Extraction, purification, characterization, anticoagulant activity, and anticoagulant mechanism of polysaccharides from the heads of Hypomesus olidus

Abstract The aim of this work was to extract, isolate, and purify polysaccharides from the heads of Hypomesus olidus and to evaluate their anticoagulant activities and anticoagulant mechanisms. Response surface methodology was used to optimize the extraction conditions for ultrasonic‐assisted extraction of polysaccharides from the heads of Hypomesus olidus. The optimal extraction conditions consisted of ultrasonic power of 275 W, ultrasonic time of 50 min, and solid–liquid ratio of 5 ml/g, giving the yield of crude polysaccharides (GYT) of 7.73 ± 0.042%. Three polysaccharide fractions, GYT‐1, GYT‐2, and GYT‐3 were purified from GYT by using DEAE‐cellulose‐52 column and Sephadex G‐100 column for anticoagulant activities. The results showed that two doses (2 and 4 mg/ml) of GYT‐1 and GYT‐3 could significantly prolong (p < .01) in partial thromboplastin time (APTT) (2.19 and 2.37 times, 2.22 and 2.44 times, respectively) and thrombin time (TT) (2.39 and 2.46 times, 2.44 and 2.80 times, respectively) compared with normal control. In particular, GYT‐3 had stronger anticoagulant activity than GYT‐1, and it was composed of arabinose, fructose, glucose, and lactose with molar ratios of 0.595:1: 2.026:0.273. However, GYT‐2 had no anticoagulant activity (p > .05). In addition, anticoagulation mechanism of polysaccharides from the heads of Hypomesus olidus (GYT‐3) was evaluated. The results showed that the anticoagulant activity of GYT‐3 was based on their binding with antithrombin AT‐III. And the inhibitory effects of GYT‐3 on factor IIa and Xa were related to the concentration of AT‐III in plasma. This study may provide a new and promising anticoagulant drug.


| INTRODUC TI ON
Cardiovascular diseases such as myocardial infarction, thrombosis, and stroke are main causes of human death, which seriously threaten human health (Kang et al., 2015;Pawlaczyk-Graja, 2018). Heparin, used in the treatment of cardiovascular diseases and prevention of thrombosis, is the most commonly used anticoagulant drug (Croles et al., 2019;Hao, Xu, Yu, & Zhang, 2019;Kawano et al., 2019). But it could produce side effects such as bleeding and thrombocytopenia (Fu, Suflita, & Linhardt, 2016;Risch, Fischer, Herklotz, & Huber, 2004;Roberts and Susan, 2018). Therefore, it is necessary to discover new and safe anticoagulant active ingredients without the risk of bleeding from natural products.
In recent years, plant-and animal-derived polysaccharides have attracted wide attention because of their strong anticoagulant activities (Song et al., 2019;Wang, Zhang, et al., 2018;Wang, Li, et al., 2018). Hypomesus olidus, a freshwater fish, is mainly distributed in northeastern China, Korea, Japan, and Northern Canada (Cho et al., 2006). Because of its fresh aroma of fresh cucumber, it is also known as "cucumber fish". The fish meat of Hypomesus olidus is often used as a delicacy, while fish heads are often discarded. However, studies have shown that by-products of aquatic products contain a variety of active substances, such as protein, polysaccharide, and fatty acid (Jayathilakan, Sultana, Radhakrishna, & Bawa, 2012). They have significant anticoagulant activities (Song et al., 2019), antioxidant activities (Choksawangkarn, Phiphattananukoon, Jaresitthikunchai, & Roytrakul, 2018), and anti-inflammatory activities. Therefore, the effective utilization of by-products can not only avoid waste of resources, but also avoid environmental pollution.
In previous studies in our laboratory, other researchers isolated a natural protein from Hypomesus olidus, which proved to have very strong anticoagulant activities (Gou, Wang, & Liu, 2016). However, studies on the anticoagulant activities of polysaccharides from the heads of Hypomesus olidus have not been reported.
In the current work, we extracted and purified polysaccharides from the heads of Hypomesus olidus, obtained three fractions (GYT-1, , and characterized their structures by ultraviolet and infrared spectroscopy. In addition, the anticoagulant activities of three fractions were evaluated by activated partial prothrombin kinase time (APTT), prothrombin time (PT), and thrombin time (TT).
Finally, the anticoagulant mechanism of the anticoagulant activities of polysaccharides from the heads of Hypomesus olidus was studied. This work may be helpful in improving the utilization value of Hypomesus olidus and providing safe anticoagulants.

| Materials
Hypomesus olidus was purchased from the aquatic product market in Changchun, China. After washing, the fish heads were taken, packaged, and stored frozen at −20°C. The fish bodies could be cooked and eaten or used for further food processing and research. DEAE-52 cellulose, Sephadex G-100, and standard monosaccharides, including rhamnose, arabinose, fructose, glucose, galactose, and lactose, were purchased from Sigma Chemical Co. Papain and trypsin were obtained from Shanghai Yuanye Biotechnology Co., Ltd. APTT, PT, and TT assay kits (fixed method) were purchased from Shanghai Sun Biotechnology Co., Ltd. Human antithrombin III (AT-III), human coagulation factor II (F II), and human coagulation factor X (F X) were purchased from Changchun Baijin Biotechnology Co., Ltd. Rabbit anti-human AT-III antibody was acquired by Beijing Boaosen Biotechnology Co., Ltd. Other reagents were analytical grade.

| Extraction of polysaccharides from the heads of Hypomesus olidus
The fish heads were degreased with petroleum ether and dried by air. The fish heads (100 g) were homogenized with twice the volume of physiological saline, then centrifuged (3,000 r/min, 20 min), and obtained supernatant. Furthermore, using 0.1 mol/L NaOH solution as alkaline solution, the precipitation after centrifugation was alkalinized by ultrasonic-assisted method using a KQ dual-frequency digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd). The supernatant was obtained by centrifugation (3,000 r/min, 20 min). The two supernatants were combined, concentrated, alcohol precipitated, centrifuged, and lyophilized to obtain crude polysaccharides from the heads of Hypomesus olidus (GYT).

| Optimization of extraction conditions of polysaccharides from the heads of Hypomesus olidus
Based on single factor tests (ultrasonic power, ultrasonic time, and solid-liquid ratio), the extraction conditions of polysaccharides from the heads of Hypomesus olidus were optimized by response surface methodology. Three factors and three levels of response surface design were used to determine the optimal conditions for ultrasonic-assisted extraction. With the polysaccharide yield as response value, the whole design consisted of 17 experimental points, including 12 factor points and 5 axis points. The level of coding test factors is shown in Table 1. The best conditions could be predicted according to the following quadratic models : where Y is the dependent variable, and X i and X j are the independent variables; A 0 , A i , A ii , and A ij are the regression coefficients of the independent variables that were estimated by the model for the intercept, linear, quadratic, and interaction terms, respectively. (1)

| Isolation and purification of GYT
The proteins in GYT were removed by trypsin combined with Sevage reagents. The GYT solution (3 mg/ml) after protein removal was loaded onto DEAE-cellulose-52 anion exchange column. Gradient elution was carried out with distilled water, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mol/L NaCl solution at a flow rate of 0.5 ml/min. The obtained fractions were further purified by Sephadex G-100 column (1.6 cm D × 60 cm H) at a flow rate of 0.5 ml/min. The polysaccharide content was determined by phenol-sulfuric acid method at 490 nm (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The same fractions were collected, concentrated, dialyzed, and lyophilized to obtain purified polysaccharide samples.

| Physicochemical properties of purified samples
Total sugar content was determined by phenol-sulfuric acid method (Dubois et al., 1956). The protein content was assessed by Bradford (1976). The sulfate content was evaluated by barium chloride-gelatine method with K 2 SO 4 as the standard (Dodgson & Prices, 1962).
With glucuronic acid as standard, the uronic acid content was determined using the method of Blumenkrantz and Asboe-Hansen (1973) with slight modification. The solubilities of purified polysaccharide samples in distilled water, acid, alkali, and some organic solvents were evaluated by Gao et al. (2015). The purified polysaccharide samples were dissolved in distilled water (1.5 mg/ml), and the pH of the polysaccharide solutions was determined by a pH meter (Mettler Toledo, Shanghai, China).

| Ultraviolet analysis
The purified polysaccharide samples were prepared into aqueous solution of an appropriate concentration and scanned in the wavelength range of 190-400 nm using TU-1901 ultraviolet-visible (UV) spectrophotometer (Beijing Pu Analysis General Instrument Co., Ltd).

| Infrared spectrum analysis
The purified polysaccharide samples (1 mg) were mixed with dry KBr powder (100 mg), grounded and blended, and then pressed.
The structure of the purified polysaccharide samples was analyzed in the range of 4000-400 cm -1 using IR-Prestuge-21 Fourier transform infrared spectrometer (Lai Chi Electronic Technology Co., Ltd.).

| Anticoagulant activity of purified polysaccharide samples
The anticoagulant activities of purified polysaccharide samples in vitro were determined by PUN-2048B semi-automatic coagulation instrument (Beijing Pulang New Technology Co., Ltd.). The three indexes (APTT, TT, and PT) were used to measure the anticoagulant activities of polysaccharides. (Sask, McClung, Berry, Chan, & Brash, 2011).

| Determination of APTT
The plasma (0.1 ml) was mixed with APTT reagent (0.1 ml), incubated at 37℃ for 5 min, and 0.1 ml of 0.025 mol/L CaCl 2 solution was added to record the results of hemagglutination analyzer.

| Determination of TT
After preheating the plasma for 3 min at 37℃, 0.2 ml plasma was taken out and mixed with 0.2 ml TT reagent at room temperature.
The results of hemagglutination test were recorded.

| Determination of PT
After preheating the plasma for 3 min at 37℃, 0.1 ml of the plasma was taken out and 0.2 ml of PT reagent was added to record the results of hemagglutination analyzer.

| Monosaccharide composition analysis
The monosaccharide composition of purified polysaccharide samples was determined by high-performance liquid chromatography.
The polysaccharide samples (10 mg) were accurately weighed and dissolved in steamed water (5 ml) and mixed with 0.25 ml concentrated HCl. Samples were hydrolyzed in autoclave at 120 ℃ for 4 hr, neutralized to neutral in NaOH solution, and diluted to 10 ml with steamed water after 48 hr of dialysis. Then, LC-20AT high-performance liquid chromatography (Shimadzu, Japan) and InertSustain NH 2 column (4.6 × 250 mm, 5 μm) were used for analysis.

| Preparation of plasma
The plasma samples were divided into the following groups: (1) normal control: normal saline mixed with plasma 1/4 (v/v); (2) positive control: heparin sodium (0.05 mg/ml) mixed with plasma 1/4 (v/v); (3) lack of AT-III plasma group: The human antithrombin Ⅲ (AT-Ⅲ) activity kit was used to detect the content of AT-Ⅲ in the plasma to determine whether the lack of AT-Ⅲ plasma was successfully prepared. Rabbit anti-human AT-III antibody was added to the normal plasma. After incubation at 37℃ for 10 min, the mixture was centrifuged (3,000 r/min, 20 min). The supernatant was taken. The content of AT-Ⅲ in plasma was detected with human antithrombin Ⅲ (AT-Ⅲ) activity kit, until AT-Ⅲ was not detected, and the lack of AT-III plasma was obtained. (4) Plasmas with different AT-III concentrations: Rabbit anti-human AT-III antibodies of 0, 2, 4, 8, 16, and 32 μL were added to 300 μL normal plasma.

| Relationship between the effects of purified polysaccharide samples on the activity of coagulation factor IIa and Xa and the concentration of AT-III
The purified polysaccharide samples with concentrations of 0, 1, 5, 10 mg/ml, and 15 mg/ml were mixed with normal plasma or lack of AT-III plasma. After incubation at 37℃ for 10 min, the mixture was centrifuged (3,000 r/min, 20 min). The supernatant was taken, and the activities of human coagulation factor IIa and Xa in plasma was determined by human coagulation factor II and X activity assay kits.

| Statistical analysis
SPSS statistical software was used for variance analysis, data processing was carried out using Graph Pad Prism 5 software and Origin Pro 7.5 software, and the results were expressed as mean ± standard deviation (SD). The acceptable level for statistical significance was p < .01.

| Effect of ultrasonic power on polysaccharide yield
The effects of different ultrasonic power (100, 200, 300, 400, and 500 W) on polysaccharide yield are shown in Figure 1a. The ultrasonic time and solid-liquid ratio were 50 min and 30 ml/g, respectively. According to Figure 1a, when the ultrasonic power was 300 W, the polysaccharide yield reached the maximum. After this power (300 W), the polysaccharide yield began to decrease. This may be due to the excessive ultrasonic power, resulting in the destruction of polysaccharide structure, thereby reducing the polysaccharide yield.

| Effect of ultrasonic time on polysaccharide yield
The effects of different ultrasonic time (30, 40, 50, 60, and 70 min) on polysaccharide yield are shown in Figure 1b. The ultrasonic power and solid-liquid ratio were 300 W and 30 ml/g, respectively.
With the increase in ultrasonic time, the yield of polysaccharides increased. When the ultrasonic time was 50 min, the polysaccharide yield was the highest, and then began to decline. This may be due to the degradation of some polysaccharides by too long ultrasonic time (Ying, Han, & Li, 2011;Zhao, Zhang, Li, Dong, & Liu, 2015).

| Effect of solid-liquid ratio on polysaccharide yield
The effects of different ultrasonic time (2, 3, 4, 5, and 6 ml/g) on polysaccharide yield are shown in Figure 1c. The other two factors are 300 W and 50 min, respectively. As shown in Figure 1c, the polysaccharide yield increased first and then decreased with the increase in solid-liquid ratio. At 5 ml/g, the polysaccharide yield was the highest. Considering that excessive liquid content could lead to waste and have adverse effects on subsequent concentration process (Wang, Zhang, et al., 2018;Wang, Li, et al., 2018), the solid-liquid ratios of 4, 5, and 6 ml/g were selected as the scope of response surface analysis.

| Statistical analysis and model fitting
In response surface model design, each combination experiment was repeated for three times, and the average polysaccharide yield in three experiments was taken as the results. The experimental results in Table 2 were analyzed by using Design Expert 8.0.5 software. The data in Table 2 were fitted by multiple regression, and the regression model equation was obtained as follows: From Table 2, it could be seen that the P value was far less than 0.01, that is, the regression model reached a very significant level.
The lack of fit (0.0784) was not significant, indicating that the regres-   (A and B), the quadratic coefficients (A 2 , B 2 , and C 2 ) and interaction coefficient (AC) were at very significant levels, which showed that they had a very significant impact on the response value.

| Physicochemical properties of fractions
In the experiment, it was found that the crude polysaccharides (GYT) were yellow brown powder. The purified polysaccharides (GYT-1, GYT-2, and GYT-3) were all white flocculent powders.
GYT, GYT-1, GYT-2, and GYT-3 were all soluble in water. During the experiment, we found that the solubility of GYT decreased with the increase in temperature, while the solubility of GYT-1, GYT-2, and GYT-3 increased with the increase in temperature.
This phenomenon may be due to the inclusion of protein and other impurities in GYT. The excessive temperature made the protein denatured to precipitate, resulting in the decrease of its solubility. In addition, the crude polysaccharides and the purified polysaccharides from the heads of Hypomesus olidus were soluble in dilute acid and alkali, and were insoluble in organic solvents such as methanol, ethanol, ether, acetone, trichloromethane, and n-butanol.  The physicochemical properties of GYT, GYT-1, GYT-2, and GYT-3 are shown in Table 3. The total sugar content and protein content of GYT increased significantly (p < .01) and decreased significantly (p < .01) after purification, indicating that the separation and purification of GYT was effective. In addition, compared with GYT, the contents of glucuronic acid and sulfate decreased significantly (p < .01). It was worth noting that GYT-1 and GYT-3 contained glucuronic acid and sulfate. And GYT-3 was significantly higher than GYT-1 (p < .01).

| UV spectrum analysis
The UV spectrum of the three components is shown in Figure 4.
The UV spectra of GYT-1 and GYT-2 were smooth (Figure 4a   GYT-3 may contain proteins binding to polysaccharides, which was in accordance with the results in Table 3.

| Infrared spectrum analysis
The composition and structure of each molecule are different, and the energy level transition of molecular vibration is also different in infrared spectroscopy (Abbott, Wolf, Wu, Butterfield, & Kleiman, 1991).
According to the characteristic absorption peaks of each functional group in the molecule, its structure can be analyzed and identified. As shown in Figure 5, the characteristic absorption peak of GYT-1, GYT-2, and GYT-3 in infrared spectra of about 3100-3500 cm −1 , 1870-1650 cm −1 , and 1400-1200 cm −1 was corresponding to the stretching vibration of O-H, C=O and the angular vibration of C-H, respectively (Jahanbin, Abbasian, & Ahang, 2017;Xu et al., 2018;Yu et al., 2017).
The signal of 1200-1000 cm −1 was assigned to the stretching vibration of C-O-C, indicating the presence of pyranose ring (Rozi et al., 2019). Infrared spectra proved that there were characteristic absorption peaks of polysaccharides in three fractions.
Moreover, in the infrared spectrum of GYT-1 and GYT-3, the absorption vibration of amino group was found near 1543 cm −1 .
The symmetrical stretching vibration of ester bond was observed at 1,411 cm −1 , indicating the existence of glucuronic acid (Wu, Zheng, Xia, & Kan, 2015). The carboxyl group was also found at 1,400 cm −1 in GYT-1, which proved the existence of glucuronic acid. The signals at 1,244 cm −1 and 1,246 cm −1 were attributed to the S=O stretching vibration absorption peak , indicating the presence of sulfate in GYT-1 and GYT-3. These results were verified in Table 3.

| Anticoagulant activity of GYT
Three indexes of anticoagulation (APTT, PT, and TT) and their changes were determined by adding different concentrations of GYT in plasma.
APTT was used to evaluate endogenous coagulation pathway and PT was used to characterize exogenous coagulation factors (Cai et al., 2016). TT is a thrombin that detects the conversion of fibrinogen to fibrin (Ye, Xu, & Li, 2012). The results are shown in Table 4 Interestingly, when the concentration was 4 mg/ml, the APTT and TT values of GYT-3 were significantly higher than that of GYT-1 (p < .01) as shown in Figure 6, indicating that the anticoagulant activity of GYT-3 was significantly higher than that of GYT-1. Studies have shown that sulfation of polysaccharides can lead to changes in their biological activities (Wang, Zhang, Yao, Zhao, & Qi, 2013). Sulfate groups play a major role in the antithrombotic activity of heparin . In this experiment, the results of physicochemical properties have proved that the sulfate content of GYT-3 was much higher than that of GYT-1. Therefore, it may be reasonable that the anticoagulant activity of GYT-3 was higher than that of GYT-1.

| Monosaccharide composition analysis
The monosaccharide composition of GYT-3 was analyzed by HPLC, and the monosaccharide composition was obtained by comparing with the peak time of standard monosaccharide. As shown in Table 5

GYT-3
The anticoagulant activity of GYT-3 in lack of AT-III plasma was significantly lower than that in normal plasma. APTT and TT in lack of AT-III plasma were significantly lower than those in normal plasma (p < .01) (Figure 8a and b). Even if the concentration of GYT-3 increased from 0 mg/ml to 4 mg/ml, the anticoagulant effects were still not obvious, which further indicated that the anticoagulant activity of GYT-3 was related to the concentration of AT-III in plasma. That is, the anticoagulant effect of GYT-3 was achieved by combining with antithrombin AT-III.

| Relationship between the effects of GYT-3 on the activity of coagulation factor IIa and Xa and the concentration of AT-III
The influence of GYT-3 on inactivation of factor IIa and Xa by AT-III were verified (Figure 9). With the increase in GYT-3 concentration, the activities of coagulation factor IIa and Xa in normal plasma gradually decreased, indicating that the addition of GYT-3 in normal plasma had effective inhibitory effects on the activities of coagulation factor IIa and Xa. However, in lack of AT-III plasma, with the increase in GYT-3 concentration, the activities of coagulation factor IIa and Xa decreased slightly, but the changes were not obvious. This indicated that the effect of GYT-3 on coagulation factor IIa and Xa was related to the presence of AT-III in plasma.
It is reported that heparin is a highly sulfated glycosaminoglycan (Zhi et al., 2018). The anticoagulant effect of heparin is based on its combination with antithrombin AT-III (Sasisekharan & Venkataraman, 2000), to form a heparin-AT-III complex, which can inactivate coagulation factors (Ⅱa, Ⅸa, Ⅹa, Ⅺa, and Ⅻa). In this experiment, the infrared spectra of GYT-3 showed that the functional group composition of GYT-3 was similar to that of heparin, so the study of anticoagulant mechanism of GYT-3 was carried out according to the anticoagulant mechanism of heparin. In this experiment, we studied the relationship between the anticoagulant activity of GYT-3 and AT-III concentration and the relationship between AT-III concentration and the activity of coagulation factor IIa and Xa. The results showed that the anticoagulant effect of GYT-3 was based on their binding with antithrombin AT-III and was dose-dependent. At the same time, the inhibition of GYT-3 on coagulation factor IIa and Xa was also related to AT-III concentration. The mechanism of action was similar to that of heparin in anticoagulation (Chuang, Swanson, Raja, & Olson, 2001). These results could preliminarily validate the previous hypothesis. These results are also helpful in designing meaningful heparin mimetic molecules to develop safe anticoagulants (Ma et al., 2018).

F I G U R E 7
Effect of GYT-3 on APTT (a) and TT (b) in plasma with different AT-III concentrations. All values are presented as the mean ± SD (n = 3). * p < .05, compared with normal plasma. ** p < .01, compared with normal plasma

| CON CLUS IONS
The polysaccharides were extracted from the heads of Hypomesus olidus by ultrasonic method; the optimal conditions were 275 W ultrasonic power, 50 min ultrasonic time, and 5 ml/g solid-liquid ratio, giving the yield of crude polysaccharides (GYT) of 7.73 ± 0.042%. The results showed that the purified fractions GYT-1 and GYT-3 could significantly inhibited coagulation through intrinsic and common pathways. GYT-3 with higher anticoagulant activity was composed of arabinose, fructose, glucose, and lactose with molar ratios of 0.595:1: 2.026:0.273. The results of anticoagulation mechanism showed that the anticoagulant activity of Hypomesus olidus head polysaccharides was based on their binding with antithrombin AT-III. In addition, the inhibitory effects of GYT-3 on factor IIa and Xa were related to the presence of AT-III in plasma. As a promising anticoagulant polysaccharide, this study provides a theoretical basis for the development of new anticoagulants and functional foods.

CO N FLI C T O F I NTE R E S T
All authors declare no conflicts of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.

R E FE R E N C E S
F I G U R E 8 Effects of AT-III concentration on APTT (a) and TT (b) of GYT-3. All values are presented as the mean ± SD (n = 3). * p < .05, compared with normal plasma. ** p < .01, compared with normal plasma F I G U R E 9 Effect of GYT-3 on the activity of coagulation factor IIa (a) and Xa (b). All values are presented as the mean ± SD (n = 3). * p < .05, compared with normal plasma. ** p < .01, compared with normal plasma