A polysaccharide of Chinese hirsutella sinensis and a preparation method and application thereof

Abstract

The present application relates to the technical field of polysaccharide extraction, and particularly relates to a Chinese hairy cap polysaccharide, a preparation method and application thereof. Polysaccharides in the Chinese hairy cap are extracted, separated and purified, and four polysaccharide components are obtained. The four polysaccharides can inhibit HIBEC-EMT, avoid liver damage, and can be used for treating and / or preventing primary biliary cholangitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide extraction, and particularly relates to a Hirsutella sinensis polysaccharide and a preparation method and application thereof. BACKGROUND

[0002] Ophiocordyceps sinensis is a traditional precious Chinese medicinal material, and is a complex of Ophiocordyceps sinensis and larvae of Hepialidae. Studies have shown that Ophiocordyceps sinensis contains polysaccharides, nucleosides, proteins or polypeptides, sterols and D-mannitol and other bioactive components, and has immunomodulatory, antitumor, antioxidant, anti-aging, hypoglycemic, antibacterial, anti-fatigue and kidney and liver protection and other pharmacological effects. As a vegetative strain of Ophiocordyceps sinensis, Hirsutella sinensis Liu, Guo, Yu et Zeng, sp. nov. has anti-fatigue, antitumor, anti-inflammatory, kidney protection and inhibition of pulmonary fibrosis and other pharmacological activities, and its fermentation products are widely used in medicine and health food. The most representative product is "Bailing Capsule" (Hirsutella sinensis Cs-C-Q80 fermentation mycelium) produced by Hangzhou East Pharmaceutical Co., Ltd. In 2010, Hirsutella sinensis was included in the Pharmacopoeia of the People's Republic of China (2010 edition) as a vegetative strain of Ophiocordyceps sinensis.

[0003] Carbohydrate compounds are the most widely existing compounds in nature and have important biological functions, including monosaccharides and derivatives thereof, oligosaccharides, polysaccharides and complex polysaccharides. Fungal polysaccharides are a kind of active polysaccharides isolated from fungal fruiting bodies, mycelium and fermentation broth. As a natural biological macromolecule, fungal polysaccharides have become a research hotspot in the fields of biology and medicine due to their wide pharmacological activities and low toxicity and side effects. Cordyceps polysaccharide (CP) has immunomodulatory, hypoglycemic, antitumor and other pharmacological effects, and is one of the main active ingredients of Ophiocordyceps sinensis. However, the research on Hirsutella sinensis polysaccharide has not been widely concerned.

[0004] Polysaccharides, as one of the main effective components of Hirsutella sinensis, have wide pharmacological activities. However, due to the complexity of the structure of polysaccharides, the research on polysaccharide components in Hirsutella sinensis is not comprehensive and in-depth enough. SUMMARY

[0005] Polysaccharides are a kind of complex sugar substances formed by the dehydration condensation of multiple monosaccharide molecules, and the structural units are connected by glycosidic bonds. Common glycosidic bonds include ɑ-1, 3 glycosidic bond, β-1, 6 glycosidic bond, β-1, 4 glycosidic bond, ɑ-1, 4 glycosidic bond and β-1, 3 glycosidic bond. Polysaccharides widely exist in animals, plants and microorganisms, and polysaccharides from different sources have different biological activities.

[0006] The polysaccharide in Chinese Trichosporon is extracted, separated and purified, and a new Chinese Trichosporon polysaccharide is obtained, which can inhibit epithelial mesenchymal transition (EMT) and has certain effect on the treatment and / or prevention of primary biliary cholangitis.

[0007] Specifically, the present application provides a Chinese Trichosporon polysaccharide component HSWP-1b, which is composed of 1,4-linked-α-D-Glcp residues and 4,6-linked-α-D-Manp residues, and the O-6 site of 1,4-linked-α-D-Glcp is branched, and the side chain is 1-linked-α-D-Glcp.

[0008] In addition to the above structural units, the Chinese Trichosporon polysaccharide component HSWP-1b of the present application also contains the following monosaccharide units: rhamnose, galacturonic acid, arabinose.

[0009] The average molecular weight of the polysaccharide is 5-15 kDa;

[0010] Further, the average molecular weight of the polysaccharide is 7-10 kDa;

[0011] Further, the average molecular weight of the polysaccharide is 8.57 kDa.

[0012] The infrared spectrum of the Chinese Trichosporon polysaccharide component HSWP-1b of the present application at least includes one or more of the following absorption peaks: 3424.33 cm -1 , 2920.28 cm -1 , 2854.17 cm -1 , 1636.73 cm -1 , 1405.36 cm -1 , 1033.51 cm -1 , 851.23 cm -1 , 766.36 cm -1 , 628.62 cm -1 .

[0013] The characteristic strong absorption band at 3424.33 cm -1 reflects the stretching vibration of O-H bond on the sugar ring; the peaks at 2920.28 cm -1 , 2854.17 cm -1 can reflect the stretching vibration of C-H and C-H2 boundary on the sugar ring; the absorption peak at 1636.73 cm -1 represents the stretching vibration of C=O boundary; the stretching vibration peak of pyranose ring is 1033.51 cm -1 , 851.23 cm-1 The absorption peaks indicate the presence of alpha configuration monosaccharide residues.

[0014] The present application provides a Chinese hairy capnoid polysaccharide component HSWP-1c, which is a galactomannoglucan consisting of 1,2,6-linked alpha-D-Galp, 1,3,4.6-linked alpha-D-Glcp, 4,6-linked beta-D-Manp, 1,4-linked alpha-D-Glcp, 1,4,6-linked alpha-D-Glcp and 1-linked alpha-D-Glcp residues.

[0015] In addition to the above structural units, the Chinese hairy capnoid polysaccharide component HSWP-1c of the present application also contains the following monosaccharide units: glucose, mannose, galactose, rhamnose and galacturonic acid.

[0016] Further, the average molecular weight of the HSWP-1c is 2-6 kDa;

[0017] Further, the average molecular weight of the HSWP-1c is 2-6 kDa;

[0018] Further, the average molecular weight of the HSWP-1c is 2.18 kDa.

[0019] The infrared spectrum of the Chinese hairy capnoid polysaccharide component HSWP-1c of the present application at least includes one or more than one of the following absorption peaks: 3432.59 cm -1 , 2926.03 cm -1 , 2845.91 cm -1 , 1639.48 cm -1 , 1599.85, 1025.25 cm -1 , 857.23 cm -1 , 620.35 cm -1 .

[0020] The characteristic strong absorption band at 3432.59 cm -1 reflects the stretching vibration of O-H bond on the sugar ring; the peak at 2845.91 cm -1 reflects the stretching vibration of C-H and C-H2 boundary on the sugar ring; the absorption peak at 1639.48 cm -1 represents the stretching vibration of C=O boundary; the stretching vibration peak of pyranose ring is 1025.25 cm -1 .

[0021] The present application provides a Chinese hairy cap polyprenol component HSWP-1d, which is composed of 1,4-linked-α-D-Glcp residues and 4,6-linked-α-D-Manp residues, and branched at O-6 position of 1,4-linked-α-D-Glcp, with 1-linked-α-D-Glcp as the side chain.

[0022] The Chinese hairy cap polyprenol component HSWP-1d of the present application also contains the following monosaccharide units: mannose, rhamnose, galacturonic acid, glucose, arabinose.

[0023] Further, the average molecular weight of the HSWP-1d is 1-5 kDa.

[0024] Further, the average molecular weight of the HSWP-1d is 1-5 kDa.

[0025] Further, the average molecular weight of the HSWP-1d is 1.09 kDa.

[0026] The infrared spectrum of the Chinese hairy cap polyprenol component HSWP-1d of the present application at least includes one or more than one of the following absorption peaks: 3421.58 cm -1 , 2923.03 cm -1 , 2851.42 cm -1 , 1639.48 cm -1 , 1405.36 cm -1 , 1025.25 cm -1 , 851.72 cm -1 , 631.37 cm -1 .

[0027] The characteristic strong absorption band at 3421.58 cm -1 reflects the stretching vibration of O-H bond on the sugar ring; the peaks at 2923.03 cm -1 , 2851.42 cm -1 reflect the stretching vibration of C-H and C-H2 boundary on the sugar ring; the absorption peak at 1639.48 cm -1 represents the stretching vibration of C=O boundary; the stretching vibration peak of pyranose ring is 1025.25 cm -1 .

[0028] The present application provides a separation method of Chinese hairy cap polyprenol component, characterized by comprising the following steps:

[0029] (1) passing the polysaccharide extract through ion exchange column chromatography to separate polysaccharide component HSWP-1;

[0030] (2) Gradient alcohol precipitation is used to separate and purify polysaccharide components HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d.

[0031] The polysaccharide extract in step (1) refers to the crude polysaccharide of Hirsutella sinensis obtained by a conventional method. The conventional method in the present application includes but is not limited to water extraction and preliminary purification after water extraction.

[0032] The water extraction includes but is not limited to heating extraction, ultrasonic extraction, microwave extraction and other conventional plant extract extraction methods.

[0033] The preliminary purification after water extraction includes but is not limited to alcohol precipitation, chitosan impurity removal and other methods.

[0034] In the purification method, auxiliary means such as protein removal, decolorization and small molecule removal can also be selectively added according to different conditions to facilitate the subsequent polysaccharide enrichment process. For example, protein removal reagents such as phenol, trichloroacetic acid, tannic acid and the like can be used; adsorbents such as cellulose, diatomite and activated carbon can be used for decolorization; and dialysis and other methods can be used to remove small molecules.

[0035] In some specific embodiments of the present application, the polysaccharide extract refers to the crude polysaccharide of Hirsutella sinensis powder obtained by water extraction, concentration and alcohol precipitation.

[0036] In the present application, if water extraction and alcohol precipitation are selected, ethanol is added to the water extract (or the concentrated solution of the water extract) until the ethanol concentration reaches 70-90% v / v, for example, 70%, 71%, 72%, 73%, 74%, 75%...80%...85%...90% v / v and the like.

[0037] In the present application, the ion exchange chromatography column used in step (1) is an anion exchange chromatography column; and is further selected from a DEAE cellulose chromatography column.

[0038] Further, the concentration of the alcohol solution used in step (2) is 30%-90%; and more further, the alcohol solution is selected from 30%, 50%, 70% and 90%.

[0039] The present application also provides a use of the above polysaccharide component in the preparation of a product for treating and / or preventing primary biliary cholangitis.

[0040] Further, the product is a product for inhibiting HIBEC-EMT (human intrahepatic bile duct epithelial cell-EMT).

[0041] Further, the product is a product for increasing or decreasing cell viability.

[0042] Further, the cell is a HIBEC cell.

[0043] Primary biliary cholangitis (PBC) is an autoimmune disease characterized by the destruction of intrahepatic small bile ducts and the presence of anti-mitochondrial antibodies, eventually developing into liver fibrosis; the main features of liver fibrosis are the loss of capillary network, the accumulation of fibrous collagen and activated myofibroblasts; during the occurrence of PBC, bile duct cells are first attacked and transformed into interstitial cells, and then liver fibrosis occurs, eventually leading to the occurrence of PBC and cirrhosis and other diseases.

[0044] The beneficial effects of the present application are as follows: four different polysaccharide components are extracted from Chinese Trichosporon, which can inhibit the HIBEC-EMT effect, and can be used for treating and / or treating diseases such as primary biliary cholangitis.

[0045] Abbreviation meaning

[0046] HIBEC: human intrahepatic bile duct epithelial cells;

[0047] PBC: primary biliary cholangitis;

[0048] EMT: epithelial-mesenchymal transition;

[0049] TGF-β1: transcription growth factor;

[0050] OCA: obeticholic acid;

[0051] HPGPC: high performance liquid permeation chromatography;

[0052] PMP: 1-phenyl-3-methyl-5-pyrazolone

[0053] TFA: trifluoroacetic acid

[0054] FT-IR: Fourier transform infrared spectrum

[0055] 1DNMR: one-dimensional nuclear magnetic resonance spectrum

[0056] 2DNMR: two-dimensional nuclear magnetic resonance spectrum

[0057] Galp: galactopyranosyl

[0058] GalA: galacturonic acid

[0059] Gal: galactose

[0060] Glc: glucose

[0061] GlcA: glucuronic acid

[0062] kDa: kilodalton

[0063] Man: Mannose

[0064] Manp: Pyranomannosyl

[0065] Xyl: Xylose

[0066] NMR: Nuclear Magnetic Resonance Spectroscopy

[0067] BSA: Bovine serum albumin. Attached Figure Description

[0068] Figure 1 For Chinese trichomoniasis polysaccharide in TSKgel G4000PW XL HPGPC elution chromatogram on column;

[0069] Figure 2 This is a standard curve of total sugar content.

[0070] Figure 3 This is a standard curve of protein content.

[0071] Figure 4 A graph showing the D-galacturonic acid content;

[0072] Figure 5 For TSKgel G4000PW XL Standard curve for molecular weight determination using chromatographic columns;

[0073] Figure 6 Fourier transform infrared spectra of Trichophyton spp. polysaccharide HSWP and HSWP-1 from China;

[0074] Figure 7 Fourier transform infrared spectra of polysaccharide components HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d from *Hymenochrysis pilosula*.

[0075] Figure 8 For HSWP-1a 1 H NMR spectrum;

[0076] Figure 9 For HSWP-1a 13 C NMR spectrum;

[0077] Figure 10 The HSQC NMR spectrum of HSWP-1a;

[0078] Figure 11 The HMBC NMR spectrum of HSWP-1a;

[0079] Figure 12 For HSWP-1a 1 H- 1 HCOSY NMR spectrum;

[0080] Figure 13 NOESY NMR spectrum of HSWP-1a;

[0081] Figure 14 C NMR spectrum of HSWP-1b; 1 H NMR spectrum of HSWP-1b;

[0082] Figure 15 HMBC NMR spectrum of HSWP-1b; 13 C NMR spectrum of HSWP-1b;

[0083] Figure 16 HSQC NMR spectrum of HSWP-1b;

[0084] Figure 17 HMBC NMR spectrum of HSWP-1b;

[0085] Figure 18 H NMR spectrum of HSWP-1b; 1 H- 1 H COSY NMR spectrum of HSWP-1b;

[0086] Figure 19 NOESY NMR spectrum of HSWP-1b;

[0087] Figure 20 C NMR spectrum of HSWP-1c; 1 H NMR spectrum of HSWP-1c;

[0088] Figure 21 C NMR spectrum of HSWP-1c; 13 C NMR spectrum of HSWP-1c;

[0089] Figure 22 HSQC NMR spectrum of HSWP-1c;

[0090] Figure 23 HMBC NMR spectrum of HSWP-1c;

[0091] Figure 24 H NMR spectrum of HSWP-1c; 1 H- 1 H COSY NMR spectrum of HSWP-1c;

[0092] Figure 25 NOESY NMR spectrum of HSWP-1c;

[0093] Figure 26 C NMR spectrum of HSWP-1d; 1 H NMR spectrum of HSWP-1d;

[0094] Figure 27 C NMR spectrum of HSWP-1d; 13 C NMR spectrum of HSWP-1d;

[0095] Figure 28HSQC NMR spectrum of HSWP-1d;

[0096] Figure 29 HMBC NMR spectrum of HSWP-1d;

[0097] Figure 30 HSWP-1d 1 H- 1 H COSY NMR spectrum of HSWP-1d;

[0098] Figure 31 NOESY NMR spectrum of HSWP-1d;

[0099] Figure 32 Cell morphology and mRNA expression of E-cadherin and vimentin under HIBEC-EMT model;

[0100] Figure 33 Cell viability values of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d at low, medium and high doses;

[0101] Figure 34 Cell morphology changes of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d at low, medium and high doses; Figure 35 Effect of Chinese Trichosporon polysaccharide components on mRNA expression of E-cadherin and vimentin in TGF-β1-induced HIBECs;

[0102] Figure 36 Effect of Chinese Trichosporon polysaccharide components on protein expression of HIBEC-EMT biomarkers;

[0103] Figure 37 Gray value analysis of Chinese Trichosporon polysaccharide components HSWP-1a (A), HSWP-1b (B), HSWP-1c (C) and HSWP-1d (D) on protein expression of HIBEC-EMT biomarkers E-cadherin, vimentin, collagen I and ZO-1;

[0104] Figure 38 Effect of Chinese Trichosporon polysaccharide components on protein expression of Smad 2 / 3, p-Smad2 / 3 and Smad 4 in TGF-β1-induced HIBEC-EMT;

[0105] Figure 39 Gray value analysis of Chinese Trichosporon polysaccharide components on protein expression of Smad 2 / 3, p-Smad2 / 3 and Smad 4 in TGF-β1-induced HIBEC-EMT. DETAILED DESCRIPTION

[0106] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. In addition, if not specifically stated, all reagents used in the following examples are commercially available or can be synthesized according to the methods described herein or known methods, and the reaction conditions not listed are also readily available to those skilled in the art.

[0107] Example 1 Extraction and separation and purification of Chinese Hirsutella polysaccharide components

[0108] (1) Extraction of Chinese Hirsutella polysaccharide (HSWP)

[0109] Take 300.0 g of Chinese Hirsutella mycelium powder, soak in 3 L of ultrapure water overnight (solid-liquid ratio 10:1), heat to boiling, cook for 1 h, centrifuge at 4000 rpm for 20 min, take the supernatant and set aside, collect the residue and continue to extract three times. Combine the supernatants obtained by three times of extraction and concentrate on an electric hot plate, concentrate to about one tenth of the original volume, cool to room temperature, and add anhydrous ethanol to the concentrated solution while stirring to make the ethanol concentration reach 80%, and stand at room temperature for 12 h. Centrifuge at 4000 rpm for 20 min, discard the supernatant and collect the precipitate, add a small amount of water to evaporate the residual ethanol, and after alcohol removal, store in a -80°C freezer overnight, then freeze-dry to obtain Chinese Hirsutella polysaccharide HSWP, with a yield of 39.6%.

[0110] (2) Separation and purification of Chinese Hirsutella polysaccharide (HSWP)

[0111] The well-soaked DEAE-cellulose filler is washed with pure water until neutral, vacuumed to remove air bubbles in the filler, and loaded into a glass column (12 cm x 60 cm). Before use, the DEAE-cellulose anion exchange chromatography column (12 cm x 60 cm) is equilibrated with two column volumes of saline and distilled water, respectively. The extracted Chinese Hirsutella polysaccharide HSWP is dissolved in distilled water to prepare a 10% polysaccharide sample solution, centrifuged (10000 rpm, 10 min) to remove insoluble substances, and loaded into the equilibrated DEAE-cellulose anion column for chromatography. The column is sealed with water, and the polysaccharide sample is incubated in the ion exchange column for 12 h to allow sufficient contact with the DEAE-cellulose anion exchange column. The elution flow rate is set at 5 mL / min, and the eluate is collected for two column volumes of distilled water elution. After heating on an electric hot plate to concentrate to one eighth of the original volume, the eluate is transferred to a beaker and further concentrated to 100-150 mL in a water bath at 80°C, then hung on the wall and stored in a -80°C freezer for 24 h, and then dried using a freeze-drying machine to obtain polysaccharide sample HSWP-1, with a yield of 34.80%.

[0112] (3) Fractionation of HSWP-1 by ethanol gradient precipitation

[0113] The polysaccharide HSWP-1 was dissolved in distilled water to prepare a 10% polysaccharide sample solution, which was stirred to dissolve. Anhydrous ethanol was added to the sample solution and mixed evenly. The ethanol concentration was measured to be 30%. The solution was left to stand overnight, centrifuged, and a white precipitate was obtained. The white precipitate was placed in a -80°C freezer overnight and freeze-dried in a freeze dryer to obtain a white and slightly yellow solid HSWP-1a. Anhydrous ethanol was continuously added to the supernatant, and the ethanol concentration was measured to be 50%. The solution was left to stand overnight in a 4°C freezer, centrifuged, and a precipitate was obtained. The supernatant was collected for later use, and the precipitate was placed in a -80°C freezer overnight and freeze-dried to obtain a uniform white flaky solid HSWP-1b. Anhydrous ethanol was added to the collected supernatant, and the ethanol concentration was measured to be 70%. The solution was left to stand overnight in a 4°C freezer, centrifuged, and a yellowish precipitate was obtained. The supernatant was collected for later use. The precipitate was placed in a -80°C freezer overnight and freeze-dried to obtain a yellowish solid powder HSWP-1c. Anhydrous ethanol was added to the supernatant, and the ethanol concentration was measured to be 90%. The solution was left to stand overnight in a 4°C freezer, centrifuged, and a yellowish-brown precipitate was obtained. The precipitate was placed in a -80°C freezer overnight and freeze-dried to obtain a dark brown solid powder HSWP-1d. The yields of HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d were 33.27%, 3.71%, 9.44%, and 9.28%, respectively.

[0114] As shown in FIG. 1, HSWP-1b, HSWP-1c, and HSWP-1d showed single symmetrical elution peaks on the elution profile of HPGPC, with retention times of about 16.63, 17.74, and 18.30 min, respectively, indicating uniformity. HSWP-1a showed continuous double elution peaks within a retention time of 12.98-14.71 min, indicating heterogeneity. Figure 1 According to a series of dextran calibration molecular weight standard curves, the average molecular weights of HSWP-1b, HSWP-1c, and HSWP-1d polysaccharides were 8.57, 2.18, and 1.09 kDa, respectively.

[0115] Example 2 Analysis of the physicochemical properties of Chinese Hirsutum polysaccharide components

[0116] 1 Determination of total sugar content of Chinese Hirsutum polysaccharide components

[0117] 1.1 Method

[0118] Phenol-sulfuric acid method was selected to determine the total polysaccharide content: sugar is dehydrated to form sugar acid compounds under the action of concentrated sulfuric acid, which is condensed with phenol to form orange red compounds, and the color depth is proportional to the sugar content. The method has small interference and the color produced is stable within 160 min.

[0119] (1) Preparation of experimental reagents

[0120] 6% phenol solution preparation: weigh phenol 37.50 g into a 250 mL brown volumetric flask (the outer layer can be wrapped with tin paper), add 100 mL of pure water, fully dissolve, and then add to the calibration line to obtain a 6% phenol solution, which is stored at 4°C in a refrigerator in the dark.

[0121] Preparation of sugar standard solution: weigh D-glucose 0.10 g into a 100 mL volumetric flask, take out 10 mL into a 100 mL volumetric flask, and dilute with water to 0.1 mg / mL, mix well and reserve.

[0122] (2) Standard curve for determination of total sugar content

[0123] Use a pipette to take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of 0.1 mg / mL standard solution into a glass test tube, and add distilled water to 1.0 mL. Repeat three times, add 0.5 mL of 6% phenol and 2.5 mL of concentrated sulfuric acid to the test tube, shake quickly and uniformly, cool to room temperature, and measure the absorbance A at λ = 490 nm. Take the absorbance A as the vertical coordinate and the sugar content C (mg) as the horizontal coordinate to obtain the standard curve A = kC. Determination of total sugar content in HSWP-1, HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d polysaccharide component: weigh HSWP-1, HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d to prepare polysaccharide sample solution with a concentration of about 0.1 mg / mL, add 0.5 mL of 6% phenol reagent and 2.5 mL of concentrated sulfuric acid, and follow the same operation as the standard curve preparation method to determine the absorbance A. According to the standard solution, calculate the total polysaccharide content in the sample.

[0124] 1.2 Results

[0125] The standard curve of total sugar content is plotted with glucose content C (mg / mL) as the horizontal coordinate and its absorbance value A at 490 nm as the vertical coordinate. The obtained standard curve is shown in Figure 2

[0126] The regression equation is A 490nm = 10.154C + 0.0084, R 2 = 0.9985

[0127] ​According to the standard curve, the total sugar content of HSWP-1, HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d of Chinese Hirsutella polysaccharide is 68.26%, 97.35%, 95.07%, 90.63% and 58.90%, respectively.

[0128] 2 Protein content determination of Chinese Hirsutella polysaccharide components

[0129] 2.1 Determination method

[0130] Bradford method (Coomassie brilliant blue) is used to determine the protein content: Coomassie brilliant blue G-250 is red in free state, and the maximum light absorption is at 488 nm. After binding with protein, it becomes blue, and the maximum absorption changes from 488 nm to 595 nm. The binding of protein and Coomassie brilliant blue reaches equilibrium in about 2 min, and remains stable within 1 h.

[0131] (1) Preparation of experimental solution

[0132] Preparation of Coomassie brilliant blue reagent: weigh 20.00 mg of bovine serum albumin (BSA), prepare a stock solution of 5 mg / mL bovine serum albumin, and dilute with water to 50 μg / mL for standby.

[0133] (2) Preparation of standard curve for protein content determination

[0134] Use a pipette to take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of 50 μg / mL protein standard solution into a glass test tube, add distilled water to 1.0 mL, repeat three times, add 4.0 mL of Coomassie brilliant blue reagent to the test tube, shake quickly and uniformly, stand for 5 min, and measure the absorbance A at λ = 595 nm. Take the absorbance A as the vertical coordinate and the bovine serum albumin content C (μg) as the horizontal coordinate to obtain the standard curve A = kC.

[0135] Determination of protein content in sample: take 1 mL of sample solution with a concentration of about 0.1 g / L, prepare it according to the standard curve preparation method, measure the absorbance, and calculate the protein content in the sample according to the standard curve and sample concentration.

[0136] 2.2 Results

[0137] The standard curve for protein content determination is drawn with the concentration C (μg / mL) of bovine serum albumin (BSA) as the horizontal coordinate and its ultraviolet absorbance value A at 562 nm as the vertical coordinate. The obtained protein standard curve is shown in Figure 3 The regression equation is A 562nm = 0.0169C + 0.0113, R 2 = 0.9989

[0138] The protein content determination results show that the protein contents of Chinese Hirsutella polysaccharides HSWP-1, HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d are 0.37%, 1.16%, 0.24%, 1.36% and 6.88%, respectively.

[0139] 3.1 Determination method

[0140] The uronic acid content of Chinese Hirsutella polysaccharide is determined by m-hydroxydiphenyl method, and the specific steps are as follows:

[0141] (1) Preparation of experimental solution

[0142] 0.5% (W / V) sodium hydroxide solution preparation: weigh 0.50 g of NaOH solid particles into a 100 mL volumetric flask, add 40 mL of distilled water, dissolve thoroughly, add pure water to the mark, mix uniformly, and reserve. Preparation of 0.3% m-hydroxydiphenyl / 0.5% (W / V) sodium hydroxide: weigh 30.0 mg of m-hydroxydiphenyl, dissolve in 10 mL of 0.5% sodium hydroxide, and store in the dark in the ice cabinet.

[0143] Preparation of saturated potassium hydroxide: weigh 5.00 g of potassium hydroxide, add 2 mL of distilled water, stir thoroughly to dissolve, and the supernatant is saturated potassium hydroxide.

[0144] Preparation of 4M sulfamic acid solution (pH 2.5): weigh 7.80 g of sulfamic acid, first add 10 mL of distilled water, drop saturated potassium hydroxide, stir or shake to dissolve, cool to room temperature. Continue to add saturated potassium hydroxide to pH 2.5, and add water to 10 mL. Store at room temperature.

[0145] Preparation of 1 mg / mL D-galacturonic acid stock solution: dissolve 10 mg of D-galacturonic acid in 10 mL of distilled water. The remaining stock solution can be stored frozen. When used, take an appropriate amount of 1 mg / mL D-galacturonic acid stock solution, dilute with distilled water to 100 μg / mL. The amount of standard solution used in each test is less than 5 mL.

[0146] (2) Preparation of uronic acid content determination standard solution

[0147] Preparation of D-galacturonic acid standard solution: Take 0, 0.05, 0.10, 0.20, 0.30, 0.40 of D-galacturonic acid standard solution respectively, add distilled water to 0.40 mL, continue to add 40 μL of sulfamic acid (pH 2.5) to each tube solution, mix well, and then add 2.5 mL of concentrated sulfuric acid to each test tube. Mix each tube solution well, heat in a boiling water bath for 20 min, quickly cool in a cold water bath to room temperature, then add 40 μL of 0.3% m-hydroxybenzene 0.5% sodium hydroxide to each tube, mix well. Place at room temperature for 15 min to 15 min-60 min. At λ = 525 nm, the A value, with D-galacturonic acid content (μg) as the abscissa and the light absorption value A as the ordinate, a standard curve is drawn.

[0148] (3) Determination of uronic acid content in Chinese Hirsutum polysaccharide sample

[0149] Take 1 mL of sample solution with a concentration of about 0.1 g / L, repeat three times, and measure the absorbance value A according to the standard curve preparation method. Calculate the uronic acid content in the sample according to the standard curve and sample concentration.

[0150] 3.2 Results

[0151] A standard curve for protein content determination is drawn with the concentration C (mg / mL) of D-galacturonic acid as the abscissa and its ultraviolet absorption value A at 525 nm as the ordinate. The obtained uronic acid standard curve is shown in Figure 4 The regression equation is A 525nm = 15.402C - 0.0078, R 2 = 0.9979

[0152] The results of protein content determination show that the uronic acid contents of Chinese Hirsutum polysaccharides HSWP-1, HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d are 0.67%, 0.66%, 0.60%, 0.74%, and 0.65%, respectively.

[0153] 4.1 Method

[0154] (1) Partial acid hydrolysis of polysaccharide

[0155] Weigh 5.0 mg of polysaccharide sample into a brown vial for partial acid hydrolysis, add 1 mL of 2M trifluoroacetic acid, hydrolyze in an oven at 120°C for 120 min, remove and cool to room temperature, transfer the cooled reaction solution to an evaporating dish, evaporate the acid at 70°C water bath, and add 0.5 mL of anhydrous ethanol, which can be added twice or three times to completely remove the trifluoroacetic acid.

[0156] (2) PMP pre-column derivatization reaction

[0157] Add 0.5 mL each of 0.5 M PMP and 0.3 M NaOH solution to the hydrolyzed sample after removing trifluoroacetic acid. After complete dissolution, incubate in a water bath at 70 °C for 30 min. Then, add 0.5 mL of 0.3 M HCl to the PMP-derived solution, mix well, and then add 1 mL of trichloroethane. Extract three times, collect the aqueous layer, and filter it through a 0.22 μm organic membrane for later use.

[0158] (3) High performance liquid chromatography detection

[0159] The filtered sample solution was analyzed using high performance liquid chromatography (HPLC) with a C-18 column (Kromasil 100-5C18 column). The mobile phase was 18% acetonitrile and 82% 0.1M phosphate buffer (pH 7.31). The injection volume was 2 μL and the detection wavelength was 245 nm.

[0160] 4.2 Results

[0161] The monosaccharide composition of the polysaccharides HSWP-1, HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d from *Trichophyton mentagrophytes* is shown in Table 1. The crude polysaccharide HSWP-1 consists of mannose (5.90%), rhamnose (1.27%), glucose (90.39%), galactose (2.00%), and arabinose (0.42%). The main components of HSWP-1a and HSWP-1b are glucose (90.12% and 85.72%, respectively), along with certain amounts of mannose, rhamnose, and galacturonic acid. HSWP-1c and HSWP-1c are mainly composed of glucose (66.71% and 78.23%), mannose (15.21% and 10.44%), a portion of galactose (7.32% and 4.65%), rhamnose (4.34% and 2.90%), and galacturonic acid (3.68% and 2.08%).

[0162] In summary, it can be concluded that the four polysaccharide components obtained from the separation and purification of the Chinese mitochondrial polysaccharide HSWP-1 mainly consist of mannose, glucose, and galactose.

[0163] Table 1. Total sugar, uronic acid, and protein content, molecular weight distribution, specific rotation, and monosaccharide composition of polysaccharides from *Trichophyton spp.* in China.

[0164]

[0165] 5. Determination of the molecular weight distribution range of polysaccharide components in *Hymenopsporum tobira* from China

[0166] 5.1 Determination Method

[0167] High performance liquid permeation chromatography was used to determine the homogeneity and molecular weight distribution range of polysaccharides.

[0168] (1) Preparation of mobile phase and sample solution

[0169] 0.15M NaCl solution: weigh 8.77g of sodium chloride, add to a 1000ml volumetric flask, pour in 200ml of ultrapure water, shake until fully dissolved, dilute to the mark, filter and exhaust, ready for use.

[0170] (2) Preparation of standard curve solution for molecular weight determination

[0171] Weigh 5mg of polysaccharide molecular weight standards of 180kDa, 4660kDa, 12.6kDa, 33.719kDa, 70.8kDa, 153kDa, and 500kDa, respectively, dissolve in 1ml of pure water to prepare a 5mg / ml solution, filter, and load into a high performance liquid chromatography system (LC-10ATvp pump, RID-10A detector), use Tsk-gel G4000PW XL column, mobile phase is 0.15M NaCl, flow rate is 0.6ml / min, column temperature is 40℃. With the elution of the mobile phase, the large molecular polysaccharide peaks first, and the small molecule peaks later. The molecular weight Mw of the standard, the molecular weight logarithm lgMw, and the retention time (RT) are plotted as Kav=(Ve-Vo) / (Vt-Vo) as the ordinate (external water volume, lgMw as the abscissa to draw the standard curve.

[0172] (3) Determination of molecular weight and uniformity of Chinese Hirsutella polysaccharide sample

[0173] Prepare a 5mg / mL polysaccharide solution, and process the same as the standard, and detect under the same liquid phase conditions. The measured retention time is substituted into the standard curve equation to calculate the molecular weight.

[0174] 5.2 Results

[0175] HPGPC determines the molecular weight and uniformity of Chinese Hirsutella polysaccharide, and the large molecules peak first and the small molecules peak later during elution. The standard curve of molecular weight distribution is plotted with the logarithm of molecular weight lgMw as the ordinate and the retention time RT as the abscissa, with different molecular weight Mw of the sugar standard as shown in Table 2. Figure 1 :

[0176] Kav=(Ve-Vo) / (Vt-Vo), the standard curve is plotted with Kav as the ordinate and lgMw as the abscissa, and the regression equation Kav=-0.2025C+1.5144, R 2 =0.9630.

[0177] Molecular weight and uniformity of HSWP-1 and HSWP-1a in TSKgel G4000PWXL The HSWP-1a showed a continuous double elution peak in the elution profile of HPGPC with the retention time of 12.98-14.71 min, indicating that HSWP-1a was not a homogeneous polysaccharide component, and the molecular weight range was 104.87-922.59 kDa (average molecular weight between two peaks). According to the calibration curve of a series of dextran molecular weight standards, the average molecular weights of HSWP-1b, HSWP-1c and HSWP-1d were 8.57, 2.18 and 1.09 kDa, respectively.

[0178] Table 2 Standard molecular weight, lgMw and retention time

[0179]

[0180] 6 Specific optical rotation determination of HSWP

[0181] 6.1 Determination method

[0182] Specific optical rotation is a physical constant of organic substances, which is used to indicate the optical rotation of the substance. 4.0 mg (±0.1) of each polysaccharide sample HSWP, HSWP-1, HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d was dissolved in 20 mL of deionized water to obtain a polysaccharide solution with a concentration of 0.020 g / 100 mL. 1 mL was taken each time and placed in an optical tube, and the bubbles were removed. The measurement was repeated 6 times to take the average value, and the optical rotation was detected by an automatic optical instrument (the length of the optical tube was 0.5000 dm, the temperature was maintained at 25°C ± 0.5°C, and the detection wavelength was 589.44 nm.

[0183] 6.2 Results

[0184] Optical rotation can reflect the structural characteristics of polysaccharides. Generally, a positive specific optical rotation represents D-glycoside, and a negative value represents L-glycoside. As shown in Table 1, the specific optical rotations of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d were 0.00°, +3.36°, +37.70° and +16.68° (C=0.02 g / mL, H2O), respectively. This high positive value indicates that D-type sugar residues dominate in them. The specific optical rotation was calculated as follows:

[0185]

[0186] a is optical rotation; T is temperature; l is the wavelength of the light source; L is the length of the polarimeter tube; C is the concentration of the polysaccharide (g / mL).

[0187] Example 3 Structural characterization of the components of the polysaccharides from P. sinensis

[0188] 1 Fourier Transform Infrared Spectroscopy (FT-IR) analysis

[0189] 1.1 Method

[0190] The Fourier Transform Infrared (FT-IR) spectra of the polysaccharide samples powders were recorded directly in the range of 4000-500 cm -1 using a Thermo IS50 Fourier Transform Infrared Spectrometer with a DTGS detector.

[0191] 1.2 Results

[0192] The strong absorption bands at 3391.29 cm -1 (HSWP), 3349.96 cm -1 (HSWP-1), 3438.10 cm -1 (HSWP-1a), 3424.33 cm -1 (HSWP-1b), 3432.59 cm -1 (HSWP-1c), 3421.58 cm -1 (HSWP-1d) reflect the stretching vibration of O-H bond on the sugar ring; the peaks at about 2928.54 cm -1 (HSWP), 2928.54 cm -1 (HSWP-1), 2923.03 cm -1 , 2848.66 cm -1 (HSWP-1a), 2920.28 cm -1 , 2854.17 cm -1 (HSWP-1b), 2923.03 cm -1 , 2845.91 cm -1 (HSWP-1c), 2923.03 cm -1 , 2851.42 cm -1 (HSWP-1d) can reflect the stretching vibration of C-H and C-H2 boundary on the sugar ring; the absorption bands at 1650.50 cm -1 (HSWP), 1647.74 cm -1 (HSWP-1), 1633.97 cm -1 (HSWP-1a), 1636.73 cm -1 (HSWP-1b), 1639.48 cm -1(HSWP-1c), 1639.48 cm -1 The absorption peak at (HSWP-1d) represents the stretching vibration of C=0 boundary; the stretching vibration peaks of pyranose ring are 1030.76 cm -1 (HSWP), 1030.76 cm -1 (HSWP-1), 1039.02 cm -1 (HSWP-1a), 1033.51 cm -1 (HSWP-1b), 1025.25 cm -1 (HSWP-1c), 1025.25 cm -1 (HSWP-1d). While 854.43 cm -1 (HSWP), 854.49 cm -1 (HSWP-1), 851.72 cm -1 (HSWP-1a), 851.23 cm -1 The absorption peak at (HSWP-1b) indicates the presence of α-configuration monosaccharide residues, and based on the above analysis, it can be known that there are α-pyranose monosaccharide residues in Chinese Hirsutum polysaccharides HSWP, HSWP-1, HSWP-1a and HSWP-1b.

[0193] 2 Nuclear Magnetic Resonance Spectroscopy (NMR) analysis

[0194] 2.1 Method

[0195] The polysaccharide sample (50 mg) was dissolved in 1.0 mL of D2O for NMR analysis, and acetone was used as an internal standard. The NMR spectra were measured 1 H NMR, 13 CNMR, 1 H- 1 H COSY, HSQC, NOESY and HMBC spectra, and the NMR spectra were recorded on a Varian Mercury 600 MHz nuclear magnetic resonance spectrometer.

[0196] 2.2 Results

[0197] (1) Analysis of NMR results of HSWP-1a

[0198] HSWP-1a in 1D( 1 H, 13 C NMR) and 2D( 1 H- 1 H COSY, 13 C- 1 H HMBC, 1 H- 1 H NOESY and 13 C- 1HHSQC) nuclear magnetic resonance signals such as Figures 8-13 As shown, its 1 H and 13 The 10⁻⁶ C NMR chemical shifts are shown in Table 3. Combined with monosaccharide composition and FT-IR analysis, the major chemical shifts were determined. 1 H- 13 C signal. Based on two-dimensional spectra from HSQC, HMBC, COSY, and NOESY, combined with literature data, the polysaccharide components were analyzed. 1 H and 13 The nuclear magnetic resonance (NMR) spectrum was used to characterize the NMR signal.

[0199] Combined with HSQC ( Figure 10 ), HMBC ( Figure 11 ), 1 H- 1 H COSY( Figure 12 ) and NOESY ( Figure 13 Two-dimensional spectra, giving HSWP-1a in 1H NMR ( Figure 8 ) and 13C NMR ( Figure 9 The nuclear magnetic resonance signal in the spectrum. For example... Figure 8 and Figure 9 As shown, HSWP-1a exhibits anodic carbon and anodic proton signal peaks at δ99.67 / 5.31, δ99.73 / 5.26, and δ98.59 / 4.88 ppm, which are attributed to 1,4-linked-α-D-Glcp(A), 1,4,6-linked-α-D-Glcp(B), and 1-linked-α-D-Glcp(C) residues, respectively. Meanwhile, the carbon signal peaks at δ76.75 and 76.88 ppm may be C-4 of the 1,4-linked-α-D-Glcp residue and the 1,4,6-linked-D-Glcp residue, respectively, and the carbon signal peak at δ69.29 ppm is assigned to C-6 of the 1,4,6-linked-α-D-Glcp residue. Figure 11 As shown, the correlation shifts of 5.31 / 76.75ppm (AH-1 / AC-4) and 3.51 / 99.67ppm (AH-4 / AC-1) indicate the presence of 1,4-linked-α-D-Glcp residues in the HSWP-1a carbohydrate chain. The correlation shifts of 5.31 / 76.85ppm (AH-1 / BC-4) and 4.88 / 69.29ppm (CH-1 / BC-6) indicate that the 1,4-glcp backbone has a terminal 1-linked-α-D-Glcp as a side chain at O-6.

[0200] (2) Analysis of NMR results of HSWP-1b

[0201] HSWP-1b in 1D ( 1 H, 13 C NMR) and 2D ( 1 H- 1 H COSY、 13 C- 1 H HMBC, 1 H- 1 H NOESY and 13 C- 1 HHSQC) nuclear magnetic resonance signals such as Figures 14-19 As shown, its 1 H and 13 The 10⁻⁶ C NMR chemical shifts are shown in Table 3. Combined with monosaccharide composition and FT-IR analysis, the major chemical shifts were determined. 1 H- 13 C signal. Based on two-dimensional spectra from HSQC, HMBC, COSY, and NOESY, combined with literature data, the polysaccharide components were analyzed. 1 H and 13 The nuclear magnetic resonance (NMR) spectrum was used to characterize the NMR signal.

[0202] like Figure 14 and Figure 15 As shown, the anomeric carbons at δ 95.58 / 4.56, 99.63 / 5.32, 99.74 / 5.27, and 98.58 / 4.89 ppm, along with the anomeric proton signal peaks, indicate that HSWP-1b is mainly composed of four sugar residues: 4,6-linked-β-D-Manp (A), 1,4-linked-α-D-Glcp (B), 1,4,6-linked-α-D-Glcp (C), and 1-1... The inked-α-D-Glcp(D) signal is shown, with δ76.53 ppm representing the signal peak at C-4 of the 1,4-linked-α-D-Glcp residues; δ76.77 and 69.21 ppm representing the signal peaks at C-4 and C-6 of the 1,4,6-linked-α-D-Glcp residues; and δ76.67 and 66.12 ppm representing the signal peaks at C-4 and C-6 of the 4,6-linked-α-D-Manp residues. Figure 17The HMBC spectrum shows that the correlation shifts at 3.88 / 66.12ppm (A H-4 / A C-6) and 3.59 / 66.12ppm (A H-6 / A C-4) indicate that the carbohydrate chain is composed of 4,6-linked-β-D-Manp residues. The correlation shifts of 5.32 / 76.53ppm (B H-1 / B C-4), 3.58 / 99.63ppm (C H-4 / B C-1), 5.32 / 76.80ppm (B H-1 / C C-4), 4.89 / 69.21ppm (DH H-1 / C-6), and 4.89 / 73.26ppm (DH H-1 / B C-6) indicate that the main chain of HSWP-1b consists of 1,4-linked-α-D-Glcp, 1,4,6-linked α-D-Glcp, and 4,6-linked β-D-Manp residues, and its side chains are formed by a single 1-linked-α-D-Glcp residue.

[0203] (3) Analysis of NMR results of HSWP-1c

[0204] HSWP-1c in 1D ( 1 H, 13 C NMR) and 2D ( 1 H- 1 H COSY、 13 C- 1 H HMBC, 1 H- 1 HNOESY and 13 C- 1 HHSQC) nuclear magnetic resonance signals such as Figures 20-25 As shown, its 1 H and 13 The 10⁻⁶ C NMR chemical shifts are shown in Table 3. Combined with monosaccharide composition and FT-IR analysis, the major chemical shifts were determined. 1 H- 13 C signal. Based on two-dimensional spectra from HSQC, HMBC, COSY, and NOESY, combined with literature data, the polysaccharide components were analyzed. 1 H and 13 The nuclear magnetic resonance (NMR) spectrum was used to characterize the NMR signal.

[0205] like Figure 20 and Figure 21As shown, HSWP-1c exhibited six anomeric carbon and proton signal peaks at δ 104.71 / 5.10, 107.83 / 5.01, 95.68 / 4.56, 99.62 / 5.32, 99.73 / 5.26, 98.51 / 4.88 ppm, which were assigned to 1,2,6-linked α-D-Galp (A), 1,3,4,6-linked α-D-Glcp (B), 4,6-linked β-D-Manp (C), 1,4-linked α-D-Glcp (D), 1,4,6-linked α-D-Glcp (E) and 1-linked α-D-Glcp (F) residues, respectively. In addition, the carbon signal peaks at δ 76.81 and 69.80 ppm were C-2 and C-6 of 1,2,6-linked-α-D-Galp (A) residue; δ 76.79, 82.64 and 62.78 ppm were C-3, C-4 and C-6 of 1,3,4,6-linked α-D-Glcp (B) residue; δ 76.84 and 66.44 ppm were C-4 and C-6 of 4,6-linked-β-D-Manp (C) residue; δ 76.75 ppm was C-4 of 1,4-linked-α-D-Glcp (D) residue; δ 76.76 and 69.31 ppm were C-4 and C-6 of 1,4,6-linked α-D-Glcp (E) residue. According to the Figure 20 and Figure 21 13 C- and 1 H-NMR spectra, Figure 24 1 H- 1 H COSY spectra and Figure 21 1 H- 13 C HSQC spectra, the other carbon / proton chemical shifts of HSWP-1c sugar residues were assigned and listed in Table 3.

[0206] From the HMBC spectrum, Figure 23 ​​​It can be seen that 5.31 / 76.51(DH-1 / DC-4), 3.57 / 99.62(EH-4 / DC-1), 5.32 / 76.81(DH-1 / BC-2), 3.56 / 99.72(DH-2 / EC-1), 5.10 / 76.75(AH-1 / DC-4), 3.56 / 60.57(EH-2 / FC-6), 3.57 / 69.80(BH-4 / AC-6), and 3.57 / 71.67(BH-4 / EC-6) are... The relevant shifts at C-2 indicate that the main chain of HSWP-1c consists of 1,4-linked-α-D-Glcp, 1,4,6-linked-α-D-Glcp, 1,2,6-linked-α-D-Galp, and 4,6-linked-β-D-Manp residues. Its side chains are formed by 1-linked-α-D-Glcp residues.

[0207] (4) Interpretation of NMR results of HSWP-1d

[0208] HSWP-1d in 1D ( 1 H, 13 C NMR) and 2D ( 1 H- 1 H COSY、 13 C- 1 H HMBC, 1 H- 1 H NOESY and 13 C- 1 HHSQC) nuclear magnetic resonance signals such as Figures 26-31 As shown, its 1 H and 13 The 10⁻⁶ C NMR chemical shifts are shown in Table 3. Combined with monosaccharide composition and FT-IR analysis, the major chemical shifts were determined. 1 H- 13 C signal. Based on two-dimensional spectra from HSQC, HMBC, COSY, and NOESY, combined with literature data, the polysaccharide components were analyzed. 1 H and 13 The nuclear magnetic resonance (NMR) spectrum was used to characterize the NMR signal.

[0209] like Figure 26 and Figure 27As shown, HSWP-1d exhibited five anomeric carbon / proton signal peaks at δ 95.75 / 4.56, 99.62 / 5.31, 99.70 / 5.26, 98.59 / 4.88, 91.84 / 5.14 ppm, which were assigned to 4,6-linked-β-D-Manp (A), 1,4-linked-α-D-Glcp (B), 1,4,6-linked-α-D-Glcp (C), 1-linked-α-D-Glcp (D) and α-D-reducding-Glcp (D) residues, respectively. The signals at δ 76.69 and 66.10 ppm were C-4 and C-6 of 4,6-linked-β-D-Manp (A) residues; δ 76.81 ppm was C-4 of 1,4-linked-α-D-Glcp (B) residues; δ 76.67 and 69.31 ppm were C-4 and C-6 of 1,4,6-linked-α-D-Glcp (C) residues. From Figure 29 From the HBMC spectrum of HSWP-1d, the relevant shifts of 3.87 / 99.62 (A H-4 / B C-1), 3.87 / 98.59 (A H-4 / D C-1), 5.31 / 76.67 (B H-1 / C C-4), 3.57 / 99.31 (C H-4 / B C-1), 3.57 / 98.59 (C H-4 / D C-1), 5.14 / 69.31 (E H-1 / D C-4), 5.14 / 69.31 (E H-1 / D C-6), 4.88 / 72.71 (D H-1 / E C-3), 4.88 / 69.31 (D H-1 / C-6) indicated that the main chain of HSWP-1d was composed of 1,4-linked-α-D-Glcp, 1,4,6-linked-α-D-Glcp and 4,6-linked-β-D-Manp residues. Its side chain was connected by 1-linked-α-D-Glcp on O-6 of 1,4-linked-α-D-Glcp residues.

[0210] Table 3. HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d 1 H and 13 Assignment of C chemical shifts

[0211]

[0212] Experimental Example 1 Effect of Chinese Hirsutia polysaccharide components on HIBEC-EMT cell survival rate and morphology

[0213] 1 Experimental method

[0214] 1.1 HIBEC cell culture

[0215] HIBECs were purchased from Zhijiao Xin Zhou (Shanghai), and the complete medium ZQ-1315 was used. The culture temperature was 37°C, and the carbon dioxide concentration was 5%.

[0216] (1) Cell recovery:

[0217] Prepare 6 mL of complete medium in a T-25 culture flask in a sterile area, take the cryopreservation tube from the liquid nitrogen tank, quickly melt it in a 37°C water bath (within 1 min), take it out of the water bath, spray 75% ethanol on the surface, transfer it to the clean bench, remove the cryopreservation solution with a pipette, add 1 mL of medium, mix well by blowing, add it to the prepared culture flask, mix gently, and place it in a 37°C, 5% CO2 incubator.

[0218] (2) Cell passage:

[0219] Under an inverted microscope, observe the cells when they are completely adherent and cover the entire culture flask, about 85%, spray 75% alcohol on the surface, open the culture flask in the clean bench, aspirate the medium, wash with PBS, add 1.0 mL of trypsin (0.05% containing EDTA) and digest for about 10-15 S, observe a thin mist, discard the trypsin, add 2.0 mL of medium to stop the digestion. Gently blow the single cell suspension with a pipette, and according to the ratio of 1:3, aspirate the single cell suspension and add it to the culture flask, add fresh medium to 6 mL, and place it in the incubator for culture.

[0220] (3) Cell cryopreservation

[0221] After cell recovery, a number of cells were cryopreserved for use during cell passage. Take the HIBECs cells in the logarithmic growth phase, discard the original culture medium, wash the remaining culture medium with 2.0 mL of PBS, digest with 1 mL of trypsin (Trypin-EDTA) for 15 S, discard the trypsin, add 2 mL of complete medium, and gently blow the single cell suspension with a pipette. Count using a hemocytometer. Add the cell suspension to the cryopreservation tube, about 1×10 6 cells per tube, centrifuge the cells (1000 rpm, 5 min), discard the supernatant, and resuspend the cells with 1 mL of cell cryopreservation solution (10% DMSO, 70% basic medium, 20% fetal bovine serum). Place the cryopreservation tube in an isopropanol gradient cooling box and store it in a -80°C freezer for 12 hours, then transfer it to liquid nitrogen for long-term storage.

[0222] (4) Seeding

[0223] Seeding is performed after harvesting and counting the cells, adjusting the cell concentration to 1×10 4HIBECs were seeded at 1 x 105cells / mL in 96-well plates (100 μL / well) and 6-well plates (3 mL / well, 3.5 x 105cells / mL). The seeded plates were incubated in the incubator and then stimulated with drugs before the induction of HIBEC-EMT model by TGF-βl for the detection of epithelial and mesenchymal markers.

[0224] (5) Cell growth curve

[0225] Generally, the cell growth curve is determined before the experiment to determine the seeding density. The cell growth curve is usually determined using 96-well plates, in which different densities of cell suspension (5000, 10000, 15000, 20000, 25000) are seeded in different wells, usually 12 wells for each density. After seeding, the cell proliferation of the cell culture plate is determined by MTT method at intervals of 12 h, 24 h, 36 h, 48 h, 60 h and 72 h.

[0226] (6) Drug administration to cells

[0227] After 24 hours of culture of human intrahepatic bile duct epithelial cells, the cells are firmly adhered, and the cells are stimulated with polysaccharide components. This period of time is usually 24 hours, but it can be adjusted according to the experimental requirements and cell characteristics. When the cells are administered, the original culture medium is discarded and replaced with a new culture medium containing drugs. After discarding the original culture medium, the culture medium containing HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d is added and cultured for 12 hours, and then the culture medium containing TGF-βl is added and induced for 12 hours.

[0228] 1.2 Cell survival rate determination

[0229] HIBECs were seeded in 96-well plates at a density of 1 x 105cells / well and cultured in a carbon dioxide incubator for 24 hours. The old culture medium was then discarded and replaced with a culture medium containing HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d (6.25, 12.5, 25 μg / mL) or OCA (99 nM) and cultured for 12 hours. Then, a culture medium containing TGF-βl was added and induced for 12 hours. After drug incubation, MTT solution (5 mg / mL) was added to each well of the 96-well plate (10 μL per well to ensure that MTT was in excess), and the plate was placed in a carbon dioxide incubator for further incubation for 4 hours. Then, 150 μL of triple solution (containing 10% SDS, 5% isopropanol and 0.01 M HCl) was added to each well, and the plate was again placed in a carbon dioxide incubator for overnight incubation. The absorbance value of each well was read at λ = 570 nm.

[0230] Cell activity (%) = (OD drug - OD Normal control ) / (OD Normal control-OD blank ).

[0231] 1.3 Cell morphology observation

[0232] Human intrahepatic bile duct epithelial cells were pre-incubated for 24 hours. Some cells were maintained for 12 hours with different concentrations of polysaccharides HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d, followed by incubation with TGF-β1 (100 ng / mL) for another 12 hours. Other cells were cultured in blank medium and then treated with TGF-β1 (100 ng / mL) or without TGF-β1 (100 ng / mL) for another 12 hours. Finally, HIBECs were observed under 100x magnification using an optical microscope (Olympus CKX41, Japan).

[0233] 2 Results Analysis

[0234] 2.1 Screening of HIBEC-EMT concentrations induced by different concentrations of TGF-β1

[0235] A stable HIBEC-EMT model was initially obtained based on cell viability, changes in cell morphology, and mRNA expression. Figure 32 As shown, when comparing the untreated cell control group with the TGF-β1-induced model group, at 50% cell viability, there was no significant difference between the two groups, but cell morphology changed, becoming thinner. At 100 ng / mL, cell morphology became thinner, cell adhesion decreased, growth rate slowed, and the viability was 80.00 ± 0.02%. The mRNA expression of the epithelial marker E-cadherin decreased by 20%, while the expression of the mesenchymal marker protein vimentin increased by 500%. At 200 ng / mL, cell damage was severe, with a viability as low as 60% and extremely slow growth.

[0236] 2.2 Effects of Chinese Trichophyton polysaccharide components on the survival rate and morphology of HIBEC-EMT cells

[0237] like Figure 33 Compared with the model group, the cell viability of the high-dose polysaccharide HSWP-1a pretreatment group was significantly enhanced, increasing by 5.66±0.03%. Compared with the model group, the cell viability of the medium-dose polysaccharide HSWP-1b pretreatment group was significantly increased by 6.71±0.03%, and the cell viability of the low-dose and high-dose polysaccharide HSWP-1c pretreatment groups were significantly increased by 7.62±0.02% and 4.48±0.01%, respectively. Compared with the model group, the cell viability of the high-dose polysaccharide HSWP-1d pretreatment group was increased, but the increase was not significant.

[0238] Experimental Example 2: Inhibitory Effect of Chinese Trichophyton spore polysaccharide components on HIBEC-EMT

[0239] 1 Experimental method

[0240] 1.1 Real-time fluorescence quantitative (RT-PCR) experiment

[0241] RT-PCR requires RNA extraction and real-time quantitative polymerase chain reaction, which is operated as follows:

[0242] (1) Extraction and quantification of RNA: total RNA was extracted from cells cultured in a 6-well plate using Mini BEST Universal RNA Extraction Kit, and after obtaining the RNA, the RNA concentration was determined using a nucleic acid protein detector, and the determination was performed in triplicate.

[0243] (2) Reverse transcription to obtain cDNA: according to the PrimeScrit TM Master Mix Kit reverse transcriptase kit instructions, dilute RNA from 200 ng RNA to further reverse transcription to obtain cDNA, the reaction system is as shown in Table 4.

[0244] 10 μL reverse transcription system:

[0245] Table 4 10 μL reverse transcription reaction system

[0246]

[0247] Reverse transcription PCR instrument time gradient setting:

[0248] First step 37℃, 15min (6X)

[0249] Second step 85℃, 5S

[0250] (3) Real-time fluorescence quantitative-RT-PCR reaction: the obtained cDNA is subjected to real-time fluorescence quantitative PCR amplification according to the QuantStudio3 instrument instructions and TB Green Premix Ex Tag II kit instructions. The primer group for detecting E-cadherin and vimentin in HIBECs is shown in Table 5, and the PCR reaction system is shown in Table 6. The relative expression level of the target gene is standardized to GAPDH, and the 2 -ΔΔCt Method evaluation, and give the ratio with the control.

[0251] Table 5 Real-time fluorescence quantitative PCR primer sequence

[0252]

[0253] Table 6 Real-time fluorescence quantitative RT-PCR 20 μL reaction system

[0254]

[0255] Real-time PCR instrument time gradient setting (40X):

[0256] First step pre-denaturation: 95°C, 30s;

[0257] Second step PCR amplification: 95°C, 5s / 60.0°C, 34s;

[0258] Third step melting curve: 95°C, 15s / 60.0°C, 1min / 95°C, 15s.

[0259] 1.2 Western Blot (WB) experiment

[0260] HIBECs were seeded in 6-well plates at a density of about 350,000 cells per well and incubated at 37°C in a 5% CO2 incubator for 24 hours. After 12 hours of stimulation with HSWP-1a, HSWP-1b, HSWP-1c, HSWP-1d (6.25, 12.5, 25 μg / mL) and OCA (99 nM), the culture medium containing the polysaccharide sample was discarded and replaced with a culture medium containing TGF-β1 for 12 hours.

[0261] (1) Protein extraction: The culture medium in the 6-well plate was discarded and washed twice with PBS to remove the protein in the culture medium. After discarding the PBS, the cell culture plate was placed on ice. 100 μL of RIPA super strong lysis solution was added to each well, and the cells and lysis solution were collected into a 1.5 mL centrifuge tube using a cell scraper, and lysed on ice for 30 min, with inversion and mixing every 3 min. The lysed cells were centrifuged at 4°C (10000g, 10 min) to collect the supernatant protein solution, which was stored on ice for later use.

[0262] (2) Protein quantification: The protein concentration of the supernatant was determined using a BCA protein concentration determination kit.

[0263] (3) Protein sample denaturation: The protein concentration was adjusted to be consistent, and 5X protein loading buffer was added in proportion. The sample was heated at 100°C for 8 min on a metal plate, cooled to room temperature, centrifuged (10000g, 5 min), and stored in a -80°C freezer.

[0264] (4) Electrophoresis: The supernatant of the denatured protein sample was used for protein gel electrophoresis (5% upper concentration gel, 8% lower gel), with 10 μL per well and a loading amount of 20 μg. The electrophoresis conditions were 80V for 30 min and 120V for 50 min.

[0265] (5) Membrane transfer: After the protein sample was separated by SDS-PAGE, it was transferred to a PVDF (0.2 μm) membrane. The PVDF membrane was activated in advance with methanol, and the transfer conditions were 250 mA for 120 min.

[0266] (6) Blocking: blocking with 5% milk buffer at room temperature for 90 min.

[0267] (7) Incubation of antibody: denatured protein samples were detected with a combination of primary antibody and horseradish peroxidase-conjugated secondary antibody, the primary antibody was incubated at 4°C for 12 h with shaking at 70 r / min, and the secondary antibody was incubated at room temperature for 1 h with shaking at 70 r / min.

[0268] (8) Development: chemical imaging development was used, and both A and B development working solutions were evenly dropped on the PVDF membrane, which was directly placed in a dark box for development and imaged on a Tan 800 chemical imager.

[0269] (9) The gray density of the protein band was normalized to the density of β-actin as an internal control, and the results were normalized to the control by using ImageJ software.

[0270] 2 Result analysis

[0271] 2.1 Effect of Chinese Hirsutella polysaccharide components on E-cadherin and vimentin mRNA expression

[0272] Real-time fluorescent quantitative PCR was used to evaluate the effect of polysaccharide components on the mRNA expression of HIBEC-EMT biomarkers, including E-cadherin and vimentin. The study was as follows Figure 35 The cytotoxicity study of HIBECs by MTT method showed that polysaccharides had a significant proliferative effect on cell viability values. TGF-β1 induced a significant increase in the expression of mesenchymal markers and a significant decrease in the expression of epithelial markers. Compared with the normal control group, the model group showed an increase of 38.94±0.19% in vimentin mRNA expression and a decrease of 11.12±0.30% in E-cadherin mRNA expression. Compared with the model group, HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d increased the expression of E-cadherin mRNA by 18.65-36.00%, 20.24-50.04%, 88.33-199.60% and 44.77-145.93%, respectively, and decreased the expression of vimentin mRNA by 10.91-49.86%, 13.65-18.86%, 6.96-11.96% and 7.38-16.77%, respectively.

[0273] 2.2 Effect of Chinese Hirsutella polysaccharide components on E-cadherin, vimentin, collagen I and ZO-1 protein expression

[0274] The results show that all different concentrations of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d can up-regulate the protein expression of epithelial cell markers such as E-cadherin, ZO-1, etc., and reduce the expression of vimentin protein and collagen I protein. At the same time, HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d have obvious inhibitory effect on all important fibrous related proteins. On the basis of these findings, by hindering the expression and accumulation of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d, these fibrosis related molecules, the anti-fibrosis effect of the four polysaccharide components of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d is proved.

[0275] Compared with the model control group, the low-dose groups of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d could reduce the expression of vimentin, and the protein expression was reduced by 17.66±0.08%, 14.58±0.04%, 24.29±0.06% and 28.00%±0.02, respectively. The medium-dose groups of the polysaccharide components of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d reduced the expression of vimentin by 17.25±0.12%, 15.15±0.13%, 24.69±0.10% and 31.79±0.08%, respectively. The high-dose groups reduced the expression of vimentin by 33.46±0.08%, 38.92±0.01%, 31.28±0.13% and 36.43±0.11%, respectively. Compared with the model group, the low-dose groups of the four polysaccharide components reduced the expression of collagen I by 43.54±0.03%, 30.76±0.04%, 51.33±0.04% and 38.58±0.05%, respectively. The medium-dose groups reduced the expression of collagen I by 50.83±0.02%, 38.27±0.04%, 46.26±0.05% and 38.00±0.04%, respectively. Compared with the model control group, the low-dose groups of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d increased E-cadherin by 47.06±0.13%, 40.98±0.12%, 35.02±0.06% and 27.31±0.12%, respectively. The medium-dose groups of the four polysaccharide components increased the expression of E-cadherin by 73.70±0.17%, 67.15±0.14%, 53.88±0.20% and 50.92%±0.14, respectively. The high-dose groups increased E-cadherin by 46.24±0.03%, 26.26±0.00%, 36.58±0.06% and 49.18±0.16%, respectively. Compared with the model control group, the low-dose groups of HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d increased ZO-1 by 52.26±0.05%, 17.37±0.01%, 28.47±0.05% and 101.21±0.06%, respectively. The medium-dose groups of the four polysaccharide components increased ZO-1 by 50.78±0.04%, 5.79±0.02%, 50.00±0.04% and 103.04±0.03%, respectively. The high-dose groups of the polysaccharide components increased ZO-1 by 55.95±0.03%, 113.94±0.03%, 69.47±0.05% and 122.07±0.06%, respectively.

[0276] 2.3 Effect of polysaccharide components of China hypsizygus marmoreus on expression of TGF-β1 / Smad signaling pathway proteins

[0277] Results as shown in Figure 38 and Figure 39 Compared with the model control group, polysaccharide components of 25 μg / mL could reduce the expression of Smad2 / 3, p-Smad2 / 3.

[0278] The TGF-β1 / Smad signaling pathway was significantly activated in the model group, and the expression of Smad2 / 3, p-Smad2 / 3 and Smad4 was increased by 24.32±0.05%, 70.88±0.11% and 69.79±0.05%, respectively, compared with the untreated control group.

[0279] During the process of TGF-β1-induced EMT, Smad2 / 3 protein expression increased significantly, and the expression of phosphorylated Smad2 / 3 increased. The ratio of p-Smad2 / 3 protein expression to Smad2 / 3 increased by 37.00%, and further reached the downstream gene Smad4. At the same time, compared with the TGF-β1 model group, the polysaccharide samples significantly reduced the expression of p-Smad2 / 3 protein and Smad2 / 3 protein, reduced the degree of mesenchymalization, and had a protective effect on HIBECs. Therefore, it is inferred that TGF-β1 induces EMT of human intrahepatic bile duct cells through the TGF-β1 / Smad signaling pathway, and China hypsizygus marmoreus polysaccharide components play an inhibitory role in EMT by reducing the expression of p-Smad2 / 3, Smad2 / 3 and Smad4 in the TGF-β1 / Smad pathway.

Claims

1. A Hirsutella sinensis polysaccharide component HSWP-1b, characterized in that, mannoglucan consisting of 1,4-linked-α-D-Glcp residues and 4, 6-linked-α-D-Manp residues branched at O-6 position of 1,4-linked-α-D-Glcp, with side chains of 1-linked-α-D-Glcp; The separation method of the Chinese hairy basidiomycete polysaccharide components comprises the following steps: (1) separating the polysaccharide extract through ion exchange column chromatography to obtain a polysaccharide component HSWP-1; (2) adopting gradient alcohol precipitation to separate and purify to obtain polysaccharide components HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d; The polysaccharide HSWP-1 is dissolved with distilled water to prepare a 10% polysaccharide sample solution, and is stirred to be dissolved; anhydrous ethanol is added to the sample solution, and is mixed uniformly; when the alcohol concentration reaches 30%, the solution is left to stand overnight, and is centrifuged to obtain a white precipitate; the white precipitate is placed in a-80℃ refrigerator to freeze overnight, and is freeze-dried in a freeze dryer to obtain a white and slightly yellow solid HSWP-1a; anhydrous ethanol is continuously added to the supernatant, and when the alcohol concentration reaches 50%, the solution is left to stand overnight in a 4℃ refrigerator, and is centrifuged to obtain a precipitate; the supernatant is collected for standby use, the precipitate is placed in a-80℃ refrigerator to freeze overnight, and is freeze-dried to obtain a uniform white flaky solid HSWP-1b; the ion exchange chromatography column used in step (1) is a cation exchange chromatography column, and the cation exchange chromatography column is selected from a DEAE cellulose chromatography column; The polysaccharide extract refers to a Chinese hairy basidiomycete crude polysaccharide obtained by water extraction, concentration and alcohol precipitation of Chinese hairy basidiomycete powder; The polysaccharide further contains the following monosaccharide units: rhamnose, galacturonic acid and arabinose; The average molecular weight of the polysaccharide is 8.57 kDa.

2. The polysaccharide according to claim 1, characterized in that, The infrared spectrum thereof includes at least one or more of the following absorption peaks: 3424.33 cm -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 .

3. A Hirsutella sinensis polysaccharide component HSWP-1c, characterized in that, galactomanno-glucan consisting of 1,2,6-linked α-D-Galp, 1,3,4.6-linked α-D-Glcp, 4,6-linked β-D-Manp, 1,4-linked α-D-Glcp, 1,4,6-linked α-D-Glcp and 1-linked α-D-Glcp residues; The separation method of the Chinese hairy basidiomycete polysaccharide components comprises the following steps: (1) separating the polysaccharide extract through ion exchange column chromatography to obtain a polysaccharide component HSWP-1; (2) adopting gradient alcohol precipitation to separate and purify to obtain polysaccharide components HSWP-1a, HSWP-1b, HSWP-1c and HSWP-1d; The polysaccharide HSWP-1 is dissolved in distilled water to prepare a 10% polysaccharide sample solution, stirred to dissolve, and then anhydrous ethanol is added to the sample solution and mixed uniformly. The ethanol concentration is measured to be 30%, and the solution is left to stand overnight, centrifuged to obtain a white precipitate, and the supernatant is collected for later use. The white precipitate is placed in a -80°C freezer overnight, and then freeze-dried to obtain a white and slightly yellow solid HSWP-1a. Anhydrous ethanol is continuously added to the supernatant, and the ethanol concentration is measured to be 50%. The solution is left to stand overnight in a 4°C freezer, centrifuged to obtain a precipitate, and the supernatant is collected for later use. The precipitate is placed in a -80°C freezer overnight, and then freeze-dried to obtain a uniform white flaky solid HSWP-1b. Anhydrous ethanol is added to the collected supernatant, and the ethanol concentration is measured to be 70%. The solution is left to stand overnight in a 4°C freezer, centrifuged to obtain a light yellow precipitate, and the supernatant is collected for later use. The precipitate is placed in a -80°C freezer overnight, and then freeze-dried to obtain a light yellow solid powder HSWP-1c. The ion exchange chromatography column used in step (1) is an anion exchange chromatography column, and the anion exchange chromatography column is selected from a DEAE cellulose chromatography column. The polysaccharide extract refers to a crude polysaccharide of Chinese Hirsutella rhodii obtained by water extraction, concentration, and alcohol precipitation of Chinese Hirsutella rhodii powder; HSWP-1c also contains the following monosaccharide units: galactose, rhamnose, and galacturonic acid; The average molecular weight of HSWP-1c is 2.18 kDa.

4. The polysaccharide component according to claim 3, characterized in that, The infrared spectrum thereof includes at least one or more of the following absorption peaks: 3432.59 cm -1 , 2926.03 cm -1 , 2845.91 cm -1 , 1639.48 cm -1 , 1599.85 cm -1 , 1025.25 cm -1 , 857.23 cm -1 , 620.35 cm -1 .

5. A Hirsutella sinensis polysaccharide component HSWP-1d, characterized in that, The mannan glucan is composed of 1,4-linked-α-D-Glcp residues and 4,6-linked-α-D-Manp residues, and the O-6 site of 1,4-linked-α-D-Glcp is branched, and the side chain is 1-linked-α-D-Glcp; The separation method of the polysaccharide components of Chinese Hirsutella rhodii includes the following steps: (1) the polysaccharide extract is separated by ion exchange column chromatography to obtain a polysaccharide component HSWP-1; (2) gradient alcohol precipitation is used for separation and purification to obtain polysaccharide components HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d; The separation method of the polysaccharide components of Chinese Hirsutella rhodii includes the following steps: (1) the polysaccharide extract is separated by ion exchange column chromatography to obtain a polysaccharide component HSWP-1; (2) gradient alcohol precipitation is used for separation and purification to obtain polysaccharide components HSWP-1a, HSWP-1b, HSWP-1c, and HSWP-1d; The polysaccharide HSWP-1 was dissolved in distilled water to prepare a 10% polysaccharide sample solution, which was stirred to dissolve. Anhydrous ethanol was added to the sample solution and mixed evenly. The ethanol concentration was measured to be 30%. The solution was left to stand overnight, centrifuged, and a white precipitate was obtained. The supernatant was collected for later use. The white precipitate was placed in a -80°C freezer overnight, and freeze-dried to obtain a white and slightly yellow solid HSWP-1a. Anhydrous ethanol was continuously added to the supernatant. When the ethanol concentration was measured to be 50%, the solution was left to stand overnight in a 4°C freezer, centrifuged, and a precipitate was obtained. The supernatant was collected for later use. The precipitate was placed in a -80°C freezer overnight, and freeze-dried to obtain a uniform white flaky solid HSWP-1b. Anhydrous ethanol was added to the collected supernatant. The ethanol concentration of the solution was measured to be 70% by alcoholometry. The solution was left to stand overnight in a 4°C freezer, centrifuged, and a yellowish precipitate was obtained. The supernatant was collected for later use. The precipitate was placed in a -80°C freezer overnight, and freeze-dried to obtain a yellowish solid powder HSWP-1c. Anhydrous ethanol was added to the supernatant. The ethanol concentration of the solution was measured to be 90% by alcoholometry. The solution was left to stand overnight in a 4°C freezer, centrifuged, and a yellowish-brown precipitate was obtained. The precipitate was placed in a -80°C freezer overnight, and freeze-dried to obtain a dark brown solid powder HSWP-1d. The ion exchange chromatography column used in step (1) was an anion exchange chromatography column, which was selected from a DEAE cellulose chromatography column. The polysaccharide extract refers to a crude polysaccharide of Chinese Hirsutella rhodii obtained by water extraction, concentration, and alcohol precipitation. HSWP-1d also contains the following monosaccharide units: rhamnose, galacturonic acid, and arabinose. The average molecular weight of HSWP-1d is 1.09 kDa.

6. The polysaccharide component according to claim 5, characterized in that, The infrared spectrum thereof includes at least one or more of the following absorption peaks: 3421.58 cm -1 , 2923.03 cm -1 , 2851.42 cm -1 , 1639.48 cm -1 , 1405.36 cm -1 , 1025.25 cm -1 , 851.72 cm -1 , 631.37 cm -1 .

7. Use of the polysaccharide component separated according to any one of claims 1-6 in the preparation of a product for treating and / or preventing primary biliary cholangitis.

8. Use according to claim 7, characterized in that, The product is a product for inhibiting HIBEC-EMT.

9. Use according to claim 8, characterized in that, The product is a product for increasing or decreasing cell viability.

10. Use according to claim 9, characterized in that, The cell is a HIBEC cell.

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