Qualitative and quantitative detection method for D-component of grifola frondosa polysaccharide

Abstract

The invention discloses a qualitative and quantitative detection method of a grifola frondosa polysaccharide D-component, which comprises the following steps: selecting defatted grifola frondosa powder from all over the country, extracting crude polysaccharide, carrying out HPLC (High Performance Liquid Chromatography) analysis and detection, and judging the existence of the grifola frondosa polysaccharide D-component according to the chromatographic retention time. Then analyzing monosaccharide composition and amino acid composition of the purified grifola frondosa polysaccharide D-component so as to further determine the composition range of monosaccharide and amino acid in the grifola frondosa polysaccharide D-component, and then detecting the HPLC peak area of the pure grifola frondosa polysaccharide D-component by HPLC analysis so as to measure the content of the grifola frondosa polysaccharide D-component.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for qualitative and quantitative detection of Grifola frondosa polysaccharide D-component. Background Art

[0002] Grifola frondosa is an edible mushroom with important nutritional and medicinal values. Its polysaccharide active components (especially D-component) have significant anti-tumor and immunomodulatory effects, so the demand in the health food and pharmaceutical markets has increased rapidly. Grifola frondosa polysaccharide D-component is one of the most intensively studied active polysaccharides in Grifola frondosa. The D-component is essentially a β-glucan complex containing protein (protein content is about 30%). Research shows that Grifola frondosa D-component has a unique β-D-glucan structure: it has a dual branched configuration with both (1→6)-main chain with (1→3)-branches and (1→3)-main chain with (1→6)-branches. The molecular weight of this component is very high (about 1.4×10 6 kDa), and the high molecular weight is considered to be one of the factors for its strong immunological activity. For example, conventional polysaccharide content detection methods cannot distinguish β-glucan from Grifola frondosa from α-polysaccharide adulterants such as starch, and currently no patents or literature on analytical methods for qualitative and quantitative detection of Grifola frondosa polysaccharide D-component have been retrieved. Therefore, there is an urgent need to establish a highly accurate and sensitive detection method for identifying the authenticity and content of Grifola frondosa D-component, so as to effectively control the product quality of Grifola frondosa polysaccharide D-component and protect the interests of consumers.

[0003] At present, the detection methods for polysaccharide components mainly include colorimetry, spectroscopy, chromatography, etc., but each has its limitations. The commonly used colorimetric methods (such as phenol-sulfuric acid method and anthrone-sulfuric acid method) can only determine the content of polysaccharides in samples. They are simple and sensitive in operation but have poor specificity, unable to provide information on the structural position of polysaccharides, nor can they distinguish polysaccharides from different sources. Infrared spectroscopy can be used to identify the characteristic peaks of functional groups of polysaccharides, such as determining whether there are characteristic absorption peaks of β-glucan in the sample, etc. However, the infrared spectra of different polysaccharides are often similar, making it difficult to accurately distinguish the Grifola frondosa D-fraction. High-performance liquid chromatography (HPLC) is used for the analysis of sugar components in traditional Chinese medicines and foods due to its high-efficiency separation. However, directly analyzing complex macromolecular polysaccharides is challenging: conventional HPLC polysaccharide analysis is mostly limited to the monosaccharide or oligosaccharide components after hydrolysis. In terms of detection means, different detectors have their own advantages and disadvantages. The refractive index detector (RID) is a commonly used detection method after HPLC separates polysaccharides, but its sensitivity is easily affected by factors such as the composition of the mobile phase and temperature, and baseline drift results in low detection sensitivity and accuracy. Since most polysaccharides lack ultraviolet absorption groups, ultraviolet detectors (UV) need to introduce chromophores through chemical derivatization before quantitative detection, which increases the operation complexity and error sources. In addition, methods such as high-performance thin-layer chromatography are also used for polysaccharide analysis, but they often require qualitative comparison with reference standards, with a large workload and difficulty in quantification. Generally speaking, the existing technologies lack a method with both high specificity and high sensitivity to accurately detect the content and authenticity of the Grifola frondosa polysaccharide D-fraction.

[0004] Based on the above background, the present invention provides a new detection scheme that can overcome the deficiencies of existing methods. In terms of accuracy, through the dual determination of chromatographic retention time and component analysis, the specificity of identifying the Grifola frondosa D-fraction is improved; in terms of sensitivity, by adopting optimized chromatographic conditions and detector configuration, low-content target components can be detected and doped non-target polysaccharides can be distinguished; in terms of repeatability, by strictly controlling the detection conditions and comparing with self-made D-fraction repeatedly, it is ensured that the results have good consistency and repeatability. Summary of the Invention

[0005] Aiming at the above technical problems existing in the prior art, the purpose of the present invention is to provide a qualitative and quantitative detection method for the Grifola frondosa polysaccharide D-fraction. The core is to analyze the Grifola frondosa polysaccharide extract by coupling an ultraviolet detector and a refractive index detector (HPLC-UV-RID) on high-performance liquid chromatography. Through this method, the presence of the Grifola frondosa D-fraction can be determined according to the chromatographic retention time, and its content can be calculated through the chromatographic peak area. At the same time, the monosaccharide composition and amino acid composition of the purified Grifola frondosa polysaccharide D-fraction are analyzed to further determine the range of monosaccharide and amino acid compositions in the Grifola frondosa polysaccharide D-fraction. This multi-dimensional determination method greatly improves the accuracy and specificity of detection.

[0006] Specifically, the method of the present invention includes the following steps: 1. Sample preparation: Select Grifola frondosa fruit bodies from all over the country for polysaccharide extraction. Use the hot water extraction method to obtain crude Grifola frondosa polysaccharides, and prepare polysaccharide samples from other sources (such as Ganoderma lucidum, Lycium barbarum, Lentinus edodes, Tremella fuciformis, Cordyceps sinensis, Tricholoma matsutake polysaccharides) as control tests.

[0007] 2. Chromatographic analysis: Configure the above-mentioned crude polysaccharide sample solution to a certain concentration and directly inject it into the high-performance liquid chromatography system. Select a gel permeation chromatography column (TSKgel G5000PWXL chromatography column with a molecular weight exclusion limit of 2.5×10 6 ), the mobile phase is deionized water, and the flow rate is about 0.6 mL / min. Connect an ultraviolet detector (detection wavelength is 280 nm to detect the absorption of possible accompanying proteins or amide bonds in the polysaccharide) and a differential refractive index detector in series on the chromatograph, and detect and record the eluted components.

[0008] 3. Qualitative determination: Determine the retention time range of the D-component according to the chromatograms of Grifola frondosa crude polysaccharides from all over the country. The characteristic peaks within the retention time range of the D-component of Grifola frondosa polysaccharides are initially used to determine whether the sample contains the D-component. In order to further determine the chromatographic peak, the present invention combines the analysis of monosaccharide and amino acid composition to characterize the target peak. The suspected D-component chromatographic peak sample purified and separated from Grifola frondosa crude polysaccharides from all over the country is analyzed for monosaccharide components by the PMP pre-column derivation method using high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD), and the amino acid components are determined by an amino acid analyzer. By measuring the monosaccharide composition and amino acid composition of the D-component of Grifola frondosa polysaccharides from all over the country, determine the composition ranges of both. If the detected monosaccharide and amino acid composition ranges are within the protection scope of the present invention, it can be confirmed that the chromatographic peak is the D-component of Grifola frondosa polysaccharides; otherwise, it can be determined that it is not the D-component or there is doping. Combining the dual determination of chromatographic retention time and chemical composition can quickly and accurately qualitatively determine the D-component of Grifola frondosa polysaccharides.

[0009] 4. Quantitative determination: Determine the content of the D-component of Grifola frondosa by the chromatographic peak area. First, prepare a series of concentration solutions of the self-made D-component of Grifola frondosa, and perform the above-mentioned HPLC analysis to plot the peak area-concentration working curve. Then calculate the area of the D-component chromatographic peak in the sample, and substitute it into the standard curve to calculate its absolute content.

[0010] The technical solution adopted by the present invention is specifically as follows: A method for qualitative and quantitative detection of the D-component of Grifola frondosa polysaccharides, comprising the following steps: 1) Select the defatted Grifola frondosa powder from fruiting bodies of Grifola frondosa across the country, obtain the crude Grifola frondosa polysaccharide by hot water extraction method, conduct HPLC analysis and detection, obtain a series of HPLC diagrams of the crude Grifola frondosa polysaccharide, determine the peak emergence time range of the D-component of Grifola frondosa polysaccharide, and preliminarily judge whether the sample contains the D-component of Grifola frondosa polysaccharide according to the characteristic peaks within the peak emergence time range; 2) The crude polysaccharides extracted from the defatted Grifola frondosa powder from various sources across the country are respectively subjected to purification and separation treatment to obtain the D-component of Grifola frondosa polysaccharide; 3) Determine the monosaccharide and amino acid compositions of the D-component of Grifola frondosa polysaccharide from various producing areas across the country, summarize the analysis results from different producing areas, determine the main monosaccharide content range and the content range of the main amino acids, that is, determine the fingerprint characteristic values of the D-component of Grifola frondosa polysaccharide, and further qualitatively judge the D-component of Grifola frondosa polysaccharide according to this characteristic value; 4) Make an HPLC analysis standard curve for the D-component of Grifola frondosa polysaccharide and quantitatively detect the content of the D-component of Grifola frondosa polysaccharide in the sample.

[0011] Furthermore, the method steps for extracting the crude Grifola frondosa polysaccharide in step 1) are as follows: the defatted Grifola frondosa powder is mixed according to a solid-liquid mass ratio of 1:10 - 30, stirred evenly, the mixed solution is heated in a heating environment at 90 - 100 °C for 2 - 4 h, after the temperature drops to room temperature, it is filtered by suction, the filtrate is concentrated by rotary evaporation and then freeze-dried to obtain the crude Grifola frondosa polysaccharide.

[0012] Furthermore, the HPLC analysis and detection conditions in step 1) are as follows: Chromatographic conditions: use a TSKgel G5000PW_XL gel permeation column, 7.8 mm × 300 mm; Use pure water as the mobile phase with a flow rate of 0.5 - 0.6 mL / min; Couple an ultraviolet detector with a differential refractive index detector, set the detection wavelength of the ultraviolet detector to 280 nm, and the temperature of the column oven is 35 °C; The HPLC analysis and detection conditions when making the HPLC analysis standard curve in step 4) are the same as those in step 1) HPLC analysis and detection conditions.

[0013] Furthermore, the steps of separation and purification in step 2) are as follows: S1: After dissolving the crude polysaccharide in pure water, it is successively passed through tubular ceramic membranes with pore sizes of 200 nm, 50 nm, and 5 nm, and the retentate is collected; S2: The retentate is concentrated through a 0.001 μm filter membrane, the concentrated solution is mixed with Sevag reagent according to a volume ratio of 1:3 - 5, Sevag reagent is a CHCl3 - CH3(CH2)3OH mixed solvent with a volume ratio of 4 - 6:1, centrifuged and separated, and the upper layer polysaccharide solution is collected and freeze-dried; S3: The freeze-dried polysaccharide is dissolved in deionized water, filtered through a 0.45 μm filter membrane, and loaded onto a DEAE Sepharose Fast Flow anion exchange column. It is eluted successively with aqueous sodium chloride solutions of different concentrations, namely 0 M, 0.25 M, and 0.5 M. The eluate of the 0.5 M aqueous sodium chloride solution is collected and dialyzed and freeze-dried. S4: The freeze-dried product is further dissolved in pure water, and semi-preparative liquid phase purification is carried out using an XK 26 / 100 Sephacryl S-300 HR column. The mobile phase is ultrapure water. The target component is collected and freeze-dried to obtain the finally purified Grifola frondosa polysaccharide D-component.

[0014] Furthermore, the monosaccharide composition of the Grifola frondosa polysaccharide D-component is determined in step 3) as follows: Step 1: The Grifola frondosa polysaccharide D-component is added to a 1.5 M - 3 M trifluoroacetic acid aqueous solution according to a feed ratio of 10 mg: 1.5 - 3 mL, added to a reactor and sealed, and hydrolyzed at a temperature of 100 - 120 °C for 5 - 10 h. After the reaction ends, methanol is added and rotary evaporation is used to remove the excess trifluoroacetic acid. Step 2: The hydrolysis product in Step 1 is dissolved in water, and a NaOH solution and an excessive amount of PMP solution are added. The final concentration of NaOH in the mixed solution is 0.05 - 0.2 M, and the reaction is heated at a temperature of 60 - 80 °C for 40 - 80 min. After cooling to room temperature, acid is added to adjust the pH to neutral, and chloroform is added to wash away the excess PMP. The aqueous layer containing the derivatized product is collected. Among them, the PMP is 1-phenyl-3-methyl-5-pyrazolone. PMP introduces a group with ultraviolet absorption or fluorescence characteristics through a derivatization reaction with the aldehyde group of the monosaccharide, enabling the monosaccharide without significant light absorption to be efficiently detected in high performance liquid chromatography.

[0015] Step 3: The aqueous layer containing the derivatized product in Step 2 is analyzed for the monosaccharide composition by high performance liquid chromatography-evaporative light scattering detector. The chromatographic column for high performance liquid chromatography is C 18 SHISEIDO, 4.6 mm × 250 mm × 5 μm, and the wavelength of the ultraviolet-visible absorption detector is 250 nm.

[0016] Furthermore, the amino acid composition of the Grifola frondosa polysaccharide D-component is determined in step 3) as follows: Step 1: The Grifola frondosa polysaccharide D-component is added to 5.0 - 6.0 M hydrochloric acid according to a feed ratio of 10 mg: 1.5 - 3 mL, added to a hydrolysis tube, deoxygenated by introducing nitrogen, sealed, and placed in a constant temperature drying oven at 100 - 110 °C. It is hydrolyzed at high temperature for 20 - 30 h to completely decompose the protein into free amino acids. Step 2: After the hydrolysis is completed, take out the hydrolysis tube, cool it to room temperature, open the sealed port, transfer the hydrolysis solution to a centrifuge tube, and blow dry the free hydrochloric acid with nitrogen at a temperature of 40 - 60 °C until the sample is dry. Step 3: Add sodium citrate buffer solution with pH 2.2 to the dried residue for reconstitution, vortex thoroughly to mix evenly, filter through a 0.22 μm polytetrafluoroethylene filter membrane, and take the filtrate to detect and analyze the amino acid composition with an amino acid automatic analyzer.

[0017] Further, in step 1), the defatted Grifola frondosa fruit body powder is respectively produced in Qingyuan County, Zhejiang Province; Qianxi County, Hebei Province; Tongzi County, Guizhou Province; Guilin City, Guangxi Zhuang Autonomous Region; and Songxi County, Fujian Province.

[0018] Further, when determining the fingerprint characteristic values of the Grifola frondosa polysaccharide D-component in step 3), the monosaccharide content and amino acid content are as follows: Monosaccharide content index: Calculated by mole percentage, glucose is 50 - 60%, galactose is 25 - 35%, mannose is 5 - 10%, and the rest are mainly glucuronic acid and rhamnose. Amino acid content index: Calculated by mole percentage, glycine is 17 - 23%, glutamic acid is 15 - 22%, aspartic acid is 7 - 12%, alanine is 5 - 9%, tyrosine is 4.5 - 8.5%, phenylalanine is 4.5 - 8.5%, threonine is 4 - 8%, serine is 4 - 8%, and the rest are leucine, valine, lysine, proline, arginine, isoleucine, histidine, methionine, and cysteine.

[0019] Further, in step 4), the process of making the HPLC analysis standard curve of the Grifola frondosa polysaccharide D-component is as follows: Use the Grifola frondosa polysaccharide D-component with a purity greater than 98% as the standard product, prepare standard solutions with five concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL with water, then perform HPLC analysis and detection, and draw a standard curve with the concentration of the Grifola frondosa polysaccharide D-component as the abscissa and the HPLC chromatographic peak of the Grifola frondosa polysaccharide D-component as the ordinate.

[0020] The present invention can accurately qualitatively and quantitatively analyze the Grifola frondosa polysaccharide D-component. Experimental verification shows that the detection limit of this method for the Grifola frondosa D-component is low, the quantitative linear relationship is good, the relative standard deviation of repeated analysis is small, and it is suitable for the quality control and authenticity identification of Grifola frondosa polysaccharide products.

[0021] Compared with the prior art, the beneficial effects achieved by the present invention are: (1)Specificity improvement: By combining the double determination of chromatographic retention time and chemical composition, the specificity of detection is greatly improved. Only the Grifola frondosa D-fraction can simultaneously meet the specific chromatographic peak time and the characteristic monosaccharide + amino acid composition, so as to accurately determine the presence of the Grifola frondosa D-fraction and avoid misidentifying polysaccharides from other sources as the D-fraction.

[0022] (2)High sensitivity: By using HPLC tandem with ultraviolet and differential refractive index detection, the signal response intensity is improved, and the low-content D-fraction can also be detected. Even if a small amount of polysaccharides from non-Grifola frondosa sources are incorporated into the sample, this method can effectively distinguish them through chromatographic detection and compositional differences, ensuring the detection of the target component. Compared with the traditional single RI detection method, the detection limit of the D-fraction in this invention is lower and the quantitative linear range is wider.

[0023] (3)Good repeatability: Verified by multiple repeated experiments, the relative standard deviation (RSD) of the D-fraction content determination of the same sample by the method of this invention is less than 2%, with good reproducibility, ensuring the stability and reliability of the detection results.

[0024] In summary, the method of this invention is significantly superior to the existing polysaccharide detection technologies in terms of specificity, sensitivity and repeatability, and can provide a reliable means for the quality evaluation of Grifola frondosa polysaccharide products, which has a positive significance for cracking down on market adulteration and maintaining product efficacy. Description of the Drawings

[0025] Figure 1 is the HPLC chromatogram of the crude Grifola frondosa polysaccharide from Qingyuan County, Zhejiang Province; Figure 2 is the HPLC chromatogram of the crude Grifola frondosa polysaccharide from Qianxi County, Hebei Province; Figure 3 is the HPLC chromatogram of the crude Grifola frondosa polysaccharide from Tongzi County, Guizhou Province; Figure 4 is the HPLC chromatogram of the crude Grifola frondosa polysaccharide from Guilin City, Guangxi; Figure 5 is the HPLC chromatogram of the crude Grifola frondosa polysaccharide from Songxi County, Fujian Province; Figure 6 is the HPLC chromatogram of the purified Grifola frondosa polysaccharide D-fraction from Qingyuan County, Zhejiang Province; Figure 7 is the standard curve of the Grifola frondosa polysaccharide D-fraction; Figure 8 is the HPLC chromatogram of the crude Coriolus versicolor polysaccharide;

[0026] Figure 9 is the HPLC chromatogram of the detection of the mixed solution after doping the wolfberry polysaccharide sample solution and the Grifola frondosa D-fraction solution in Example 8. Detailed Embodiments

[0027] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0028] The steps for extracting and processing the fruiting bodies of Grifola frondosa to obtain defatted Grifola frondosa powder are as follows: Mechanically crush the fruiting bodies of Grifola frondosa using a pulverizer, screen the crushed material through an 80-mesh sieve to obtain the required powder, and then store it in a sealed manner for later use. Add the Grifola frondosa fruiting body powder to 95% ethanol with a volume 5 times that of the powder, stir well and mix, and then soak for 24 h. During this process, ethanol can effectively dissolve and remove impurities such as lipids and pigments in the Grifola frondosa powder. After soaking, separate the ethanol liquid through a suction filtration device, take out the filter residue and place it in a well-ventilated place to naturally volatilize the ethanol. After the ethanol has completely volatilized, store the filter residue in a sealed manner to ensure its dryness and no ethanol residue, that is, obtain defatted Grifola frondosa powder.

[0029] Example 1, Step 1, Sample preparation: Weigh 500 g of defatted Grifola frondosa powder, add deionized water, with a solid-liquid ratio of 1:20 (w / w), and stir well. Heat and reflux the mixture at 100 °C for 3 h. After the temperature drops to room temperature, use a suction filtration device to separate the filter residue, collect the supernatant, concentrate it at 55 °C using a rotary evaporator, and freeze-dry to obtain crude Grifola frondosa polysaccharide. Accurately weigh 50 mg of the crude polysaccharide and dissolve it in 10 mL of ultrapure water to fully dissolve and obtain a test solution with a concentration of 5 mg / mL.

[0030] Step 2, Qualitative analysis: Perform HPLC analysis on the test solution of the crude polysaccharide prepared in Step 1. HPLC chromatographic conditions: Use a TSKgel G5000PW_XL gel exclusion column (7.8 mm × 300 mm, the average particle size of the porous gel microsphere filler in the chromatographic column is 10 µm) as the chromatographic column for separation, use pure water as the mobile phase, with a flow rate of 0.6 mL / min, and an injection volume of 20 μL. A UV detector is combined with a refractive index detector, the wavelength is set at 280 nm, and the column oven temperature is 35 °C.

[0031] According to the operation procedures of Step 1 and Step 2 in Example 1, when the producing areas of the defatted Grifola frondosa powder are Qingyuan County, Zhejiang Province; Qianxi County, Hebei Province; Tongzi County, Guizhou Province; Guilin City, Guangxi Zhuang Autonomous Region; and Songxi County, Fujian Province respectively, the HPLC chromatogram results of the corresponding test solutions of the crude polysaccharide are shown in Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5As shown, they each showed a main chromatographic peak at retention times of 6.98, 6.72, 6.62, 6.92, and 6.5 min in sequence. Subsequently, miscellaneous peaks appeared. The retention times of this peak in samples from different origins were slightly different but all fell within the range of 6.50 min - 6.98 min. As Figures 1 - 5 shown, the retention time range of 6.50 min - 6.98 min for all main peaks was used as the chromatographic identification of the Grifola frondosa polysaccharide D-component.

[0032] Figures 1 - 5 In [reference], the left figure corresponds to the detection result under a differential refractive index detector, and the right figure corresponds to the detection result under an ultraviolet detector.

[0033] Step 3: The steps for purifying and separating the Grifola frondosa polysaccharide D-component are as follows: The Grifola frondosa polysaccharide extract was successively passed through tubular ceramic membranes with pore sizes of 200 nm, 50 nm, and 5 nm, and the retentate was collected. Then, protein was removed by the Sevag method. The retentate was concentrated through a 0.001 μm filter membrane. The concentrated solution was mixed with Sevag reagent at a volume ratio of 1:4. The Sevag reagent was CHCl3 - CH3(CH2)3OH with a volume ratio of 5:1. The mixture was centrifuged at a speed of 4000 r / min for 5 min, and the upper-layer polysaccharide solution was collected and freeze-dried.

[0034] Subsequently, the freeze-dried polysaccharide was dissolved in deionized water, filtered through a 0.45 μm filter membrane, and loaded onto a DEAE Sepharose Fast Flow anion exchange column. The column was a fast-flow column (60.6 × 218.5 mm). Then, it was eluted successively with aqueous sodium chloride solutions of different concentrations: 0 M, 0.25 M, and 0.5 M, with a flow rate of 10 mL / min. The eluate eluted with 0.5 M aqueous sodium chloride solution was collected and dialyzed (the eluate was placed in a 10000 Da dialysis bag, and the 10000 Da dialysis bag was placed in a bucket of ultrapure water and stirred with a rotor for dialysis). Finally, further purification was carried out using an XK 26 / 100 Sephacryl S-300 HR column (column size: 26 × 1000 mm). The mobile phase was ultrapure water, with a flow rate of 1 mL / min. The target component was eluted, collected, and freeze-dried to obtain the finally purified Grifola frondosa polysaccharide D-component with a purity of over 98%.

[0035] According to the operation process of Step 3 in Example 1, the crude Grifola frondosa polysaccharide from Qingyuan County, Zhejiang Province was purified and separated to obtain the pure Grifola frondosa polysaccharide D-component, and HPLC chromatographic analysis and detection were carried out on it. The detection conditions repeated Step 2 of Example 1, and the detection results are shown in Figure 6 . Figure 6Among them, the upper figure corresponds to the detection result under the differential refractive index detector, and the lower figure corresponds to the detection result under the ultraviolet detector.

[0036] Step 4: Further determine the monosaccharide and amino acid compositions of the Grifola frondosa crude polysaccharide purified and separated from all over the country.

[0037] The steps for detecting the monosaccharide content of the polysaccharide D-component of Grifola frondosa from all over the country are as follows: 1) Weigh 10 mg of the purified polysaccharide D-component of Grifola frondosa from each place and put it into a stoppered test tube. Add 2 mL of 2.0 mol / L trifluoroacetic acid aqueous solution, seal it, and hydrolyze it at 110 °C for 8 h. After the reaction is completed, add 6 mL of methanol solution, and rotate and evaporate to remove the excess trifluoroacetic acid. Repeat the operation of adding 6 mL of methanol and spinning it dry 3 - 5 times.

[0038] 2) Then perform PMP derivatization on the sample: Weigh 25 mg each of 10 standards including D-galactose, L-rhamnose, D-glucuronic acid, D-galacturonic acid 5, N-acetyl-D-glucosamine, D-glucose, D-mannose, D-xylose, D-arabinose, and D-fucose, and make up the volume to 5 mL with water in a PE tube, that is, the concentration of each monosaccharide reference substance is 5 mg / mL. Take 100 μL of each monosaccharide standard solution and mix them to obtain a mixed standard solution of 10 monosaccharide standard solutions, which is the mixed standard solution.

[0039] Take 25 mg of the polysaccharide D-component of Grifola frondosa with a purity of over 98% extracted in Example 1, and make up the volume to 5 mL with water in a PE tube, that is, obtain a sample solution to be measured with a concentration of 5 mg / mL of the polysaccharide D-component of Grifola frondosa.

[0040] Take 200 μL of the sample solution to be measured, single standard solution, and mixed standard solution respectively, add an equal volume of 0.3 mol / L NaOH solution and 0.5 mol / L PMP methanol solution, heat at 70 °C for 60 min, and after cooling to room temperature, add 0.3 mol / L HCl solution for neutralization to adjust the pH to 7.0. Add an equal volume of chloroform, vortex and mix well, remove the chloroform layer, and repeat the operation of adding chloroform and vortexing to remove the chloroform layer 3 - 5 times to remove the excess PMP, and then analyze the monosaccharide composition by the method of high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD).

[0041] The detection conditions of high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) are: the chromatographic column is C 18SHISEIDO (4.6 mm × 250 mm × 5 μm); the column temperature was 25 °C; the wavelength of the ultraviolet-visible absorption detector was 250 nm; the mobile phase was acetonitrile-phosphate buffer solution (KH2PO4, 0.05 mol / L, pH 6.9) with a volume ratio of 19:81, the sample solution detection volume was 10 μL, and the mobile phase flow rate was 1 mL / min.

[0042] For the D-component of Grifola frondosa polysaccharide from the production areas of Qingyuan in Zhejiang, Qianxi in Hebei, Tongzi in Guizhou, Guilin in Guangxi, and Songxi in Fujian, after the monosaccharide content detection was carried out according to the above process, the monosaccharide content ranges released after the hydrolysis of the D-component of Grifola frondosa polysaccharide were summarized in Table 1. It can be seen that: mainly glucose (molar ratio 50%-60%), and also contained galactose (25%-35%), mannose (5%-10%), glucuronic acid and rhamnose (2%-5%). Other monosaccharide components such as arabinose and fucose were not detected.

[0043] Table 1. Monosaccharide composition ranges of the D-component of Grifola frondosa polysaccharide

[0044] For the determination of the amino acid content of the D-component of Grifola frondosa polysaccharide from all over the country, the steps were as follows: 2.0 mL of 6 mol / L hydrochloric acid was added to 10 mg of the D-component of Grifola frondosa polysaccharide and added to a hydrolysis tube. Deoxygenation treatment was carried out by introducing nitrogen. After sealing, it was placed in a constant temperature drying oven at 110 °C and hydrolyzed at high temperature for 24 h to completely decompose the protein into free amino acids. After the hydrolysis was completed, the hydrolysis tube was taken out and cooled to room temperature, and then the sealing was opened. The hydrolysis solution was transferred to a centrifuge tube and dried under nitrogen at 50 °C to remove the free hydrochloric acid until the sample was dry. 1.0 mL of sodium citrate buffer solution (0.02 mol / L) with pH 2.2 was added to the dried residue for reconstitution. After thorough vortex mixing, it was filtered through a 0.22 μm polytetrafluoroethylene filter membrane, and the filtrate was taken for subsequent detection by an amino acid automatic analyzer.

[0045] For the Grifola frondosa polysaccharide D-component from Qingyuan in Zhejiang, Qianxi in Hebei, Tongzi in Guizhou, Guilin in Guangxi, and Songxi in Fujian, after detecting the amino acid content according to the above process, the ranges of monosaccharide contents released after hydrolysis of the Grifola frondosa polysaccharide D-component are summarized in Table 2. As a result, 17 amino acids, namely aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), alanine (Ala), threonine (Thr), serine (Ser), tyrosine (Tyr), phenylalanine (Phe), valine (Val), leucine (Leu), lysine (Lys), arginine (Arg), proline (Pro), cysteine (Cys), methionine (Met), isoleucine (Ile), and histidine (His), were detected. Glycine, glutamic acid, and aspartic acid are the main amino acids in the Grifola frondosa polysaccharide D-component. The above amino acids account for about 20%-30% of the dry weight of the Grifola frondosa polysaccharide D-component standard, which is consistent with the literature reports on the protein content of the Grifola frondosa D-component. The above compositional differences in the Grifola frondosa D-component from different sources are extremely small and all fall within the characteristic range of the Grifola frondosa D-component.

[0046] Table 2. Amino acid composition ranges of the Grifola frondosa polysaccharide D-component

[0047] This example established the fingerprint characteristic values of the Grifola frondosa D-component: retention time of 6.50 min - 6.98 min, glucose-dominated monosaccharide composition, and an amino acid profile with high glycine. This characteristic provides a standard reference for the identification of subsequent other samples.

[0048] Step Five: Quantitative calculation: Respectively take the Grifola frondosa polysaccharide D-component standard with a purity greater than 98% from Qingyuan County, Zhejiang, which was extracted in Step Three, dissolve and dilute it with water to prepare standard solutions with five concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL, and repeat the injection analysis. The analysis conditions are the same as the HPLC analysis conditions in Step Two. Use the peak area of the Grifola frondosa polysaccharide D-component as the ordinate and the concentration of the Grifola frondosa polysaccharide D-component as the abscissa to make a standard curve. The results of the standard curve are shown in Figure 7 , and its linear regression equation is y = 14969398x + 2665674 (correlation coefficient R 2 = 0.9996).

[0049] Repeatability verification: Prepare solutions with the same concentration of the Grifola frondosa polysaccharide D-component from Qingyuan, Zhejiang, and perform 5 injections according to the same chromatographic analysis method as above. The detection results of the peak time and peak area are shown in Table 3: Table 3. HPLC detection results of the Grifola frondosa polysaccharide D-component

[0050] (1)RSD of peak emergence time: Retention time = 6.75 min, SD = 0.124, RSD = 1.84% (2)RSD of peak area: Peak area = 77911144 mAu·s, SD = 614846, RSD = 1.12% The RSDs calculated from the peak emergence time and peak area of the Grifola frondosa polysaccharide D - fraction standard are both < 2%, meeting the high - precision requirements. It is determined that the peak emergence time of the Grifola frondosa polysaccharide D - fraction within the reasonable range of 6.50 min - 6.98 min can be used as a qualitative standard.

[0051] Example 2, Sample preparation: The method for extracting Lentinan is the same as that in Example 1. Accurately weigh 50 mg of crude Lentinan and dissolve it in 10 mL of ultrapure water to obtain a control solution with a concentration of 5 mg / mL. Perform detection according to the same HPLC analysis method as in Step 2 of Example 1.

[0052] Qualitative detection: The HPLC chromatogram of the Lentinan sample does not show the characteristic peak of the Grifola frondosa D - fraction within the range of 6.50 min - 6.98 min; its main peak emergence time is significantly shifted, and the main elution peak in the chromatogram appears at about 7.45 min.

[0053] In addition, after the Lentinan is separated and purified according to the method in Step 3 of Example 1, the monosaccharide and amino acid compositions are determined according to the method in Step 4 of Example 1: By monosaccharide composition analysis, Lentinan is mainly composed of glucose, but the proportion is significantly higher than that of the Grifola frondosa D - fraction. The detection results show that glucose accounts for 82.6%, mannose 7.8%, galactose 4.5%, and there are also small amounts of xylose 3.3% and arabinose 1.0%. Compared with the Grifola frondosa D - fraction, the proportion of glucose in Lentinan is much higher and it is almost the single main component. This is consistent with the known fact that Lentinan is mainly β - 1,3 - glucan and its monosaccharides are almost all glucose. In terms of amino acid composition, the protein content of the Lentinan sample is very low (< 2%), and only trace amounts of amino acids are detected after hydrolysis: glycine is about 2%, glutamic acid is about 3.5%, and the others such as alanine and arginine are all less than 2%. None of the amino acid contents are close to the characteristic range of the Grifola frondosa D - fraction. In conclusion, the Lentinan sample does not conform to the fingerprint characteristics of the Grifola frondosa D - fraction in terms of retention time, monosaccharide composition, or amino acid spectrum, proving that it does not contain the D - fraction.

[0054] Example 3, Sample preparation: Take 50 g of dried Tremella fuciformis and extract the water - soluble polysaccharide for analysis by the same method as in Example 1. Prepare an aqueous solution of Tremella polysaccharide with a concentration of 5 mg / mL as the control solution and perform detection according to the same HPLC analysis method as in Step 2 of Example 1.

[0055] Qualitative analysis: The main peak of the Tremella polysaccharide sample in the HPLC chromatogram appears at 8.48 min, and no obvious peak is observed in the range of 6.50 min - 6.98 min.

[0056] In addition, after the Tremella polysaccharide is separated and purified according to the method of Step 3 of Example 1, the monosaccharide and amino acid composition is determined according to the method of Step 4 of Example 1: The monosaccharide composition analysis shows that the composition of the Tremella polysaccharide is quite different from that of the Grifola frondosa D-fraction. Mannose and glucuronic acid are the main monosaccharides, accompanied by relatively high proportions of xylose and arabinose. Specifically measured: mannose accounts for 37%, glucuronic acid accounts for 15.4%, galactose accounts for 24.6%, arabinose accounts for 8.3%, and there is also 3.1% fucose, while the glucose content is only 4.1%. This composition is consistent with the structure of the Tremella polysaccharide reported in the literature. Obviously, the Tremella polysaccharide lacks the high glucose content characteristic of the Grifola frondosa D-fraction. The amino acid analysis results show that the protein content of the Tremella polysaccharide is extremely low (<1%), only a small amount of amino acids are detected, and the total content is less than one-tenth of the Grifola frondosa polysaccharide D-fraction. The proportion distribution of each amino acid has no obvious prominent one (all fluctuate within the range of 1% - 3%). Therefore, both the chromatographic retention time and chemical composition of the Tremella polysaccharide are not within the characteristic range of the D-fraction.

[0057] Example 4, Sample preparation: Take 50 g of dried Cordyceps sinensis, extract it under the same conditions as in Example 1 to obtain a crude polysaccharide solution. Prepare an aqueous solution with a Cordyceps polysaccharide concentration of 5 mg / mL as the control solution, and perform detection according to the same HPLC analysis method as in Step 2 of Example 1.

[0058] Qualitative analysis: The retention time of the main peak of the Cordyceps polysaccharide sample on HPLC is 11.20 min, and it does not enter the D-fraction window of 6.50 min - 6.98 min.

[0059] After the Cordyceps polysaccharide is separated and purified according to the method of Step 3 of Example 1, the monosaccharide and amino acid composition is determined according to the method of Step 4 of Example 1: Its monosaccharide composition has multiple components and is relatively average. Detection shows that: mannose accounts for 21.5%, glucose 35.0%, galactose 25.5%. In addition, arabinose is 10.2% and rhamnose is 5.6%. It can be seen that the galactose proportion of the Cordyceps polysaccharide is within the D-fraction range, while the glucose content is significantly reduced, and arabinose not contained in the D-fraction is detected at the same time. The amino acid composition analysis shows that the highest contents among the more than a dozen detected amino acids are glutamic acid and aspartic acid, with a content of 15%, glycine 3%, and the others such as alanine and leucine are all between 1% - 3%. Thus, it is confirmed that the fingerprint characteristics of the Cordyceps polysaccharide do not match those of the Grifola frondosa polysaccharide D-fraction and do not contain the D-fraction.

[0060] Example 5, Sample Preparation: Select dried Coriolus versicolor, extract water-soluble polysaccharides for analysis by the same method as in Example 1, prepare an aqueous solution with a Coriolus versicolor polysaccharide concentration of 5 mg / mL as the control solution, and perform detection by the same HPLC analysis method as in Step 2 of Example 1. The HPLC chart of the crude Coriolus versicolor polysaccharide is shown in Figure 8 .

[0061] Qualitative Analysis: The main peak of Coriolus versicolor polysaccharide appears in the HPLC chromatogram at 6.78 min, clearly falling within the retention time window (6.50 min - 6.98 min) of the Grifola frondosa D-fraction defined by the present invention. Therefore, only from the perspective of chromatographic retention time, the elution behavior of Coriolus versicolor polysaccharide is extremely similar to that of the D-fraction.

[0062] After the Coriolus versicolor polysaccharide is separated and purified by the method in Step 3 of Example 1, the monosaccharide and amino acid compositions are determined by the method in Step 4 of Example 1: The results of monosaccharide composition analysis show that glucose accounts for 55% in Coriolus versicolor polysaccharide, galactose is 28%, glucuronic acid is 10%, rhamnose is 5% and mannose is 2%. The monosaccharide composition is the same as that of the D-fraction, and the proportions of glucose and galactose both fall within the monosaccharide composition range of the D-fraction, while there are obvious differences in glucuronic acid, rhamnose and mannose. In terms of amino acid composition, Coriolus versicolor polysaccharide has typical glycoprotein characteristics, and the total protein content is about 20%. 12 common amino acids are detected, and their distribution is very close to that of the Grifola frondosa D-fraction. Among them: glutamic acid is 20%, aspartic acid is 12.5%, glycine is 18%, proline is 9%, threonine is 7%, alanine is 6%, lysine is 5%, serine is 5%, arginine is 4.5%, and leucine is 4.5%. Compared with the Grifola frondosa D-fraction, the main differences in Coriolus versicolor polysaccharide are as follows: the content of phenylalanine is relatively high, being 12%, which exceeds the upper limit (8.5%) defined by the D-fraction; the content of cysteine is on the high side, being 2.5%, higher than the maximum value of 1.0% specified by the D-fraction.

[0063] Therefore, although Coriolus versicolor polysaccharide is highly similar to the Grifola frondosa D-fraction in terms of retention time, monosaccharide composition and most amino acid proportions, there are still significant differences in specific indicators. This difference can be used as an auxiliary index for further discrimination, thus effectively avoiding misjudgment caused by overlapping retention times and ensuring the specificity of the method of the present invention.

[0064] Example 6, Sample Preparation: Take 50 g of dried Ganoderma lucidum fruit body powder, extract water-soluble polysaccharides for analysis by the same method as in Example 1, prepare an aqueous solution with a Ganoderma lucidum polysaccharide concentration of 5 mg / mL as the control solution, and perform detection by the same HPLC analysis method as in Step 2 of Example 1.

[0065] Qualitative analysis: The Ganoderma lucidum polysaccharide sample was analyzed by HPLC according to the method of the present invention. The results were significantly different from those of the Grifola frondosa sample: no characteristic peaks appeared within the retention time range of the D-component. The main peaks in the chromatogram of Ganoderma lucidum polysaccharide appeared within 10.07 min, indicating that the molecular weight distribution of Ganoderma lucidum polysaccharide was lower than that of the D-component of Grifola frondosa. At the same time, no obvious absorption peak was observed in the UV detection signal of Ganoderma lucidum polysaccharide, only a weak signal intensity existed, which was also different from the characteristic of the D-component of Grifola frondosa that bound a small amount of protein and showed a weak absorption at 280 nm.

[0066] After the Ganoderma lucidum polysaccharide was separated and purified according to the method of Step 3 of Example 1, the monosaccharide and amino acid compositions were determined according to the method of Step 4 of Example 1: To further verify the specificity, the monosaccharide composition analysis of the main chromatographic peaks of the Ganoderma lucidum sample was carried out. The results showed that the main products of the hydrolysis of Ganoderma lucidum polysaccharide were glucose (40%) and galactose (45%). Rhamnose and glucuronic acid components were not detected, but arabinose (10%) and fucose (5%) that were not present in the D-component were detected. This was significantly different from the monosaccharide composition characteristics of the D-component of Grifola frondosa (containing rhamnose and glucuronic acid). Thus, it can be seen that the method of the present invention will not produce a D-component peak determination in the Ganoderma lucidum polysaccharide sample, proving the specificity of the method.

[0067] Example 7, Sample preparation: Take 50 g of dried Ningxia wolfberry fruits, extract the water-soluble polysaccharide for analysis according to the same method as in Example 1, prepare an aqueous solution with a concentration of 5 mg / mL of wolfberry polysaccharide as the control solution, and detect it according to the same HPLC analysis method as in Step 2 of Example 1.

[0068] Qualitative analysis: The wolfberry polysaccharide sample was detected according to the same HPLC analysis method. No characteristic chromatographic peak of the D-component of Grifola frondosa polysaccharide was found at 6.50 min - 6.98 min. The peak time of wolfberry polysaccharide was at 11.32 min, and the peak shape was relatively wide, presumably composed of a series of pectin polysaccharides with a small molecular weight. Wolfberry polysaccharide had a low absorption at 280 nm, which might be due to the residual trace protein without purification, in line with its characteristic of being a pure polysaccharide with almost no protein.

[0069] After the wolfberry polysaccharide was separated and purified according to the method of Step 3 of Example 1, the monosaccharide and amino acid compositions were determined according to the method of Step 4 of Example 1: The wolfberry polysaccharide sample was hydrolyzed and analyzed for monosaccharides. It was found that its main monosaccharides were galactose (30%), arabinose (25%), and galacturonic acid (20%) (in line with the typical plant pectin-type polysaccharide composition). Among them, the content of galacturonic acid was 20%, which was much higher than that of the D-component. There was a small amount of glucose (15%) and rhamnose (10%). This composition was significantly different from the monosaccharide spectrum of Grifola frondosa D-component. The highest was glutamic acid at 9.2%, aspartic acid at 8.5%, glycine only at 3.1%, serine at 7.4%, proline at 6.8%, lysine at 5.0%, and the others were all below 5%. In contrast, the glycine and glutamic acid in the Grifola frondosa D-component were much higher than these levels. Therefore, the wolfberry polysaccharide itself did not contain the Grifola frondosa D-component, and its compositional characteristics were completely outside the D-component fingerprint range. It could be determined that the wolfberry polysaccharide sample did not contain the Grifola frondosa D-component.

[0070] Example 8 (Verification of Adulteration Model) Experimental Purpose: To verify the detection ability and accuracy of the method of the present invention for Grifola frondosa D-component in complex matrices.

[0071] Experimental Method: Take 90 mL of the wolfberry polysaccharide sample solution (without Grifola frondosa D-component) in Example 7, add 10 mL of the Grifola frondosa D-component solution with a concentration of 5.0 mg / mL prepared in Step 5 of Example 1, and mix the two evenly to form an "adulterated sample", simulating the situation where a certain proportion of Grifola frondosa polysaccharide D-component is adulterated in the actual product. The theoretical addition amount of Grifola frondosa D-component in the mixed sample after mixing was 10% (volume ratio), and the corresponding concentration of D-component in the solution was about 0.5 mg / mL. Take the adulterated sample and the unadulterated wolfberry polysaccharide sample respectively for HPLC analysis and comparison.

[0072] Results and Analysis: The wolfberry polysaccharide sample without D-component addition had no chromatographic peak at 6.5 min - 6.98 min (as shown in Example 7). However, a clear new chromatographic peak appeared at the retention time of 6.82 min in the chromatogram of the mixed sample after adding Grifola frondosa D-component. See Figure 9, and its retention time is consistent with that of the Grifola frondosa D-fraction peak. The ultraviolet spectrum and differential refractive index response of this peak also conform to the characteristics of the D-fraction. Calculate the peak area of this peak, convert it into the content of Grifola frondosa D-fraction, and the result is 0.47 mg / mL, which is converted back to the proportion in the total amount of the mixed solution as 9.4%. Compared with the theoretically added 0.5 mg / mL (10%), the recovery rate is 94%. Considering a small amount of sample loss and operation errors, this result is within the allowable range, indicating that this method has a high quantitative accuracy for Grifola frondosa D-fraction. Thus, even in the complex matrix of Lycium barbarum polysaccharide, the method of the present invention can still accurately identify and quantify Grifola frondosa D-fraction without being interfered by other polysaccharide components in the matrix. This example simulates the possible adulteration situations in the market, proving that the method of the present invention can be used to detect the artificially added Grifola frondosa D-fraction in other polysaccharide products, and has good applicability and accuracy.

[0073] Figures 8 - 9 In it, the upper figure corresponds to the detection result under the differential refractive index detector, and the lower figure corresponds to the detection result under the ultraviolet detector.

[0074] In summary, through the verification of the above-mentioned various embodiments, the qualitative and quantitative detection method of Grifola frondosa polysaccharide D-fraction of the present invention has high specificity, sensitivity and reliability. It can effectively distinguish Grifola frondosa D-fraction from polysaccharides from other sources and accurately determine its content, which has important value for the quality control, authenticity identification and scientific research analysis of Grifola frondosa polysaccharide products.

[0075] The content described in this specification is only a list of the implementation forms of the inventive concept, and the protection scope of the present invention should not be regarded as limited to the specific forms stated in the embodiments.

Claims

1. A qualitative and quantitative detection method for Grifola frondosa polysaccharide D-component, characterized in that It includes the following steps: Step 1: Select defatted Grifola frondosa powder from fruiting bodies of Grifola frondosa across the country, obtain crude Grifola frondosa polysaccharide by hot water extraction method, conduct HPLC analysis and detection, obtain a series of HPLC charts of crude Grifola frondosa polysaccharide, determine the elution time range of Grifola frondosa polysaccharide D-component, and preliminarily judge whether the sample contains Grifola frondosa polysaccharide D-component according to the characteristic peaks within the elution time range; Step 2: Purify and separate the crude polysaccharides extracted from defatted Grifola frondosa powder from various sources across the country to obtain Grifola frondosa polysaccharide D-component; Step 3: Determine the monosaccharide and amino acid compositions of Grifola frondosa polysaccharide D-component from various producing areas across the country, summarize the analysis results from different sources of production areas, determine the main monosaccharide content range and the content range of main amino acids, that is, determine the fingerprint characteristic values of Grifola frondosa polysaccharide D-component, and further qualitatively judge Grifola frondosa polysaccharide D-component according to this characteristic value; Step 4: Make an HPLC analysis standard curve for Grifola frondosa polysaccharide D-component and quantitatively detect the content of Grifola frondosa polysaccharide D-component in the sample.

2. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component as described in claim 1, characterized in that The method steps for extracting crude Grifola frondosa polysaccharide in Step 1 are as follows: Mix defatted Grifola frondosa powder according to a solid-liquid mass ratio of 1:10 - 30, stir evenly, heat the mixture in a heating environment at 90 - 100 °C for 2 - 4 h, after the temperature drops to room temperature, filter by suction, concentrate the filtrate by rotary evaporation and then freeze-dry to obtain crude Grifola frondosa polysaccharide.

3. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component according to claim 1, characterized in that The HPLC analysis and detection conditions in Step 1 are as follows: Chromatographic conditions: Use a TSKgel G5000PW_XL gel exclusion column, 7.8 mm × 300 mm; Use pure water as the mobile phase with a flow rate of 0.5 - 0.6 mL / min; Couple an ultraviolet detector with a differential refractive index detector. Set the detection wavelength of the ultraviolet detector to 280 nm and the temperature of the column oven to 35 °C; The HPLC analysis and detection conditions when making the HPLC analysis standard curve in Step 4) are the same as those in Step 1).

4. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component according to claim 1, characterized in that The steps for separation and purification in Step 2 are as follows: S1: Dissolve the crude polysaccharide in pure water and sequentially pass it through tubular ceramic membranes with pore sizes of 200 nm, 50 nm, and 5 nm, and collect the retentate; S2: Concentrate the retentate through a 0.001 μm filter membrane, mix the concentrated solution with Sevag reagent according to a volume ratio of 1:3 - 5. Sevag reagent is a CHCl3-CH3(CH2)3OH mixed solvent with a volume ratio of 4 - 6:1, centrifuge and separate, and collect the upper polysaccharide solution and freeze-dry it; S3: Dissolve the freeze-dried polysaccharide in deionized water, filter it through a 0.45 μm filter membrane, load it onto a DEAE Sepharose Fast Flow anion exchange column, and elute it sequentially with sodium chloride aqueous solutions with different concentrations of 0 M, 0.25 M, and 0.5 M. Collect the eluate of 0.5 M sodium chloride aqueous solution and dialyze and freeze-dry it; S4: The freeze-dried product was further dissolved in pure water and subjected to semi-preparative night phase purification using an XK 26 / 100 Sephacryl S-300 HR column with ultrapure water as the mobile phase. The target fraction was collected and freeze-dried to obtain the final purified Grifola frondosa polysaccharide D-fraction.

5. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component as claimed in claim 1, characterized in that In step 3, the monosaccharide composition of the D-component of Grifola frondosa polysaccharide is determined according to the following steps: Step 1: Add the D-component of Grifola frondosa polysaccharide to a 1.5M-3M trifluoroacetic acid aqueous solution at a feed ratio of 10 mg: 1.5-3 mL, add it to a sealed reactor, heat it at 100-120°C for hydrolysis for 5-10 hours, wait for the reaction to end, add methanol and remove excess trifluoroacetic acid by rotary evaporation; Step 2: The hydrolyzate of step 1 is dissolved in water, and NaOH solution and excess PMP solution are added. The final concentration of NaOH in the mixed solution is 0.05-0.2M. The mixture is heated at 60-80°C for 40-80 minutes. After cooling to room temperature, acid is added to adjust the pH to neutral. Chloroform is added to wash and remove excess PMP, and the water layer containing the derivatized product is collected; Step 3. Analyze the monosaccharide composition of the aqueous layer containing the derivatization product by high performance liquid chromatography-evaporative light scattering detector. The chromatographic column for high performance liquid chromatography is C 18 SHISEIDO, 4.6 mm×250 mm×5 μm, and the wavelength of the ultraviolet-visible absorption detector is 250 nm.

6. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component according to claim 1, characterized in that In step 3, the amino acid composition of the D-component of Grifola frondosa polysaccharide is determined according to the following steps: Step 1: Add the D-component of Grifola frondosa polysaccharide to 5.0-6.0M hydrochloric acid at a feed ratio of 10mg:1.5-3mL, add it to a hydrolysis tube, deoxygenate it by passing nitrogen, seal it, place it in a 100-110°C constant temperature drying oven, and hydrolyze it at high temperature for 20-30h to completely decompose the protein into free amino acids; Step 2: After the hydrolysis is completed, take out the hydrolysis tube, cool it to room temperature, open the sealed port, transfer the hydrolyzate to a centrifuge tube, and blow dry with nitrogen at 40-60°C to remove free hydrochloric acid until the sample is dry; Step 3: Add pH 2.2 sodium citrate buffer to the dry residue for re-dissolution, vortex mix thoroughly and filter through a 0.22 μm polytetrafluoroethylene filter membrane, and use an amino acid automatic analyzer to detect and analyze the amino acid composition of the filtrate.

7. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component according to claim 1, characterized in that Step 1: The defatted Grifola frondosa powder of Grifola frondosa fruiting bodies is produced in Qingyuan County, Zhejiang Province, Qianxi County, Hebei Province, Tongzi County, Guizhou Province, Guilin City, Guangxi Province and Songxi County, Fujian Province.

8. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component according to claim 1, characterized in that When determining the fingerprint characteristic value of the D-component of Grifola frondosa polysaccharide in step 3, the monosaccharide content and amino acid content are as follows: Monosaccharide content index: in terms of molar percentage, glucose 50-60%, galactose 25-35%, mannose 5-10%, and the rest are mainly glucuronic acid and rhamnose; Amino acid content indicators: in terms of molar percentage, glycine 17-23%, glutamic acid 15-22%, aspartic acid 7-12%, alanine 5-9%, tyrosine 4.5-8.5%, phenylalanine 4.5-8.5%, threonine 4-8%, serine 4-8%, and the rest are leucine, valine, lysine, proline, arginine, isoleucine, histidine, methionine and cysteine.

9. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component according to claim 1, characterized in that The process of preparing the HPLC analysis standard curve of Grifola frondosa polysaccharide D-component in Step 4 is as follows: Using Grifola frondosa polysaccharide D-component with a purity greater than 98% as the standard product, prepare standard solutions with five concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL with water, and then conduct HPLC analysis and detection. Draw a standard curve with the concentration of Grifola frondosa polysaccharide D-component as the abscissa and the peak area of the HPLC chromatographic peak of Grifola frondosa polysaccharide D-component as the ordinate.

10. The qualitative and quantitative detection method of Grifola frondosa polysaccharide D-component according to claim 1, characterized in that The elution time range of Grifola frondosa polysaccharide D-component in Step 1 is 6.50 min - 6.98 min.

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