Characterization of an Acidic Polysaccharides from Carrot and Its Hepatoprotective Effect on Alcoholic Liver Injury in Mice

Xiangying Kong,a Wei Liang,a Xinyue Li,a Meng Qiu,a Wenjun Xu,a and Hongman Chen*a
a Bioscience and Biotechnology College, Shenyang Agriculture University, 120 Dongling Road,
Shenyang 110866, P. R. China,
e-mail: [email protected]

The characteristics of acidic polysaccharides extracted from Daucus carota L. var. sativa Hoffm were investigated and its hepatoprotective effects on alcoholic liver injury were determined in the mice model. A carrot polysaccharide (CPS-I: Carrot polysaccharide-I) with the molecular weight of 3.40 × 104 kDa was isolated from Daucus carota L. and purified by diethylaminoethyl-52 and Sephadex G-150 column chromatography. The components were analyzed by HPLC, which revealed that CPS-I consisted of galacturonic acid, rhamnose, xylose, arabinose, fructose, and galactose at a relative ratio of 1 : 3.16 : 1.13 : 5.53 : 3.45 : 7.76. Structural characterization analysis suggested that CPS-I was mainly composed of 6)-β-D-Galp-(1 and 5)-α-L-Araf-(1 . The hepatoprotective effect of CPS-I was evaluated by alcoholic liver injury mice model.
The results showed that the administration of CPS-I (300 mg/kg/day) alleviated the alcoholic liver injury in mice by increasing the levels of ADH and ALDH and reducing oxidative stress. CPS-I ameliorated the pathological changes of liver characterized by lipid accumulation, and reduced the number of lipid droplets.

1. Introduction
Frequent alcoholic consumption is one of the main contributors of hepatic injury, which has been a global healthy problem.[1] Excessive drinking often leads to abnormal liver changes, such as hepatocytes, necrosis, fibrosis and even cirrhosis.[2] Among many illnesses caused by alcohol consumption, alcohol-induced liver injury is the most common in the world. Accumulating evidence indicated that one of the pathogenesis of alcoholic liver injury was oxidative stress.[3] Alcohol metabolism in liver is mainly dependent on ethanol dehydrogenase (ADH) pathway. Excessive alcohol consumption can lead to the generation of oxidative metabolites including acetaldehyde and free radicals, which play a predominant role in the pathological progress of the alcoholic liver.[4] At present, the first- line drugs such as glucocorticoid available for the treatment of Alcoholic liver disease (ALD) are limited, and some of them exhibit adverse side effects.[5] Natural antioxidants have always been potential liver- protecting agents.[6]
Polysaccharides with antioxidant properties are natural products with many bioactivities.[7] Some polysaccharides in plants, such as polysaccharides extracted from Dendrobium huoshanense, Gynostemma pentaphyllum, Lycium barbarum and Radix Codonopsis lanceolatae exhibited the anti-ALD activity,[8–11] Den- drobium huoshanense polysaccharides could attenuate early steatosis and inflammation by regulating fatty acid metabolism pathway in mice with sub-acute alcoholic liver injury. Polysaccharide (TASP), isolated from (Triticum aestivum. L), displayed hepatoprotective effects against ALD by inhibiting ethanol-induced CYP2E1 activation and hindering the expression of NADPH oxidase genes in ethanol fed mice.[12] How- ever, few studies concerning the hepatoprotective activities of polysaccharides from Daucus carota L. was reported.
Carrot (Daucus carota L.), commonly known as ‘clove turnip ‘ or ‘yellow turnip’, one of the world’s top ten vegetable crops, is an umbelliferae herbaceous plant grown worldwide.[13] The fresh roots of carrot are consumed as vegetables or fruits in different recipes ofthe world. Carrot has been used to promote visual acuity eyesight, heat-clearing and detoxication in China.[14] In recent years, carrots have attracted much attention due to the dietary nutrition of the bioactive compounds including carbohydrates (sugars, pectins and fibers), α- and β-carotene, fat-soluble vitamins as well as minerals.[15–17] Polysaccharides, one of the most abundant carbohydrates in carrots, play pharma- cological roles such as antioxidant, anti-inflammatory and anti-hyperglycemia.[18–20] Previous literature re- vealed that the polysaccharides extracted from the carrots exhibited antioxidant and hypoglycemic ef- fects. In the present study, the hepatoprotective effect of carrot polysaccharide on alcohol-induced acute liver injury in mice was investigated, this study would be helpful to understand the protective mechanisms of polysaccharides against alcoholic liver disease.

2. Results and Discussion
2.1. Isolation and Purification of Polysaccharide from Carrot
The crude polysaccharide was extracted with distilled water at 80 °C from carrot (Daucus carota L.) and precipitated with 95 % (v/v) ethanol. After the precip- itate was dissolved, along with deproteination and lyophilized to produce a light brown powder. The yield of crude polysaccharide was 14.6 %. Then the compo- nents of CPS were isolated by ion exchange chroma- tography on the DEAE-52 column, eluted with 0.1 mol/ L, 0.2 mol/L and 0.3 mol/L NaCl solution, respectively. Three fractions (Figure 1A) were collected in the chromatogram, which were named CPS I, II and III. The relative carbohydrate content of fraction I, II and III was 61.17 %, 15.23 % and 10.58 %, respectively. Mean- while, the scavenging ability of superoxide anion radical of three fractions were investigated, indicating the value of C50 of I, II and III were 0.38 mg/mL,1.2 mg/mL, and 3.2 mg/mL, respectively. The result revealed that fraction I was the main component of CPS and exhibited the highest antioxidant capacity.
The collected fraction I was further purified by Sephadex G-150 gel filtration chromatography as the chromatogram showed only one peak, which was named CPS-I (Figure 1B). The results showed that the yield of CPS-I was 3.51 %. The content of polysacchar- ide in CPS-I detected by the phenol sulfuric acid method was 97.6 %, whereas the content of uronic acid in CPS-I was 4.53 %. The UV absorption spectra of CPS-I revealed that there was no protein and nucleic acid in polysaccharides

2.2. Structural Characterizations of CPS-I
The HPGPC profile of CPS-I had a single peak, which indicated that it was a homogeneous polysaccharides (Figure 2B). According to calibration with standard dextrans (lgMw= 0.3135 Rt+ 7.8465, R2 = 0.9755),the molecular weight (Mw) of CPS-I was 3.40 × 104 kDa. The monosaccharide compositions of CPS-I were investigated by HPLC analysis. As shown in Figure 2A, the CPS-I was primarily composed of D-galacturonic acid, D-xylose, L-rhamnose, D-arabinose, D-galactose, and D-fructose in the mole ratios of 1: 1.13: 3.16: 5.53:7.76: 3.45.Analysis of CPS-I through FT-IR spectra at the range of 4000 –400 cm—1 was shown in Figure 2C. The absorption peak, appeared at 3420 cm—1,was ascribed to the typical OH stretching vibration. The weak peaks at 2937 cm—1 and 1736 cm—1 were attributed to C H stretching of the CH2 groups and CHO stretch-ing vibration, respectively. The absorption peak found at 1615 cm—1 was corresponding with C=O asymmetric stretching vibrations of the carboxylate groups. In addition, the peak, appeared at 1423 cm—1, was assigned to bending vibration. Moreover, the stretch- ing peaks at 1045 cm—1 indicated the presence of pyranoside.
The 13C-NMR spectrum of CPS-I can provide further structural information (Figure 3B). The chemical shifts at 104.70, 81.67, 84.36, 76.96 and 60.79 were attributed to 5)-α-L-Araf-(1 . The chemical shifts around103.15 –70.58 ppm were ascribed to 6)-β-D-Galp- (1 and the signals around 107 –110 ppm indicated the presence of α-L-p arabinose absorption. The chemical shifts around 80 – 85 ppm, indicated 1,2,3,5linked galactose. A peak in the region of 99.32 ppm pointed to the C-1 position of the α-L- p-rhamnose. The NMR spectrum results suggested that there might be 1,2 or 1,4 linked rhamnose exited in the poly- saccharide.

2.3. Serum ALT Activities and Liver Index
Liver index refers to the ratio of wet liver weight to the body weight of mice, which can partly reflect the degree of the liver injury. As shown in Figure 4A, compared with normal, acute alcohol absorption led to a significant increase in liver index (P < 0.01). In comparison with the model group, the liver index in the CPS-I-100 group was decreased by 24.95 % (P < 0.01), the CPS-I-200 group was reduced by 29.47 % (P < 0.01), and the CPS-I-300 group was reduced by 36.16 % (P < 0.01). Elevated ALT levels can indicate whether mice are damaged by alcohol. Even small amounts of cell necrosis or changes in membrane permeability can result in the release of ALT into the blood and increase ALT activity in the serum. As shown in Figure 4B, compared with normal group, the Levels of serum ALT increased significantly in the model group (P < 0.01), which indicated that the mice model of alcohol- induced liver injury was successfully constructed. Compared with the model group, the administration with CPS-I groups showed a decreasing trend with theincrease of dose, and ALT level was significantly reduced in the high dose group (P < 0.05). 2.4. The Effect of CPS-I on ADH, ALDH, GSH-PX, SOD and MDA in ALD Mice The alcohol dehydrogenase system (ADH) in the liver is responsible for alcohol decomposition. The effect of CPS-I on ADH in mice was investigated. As shown in Figure 5A, in comparison with the model group, thetreatment with CPS-I (200 mg/kg/day and 300 mg/kg/ day) significantly increased ADH levels (P < 0.05). ALDH is responsible for the removal of acetaldehyde, a highly toxic intermediate product of alcohol metabo- lism. Compared with the model group, the ALDH levels in the middle and high dose of CPS-I groups were increased significantly, respectively (P < 0.01) (Figure 5B), indicating that CPS-I improved ethanol metabolism by increasing ADH and ALDH levels. Furthermore, the effects of CPS-I on antioxidant ability in mice liver were investigated. The effect of CPS-I on SOD and GSH-PX activities in liver was shown in Figure 5C and 5D. Compared with the normal group, a significant decrease appeared in the activities of SOD and GSH-px in model group mice (SOD was 81.11 % of normal and GSH-px was 67.89 % of normal, respec- tively. (P < 0.01)). After the administrated of CPS-I for 4weeks, GSH-PX and SOD levels in the CPS-I-200 group and the CPS-I-300 group were all increased by 29.34 %, 31.52 %,19.82 %, 22.41 %, respectively (P < 0.05). The positive control administrated group showed similar results. Meanwhile, the level of MDA also varied among groups, compared with the normal group, MDA level in ALD group was significantly increased by 103.70 % (P < 0.01), compared with the model group, the MDA content in ALD mice treated with low, medium, and high dosages of CPS-I were decreased by 14.54 %,29.09 % and 38.18 %, respectively (Figure 5E). The results indicated that CPS-I could protect against lipid peroxidation caused by alcohol metabolism. 2.5. Histopathological Observations H&E staining of liver tissue provided consistent evidence of biochemical analysis. In the normal group, radially arranged hepatic cords from the central vein was clear, and the polygonal outline of liver cells was intact (Figure 6A). However, pathological changes induced by ethanol, such as cavitation, early steatosis and nuclear shrinkage in liver tissues emerged in model group mice (Figure 6B). Compared with the model group, positive group and CPS-I treatment group alleviated the inflammatory cells, moreover, vacuolization, hepatocyte necrosis, and fibrosis gradu- ally decreased with the increase of polysaccharide dose (Figure 6D, 6E and 6F). In particular, the hepaticarchitecture in the CPS-I-300 group was almost similar to that of the normal hepatic group (Figure 6F). 2.6. Effect of CPS-I on TC, TG, LDL-C, HDL-C in Serum of ALD Mice The indexes related serum lipid including TC, TG, LDL- c, and HDL-c in each group were shown in Table 1. In comparison with the normal group, consuming large amounts of alcohol lead to a significant increase in TC, TG, LDL-c and decrease in HDL-c (P < 0.01). In compar- ison with the model group, the levels of TC, TG, and LDL-c in the CPS-I-200 group were reduced by 34.18 %, 17.69 % and 23.27 %, and 48.71 %, 30.08 % and 56 % ithe CPS-I-300 group (P < 0.01), respectively. Mean- while, the levels of HDL-c in the CPS-I-200 group andthe CPS-I-300 group were all increased by 50.93 % and 55.64 %, respectively, which indicated that CPS-I could improve the blood lipid of mice with alcohol liver injury. 2.7. TEM Observation In order to detect hepatocyte injury, the morpholog- ical changes of subcellular structure in the liver of different groups were observed by transmission elec- tron microscopy (TEM). The architecture of hepatic cells in the normal group was intact, the peripheral morphology and striated cristaes of mitochondria were clear. Meanwhile the rough endoplasmic retic- ulum with lamellar structure were widely distributed in the cells, with occasional few or no lipid droplets (Figure 7A). In contrast, the hepatocyte ultrastructure of the model group was arranged in disorder. A large number of lipid droplets and swollen mitochondria appeared, the structural damage of cristaes and rough endoplasmic reticulum was also observed (Figure 7B). In comparison with the model group, the CPS-I-100 treatment reduced mitochondrial swelling and ne- crosis, but fat droplets abounded (Figure 7D). The mitochondria morphology with medium and high dosages of CPS-I treatment was similar to that of the normal group, and the rough endoplasmic reticulum was clearly visible (Figure 7E and 7F). Although there were still fat droplets in the CPS-I-300 group, the number of fat droplets decreased significantly (Fig- ure 7F). The positive group had a similar effect (Fig- ure 7C). These results indicated that CPS-I alleviated hepatic cells injury induced by ethanol. Carrot is a kind of medicinal and edible vegetable with a variety of secondary metabolites, known as “little ginseng” in China. Polysaccharide is the main bioactive compound in carrot. Our results indicated the presence of an active polysaccharide which named CPS-I in carrots. The CPS-I was polymerized withgalacturonic acid, rhamnose, xylose, arabinose, fruc- tose, and galactose, and with a molecular weight of3.40 × 104 kDa. Previous studies revealed that carrot polysaccharides possessed many important functions including immune regulation, antioxidant capacity and intestines health care.[21] However, few have been reported the protective effect of carrot polysaccharides against liver damage caused by excessive alcohol consumption. Alcohol dependence are serious problems in the world. The pathogenesis of alcoholic liver injury is complex, and the metabolic disorder caused by ethanol and its toxic metabolites are the main contributors.[22–23] After entering the body, ethanol is oxidized to acetaldehyde by hepatic ethanol dehydro- genase. Acetaldehyde can damage liver cells by activating a series of oxidase systems in the liver and cause lipid peroxidation, resulting in increased liver lipid production, lipid droplet accumulation and chronic inflammation.[24–26] Long-term intake in mice caused hepatauxe, asso- ciated with significant increase in the levels of serum TC, TG and LDL-c in ALD mice, while serum HDL-c level was significantly decreased.[27–28] Our studies showed that CPS-I could decrease liver index in alcohol induced mice. CPS-I also decreased serum ALT, AST levels, as well as the indexes of abnormal lipid metabolism such as serum TC, TG, LDL-c levels, suggesting CPS-I had reversal effects on alcoholic liver injury. Many studies have shifted emphasis on improv- ing the body’s antioxidation action to prevent oxida- tive stress which can lead to alcohol damage.[29–30] SOD can remove free radicals and protect the liver from damage.[31] GSH-px specifically catalyzes the reduction reaction of hydrogen peroxide and plays an important role in protecting the structure and function integrity of the cell membrane.[32] MDA is a degrada- tion product of polyunsaturated fatty acid peroxides, which has strong toxicity after being cross-linked withlipoprotein and can destroy cell membrane structure.[33] This study suggested that CPS-I could protect liver against alcoholic injury in mice by promoting antioxidant capacity. Recent studies showed that liver lipid deposition was an important manifestation of liver injury caused by alcohol, and reducing lipid deposition is also a tragedy of liver protection.[34] Transmission electron microscope scanning of liver illustrated that there were a certain number of fat droplets and vacuolated mitochondria in the liver of model group mice. After the treatment with CPS-I, the number of round lipid droplets in the liver was reduced and mitochondrial damage was alleviated, demonstrating that CPS-I could protect and treat the alcoholic liver injury by reducing lipid droplet deposition. In addition, CPS-I could significantly improve the activities of ADH and ALDH, which further confirmed that CPS-I had protec- tive effect on alcoholic liver injury through multiple pathways. In the future, we will focus on the signaling pathways related to lipid droplet generation in the liver and further explain the protective mechanism against alcoholic liver injury. 3. Conclusion In this study, the water soluble polysaccharide CPS-I was extracted and purified from carrot. The structural characterization of CPS-I showed that it was a homogenous polysaccharide with a molecular weight of 3.40 × 104 kDa, and primarily composed of D- galacturonic acid, L-rhamnose, D-xylose, D-arabinose, D-fructose, and D-galactose. CPS-I exhibited protective effects on liver injury induced by alcohol in mice. The mechanism of CPS-I hepatoprotection effect might be related to elevating the alcohol dehydrogenase, which were involved in alcohol metabolism. Meanwhile, CPS-I could attenuate oxidative stress by promoting hepatic levels of SOD and GSH-px. Additionally, CPS-I regulated lipid metab- olism by decreasing levels of TG and TC in serum, and improves pathological changes of liver caused by lipid accumulation. The above results suggested that CPS-I could be a potential pharmaceutical ingredients to prevent alcoholic liver. Experimental Section Materials and Methods Materials and Chemicals Carrots (Daucus carota L. var. sativa Hoffm) purchased from the agro-product market in Shenhe District Shenyang, were kept in a constant temperature incubator at 60 °C after cleaning and cutting into filaments. The carrot powder used in this study was processed by grinding carrot filaments with a pulver- izer and grading with a 40-mesh sieve. Silymarin, cellulase, DEAE-52 and Sephadex G150 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Biochemical analysis kits for SOD, ADH, ALDH, GSH-Px and MDA were obtained from Jiancheng Bioengineering Institute (Nanjing, China). Preparation and Purification of Polysaccharides from Carrots 50 g carrot powder was suspended in 0.05 mM citrate buffer (pH 4.5, 1 : 9 w/v) and added cellulase (0.8 % w/ v) at 50 °C for 2 h, then, the reaction mixture was centrifuged (8000 ×g) for 10 min. After discarded residues, the supernatant was concentrated to one- third of the initial volume by vacuum-rotary evapo- ration. The concentrated filtrate was added absolute ethyl alcohol to a final concentration of 90 % (v/v) for precipitating overnight. The precipitate was collected by centrifugation (3000 ×g, 20 min) and deproteinized by the Sevage method. The precipitate was redis- solved in distilled water and lyophilized to obtained crude carrot polysaccharide (CPS). 500 mg of freeze-dried powder of CPS was dis- solved in 5 mL of distilled water and loaded on a DEAE-52 cellulose column (1.6 cm× 25 cm). The solu- tion was eluted with distilled water, 0.1, 0.3 and 0.5 M NaCl (flow rate, 1 mL/min), respectively. The phenol- sulfuric acid method was used to monitored poly- saccharide content of each collected fraction. The purified polysaccharide fractions were applied on a Sephadex G-150 column (1.0 cm × 35 cm) and eluted with distilled water. After determined by the phenol- sulfuric acid method, the elution of polysaccharide was gathered, concentrated, and freeze-dried for further study. Detection of Monosaccharide Composition 5 mg polysaccharide sample was hydrolyzed with 2 mol/L trifluoroacetic acid (4 mL) in an ampoule (10 mL) at 110 °C for 8 h. The hydrolysate was added to methanol (4 mL) and dried in a vacuum at 40 °C. The dried product was dissolved in distilled water (800 μL) for analysis of HPLC system (Waters 240, Japan), which equipped with SPD-10A differential refractive index dector and an Amino column (3.0 mm× 150 mm). The chromatography was carried out as follows: mobile phase was a mixture of acetonitrile and distilled water (80 : 20, v/v) at flow rate of 0.6 mL/min. The injection volume was 5 μL. The monosaccharide composition was identified by com- paring eight sugar standards, which included D- galacturonic, D-arabinose, L-rhamnose, D-mannose, D- xylose, D-galactose, D-glucose, D-fructose. Molecular Weight Determination The molecular weight of polysaccharide was examined by a Waters515 high-performance gel permeation chromatography (HPGPC) system (Waters, USA) equipped with a Waters 2410 Refractive Index Detec- tor, and a UltharydorgelTMLinear column (7.8 mm× 300 mm). In the process of chromatography, the mobile phase was ultrapure water at a flow rate of 1 mL/min. The dextran standards (T-2000, T-500, T-70, T-10, T-5) were applied to establish a calibration curve. Spectrum Analysis Fourier Transform Infrared (FT-IR) Analysis The polysaccharide sample (3 mg) was mixed with KBr powder and pressed into a 1 mm disc, which was examined with a Equinox 55 FT-IR spectrophotometer (Bruker Inc. Germany) in the range of 500 –4000 cm—1. 1H- and 13C-NMR Analysis 30 mg of polysaccharide sample was dissolved in D2O (2.0 mL, 99.9 %) at 25 °C, then, the analysis of 1H (600 Mz) and 13C (150 Mz) spectra of DCP-II were carried out by ARX-500 spectrometer (Bruker Inc. Germany). Animal Experiments Animals Male Kunming mice (SPF, 8 weeks old) were housed under constant conditions with a room temperature of23 2 °C, the humidity of 55 5% and 12 h light-dark cycles. After 1 week acclimatized in a standard labo- ratory diet, all mice were randomly divided into different groups. The procedures were approved by the Institutional Animal Care and Use Committee of Shenyang Agricultural University (Shenyang, China permission number: 201612001). Experimental Scheme SPF Kunming mice weighing 20 –22 g were housed under environmental conditions with a room temper- ature of 23 2 °C, 12 h light-dark cycles and humidity of 55 5%. After provided with standard diet for a week of acclimatization, all experimental mice were divided into 6 groups, and ten mice in each group: normal group (saline solution), model group (saline solution + 0.5 % (v/v) ethanol), CPS-I treatment group (100 mg, 200 mg, and 300 mg CPS-I/kg body weight+ 0.5 % (v/v) ethanol) and positive group (100 mg silymarin/kg body weight + 0.5 % (v/v) ethanol). All mice were administered once daily for 28 days. At 8 h after the final administration, all mice were provided intragastrically with 50 % ethanol (12 mL/kg body weight) for inducing subacute alcoholic liver injury, except the normal group given an equal amount of saline solution. All mice were deeply anesthetized following the ethanol administration, then, the blood was obtained to harvest serum by centrifugation. 10 % neutral formalin and 2.5 % glutaraldehyde were used to fix the left lobes of livers for histological and transmission electron microscope (TEM) observation. The homogenate of right lobes of the livers was applied for further analysis. Determination of Liver Index and Serum Alanine Amino- transferase (ALT), Aspartate Aminotransferase (AST), Alcohol Dehydrogenase (ADH) and Acetaldehyde Dehy- drogenase (ALDH) Activities Calculation of mice liver index was according to the following formula:[35] liver index ¼ liver weight=body weight � 100 % Assay of the activities of ALT, AST, ADH and ALDH, and the measurement of TC, TG in serum was detected according to corresponding kits. Hepatic Biochemical Parameters Assay The liver tissues were mixed with physiological saline (w/v, 1/9) and homogenized at 0 °C to prepared 10 % homogenate. The supernatant was obtained after centrifuged at 3000 rpm for 10 min at 4 °C and was used to assay activities of TC, TG, LDL-c, HDL-c, ADH, ALDH, SOD, GSH-Px and MDA according to related kits. Histopathological Observation The liver tissues fixed with 10 % formalin were embedded in paraffin, then, sliced into 5 μm pieces. After stained with hematoxylin and eosin (H&E), the liver slices were observed and imaged with a light microscope (WV-GP230, Panasonic, Japan). TEM Examination The liver samples were taken from 2.5 % glutaralde- hyde, then, fixed with 1 % osmium acid. After embedded and polymerized, the samples were sec- tioned with a thickness of 50 –70 nm and stained with uranium dioxo acetate-lead citrate according to the corresponding procedure. The degree of liver injury was assayed based on TEM observation by H-7650 electron microscope (Hitachi, Japan). Statistical Analysis SPSS 19.0 software was used for statistical analysis of the data. Comparison between groups was performed by one-way analysis of variance (ANOVA). All the experimental results were showed as the mean standard deviation (SD). References [1] J. Aron-Wisnewsky, B. Gaborit, A. Dutour, K. 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