Hypolipidemic and anti‐atherogenic activities of crude polysaccharides from abalone viscera

Abstract This study was performed to evaluate the hypolipidemic and anti‐atherogenic activities of the crude polysaccharides extracted from abalone viscera (AVCP). The major functional groups of purified polysaccharides were analyzed by infrared spectroscopy (IR). Male Kunming mice (SPF) were divided into six groups and were treated with normal diet or high‐fat diet with AVCP or Xuezhikang (hypotensive drug) for 5 weeks. Physicochemnical analysis of AVCP showed the presence of 60.4% polysaccharides, 17.9% protein, 6.0% fat and 10.9% moisture. The IR analysis of AVP showed the presence of functional groups of sugar moiety and sulfate groups. The results demonstrated that AVCP not only led to significant reduction of total cholesterol (TC), triglycerides (TG), low‐density lipoprotein cholesterol (LDL‐C), and increase of high‐density lipoprotein cholesterol (HDL‐C) in plasma, but also to significant increments of malondialdehyde (MDA) and superoxide dismutase (SOD) activities. However, AVCP played no role in mice weight. Furthermore, the results of the photomicrograph of liver tissue showed that AVCP reduced lipid droplets and prevented the disordered structure of the liver. The results suggested that AVCP exhibited significantly hypolipidemic and anti‐atherogenic activities.

major risk factors in the pathogenesis of atherosclerosis (Morishita, Iwahashi, & Kido, 1986;Wu, 2007). Moreover, lipid peroxidation is also associated with the development of atherosclerosis (Chanet et al., 2012). Nowadays, atorvastatin drugs are widely used to treat atherosclerosis since it can decrease the level of plasma LDL-C and has a strong antioxidant effect. Unfortunately, these drugs have adverse effects, such as a headache, abdominal distension, and insomnia (Lusis, 2000), which are harmful to the human body. Therefore, alternative methods are needed to against atherosclerosis.
It has been indicated that natural polysaccharides extracted from plants and microorganisms have hypolipidemic activity in general and can be developed as novel potential hypolipidemic agents (Lankin et al., 2003;Lee & Park, 2013). The polysaccharides of Lonicera japonica have effect on lowering blood lipids in vivo and scavenging superoxide and hydroxyl radicals in vitro (Chen et al., 2011;Li, Zhang, & Ma, 2010). However, as far as our literature survey could ascertain, hypolipidemic and anti-atherogenic activities of AVCP have not been studied.
Therefore, the objective of the study was to investigate the effect of AVCP on the hypolipidemic and anti-atherogenic activities.

| Materials and equipment
Frozen abalone (Haliotis discus hannai )viscera, stored at −18°C, were kindly provided by Zhangzhou Ousheng Food Co., Ltd. The frozen abalone viscera was thawed at room temperature prior to experiment.
Pulsed electric field (PEF) system ( Figure 1) was self-designed by Professor Jinquan Chen in Fujian Agriculture and Forestry University, China. It consists of a high-voltage pulse generator, an oscillograph, a coaxial liquid material treatment chamber, a pump, and an ice-water bath. The structure diagram of the PEF treatment chamber is shown in Figure 2. There was a pair of parallel electrode bars, locating at two ends of the treatment chamber. The positive electrode received high-voltage pulse power and was grounded at the negative electrode. Pulse shape (square shape bipolar) was monitored online with an oscilloscope during treatment. The diameter of the treatment zone was 6 mm.
All the chemicals were of analytical grade. Triglyceride kits, total cholesterol reagent kits, high-density lipoprotein cholesterol kits, low-density lipoprotein cholesterol kits, superoxide dismutase kit, and malondialdehyde test kit were obtained from Nanjing Jiancheng Biology Engineering Institute.

| Experimental animals
Specific pathogen-free (SPF) male Kunming mice (15 ± 2 g) were obtained from the Wu Laboratory Animal Center. Mice were acclimatized for 1 week before the commencement of experiments under standard environmental conditions of temperature at 25 ± 2°C and 50%-60% of relative air humidity and 12 hr dark/light cycle with F I G U R E 1 Schematic of high-intensity pulsed electric fields processing apparatus free access to normal diet and water. The animal facilities and all experimental procedures were carried out according to the National Institutes of Health (NIH) guidelines.

| Experimental diets and drugs
Normal diet was purchased from the local market, the nutritional ingredients of which were in accordance with nutritional standards. The high-fat feeding was self-made which contained 80.8% normal feeding, 5% lard, 4% cholesterol, 0.2% bile salt, and 10% protein powder. Xuezhikang capsule (an approved traditional Chinese medicine for the treatment of hyperlipidemia bought from pharmacy) was obtained from Beijing Beida Weixin Biological Technology Co., Ltd.

| AVCP extraction
One-kilogram abalone viscera mixed with 2 kg of water was pulped, removing impurities and connective tissues. The abalone viscera solution was filtered, homogenized, and centrifuged successively.
One liter of supernatant solution was treated by PEF with a flow velocity of 100 ml/min, and the electric field intensity and pulse number were 30 kV/cm and 12, respectively. After processing for 10 min, the solution was hydrolyzed by the neutral protease (2.4 × 10 4 U/g) for 2.5 hr at 50°C. The enzyme was inactivated by increasing the temperature to 100°C and kept for 5 min. Afterward, the hydrolyzate was centrifuged at 2,670 g for 15 min. The supernatant was precipitated via adding 95% alcohol with the ratio of 1:3 (v/v) and stored at 4°C overnight. The precipitates were centrifuged at 2,670 g for 15 min to obtain the sediment, which was then dried by vacuum freeze dryer to yield the abalone viscera crude polysaccharides (AVCP) powder.

| AVCP purification
Abalone viscera crude polysaccharide (1 mg/ml) was mixed with Sevag solvent (chloroform:n-butanol = 4:1 (v/v)) at ratio of 3:1. The mixture was thoroughly stirred with a magnetic stirrer for 30 min and then centrifuged at 2,809 g for 10 min. The solution was separated into three layers. The upper layer was harvested and treated again as mentioned above until the middle layer with protein precipitates disappeared. The supernatant was dialyzed using MD34-3500 membrane (Beijing Solarbio Science & Technology Co., Ltd) against running tap water for 48 hr and distilled water for another 24 hr at ambient temperature, changing water per 2 hr. The dialysate was dried by the vacuum freeze dryer to obtain the preliminary purified polysaccharides powder.
One hundred milligrams of the preliminary purified polysaccharides was dissolved in distilled water. The mixture was centrifuged to discard the sediment. The supernatant was loaded into a DEAE-52 chromatography column (Beijing Solarbio Science & Technology Co., Ltd), eluted with distilled water, 0.1, 0.3, and 0.6 mol/L of NaCl solution in sequence at flow rate of 0.5 ml/min. The solution out of column was collected in each tube per 6 min.
The content of polysaccharides in each tube was determined by the phenol-sulfuric acid method (Wang, Wang, & Pang, 2007

Metal conductor
Liquid alcohol, and centrifuged. The precipitate was dissolved in distilled water and then dried to obtained the purified polysaccharides powder (AVP).

| The content of crude polysaccharides in AVCP
Total sugar content and the reducing sugar content of AVCP were individually measured using the phenol-sulfuric acid method and 3,5-dinitrosalicylic acid method (DNS; Zhao, Xue, & Li, 2008). The percentage of crude polysaccharides in AVCP was calculated using Equation 1: where C t and C r represent the total sugar content (%) and reducing sugar content (%), respectively.

| The content of protein in AVCP
Protein in AVCP powder was analyzed with an Automatic Kjeldahl Analyzer (Shanghai HongJi Instrument Co., Ltd). One hundred fifty milligram of AVCP powder was placed into a 500-ml Kjeldahl nitrogen digestive tube and mixed with 0.2 g of anhydrous cupric sulfate, 6.0 g of potassium sulfate, and 10 ml concentrated sulfuric acid. Another digestive tube without AVCP but with other chemical agents, as control, was prepared. The tubes were transferred into the digestive furnace and digested at 250°C until the clear bluegreen liquid appeared. After cooling to ambient temperature, the content of protein was determined according to national standards (SAC, 2016). All assays were performed triplicate.

| Gas chromatography analysis of AVP
Ten milligrams of each standard monosaccharide (L-rhamnose, D-xylose, D-mannose, D-glucose, D-galactose, fructose, glucuronic acid, fucose) was mixed with 10 mg of hydroxylamine hydrochloride and dissolved in 0.5 ml of pyridine. The solution was kept in water bath at 90°C for 30 min. After cooling down, the solution was added with 0.5 ml of anhydrous acetic anhydride and reacted in water bath at 90°C for 30 min. The reaction product was analyzed by gas chromatography (GC; Bai & Zhang, 2011).

| Infared spectroscopy analysis of AVP
The purified abalone viscera polysaccharide was mixed with KBr powder at ratio of 1:100 (g/g) and pressed into a pellet.

| Animal grouping and treatment schedule
A total of 60 mice were weighed and randomly divided into six groups of 10 mice: blank control group (A), positive control group (B), negative control group (C), low-dose group (D), middle-dose group (E), and high-dose group (F), and fed as shown in Table 1. All mice had free access to potable drinking water, and each mouse was weighed once a week during the experiment. After 5 successive weeks for administration, eyeball blood was collected in heparin (anticoagulant) tubes from each mouse after gavage for 1 hr and centrifuged immediately at 500 g for 10 min. The supernatant serum was obtained for biochemical analysis. The animals were thereafter killed by cervical dislocation. The livers were harvested and perfused with normal saline (0.9%) at 4°C. After then, the hepatic tissues were sectioned (5 mm × 5 mm × 2 mm) and immersed in a 10% formaldehyde solution for histopathological assessment.

| Determination of lipid and lipid peroxidation index
The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) activities in serum were individually determined using Malondialdehyde kit and Superoxide dismutase kit. The levels of TG, TC, LDL-C, and HDL-C were separately analyzed using triglyceride kit, total cholesterol reagent kit, low-density lipoprotein cholesterol kit, and high-density lipoprotein cholesterol kit.

| Histopathological assessment
Hepatic tissues were embedded in paraffin and stained with hematoxylin and eosin, and then examined by an electronic microscope. (1)

| Statistical analysis
Statistical analysis was performed using SPSS, Version 13.0 (SPSS, Inc.). All the results were presented as mean ± standard deviation (SD) for ten mice in each group. p < .05 means significant (data were subjected to the analysis of variance [ANOVA], followed by mean comparisons by Duncan's multiple range test at p < .05).
The monosaccharide composition of AVP was qualitatively determined by GC (Table 3)

| IR analysis of AVP
The band characteristics of AVP-1, AVP-2 and AVP-3 are illustrated in Table 4 and Figure

| Effect of AVCP on body weight of animals
After 5 weeks, the weights of mice in each group are shown in Figure 5, and it indicated that the various treatment interventions demonstrated no significant difference in the body weight of mice.

Effect of AVCP on the level of TC
After 5 weeks fed, the results in Table 5  close to that in group A (14.02 ± 0.91 mmol/L). As shown in Table 5, with increasing dose of AVCP, the level of TG was found to be correspondingly decreased. The results indicated that AVCP played a role in reducing TG level, relating to AVCP dose.

Effect of AVCP on the level of HDL-C
Xuezhikang capsule in group B caused a significant increment (p < .05) in HDL-C level compared to group C. As compared with group C, the same trends were obtained in groups D-F with AVCP doses of 200, 400, and 600 mg/kg, indicating that AVCP had a significant effect on increasing HDL-C level (p < .01; Table 5). With the dose of 200 mg/kg of AVCP, a significant rise in the level of HDL-C (20.42%) was noted when compared with group C (p < .01), which was higher than that with 400 and 600 mg/kg of AVCP (19.47% and 15.93%), suggesting that there was an inverse correlation between the dose of AVCP and the level of HDL-C.

| Effect of AVCP on lipid peroxidation
Effect of AVCP on the concentration of MDA As shown in Table 6, compared to group C, the concentrations of MDA were significantly (p < .01) decreased by 17.36%, 25.67%, and 36.92% in the mice treated with AVCP at the doses of 200, 400, and 600 mg/ kg (group D, group E, and group F), respectively. However, the levels of MDA in groups D and E were higher than the control group, group B, and group F, suggesting that the dose of AVCP ≥ 400 mg/kg would reduce the concentration of MDA to a normal level.

Effect of AVCP on SOD activity
Hyperlipidemic diets (group C) significantly decreased the activity of antioxidant enzyme SOD compared to normal diets (group A; p < .01). The data in Table 6

| Histopathological assessment
Microscopic images of liver tissues are shown in Figure 6A  with central nucleus ( Figure 6A). However, group C showed the disordered liver structure with hepatocellular necrosis and extensive vacuolization which are indicated with the black arrow in Figure 6C.
Xuezhikang capsule significantly attenuated lipid vacuolization ( Figure 6B). With the treatments of 400 and 600 mg/kg AVCP, the disordered liver tissues returned to normalcy ( Figure 6E,F). However, with low dose of AVCP in group D ( Figure 6D), liver cell architecture was better than that in negative group C but worse than that in other groups. These results demonstrated that AVCP could reduce the accumulation of lipid droplets in hepatic tissue cells of hyperlipidemia mice and prevent cardiovascular disease. Moreover, the extent of liver tissue recovery was correlated with the dose of AVCP.

| D ISCUSS I ON
In this study, AVCP could be extracted by combination Atherosclerosis is the main contributor to the pathogenesis of myocardial and cerebral infarction and highly related to blood lipids.
Hyperlipidemia may be responsible for liver damage. In animal models of the high-fat diet, a dramatic increase in serum low-density lipoprotein (Morishita et al., 1986), triglyceride, and total cholesterol levels was reported with relatively slight changes in high-density lipoprotein. Then, elevated levels of plasma TC and LDL-C, accompanied by reducing TG and HDL levels, are often associated with an increased risk of atherosclerosis (Chander, Kapoorn, & Singh, 1988;Wu, 2007).

TA B L E 5
Effect of AVCP on TG, TC, HDL-C, and LDL-C level in rat serum and a decrease in HDL-C (Ban, Rico, Um, & Kang, 2012;Chawda, Mandavia, Parmar, Baxi, & Tripathi, 2014;Zhang, Zhang, Jiang, & Xia, 2013). In the present study, feeding mice with high-fat diet resulted in upregulation of TC, TG, and LDL-C and down-regulation of HDL-C level. In animal studies, it has been reported that some polysaccharides inhibit low-density lipoprotein oxidation and have an overall positive effect on lipid metabolism and cholesterol (Liu et al., 2012).
In the present study, we found that daily oral administration of different doses of AVCP inhibited serum TC, TG, and LDL-C accumulation after 5 weeks and up-regulated the level of HDL-C, indicating that AVCP might be beneficial for degenerative diseases, as well as atherosclerosis.
Oxidative stress, the disturbance of the delicate balance between oxidants and antioxidants, could impair the antioxidant defense systems (Li et al., 2010). It was generally caused by the increasing levels of free radicals. Current studies have reported that the development of atherosclerosis was related to oxidative stress in plasma (Lusis, 2000). The major antioxidant enzymes, including SOD, GSH-Px, and CAT, used as biomarkers to indicate free radical species (ROS) production, are regarded as the primary defense system against ROS generated in vivo during oxidative stress (Akindele, Otuguor, Singh, Ota, & Benebo, 2015;Tang et al., 2013). SOD is the only enzyme that disrupts oxygen free radicals and exists in all cells with high amounts in erythrocytes. Higher the SOD activity, faster the free radicals were scavenged (Fan et al., 2016). Previous studies in vivo indicated that feeding the animal with a high-fat diet could lead to an increase of free radical production (Dobrian, Davies, Prewitt, & Lauterio, 2000;Feng, Yu, Ying, Hua, & Dai, 2011;Rony, Ajith, Nima, & Janardhanan, 2014) and a decrease of SOD activity (Bagchi, Bagchi, Hassoun, & Stohs, 1995), followed by hypercholesterolemia and oxidative stress (Ohkawa, Ohishi, & Yagi, 1979).
Malondialdehyde (MDA), one of the end products of tissue lipid peroxidation, is regarded as a marker of lipid peroxidation and an index of the level of oxygen free radicals as well. Lower MDA level suggests less lipid peroxidation and weaker oxidant stress (Padmavathi, Senthilnathan, Chodon, & Sakthisekaran, 2006), followed by the reduction of atherosclerosis (Illingworth, 1993). The obtained results in this study indicated that high-fat diet caused a marked reduction in serum SOD activity and an obvious increase in the concentration of MDA, suggesting a detrimental effect of intrinsic antioxidant defense system and endothelial cell injuries in mice. After 5 weeks of treatment of AVCP and Xuezhikang capsule (cholesterol-lowering drug), the activities of SOD were significantly increased, and the levels of MDA were dramatically reduced. AVCP could preserve the activity of the antioxidant enzyme in high-lipid organs, rectify the metabolic disturbance of free radicals, and maintain the dynamic balance of the oxidation and antioxidative systems.

| CON CLUS ION
This study demonstrated the potential lipid-lowering effects of AVCP. By intake of AVCP, serum lipids of TG, TC, and LDL-C and HDL-C level was controlled, and SOD activity and the level of MDA were adjusted. The results implied that further study should be conducted to identify the possible mechanism and the therapeutic effect in the treatment of hyperlipidemia-related diseases, such as atherosclerosis.

CO N FLI C T O F I NTE R E S T
Authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
This study involving animal testings was approved by the Institutional Review Board of Fujian Medical University.