion

Alcoholic liver injury is one of the most common causes of liver disease and has become an increasingly important public health problem worldwide. Therefore, research and development on effective therapies for protecting alcohol-induced acute liver injury have received more and more attention [28]. Pervious literature has demonstrated that the possible mechanisms of ALD from alcohol consumption may include oxidative stress, lipid peroxidation, inflammatory factors and

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structural-functional cellular changes. Oxidative stress and inflammation play an important role in the pathogenesis of alcoholic liver injury. The polysaccharides have been found to show hepatoprotective effects by regulating oxidative stress and activating antioxidant enzyme activities [29, 30]. However, few reports on Chimonanthus nitens Oliv. polysaccharides have been published until now. The aim of the present work was to investigate the antioxidant and hepatoprotective

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effects of COP against ALD.

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Hepatocytes synthesize most serum proteins, accordingly, serum level of hepatocyte protein constitute important biomarker reflecting system process and liver status [31]. The activities of

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AST and ALT in the serum are commonly used as reliable markers for monitoring liver damage

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clinically, because they could leach out of hepatocytes into the blood circulation when liver

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damage occurs [32, 33]. Experimental results have shown that acute alcohol intake can

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successfully induce liver damage as evidenced by the increased activity of serum ALT and AST. However, polysaccharide treatment significantly reduced serum ALT and AST activities,

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suggesting that COP had the potential to increase cell membrane stability and thus maintain structural integrity of liver cells. The above results were also supported by pathological observations.

It has been proved that alcoholic liver injury is associated with oxidative stress and lipid peroxidation [34, 35]. Excessive production of ROS including hydroxyl, superoxide anion, DPPH radicals and hydrogen peroxide caused by excessive drinking plays an important role in the development of ALD [36, 37]. Therefore, improving the scavenging ability of ROS is important for the treatment and prevention of ALD. In vivo, SOD, GSH-Px and other antioxidant enzymes are important defense enzymes against alcohol-induced oxidative damage, which can balance the

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antioxidant and oxidation systems. The transcription of GSH-Px and SOD can be up-regulated by nuclear factor erythrocyte 2-related factor -2, thereby inducing the production of antioxidant enzyme [38]. In this experiment, COP significantly increased the activity of SOD and GSH-Px in serum and liver of mice compared with MC group, which may be caused by COP activating erythrocyte 2 related factor -2 in mice. These results further suggest that the activity of antioxidant

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enzymes may be one of the mechanisms promoting the protective effect of COP on alcoholic liver

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injury. In addition, lipid peroxidation, as a major indicator of oxidative damage, can damage the structure and/or function of cell membranes [39]. MDA is an important product of lipid

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peroxidation formed by the interaction between ROS and polyunsaturated fatty acids [40]. In this

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study, COP could reverse the increase of malondialdehyde induced by alcohol.

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Because alcohol-induced inflammation can stimulate liver disease, inflammatory cytokines

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(IL-1β, TNF-α, and IL-6) were tested in this study. Inflammatory cytokines, TNF-α, can accelerate neutrophil migration, promoting neutrophil production of proteolytic enzymes and the generation

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of ROS, and it can lead to liver injuries [41]. In addition, IL-1β and IL-6 play an important role in the accumulation of liver injury, and their elevated levels can lead to some inflammatory diseases and malignancies [42]. IL-1β, TNF-α, and IL-6 in plasma and liver decreased after treatment with COP, suggesting that polysaccharides from Chimonanthus nitens Oliv. can protect liver from alcohol damage.

5. Conclusions

This study showed that COP had a protective effect on acute liver injury induced by alcohol, and alcohol induced acute liver injury by inhibiting oxidative stress, lipid peroxidation and

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enhancing antioxidant capacity. COP inhibited the release of TNF- alpha IL-1β and IL-6 in mice with acute liver injury, showing anti-inflammatory ability. To our knowledge, this is the first time to provide experimental evidence for the liver protective effect of COP, which can be used as a potential natural non-toxic functional food to prevent and alleviate ALD and its complications.

Conflict of interests

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The authors declared no conflicts of interest

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Acknowledgements

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The authors gratefully acknowledge the financial supports by a grant from the Natural Science

(20182BCB22003),

Jiangxi

Modern

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Foundation of China (31560459), Jiangxi Provincial Academic and Technical Leaders Program Agricultural

Scientific

Research

Innovation

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China (YC2018-S203).

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(JXXTCX201703-03), and the Graduate Innovative Special Fund Projects of Jiangxi Province,

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Table. 1. Effects of COP on the body weights, liver weight and liver index Body weight (g)

Liver weight (g)

Liver index (%)

NC

42.40±1.43

1.50±0.09

3.60±0.52

MC

38.10±1.45**

2.04±0.22**

5.30±0.68**

COP-L

38.90±1.66**

1.76±0.17**

4.60±0.52**

COP-M

38.80±1.75**

1.69±0.12**

4.40±0.52**

COP-H

38.40±2.37**

1.72±0.16**

PC

40.80±3.49

1.62±0.14

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Groups

4.50±0.53** 3.90±0.57

The values were reported as the Mean ± S.D. of 8 mice in each group. NC group: saline water; MC group: saline

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water + alcohol (56%, 12 mL/kg body weight); PC group: bifendate (150 mg/kg body weight; **P < 0.01

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compared to the normal mice.

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Table. 2. Effects of COP on the body weights, liver weight and liver index Body weight (g)

Liver weight (g)

Liver index (%)

NC

42.40±1.43**

1.50±0.09**

3.60±0.52**

MC

38.10±1.45

2.04±0.22

5.30±0.68

COP-L

38.90±1.66

1.76±0.17**

4.60±0.52**

COP-M

38.80±1.75

1.69±0.12**

4.40±0.52**

COP-H

38.40±2.37

1.72±0.16**

PC

40.80±3.49**

1.62±0.14**

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Groups

4.50±0.53** 3.90±0.57**

The values were reported as the Mean ± S.D. of 8 mice in each group. NC group: saline water; MC group: saline

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water + alcohol (56%, 12 mL/kg body weight); PC group: bifendate (150 mg/kg body weight; **P < 0.01

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compared to the MC group.

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Highlights

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In this study we conclude that polysaccharides from Chimonanthus nitens Oliv. leaves (COP) has a protective effect on acute liver injury induced by alcohol. In vivo COP significantly reduced the levels of ALT, AST and MDA in serum, and significantly increased the activity of SOD and GSH-Px. Mice pretreated with COP had a higher SOD and GSH-Px activity in liver and lower content of TNF-α, IL-6 and IL-1β in the liver and serum compared with alcohol exposure. Liver histopathological changes induced by alcohol returned to normal in the COP pretreatment group.

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