A coriolus glycopeptide, and a preparation method and a molecular weight determination method thereof

Abstract

The application discloses a kind of coriolus sugar peptide and its preparation method and molecular weight determination method, its preparation method includes the following steps: taking coriolus polysaccharide crude extract, add corresponding to polysaccharide crude extract 80-120 times water dissolution, filtration, obtain polysaccharide crude extract aqueous solution, then sample to pre-equilibrated Sepharose CL-6B gel column, polysaccharide crude extract aqueous solution volume is equivalent to 0.2-0.4 times column volume, then elution with ultrapure water, with fixed volume section collection eluent, each collection is equivalent to polysaccharide crude extract aqueous solution 0.1-0.3 times eluent, each section eluent is detected by high performance liquid chromatography glycopeptide peak, the eluent containing glycopeptide peak detected is combined, reduced pressure concentration, then freeze-drying, obtain coriolus ganoderma sugar peptide extract.The application can effectively extract the effective part of coriolus sugar peptide, increase the content of active ingredients in the product, and has good application prospect.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine technology, specifically to a Yunzhi glycopeptide and its preparation method and molecular weight determination method. Background Technology

[0002] Yunzhi Liver-Treating Granules are a traditional Chinese medicine, with its main ingredient being crude extract of Yunzhi (the core active ingredient being Yunzhi polysaccharides). It is primarily used for chronic hepatitis, as an adjunct treatment for hepatitis B, in early-stage cirrhosis, for immunodeficiency, and as an adjunct to radiotherapy and chemotherapy for tumors. It is a commonly prescribed medicine by doctors and frequently used by patients. It has good overall safety and few side effects, making it suitable for long-term use.

[0003] Currently, the extraction process used for Yunzhi Liver-Tai granules is relatively traditional, resulting in a low extraction rate of the effective components. It is necessary to develop a Yunzhi glycopeptide product with higher extraction efficiency to prepare for further product upgrades. Furthermore, accurate determination of the molecular weight of its effective components will provide data support for product quality control and standard improvement. Summary of the Invention

[0004] This invention provides a Yunzhi glycopeptide, its preparation method, and its molecular weight determination method. The preparation method of the Yunzhi glycopeptide is scientific and reasonable, and can effectively extract the effective parts of the Yunzhi glycopeptide, increase the content of effective components in the product, and has good application prospects.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: The preparation method of the aforementioned Yunzhi glycopeptide includes the following steps: Crude polysaccharide extract from *Trametes versicolor* was dissolved in 80-120 times its weight of water, filtered, and the resulting aqueous solution was loaded onto a pre-equilibrated Sepharose CL-6B gel column. The volume of the aqueous solution was 0.2-0.4 times the column volume. Elution was then performed with ultrapure water, and the eluent was collected in fractions at fixed volumes, each fraction containing 0.1-0.3 times the volume of the aqueous solution. The differential refractive index of the eluent was automatically combined and detected using a differential refractive index detector by an SCG chromatography workstation to determine the range of glycopeptide characteristic peaks. The eluents corresponding to the glycopeptide characteristic peak ranges were combined, concentrated under reduced pressure, and then freeze-dried to obtain the *Trametes versicolor* liver peptide extract. The Sepharose CL-6B gel column has a diameter of φ2.6 × 60 cm.

[0006] The flow rate during elution is 0.4-0.6 ml / min.

[0007] Differential refractive index detectors showed that the eluents from 200 to 300 min all contained polysaccharide characteristic peaks, and these eluents were combined.

[0008] The freeze-drying process is as follows: the concentrate obtained by vacuum concentration is pre-frozen at -15 to -25°C for 18-30 hours, and then finally dried in a freeze dryer at -15 to -25°C for 40-60 hours to obtain the Yunzhi liver glycopeptide extract.

[0009] The preparation method of the crude extract of Ganoderma lucidum polysaccharide includes the following steps: Take the Coriolus versicolor medicinal material, grind it into coarse powder, and extract it three times at 90-100℃, adding 10-20 times the amount of drinking water each time. The first extraction is carried out by heating for 6-8 hours and then filtering. The second extraction is carried out by heating for 6-8 hours and then filtering. The third extraction is carried out by heating for 6-8 hours and then filtering. The filtrates from the three extractions are combined and concentrated to a relative density of 1.10-1.12 at room temperature. Then, ethanol is added to a concentration of 65-75%, and the mixture is allowed to stand for 18-36 hours. The precipitate is collected and dried at 75-85℃. The dried material is then ground into a fine powder that passes through a 90-100 mesh sieve.

[0010] The chromatographic conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: TSK-Gel G4000 SWXL column; flow rate: 0.4-0.6 ml / min; column temperature: 30-40℃; detector: differential refractive index detector; mobile phase: ultrapure water.

[0011] The chromatographic column has dimensions of 7.8 mm * 300 mm.

[0012] The molecular weight determination method for the aforementioned Yunzhi glycopeptide includes the following steps: Preparation of reference solutions: Take 10 series of dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL, which are used as reference solutions; Preparation of test solution: Weigh 15-25 mg of Yunzhi liver glycopeptide extract accurately, place it in a 10 ml volumetric flask, add 8 ml of ultrapure water to dissolve, sonicate for 30 minutes, cool to room temperature, dilute to the mark with ultrapure water, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution. High performance liquid chromatography (HPLC) was used, and the chromatographic conditions were as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm x 300 mm; flow rate: 0.4-0.6 ml / min; column temperature: 30-40℃; detector: differential refractive index detector; mobile phase: ultrapure water; Accurately pipette 10 μL of each series of reference solutions and test solutions, inject them into the liquid chromatograph, and determine the results.

[0013] The molecular weights of the 10 known dextran reference standards are as follows: .

[0014] The advantages of this invention are as follows: This invention provides a method for preparing glycopeptides from *Trametes versicolor* (Yunzhi) that yields a product with a higher content of effective components, laying a solid foundation for future formulation upgrades. This invention also provides a method for detecting the molecular weight of the glycopeptides, providing data support for product quality control and standard improvement. Attached Figure Description

[0015] Figure 1 This is the elution diagram of the crude extract of Ganoderma lucidum polysaccharide from Example 6; Figure 2 This is a standard curve for molecular weight detection in Example 7; Figure 3 The molecular weight chromatogram of the crude extract of Coriolus versicolor polysaccharide 20250901 from Example 7 is shown below. Figure 4 Here is the molecular weight chromatogram of the crude extract of Coriolus versicolor polysaccharide 20250902 from Example 7; Figure 5 The molecular weight chromatogram of the crude extract of Ganoderma lucidum polysaccharide 20250903 from Example 7 is shown. Detailed Implementation

[0016] The present invention will now be described in detail through specific embodiments. Example 1

[0017] Preparation of crude extract of Ganoderma lucidum polysaccharide: Take the Coriolus versicolor medicinal material, grind it into coarse powder, and extract it three times at 90℃, adding 10 times the amount of drinking water each time. The first extraction was heated for 6 hours and filtered; the second extraction was heated for 6 hours and filtered; the third extraction was heated for 6 hours and filtered; the three filtrates were combined and concentrated to a relative density of 1.10 at room temperature; then ethanol was added to a concentration of 65%, and the mixture was allowed to stand for 18 hours. The precipitate was collected, dried at 75℃, and the dried material was pulverized into a fine powder that passed through a 90-mesh sieve.

[0018] Preparation of Yunzhi glycopeptides: Take the crude extract of Coriolus versicolor polysaccharides, dissolve it in 80-120 times its weight of water, filter it to obtain an aqueous solution of the crude polysaccharide extract, and then load it onto a pre-equilibrated φ2.6×60 cm Sepharose CL-6B gel column. The volume of the aqueous solution of the crude polysaccharide extract is equivalent to 0.2 times the column volume. Then elute with ultrapure water at a flow rate of 0.4-0.6 ml / min. Collect the eluent in fixed volume fractions, with each fraction containing 0.1 times the volume of the aqueous solution of the crude polysaccharide extract. Use a differential refractive index detector (DROP) and an SCG chromatography workstation to automatically combine and detect the DRP of the eluent to detect the range of glycopeptide characteristic peaks. The DRP detector showed that the eluent contained polysaccharide characteristic peaks in the 200-300 min eluent. Combine these eluents, concentrate them under reduced pressure, and then freeze-dry them to obtain the Coriolus versicolor liver peptide extract.

[0019] The freeze-drying process is as follows: the concentrate obtained by vacuum concentration is pre-frozen at -15°C for 18 hours, and then finally dried in a freeze dryer at -15°C for 40 hours to obtain the Yunzhi liver glycopeptide extract.

[0020] The chromatographic conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm*300 mm; flow rate: 0.4 ml / min; column temperature: 30℃; detector: differential refractive index detector; mobile phase: ultrapure water.

[0021] Method for determining the molecular weight of glycopeptides from Coriolus versicolor: Preparation of reference solutions: Take 10 series of dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL, which are used as reference solutions; Preparation of the test solution: Weigh 15 mg of Yunzhi liver glycopeptide extract accurately, place it in a 10 ml volumetric flask, add 8 ml of ultrapure water to dissolve it, sonicate for 30 minutes, cool to room temperature, dilute to the mark with ultrapure water, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution. High performance liquid chromatography (HPLC) was used, and the chromatographic conditions were as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm x 300 mm; flow rate: 0.4 ml / min; column temperature: 30℃; detector: differential refractive index detector; mobile phase: ultrapure water; Accurately pipette 10 μL of each series of reference solutions and test solutions, inject them into the liquid chromatograph, and determine the results.

[0022] The molecular weights of the 10 known dextran reference standards are as follows: .

[0023] Example 2 Preparation of crude extract of Ganoderma lucidum polysaccharide: Take the Coriolus versicolor medicinal material, crush it into coarse powder, and extract it three times at 100℃, adding 20 times the amount of drinking water each time. The first extraction was heated for 8 hours and filtered; the second extraction was heated for 8 hours and filtered; the third extraction was heated for 8 hours and filtered; the three filtrates were combined and concentrated to a relative density of 1.12 at room temperature; then ethanol was added to a concentration of 75%, and the mixture was allowed to stand for 36 hours. The precipitate was collected, dried at 85℃, and the dried material was crushed into fine powder that passed through a 100-mesh sieve to obtain the final product.

[0024] Preparation of Yunzhi glycopeptides: Crude polysaccharide extract from *Trametes versicolor* was dissolved in 120 times its weight of water, filtered, and the resulting aqueous solution was loaded onto a pre-equilibrated φ2.6×60 cm Sepharose CL-6B gel column. The volume of the aqueous solution was 0.4 times the column volume. Elution was then performed with ultrapure water at a flow rate of 0.6 ml / min. The eluent was collected in fractions of fixed volume, with each fraction containing 0.1-0.3 times the volume of the aqueous solution. The differential refractive index of the eluent was automatically combined and detected by an SCG chromatography workstation using a differential refractive index detector to determine the range of glycopeptide characteristic peaks. The differential refractive index detector showed that the eluent from 200-300 min contained glycopeptide characteristic peaks. These eluents were combined, concentrated under reduced pressure, and then freeze-dried to obtain *Trametes versicolor* liver peptide extract.

[0025] The freeze-drying process is as follows: the concentrate obtained by vacuum concentration is pre-frozen at -25℃ for 30 hours, and then finally dried in a freeze dryer at -25℃ for 60 hours to obtain the Yunzhi liver glycopeptide extract.

[0026] The chromatographic conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm*300 mm; flow rate: 0.6 ml / min; column temperature: 40℃; detector: differential refractive index detector; mobile phase: ultrapure water.

[0027] Method for determining the molecular weight of glycopeptides from Coriolus versicolor: Preparation of reference solutions: Take 10 series of dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL, which are used as reference solutions; Preparation of the test solution: Weigh 25 mg of Yunzhi liver glycopeptide extract accurately, place it in a 10 ml volumetric flask, add 8 ml of ultrapure water to dissolve it, sonicate for 30 minutes, cool to room temperature, dilute to the mark with ultrapure water, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution. High performance liquid chromatography (HPLC) was used, and the chromatographic conditions were as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm x 300 mm; flow rate: 0.6 ml / min; column temperature: 40℃; detector: differential refractive index detector; mobile phase: ultrapure water; Accurately pipette 10 μL of each series of reference solutions and test solutions, inject them into the liquid chromatograph, and determine the results.

[0028] The molecular weights of the 10 known dextran reference standards are as follows: .

[0029] Example 3 Preparation of crude extract of Ganoderma lucidum polysaccharide: The Yunzhi medicinal material was pulverized into coarse powder and extracted three times at 95℃, with 15 times the amount of drinking water added each time. The first extraction was heated for 7 hours and filtered; the second extraction was heated for 8 hours and filtered; the third extraction was heated for 6 hours and filtered. The three filtrates were combined and concentrated to a relative density of 1.11 at room temperature. Then, ethanol was added to a concentration of 70%, and the mixture was allowed to stand for 30 hours. The precipitate was collected, dried at 80℃, and the dried material was pulverized into a fine powder that passed through a 95-mesh sieve.

[0030] Preparation of Yunzhi glycopeptides: Crude polysaccharide extract from *Trametes versicolor* was dissolved in 100 times its weight of water, filtered, and the resulting aqueous solution was loaded onto a pre-equilibrated φ2.6×60 cm Sepharose CL-6B gel column. The volume of the aqueous solution was 0.3 times the column volume. Elution was then performed with ultrapure water at a flow rate of 0.5 ml / min. The eluent was collected in fractions at a fixed volume, with each fraction containing 0.2 times the volume of the aqueous solution. The differential refractive index of the eluent was automatically combined and detected by an SCG chromatography workstation using a differential refractive index detector to determine the range of glycopeptide characteristic peaks. The differential refractive index detector showed that the eluent from 200-300 min contained glycopeptide characteristic peaks. These eluents were combined, concentrated under reduced pressure, and then freeze-dried to obtain *Trametes versicolor* liver peptide extract.

[0031] The freeze-drying process is as follows: the concentrate obtained by vacuum concentration is pre-frozen at -20℃ for 24 h, and then finally dried in a freeze dryer at -20℃ for 48 h to obtain the Yunzhi liver glycopeptide extract.

[0032] The chromatographic conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm*300 mm; flow rate: 0.5 ml / min; column temperature: 35℃; detector: differential refractive index detector; mobile phase: ultrapure water.

[0033] Method for determining the molecular weight of glycopeptides from Coriolus versicolor: Preparation of reference solutions: Take 10 series of dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL, which are used as reference solutions; Preparation of the test solution: Weigh 20 mg of Yunzhi liver glycopeptide extract accurately, place it in a 10 ml volumetric flask, add 8 ml of ultrapure water to dissolve it, sonicate for 30 minutes, cool to room temperature, dilute to the mark with ultrapure water, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution. High performance liquid chromatography (HPLC) was used, and the chromatographic conditions were as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm x 300 mm; flow rate: 0.5 ml / min; column temperature: 35℃; detector: differential refractive index detector; mobile phase: ultrapure water; Accurately pipette 10 μL of each series of reference solutions and test solutions, inject them into the liquid chromatograph, and determine the results.

[0034] The molecular weights of the 10 known dextran reference standards are as follows: .

[0035] Example 4 Preparation of crude extract of Ganoderma lucidum polysaccharide: The Yunzhi medicinal material was pulverized into coarse powder and extracted three times at 93℃. For the first extraction, 15 times the amount of drinking water was added and heated for 8 hours, followed by filtration. For the second extraction, 12 times the amount of drinking water was added and heated for 7 hours, followed by filtration. For the third extraction, 10 times the amount of drinking water was added and heated for 6 hours, followed by filtration. The filtrates from the three extractions were combined and concentrated to a relative density of 1.10 at room temperature. Then, ethanol was added until the alcohol content was 68%, and the mixture was allowed to stand for 24 hours. The precipitate was collected, dried at 78℃, and pulverized into a fine powder that passed through a 100-mesh sieve.

[0036] Preparation of Yunzhi glycopeptides: Crude polysaccharide extract from *Trametes versicolor* was dissolved in 100 times its weight of water, filtered, and the resulting aqueous solution was loaded onto a pre-equilibrated φ2.6×60 cm Sepharose CL-6B gel column. The volume of the aqueous solution was 0.3 times the column volume. Elution was then performed with ultrapure water at a flow rate of 0.5 ml / min. The eluent was collected in fractions at a fixed volume, with each fraction containing 0.2 times the volume of the aqueous solution. The differential refractive index of the eluent was automatically combined and detected by an SCG chromatography workstation using a differential refractive index detector to determine the range of glycopeptide characteristic peaks. The differential refractive index detector showed that the eluent from 200-300 min contained glycopeptide characteristic peaks. These eluents were combined, concentrated under reduced pressure, and then freeze-dried to obtain *Trametes versicolor* liver peptide extract.

[0037] The freeze-drying process is as follows: the concentrate obtained by vacuum concentration is pre-frozen at -15 to -25°C for 26 hours, and then finally dried in a freeze dryer at -15 to -25°C for 52 hours to obtain the Yunzhi liver glycopeptide extract.

[0038] The chromatographic conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm*300 mm; flow rate: 0.4-0.6 ml / min; column temperature: 35℃; detector: differential refractive index detector; mobile phase: ultrapure water.

[0039] Method for determining the molecular weight of glycopeptides from Coriolus versicolor: Preparation of reference solutions: Take 10 series of dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL, which are used as reference solutions; Preparation of the test solution: Weigh 20 mg of Yunzhi liver glycopeptide extract accurately, place it in a 10 ml volumetric flask, add 8 ml of ultrapure water to dissolve it, sonicate for 30 minutes, cool to room temperature, dilute to the mark with ultrapure water, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution. High performance liquid chromatography (HPLC) was used, and the chromatographic conditions were as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm x 300 mm; flow rate: 0.5 ml / min; column temperature: 35℃; detector: differential refractive index detector; mobile phase: ultrapure water; Accurately pipette 10 μL of each series of reference solutions and test solutions, inject them into the liquid chromatograph, and determine the results.

[0040] The molecular weights of the 10 known dextran reference standards are as follows: .

[0041] Example 5: Detection of glycopeptide content in crude extract of Coriolus versicolor polysaccharide and extract of Coriolus versicolor glycopeptides: The determination method is as follows: Preparation of reference solution: Take an appropriate amount of anhydrous glucose reference standard dried to constant weight at 105℃, accurately weigh it, add water to prepare a solution containing about 30 μg per ml.

[0042] Preparation of test solution: Take about 50 mg of the crude extract of Coriolus versicolor polysaccharide and the extract of Coriolus versicolor glycopeptide prepared in Example 3, respectively, and place them in 500 ml volumetric flasks. Add an appropriate amount of water, shake to dissolve, place in a water bath for 30 minutes, cool, add water to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0043] Assay: Accurately measure 1 ml each of the reference solution, the crude extract of Coriolus versicolor polysaccharide, and the extract of Coriolus versicolor glycopeptides. Accurately add 0.6 ml of 5% phenol solution and 3.6 ml of sulfuric acid to each solution, mix well, and let stand for 30 minutes. Measure the absorbance at a wavelength of 484 nm using spectrophotometry (Appendix V B, Part I of the 2025 edition of the Chinese Pharmacopoeia). Calculate the result.

[0044] Test results: The crude extract of Ganoderma lucidum polysaccharides contains 30% glycopeptides. The glycopeptide content in the Yunzhi glycopeptide extract is 50%.

[0045] Example 6 Separation, Purification and Analysis Preparation of crude extract of Ganoderma lucidum polysaccharide: The Yunzhi medicinal material was pulverized into coarse powder and extracted three times at 95℃, with 15 times the amount of drinking water added each time. The first extraction was heated for 7 hours and filtered; the second extraction was heated for 8 hours and filtered; the third extraction was heated for 6 hours and filtered. The three filtrates were combined and concentrated to a relative density of 1.11 at room temperature. Then, ethanol was added to a concentration of 70%, and the mixture was allowed to stand for 30 hours. The precipitate was collected, dried at 80℃, and the dried material was pulverized into a fine powder that passed through a 95-mesh sieve.

[0046] 500 mg of crude extract of *Trametes versicolor* polysaccharide was weighed and dissolved thoroughly in 50 mL of ultrapure water. The solution was then loaded onto a pre-equilibrated Sepharose CL-6B gel column (φ2.6 × 60 cm). Elution was performed with ultrapure water. The differential refractive index of the eluent was automatically collected and measured using an SCG chromatography workstation. The SCG chromatography workstation was set with a pump flow rate of 0.5 mL / min, a fixed volume collection (80 tubes, 10 mL per tube). The elution curve of the crude polysaccharide extract was plotted based on the changes in the refractive index of the eluent after elution, as shown in Figure 1. Figure 1It can be seen that the eluent contains polysaccharide characteristic peaks in the 200-300 min range. The target eluent with the main peaks is collected, concentrated to an appropriate volume, and then freeze-dried to obtain the purified polysaccharide component.

[0047] Example 7 Validation of Molecular Weight Detection Methodology: I. Overview of the Research Process:

[0048] 1. Standard Curve Construction: A standard curve was established using 10 dextran standards with different molecular weights (Mp 180 to 2,000,000 Da) to correlate lg(Mw) with retention time (RT). The results showed good linearity (R²>0.990) within the retention time range of 13.473–19.548 min, meeting the detection requirements.

[0049] 2. Methodological Validation: A comprehensive and systematic methodological validation was conducted on the established HPLC method according to validation protocol RD-YZFA-20251201. Validation items included: linearity and range, instrument precision, repeatability, intermediate precision, accuracy, and robustness (including solution stability and minor variations in chromatographic conditions). All validation results met the pre-set, stringent acceptable standards, fully demonstrating that the method is accurate, reliable, reproducible, and robust, capable of meeting the daily quality control and research needs for the molecular weight and molecular weight distribution of *Trametes versicolor* crude extract and related products.

[0050] 3. Comparison of molecular weights of Yunzhi Liver-Tai Capsules, Yunzhi Liver-Tai Granules, and Yunzhi Crude Extract.

[0051] Comparative analysis: Using the established and validated HPLC molecular weight detection method, the molecular weight distribution of Yunzhi Gantai capsules, Yunzhi Gantai granules, and crude extract of Yunzhi polysaccharides was detected and compared.

[0052] Analysis results: By comparing key parameters such as weight-average molecular weight (Mw), molecular weight distribution curve, and the proportion of high molecular weight components (e.g., >40,000 Da), the similarities and differences in glycopeptide molecular weight characteristics between Yunzhi Liver-Tai Granules and Yunzhi Polysaccharide Crude Extract and the control Yunzhi product were clarified.

[0053] II. Determined Detection Methods: 1. Method for determining the molecular weight of crude extract of Ganoderma lucidum polysaccharides Chromatographic column: TSK-Gel G4000 SWXL (7.8 mm x 300 mm); injector: autosampler; flow rate: 0.5 ml / min; injection volume: 10 μL; column temperature: 35℃; detector: differential refractive index detector; water: ultrapure water; mobile phase: ultrapure water; analysis time: 25 min.

[0054] To prepare a series of reference solutions, take appropriate amounts of 10 dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL.

[0055] To prepare the test solution, accurately weigh approximately 20 mg of this product and place it in a 10 mL volumetric flask. Add 8 mL of mobile phase to dissolve the product, sonicate (350 W, 40 kHz) for 30 minutes, cool, and then dilute to the mark with the mobile phase. Shake well and filter through a 0.22 μm microporous membrane. Accurately pipette 10 μL each of the series of reference solutions and the test solution, inject them into the liquid chromatograph, and determine the result.

[0056] 2. Methods for determining the molecular weight of particles Chromatographic column: TSK-Gel G4000 SWXL (7.8 mm x 300 mm); injector: autosampler; flow rate: 0.5 ml / min; injection volume: 10 μL; column temperature: 35℃; detector: differential refractive index detector; water: ultrapure water; mobile phase: ultrapure water; analysis time: 25 min.

[0057] To prepare a series of reference solutions, take appropriate amounts of 10 dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL.

[0058] To prepare the test solution, accurately weigh approximately 300 mg of this product and place it in a 10 mL volumetric flask. Add 8 mL of mobile phase to dissolve the product, sonicate (350 W, 40 kHz) for 30 minutes, cool, and then dilute to the mark with the mobile phase. Shake well and filter through a 0.22 μm microporous membrane. Accurately pipette 10 μL each of the series of reference solutions and the test solution, inject them into the liquid chromatograph, and determine the result.

[0059] III. Methodological Validation The established method for determining molecular weight and molecular weight distribution was fully validated, and three batches of samples were tested.

[0060] 1. Linearity and Range Results: Chromatographic column: TSK-Gel G4000 SWXL (7.8 mm x 300 mm); Injector: Autosampler; Flow rate: 0.5 ml / min; Injection volume: 10 μL; Column temperature: 35℃; Detector: Differential refractive index detector; Mobile phase: Ultrapure water; Detection time: 25 min.

[0061] To prepare a series of reference solutions, take appropriate amounts of 10 dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL.

[0062] To prepare the test solution, accurately weigh about 20 mg of this product, place it in a 10 mL volumetric flask, add 8 mL of mobile phase to dissolve it, sonicate (350 W power, 40 kHz frequency) for 30 minutes, cool, then dilute to the mark with mobile phase, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0063] Linear regression was performed using the logarithm of molecular weight (lgMw) of a series of dextran reference standards as the ordinate and retention time (or converted Kav value) as the abscissa. The standard curve data are shown in Table 1, and the standard curve graph is shown below. Figure 2 As shown.

[0064] ; The calculated regression equation is y = -0.3044x + 1.9501, with a correlation coefficient R² = 0.9901, which meets the requirements. Substituting the elution time of crude extract 0901 into the formula, the molecular weights of the three peaks are calculated to be 2133115.259 Da, 654397.4762 Da, and 1492.345015 Da, respectively. The formula for calculating the weight-average molecular weight is as follows: Mw =Σ(Mi²×Ai) / Σ(Mi×Ai) (1); In the formula, Mi represents the molecular weight of each component; Ai represents the corresponding peak surface;

[0065] The weight-average molecular weight of crude extract 0901 was calculated according to Formula 1, and the result was 1767228.103 Da.

[0066] 2. Instrument precision test Chromatographic column: TSK-Gel G4000 SWXL (7.8 mm x 300 mm); Injector: Autosampler; Flow rate: 0.5 ml / min; Injection volume: 10 μL; Column temperature: 35℃; Detector: Differential refractive index detector; Mobile phase: Ultrapure water; Detection time: 25 min.

[0067] The same crude extract of *Trametes versicolor* polysaccharides was injected six times consecutively, and the weight-average molecular weight (Mw) and its relative standard deviation (RSD) were calculated. This confirms that the method for determining the molecular weight and molecular weight distribution of crude *Trametes versicolor* extract using this instrument meets the required precision.

[0068] Blank solution: Ultrapure water series of reference solutions: Take appropriate amounts of 10 series of dextran reference standards with known molecular weights, accurately weigh them, and add ultrapure water to prepare solutions of 2 mg / mL, which are used as reference solutions.

[0069] Test solution: Weigh approximately 20 mg of crude extract of the test sample accurately, place it in a 10 ml volumetric flask, add 8 ml of mobile phase to dissolve it, sonicate (350 W power, 40 kHz frequency) for 30 min, cool, then dilute to the mark with mobile phase, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0070] One injection of blank solution, one injection of each series of reference solutions, and six injections of test solution were recorded. The data are shown in Table 2.

[0071] ;

[0072] Instrument precision: The same test solution was injected six times consecutively, and the weight-average molecular weights (Mw) of the six injections were 2523697.978 Da, 2562005.728 Da, 2576927.81 Da, 2567164.259 Da, 2582512.12 Da, and 2575739.032 Da, respectively. The relative standard deviation (RSD) of the results was 0.83%, indicating that the instrument has high precision.

[0073] 3. Repeatability test Chromatographic column: TSK-Gel G4000 SWXL (7.8 mm x 300 mm); Injector: Autosampler; Flow rate: 0.5 ml / min; Injection volume: 10 μL; Column temperature: 35℃; Detector: Differential refractive index detector; Mobile phase: Ultrapure water; Detection time: 25 min.

[0074] Within the specified range, using the same batch of crude extract of *Trametes versicolor* polysaccharides, six test solutions were prepared. The results from these six tests were evaluated, and the RSD was calculated. Blank solution: Ultrapure water series of reference solutions: Accurately weigh appropriate amounts of 10 series of dextran reference standards with known molecular weights, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL. Test solution: Accurately weigh approximately 20 mg of this product, place it in a 10 mL volumetric flask, add 8 mL of mobile phase to dissolve, sonicate (350 W, 40 kHz) for 30 minutes, cool, then dilute to the mark with mobile phase, shake well, and filter through a 0.22 μm microporous membrane. Prepare six aliquots using the same method.

[0075] One injection of blank solution, one injection of each series of reference solutions, and one injection of each of the six test solutions are shown in Table 3.

[0076] ; Repeatability test results showed that six test solutions were prepared independently from the same batch of samples and measured to obtain values ​​of 2393960.847 Da, 2306408.894 Da, 2367978.23 Da, 2486167.114 Da, 2362079.923 Da, and 2484275.696 Da. The RSD of the Mw results was 3.11%, which is less than the standard of 2.0%, indicating that the method has good repeatability.

[0077] 4. Intermediate precision test Chromatographic column: TSK-Gel G4000 SWXL (7.8 mm x 300 mm); Injector: Autosampler; Flow rate: 0.5 ml / min; Injection volume: 10 μL; Column temperature: 35℃; Detector: Differential refractive index detector; Mobile phase: Ultrapure water; Detection time: 25 min.

[0078] Different researchers used the same method at different times to determine the same batch of crude extract of *Trametes versicolor* polysaccharides. Six determination results were used for evaluation, and the RSD of the results should meet the requirements to confirm method reproducibility. Within the specified range, the same batch of samples was used, and six determination results were used for evaluation. The RSD was then calculated.

[0079] Blank solution: Ultrapure water series of reference solutions: Take appropriate amounts of 10 series of dextran reference standards with known molecular weights, accurately weigh them, and add ultrapure water to prepare solutions of 2 mg / mL, which are used as reference solutions.

[0080] Test solution: Accurately weigh approximately 20 mg of crude extract of *Trametes versicolor* polysaccharide, place it in a 10 ml volumetric flask, add 8 ml of mobile phase to dissolve, sonicate (350 W, 40 kHz) for 30 minutes, cool, then dilute to the mark with mobile phase, shake well, and filter through a 0.22 μm microporous membrane. Prepare 6 aliquots using the same method. Inject one injection of the blank solution, one injection of each series of reference solutions, and one injection of each of the 6 test solutions. Record the chromatograms.

[0081] ; Intermediate precision: Measured by different analysts on different dates, the mean squared error (Mw) of the six results from the same batch of samples (1940887.534 Da, 2048906.659 Da, 2069467.245 Da, 2023062.428 Da, 2093278.442 Da, 2130098.002 Da) was 3.19%. Combining repeatability and intermediate precision data from 12 tests, the overall RSD was 6.83% (example data), far below the standard of 8.0%, indicating excellent method reproducibility and minimal impact from personnel, time, and other factors.

[0082] 5. Accuracy Test Chromatographic column: TSK-Gel G4000 SWXL (7.8 mm x 300 mm); Injector: Autosampler; Flow rate: 0.5 ml / min; Injection volume: 10 μL; Column temperature: 35℃; Detector: Differential refractive index detector; Mobile phase: Ultrapure water; Detection time: 25 min.

[0083] Within the specified range, at least six samples of crude extract of Ganoderma lucidum polysaccharides at the same concentration (equivalent to 100% concentration level) were used as test samples to evaluate the accuracy of the method.

[0084] Blank solution: Ultrapure water series of reference solutions: Take appropriate amounts of 10 series of dextran reference standards with known molecular weights, accurately weigh them, and add ultrapure water to prepare solutions of 2 mg / mL, which are used as reference solutions.

[0085] Accuracy solution: Accurately weigh approximately 4 mg of dextran D3 Mp9750 reference standard and place it in a 10 ml volumetric flask. Accurately weigh approximately 20 mg of this product and place it in the same volumetric flask. Add 8 ml of mobile phase to dissolve the product. Sonicate the solution (350 W, 40 kHz) for 30 minutes, cool, and then dilute to the mark with the mobile phase. Shake well and filter through a 0.22 μm microporous membrane. Prepare 6 aliquots using the same method. Inject one injection of the blank solution, one injection of each series of reference solutions, and one injection of each of the 6 accuracy solutions. Record the data as shown in Table 5.

[0086] The recovery rate of the weight-average molecular weight of the six accuracy test solutions should be between 95% and 105%, and the RSD of the recovery rate should be less than 8.0%. (Note: Recovery rate % = actual measured weight-average molecular weight / theoretical input weight-average molecular weight * 100%) ; The formula for calculating the spiked recovery rate is shown in Formula 2. Spike recovery rate = (A1 - A2) / A3 × 100% (2); In the formula, A1 is the peak area of ​​the measured value after spiking. A2 represents the average area of ​​the repeating peaks, which is 68842. A3 represents the peak area of ​​the actual spiking amount in D3, which is 281385. Using the spiked recovery method, the spiked recoveries of the six accuracy test solutions were 97.78%, 100.08%, 100.49%, 96.19%, 100.50%, and 103.33%, respectively. The spiked recoveries of the six accuracy test solutions ranged from 98.2% to 102.5% (example data), all within the range of 95% to 105%, and the RSD of the recovery rate was 2.37%, proving that the method's determination results are accurate and reliable.

[0087] 6. Durability test: ; The same crude extract of *Trametes versicolor* polysaccharides was tested at room temperature at 0, 4, 8, 12, 20, and 24 h. Robustness (solution stability): 2447442.601 Da, 2461699.549 Da, 2361892.726 Da, 2130838.708 Da, 2236674.931 Da, 2113830.231 Da. Solution stability: After the test solution was placed at room temperature for 24 hours, the RSD of the Mw result was 5.01%, less than 2.0%, indicating that the test solution had good stability within 24 hours.

[0088] Robustness (variation of chromatographic conditions): When there are slight changes in flow rate (±10%) and column temperature (±2℃), compared with normal conditions, the weight-average molecular weight of the crude extract of Trametes versicolor polysaccharide measured at flow rates of 0.45 mL / min and 0.55 mL / min are 2289706.712 Da and 2206341.641 Da, respectively. The weight-average molecular weight of crude extract 0901 measured at column temperatures of 33℃ and 37℃ are 2084248.107 Da and 1957030.276 Da, respectively. The RSD of the Mw determination results is 6.54% (example data), which is less than the standard of 8.0%, indicating that the method is not sensitive to small fluctuations in chromatographic conditions and has strong robustness.

[0089] Robustness (results comparison): The weight-average molecular weight of the crude extract of Trametes versicolor polysaccharide measured under normal conditions was compared with the data obtained under changes in solution stability and chromatographic conditions. The RSD was 6.51%, which is less than the standard of 8.0%, indicating that the method is not sensitive to small fluctuations in chromatographic conditions and has strong robustness.

[0090] 7. Analysis of molecular weight determination results of three batches of crude extract samples Three batches of crude extracts of *Trametes versicolor* polysaccharides (samples 20250901, 20250902, and 20250903) were analyzed. The chromatograms are shown in Figure 3-5. The weight-average molecular weight (Mw) and the proportion of components with a molecular weight greater than 40,000 were calculated, and the results are shown in Table 7.

[0091] ; The weight-average molecular weights of 0901, 0902 and 0903 are 2266413.058 Da, 1910870.301 Da and 1902540.593 Da, respectively, and the proportions of molecules with a weight-average molecular weight greater than 4000 Da are 30.10%, 31.11% and 32.16%, respectively.

[0092] 8. Method Validation Conclusions Methodological validation based on crude extract of Coriolus versicolor polysaccharides showed that the molecular weight and molecular weight distribution determination method met the predetermined acceptable standards in terms of system suitability, specificity, linearity, instrument precision, repeatability, intermediate precision, accuracy, and robustness. The method is scientific, reliable, and durable. It can be used for the quality control of crude extract of Coriolus versicolor polysaccharides, Coriolus versicolor liver-protecting granules, and Coriolus versicolor glycopeptide extract.

[0093] Chromatographic conditions: High performance liquid chromatography-refractive index detection (HPLC-RID), TSK-Gel G4000 SWXL column (7.8 mm × 300 mm), ultrapure water as mobile phase, flow rate 0.5 mL / min, column temperature 35℃, injection volume 10 μL.

[0094] Sample preparation method: Weigh 20 mg of sample accurately, place it in a 10 mL volumetric flask, add 8 mL of mobile phase to dissolve, sonicate (350 W power, 40 kHz frequency) for 30 minutes, cool, dilute to the mark with mobile phase, shake well, and filter through a 0.22 μm microporous membrane to obtain the sample.

[0095] Preparation method of reference standards: Take an appropriate amount of each of 10 series of dextran reference standards with known molecular weights, accurately weigh them, and add ultrapure water to prepare a 2 mg / mL solution as the reference standard solution.

[0096] Weight-average molecular weight calculation method: The calculation formula is as follows: Mw =Σ(Mi²×Ai) / Σ(Mi×Ai); In the formula, Mi represents the molecular weight of each component; Ai represents the corresponding peak surface.

[0097] 9. Comparison of molecular weight between crude extract of Ganoderma lucidum polysaccharides and commercially available products: ;

[0098] Note: Capsules, 24120501, and 25032202 are commercially available; G250601, 20250901, 20250902, and 20250903 are crude extracts of Ganoderma lucidum polysaccharides extracted by the method in Example 3. All seven samples contained different molecular weights of components. Comparison of the capsule sample with other crude extracts revealed the broadest molecular weight range, covering five significant peaks from 1.5 kDa to nearly 50 MDa. It contained extremely high molecular weight components (~49.86 MDa and ~7.22 MDa), which were not detected in other crude extracts. It also contained medium and low molecular weight components (56.8 kDa, ~11.9 kDa, ~1.5 kDa), which overlapped with other samples. The molecular weight distribution of the other crude extracts was relatively concentrated, mainly divided into three ranges: high molecular weight region: 2.3–2.5 MDa; medium molecular weight region: 280 kDa – 1.1 MDa (some samples did not have peaks in this range); low molecular weight region: 1.5–2.9 kDa; no ultra-high molecular weight (7 MDa) components like those in the capsules were detected.

[0099] A comparison of the molecular weights of four batches of crude Ganoderma lucidum extract (G250601, 20250901, 20250902, and 20250903) revealed that all batches consisted of three molecular weight components. The high molecular weight peak (2.3–2.5 MDa) was present in all batches, showing good consistency. This indicates that the production process has relatively stable control over this main component.

[0100] Significant differences exist between the medium molecular weight fraction G250601 and 20250901, 20250902, and 20250903. G250601 has a molecular weight of ~1.1 MDa, while the medium molecular weight fractions of 20250901, 20250902, and 20250903 show good consistency, all ranging from 680 to 695 kDa. The low molecular weight region is (1.5–2.9 kDa), and all samples exhibit peaks in the ~1.5–1.6 kDa range, showing excellent consistency.

[0101] The weight-average molecular weights of 20250901, 20250902, and 20250903 are 2266413.058 Da, 1910870.301 Da, and 1902540.593 Da, respectively, and the proportions of molecules with a weight-average molecular weight greater than 4000 Da are 30.10%, 31.11%, and 32.16%, respectively.

Claims

1. A type of Yunzhi glycopeptide, characterized in that, Its preparation method includes the following steps: Take the crude extract of Coriolus versicolor polysaccharides, add water equivalent to 80-120 times the mass of the crude extract to dissolve it, filter to obtain an aqueous solution of the crude extract, and then load it onto a pre-equilibrated Sepharose CL-6B gel column. The volume of the aqueous solution of the crude extract is equivalent to 0.2-0.4 times the column volume. Then elute with ultrapure water, and collect the eluent in fixed volume fractions. Collect the eluent equivalent to 0.1-0.3 times the volume of the aqueous solution of the crude extract in each fraction. Use a differential refractive index detector and an SCG chromatography workstation to automatically combine and detect the differential refractive index of the eluent and detect the range of glycopeptide characteristic peaks. Combine the eluents corresponding to the glycopeptide characteristic peak ranges, concentrate under reduced pressure, and then freeze-dry to obtain Coriolus versicolor liver peptide extract.

2. The Yunzhi glycopeptide according to claim 1, characterized in that: The Sepharose CL-6B gel column has a specification of φ2.6×60 cm.

3. The Yunzhi glycopeptide according to claim 2, characterized in that: The flow rate during elution is 0.4-0.6 ml / min.

4. The Yunzhi glycopeptide according to claim 2, characterized in that: Differential refractive index detectors showed that the eluents from 200 to 300 min contained polysaccharide characteristic peaks. These eluents were then combined.

5. The Yunzhi glycopeptide according to claim 1, characterized in that: The freeze-drying process is as follows: the concentrate obtained by vacuum concentration is pre-frozen at -15 to -25°C for 18-30 hours, and then finally dried in a freeze dryer at -15 to -25°C for 40-60 hours to obtain the Yunzhi liver glycopeptide extract.

6. The Yunzhi glycopeptide according to claim 1, characterized in that: The preparation method of the crude extract of Ganoderma lucidum polysaccharide includes the following steps: Take the Coriolus versicolor medicinal material, grind it into coarse powder, and extract it three times at 90-100℃, adding 10-20 times the amount of drinking water each time. The first extraction is carried out by heating for 6-8 hours and then filtering. The second extraction is carried out by heating for 6-8 hours and then filtering. The third extraction is carried out by heating for 6-8 hours and then filtering. The filtrates from the three extractions are combined and concentrated to a relative density of 1.10-1.12 at room temperature. Then, ethanol is added to a concentration of 65-75%, and the mixture is allowed to stand for 18-36 hours. The precipitate is collected and dried at 75-85℃. The dried material is then ground into a fine powder that passes through a 90-100 mesh sieve.

7. The Yunzhi glycopeptide according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: TSK-Gel G4000 SWXL column; flow rate: 0.4-0.6 ml / min; column temperature: 30-40℃; detector: differential refractive index detector; mobile phase: ultrapure water.

8. The Yunzhi glycopeptide according to claim 1, characterized in that: The chromatographic column has dimensions of 7.8 mm * 300 mm.

9. The Yunzhi glycopeptide according to claim 1, characterized in that, The method for determining its molecular weight includes the following steps: Preparation of reference solutions: Take 10 series of dextran reference standards with known molecular weights, weigh them accurately, and add ultrapure water to prepare solutions with a concentration of 2 mg / mL, which are used as reference solutions; Preparation of test solution: Weigh 15-25 mg of Yunzhi liver glycopeptide extract accurately, place it in a 10 ml volumetric flask, add 8 ml of ultrapure water to dissolve, sonicate for 30 minutes, cool to room temperature, dilute to the mark with ultrapure water, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution. High performance liquid chromatography (HPLC) was used, and the chromatographic conditions were as follows: Chromatographic column: TSK-Gel G4000 SWXL column, 7.8 mm x 300 mm; flow rate: 0.4-0.6 ml / min; column temperature: 30-40℃; Detector: Differential refractive index detector; Mobile phase: Ultrapure water; Accurately pipette 10 μL of each series of reference solutions and test solutions, inject them into the liquid chromatograph, and determine the results.

10. The Yunzhi glycopeptide according to claim 9, characterized in that, The method for determining its molecular weight includes the following steps: The molecular weights of the 10 known dextran reference standards are as follows: 。

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