Cs-4 polysaccharide, and preparation method and application thereof

Abstract

The application discloses a kind of Cs-4 polysaccharide and its preparation method and its application in the prevention and treatment of high uric acid and gout drug, belong to the field of prevention and treatment of high uric acid and gout in traditional Chinese medicine, the preparation method of the present application is as follows: after fermentation cordyceps sinensis powder (Cs-4) is defatted, papain is used for enzymolysis, supernatant is collected by centrifugation, dialysis, alcohol precipitation is obtained Cs-4 polysaccharide.The preparation method of the present application is as follows: Cs-4 polysaccharide is filled into 2# capsule by powder direct filling capsule process, control environment relative humidity is below 65%RH, each grain is loaded 0.38g, namely obtained.A kind of drug or health food composition is prepared by the extraction method of Cs-4 polysaccharide.The in vivo animal experiment results show that when Cs-4 polysaccharide is administered to hyperuricemia rats by gavage, the uric acid level in serum can be reduced, and the uric acid content of Cs-4 polysaccharide 40mg / kg group is significantly reduced, which is equivalent to the effect of positive drug dibenzyline tablet group, and has good application prospect.

Description

Technical Field

[0001] This invention relates to a fermented Cordyceps militaris powder (Cs-4) polysaccharide, its preparation method, and its application in the prevention and treatment of hyperuricemia and gout. This invention belongs to the field of traditional Chinese medicine for the prevention and treatment of hyperuricemia and gout. Background Technology

[0002] Cordyceps sinensis is a rare and precious traditional Chinese medicine unique to my country. Fermented Cordyceps sinensis powder (Cs-4), derived from Cordyceps sinensis, is superior to it. Existing literature reports that fermented Cordyceps sinensis powder (Cs-4) has the closest chemical composition to natural Cordyceps sinensis. Fermented Cordyceps sinensis powder (Cs-4) is produced by Jiangxi Guoyao Co., Ltd. (formerly Jiangxi Guoyao Factory) using room-temperature bio-fermentation technology. It is extracted from natural Cordyceps sinensis, fermented, pulverized, and refined. This invention defines this fermented Cordyceps sinensis powder as fermented Cordyceps sinensis powder (Cs-4), also known as Cordyceps sinensis powder.

[0003] The incidence of hyperuricemia is increasing year by year, and the age of onset is becoming younger. This disease is a metabolic disorder caused by excessive uric acid production or / or decreased uric acid excretion due to purine metabolism disorders. It can lead to renal insufficiency and is an important factor in inducing gout, kidney disease, and hypertension. Currently, drugs for treating hyperuricemia include allopurinol, colchicine, and benzbromarone, but these often cause liver and kidney damage and gastrointestinal adverse reactions. Therefore, finding safe and non-toxic uric acid-lowering drugs has become a current research hotspot.

[0004] Fermented Cordyceps sinensis powder (Cs-4), a medicine with Chinese characteristics, represents a new achievement in my country's drug research and application. Its formulation, Jinshuibao capsules / tablets, has the effects of tonifying the lungs and kidneys, replenishing essence and qi, and is widely used clinically in the treatment of kidney diseases. However, its effects on the prevention and treatment of hyperuricemia and gout are rarely reported or studied.

[0005] According to literature reports, fermented Cordyceps militaris powder (Cs-4) mainly contains small molecules such as nucleosides, sterols, fatty acids, and amino acids, as well as large molecules such as polysaccharides and polypeptides. Traditionally processed Jinshuibao products have shown significant clinical efficacy, but the specific effective components and mechanisms of action are not yet fully understood. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide the preparation of fermented Cordyceps militaris powder (Cs-4) polysaccharide and its application in drugs for the prevention and treatment of hyperuricemia and gout.

[0007] The technical solution of the present invention is as follows:

[0008] To address the shortcomings of existing technologies, this invention provides a method for preparing Cs-4 polysaccharide and its application in drugs for the prevention and treatment of hyperuricemia and gout.

[0009] In a first aspect, the present invention provides a method for preparing Cs-4 polysaccharide. According to an embodiment of the present invention, the method includes: S1: defatting fermented Cordyceps militaris powder (Cs-4) with ethanol, discarding the ethanol extract to obtain a defatted residue; S2: extracting the residue using an enzymatic method to obtain crude Cs-4 polysaccharide; S3: subjecting the crude Cs-4 polysaccharide to ultrafiltration to obtain Cs-4 polysaccharide. The method according to the embodiment of the present invention is simple to operate, has high extraction efficiency, and the purity of the extracted polysaccharide reaches over 80%.

[0010] Preferably, the preparation method may further include at least one of the following additional technical features:

[0011] The degreasing process is performed three times, each time lasting 0.5-1.5 hours.

[0012] In the defatting process, the amount of ethanol is 8-12 times the mass of the fermented Cordyceps militaris powder (Cs-4).

[0013] The defatted ethanol is 85-100% ethanol by volume.

[0014] The enzymatic extraction process was carried out under the condition that the added enzyme was papain.

[0015] The experiment was conducted under conditions where 0.8-3.0 wt% of papain was added.

[0016] The extraction process was carried out at a temperature of 38℃-42℃.

[0017] The extraction time for the extraction process is 0.5-3 hours.

[0018] The enzyme solution added during the extraction process is 8-12 times its volume.

[0019] The ultrafiltration filter has a filter cutoff of 10 kDa to 150 kDa.

[0020] In another aspect, the present invention also provides a pharmaceutical or health food composition. The pharmaceutical composition comprises the aforementioned Cs-4 polysaccharide. Its formulation is selected from, but is not limited to, any one of the following: tablets, suspensions, granules, pills, powders, drop pills, syrups, mixtures, elixirs, effervescent tablets, pastes, emulsions, teas, powders, injections, gels, plasters, ointments, creams, lotions, suppositories, and capsules.

[0021] In another aspect, the invention also proposes the use of the aforementioned Cs-4 polysaccharide in the preparation of a pharmaceutical for the prevention or treatment of hyperuricemia or gout. The inventors have found that this polysaccharide has superior efficacy in preventing or treating hyperuricemia, achieving better results with a lower dosage compared to Jinshuibao (a traditional Chinese medicine).

[0022] In another aspect, the present invention also proposes a formulation molding process for the aforementioned Cs-4 polysaccharide, wherein the formulation is a hard capsule, and the pharmaceutical powder is directly filled into No. 2 capsules using a Cs-4 polysaccharide powder filling process, with each capsule containing 0.38g, and the relative humidity of the environment is controlled below 65%RH.

[0023] Preferably, the capsule filling process involves direct filling with medicinal powder.

[0024] Preferably, the capsule size is 2# capsule.

[0025] Preferably, each capsule contains 0.38g.

[0026] Preferably, the relative humidity of the environment is controlled to be below 65%RH.

[0027] 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 terms "about" or "approximately" mean within an acceptable standard error 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.

[0028] The beneficial effects of this invention are:

[0029] The polysaccharide component obtained from fermented Cordyceps militaris powder (Cs-4) through a series of extraction, separation, and purification processes can be used to prevent or treat hyperuricemia or gout. Moreover, this polysaccharide component has a superior effect in preventing or treating hyperuricemia, achieving better results with a lower dosage compared to Jinshuibao (a traditional Chinese medicine). Attached Figure Description

[0030] Figure 1 This is the HPSEC pattern of Cs-4 polysaccharide according to Example 2 of the present invention;

[0031] Figure 2 This is a monosaccharide composition diagram of Cs-4 polysaccharide according to Example 2 of the present invention;

[0032] Figure 3This is a graph showing the evaluation results of the uric acid-lowering efficacy of Cs-4 polysaccharide according to Example 4 of the present invention;

[0033] Figure 4 The polysaccharide hygroscopicity curve of Cs-4 polysaccharide according to Example 5 of the present invention;

[0034] Figure 5 This is a critical relative humidity curve of Cs-4 polysaccharide according to Example 5 of the present invention. Detailed Implementation

[0035] 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.

[0036] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0037] The abbreviations used in this invention are as follows:

[0038] Fermented Cordyceps sinensis powder (Cs-4): This is the name of the raw material and a proprietary name.

[0039] Cs-4 polysaccharide is the polysaccharide name in this application, UA represents uric acid, and MTC represents minimum toxic concentration.

[0040] Example 1: Preparation method of Cs-4 polysaccharide

[0041] 1. Degreasing: Fermented Cordyceps mycelium powder (Cs-4) is heated to boiling (78℃) with 10 times the amount (mass) of 85% ethanol and extracted three times at a gentle boil for 1 hour each time. The ethanol extract is discarded, and the mycelium powder is dried after defatting and set aside for later use.

[0042] 2. Enzymatic extraction of polysaccharides: Take the defatted bacterial powder above, add 10 times the volume of 1.00wt% papain solution (HAc-NaAc buffer to control pH 5), extract in a 40℃ water bath for 1 hour, then heat to 100℃ and continue for 1 hour to inactivate the enzyme, centrifuge, take the supernatant, concentrate and dry to obtain Cs-4 crude polysaccharide.

[0043] 3. Ultrafiltration: Take the above crude polysaccharide, redissolve it in water, centrifuge, and take the supernatant to be treated by 150kDa and 10kDa ultrafiltration columns in sequence. Concentrate the 10kDa retentate and dry it to obtain the refined Cs-4 polysaccharide.

[0044] Example 2: Cs-4 polysaccharide content, molecular weight distribution, and monosaccharide composition

[0045] 1. Polysaccharide content determination:

[0046] 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.

[0047] 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 wt% 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.

[0048] Test sample detection: Weigh 50 mg of Cs-4 polysaccharide accurately, place it in a 10 mL volumetric flask, sonicate to dissolve, add water to the mark, and shake well. Take 0.2 mL of the above prepared solution into a 20 mL stoppered test tube, add water to 1.0 mL. Add distilled water to 1.0 mL, add 1.0 mL of freshly prepared 5 wt% phenol solution and 5.0 mL of concentrated sulfuric acid, shake well, let stand at room temperature for 10 min, then vortex to fully mix the reaction solution, and then place the test tube in a 30℃ water bath for 20 min, and measure the absorbance at 490 nm.

[0049] The results showed that the Cs-4 polysaccharide content was 83.2%.

[0050] 2. Molecular weight determination

[0051] 2.1 Reagent Preparation

[0052] Take 0.1 mg of sample, add 1 mL of mobile phase, and analyze the HPSEC spectrum of polysaccharide using gel size exclusion chromatography-multi-angle laser light scattering-refractive index detector (HPSECMALLS-RI). The molecular weight distribution of polysaccharide was also analyzed.

[0053] 2.2 Chromatographic conditions

[0054] The chromatographic column was a TSK-GEL G6000PWXL tandem with a TSK-GEL G4000PWXL; a 2414 differential refractive index detector (Waters, USA) and a DAWN 8+ laser detector (Wyatt, USA); the mobile phase was 0.15 mol / L NaNO3 and 0.05 mol / L NaH2PO4·2H2O (pH 7.0), the flow rate was 0.5 mol / L, and the sample loading volume was 100 μL. Molecular weight was calculated using ASTRA 6.1, corrected with 5 mg / mL pullulan standard. The refractive index increment (dn / dc) of the polysaccharide in solution was calculated as 0.146 mL / g. Each sample was repeated twice.

[0055] Test results: such as Figure 1 The Cs-4 polysaccharide shown contains two polysaccharide fractions, with a main peak weight-average molecular weight of 4.540 × 10⁻⁶. 4 (±0.710%), the second peak weight-average molecular weight is 6.460×10⁻⁶. 6 (±0.164%).

[0056] 3. Determination of monosaccharide composition

[0057] Detection method: 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℃ for 3 h, cool to room temperature, dry with nitrogen at 40℃, 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 make up 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.

[0058] Test results: such as Figure 2 As shown, the monosaccharide composition of Cs-4 polysaccharide is galactose: glucose: mannose ≈ 4:3:2, and it also contains a small amount of arabinose.

[0059] Example 3: Evaluation of the uric acid-lowering efficacy in a mouse model with hyperuricemia.

[0060] Test method:

[0061] Seventy qualified ICR mice, SPF grade, half male and half female, weighing 17.2–23.2 g, were randomly divided into 7 groups according to sex and weight: a blank control group, a model control group, a benzbromarone tablet group (13 mg / kg), a Cs-4 polysaccharide group (20 mg / kg), a Cs-4 polysaccharide group (40 mg / kg), a Cs-4 polysaccharide group (80 mg / kg), and a Cs-4 bacterial powder group (1 g / kg), with 10 animals in each group. Each group was administered the corresponding concentration orally at 20 mL / kg via gavage. The blank control group and the model control group received an equal volume of pure water. Administered once daily for 7 consecutive days. Thirty minutes before the last administration, 400 mg / kg of potassium oxonate was injected intraperitoneally at 20 mL / kg. One hour after administration, blood was collected from the orbital venous plexus to measure serum uric acid (UA) levels.

[0062] Experimental results:

[0063] As shown in Table 1, compared with the normal control group, the serum UA level in the model control group was significantly increased (P≤0.01), suggesting that intraperitoneal injection of 400 mg / kg potassium oxonate significantly increased the uric acid level in mice. Compared with the model control group, the UA levels in the benzbromarone tablet group, each dose group of Cs-4 polysaccharide, and Cs-4 bacterial powder (1 g / kg) were significantly decreased (P≤0.05 or P≤0.01).

[0064] Table 1. Significant effects of the test sample on potassium oxonate-induced hyperuricemia in mice.

[0065]

[0066] Note: Compared with the normal control group ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0067] In summary, under the conditions of this experiment, the uric acid-lowering effect of Cs-4 polysaccharide was evaluated based on uric acid levels in mice. The results showed that the model group was successfully established. Compared with the model group, oral administration of 20 mg / kg, 40 mg / kg, and 80 mg / kg of Cs-4 polysaccharide to hyperuricemic rats reduced serum uric acid levels. The 40 mg / kg Cs-4 polysaccharide group showed a significant reduction in uric acid levels, comparable to the effect of the positive control drug allobenzbromarone tablets.

[0068] Example 4: Evaluation of the effect of allopurinol on the zebrafish hyperuricemia model

[0069] Experimental instruments and reagents:

[0070] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); Multifunctional microplate reader (SPARK, TECAN, Switzerland); 6-well plate (Nest Biotech, China); 96-well microplate (Costar, USA).

[0071] Dimethyl sulfoxide (DMSO, lot number BCBZ1685, Sigma, USA); potassium oxonate (lot number C1808048, Shanghai Aladdin Biochemical Technology Co., Ltd., China); sodium xanthine (lot number SLBV3159, Sigma-Aldrich (Shanghai) Trading Co., Ltd., China); Amplex™ Red Uric Acid / Uricase Assay Kit (lot number 2194976, Thermo Fisher Scientific, USA).

[0072] Test method:

[0073] MTC determination: Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in 6-well plates, with 20 zebrafish treated in each well (experimental group). Samples were administered in water at concentrations of 10, 25, 50, 100, and 200 μg / mL, respectively. A normal control group (treated with E3 medium) and a model control group were also included, with a well volume of 3 mL. Except for the normal control group, all experimental groups were induced with potassium oxonate combined with sodium xanthine to establish a hyperuricemic model in zebrafish. After treatment at 28 ℃ for 24 h, the number of dead zebrafish and phenotypic characteristics were recorded in each experimental group, and the MTC of the samples in the model zebrafish was determined.

[0074] 2) Evaluation of uric acid-lowering efficacy: Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in 6-well plates, with 20 zebrafish treated in each well. Samples were administered in water at concentrations of 50, 100, and 200 μg / mL, respectively, with a positive control of allopurinol at 136 μg / mL. A normal control group and a model control group were also included. Each well contained 3 mL of liquid. Except for the normal control group, all other experimental groups were induced to develop a hyperuricemia model using potassium oxonate combined with sodium xanthine. After treatment at 28 ℃ for 24 h, the uric acid content in the zebrafish was detected using a uric acid fluorescence detection kit. The uric acid fluorescence value of the zebrafish was analyzed, and the uric acid-lowering efficacy of the samples was evaluated based on the statistical analysis results. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0075] Experimental results:

[0076] 1) MTC determination: Under the experimental conditions, no zebrafish died in any group, and the condition of the zebrafish was similar to that of the model group, indicating that the MTC of Cs-4 polysaccharide in zebrafish was greater than 200.0 μg / ml. Therefore, the concentrations for the uric acid-lowering evaluation test were set at 200, 100, and 50 μg / ml.

[0077] 2) Evaluation of uric acid-lowering efficacy: Under the experimental conditions, Cs-4 polysaccharide at 200.0 μg / ml showed comparable uric acid-lowering effects to the positive control drug, both being statistically significant (p<0.05). At 50 and 100 μg / ml, it also showed a trend towards lowering uric acid (see reference). Figure 3 ).

[0078] In summary, under the experimental conditions, the uric acid-lowering effect of Cs-4 polysaccharide was evaluated based on the number of deaths, phenotypic characteristics, and uric acid content in zebrafish during the experiment. The results showed that the model group was successfully established. Compared with the model group, the uric acid content in the Cs-4 polysaccharide 200 μg / ml group was significantly reduced, and the effect was comparable to that of the positive control drug allopurinol.

[0079] Example 5: Study on the molding process of Cs-4 polysaccharide

[0080] 1. Dosage form selection

[0081] Preliminary pharmacological studies have confirmed the effectiveness of Cs-4 polysaccharide at doses of 20 mg / kg, 40 mg / kg, and 80 mg / kg. The 40 mg / kg group showed a significant reduction in uric acid levels, comparable to the positive control group (allebenzbromarone tablets). This dose translates to an effective adult dose (60 kg) of 380 mg / day. Since Cs-4 polysaccharide is the effective component, the daily effective adult dose is relatively small, making it suitable for various conventional oral dosage forms. Analysis of the prepared Cs-4 polysaccharide powder revealed good flowability and low hygroscopicity. Therefore, considering both formulation development and production costs, it is proposed to directly encapsulate it into capsules, with each capsule weighing 0.38 g.

[0082] 2. Study on the properties of Cs-4 polysaccharide powder

[0083] The prepared Cs-4 polysaccharide was partially sieved into two parts: one larger than 80 mesh and the other smaller than 80 mesh. A portion of the powder was granulated with 90% ethanol (by volume) for later use.

[0084] 2.1 Material Flowability

[0085] Using an ERWEKA powder particle flowability tester, the above-mentioned powder was poured along the funnel wall into the upper funnel until the powder at the bottom formed a cone. The diameter of the bottom of the cone was measured, and the angle of repose was calculated according to tga=H / R. The results are shown in Table 2 below.

[0086] Table 2. Results of the repose angle of Cs-4 polysaccharide

[0087]

[0088] The angle of repose of Cs-4 polysaccharide powder is 28°. The angle of repose after sieving to less than 80 mesh and greater than 80 mesh, as well as after granulation, is around 30°. The powder has good flowability and is suitable for direct filling production in large-scale capsule and other equipment without further processing.

[0089] 2.2 Material bulk density

[0090] A certain amount of powder was accurately weighed and placed in a 100mL graduated cylinder to read its volume. An ERWEKA bulk density tester was used, with an amplitude of 14mm and a vibration frequency of 300 times, to read the tapped volume. The loose density and tapped density were calculated using the formula: Bulk density ρ (g / ml) = Particle weight (g) / Volume (mL). The results are shown in Table 3 below.

[0091] Table 3. Cs-4 polysaccharide density results

[0092]

[0093] Preliminary pharmacological studies have confirmed the effectiveness of Cs-4 polysaccharide at doses of 20 mg / kg, 40 mg / kg, and 80 mg / kg. The 40 mg / kg group showed a significant reduction in uric acid levels, comparable to the positive control drug allobenzbromarone tablets. This dose translates to an effective adult dose (60 kg) of 380 mg / day. Due to its good flowability, Cs-4 polysaccharide can be directly filled into capsules. The amount of Cs-4 polysaccharide directly filled into each capsule size is shown in Table 4 below.

[0094] Table 4. Cs-4 polysaccharide content in different capsule shell models

[0095]

[0096] Note: Capsule numbers are derived from the national standard YBX-2000-2007 for gelatin empty capsules, and capsule volume is provided by the capsule manufacturer.

[0097] Based on the bulk density results of Cs-4 polysaccharide and the volume calculation of various capsule types, it can be seen that when the effective dose for an adult (60kg) is 380mg / day, Cs-4 polysaccharide is suitable for direct filling with No. 2 capsules, with each capsule containing 0.38g. This results in a daily dose of one capsule, which can reduce the amount and frequency of medication taken by patients and improve their clinical compliance.

[0098] 2.3 Critical relative humidity of materials

[0099] Moisture absorption curve: A small amount of powder was added to a weighing bottle, spreading it evenly to a thickness of about 2 mm at the bottom. It was dried at 105℃ to constant weight, and the weight was accurately measured. The weighing bottle was then placed in a desiccator containing a supersaturated sodium chloride solution and kept at room temperature. The weight was measured periodically, and the moisture absorption percentage was calculated. The results are shown below. Figure 4 See Table 5.

[0100] Table 5 Results of the hygroscopic curve of Cs-4 polysaccharide

[0101]

[0102] The hygroscopicity of Cs-4 polysaccharide powder and granules of various particle sizes showed little difference, all reaching equilibrium on the sixth day, with a final moisture content of approximately 20% at 75% humidity. As shown in the figure above, the hygroscopic equilibrium time for Cs-4 polysaccharide powder is 6 days. To ensure the effectiveness of the hygroscopic equilibrium, the critical relative humidity observation period is planned to be 7 days.

[0103] Critical relative humidity: Weigh 8 portions of Cs-4 polysaccharide powder, 1.5g each, and spread them evenly in flat weighing bottles dried to constant weight. After drying to constant weight, accurately weigh each bottle. Open the caps and place each bottle in a glass desiccator containing concentrated sulfuric acid solution of different concentrations. Store in a constant temperature oven at 25℃ for 7 days. Remove and weigh each bottle accurately. Calculate the moisture absorption percentage. Plot the moisture absorption percentage against relative humidity to obtain a moisture absorption equilibrium curve. Draw two tangents at the inflection point. The relative humidity corresponding to the focus of the tangents is the critical relative humidity. The results are shown below. Figure 5 See Table 6.

[0104] Table 6. Critical Relative Humidity Results for Cs-4 Polysaccharide

[0105]

[0106] The experimental results show that the critical relative humidity of Cs-4 polysaccharide powder is 65%RH. Therefore, the relative humidity of the environment should be controlled below 65%RH during the preparation process.

[0107] 3. Capsule filling research

[0108] The aforementioned powder properties study indicates that Cs-4 polysaccharides can be directly filled into capsules, with the most suitable capsule shell size being #2. This study examines the direct filling of #2 capsules with different Cs-4 polysaccharides. Since no excipients were added, there is no need to measure the capsule content. The feasibility of direct capsule filling is evaluated based on the average fill weight.

[0109] A portion of the prepared Cs-4 polysaccharide powder was sieved into two parts: one larger than 80 mesh and the other smaller than 80 mesh. Another portion of the powder was granulated with 90% ethanol (by volume) for later use. The powder was then filled into #2 hard capsule shells using capsule blister packs, and the fill weight variation was measured. The results are shown in Table 7 below.

[0110] Table 7 Results of the study on Cs-4 polysaccharide direct-filling capsules

[0111]

[0112] The results in the table above show that the filling weight of powders and granules of different particle sizes after filling hard capsules is relatively small, which meets the requirements of the pharmacopoeia. The process of directly filling Cs-4 polysaccharide powder into No. 2 capsules is feasible, and the filling weight meets expectations.

[0113] In summary, the molding process involves preparing CS-4 polysaccharide into hard capsules, and using the powder to directly fill No. 2 capsules, with each capsule containing 0.38g, while controlling the relative humidity of the environment to below 65%RH.

[0114] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. The application of a Cs-4 polysaccharide in the preparation of drugs for the prevention or treatment of hyperuricemia or gout, characterized in that, The preparation method of the Cs-4 polysaccharide includes the following steps: S1: Degrease the fermented Cordyceps militaris powder Cs-4 with ethanol, discard the ethanol extract, and obtain the defatted residue. S2: Extract the residue using an enzymatic method to obtain crude Cs-4 polysaccharide. S3: Perform ultrafiltration on the crude polysaccharide from the fermented Cordyceps militaris powder to obtain Cs-4 polysaccharide. Details are as follows: Degreasing: Fermented Cordyceps sinensis powder Cs-4 was heated to boiling with 10 times its weight in 85% ethanol and extracted three times at a gentle boil for one hour each time. The ethanol extract was discarded, and the defatted powder was dried for later use. Enzymatic extraction of polysaccharides: Take the defatted bacterial powder above, add 10 times the volume of a 1.00wt% papain solution and HAc-NaAc buffer to control the pH to 5, extract in a 40℃ water bath for 1 hour, heat to 100℃ and continue for 1 hour to inactivate the enzyme, centrifuge, take the supernatant, concentrate and dry to obtain Cs-4 crude polysaccharide. Ultrafiltration: Take the above-mentioned crude Cs-4 polysaccharide, redissolve it in water, centrifuge, and take the supernatant to be treated by 150kDa and 10kDa ultrafiltration columns in sequence. Concentrate the 10kDa retentate and dry it to obtain the purified Cs-4 polysaccharide. The Cs-4 polysaccharide composition includes: galactose, mannose, glucose, and arabinose; The mass ratio of galactose, mannose, and glucose is 4:3:

2.

2. A pharmaceutical or health food composition, characterized in that, It contains the Cs-4 polysaccharide as described in claim 1.

3. The pharmaceutical or health food composition according to claim 2, characterized in that, Its formulation can be selected from any one of tablets, suspensions, granules, syrups, gels, plasters, and capsules.

4. The application of the Cs-4 polysaccharide as described in claim 1 in capsule formulations, characterized in that, The process of directly filling No. 2 capsules with Cs-4 polysaccharide powder is adopted, with each capsule containing 0.38g, and the relative humidity of the environment is controlled below 65%RH.

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