Cs-4 fermented mycelium heteropolysaccharide, and preparation method and use thereof

Abstract

A Cs-4 fermentation mycelium heteropolysaccharide, a preparation method and use thereof, the polysaccharide structure is as shown below, n is selected from 6-104, the polysaccharide can be used for preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, preventing or treating acute kidney injury.

Description

[0001] This application claims priority to Chinese patent application 2021110438722, filed on September 7, 2021. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of traditional Chinese medicine pharmaceuticals, specifically relating to a Cs-4 fermented mycelium heteropolysaccharide prepared from the mycelium of *Paecilomyces hepiali* (Cs-4), its preparation method, and its uses. Background Technology

[0003] Cordyceps sinensis, a complex of the fruiting body of the fungus Cordyceps sinensis (Berk.) Sacc. (family Clavicipitaceae) parasitizing the larvae of insects in the family Hepialidae (family Hepialidae) and the larval corpse, is a precious traditional Chinese medicine (hereinafter referred to as Cordyceps). Due to the special growing environment and high price of natural Cordyceps, the market demand cannot be met. Therefore, modern bio-fermentation technology is used to cultivate Cordyceps mycelium to replace natural Cordyceps. In 1982, the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, isolated more than a dozen strains from Cordyceps sinensis samples collected in Diqing Tibetan Autonomous Prefecture, Yunnan Province, and cultured and morphologically studied four of them, naming them Paecilonyces hepialid chen & Dai. In 1982, the Institute of Materia Medica, Chinese Academy of Medical Sciences, isolated strain Cs-4 from fresh Cordyceps sinensis collected in Hualong County, Qinghai Province, which was also identified as Paecilonyces hepialid chen & Dai. Fermented Cordyceps mycelium powder (Cs-4), hereinafter referred to as Cs-4, is the dried powder of mycelium obtained by fermentation of the isolated Paecilomyces hepiali strain Cs-4. It has been successfully marketed as a raw material for making medicines, with the medicines named Jinshuibao capsules and Jinshuibao tablets.

[0004] Jinshuibao, as a representative variety of artificially cultivated Cordyceps, has the effects of tonifying the lungs and kidneys, replenishing essence and qi. Clinically, it has been widely used in the treatment of kidney diseases, respiratory diseases, metabolic diseases, cardiovascular diseases, endocrine diseases, and as an adjuvant therapy for tumors. However, the material basis of its pharmacological effects is not clear.

[0005] Cordyceps polysaccharides, as one of the main active ingredients in Cordyceps, have attracted much attention from researchers both at home and abroad. However, current research focuses on the activity screening of crude Cordyceps polysaccharides, while polysaccharide structure research is still in its early stages. Most studies only cover monosaccharide composition and molecular weight information, or simply grade polysaccharides without complete purification, thus failing to accurately explore the chemical structure of Cordyceps polysaccharides. This seriously limits the quality control and subsequent clinical translation of active Cordyceps polysaccharides. Summary of the Invention

[0006] There is a lack of research on Cs-4 fermented mycelial polysaccharides in the existing technology. The Cs-4 fermented mycelial polysaccharides extracted by the existing technology have low purity, and there is limited research on the fine structure of the polysaccharides. Based on this, the present invention has carried out in-depth research on Cs-4 fermented mycelial polysaccharides, and the extracted polysaccharides have high purity. The specific monosaccharide composition, relative molecular mass, glycosidic bond linkage mode, etc. of the polysaccharides have been studied in detail. Comparative experiments were carried out with fermented Cordyceps militaris powder in animal models. It can be seen that the polysaccharides of the present invention achieve better effects with lower dosage compared with fermented Cordyceps militaris powder.

[0007] In a first aspect, the present invention provides a Cs-4 fermented mycelium heteropolysaccharide. According to an embodiment of the invention, the structural formula is as follows:

[0008]

[0009] Among them, Manp is pyranose-type mannose, Galp is pyranose-type galactose, and Glcp is pyranose-type glucose, and n is selected from 6-104. The inventors extracted and identified this polysaccharide for the first time. This polysaccharide has superior effects in preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury. Compared with fermented Cordyceps mycelium powder, it achieves better effects with a lower dosage.

[0010] According to embodiments of the present invention, the above-mentioned polysaccharide may further include one of the following additional technical features:

[0011] According to embodiments of the present invention, Manp is pyranose-type mannose, Galp is pyranose-type galactose, Glcp is pyranose-type glucose, and n is selected from 6-68. According to embodiments of the present invention, n is selected from 15-21.

[0012] According to an embodiment of the present invention, n is selected from 15-104.

[0013] According to an embodiment of the present invention, n is selected from 21-104.

[0014] According to an embodiment of the present invention, n is selected from 6-15.

[0015] According to an embodiment of the present invention, n is selected from 6-21.

[0016] According to an embodiment of the present invention, n is selected from 15-68.

[0017] According to an embodiment of the present invention, n is selected from 21-68.

[0018] According to an embodiment of the present invention, n is selected from any integer between 6 and 104.

[0019] According to an embodiment of the present invention, n is selected from any integer between 6 and 68.

[0020] According to an embodiment of the present invention, n is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 100, 101, 102, 103, 104, 105, 106, 107.

[0021] According to embodiments of the present invention, the relative molecular weight of the polysaccharide is selected from 10-152 kDa. It should be noted that the relative molecular weight used in this invention refers to the weight-average molecular weight, which is the average weight of the molecules, i.e., the weight-weighted average of the molecular weight.

[0022] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-100 kDa.

[0023] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-23.25 kDa.

[0024] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-30.12 kDa.

[0025] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-151.15 kDa.

[0026] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 23.25-30.12 kDa.

[0027] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 23.25-100 kDa.

[0028] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 30.12-100 kDa.

[0029] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 23.25-151.15 kDa.

[0030] According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 30.12-151.15 kDa.

[0031] According to embodiments of the present invention, the relative molecular weight of the polysaccharide is selected from 23.15 kDa, 30.12 kDa, or 151.15 kDa.

[0032] According to an embodiment of the present invention, the polysaccharide composition includes glucose, galactose and mannose, wherein the molar ratio of glucose, galactose and mannose is 1:(1.0-2.0):(1.5-2.5).

[0033] According to an embodiment of the present invention, the polysaccharide composition includes glucose, galactose and mannose, wherein the molar ratio of glucose, galactose and mannose is 1:(1.5-1.8):(2.0-2.5).

[0034] According to an embodiment of the present invention, the polysaccharide composition includes glucose, galactose and mannose, wherein the molar ratio of glucose, galactose and mannose is 1:(1.71-1.74):(2.09-2.44).

[0035] In a second aspect, the present invention provides a method for extracting heteropolysaccharides from the aforementioned Cs-4 fermentation mycelium. According to an embodiment of the present invention, the method includes: 1) defatting Cs-4 mycelium powder with ethanol, discarding the ethanol extract, and obtaining a defatted residue; 2) extracting the residue in water to obtain an aqueous extract; 3) subjecting the aqueous extract to alcohol precipitation, wherein the solvent for the alcohol precipitation is ethanol; and 4) purifying the precipitate obtained from the alcohol precipitation in step 3) to obtain the fermentation mycelium heteropolysaccharides. The method according to the embodiment of the present invention is simple to operate, has high extraction efficiency, and yields polysaccharides with a purity exceeding 94%.

[0036] According to embodiments of the present invention, the method may further include at least one of the following additional technical features:

[0037] According to an embodiment of the present invention, the degreasing treatment is carried out at a temperature of 100°C.

[0038] According to an embodiment of the present invention, the degreasing treatment is performed three times, each time for 1 hour.

[0039] According to an embodiment of the present invention, the amount of ethanol in the defatting process is 10 times that of Cs-4.

[0040] According to an embodiment of the present invention, the ethanol used for defatting is 85%-100% ethanol.

[0041] According to an embodiment of the present invention, the extraction process is carried out at a temperature of 75°C-78°C.

[0042] According to an embodiment of the present invention, the extraction process is performed three times, each time lasting one hour.

[0043] According to an embodiment of the present invention, the amount of water in the extraction process is 10 times that of the residue.

[0044] According to an embodiment of the present invention, the ethanol undergoing the alcohol precipitation treatment is 80% ethanol.

[0045] In another aspect of the invention, a method for extracting the aforementioned Cs-4 fermentation mycelium heteropolysaccharide is provided. According to an embodiment of the invention, the method comprises: 1) extracting Cs-4 three times with 10 times the volume of 85% ethanol, each time for 1 hour, discarding the ethanol extract, and obtaining a residue; 2) extracting the residue three times with 10 times the volume of water, each time for 1 hour, and combining the aqueous extracts; 3) precipitating the aqueous extract with 80% ethanol to obtain a precipitate Cs-4-P; 4) purifying the precipitate Cs-4-P to obtain the fermentation mycelium heteropolysaccharide Cs-4-P1, and further purifying to obtain three homogeneous polysaccharides, named Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3.

[0046] According to embodiments of the present invention, the method may further include at least one of the following additional technical features:

[0047] According to embodiments of the present invention, the purification process includes protein removal, destaining, and column purification. It should be noted that protein removal, destaining, and column purification can be conventional methods, and appropriate methods can be selected according to specific needs.

[0048] In another aspect, the present invention also provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the Cs-4 fermentation mycelium heteropolysaccharide described above or the Cs-4 fermentation mycelium heteropolysaccharide Cs-4-P1 obtained according to the method described above, and three homogeneous polysaccharides (Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3) obtained by further purification.

[0049] In another aspect, the present invention also proposes the use of the aforementioned Cs-4 fermented mycelium heteropolysaccharide, or the Cs-4 fermented mycelium heteropolysaccharide obtained according to the aforementioned method, or the aforementioned pharmaceutical composition, in the preparation of a medicament for the prevention or treatment of chronic renal failure, suppression of immune rejection, prevention or treatment of hyperlipidemia, and prevention or treatment of acute kidney injury. The inventors have found that this polysaccharide has superior effects in preventing or treating chronic renal failure, suppressing immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury, achieving better effects at a lower dosage compared to Jinshuibao.

[0050] In one embodiment of the invention, the acute kidney injury is selected from cisplatin-induced acute kidney injury and / or LPS-induced acute kidney injury. The pharmaceutical composition according to the invention comprises the Cs-4 fermentation mycelium heteropolysaccharide described above or the Cs-4 fermentation mycelium heteropolysaccharide obtained according to the methods described above. The inventors have found that this polysaccharide has superior efficacy in preventing or treating acute kidney injury, comparable to that of the positive control drugs amifostine and dexamethasone.

[0051] In another aspect, the present invention also provides a pharmaceutical composition for the prevention or treatment of hyperlipidemia. According to embodiments of the present invention, the pharmaceutical composition comprises 1.0-10.0 μg / mL of the aforementioned Cs-4 fermented mycelium heteropolysaccharide or the Cs-4 fermented mycelium heteropolysaccharide obtained according to the aforementioned method. The inventors have found that this polysaccharide has superior efficacy in the prevention or treatment of hyperlipidemia, achieving better results at a lower dosage compared to Jinshuibao.

[0052] According to embodiments of the present invention, the above-described pharmaceutical composition may further include at least one of the following additional technical features:

[0053] According to an embodiment of the present invention, the concentration of the polysaccharide is 3.0 μg / mL. The inventors have found that at a polysaccharide concentration of 3.0 μg / mL, its effect in preventing or treating hyperlipidemia is better, and significantly higher than that of Jinshuibao and the classic drug atorvastatin calcium.

[0054] In another aspect, the present invention provides at least one of the following technical effects:

[0055] 1) This invention is the first to extract and identify polysaccharides with repeating structural units (abbreviated as Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3);

[0056] 2) The extraction method of the present invention is simple to operate;

[0057] 3) The heteropolysaccharide of the present invention has high purity, reaching over 94%;

[0058] 4) The heteropolysaccharide of the present invention is more effective than fermented Cordyceps sinensis powder Cs-4 (1600mg / kg / day) and Abelmoschus manihot capsules (1000mg / kg / day) in preventing or treating chronic renal failure at low doses (16mg / kg / day), and is comparable to or better than the classic clinical drug dexamethasone. The present invention aims to provide a new alternative drug for the prevention or treatment of chronic renal failure.

[0059] 5) The heteropolysaccharide of the present invention is more effective than fermented Cordyceps sinensis powder Cs-4 in inhibiting immune rejection, and its efficacy is comparable to or better than that of the classic clinical drug cyclosporine, with better safety. The present invention aims to provide a new alternative drug for inhibiting immune rejection.

[0060] 6) When preventing or treating hyperlipidemia, the heteropolysaccharide of the present invention can lower triglycerides and total cholesterol at low doses (1.0-10.0 μg / mL) with better efficacy than Jinshuibao, and its efficacy is comparable to or better than that of the classic drug atorvastatin calcium. The present invention aims to provide a new alternative drug for the prevention or treatment of hyperlipidemia.

[0061] It should be noted that, in the context of this invention, when the terms "about" or "approximately" are used, whether or not they are used, it means within 10% of a given value or range, appropriately within 5%, and particularly within 1%. Alternatively, for those skilled in the art, the term "about" or "approximately" means within an acceptable standard error range of the average value. Whenever a number with a value of N is disclosed, any number having a value within N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, or N+ / -10% is explicitly disclosed, where "+ / -" refers to addition or subtraction. Attached Figure Description

[0062] Figure 1 This is an HPGPC map according to an embodiment of the present invention.

[0063] Figure 2 The image shows the infrared spectrum of the methylated sample according to an embodiment of the present invention.

[0064] Figure 3 This is the total ion current spectrum of the Cs-4-P1-2 derivative according to an embodiment of the present invention.

[0065] Figure 4 It is Cs-4-P1-2 according to an embodiment of the present invention. 1 H NMR spectrum.

[0066] Figure 5 It is Cs-4-P1-2 according to an embodiment of the present invention. 13 C NMR spectrum.

[0067] Figure 6 It is Cs-4-P1-2 according to an embodiment of the present invention. 1 H- 1 H COSY spectrum.

[0068] Figure 7 It is Cs-4-P1-2 according to an embodiment of the present invention. 1 H- 1 H TOCSY diagram.

[0069] Figure 8 According to an embodiment of the present invention 1 H- 13 C HSQC diagram.

[0070] Figure 9 According to an embodiment of the present invention 1 H- 13 C HMBC map.

[0071] Figure 10 According to an embodiment of the present invention 1 H- 1 H NOESY spectrum.

[0072] Figure 11 These are pathological slide images according to embodiments of the present invention. Detailed Implementation

[0073] The present application is described in detail below with reference to embodiments, but this does not imply any adverse limitations on the present application. The present application has been described in detail herein, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific implementations of the present application without departing from the spirit and scope thereof.

[0074] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0075] Dexamethasone acetate tablets (batch number: 015200406, Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd.), Abelmoschus manihot (batch number: 20110210, Jiangsu Suzhong Pharmaceutical Group Co., Ltd.), Fermented Cordyceps sinensis powder (batch number: 190800810-1, Jiangxi Guoyao Co., Ltd.), and Neosiprine cyclosporine soft capsules (batch number: 200825, Hangzhou Sino-American East China Pharmaceutical Co., Ltd.).

[0076] The abbreviations used in this invention are as follows:

[0077] Glcp represents glucose pyranose, Galp represents galactopyranose, Manp represents mannopyranose, h represents hour, min represents minute, W represents week, rpm represents the number of rotations per minute, DEAE represents diethylaminoethyl cellulose, Seph represents dextran gel, HPGPC represents high performance gel electrophoresis, Mw represents weight-average molecular weight, Mp represents peak molecular weight, t R The numbers represent retention time, BUN represents blood urea nitrogen, CREA represents serum creatinine, UA represents uric acid, CsA represents cyclosporine, MTC represents minimum toxic concentration, and LPS represents lipopolysaccharide.

[0078] Example 1: Preparation method of Cs-4-P, Cs-4-P1, Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3

[0079] 1. Degreasing: Fermented Cordyceps sinensis powder Cs-4 is heated to boiling (78℃) with 10 times the amount of 85% ethanol and extracted three times at a gentle boil, one hour each time. The ethanol extract is then discarded.

[0080] 2. Water extraction and alcohol precipitation: The remaining residue was heated to boiling (100℃) with 10 times the amount of water and extracted three times at a gentle boil, each time for 1 hour. The water extracts were combined and concentrated under reduced pressure at 70℃ to obtain a clear extract (density 1.05-1.07). 95% ethanol was added to the clear extract to a concentration of 85% alcohol, stirred for 30 minutes, and allowed to stand for 24 hours. The supernatant was filtered / concentrated, the precipitate was collected, dried under reduced pressure at 65℃, and pulverized to obtain crude polysaccharide Cs-4-P (yield 21%).

[0081] 3. Deproteinization and decolorization: 2% water extract crude polysaccharide aqueous solution, equal volume of Sevage reagent, vigorous shaking for 10 minutes each time, 4000 rpm, 10 min, repeat decolorization 3-4 times until no obvious white layer appears, combine the upper layer solutions.

[0082] 4. Decolorization: Add 2% activated carbon powder to the monosaccharide solution, keep warm at 50℃ for 30 minutes, filter twice with double-layer filter paper, pass through a 0.45-micron membrane, and concentrate until there is no organic reagent odor, ready for the next purification step.

[0083] 5. DEAE-52 column chromatography: elution with pure water yielded neutral polysaccharide Cs-4-P1;

[0084] 6. Seph G-100 column chromatography separation and purification: elution with pure water yielded three homogeneous polysaccharides, named Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3, with Cs-4-P1-1 having the lowest yield.

[0085] Example 2: Content, molecular weight, and monosaccharide composition of Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3.

[0086] Polysaccharide content determination:

[0087] This method refers to the polysaccharide determination method in SN / T 4260-2015, "Determination of Crude Polysaccharides in Exported Plant-Derived Foods by Phenol-Sulfuric Acid Method". This method utilizes the hydrolysis of polysaccharides into monosaccharides under concentrated acid, followed by dehydration condensation to form uronic acid derivatives, which then combine with phenol for colorimetric development. The total sugar content is calculated by measuring the absorbance value after color development. When using this method to determine polysaccharide content, a monosaccharide must be selected as a reference to construct a standard curve.

[0088] Standard curve preparation: Accurately weigh 5.417 mg of glucose and place it in a 50 mL volumetric flask. Dissolve and dilute to volume with water to obtain a 0.1 mg / mL glucose standard solution. Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution into 20 mL stoppered test tubes, respectively. Add distilled water to a final volume of 1.0 mL, add 1.0 mL of freshly prepared 5% phenol solution and 5.0 mL of concentrated sulfuric acid, shake well, and allow to stand at room temperature for 10 min. Then, vortex the mixture to ensure thorough mixing. Place the test tubes in a 30°C water bath for 20 min and measure the absorbance at 490 nm. Plot glucose concentration on the x-axis and absorbance on the y-axis to perform a linear regression to obtain the standard curve equation.

[0089] Test sample detection: Accurately weigh 50 mg of Cs-4-P1 and place it in a 10 mL volumetric flask. Dissolve by sonication, add water to the mark, and shake well. Take 0.2 mL of the above-prepared solution into a 20 mL stoppered test tube and add water to bring the volume to 1.0 mL. Add distilled water to the mark, add 1.0 mL of freshly prepared 5% phenol solution and 5.0 mL of concentrated sulfuric acid, shake well, and let stand at room temperature for 10 min. Then, use a vortex mixer to thoroughly mix the reaction solution. Place the test tube in a 30℃ water bath for 20 min and measure the absorbance at 490 nm.

[0090] The polysaccharide content of Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3 was determined to be 94.61%, 98.61%, and 99.52%, respectively.

[0091] 2. Molecular weight determination

[0092] High-performance gel permeation chromatography (HPGPC) can be used to determine the relative molecular mass and distribution of macromolecules, and is the preferred method for determining the relative molecular mass of polysaccharides. Its principle is based on the relationship between the relative molecular mass of polysaccharides on the gel column and the elution retention time (t). R To determine the relationship between the properties of the samples, a standard curve is first prepared using standard samples with known relative molecular masses. Then, the t-values ​​of the samples are used to determine the relationship between the properties of the samples and the relative molecular masses. R The relative molecular mass is obtained from the curve. The peak molecular weight Mp represents the molecular weight corresponding to the retention time of the highest peak, while the peak start and peak end molecular weights represent the molecular weights corresponding to the peak start and peak end times, respectively. The weight-average molecular weight Mw is obtained by statistically averaging the molecular weights, representing molecules of equal weight distributed on both sides.

[0093] 2.1 Reagent Preparation

[0094] Preparation of 0.71% sodium sulfate: Weigh 14.20g of sodium sulfate into a 2L beaker, add 2000ml of purified water to dissolve and dilute, filter through a 0.22um microporous membrane, and degas by sonication for about 10 minutes to obtain the solution.

[0095] Preparation of test solution: Accurately weigh 50 mg of each sample into a 10 ml volumetric flask, add an appropriate amount of the filtered mobile phase obtained above, sonicate for 10 min, then dilute to the mark and shake well to obtain the test solution. Store the test solution at 4℃.

[0096] Preparation of reference solution: Weigh 10 mg each of dextran D0, D1, D2, D3, D4, D5, D6, D7, D8, D2000, and dextran 410000 into each 1.5 ml disposable centrifuge tube. Accurately weigh each tube and accurately transfer 1 ml of 0.71% sodium sulfate solution into each centrifuge tube. Shake well to obtain the reference solution.

[0097] 2.2 Chromatographic conditions

[0098] Chromatographic column: ZRD-LC-53TOSOH TSKgel guardcolumn PWXL (6*40mm, 12um); ZRD-LC-54TOSOH TSKgel G3000PWXL (7.8*300mm, 7um); ZRD-LC-55TOSOH TSKgel G4000 PWXL(7.8*300mm,10um); ZRD-LC-62TOSOH TSKgel G5000PWXL(7.8*300mm,10um).

[0099] Elution conditions: 0.71% sodium sulfate solution, isocratic elution.

[0100] The RID detector was used, with a column temperature of 35℃, a flow rate of 0.8 mL / min, and an injection volume of 20 μL.

[0101] A standard curve was plotted with retention time on the x-axis and the corresponding logarithmic molecular weight on the y-axis. The relative molecular weights of samples Cs-4-P1, Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3 were calculated using the regression equation. The results are as follows: Figure 1 As shown in Table 1, the weight-average molecular weights (Mw) of Cs-4-P1, Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3 range from 23.25 to 151.15 kDa.

[0102] Table 1

[0103]

[0104] 3. Determination of monosaccharide composition

[0105] Weigh 2 mg of sample into a reaction flask, add 3 mL of 2 mol / L trifluoroacetic acid (TFA), heat in an oil bath at 110 °C for 3 h, cool to room temperature, dry under nitrogen at 40 °C, add 3 mL of methanol, dry again, repeat 4–5 times to completely remove TFA. Dissolve the sample in the reaction flask with ultrapure water and bring the volume to 100 mL. Centrifuge a portion of the solution at 12000 g for 20 min, collect the supernatant, and determine its monosaccharide composition using high performance anion chromatography (HPAEC), with a monosaccharide mixed standard as a reference. As shown in Table 2, the monosaccharide compositions of Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3 are identical, possessing the same constituent units.

[0106] Table 2

[0107] sample Glcp Galp Manp Cs-4-P1-1 1.00 1.71 2.44 Cs-4-P1-2 1.00 1.73 2.34 Cs-4-P1-3 1.00 1.74 2.09

[0108] Example 3: Structural Analysis of Cs-4-P1-2

[0109] 1. Polysaccharide methylation

[0110] After methylation, the polysaccharide was analyzed and its infrared spectrum was measured in the range of 3100-3600 cm⁻¹. -1 The disappearance of the hydroxyl absorption peak between the two indicates that the polysaccharide has been completely methylated. Figure 1 The fully methylated samples underwent hydrolysis, reduction, and acetylation treatments, followed by GC-MS analysis. The total ion chromatogram for the methylation analysis of Cs-4-P1-2 polysaccharide is shown below. Figure 2 The methylation analysis results are shown in Table 3.

[0111] Table 3

[0112] Methylated sugars Connection method Mole ratio <![CDATA[2,3,4,6-Me4-Glcp]]> Terminal Glcp 1.77 <![CDATA[2,3,4,6-Me4-Galp]]> Terminal Galp 1.00 <![CDATA[2,3,6-Me3-Galp]]> 1,4-Linked Galp 2.04 <![CDATA[2,3,4-Me3-Manp]]> 1,6-Linked Manp 2.33 <![CDATA[2,3-Me2-Manp]]> 1,4,6-Linked Manp 2.96

[0113] 2. Spectral Analysis

[0114] from 1 H NMR ( Figure 3 As can be seen from the data, there are 5 resonance signal peaks in the anomeric hydrogen region. Based on the peak area ratio of the anomeric hydrogens, a total of 9 sugar residue signal peaks are determined, appearing at δ4.92ppm (3 H), δ4.89ppm (2 H), δ4.58ppm, δ4.46ppm (2 H), and δ4.40ppm, respectively. This indicates that the sugar residues are in the β configuration. According to the decreasing order of chemical shift of the anomeric hydrogens, the sugar residues are numbered A, B, C, D, and E. 13 C NMR ( Figure 4 In the spectrum, the signal in the anomeric carbon signal region is δ102.1ppm~δ107.1ppm, further indicating that the glucose residue is in the β configuration.

[0115] To further elucidate the chemical structure of the polysaccharide, two-dimensional nuclear magnetic resonance (NMR) spectra (COSY, TOCSY, HMQC, HMBC) were performed, and the chemical shifts of each sugar residue were assigned.

[0116] Sugar residue A: According to 1 The chemical shift of the hydrogen at position 1 (H-1) of sugar residue A, determined by ¹H NMR, was obtained by COSY spectroscopy. Figure 5 The H-2 and H-3 signals were identified, and the H-2 and H-3 chemical shifts of sugar residue A were assigned to δ3.53 ppm and δ3.72 ppm, respectively. This was combined with COSY and TOSCY spectra. Figure 6 The chemical shifts of H-4 (δ 3.92 ppm), H-5 (δ 3.65 ppm), and H-6 (δ 3.95 ppm, δ 3.75 ppm) signals were assigned. After assigning the chemical shifts of H, the results could be obtained using HSQC spectroscopy (HSQC). Figure 7 This is assigned to the chemical shifts of the carbon atoms on the sugar ring. According to...

[0117] The chemical shifts of C and H in Table 4 determine that sugar residue A is →4,6)-β-D-Manp-(1→).

[0118] Table 4

[0119]

[0120] Based on the above method, the chemical shifts of carbon (C) and hydrogen (H) signals in sugar residues B, C, D, and E were sequentially assigned. The complete assignment results are shown below.

[0121] Table 4. By comparison, the connection modes of sugar residues B, C, D, and E were determined to be →4)-β-D-Galp-(1→, β-D-Galp-(1→, β-D-Glcp-(1→, →6)-β-D-Manp-(1→).

[0122] After the attribution was completed, the HMBC spectrum was further utilized ( Figure 9 The sites and order of connections between each sugar residue were identified. From the HMBC signal, it was found that there were significant correlations between H-1 of residue A, C-4 of residue B, and C-6 of residue E; H-1 of residue B and C-6 of residue A; H-1 of residue C and C-4 of residue B; H-1 of residue D and C-4 of residue A; and H-1 of residue E and C-4 of residue A. The long-range correlation data for each sugar residue are shown in Table 5.

[0123] Table 5

[0124] sugar residues hydrogen proton Related A:→4,6)-β-D-Manp-(1→ H-1 80.4 (B; C-4), 68.9 (E; C-6) B:→4)-β-D-Galp-(1→ H-1 68.7 (A; C-6) C:β-D-Galp-(1→ H-1 80.4(B; C-4) D:β-D-Glcp-(1→ H-1 79.7 (A; C-4) E:→6)-β-D-Manp-(1→ H-1 79.3(A:C-4)

[0125] Then, based on the NOESY spectrum ( Figure 10 The NOE spectrum was cross-validated with the HMBC spectrum. The NOE spectrum also showed that there were correlation points between H-1 of residue A, H-4 of residue B, and H-6 of residue D; H-1 of residue B and H-6 of residue A; H-1 of residue D and H-4 of residue A; and H-1 of residue E and H-4 of residue A. The NOE data are shown in Table 6.

[0126] Table 6

[0127] sugar residues hydrogen proton NOE related A:→4,6)-β-D-Manp-(1→ H-1 4.02 (B; H-4), 3.87 (E; H-6) B:→4)-β-D-Galp-(1→ H-1 3.75 (A; H-6) D:β-D-Glcp-(1→ H-1 3.90(A; H-4) E:→6)-β-D-Manp-(1→ H-1 3.92(A; H-4)

[0128] Based on the cross-validation results of HMBC and NOESY spectra, the connection mode between sugar residues can be determined. It can be inferred that position 1 of residue A is connected to position 4 of residue B and position 6 of residue E; position 1 of residue B is connected to position 6 of residue A; position 1 of residue C is connected to position 4 of residue B; and positions 1 of residues D and E are connected to positions 4 of residue A, respectively. Combining this with the methylation results showing a sugar residue ratio of A:B:C:D:E = 3:2:1:2:1, the repeating unit of the polysaccharide component is confirmed as:

[0129]

[0130] This polysaccharide is a heteropolysaccharide with alternating main chains of β-(1→4)-D-mannose, β-(1→6)-D-mannose, and β-(1→4)-D-galactose, and β-D-galactose and β-D-glucose side chains linked at positions 6 and 4 of the mannose, respectively. This repeating fragment is a 9-saccharide. Based on this, and using the weight-average molecular weight (Mw) of Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3 in Table 1, their n values ​​are approximately 104, 21, and 16, respectively.

[0131] Example 4: Study on the effect of adenine on chronic renal failure in rats

[0132] Experimental Methods: Seventy SD rats, half male and half female, were randomly divided into four groups of 10 rats each, based on body weight: normal control group, model group, Cs-4-P1-2 group, fermented Cordyceps militaris powder group, Abelmoschus manihot capsule group, and dexamethasone group. Before the experiment, rats were placed in metabolic cages and fasted for 24 hours (with normal water intake). Urine output, urinary protein, and urinary creatinine were recorded over 24 hours. After weighing the rats, 1 mL of blood was collected from the orbital sinus and placed in a 1.5 mL centrifuge tube. The blood was centrifuged at 3000 rpm for 10 min, and blood urea nitrogen (BUN), creatinine (Scr), and uric acid were measured as baseline values. Except for the normal control group, rats in the other groups were administered adenine (dissolved in physiological saline) 200 mg / kg by gavage once daily for 4 weeks. In the last week, no model was established, only the drug was administered. Blood samples were collected from the orbital sinus of rats at 7, 14, 21, 28, and 35 days of age. The samples were centrifuged at 3000 rpm for 10 min to obtain serum for testing blood urea nitrogen (BUN), creatinine (Scr), and uric acid levels. Urine output over 24 hours was recorded, and urinary protein and creatinine levels were measured to calculate creatinine clearance. At the experimental endpoint, the rats were dissected, and the kidneys were removed. The renal cortex was separated on ice, weighed, and organ indices were calculated. The kidneys were then fixed in 4% neutral formaldehyde solution, embedded in paraffin, stained with hematoxylin and eosin (HE), examined under a light microscope, and the slides were read.

[0133] Experimental results: 1) Animal body weight changes are as follows

[0134] As shown in Table 7.

[0135] Table 7

[0136]

[0137]

[0138] Note: The number of rats on day 0 was 10 per group, with half males and half females, compared with the model group. * P≤0.05, ** P≤0.01, *** P≤0.001

[0139] Depend on

[0140] Table 7 shows that, compared with the model group, the weight of rats in the normal control group increased significantly on days 7, 11, and 14 of the experiment; the weight of rats in the normal control group increased significantly on days 18, 21, and 28; and the weight of rats in the normal control group increased very significantly on days 25 and 32 (P≤0.001). The weight of rats in the dexamethasone group decreased compared with the model group starting from day 11, with significant decreases on days 18 and 28, suggesting that it is related to the side effects of hormones. No significant effect was observed on weight in the Cs-4-P1-2 group, the Cs-4-P group, and the fermented cordyceps powder group.

[0141] 2) The kidney weight and organ coefficient of rats are shown in Table 8 below.

[0142] Table 8

[0143] Group 5w weight 5w kidney weight 5w organ coefficient normal control group <![CDATA[333.55±88.76 *** ]]> <![CDATA[2.40±0.74 *** ]]> <![CDATA[0.72±0.09 *** ]]> Model group 229.44±62.94 9.24±3.37 3.98±0.90 Dexamethasone (0.1 mg / kg / day) 175.08±14.54 <![CDATA[5.72±0.68 * ]]> 3.29±0.51 Okra (1g / kg / day) 266.80±45.23 9.89±4.33 3.66±1.52 Cs-4-P1-2 (16 mg / kg / day) 230.99±52.21 6.99±3.43 <![CDATA[2.94±0.92 * ]]> Cs-4-P (448 mg / kg / day) 261.16±57.00 8.69±2.69 3.35±0.92 Fermented Cordyceps sinensis powder (1600mg / kg / day) 233.04±57.48 7.02±2.54 <![CDATA[2.97±0.60 * ]]>

[0144] Note: Male and female rats were counted together and compared with the model group. * P≤0.05, ** P≤0.01, *** P≤0.001

[0145] As shown in Table 8 above, the body weight of the model group was lower than that of the normal control group, while the kidney weight and renal organ coefficient were higher than those of the normal control group, all with highly significant differences (P≤0.001). Compared with the model group, the kidney weight of rats in the dexamethasone group was significantly decreased (P≤0.05), and the kidney weight of rats in the Cs-4-P1-2, Cs-4-P, and fermented cordyceps powder groups showed a decreasing trend. Compared with the model group, the renal coefficient of the normal control group, the Cs-4-P1-2, and fermented cordyceps powder groups was significantly reduced (P≤0.05), and the Cs-4-P group showed a decreasing trend.

[0146] 3) The changes in serum BUN in rats during the experiment are shown in Table 9 below.

[0147] Table 9

[0148]

[0149] Note: Male and female rats were combined for statistical analysis. Compared with the model group, * P≤0.05, ** P≤0.01, *** P≤0.001

[0150] Blood urea nitrogen (BUN) in serum is an important indicator of glomerular filtration function in clinical practice. Gavage administration of adenine can lead to an increase in serum BUN in rats. As shown in Table 9, compared with the model group, the BUN level in the normal control group was 5.22±1.16 mmol / L one week after modeling, while the BUN level in the model group ranged between 13.99±2.95 mmol / L and 17.00±6.31 mmol / L, with a highly significant difference between the two groups (P≤0.001). At 2 weeks (one week of administration), the BUN levels in the *Abelmoschus manihot* group and the Cs-4-P1 group were significantly reduced (P≤0.05). At 4 weeks (three weeks of administration), the BUN levels in the *Abelmoschus manihot* group, the Cs-4-P1-2 group, and the fermented *Cordyceps militaris* powder group were significantly reduced (P≤0.05). At 5 weeks (four weeks of administration), the BUN levels in the dexamethasone group and the Cs-4-P1-2 group were significantly reduced (P≤0.05). During the administration period, the BUN levels in the Cs-4-P group and the fermented *Cordyceps militaris* powder group showed a decreasing trend. Therefore, it can be concluded that Cs-4-P1-2 can improve serum BUN in rats with chronic renal failure and has a significant therapeutic effect on chronic renal failure.

[0151] 4) The changes in serum CREA during the experiment are shown in Table 10 below.

[0152] Table 10

[0153]

[0154] Note: Male and female rats were combined for statistical analysis. Compared with the model group, * P≤0.05, ** P≤0.01, *** P≤0.001

[0155] Serum creatinine (CREA) is an important indicator of kidney function, and elevated CREA indicates impaired kidney function. As shown in Table 10, compared with the model group, the CREA level in the normal control group was 27.20±5.73 μmol / L one week after modeling, while the levels in the model group ranged between 58.28±13.40 μmol / L and 65.86±11.13 μmol / L, showing a highly significant difference between the two groups (P≤0.001). At week 2 (one week of administration), the CREA level in the dexamethasone group was significantly reduced (P≤0.001), while the CREA levels in the okra group and the Cs-4-P1-2 group were significantly reduced (P≤0.05). At week 3 (two weeks of administration), the CREA level in the dexamethasone group was significantly reduced (P≤0.01), and the other groups showed a decreasing trend. At week 4 (three weeks of administration), the CREA levels in all groups showed a decreasing trend. At week 5 (four weeks of administration), the CREA level in the dexamethasone group was significantly reduced, and the other groups showed a decreasing trend. Therefore, it can be concluded that Cs-4-P1-2 can improve serum CREA in rats with chronic renal failure and has a significant therapeutic effect on chronic renal failure.

[0156] 5) The changes in UA during the experiment are shown in Table 11 below.

[0157] Table 11

[0158]

[0159] Note: Male and female rats were combined for statistical analysis. Compared with the model group, * P≤0.05, ** P≤0.01, *** P≤0.001

[0160] Table 11 shows that, compared with the model group, the UA level in the normal control group was 91.00±12.51 μmol / L one week after modeling, while the UA level in the model group ranged between 113.75±24.84 μmol / L and 133.29±18.50 μmol / L, with a highly significant difference between the two groups (P≤0.001). Two weeks into the experiment, i.e., one week after administration, the UA level in the normal control group was significantly lower, while the UA level in the dexamethasone group was significantly lower (P≤0.001). The levels in the okra group and Cs-4-P group were also significantly lower. The levels of UA in the dexamethasone group were significantly reduced (P≤0.05), and the levels of UA in the Cs-4-P1-2 and fermented Cordyceps mycelium powder groups were significantly reduced (P≤0.01). In the 3-week experiment (two weeks of administration), the UA level in the dexamethasone group was significantly reduced, while the other groups showed a decreasing trend. In the 4-week experiment (three weeks of administration), the UA level in the fermented Cordyceps mycelium powder group showed a significant trend, while the other groups showed a decreasing trend. In the 5-week experiment (four weeks of administration), the UA level in the dexamethasone group was significantly increased, the Cs-4-P1-2 level was significantly increased, and the other groups showed an increasing trend.

[0161] 6) Kidney pathology.

[0162] Adenine-induced chronic renal failure can cause severe renal tissue lesions, simulating the end-stage renal failure state. Histopathological sections of the kidneys stained with hematoxylin and eosin (HE) and observed under a light microscope showed the following results: In the normal control group, the kidney tissue structure of rats was normal, with thin and clearly defined glomerular vascular loops, normal endothelial and mesangial cell numbers, and normal peripheral tubules. Compared with the normal control group, the model group showed glomerular fibrosis, tubular atrophy or dilation, foreign body filling within the tubules and glomeruli, and extensive fibrous tissue proliferation and inflammatory cell infiltration in the interstitium. Compared with the model group, the Cs-4-P1-2 treatment group showed an increase in residual glomerular units and a significantly lower pathological score (P≤0.01), while the Abelmoschus manihot and fermented Cordyceps militaris powder groups showed significantly lower pathological scores (P≤0.05). This indicates that Cs-4-P1-2 and fermented Cordyceps militaris powder have a mitigating and restorative effect on renal pathological changes induced by long-term adenine gavage, with Cs-4-P1-2 showing the best effect.

[0163] Table 12

[0164] Group Pathological score normal control group <![CDATA[0.00±0.00 *** ]]> Model group 9.00±0.00 Dexamethasone (0.1 mg / kg) 8.60±0.55 Okra (1g / kg / day) <![CDATA[8.43±0.45 * ]]> Cs-4-P1-2 (16 mg / kg / day) <![CDATA[8.31±0.37 ** ]]> Cs-4-P 8.58±0.49 Fermented Cordyceps mycelium powder group (1600mg / kg / day) <![CDATA[8.31±0.88 * ]]>

[0165] Note: Comparison with model group * P≤0.05, ** P≤0.01, *** P≤0.001

[0166] The effects of Cs-4-P1 on the kidneys were assessed based on the remaining glomeruli and nephrons. Table 12 shows that, compared to the model group, the pathological score of the normal control group was 0, while the pathological scores of rats in the *Abelmoschus manihot* group and the fermented *Cordyceps militaris* powder group were reduced. The Cs-4-P group showed a decreasing trend, and the pathological score of rats in the Cs-4-P1-2 group was significantly reduced.

[0167] 7) The rat mortality rate at the experimental endpoint is shown in Table 13 below.

[0168] Table 13

[0169] Group Original number Number of survivors mortality rate(%) normal control group 10 10 0.00 Model group 10 7 30.00 Dexamethasone (0.1 mg / kg) 10 5 50.00 Okra (1g / kg / day) 10 7 30.00 Cs-4-P1-2 (16 mg / kg / day) 10 8 20.00 Cs-4-P 10 6 30.00 Fermented Cordyceps mycelium powder group (1600mg / kg / day) 10 8 20.00

[0170] Table 13 shows that all model groups experienced mortality during the experiment, with the dexamethasone group exhibiting the highest mortality rate at 50%, which was attributed to the side effects of the hormone. The mortality rates of the Cs-4-P1-2 group and the fermented Cordyceps powder group were both 20%, lower than those of the model group.

[0171] In summary, under the conditions of this experiment, the effects of fermented Cordyceps militaris powder components on adenine-induced chronic renal failure were studied from several aspects, including observation during the rat experiment, body weight, serum biochemical indicators, and total pathological score. The experimental results showed that the model group was successfully established. Compared with the model group, the Cs-4-P1-2 group showed relatively effective serum biochemical indicators, reduced mortality, and could alleviate the effects of adenine-induced chronic renal failure. Furthermore, it can be seen that the effect of Cs-4-P1-2 of this invention on adenine-induced chronic renal failure is significantly better than that of dexamethasone, okra, and fermented Cordyceps militaris powder and its crude polysaccharide (Cs-4-P).

[0172] Example 5: Pharmacodynamic study of inhibiting immune rejection response

[0173] Test method:

[0174] (1) Establishment of a skin grafting model

[0175] According to the reference (Cai Chunxiao, Ma Chunmei, et al. Establishment of a mouse xenogeneic skin transplantation model and evaluation of the efficacy of immunosuppressive drugs. Chinese Journal of Pharmacology, 2016, 32(11):1613-1619.), BALB / c mice and C57 / BL6 mice were anesthetized with sodium pentobarbital (40 mg / kg), and the ears of the donor mice and the back skin of the recipient mice were disinfected with 70% alcohol. The ears of the donor mice were removed from the base and placed in cold sterile PBS for later use. The epidermis of one side of the back of the recipient mouse was cut off (about 1 cm in diameter). The inner and outer skin of the donor mouse's ear were separated, and the inner skin was taken, with the separated side facing down, and attached to the back of the recipient mouse. The skin that was not completely aligned was removed and repaired with scissors, and then a sterile wound dressing was applied and sutured in place with silk thread. After 2 days, the wound dressing was gently cut off to expose the transplantation site, and the transplant rejection reaction was evaluated. In this experiment, xenografting involved transplanting skin from C57 / BL6 mice into BALB / c mice, while isografting involved transplanting skin from BALB / c mice into BALB / c mice.

[0176] (2) Graft Rejection Score

[0177] According to the literature, the rejection reaction of transplanted skin is divided into grades 0-5 (as shown in Table 14).

[0178] Table 14

[0179] level transplant rejection 0 The skin is intact and there is no rejection reaction. 1 Rejection has just occurred 2 >25% skin necrosis 3 >50% skin necrosis 4 >75% skin necrosis 5 Complete skin necrosis (>95%)

[0180] Note: Transplant rejection is expressed as the percentage of necrotic skin to total skin. Skin damage caused by sutures, suture materials, skin displacement, and other traumas is not included in the statistics.

[0181] When the rejection level of a skin graft reaches grade 5, it can be defined as the death of the transplanted skin.

[0182] (3) Dosage design and basis

[0183] The clinical human dosage of cyclosporine as an immunosuppressant is 15 mg / kg / day, and the mouse dosage is 150 mg / kg. The human dosage of fermented cordyceps powder is 3 g to 6 g / day, and the dosage of fermented cordyceps powder is set at 3000 mg / kg / day for mice. Based on the yield, the dosage of Cs-4-P1-2 is set at 30 mg / kg / day for mice.

[0184] Table 15

[0185]

[0186] **P<0.01 vs. Model** *P<0.05 vs. Model##P<0.01 vs. Blank#P<0.05 vs. Blank

[0187] The skin will be scored starting from the fourth day.

[0188] Table 15 shows that after transplantation, the scores of the blank (allogeneic skin transplant) group were lower than those of the model group, and there was a highly significant difference between the two groups after 14 days (P<0.01). The scores of the cyclosporine group were consistently lower than those of the model group from 6 to 13 days, with a highly significant difference (P<0.01). After 14 days, the transplanted skin began to scorch extensively, showing no difference from the model group. The scores of the fermented cordyceps powder group were consistently lower than those of the model group from 6 to 12 days, with a highly significant difference. After 13 days, the transplanted skin began to scorch extensively, showing no difference from the model group. The scores of the Cs-4-P1-2 group were lower than those of the model group from 10 to 13 days, with a highly significant difference (P<0.01). After 14 days, the transplanted skin began to scorch extensively, showing no difference from the model group. This indicates that cyclosporine, Cs-4-P1-2, and fermented cordyceps powder have a significant inhibitory effect on the immune rejection response of mouse skin transplantation, with Cs-4-P1-2 showing a dose advantage.

[0189] 2) Comparison of body weights of each group

[0190] Table 16

[0191]

[0192]

[0193] **P < 0.01 vs model; *P < 0.05 vs model; ##P < 0.01 vs blank; #P < 0.05 vs blank

[0194] As can be seen from Table 16, after transplantation, there was no difference in body weight between the blank (allogeneic skin transplantation) group, the Cs-4-P1-2 group and the model group; the body weight of the mice in the cyclosporine group increased slowly after transplantation and was always lower than that of the model group, with a highly significant difference (P < 0.01); the body weight of the mice in the fermented cordyceps mycelium powder group was lower than that of the model group within 7 - 12 days, with a significant difference. It is suggested that compared with cyclosporine, Cs-4-P1-2 has less side effects and higher safety.

[0195] Study on the effect of Example 6 on hyperlipidemic zebrafish

[0196] Test method:

[0197] (1) Experimental animals

[0198] The zebrafish were all raised in fish culture water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; the pH was 6.5 - 8.5; the hardness was 50 - 100 mg / L CaCO3), provided by the fish culture center of our company, and the license number for the use of experimental animals was: SYXK(Zhe)2012 - 0; the feeding management met the requirements of international AAALAC certification (certification number: 001458).

[0199] The melanin allelic mutant translucent Albino strain zebrafish were bred in the way of natural paired mating. The zebrafish at 7 days post-fertilization (7 dpf) were used for the determination of the maximum test concentration (MTC) of lipid-lowering efficacy.

[0200] The melanin allelic mutant translucent Albino strain zebrafish were bred in the way of natural paired mating. The zebrafish at 5 dpf were used for the evaluation of lipid-lowering efficacy.

[0201] (2) Detection method

[0202] a. Determination of MTC

[0203] 7dpf melanin allele mutant translucent Albino zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water solution (concentrations shown in Table 17), and a normal control group was also included. The volume of each well was 3 mL. After treatment at 28℃ for 48 h, the MTC of the samples compared to normal zebrafish was measured.

[0204] b. Evaluation of lipid-lowering efficacy

[0205] A hyperlipidemia model was established in zebrafish of the Albino strain (5 dpf melanin allele mutant) by randomly selecting zebrafish and administering water-soluble egg yolk powder. After 16 h of feeding, the zebrafish were randomly assigned to 6-well plates, with 30 zebrafish per well. Samples (concentrations shown in Tables 17-18) were administered water-soluble, along with a positive control of atorvastatin calcium at a concentration of 0.240 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. After 48 h of treatment at 28℃, the zebrafish homogenate was homogenized, and the supernatant was collected. Triglyceride and total cholesterol reagent kits were used for reaction, and the OD values ​​of triglycerides and total cholesterol in each experimental group were measured using a multi-functional microplate reader. The triglyceride content (C1) and total cholesterol content (C2) were analyzed, and the lipid-lowering efficacy of the samples was evaluated based on statistical analysis of these indicators. Statistical results are expressed as mean ± SE. The formula for calculating the lipid-lowering efficacy is as follows:

[0206] 1) Triglyceride-lowering effect:

[0207]

[0208] 2) Effect of lowering total cholesterol:

[0209]

[0210] Statistical analysis was performed using SPSS 26.0 software. p<0.05 indicated that the difference was statistically significant.

[0211] Test results

[0212] Under the experimental conditions, the MTC (minimum toxic concentration) of fermented Cordyceps militaris powder and Cs-4-P1-2 in normal zebrafish were 300 and 10.0 μg / mL, respectively. See Table 17 for details.

[0213] Table 17

[0214]

[0215] Evaluation of lipid-lowering efficacy

[0216] Under the experimental conditions, fermented Cordyceps militaris powder and Cs-4-P1-2 have the effect of lowering triglycerides; Cs-4-P1-2 has the effect of lowering total cholesterol, as detailed in Table 18.

[0217] Table 18

[0218]

[0219] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0220] Table 19

[0221]

[0222] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0223] As shown in Table 19 above, Cs-4-P1-2 exhibits superior triglyceride-lowering efficacy when the concentration is between 1.0 and 10.0 μg / mL, with the best triglyceride-lowering efficacy at 3.00 μg / mL. Its triglyceride-lowering activity is superior to that of atorvastatin calcium.

[0224] Table 20

[0225]

[0226] Compared with the model control group, *p<0.05

[0227] Table 21

[0228]

[0229] Compared with the model control group, *p<0.05, **p<0.01

[0230] As shown in Table 21 above, Cs-4-P1-2 has better total cholesterol-lowering effects when the concentration is between 1.0 and 10.0 μg / mL, and the total cholesterol-lowering effect is best when the concentration is between 1.00 and 3.00 μg / mL, which is significantly better than fermented cordyceps powder and atorvastatin calcium.

[0231] Example 7: Effect of cisplatin on acute kidney injury in mice

[0232] Test method:

[0233] Mice were fasted for 12 hours before surgery but had free access to water. Except for the control group, all other groups received a single intraperitoneal injection of cisplatin solution at a dose of 10 mg / kg, while the control group received an equal volume of physiological saline. Fifteen minutes later, mice were administered amifostine, Cs-4-P1, and Cs-4-P1-2, respectively, according to their assigned groups. Blood samples were collected on days 1, 3, and 5 post-administration to measure serum urea nitrogen (BUN) and creatinine (Scr).

[0234] Results: As shown in Table 22, serum urea nitrogen and creatinine in the control group tended to stabilize on days 1, 3 and 5. Serum urea nitrogen and creatinine in the model group began to rise on day 3 and remained at a high level on day 5. Compared with the control group, serum urea nitrogen and creatinine in the model group showed statistically significant differences on days 3 and 5 (p<0.05, p<0.01). On days 3 and 5 after administration of the positive control drug amifostine (300 mg / kg), the serum urea nitrogen and creatinine levels in mice significantly decreased compared to the model group (p<0.05, p<0.01). Similarly, on days 3 and 5 after administration of the test drug Cs-4-P1 (160 mg / kg), the serum urea nitrogen and creatinine levels in mice significantly decreased compared to the model group (p<0.05, p<0.01). After administration of Cs-4-P1-2 (160 mg / kg and 80 mg / kg), the serum urea nitrogen and creatinine levels in mice significantly decreased on days 3 and 5, showing statistically significant differences compared to the model group (p<0.05, p<0.01). Therefore, the effect of Cs-4-P1-2 at a dose of 160 mg / kg on improving serum urea nitrogen and creatinine is consistent with that of the positive control drug amifostine (300 mg / kg). It is evident that Cs-4-P1-2 has therapeutic and ameliorative effects on acute kidney injury in mice.

[0235] Table 22

[0236]

[0237] Compared with the control group, ## p < 0.01; compared with the model group, *p < 0.05, **p < 0.01

[0238] Example 8: Effect of LPS on acute kidney injury in mice

[0239] Test method:

[0240] Mice were fasted for 12 hours before surgery but had free access to water. Except for the control group, all other groups received a single intraperitoneal injection of 20 mg / kg LPS solution, while the control group received an equal volume of physiological saline. Fifteen minutes later, dexamethasone and Cs-4-P1-2 were administered according to the respective groups. Animals were sacrificed 36 hours later, and serum creatinine and blood urea nitrogen were measured.

[0241] Experimental results:

[0242] The results are shown in Table 23. Compared with the control group, the serum urea nitrogen and creatinine levels of the model group mice were 17.69±1.13mmol / L and 45.35±5.85μmol / L, respectively, showing statistical differences (p<0.01). Mice administered 10 mg / kg of the positive control drug dexamethasone showed significantly lower serum urea nitrogen and creatinine levels (p<0.05) compared to the model group. In the low, medium, and high dose Cs-4-P1-2 groups, serum urea nitrogen levels decreased to 14.41±1.73, 13.20±1.72, and 12.72±0.55 mmol / L, respectively, and serum creatinine levels decreased to 35.15±26.22, 26.34±2.75, and 24.33±9.65 μmol / L, respectively. Compared to the model group, the medium and high dose Cs-4-P1-2 groups showed statistically significant differences in serum urea nitrogen and creatinine levels (p<0.05, p<0.01, p<0.001). It is evident that the high-dose Cs-4-P1-2 group had effects on serum urea nitrogen and creatinine in LPS-induced acute kidney injury mice comparable to those of the positive control drug dexamethasone. Therefore, Cs-4-P1-2 has a therapeutic and ameliorative effect on acute kidney injury in mice.

[0243] Table 23

[0244]

[0245] Figure 8 According to an embodiment of the present invention 1 H- 13 C HSQC diagram. Figure 11 These are pathological slide images according to embodiments of the present invention.

Claims

1. A Cs-4 mycelial heteropolysaccharide of the genus Fer- mentiiporia, characterized in that, The structural formula is shown as follows: ， wherein Manp is pyranose mannose, Galp is pyranose galactose, Glcp is pyranose glucose, and n is selected from 6-104.

2. Cs-4 fermenting mycelial heteropolysaccharide according to claim 1, characterized in that, The relative molecular weight of the polysaccharide is selected from 10-152 kDa.

3. Cs-4 fermenting mycelial heteropolysaccharide according to claim 1 or 2, characterized in that, The polysaccharide is composed of glucose, galactose and mannose, and the molar ratio of the glucose, galactose and mannose is 1:(1.0-2.0):(1.5-2.5).

4. A method for extracting the Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1-3, characterized in that, 1) defatting treatment of the Cs-4 mycelium powder with ethanol, discarding the ethanol extract, and obtaining a defatted residue, 2) extraction treatment of the residue in water, and obtaining a water extract, 3) alcohol precipitation treatment of the water extract, and the solvent for the alcohol precipitation treatment is ethanol, 4) purification treatment of the precipitate after the alcohol precipitation treatment in step 3), and obtaining the Cs-4 fermented mycelium heteropolysaccharide.

5. The method of claim 4, wherein, The defatting treatment is performed at a temperature of 100°C; Optionally, the defatting treatment is performed three times, each time for 1 hour; Optionally, the amount of ethanol in the defatting treatment is 10 times the amount of Cs-4; Optionally, the ethanol in the defatting treatment is 85%-100% ethanol; Optionally, the extraction treatment is performed at a temperature of 75°C-78°C; Optionally, the extraction treatment is performed three times, each time for 1 hour; Optionally, the amount of water in the extraction treatment is 10 times the amount of residue; Optionally, the ethanol in the alcohol precipitation treatment is 80% ethanol.

6. A method for extracting the Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1-3, characterized in that, 1) defatting treatment of the Cs-4 mycelium powder with 10 times the amount of 85% ethanol, heating extraction for 3 times, each time for 1 hour, discarding the ethanol extract, and obtaining a residue; 2) extraction treatment of the residue in 10 times the amount of water, heating extraction for 3 times, each time for 1 hour, and combining the water extract; 3) alcohol precipitation treatment of the water extract with 80% ethanol, and obtaining a precipitate; 4) purification treatment of the precipitate, and obtaining the Cs-4 fermented mycelium heteropolysaccharide.

7. The extraction method according to any one of claims 4-6, characterized in that, The purification treatment includes deproteinization treatment, decolorization treatment, and column purification treatment.

8. A pharmaceutical composition, characterized by, The Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1-3 or obtained by the method according to any one of claims 4-7.

9. Use of the Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1-3 or obtained by the method according to any one of claims 4-7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, and / or preventing or treating acute kidney injury.

10. A pharmaceutical composition for preventing or treating hyperlipemia, characterized by, The Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1-3 or obtained by the method according to any one of claims 4-7 is contained in an amount of 1.0-10.0 µg / mL.

11. The pharmaceutical composition of claim 10, wherein, The concentration of the polysaccharide is 3.0 µg / mL.

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