The fruiting body of S. vaninii was harvested from Zhejiang Academy of Agricultural Science farm after it was grown for 4 months. The mushroom was dried at 60°C in an oven, pulverized and passed through a 60-mesh screen for subsequent use. Dextrans (Dextran T1000, T500, T70, T40, and T10) were purchased from the American Polymer Standards Corp (Mentor, OH, USA). Monosaccharide standards (arabinose, fucose, mannose, rhamnose, glucose, galactose, xylose, galacturonic acid, and glucuronic acid), Coomassie Brilliant Blue G-250, trifluoroacetic acid and simvastatin were obtained from Sigma-Aldrich (St. Louis, MO, USA). DEAE cellulose-52 and Sephacryl S-400HR were supplied by Baoman Biotechnology (Shanghai, China). All chemicals and solvents were reagent grade or better.

Ground S. vaninii powder (1.0 g) was heated at reflux in a round-bottom flask with preheated ethanol (92.5 wt%) and hydrochloric acid (37%) in different ratios (8.5 ml:1.5 ml, 9.0 ml:1 ml, and 9.5 ml: 0.5 ml) for various durations (0.5 h, 1 h, and 1.5 h) at different temperatures (60, 70, and 80°C). The mixture was cooled down in an ice-water bath and neutralized with sodium bicarbonate (15%, w/v) following the treatment. The treated mushrooms were separated by centrifugation at 6,000 rpm for 15 min. Subsequent procedures were similar to those for our reported method (8). The yield of polysaccharide was calculated according to the following equation:

BBD was employed to find the optimum conditions based on the preliminary range of extraction variables. Processing temperature (A), processing time (B) and the amount of HCl (C) were chosen to investigate their combined effect. The range and levels of independent variables are presented in Table 1A. Design Expert software (8.0.6) was used for the experimental design, and the variance and regression coefficients of individual linear, quadratic and interaction terms. Seventeen combinations including five replicates of the center point were included in this design. All trials were performed in triplicate and the average polysaccharide yield was taken as the result.

Conventional hot water (HWE) and ultrasonic-assisted extraction (UAE) were conducted as control experiments using the previously optimized extraction conditions. For conventional hot water extraction, distilled water (30 ml) was added to pre-treated mushroom powder (1.0 g) and the mixture was heated at reflux with boiling water for 1 h. The supernatant was separated from the solid residue by centrifugation and the above extraction was repeated twice (9). The subsequent procedure was similar to that for the acid-ethanol pretreatment. For ultrasonic-assisted extraction (UAE), distilled water (30 ml) was added to pre-treated mushroom powder (1.0 g) and the mixture was extracted in an ultrasonic cleaner (KQ-500E, Kunshan Ultrasound Instrument Co., Ltd., Jiangsu, China, 40 kHz) at 50°C for 25 min (10). The subsequent procedure was similar to that for the acid-ethanol pretreatment.

The crude PFSV was first purified through a DEAE-52 cellulose column (2.6 × 60 cm) using H₂O₂ and different concentrations of NaCl solutions (0–0.5 M) at 1.0 ml·min⁻¹. The fractions were collected in 10-ml aliquots per tube, and the sugar content was determined using the phenol-sulfuric acid assay (11). The second fraction was further purified by a Sephacryl S-400HR column (1.6 × 100 cm) at 0.2 ml·min⁻¹ for further investigation (PFSV-2). The main fractions were collected, dialyzed and lyophilized for further use.

For FT-IR spectroscopy, PFSV-2 was mixed with KBr and the spectra were recorded from 4,000 to 500 cm⁻¹ with a Nicolet 6700 FT-IR spectrometer (Thermo Electron Corp., Waltham, MA, USA).

The monosaccharide composition of PFSV-2 was determined by high-performance anion-exchange chromatography (Dionex LC₃₀ equipped with a CarboPac^TM PA20 column and a pulsed amperometric detector) (12).

The molecular weight of PFSV-2 was determined by high-performance gel permeation chromatography (HPGPC), using a refractive index detector and a multi-angle laser scattering detector according to the reported method (13).

The methylation of PFSV-2 was performed according to the method of Huang et al. (14). After methylation, derivatives for gas chromatography-mass spectrometric analysis were obtained by hydrolysis, reduction and acetylation of the completely methylated polysaccharide.

The freeze-dried PFSV-2 (50 mg) was dissolved in 500 μl of deuterium oxide (D₂O). ¹H, ¹³C, ¹H-¹H COSY, NOESY, HSQC and HMBC NMR spectra of PFSV-2 were obtained using a Bruker AV-400 MHz (¹³C) spectrometer, using tetramethylsilane (TMS) as an internal standard according to a previously published method (15).

Data are shown as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used as the statistical analysis technique. The statistical significance of differences between the groups was assessed using the Student's t-test.

To achieve the highest PFSV yield, BBD constituted 17 experiments for optimization of three individual parameters were performed. The observed and predicted responses of these experiments are presented in Table 1A.

The response variable Y was characterized by the following second-order polynomial equation through analysis of the experimental data with Design-Expert 8.05:

Table 1B summarized the results of the analysis of variance, goodness-of-fit and the adequacy of the model. The high F-value (22.84) and low p-value (0.0002) suggested that the regression model was significant, whereas the lack-of-fit value was insignificant. These results indicated that the model was sufficiently accurate for predicting the variations. The high values of R² (0.9671) and Radj2 (0.9647) indicated a high degree of correlation between the experimental and predicted values. The low CV (2.91%) and high value of adequate precision (18.457) suggested that the experiment had a very high degree of precision and reliability (17).

According to this model, the linear terms of processing temperature and amount of HCl were the significant factors impacting the yield of PFSV, with the quadratic terms of processing time and amount of HCl indicating that these variables had the largest effects. The coefficients of the other terms were not significant. The 3D response surface (Figures 1A–C) and 2D contour plots (Figures 1a–c) provided a vivid portrayal of the effects of the variables as well as their interactions on the yield of polysaccharides. By analyzing the regression equation and the response surface contour plots, the optimal extraction conditions of PFSV were determined as follows: processing temperature, 80°C; processing time 0.81 h; amount of HCl, 1.5 ml. The maximum predicted extraction yield of PFSV was 6.02%, which corresponded well with the actual yield (5.94 ± 0.16%, n = 3). The above results indicated that the model designed in this study was valid.

The yields of PFSV, which could be achieved by the acid-ethanol pretreatment using the optimized procedure, were compared with those obtained by traditional extraction methods (HWE and UAE). The yields of polysaccharides obtained by HWE and UAE were 1.31 ± 0.15% and 2.04 ± 0.25%, respectively. Therefore, the PFSV yield obtained by acid-ethanol pretreatment in this study was 3.53-fold and 1.91-fold higher compared with conventional HWE and UAE, respectively (Supplementary Figure S1). To summarize, the yields of PFSV obtained by acid-ethanol pretreatment were much higher than those obtained using the other two traditional extraction methods.

Using DEAE-52 cellulose column chromatography, PFSV was fractionated to obtain three fractions (Figure 2A). The second fraction was the main fraction, containing 89.8% of PFSV. This was further purified on a Sephacryl S-400HR column to obtain PFSV-2. High-performance anion-exchange chromatography analysis revealed that PFSV-2 was composed of fucose, rhamnose, galactose, glucose, xylose, and mannose in molar ratios of 13.50, 0.27, 32.59, 7.01, 0.85, and 15.82, respectively. Fucose, galactose, and mannose were the most abundant monosaccharides. The FT-IR spectra of PFSV-2 are shown in Figure 2B. The peak at 3,348 cm⁻¹ (O–H groups) corresponded to the main characteristic polysaccharides peak (18). The absorption peaks at 2,930 and 1,417 cm⁻¹ were assigned to the asymmetric stretching vibrations of C–H. The absorption at 1,640 cm⁻¹ corresponded to the C=O stretching vibration. The broad peak around 1,404 cm⁻¹ indicated the presence of uronic acid in PFSV-2. The absorptions at 1,078 and 876 cm⁻¹ indicated that α- and β-configurations were both present. The skeletal modes of the pyranose rings were reflected in the bands in the 350–600 cm⁻¹ range. The molecular weight parameters of PFSV-2 were determined by HPSEC-MALLS-RI. As shown in Figure 2C, the Mn, Mp, Mw, and Mz values of PFSV-2 were 17.497, 20.377, 19.256, and 21.193 KDa, respectively. The polydispersity indices (Mw/Mn and Mz/Mn) were 0.99 and 1.21, indicating that PFSV-2 was a homogeneous polysaccharide with a relatively narrow molecular weight distribution.

Methylation and GC-MS methods were used to determine the glycosidic bond types in PFSV-2. The sugar residues of PFSV-2 are summarized in Table 2 and their corresponding mass spectra are shown in Supplementary Figure S3. Nine derivatives were identified from the PFSV-2 methylation products. Among these monosaccharide residues, 6-linked Galp accounted for the largest proportion of the total sugar residues, suggesting that the backbone of PFSV-2 may be composed of 6-linked Galp.

The chemical structure of PFSV-2 was further determined using ¹H, ¹³C, COSY, HSQC, and HMBC NMR spectroscopy (Figure 3). In the ¹H spectrum (Figure 3A), the 3.2–5.3 ppm anomeric region was crowded. Six coupling signal peaks were identified from 4.4–5.4 ppm, indicating that there were at least six types of sugar residues in PFSV-2. The resonances between 3.3 and 4.1 ppm were assigned to the ring protons and highlighted the presence of the pyranose form (19).

The anomeric configuration of each residue in PFSV-2 was elucidated using the ¹³C NMR spectroscopy (Figure 3B). The chemical shifts of the anomeric carbons ranged from 95 to 110 ppm. Three strong signal peaks appeared at 102.23, 100.78, and 97.8 ppm, indicating that there was overlap between the anomeric carbon signals of the residues. The strong ¹³C signal at 56 ppm was assigned to the –O-CH₃, which indicated its high content. It is more likely to link the → 6)-α-Galp-(1 → at C-2, C-3 or C-4. In combination with the results of methylation analysis, HSQC, and ¹³C NMR and the residue classification, the anomeric carbon signals of the residues A, B, C and D (Figure 3C) were identified. Residues A, B, C and D were → 6)-α-Galp-(1 → and → 2,6)-β-Manp-(1 → , t-α-Manp-(1 → , and → 2)-α-Fucp-(1 → , respectively. The chemical shifts of these glycosidic linkages in PFSV-2 were assigned and are listed in Table 2.

Based on the chemical shifts of all the sugar residues and in combination with the HMBC results, the linkage sites and sequences of different sugar residues in PFSV-2 were analyzed. As suggested by the HMBC spectrum of PFSV-2 (Figure 3D), the correlation peaks at 4.91/69.07 ppm (A H-1/A C-6), 3.57/97.93 ppm (B H-6/A C-1), 3.81/102.23 ppm (A H-6/B C-1), 4.98/69.91 ppm (C H-1/B C-2), and 5.50/69.07 ppm (D H-1/A C-6) indicated that the O-1 of residue A was linked to C-6 of A, O-6 of residue B was linked to the C-1 of residue A, O-6 of residue A was linked to the C-1 of residue B, O-1 of residue C was linked to the C-2 of residue B, and O-1 of residue D was linked to the C-6 of residue A. Furthermore, in the NOESY results (Figure 3E), cross-peaks appeared at (A H1, A H6, D H2), (B H1 and A H6) and (D H1 and A H6). In combination with the previous results, this enabled the verification of the linkage sites and sequences of different sugar residues in PFSV-2 and the primary structure of PFSV-2 to be deduced, as shown in Figure 3F. The backbone of PFSV-2 was composed of → 6)-α-Galp-(1 → and → 2,6)-β-Manp-(1 → and → 2)-α-Fucp-(1 → and branched of t-α-Manp-(1 → at position 2 of residue B.

The zebrafish hyperlipidemia model is commonly used for the assessment of hypolipidemic drugs, as zebrafish larvae possess important organs for energy balance and metabolism, such as lipid storage in white adipocytes (20). ORO staining can be used to visualize neutral lipid localization in whole zebrafish (21). As shown in Supplementary Figure S3, minimal ORO staining was found in the control group (without feeding egg yolk) throughout the culture period. In contrast, in the egg yolk group, the lipid was abundant in the gut, yolk sac and blood vessels. As expected, compared to the egg yolk control, PFSV-2 at concentrations of 0.5 and 1.0 mg·ml⁻¹ significantly decreased the lipid content. For the positive group, simvastatin also remarkably lowered the lipid level compared to the egg yolk group. These results manifested that PFSV-2 is a potent hypolipidemic agent.

As shown in Figures 4A, B, compared with the control group, the TG and TC content of the egg yolk group was significantly increased (p < 0.05). Following treatment with PFSV-2 for 3 days, the content of TG and TC was significantly reduced as compared to the egg yolk group. Interestingly, the lipid-modulating effect of PFSV-2 showed concentration dependence. This result confirmed the results of ORO staining; that is, the decrease in lipid levels following treatment by PFSV-2.

Abnormal lipid accumulation and deposition can be induced by an imbalance between lipogenesis and lipolysis. Therefore, the hypolipidemic activity of PFSV-2 depends on the facilitation of lipolysis or inhibition of lipogenesis. For lipogenic genes, the effects of PFSV-2 on the expression levels of peroxisome proliferator-activated receptor gamma (pparg), fatty acid synthase (fasn) and microsomal triglyceride transfer protein (mtp) were determined. As shown in Figure 4C, the expressions of the fatty acid synthesis related genes pparg and fasn were upregulated significantly in egg yolk group compared with the control group. Importantly, the expression of these genes was significantly downregulated by PFSV-2 in yolk-treated zebrafish larvae. However, there was no significant difference in mtp gene expression between the egg yolk group and PFSV-2 treatment groups.

To investigate the effects of PFSV-2 on cholesterol biosynthesis genes, the mRNA levels of HMGCRa and HMGCRb (the rate-limiting enzymes of cholesterol synthesis in zebrafish) were investigated (Figure 4D). The results indicated that HMGCRb expression were notably elevated in the egg yolk group but remarkably decreased in the PFSV-2 treatment group. However, the HMGCRa gene expression was not significantly different in any of the tested groups. Thus, PFSV-2 may exert its hypocholesterolemic effect by decreasing the levels of HMGCRb.

To explore the effects of PFSV-2 on lipolysis, mainly beta-oxidation, the mRNA expression levels of peroxisome proliferator-activated receptor alpha b (pparab) and acetyl-CoA carboxylase alpha (acaca) were determined (Figure 4E). The expressions of pparab and acaca were remarkably decreased in the egg yolk group but significantly upregulated by PFSV-2.

Various methods have been reported for the extraction of polysaccharide from S. vaninii. Chan et al. (22) extracted polysaccharides from S. vaninii using hot water, microwave-assisted extraction, maceration, and ultrasonic-assisted extraction, achieving yields of 2.17%, 2.25%, 2.02%, and 2.11%, respectively. In the present study, the yields of PFSV-2 extracted by HWE and UAE were 1.31 ± 0.15% and 2.04 ± 0.25%, respectively. These results were similar to those reported above. However, the PFSV-2 yield obtained by acidic ethanol pretreatment reached 5.94 ± 0.16%. This result confirmed that the acid-ethanol pretreatment plays an important role when aiming to high polysaccharide yield. Disruption not only enabled solvent to penetrate the cell wall but also enhanced polysaccharide dissolution.

In the present study, the zebrafish was used to investigate the lipid-lowering activity of PFSV-2. Zebrafish on a high-calorie diet showed hyperlipidemia, as verified by ORO staining and high TG and TC levels. PFSV-2 treated larvae showed smaller area of ORO staining and lower TG and TC content. These results indicated that PFSV-2 to be a potent lipid-lowering agent.

To investigate the underlying mechanism of the hypolipidemic properties of PFSV-2, seven lipid metabolism-related genes expression of pparg, fasn, mtp, HMGGRa, HMGGRb, pparab and acaca were investigated by RT-PCR. As one of the key factors driving adipogenesis, pparg can promote lipid storage in white adipose tissue as well as preadipocyte differentiation to mature adipocytes (23). The activation of pparg ultimately promotes the uptake, synthesis, esterification and storage of fatty acid in newly formed adipose cells (24). In the present study, the expression of pparg was significantly increased in the egg yolk group, which was in disagreement with the results of Wang et al. (25), who found that there were no changes to pparg in the egg yolk group. The low levels of pparg in PFSV-2 treated zebrafish larvae showed that PFSV-2 antagonized lipid storage in adipose tissue and impeded preadipocytes differentiation to mature adipocytes.

The gene fasn can facilitate the synthesis of long-chain fatty acids. It is minimally expressed in normal cells and highly expressed in various pathological conditions (26). The findings of the present study were consistent with previous reports (27), that is, the expression of fasn was significantly increased in the egg yolk group and suppressed by PFSV-2, compared with the control. These results suggested that the curative properties of PFSV-2 against hyperlipidemia are partly caused by suppression of the lipogenesis gene fasn.

HMGCRb is the key enzyme involved in lipid metabolism regulation and cholesterol biosynthesis in zebrafish larvae and an essential involved gene in the synthesis of TC (28). HMGCRb is the target of a cholesterol-lowering drugs-statins (29). In the present study, PFSV-2 significantly downregulated the elevated mRNA levels of HMGCRb in zebrafish larvae. Thus, PFSV-2 may exert its hypocholesterolemic effect by decreasing the level of HMGCRb.

The acaca gene can regulate β-oxidation of mitochondrial fatty acid and affect the expression of lipid metabolism (30). Additionally, the activation of pparab can decrease lipid levels via the enhancement of fatty acid β-oxidation in the liver (31). In the present study, the expression of pparab and acaca was remarkably decreased in the egg yolk group but significantly upregulated by PFSV-2. These results indicated that PFSV-2 may promote β-oxidation of fatty acid through upregulating the expression of pparab and acaca.

The hypolipidemic effects of polysaccharides have been demonstrated in many studies. Polysaccharides from cassia seeds can bind to bile acids, thereby decreasing the absorption of dietary cholesterol (32). Polysaccharides from Auricularia auricular displayed a hypolipidemic effect against high-fat emulsion-induced hyperlipidemia (33). Polysaccharides derived from Grifola frondose and Catathelasma ventricosum showed hypolipidemic activity in streptozotocin-induced diabetic mice (34, 35). And polysaccharide from Agaricus bisporus can impede the lipid accumulation in zebrafish models mainly through the regulation of adipogenesis (36).

Hypolipidemic activity of polysaccharide mainly depends on their molecular weight, sulfate content, sulfate position, type of linkage and molecular geometry (37). Laminarin sulfates with highest sulfate content have shown maximum hypolipidemic activity (38). Polysaccharides from Gastrodia elata Blume, which consisted mainly of glucose, exhibited a marked hypolipidemic effect in rats (39). Molecular weight has been demonstrated to affect the bile acid-binding ability of polysaccharides from Plantago asiatica seeds (40). The high choate-binding ability of polysaccharide was reported due to its lower molecular weight and more exposed hydroxyl groups (34). Zeng et al. (41) reported that the hypolipidemic effect of polysaccharide from Fortunella margarita (Lour.) Swingle was affected by a combination of multiple structural factors. It was also reported that the potent lipid-lowering potential of the polysaccharide partly by alleviating oxidative stress (42). The inherent mechanism underlying the hypolipidemic activity of PFSV-2 will be investigated in future research.