Structural analysis, in vitro antioxidant and lipid-lowering activities of purified Tremella fuciformis polysaccharide fractions

doi:10.21203/rs.3.rs-2327340/v1

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Structural analysis, in vitro antioxidant and lipid-lowering activities of purified Tremella fuciformis polysaccharide fractions

https://doi.org/10.21203/rs.3.rs-2327340/v1

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published 30 Sep, 2023

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In order to study the composition, structure and physicochemical properties of Tremella fuciformis polysaccharide (TFPS), four new polysaccharide fractions (TFPS1, TFPS2, TFPS3 and TFPS4) were successfully isolated from the hot-water extracted crude polysaccharides by DEAE-52 cellulose column. Their structures were analyzed by FT-IR, SEM, HPGPC, monosaccharide composition, Congo red and I₂-KI experiments. TFPS1 was a heteropolysaccharide with a molecular weight of 742 Da for the major polysaccharide fragment. TFPS2, TFPS3 and TFPS4 were single component homogeneous polysaccharides with 350774 Da, 622675 Da and 624724 Da, respectively. Glucose was the major monosaccharide of TFPS1 and TFPS2, while Mannose was the major monosaccharide of TFPS3 and TFPS4. TFPS1 and TFPS2 displayed a stable triple helix structure at aqueous solution, while TFPS3 and TFPS4 presented longer side chains and more branched chains. TFPS1, TFPS2, TFPS3 and TFPS4 all possessed in vitro antioxidant activities. Moreover, the four polysaccharide fractions all demonstrated significant cholesterol binding ability, as well as obvious sodium taurocholate binding ability. The results of this study may provide some guidance on the medicinal value of TFPS.

Tremella fuciformis polysaccharide

Isolation

Lipid-lowering activity

Structural analysis

Antioxidant activity

As the fruiting body of Basidiomycota fungus [1], Tremella fuciformis is not only an edible fungus with economic value, but also a long-established medicine in Chinese medicine [2]. It is rich in protein, polysaccharides, dietary fiber, many minerals, trace elements and vitamins [3, 4]. Among them, the main active ingredient of Tremella fuciformis is Tremella fuciformis polysaccharide (TFPS) [5]. Related studies have demonstrated that TFPS has anti-tumor [6], antioxidant [7], moisturizing [8–10], hypolipidemic [11], anti-inflammatory [12, 13] and antibacterial functions [11]. TFPS can be used in a variety of applications such as pharmaceuticals [14], cosmetics [15] and food [16, 17]. It exhibited inhibitory effect on cholesterol micelles in a concentration-dependent manner in vitro [18]. Moreover, Dextran sodium sulfate-induced colitis can be alleviated by high-dose TFPS immunomodulation. TFPS can reduce colon tissue damage, promote the production of anti-inflammatory cytokines, and restore the intestinal microbiota and microbial metabolites [19, 20]. Furthermore, TFPS solution at certain concentration can effectively reduce UVA damage to skin fibroblasts [21]. In addition, Hepatic fatty acid synthesis can be regulated by TFPS, which suppresses NAFLD mainly by decreasing the expression of fatty acid synthesis regulators [22].

TFPS is a kind of heterosaccharide, different fractions of polysaccharides should have different biological activities [23]. Although bioactivities of the heterosaccharide have been extensively studied, it is not very clear about the relationship between structure, conformation and bioactivity of their isolated and purified fractions. In this study, TFPS were separated by ion exchange column chromatography. And four polysaccharide fractions (TFPS1, TFPS2, TFPS3 and TFPS4) were isolated and purified from the crude TFPS. They were physically characterized by FT-IR and SEM, and their structural features were tested in combination with monosaccharide composition, Congo red and I₂-KI experiments. The in vitro antioxidant activity, cholesterol binding and sodium taurocholate binding assays were also tested for the four polysaccharide fractions. The information obtained can provide a theoretical basis for better development and utilization of TFPS.

2.1 Materials and Reagents

Tremella fuciformis were commercially available from Gutian, Fujian (China). DEAE-52 cellulose was purchased from Shanghai Dipper Biotechnology Co. Sulfuric acid (H₂SO₄) and sodium hydroxide (NaOH) were bought from Tianjin Windship Chemical Reagent Technology Co. Congo red and sodium chloride (NaCl) were obtained from Aladdin Biochemical Technology Co. Phenol (C₆H₆O) was acquired from Tianjin Yongda Chemical Reagent Co. Salicylic acid (C₇H₆O₃) was procured from Zhengzhou Jinbei Chemical Co. Anhydrous ethanol (C₂H₆O) was received from Shandong Bosheng Chemical Co. Standard monosaccharides (including Mannose ( Man), ribose (Rib), rhamnose (Rha), glucuronic acid (Glc-UA), galacturonic acid (Gal-UA), N-acetyl-aminoglucose (Glc-NAc), glucose (Glc), N-acetyl-aminogalactose (Gal-NAc), galactose (Gal), xylose (Xyl), arabinose (Ara) and fucose (Fuc)) were purchased from Shanghai Yuanye Biotechnology Co. The water used in the experiments was distilled water. All chemicals were of analytical grade and used straight away without further purification.

2.2 Experimental Methods

2.2.1 Extraction of TFPS

Extraction of TFPS was carried out by hot water extraction method. The dried Tremella fuciformis (3 g) was weighed for crushing and placed in a beaker. The obtained powder was stirred in 180 mL distilled water and refluxed for 7 h. After filtering through a double layer of filter cloth, 200 ml of distilled water was added to the filter residue, heated and stirred all the time for 2 h. After filtration, the two filtrates were combined and concentrated under vacuum to 40 mL. To the filtrate, four times the amount of ethanol (90%) was added. The filtrate was left overnight at 4°C in a refrigerator and separated by centrifugation (8000 r/min, 8 min). The obtained precipitate was dissolved with of water. Proteins were removed by static adsorption with AB-8 macroporous resin. Then, the TFPS solution was dialyzed (8000 Da) to remove small molecules and inorganic ions. Finally, the dialysate was vacuum concentrated to 30 mL and lyophilized using an lyophilizer (FD-1A-50, Shanghai, China), then the TFPS solid was obtained.

2.2.2 Separation and purification

The flow chart for the preparation of TFPS1, TFPS2, TFPS3 and TFPS4 were shown in Fig. S1. Separation was performed by DEAE-52 cellulose column. The TFPS was prepared as a 10 mg/mL aqueous solution sample with a loading volume of 100 mL. The sample was eluted with distilled water, 0.2, 0.4, 0.6 and 2 mol/L NaCl solution in a gradient at a flow rate of 4 mL/min, and one tube was collected every 10 mL. The purified fractions corresponding to the peaks were collected under the monitoring of phenol-sulfuric acid method [24]. The four purified polysaccharide fractions were dialyzed for 72 h and then lyophilized before further study.

2.3 Analysis of total sugar content and yield

The total sugar content was measured by the sulfuric acid-phenol method using glucose as the standard [25].

$$\text{Y}\text{i}\text{e}\text{l}\text{d}（\text{%}）={\text{M}}_{1}/{\text{M}}_{2}\times 100\text{%}$$

$$\text{T}\text{o}\text{t}\text{a}\text{l} \text{s}\text{u}\text{g}\text{a}\text{r} （\text{%}）=(\text{A}\times \text{B}\times \text{D})/{\text{M}}_{3}\times 100\text{%}$$

Where: M₁ indicates the mass of each fraction after separation and purification, mg; M₂ represents the mass of polysaccharide before separation and purification, mg; A is the mass concentration of the solution calculated from the absorbance value, mg/mL; B shows the dilution of the sample; D means the volume of the test solution, mL; M₃ expresses the mass of the sample, mg.

2.4 UV analysis

The UV spectra of TFPS1, TFPS2, TFPS3 and TFPS4 were recorded in the range of 200 ~ 400 nm using an Ultraviolet spectrophotometer (UV, TU-1810, Beijing, China) [26].

2.5 Structural Analysis

2.5.1 Molecular weight determination

The average molecular weights of TFPS1, TFPS2, TFPS3 and TFPS4 were determined by high performance gel permeation chromatography (HPGPC) on a gel chromatograph (GPC, Model 515, Waters, USA) [27]. The analytical columns of this instrument were Waters Ultrahydrogel 500 and 120 in two tandem, mobile phase: 0.1 mol/L aqueous NaNO₃ solution at a flow rate of 1 mL/min.

2.5.2 Monosaccharide composition analysis

The monosaccharide compositions of TFPS1, TFPS2, TFPS3 and TFPS4 were examined by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization [28]. First, weigh 20 mg of polysaccharide sample in a 10 ml ampoule, added 3 ml of trifluoroacetic acid (TFA, 2 mol/L), acid digestion at 120 ℃ for 4 h. Added methanol nitrogen blowing to evaporate the TFA, then added water to redissolve to get 1 mg/ml acid hydrolysis sample solution. Mix 250 uL of sample solution, 250 uL of 0.6 mol/L NaOH and 500 uL of 0.4 mol/L LPMP-methanol, and reacted at 70°C for 1 h. After cooling for 10 min, added 500 uL of 0.3 mol/L HCl to neutralize, and then added 1 mL of chloroform to extract 3 times. The supernatant was taken and used for monosaccharide composition analysis. The analysis was performed at 30°C using a high performance liquid chromatograph (HPLC, Shimadzu LC-20AD, Japan) with an Xtimate C18 (4.6*200 mm 5 um). The control standard monosaccharides were Man, Rib, Rha, Glc-UA, Gal-UA, Glc-NAc, Glc, Gal-NAc, Gal, Xyl, Ara and Fuc.

2.5.3 FT-IR spectral analysis

The functional groups of TFPS1, TFPS2, TFPS3 and TFPS4 were examined using FT-IR [29]. A sample of 5 mg polysaccharide was weighed, mixed with 100 mg KBr and pressed. A spectrometer (FT-IR, Nicolet iS50, ThermoFisher, China) was used in the wavenumber range of 4000 ~ 600 cm^− 1.

2.5.4 SEM text

The surface morphology of polysaccharides is frequently analyzed by SEM. TFPS1, TFPS2, TFPS3 and TFPS4 were attached to the sample stage using conductive adhesive followed by spraying a thin gold layer [30]. The gold sprayed samples were placed in a scanning electron microscope (SEM, SU3500, HITACHI, Japan) and the images were magnified at 1000×.

2.5.5 Iodine-potassium iodide (I₂-KI) experiments

The side chain structures of TFPS1, TFPS2, TFPS3 and TFPS4 were determined by I₂-KI experiments [31]. The sample solution (1 mg/mL) was mixed with iodine reagent (0.2% KI solution containing 0.02% I₂), which was scanned in the UV-vis light range from 300 to 700 nm.

2.5.6 Congo red experiment

The triple helix structures of TFPS1, TFPS2, TFPS3 and TFPS4 were identified using Congo red experiments [32]. The sample solution (2 mg/mL) was mixed with Congo red solution dissolved in different concentrations of NaOH with UV-vis light scanning in the range of 300 ~ 700 nm.

2.6 Antioxidant studies in vitro

TFPS1, TFPS2, TFPS3 and TFPS4 samples were prepared as 2 mg/mL solutions as well as diluted into 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, 1 mg/mL and 1.5 mg/mL sample solutions, respectively.

2.6.1 Determination of ferric ion reducing power

The reduction power of TFPS1, TFPS2, TFPS3 and TFPS4 was tested according to the previous reported method [33], but with minor modifications. Mix 1 mL of sample solution at different concentrations, 2.5 mL of phosphate buffer at pH 6.6 (0.2 mol/L) and 2.5 mL of 1% potassium ferricyanide, and react in a water bath at 50°C for 20 min. Afterwards, 2.5 mL of 10% (W/V) trichloroacetic acid was added and centrifuged at 4000 r/min for 10 min. Then, 2.5 mL of supernatant was taken, 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl₃ were added and mixed, and the sample was left for 10 min. Finally, the absorbance of the reaction solution was measured at 700 nm and zeroed using distilled water instead of FeCl₃ solution as a blank. The higher is the value, the higher is the reducing power of the sample. The reducing power was calculated by Eq. (3):

$${\text{A}}_{1}={\text{A}}_{\text{X}}-{\text{A}}_{0}$$

Where: A₁ indicates reducing power of Fe³⁺; A_X denotes the absorbance value of the sample group; A₀ means the absorbance of the blank control group.

2.6.2 Scavenging capacity of DPPH free radicals

The DPPH radical scavenging ability of TFPS1, TFPS2, TFPS3 and TFPS4 was examined based on previously reported methods with appropriate modifications [34, 35]. The samples were mixed and shaken with 3 mL of polysaccharide solution and 1 mL of DPPH (0.1 mmol/mL) in anhydrous ethanol, and then reacted for 1 h at room temperature and protected from light. The absorbance value of the supernatant was measured at 517 nm and recorded as A_Sample, followed by an equal volume of distilled instead of the sample to be measured and recorded as A_Blank. As well as replacing the DPPH solution with an equal volume of anhydrous ethanol, the absorbance value was measured as A_Control. DPPH scavenging activity was calculated by Eq. (4):

${\text{A}}_{2} （\text{%}）=[1-({\text{A}}_{\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}}-{\text{A}}_{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}})/{\text{A}}_{\text{B}\text{l}\text{a}\text{n}\text{k}}]\times 100\text{%}$ ()

Where: A₂ denotes DPPH radical scavenging rate; A_Sample represents the absorbance value of sample group; A_Control indicates the absorbance value of sample control group; A_Blank means the absorbance value of blank control group.

2.6.3 Scavenging capacity of hydroxyl radicals

The hydroxyl radical scavenging ability of TFPS1, TFPS2, TFPS3 and TFPS4 was determined on the basis of the reported method with slight modifications [36]. Different concentrations of 1 mL of sample solution, 1 ml (9 mmol/L) salicylic acid-ethanol solution, 1 mL (9 mmol/L) FeSO₄ and 1 ml (8.8 mmol/L) H₂O₂ solution were mixed and reacted in a water bath at 37°C for 1 h. After the reaction solution was cooled to room temperature, its absorbance value was measured at a wavelength of 510 nm. Under the same conditions the absorbance values of the blank control solution were determined using distilled water instead of the polysaccharide solution. The absorption values of the background solution were recorded without the addition of H₂O₂ solution. The hydroxyl radical scavenging activity was calculated by Eq. (5).

${\text{A}}_{3} （\text{%}）={[\text{A}}_{\text{c}}-{(\text{A}}_{\text{i}}-{\text{A}}_{\text{j}}\left)\right]/{\text{A}}_{\text{c}}\times 100\text{%}$ ()

Where: A₃ denotes the hydroxyl radical scavenging rate; A_i indicates the absorbance value of sample solution; A_j represents the absorbance value of the background solution; A_c refers to the absorbance value of blank control solution.

2.7 In vitro hypolipidemic activity assay

2.7.1 Determination of cholesterol binding amount

TFPS1, TFPS2, TFPS3 and TFPS4 samples were prepared as 10 mg/mL solutions. 5 mL of different sample solutions were mixed with 10 mL of cholesterol solution (1 mg/mL). In order to simulate the gastrointestinal environment, the pH values were adjusted to 2 and 7, respectively, and shaken for 2 h at a constant speed of 80 r/min at 37°C. Then, 0.4 mL of the mixed solution was taken and mixed thoroughly with 0.2 mL of o-phthalaldehyde solution and 4 mL of mixed acid solution (V_{sulfuric acid} : V_{hydrochloric acid} = 1:1) in a 37°C water bath for 10 minutes. Afterwards, the supernatant was centrifuged at 4000 r/min for 20 min and the absorbance value of the solution was measured at 550 nm. The values were determined by the cholesterol standard curve, which was developed at the concentrations of 0.05, 0.075, 0.1, 0.15 and 0.2 mg/mL of standard cholesterol solution [37]. The measured cholesterol standard curve was: Y = 4.6X + 0.045, R² = 0.988. The cholesterol binding was calculated by Eq. (6):

$$\text{B}\text{i}\text{n}\text{d}\text{i}\text{n}\text{g} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} (\text{m}\text{g}/\text{g})={(\text{C}}_{1}-{\text{C}}_{2})/{\text{M}}_{4}$$

Where: C₁ indicates the amount of cholesterol added, mg; C₂ shows the amount of cholesterol remaining, mg; M₄ indicates the sample mass, g.

2.7.2 Determination of the binding rate of sodium taurocholate

TFPS1, TFPS2, TFPS3 and TFPS4 samples were prepared as 2 mg/mL solutions. The sample solution (2 mg/mL) was mixed with 1 ml of HCl solution (0.01 mol/L) and allowed to shake at 37°C for 1 h. Subsequently, 4 mL of trypsin (10 mg/mL) was added and shaken for 1 h. Followed by 4 mL of sodium taurocholate solution (2 mg/mL) was added and shaken again for 1 h. Then, 2.5 mL of supernatant was centrifuged at 5 000 r/min for 20 min, and 2.5 mL of supernatant was mixed with 7.5 mL of sulfuric acid solution (60%), incubated at 70°C for 30 min and then was cooled to room temperature. Finally, the absorbance value of each sample was measured at a wavelength of 387 nm. The values were determined by the standard curve of sodium taurocholate, which was developed at the concentrations of 0.05, 0.075, 0.1, 0.15, and 0.2 mg/mL of standard sodium taurocholate solution [38]. The measured standard curve of sodium taurocholate was: Y = 3.7738X + 0.028, R² = 0.9823. The binding rate of sodium taurocholate was calculated by Eq. (7):

$$\text{B}\text{i}\text{n}\text{d}\text{i}\text{n}\text{g} \text{r}\text{a}\text{t}\text{e} \left(\text{%}\right)=[1-(\text{C}\times \text{V})/4]\times 100\text{%}$$

Where: C denotes the concentration of sodium taurocholate in the supernatant obtained by the standard curve, mg/ml; V stands for the volume of the supernatant, ml.

3.1 Isolation and purification of TFPS

100ml TFPS solution (10 mg/mL) was added to the DEAE-52 cellulose column, and five elution peaks were obtained by sequential elution with deionized water and different concentrations of NaCl aqueous solution, as shown in Fig. 1. The four purified polysaccharide fractions (TFPS1, TFPS2, TFPS3 and TFPS4) eluted with 0, 0.2, 0.4 and 0.6 mol/L NaCl solutions were collected, respectively. The fifth fraction eluted with 2 mol/L NaCl solution was not collected because of its very low polysaccharide content and the feasibility of subsequent operations. It was observed that TFPS1 was a light yellow loose flocculent polysaccharide, while TFPS2, TFPS3 and TFPS4 were all white loose flocculent polysaccharides. The solubility analysis revealed that the four purified polysaccharide fractions were soluble in hot water, but insoluble in cold water and in organic solvents such as ethanol, methanol and acetone.

3.2 Analysis of total sugar content and yield

The total sugar content of the four purified polysaccharide fractions were determined by sulfuric acid-phenol method. The glucose standard curve was shown in Fig. S2 (a). The yield and total sugar content were calculated according to Equations (1) and (2). The yields of TFPS1, TFPS2, TFPS3 and TFPS4 were 5.7%, 6.19%, 66.68% and 16.54%, respectively. And the total sugar contents of the four purified polysaccharide fractions were 88.79%, 83.43%, 98.88% and 94.33%, respectively.

3.3. UV analysis

The UV scan showed that TFPS1, TFPS2, TFPS3 and TFPS4 had no obvious absorption peaks at 260 nm and 280 nm (As shown in Fig. S2 (b)). The results indicated that none of the four purified polysaccharide fractions contained protein and nucleic acid-like substances [29].

3.4 Structural Analysis

3.4.1 Analysis of molecular weight

The homogeneity and molecular weights of TFPS1, TFPS2, TFPS3 and TFPS4 were determined by HPGPC. Figure 2 showed the molecular weight results for the four purified polysaccharide fractions. TFPS1 had a significant peak and two small peaks. The main component of TFPS1 was a small-sized polysaccharide fragment with a molecular weight of 742 Da. In addition, there were two small amounts of large fragments with molecular weights of 18938 Da and 200468 Da. TFPS2, TFPS3 and TFPS4 were single component homogeneous polysaccharides with 350774 Da, 622675 Da and 624724 Da, respectively.

3.4.2 Monosaccharide composition analysis

According to the relevant literature [39], TFPS1, TFPS2, TFPS3, and TFPS4 were hydrolyzed to monosaccharides by trifluoroacetic acid and NaOH, and subsequently derivatized by LPMP methanol. Then their monosaccharide compositions were known by HPLC analysis (as shown in Fig. S3). The compositions and molar ratios were shown in Table 1. The monosaccharide compositions of the four purified polysaccharides were slightly different. Glc was the main monosaccharide of TFPS1 and TFPS2, while Man was the major monosaccharide of TFPS3 and TFPS4.

Table 1

Composition and molar ratio of polysaccharide monosaccharides
	Man	Rib	Rha	Glc-UA	Gal-UA	Glc	Gal	Xyl	Ara	Fuc
TFPS1	3.407	0.01	0	0.399	0.014	6.71	0.194	1.298	0.056	0.546
TFPS2	2.122	0.048	0	0.446	0.021	25.741	0.609	0.565	0.072	0.186
TFPS3	10.553	0.01	0.02	1.819	0.056	2.962	0.093	3.136	0.072	2.281
TFPS4	9.18	0.1	0.016	1.785	0.035	0.994	0.057	2.553	0.068	2.021

3.4.3 FT-IR spectral analysis

FT-IR can be used to analyze the type of functional groups in polysaccharides because of their high characterization. Figure 3 displayed the scanned FT-IR spectra of TFPS1, TFPS2, TFPS3 and TFPS4. The broad peak near the wave number 3320 cm^− 1 was caused by the O-H stretching vibration in the sugar molecule, indicating the existence of inter- or intramolecular hydrogen bonds in the polysaccharide. The characteristic peaks at wave numbers 2931 cm^− 1 represented the C-H stretching vibrations of methyl or methylene groups [40]. The characteristic peaks at wave number 1415 cm^− 1 showed the C-H stretching vibrations of carboxyl groups and variable angle vibrations of C-H. While the characteristic peak at wave number 1248 cm^− 1 indicated the contraction vibration of S = O, suggesting that the polysaccharide contains sulfate radicals [41, 42]. Furthermore, the characteristic peaks near the wave number 1603 cm^− 1 were expressed as C = O asymmetric stretching vibration of COO- and symmetric stretching vibration of C = O, indicating the presence of uronic acids in polysaccharides [43]. Meanwhile, the absorption peak at 1019 cm^− 1 corresponds to the absorption peak of the stretching vibration formed by the C-O-H and C-O-C of the pyranose ring. What’s more, the absorption peaks between 947 and 865 cm^− 1 may represent the presence of β-glycosidic bonds in all four purified polysaccharides [44]. In addition, the characteristic absorption peak of the α-glycosidic bond was demonstrated at 844 cm. The presence of α-D-mannose was demonstrated by an absorption peak near 800 cm^− 1 [45]. Therefore, it can be inferred that TFPS1 was a neutral polysaccharide with α-D-mannose as the main chain. TFPS2 was an acidic polysaccharide with α-mannose as the main chain. TFPS3 and TFPS4 were both acidic polysaccharides with α-D-mannose as the main chain. Moreover, all four purified polysaccharide fractions had both α-type and β-type glycosidic linkages.

3.4.5 SEM analysis

The microstructures of TFPS1, TFPS2, TFPS3 and TFPS4 were investigated by SEM. As showed in Fig. 4, there revealed differenct surface morphologies in size and shape. The TFPS1 exhibited powder accumulation, whose surface morphology showed a dense spherical shape. The surface of TFPS2 demonstrated interlaced extensions in the form of reticular aggregates. The surface of TFPS3 manifested staggered extensions of filaments and curled aggregates. As for TFPS4, there was smooth interlaced extension in the form of lamellar aggregates. The TFPS1 showed poor film-forming properties because it was a small molecular weight polysaccharide fraction. However, TFPS2, TFPS3 and TFPS4 were large molecular weight polysaccharide fractions and thus had good film-forming properties.

3.4.6 Analysis of I₂-KI experiments

The conformations of the polysaccharides could be elucidated by the absorption spectra of their solution reacted with I₂-KI. If the polysaccharide had longer side chains and more branched chains, the spectrum should have an absorption peak at 350 nm. If there was an absorption peak at 565 nm, the polysaccharide should have shorter side chains and fewer branched chains [46, 47]. The UV-vis spectra of the four purified polysaccharide fractions reacted with I₂-KI solution were shown in Fig. 5. TFPS3 and TFPS4 exhibited absorption peaks at 350 nm, indicating that the two purified polysaccharide fractions possessed longer side chains and more branched chains. In contrast, TFPS1 showed small absorption peaks at 565 nm, suggesting that the purified polysaccharide fraction may manifest shorter side chains and fewer branched chains. However, TFPS2 exhibited no absorption peak at 350 nm and 565 nm, which could only indicate that its conformation is different from the other three purified polysaccharide fractions.

3.4.7 Analysis of Congo red experiment

Polysaccharides with triple helix structure could complex with Congo red solution. For the Congo red-polysaccharide complex solution, their maximum absorption wavelength of the complex should shift to the long-wave direction at a certain range of NaOH concentration, compared with that of the pure Congo red solution. When the concentration of NaOH is greater than 0.3 mol/L, the triple helix conformation of the complex may be disrupted and its maximum absorption wavelength should be gradually reduced [48]. The interactions of the four purified polysaccharide fractions with Congo red solution were shown in Fig. 6. For the solutions of the purified polysaccharide fractions and Congo red, the maximum absorption wavelengths gradually shifted to the long-wave direction with the increase of NaOH concentration, indicating that the complexes had been formed. Compared with the maximum absorption wavelengths of the pure Congo red solution, those of Congo red-TFPS1 and Congo red-TFPS2 solutions decreased significantly when the NaOH concentration was greater than 0.3 mol/L. The phenomenon indicated that TFPS1 and TFPS2 possessed the triple helix structure. However, the maximum absorption wavelengths of Congo red-TFPS3 and Congo red-TFPS4 solutions were not significantly reduced, suggesting that the triple helix structure may not be existed at the aqueous solution of TFPS3 and TFPS4.

3.5 In vitro antioxidant activity analysis of TFPS1, TFPS2, TFPS3 and TFPS4

3.5.1 The reducing power of ferric ion

TFPS can reduce Fe³⁺ of K₃[Fe(CN)₆] to Fe²⁺, and Fe²⁺ further reacts with FeCl₃ to form Fe₃[Fe(CN)₆]₂, which has the maximum absorbance at 700 nm. So, the absorbance was usually measured at 700 nm for the TFPS-Fe³⁺ reacting solutions [49]. The higher was the absorbance, the stronger did the TFPS display reduction ability. The results of the four TFPS reducing power test were illustrated in Fig. 7 (a). All the four purified polysaccharide fractions displayed certain reducing power. In a certain concentration range, the reducing power exhibited a quantitative influence relationship with concentration, and the reducing power increased with the increase of concentration. When the polysaccharide concentration reached 2 mg/mL, the four polysaccharides had the strongest reducing ability, and the reducing power was ranked from largest to smallest as TFPS1, TFPS4, TFPS3, and TFPS2. Therefore, TFPS1 has the best reducing ability. Its stronger reducing power may be related to the sulfate content [50–52].

3.5.2 Scavenging capacity of DPPH free radicals

The anhydrous ethanol solution of DPPH• has a maximum absorption wavelength at 517 nm. Polysaccharides can scavenge DPPH• and decrease the absorbance of DPPH•, from which the DPPH• radical scavenging rate can be evaluated [53, 54]. The results of the four purified polysaccharide fractions scavenging DPPH• radical were summarized in Fig. 7 (b). It is obviously that TFPS1, TFPS2, TFPS3 and TFPS4 all possessed certain DPPH• radical scavenging ability. The DPPH• scavenging ability of TFPS4, TFPS3 and TFPS2 increased with the increase of polysaccharide concentrations. When the polysaccharide concentration reached 2 mg/mL, their scavenging rate were 48.07%, 46.53% and 31.1%, respectively. However, TFPS1 displayed the best DPPH• radical scavenging ability when the concentration was 1.5 mg/ml. The scavenging rate was 46.71%. In addition, TFPS2 displayed significantly smaller DPPH• scavenging ability than the other three TFPS fractions. The variation in DPPH• radical scavenging activity may be attributed to the differences in their sulfate content [32, 55, 56].

3.5.3 Scavenging capacity of hydroxyl radicals

H₂O₂ and Fe²⁺ gave rise to hydroxyl radicals, which could react with salicylic acid and produce 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. The formed two acids have the maximum absorption wavelength at 510 nm [57]. When polysaccharides scavenged the hydroxyl radicals, the absorbance at 510 nm should decrease, from which the hydroxyl radical scavenging ability can be evaluated [58]. As shown in Fig. 7 (c), all the four purified polysaccharide fractions exhibited certain hydroxyl radical scavenging ability. And the hydroxyl radical scavenging rate increased with the increasing polysaccharide concentrations. When the polysaccharide concentration reached 2 mg/mL, the maximum hydroxyl radical scavenging rates for TFPS1, TFPS2, TFPS3 and TFPS4 were 64.13%, 38.76%, 37.66% and 41.18%, respectively. Thus, the results indicated that the four purified TFPS demonstrated significant free hydroxyl radical scavenging ability. The hydroxyl radical scavenging ability of TFPS1 was obviously higher than those of TFPS2, TFPS3 and TFPS4, probably owing to the better hydroxyl radical scavenging ability of polysaccharides with small molecular weight [59, 60].

3.6 Analysis of the in vitro hypolipidemic activity of TFPS1, TFPS2, TFPS3 and TFPS4

3.6.1 Cholesterol binding capacity in vitro

Many polysaccharides showed the potential to lower blood cholesterol [61–63]. So, cholesterol binding tests were also performed for the four purified polysaccharide fractions in this paper. The total cholesterol content was determined by the o-phthalaldehyde method [64]. Cholesterol exists in the body as both free cholesterol and cholesterol esters, which can produce a purplish red substance in the presence of sulfuric acid and o-phthalaldehyde. The purplish red substance has a maximum absorption at 550 nm wavelength. So, the cholesterol binding capacity of the four TFPS were tested by detecting the absorption intensity at 550 nm of their mixed solutions, in the presence of sulfuric acid and o-phthalaldehyde. Since the pH value of the human stomach environment is 2 and that of the intestinal environment is 7, the pH values of the mixed solutions were adjusted to 2 and 7 to simulate the human environmental. As shown in Fig. 8 (a, b), pH had a great influence on the cholesterol binding amounts for the four purified polysaccharide fractions. The cholesterol binding amounts at pH = 7 was significantly higher than those at pH = 2. Among the four TFPS, TFPS2 showed the highest potential to lower cholesterol. At pH = 7, the cholesterol binding amount of TFPS2 reached 113.52 mg/g, followed by 97.28 mg/g for TFPS4 and 94.74 mg/g for TFPS1, and lowest in 91.74 mg/g for TFPS3. However, at pH = 2, the order of cholesterol binding capacity is TFPS2, TFPS1, TFPS4 and TFPS3.

3.6.2 Sodium taurocholate binding capacity in vitro

In the human body, the removal of cholesterol is also dependent on the synthesis bile acids by cholesterol and the secretion of bile acids. Moreover, bile acids are primarily present in the form of bile salts in the body. As bile salts combines with other substances and the free bile salts amount decreases, cholesterol can be further turned into bile acids. Therefore, cholesterol depletion can also be determined based on the binding ability of bile salts. Among the many bile salts, sodium taurocholate is the most difficult to bind, so the binding capacity with sodium taurocholate is usually used to determine the level of cholesterol-lowering capacity [65–67]. Consequently, sodium taurocholate binding tests were also performed on TFPS1, TFPS2, TFPS3, and TFPS4 in this paper. The in vitro sodium taurocholate binding rates of the four purified polysaccharide fractions were presented in Fig. 8 (c). TFPS1, TFPS2, TFPS3 and TFPS4 expressed obvious binding ability to sodium taurocholate, and their sodium taurocholate binding rates were similar. The binding ability of the four purified polysaccharide fractions were ranked from largest to smallest as TFPS4, TFPS3, TFPS2, and TFPS1, with binding rates of 46.58%, 46.55%, 45.93%, and 45.86%, respectively.

The four purified polysaccharide fractions (TFPS1, TFPS2, TFPS3 and TFPS4) were isolated from the crude TFPS by DEAE-52 cellulose column. The monosaccharide composition analysis indicates that their monosaccharide compositions are slightly different. Glc was the major monosaccharide of TFPS1 and TFPS2, while Man was the major monosaccharide of TFPS3 and TFPS4. FI-IR analysis showed that the main chain of all four purified polysaccharides was pyranose. TFPS1 and TFPS2 exhibited stable triple helix structures, while TFPS3 and TFPS4 showed longer side chains and more branched chains. TFPS1, TFPS2, TFPS3 and TFPS4 all possessed significant DPPH• radical and hydroxyl radical scavenging effects and Fe³⁺ reducing power. Among them, TFPS1 had the strongest Fe³⁺ reduction and hydroxyl radical scavenging ability, while TFPS4 presented the strongest DPPH• radical scavenging ability. In addition, the four polysaccharide fractions all conferred obvious cholesterol and sodium taurocholate binding capacity, suggesting that they all exerted certain hypolipidemic activity. The cholesterol binding capacity at pH = 7 was higher than that at pH = 2. TFPS2 had the highest cholesterol binding rate, while TFPS4 expressed the highest sodium taurocholate binding rate. These may provide a theoretical basis and technical support for the purified TFPS’s application in the food and pharmaceutical industries.

Author contributions

Xige Li: Methodolog, Data curation, Writing and Editing.

Yanhui Hou: Methodolog, Writing-Reviewing and Editing, Funding acquisition.

Fengyi Zhang: Data curation.

Chaoxian Jiang: Data curation.

Jun Jiang: Data curation.

Funding

This work was supported by the small pilot project of PetroChina. LTD (PRlKY17031) and the Scientific research and development project of Zhengzhou Kema Biotechnology Co. LTD (ST22059).

Data availability All data generated or analysed during this study are included in this published article.

Compliance with ethical standards

Conflict of interests There are no conflicts of interest to declare.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors

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Structural analysis, in vitro antioxidant and lipid-lowering activities of purified Tremella fuciformis polysaccharide fractions

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Abstract

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1. Introduction

2. Materials And Methods

2.1 Materials and Reagents

2.2 Experimental Methods

2.2.1 Extraction of TFPS

2.2.2 Separation and purification

2.3 Analysis of total sugar content and yield

2.4 UV analysis

2.5 Structural Analysis

2.5.1 Molecular weight determination

2.5.2 Monosaccharide composition analysis

2.5.3 FT-IR spectral analysis

2.5.4 SEM text

2.5.5 Iodine-potassium iodide (I2-KI) experiments

2.5.6 Congo red experiment

2.6 Antioxidant studies in vitro

2.6.1 Determination of ferric ion reducing power

2.6.2 Scavenging capacity of DPPH free radicals

2.6.3 Scavenging capacity of hydroxyl radicals

2.7 In vitro hypolipidemic activity assay

2.7.1 Determination of cholesterol binding amount

2.7.2 Determination of the binding rate of sodium taurocholate

3. Results And Discussion

3.1 Isolation and purification of TFPS

3.2 Analysis of total sugar content and yield

3.3. UV analysis

3.4 Structural Analysis

3.4.1 Analysis of molecular weight

3.4.2 Monosaccharide composition analysis

3.4.3 FT-IR spectral analysis

3.4.5 SEM analysis

3.4.6 Analysis of I2-KI experiments

3.4.7 Analysis of Congo red experiment

3.5 In vitro antioxidant activity analysis of TFPS1, TFPS2, TFPS3 and TFPS4

3.5.1 The reducing power of ferric ion

3.5.2 Scavenging capacity of DPPH free radicals

3.5.3 Scavenging capacity of hydroxyl radicals

3.6 Analysis of the in vitro hypolipidemic activity of TFPS1, TFPS2, TFPS3 and TFPS4

3.6.1 Cholesterol binding capacity in vitro

3.6.2 Sodium taurocholate binding capacity in vitro

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

2.5.5 Iodine-potassium iodide (I₂-KI) experiments

3.4.6 Analysis of I₂-KI experiments