Active ingredient and method for identifying structural integrity of polysaccharide preparations with a beta-1,3 glycosidic bond backbone

Abstract

The application provides a method for identifying active ingredients and structural integrity of a polysaccharide preparation with a beta-1,3 glycosidic bond as a main chain, comprising: S1: performing differential acidolysis of sample crude polysaccharide extract based on glycosidic bond stability difference; S2: performing chromatographic analysis on the acidolysis product to obtain a characteristic degradation chromatogram of the sample; S3: performing beta-1,3-glycosidic bond specific enzymolysis on the sample, and self-assembling a mixture of glucans; S4: preparing the self-assembled mixture into a solidified film, and performing Raman spectrum analysis to obtain a Raman spectrum diagram containing sample characteristic peaks; and S5: judging the polysaccharide active ingredients, content and structural integrity contained in the sample according to the characteristic degradation chromatogram and the Raman spectrum characteristic peaks. The method of the application effectively eliminates the interference of alpha-glucan adjuvants through double verification of "chemical + physical" fingerprints, accurately evaluates the integrity of the core structure of polysaccharides, and improves the detection efficiency and comprehensiveness of the detection results of polysaccharides.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality testing technology, specifically relating to a quality control method for traditional Chinese medicine formula granules. It is particularly applicable to the specific identification of target active polysaccharide components and the assessment of the integrity of their core structural units in traditional Chinese medicine formula granules containing β-glucan active ingredients and maltodextrin (α-1,4-glucan) excipients. Background Technology

[0002] The core active ingredients of many traditional Chinese medicines, such as Poria cocos, shiitake mushrooms, and Astragalus membranaceus, are polysaccharides (especially β-glucan). To improve the formulation's formability and flowability, large amounts of maltodextrin are often added as excipients during production. However, existing technologies have the following problems: Difficulty in effectively distinguishing between active ingredients and excipients: Both active polysaccharides (such as β-1,3-glucan) and excipient maltodextrin (α-1,4-glucan) are polysaccharides with highly similar physicochemical properties (such as solubility and molecular weight range). Conventional methods for determining total polysaccharide content (such as the sulfuric acid-anthrone method) cannot distinguish between the two, resulting in test results that do not accurately reflect the content of active ingredients, and may even be artificially inflated with increasing excipient addition, thus losing their significance for quality evaluation.

[0003] There is a lack of effective evaluation of the core structural units of polysaccharides: Existing technologies mostly focus on the content or total sugar composition of polysaccharides, while the complex spatial structure and specific glycosidic bond linkages of polysaccharides are the basis of their activity. Existing methods cannot directly and effectively assess the damage caused to the core structural units (such as β-1,3-glycosidic bonds) necessary for activity during the production process (such as high-temperature concentration, drying, and granulation).

[0004] The detection methods are either time-consuming or lack specificity: some methods (such as specific enzymatic digestion combined with chromatography) can perform identification, but the procedures are cumbersome and time-consuming; other spectroscopic methods are often difficult to promote and apply due to unstable signals and poor reproducibility. For highly similar excipient interference systems, there is a lack of a rapid, specific, and comprehensive quality control method that can assess the integrity of the active structure.

[0005] Cao Yuxin et al. (Chinese Traditional and Herbal Drugs, 2020, 51(21): 5598-5606.) disclosed the use of "partial acid hydrolysis-hydrophilic interaction chromatography" for fingerprinting of Astragalus polysaccharides, but their sample was pure medicinal polysaccharide, which did not involve the interference of high content of maltodextrin excipients in the formulation granules, and did not propose a structural integrity assessment. CN116380863B disclosed the Raman detection of polysaccharides using metal-organic framework thin films, but it relies on dye labeling and surface-enhanced Raman, which is cumbersome and complex to operate, and can only reflect the polysaccharide content, lacking a structural integrity assessment.

[0006] Therefore, developing a quality control method that can effectively eliminate the interference of excipients and quickly evaluate the integrity of the core structural units of active polysaccharides is of great application value and universal significance for improving the product quality of traditional Chinese medicine formula granules and ensuring clinical efficacy.

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a quality control method for traditional Chinese medicine (TCM) formula granules based on characteristic degradation sugar spectra and Raman fingerprinting. This method aims to solve the problem of specifically identifying whether TCM formula granules contain β-glucan active ingredients derived from medicinal materials under the interference of high-content excipients (especially α-glucan maltodextrin excipients), and to quantitatively evaluate the integrity of the core structural units (such as β-1,3-glycosidic bonds) of polysaccharide active ingredients after the formulation process. Summary of the Invention

[0008] To achieve the above objectives, the present invention first provides a method for identifying the active ingredients and structural integrity of polysaccharide preparations with β-1,3 glycosidic bonds as the main chain, the method comprising the following steps: S1: Obtain the crude polysaccharide extract of the sample to be tested, and use an acid solution to perform differential acid hydrolysis (also referred to as partial acid hydrolysis in this invention) on the crude polysaccharide extract based on the difference in glycosidic bond stability, to obtain the partially acid hydrolysis products that selectively break the β-1,3-glycosidic bonds in the polysaccharide molecules while basically retaining other types of glycosidic bonds, such as β-1,6-glycosidic bonds and α-1,4-glycosidic bonds. S2: The partial acid hydrolysis products obtained in S1 are subjected to derivatization based on chromatographic analysis and hydrophilic chromatographic analysis to obtain the characteristic degradation chromatogram of the sample to be tested. S3: Obtain the crude polysaccharide extract of the sample to be tested, and perform enzymatic hydrolysis using a β-1,3-glycosidic bond-specific hydrolase to obtain a temperature-controlled β-1,3-glycosidic bond-based glucosinolate self-assembled mixture. The self-assembly described in this invention refers to the process in which β-1,3-glucan is cleaved into a series of short-chain oligosaccharides after enzymatic hydrolysis. These oligosaccharide fragments retain the conformational "memory" of the original polysaccharide chain and can spontaneously form an ordered structure in solution through intermolecular forces such as hydrogen bonds. The nanostructures (such as layered crystals) formed by self-assembly have a highly regular periodicity and provide a very clear Raman "fingerprint". By analyzing the Raman spectrum of the self-assembled product, the characteristic peaks at specific wavelengths can be clearly attributed to a certain glycosidic bond as "diagnostic peaks", and the original structural information of the oligosaccharide fragments that build these structures can be inferred in reverse. S4: The β-1,3-glucan self-assembled mixture obtained in S3 was used to prepare a cured film, and Raman spectroscopy analysis was performed to obtain a Raman spectrum containing the characteristic peaks of the sample to be detected. S5: Determine the polysaccharide active ingredients and their content in the sample based on whether the characteristic chromatographic peaks of β-1,3-glycosidic bonds are present in the characteristic degradation chromatogram of the sample obtained in S2 and their peak height / area ratio; determine the structural integrity of the polysaccharide active ingredients in the sample based on whether the Raman spectral characteristic peaks of β-1,3-glycosidic bonds are present in the sample obtained in S4.

[0009] In the method of the present invention, the numbering of S1 and S3 is only used to distinguish different steps and does not mean that S1 must be performed first. That is, in an optional scheme, S1 and S3 can be performed simultaneously, or S3 can be performed before S1. Both are optional technical solutions of the present invention.

[0010] In a preferred embodiment of the present invention, the polysaccharides with β-1,3 glycosidic bonds as the main chain include Poria cocos polysaccharide, Lentinus edodes polysaccharide, Grifola frondosa polysaccharide, Ganoderma lucidum polysaccharide, Trametes versicolor polysaccharide, and Polyporus umbellatus polysaccharide. These polysaccharides, in addition to having β-1,3 glycosidic bonds as the main chain, also contain other types of branches in varying numbers, such as β-1,6 branches. Although the present invention provides specific embodiments of Poria cocos polysaccharide and Lentinus edodes polysaccharide, those skilled in the art will understand that the identification methods provided by the present invention are applicable to these polysaccharide preparations with β-1,3 glycosidic bonds as the main chain.

[0011] In specific embodiments of the present invention, examples of dosage forms of Poria cocos polysaccharide formulation granules and decoction pieces are provided. These are common dosage forms in traditional Chinese medicine manufacturing. The method of the present invention is also applicable to other dosage forms of traditional Chinese medicine in this field, such as pills, powders, ointments, medicated wines, and elixirs.

[0012] In a preferred embodiment of the present invention, the acid solution in S1 is a trifluoroacetic acid (TFA) solution, and the reaction conditions for the differentiated acid hydrolysis are set as follows: a trifluoroacetic acid solution with a trifluoroacetic acid concentration of 0.2 to 0.7 mol / L, an acid hydrolysis temperature of 75 to 85°C, and an acid hydrolysis time of 1.0 to 2.5 hours. After the acid hydrolysis is completed, the trifluoroacetic acid is removed by rotary evaporation and then reconstituted to obtain the acid hydrolysis product.

[0013] In a specific implementation of this invention, the differentiated acid hydrolysis reaction conditions are a core technical feature for preferentially breaking β-1,3-glycosidic bonds in polysaccharide molecules. For example, the preferred reaction conditions for differentiated acid hydrolysis of Poria cocos polysaccharide are a trifluoroacetic acid concentration of 0.5 mol / L, an acid hydrolysis temperature of 80°C, and a preferred acid hydrolysis time of 2 hours. Since the structure of lentinan is slightly different, the acid hydrolysis conditions can be fine-tuned, for example, by using a TFA concentration of 0.2~0.4 mol / L to produce stable and characteristic oligosaccharide fragments.

[0014] In a preferred embodiment of the present invention, the derivatization treatment in S2 is ethyl 4-aminobenzoate (4-ABEE) and sodium cyanoborohydride derivatization. The derivatized sample is analyzed using hydrophilic interaction chromatography-fluorescence detection. Specific chromatographic conditions are: amide column, water-acetonitrile mobile phase, gradient elution; excitation wavelength of 305 nm, and emission wavelength of 360 nm. The retention time of the characteristic peak of the β-1,3 glycosidic bond in the hydrophilic chromatographic analysis is 4.8–6.5 minutes. If a characteristic peak appears in the chromatogram at a retention time of 4.8–6.5 minutes, it indicates that the sample contains characteristic oligosaccharide components derived from the target active polysaccharide, achieving specific identification under high excipient interference.

[0015] In specific implementations of this invention, the retention times of the characteristic peaks of β-1,3-glycosidic bonds in hydrophilic chromatographic analysis of different polysaccharides vary slightly. For example, the retention time for Poria cocos polysaccharide is 4.8–5.2 minutes, more preferably 5 minutes. This characteristic peak does not interfere with the analysis of seven easily confused medicinal materials (Platycodon grandiflorus, Polygonatum sibiricum, Dioscorea opposita, Panax ginseng, Astragalus membranaceus, Polyporus umbellatus, and Lentinus edodes polysaccharides). Figure 3 ), with strong exclusivity.

[0016] The retention time for lentinan may be slightly adjusted, but is preferably 6.0 to 6.5 minutes.

[0017] In a preferred embodiment of the present invention, in step S5, the peak height / area ratio is used as an upper or lower threshold for determining the content and amount of polysaccharide active ingredients in the sample to be tested, within the range of 4.21% to 7.81%. The threshold is preferably used for determining the content and amount of polysaccharide active ingredients in Poria cocos polysaccharide preparations.

[0018] Based on common technical knowledge in this field, acid hydrolysis is universally applicable, and both α-1,4 and β-1,3 glycosidic bonds will be hydrolyzed. The concept of this invention lies in separating the complex oligosaccharide mixture produced after hydrolysis according to their polarity differences using hydrophilic interaction chromatography, thus forming a polarity distribution map of the oligosaccharides. In this distribution map, medicinal materials such as Poria cocos will produce a characteristic, specific oligosaccharide chromatographic peak (RT≈5min), which is not present in maltodextrin excipients and many easily confused medicinal materials (see...). Figure 3 This enables "fingerprint"-like specific identification even under the interference of auxiliary materials.

[0019] In this invention, the peak height / area of ​​the characteristic peak shows a good linear relationship with the sample concentration within a certain range, and can be used as a semi-quantitative indicator of active oligosaccharide fragments to monitor batch-to-batch consistency. Figure 4 (D), which can semi-quantitatively monitor batch consistency.

[0020] Universality: For other traditional Chinese medicines with β-glucan as the active ingredient (such as shiitake mushroom, astragalus, and poria cocos), their specific characteristic degradation sugar profiles can be established by optimizing acid hydrolysis conditions.

[0021] In a preferred embodiment of the present invention, the β-1,3-glycosidic bond-specific hydrolase in S3 is β-1,3-endoglucanase, and the enzymatic hydrolysis reaction conditions are set as follows: enzyme / substrate ratio 0.3~0.7 (w / w), pH 4.5~5.5, and enzymatic hydrolysis at 35~40℃ for 2~4 hours. After enzymatic hydrolysis, the product is obtained by ultrafiltration, dialysis, and lyophilization.

[0022] In a more preferred embodiment of the present invention, the reaction conditions for the enzymatic hydrolysis are set as follows: pH 5.0, enzyme / substrate ratio 0.5, enzymatic hydrolysis for 3 hours, 3 kDa ultrafiltration and 500 Da dialysis followed by lyophilization.

[0023] In a preferred embodiment of the present invention, the cured film in step S4 is prepared by mixing the aqueous solution of the enzymatic hydrolysis product obtained in step S3 with a composite solvent of water-ethanol-0.1% Tween 20, spotting the mixture onto a glass slide, and drying it to form a film. Water is the solvent, ethanol is used to accelerate evaporation and aid drying, and a trace amount of Tween 20 is used to reduce surface tension and improve film uniformity. The synergistic effect of these three solvents is superior to that of a simple aqueous solution. The specific ratio can be fine-tuned according to the actual effect to achieve a clear film formation without interfering with detection.

[0024] In a preferred embodiment of the present invention, the ratio of water:ethanol:0.1%Tween 20 in the composite solvent is 13:10:7.

[0025] In a preferred embodiment of the present invention, the Raman spectroscopy analysis in S4 uses a 785 nm wavelength laser with a scanning range of 200 to 2000 cm⁻¹. -1 Three to five sampling points are used to accurately reflect the Raman characteristics.

[0026] In a preferred embodiment of the present invention, in step S5, the Raman spectral characteristic peak of the β-1,3-glycosidic bond is determined to be at 1000 cm⁻¹. -1 The peak intensity is ≥50 au. The position and intensity of the characteristic peak are preferably determined based on the structural integrity of the polysaccharide active ingredients contained in the Poria cocos polysaccharide preparation.

[0027] In this invention, the self-assembly properties of polysaccharides in specific solvent systems are utilized to physically enrich and arrange oligosaccharide molecules through a "curing film" technique, thereby greatly enhancing their Raman signals and achieving the desired Raman signal for core structural units (such as β-1,3-glycosidic bonds, with characteristic peaks around 1000 cm⁻¹). -1 Visualization and dynamic tracking of structural changes are possible. This is more intuitive than macroscopic spectral methods such as near-infrared spectroscopy and can directly reflect structural changes.

[0028] This invention uses Poria cocos polysaccharide decoction pieces as a characteristic oligosaccharide reference standard, and the enzymatic hydrolysis products of the medicinal decoction pieces themselves as a reference standard. This solves the problems of difficult and expensive preparation of polysaccharide component standards, and better meets the actual quality control needs of enterprises. In one specific embodiment of this invention, the Raman spectral changes of the solidified film of the characteristic oligosaccharide reference standard from the enzymatic hydrolysis of Poria cocos polysaccharide decoction pieces are dynamically monitored after 1 day, 3 days, and 5 days; the Raman spectral changes at 1000 cm⁻¹ are used when the reference standard is fully assembled (usually after 3 days). -1 The intensity of the characteristic peak appearing at a certain point is used as a judgment threshold, which is preferably ≥50 au. This invention has found that at 1000 cm⁻¹... -1 Raman peaks are a specific physical fingerprint reflecting the integrity of the core structure of β-1,3-glycosidic bonds. By comparing them with the threshold of enzymatic hydrolysis products (e.g., ≥50 au), it is possible to directly and quantitatively determine whether the production process has damaged the key structural units of the active polysaccharide.

[0029] Comparing the acid hydrolysis products of medicinal decoction pieces (relatively intact structure, high peak intensity, 2000-8000 au) with the acid hydrolysis products of formulated granules (structural damage after processing, decreased peak intensity, such as 65-72 au), a decrease was observed between the two. The enzymatic hydrolysis control represents "the minimum effective strength of the core structural units required to maintain basic self-assembly capability."

[0030] When determining the results, the solidified film of the sample to be tested (such as the acid hydrolysis product of Poria cocos granules) was analyzed at 1000 cm⁻¹. -1 The intensity of the characteristic peak at a certain point is used to determine whether the core structural unit of the β-1,3-glycosidic bond in the sample is intact. If the peak intensity is not lower than the threshold mentioned above (e.g., ≥50 au), the core structural unit is considered to be intact. If it is lower than the threshold, the core structural unit is considered to be damaged. The peak intensity of the acid hydrolysis product of the medicinal material (usually 2000–8000 au) is used as a reference range for "good structure".

[0031] In the technical solution of this invention, the partial acid hydrolysis + fluorescence chromatography analysis completed in S1-S2 to determine the presence or absence of the characteristic structure of active polysaccharides is essentially the qualitative chemical fingerprint of the characteristic structure of active polysaccharides. The partial enzymatic hydrolysis + Raman spectroscopy analysis completed in S3-S4 to determine the integrity of the characteristic structure of active polysaccharides is essentially the verification of the physical fingerprint structure. The collaborative quality control strategy of "chemical fingerprint qualitative analysis - physical fingerprint structure verification" constructed in this invention can be implemented simultaneously, i.e., unified sample processing and dual-channel detection: Take the same batch of Chinese herbal medicine formula granule samples and divide them into two parallel portions. The first portion undergoes partial acid hydrolysis-derivativeization-HILIC-FLD to establish a characteristic degradation sugar spectrum. If a characteristic peak appears in the chromatogram at a specific retention time window (e.g., 4.8–5.2 minutes), the chemical fingerprint of the sample is deemed qualified (indicating that the sample contains characteristic oligosaccharide components from the medicinal material, excluding excipient interference). This is the chemical fingerprint determination. The second portion undergoes partial enzymatic hydrolysis-curing film-Raman spectroscopy to collect the Raman fingerprint and measure it at 1000 cm⁻¹. -1 The intensity of the characteristic peak at 1000 cm⁻¹ will be measured. -1 The Raman peak intensity is compared with a threshold determined by a reference standard (e.g., ≥50 au). If the intensity is not lower than the threshold, the core structural unit of the sample is considered intact (β-1,3-glycosidic bonds are well preserved); if it is lower than the threshold, the core structural unit is considered damaged.

[0032] Finally, a comprehensive quality evaluation is conducted: only samples that simultaneously meet the criteria of "chemical fingerprint qualified (characteristic peak detected)" and "physical fingerprint qualified (1000 cm⁻¹)" will be evaluated. -1 When the peak intensity is greater than or equal to the threshold, the batch of products is ultimately determined to be qualified; if any condition is not met, it is determined to be unqualified.

[0033] In the aforementioned collaborative quality control strategy of "chemical fingerprinting for qualitative analysis + physical fingerprinting for structural verification," chemical fingerprinting provides the answer to "what it is" (identification of specific components), while physical fingerprinting provides the answer to "is the structure intact?" (assessment of the integrity of core structural units). Both are indispensable: if only chemical fingerprinting is performed, structural damage during the production process cannot be assessed; if only Raman spectroscopy analysis is performed, it is impossible to distinguish whether the signal originates from active polysaccharides or excipients (although maltodextrin shows a similar signal at 1000 cm⁻¹). -1 While no characteristic peaks were observed, polysaccharides from other sources may cause interference, and chemical fingerprinting provides specific identification. The two work synergistically to construct a complete chain of evidence from "presence of component" to "intact active structure," overcoming the limitations of single detection principles (chromatography alone or spectroscopy alone).

[0034] The technical solution of this invention verifies the synergistic antitumor effect of the sample combined with cisplatin on human hepatocellular carcinoma Huh-7 cells by determining the free radical scavenging rate of the sample using the DPPH method and / or by using the CCK-8 method. This demonstrates that the determination of the active ingredient, its content, and structural integrity is consistent with its biological activity, and it has high reliability for clinical application. These steps can be preferred as in vitro biological activity verification in a quality control strategy, but are not mandatory steps in routine quality control.

[0035] Secondly, this invention provides a system for identifying the active ingredients and structural integrity of polysaccharide preparations with β-1,3 glycosidic bonds as the main chain, using the above-mentioned method. The system includes the following modules: (1) Sample processing module, wherein the sample processing module is used to prepare the sample to be tested into a crude polysaccharide extract; (2) Chromatographic analysis module, wherein the chromatographic analysis module is used to derivatize the crude polysaccharide extract and perform chromatographic analysis; (3) Raman spectroscopy analysis module, wherein the Raman spectroscopy analysis module is used to cure the crude polysaccharide extract into a film and perform Raman spectroscopy analysis; (4) Result analysis and output module, which is used to analyze the results output by the chromatography analysis module and the Raman spectroscopy analysis module, and to make a judgment on the polysaccharide active ingredients, content and structural integrity contained in the sample to be tested and output the judgment results.

[0036] Compared with the prior art, the present invention has the following beneficial effects: High specificity: Effectively eliminates serious interference from α-glucan excipients such as maltodextrin, and directly targets the β-glucan active ingredients derived from medicinal materials for detection.

[0037] Structural integrity can be quantified: The integrity of the core structural units of polysaccharides can be evaluated by using the "cured film-Raman spectroscopy" technique and setting thresholds, providing a new dimension for the quality control of polysaccharide drugs.

[0038] Collaborative quality control ensures high reliability: "Chemical + Physical" dual verification overcomes the limitations of single detection methods, resulting in more reliable results.

[0039] Label-free, self-assembly enhanced signal: Unlike existing technologies that rely on metal-organic frameworks and / or dye labeling, this invention utilizes the self-assembly properties of polysaccharides in specific solvent systems to physically enrich target molecules, greatly enhancing the Raman signal (without SERS enhancement). This "physical self-assembly enhancement" method, which dynamically tracks conformational changes, overcomes the shortcomings of existing Raman detection techniques for polysaccharides.

[0040] The method is easy to operate and widely applicable: the steps are clear, the time required is short (approximately 3-4 hours), and it uses endogenous reference standards. This strategy is not limited to Poria cocos; it can be applied to the quality control of other traditional Chinese medicine formulas containing β-glucan active structures, such as Lentinus edodes, Astragalus membranaceus, and Polyporus umbellatus, by adjusting and limiting specific parameters (such as trifluoroacetic acid concentration, temperature, and time) and chromatographic gradients. Attached Figure Description

[0041] Figure 1 This is a schematic flowchart of the polysaccharide detection method based on characteristic degradation sugar spectrum and Raman fingerprint spectrum of the present invention; Figure 2 This is a chromatogram of Poria cocos slices and Poria cocos granules under the preset acid hydrolysis conditions of the present invention: acid hydrolysis temperature of 80℃, acid hydrolysis time of 1h, and 0.25mol / L trifluoroacetic acid. Figure 3 This is a chromatogram of Poria cocos slices and Poria cocos granules under the preset acid hydrolysis conditions of the present invention: acid hydrolysis temperature of 80℃, acid hydrolysis time of 1h, and 0.5mol / L trifluoroacetic acid. Figure 4 This is a chromatogram of Poria cocos slices and Poria cocos granules under the preset acid hydrolysis conditions of the present invention: acid hydrolysis temperature of 80℃, acid hydrolysis time of 2h, and 0.5mol / L trifluoroacetic acid. Figure 5 These are chromatograms of Poria cocos slices under various preset acid hydrolysis conditions according to the present invention; Figure 6 This is a specificity and batch-to-batch reproducibility spectrum of the characteristic oligosaccharide peaks of Poria cocos in this invention, wherein (A) a1 Lentinus edodes polysaccharide and a2 Platycodon grandiflorus, a3 Polygonatum sibiricum, a4 Dioscorea opposita, a5 Ginseng polysaccharide formulation granules; (B) b1 Poria cocos slices and b2 formulation granules, b3 maltodextrin and b4 Astragalus membranaceus, b5 Polyporus umbellatus formulation granules; (C) and (D) Poria cocos formulation granules from different batches; the characteristic oligosaccharide peaks with RT≈5 min are shown by the dashed lines; Figure 7 The graph shows the conditional stability and linear relationship of characteristic peaks of Poria cocos. Among them, (A) different batches of Poria cocos slices; (B) Poria cocos formula granules with different alcohol precipitation concentrations, 1~3 are parallel experiments; (C) Poria cocos formula granules under acid concentration fluctuations; (D) Poria cocos formula granules under different injection concentrations; the characteristic peak at RT≈5 min and the unstable peak at RT≈6 min are shown by the dashed line. Figure 8 This is the mass spectrum of the total oligosaccharides from the partial acid hydrolysis of Poria cocos slices according to the present invention; Figure 9 This is the mass spectrum of the total oligosaccharides from the acid hydrolysis of the Poria cocos formula granules of this invention; Figure 10These are microscopic morphological changes of the three oligosaccharide-cured films of this invention over time. Note: (A1-3) acid hydrolysis products of particles; (B1-3) acid hydrolysis products of medicinal slices; (C1-3) enzymatic hydrolysis products of medicinal slices; all observed under a 10x objective lens. Figure 11 These are the Raman spectra of the three oligosaccharides of this invention as a function of storage time; wherein, (A1-3) are acid hydrolysis products of granules; (B1-3) are acid hydrolysis products of medicinal slices; (C1-3) are enzymatic hydrolysis products of medicinal slices; 1-3 of A, B, and C are parallel experiments; Figure 12 This is a specificity verification spectrum of the characteristic Raman peak of Poria cocos in the embodiments of the present invention, wherein (A1) Poria cocos slices of different concentrations; (B1) maltodextrin as an excipient; (C1) partially acid hydrolysed products of maltodextrin; (A2) partially acid hydrolysed products of Astragalus membranaceus granules; (B2) partially acid hydrolysed products of Lentinus edodes polysaccharide; (C2) partially acid hydrolysed products of Polyporus umbellatus granules; (A3) partially acid hydrolysed products of Platycodon grandiflorus granules; (B3) partially enzymatic hydrolysed products of Lentinus edodes polysaccharide; (C3) partially enzymatic hydrolysed products of Polyporus umbellatus granules; 1-3 in A, B, and C are parallel experiments; Figure 13 This is a molecular weight and particle size distribution map of the directional degradation products of Poria cocos slices and formula granules in the embodiments of the present invention; wherein, (A) molecular weight distribution of directional degradation products of Poria cocos, a1 enzymatic hydrolysis of Poria cocos slices, a2 acid hydrolysis of Poria cocos formula granules, a3 acid hydrolysis of Poria cocos slices; (B) particle size distribution of acid hydrolysis products of formula granules; (C) particle size distribution of acid hydrolysis products of slices; (D) particle size distribution of partially acid hydrolysis products of formula granules; 1 to 4 in B, C, and D are parallel experiments; Figure 14 The synergistic effect of the combination of poria cocos formula granules hydrolyzed oligosaccharides and cisplatin on Huh-7 liver cancer cells is shown in the figure. (A) Survival rate of Huh-7 cells after 24 h of treatment with oligosaccharides alone; (B) Survival rate of Huh-7 cells after 24 h of treatment with cisplatin alone; (C) Survival rate of Huh-7 cells after 24 h of treatment with a fixed cisplatin concentration (1.21 μg / mL) and different concentrations of oligosaccharides; (D) Survival rate of Huh-7 cells after 24 h of treatment with a fixed oligosaccharide concentration (400 μg / mL) and different concentrations of cisplatin. "*" indicates P<0.05 compared with the control group (0 μg / mL), and "****" indicates P<0.0001 compared with the control group (0 μg / mL). Figure 15This is a graph showing the in vitro antioxidant activity results of Poria cocos polysaccharides and oligosaccharides. (A) Free radical scavenging rate of crude polysaccharides from Poria cocos slices; (B) Free radical scavenging rate of crude polysaccharides from Poria cocos granules; (C) Free radical scavenging rate of enzymatically hydrolyzed oligosaccharides from Poria cocos slices; (D) Free radical scavenging rate of acid-hydrolyzed oligosaccharides from Poria cocos slices; (E) Free radical scavenging rate of acid-hydrolyzed oligosaccharides from Poria cocos granules; (F) Free radical scavenging rate of vitamin C (Vc) in the positive control. "*" indicates a difference of P < 0.05 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL), "**" indicates a difference of P < 0.01 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL), "***" indicates a difference of P < 0.001 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL), and "****" indicates a difference of P < 0.001 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL). (mg / mL or 3.125 μg / mL), P<0.0001. Detailed Implementation

[0042] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0043] Figure 1 This is a schematic flowchart of the polysaccharide detection method based on characteristic degradation sugar spectrum and Raman fingerprint spectrum in an embodiment of the present invention; the method will be specifically described below with reference to specific embodiments.

[0044] Example 1: Characteristic degradation glycosylation analysis (specific identification) of Poria cocos formula granules 1. Glycosyl degradation profile analysis of Poria cocos granules Preparation of the test sample: Take an appropriate amount of Poria cocos granules (about 20g), dissolve in water, precipitate with alcohol (e.g., 80% ethanol) to obtain crude polysaccharide, remove protein, and freeze-dry for later use. Partial acid hydrolysis and derivatization: Take the above crude polysaccharide, add 0.3~0.7 mol / L (preferably 0.5 mol / L) trifluoroacetic acid (TFA), and hydrolyze at 75-85℃ (preferably 80℃) for 1.5~2.5 hours (preferably 2 hours). After removing TFA by rotary evaporation, redissolve the hydrolysate, add ethyl 4-aminobenzoate (4-ABEE) and sodium cyanoborohydride for derivatization. Chromatographic analysis: Use an amide column (e.g., XBridge BEH Amide), with water-acetonitrile as the mobile phase for gradient elution, excitation wavelength 305 nm, and emission wavelength 360 nm. Results are as follows. Figures 2-5 As shown. Among them, Figure 2The chromatograms are of a1 Poria cocos slices and a2 Poria cocos granules under the preset acid hydrolysis conditions of 80℃ acid hydrolysis temperature, 1h acid hydrolysis time, and 0.25mol / L trifluoroacetic acid. Figure 3 The chromatograms are of b1 Poria cocos slices and b2 Poria cocos granules under the preset acid hydrolysis conditions of acid hydrolysis temperature of 80℃, acid hydrolysis time of 1h, and 0.5mol / L trifluoroacetic acid. Figure 4 The chromatograms are of C1 Poria cocos slices and C2 Poria cocos granules under the preset acid hydrolysis conditions of acid hydrolysis temperature of 80℃, acid hydrolysis time of 2h, and 0.5mol / L trifluoroacetic acid. Figure 5 Chromatograms of Poria cocos slices under various preset acid hydrolysis conditions. Figures 2 to 5 The dashed line in the figure represents the characteristic peak, the horizontal axis represents the retention time t (min), and the vertical axis represents the response intensity (mV).

[0045] like Figures 2 to 5 The image shows chromatograms obtained after acid hydrolysis of Poria cocos granules and processed Poria cocos slices under various preset acid hydrolysis conditions. According to... Figure 4 and Figure 5 It can be seen that the optimal acid hydrolysis conditions are: a large number of characteristic oligosaccharide peaks, sharp and symmetrical peak shapes, the largest peak height / peak area, few impurity peaks, complete polysaccharide degradation, and no excessive hydrolysis into monosaccharides. These conditions are: acid hydrolysis temperature of 80℃, acid hydrolysis time of 2h, and 0.5mol / L trifluoroacetic acid.

[0046] 2. Characteristic degradation glycosylation analysis of multiple oligosaccharide peaks and verification of batch-to-batch reproducibility Experimental Procedure: The optimal acid hydrolysis conditions were selected as 0.5 mol / L trifluoroacetic acid (TFA), 80 °C, 2 h. The hydrolysate was evaporated to dryness with methanol to remove TFA, then reconstituted with water to 4 mg / mL. An equal volume of derivatization reagent (methanol:acetic acid = 9:1, containing 90 mg / mL 4-ABEE and 9 mg / mL sodium borohydride cyanide) was added, and the reaction was carried out at 80 °C for 20 min. The mixture was extracted five times with diethyl ether, filtered through a 0.22 μm filter, and analyzed by HPLC-FLD. Column: XBridge BEHAmide. Mobile phase: water (A) - acetonitrile (B); gradient elution program: 0 min, 20% A; 5 min, 25% A; 30 min, 30% A; 40 min, 35% A; 50 min, 90% A; 60 min, 90% A; 80 min, 20% A; 110 min, 20% A; flow rate: 1.0 mL / min; column temperature: 30℃; FLD excitation wavelength: 305 nm; emission wavelength: 360 nm.

[0047] Result determination: such as Figure 6As shown in the chromatogram of the test samples, under optimal acid hydrolysis conditions, at a retention time (RT) of 4.8–5.2 minutes (RT≈5 min), only the samples derived from Poria cocos (b1 Poria cocos slices and b2 granules) exhibited a characteristic peak at this point. Maltodextrin and eight reference standards (a1 Lentinus edodes polysaccharide, a2 Platycodon grandiflorus, a3 Polygonatum sibiricum, a4 Dioscorea opposita, a5 Ginseng polysaccharide granules, b3 maltodextrin, b4 Astragalus membranaceus, and b5 Polyporus umbellatus granules) were not detected. Figure 6 A, Figure 6 Peak B indicates that this peak has good specificity for Poria cocos. This peak was stably reproduced in 10 batches of Poria cocos granules. Figure 6 C Figure 6 The peak (D) indicates that it is not affected by batch size or preparation process. This peak is formed based on the polarity difference of oligosaccharides after acid hydrolysis and is not present in maltodextrin or many other medicinal herb formulations, indicating that this method can effectively eliminate interference from excipients and coexisting substances, and has specific identification ability for active oligosaccharide fragments in Poria cocos formulations. The peak height percentage can be used as a semi-quantitative indicator (the control range can be set to the average value ± 30%, such as 4.21%~7.81%).

[0048] 3. Conditional stability and linear relationship analysis of characteristic peaks of Poria cocos Figure 7 The results of three parallel experiments (1-3) were performed on the granules of Poria cocos using 55% and 80% alcohol precipitation. The acid hydrolysis chromatograms of different batches of the medicinal slices showed a high degree of consistency. Figure 7 (A) This confirms good batch-to-batch reproducibility. Precipitation experiments with 55% and 80% ethanol showed that the unstable peak (RT≈6min) was affected by the proportion of excipients, while the characteristic peaks were all stable. Figure 7 (B), confirming that it originates from Poria cocos polysaccharide. When the acid hydrolysis concentration fluctuates in the range of 0.3-0.7 mol / L, the peak height is highest at 0.5 mol / L. Figure 7 (C); Within the injection concentration range of 1.0-5.0 mg / mL, the peak height percentage showed a certain linear relationship with the injection concentration (R²=0.965). Figure 7 (D), indicating that this method can be used for semi-quantitative analysis of characteristic oligosaccharides.

[0049] 4. Comparison of mass spectra of total oligosaccharides from partial acid hydrolysis of Poria cocos slices and formula granules. The total oligosaccharides from acid hydrolysis of Poria cocos slices and formulated granules were analyzed by electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS). Positive ion mode; drying gas temperature 320℃; drying gas flow rate 8 L / min; nebulizer pressure 35 psi; sheath gas temperature 350℃; capillary voltage 3500 V; fragmentation voltage 135 V; scan range m / z 100-1200.

[0050] Mass spectra of partially acid-hydrolyzed total oligosaccharides from Poria cocos slices and Poria cocos granules are compared. Figure 8 and Figure 9 As shown, where, Figure 8 The peak for total oligosaccharides in Poria cocos slices was observed on mass spectrometry. Figure 9 The mass spectrometry peaks of total oligosaccharides in Poria cocos formula granules are shown. Figure 8 and Figure 9 The horizontal axis represents the mass-to-charge ratio (m / z) of oligosaccharide molecules at different retention times, while the vertical axis represents the relative abundance, which indicates the strength of the oligosaccharide ion signal corresponding to that mass-to-charge ratio and reflects the relative content of the oligosaccharide in the sample.

[0051] Figure 8 and Figure 9 This indicates that, under the same optimal trifluoroacetic acid hydrolysis conditions, the acid hydrolysis scheme of this invention can simultaneously release oligosaccharides from both types of samples when treating Poria cocos slices and Poria cocos formulation granules, and can visually distinguish the differences in oligosaccharide profiles between the two. Both samples exhibit a maximum peak at a retention time of 4.2 min, but the peak shapes differ significantly. The maximum peak of the oligosaccharide mass spectrum from the slices is symmetrical and shows a gradient converging towards the center; while the maximum peak from the formulation granules is incomplete on the left half, with only a small sharp peak appearing around 4 min, and the right half showing a steep peak shape. The quasi-molecular ion peak corresponding to the maximum peak is m / z 588.19 [M+H]⁺. Furthermore, the number of oligosaccharide mass spectrum peaks is roughly the same for both samples within 6 min, but after 6 min, the number of peaks in the slices sample is significantly higher than that in the formulation granules, suggesting the absence of some oligosaccharide fragments. The maximum peaks and differences revealed by mass spectrometry provide a molecular-level basis for the preparation of enzymatic hydrolysis control standards and subsequent physical characterization.

[0052] Example 2: Characteristic Raman Spectroscopic Analysis of Poria Cocos Formula Granules (Integrity Identification) 1. Preparation of characteristic oligosaccharide reference standards and determination of Raman threshold Enzymatic hydrolysis preparation: Take the crude polysaccharide from Poria cocos slices, add β-1,3-endoglucanase (enzyme / substrate ratio 0.3~0.7, preferably 0.5), and hydrolyze at pH 4.5~5.5 and temperature 35~40℃ for 2~4 hours (preferably 3 hours). The hydrolysate is then ultrafiltered, dialyzed, and lyophilized to obtain the characteristic oligosaccharide reference standard. Preparation of the solidified membrane: Dissolve the sample to be tested in water and mix with a composite solvent (water:ethanol:0.1% Tween 20 = 13:10:7).

[0053] The glass slide was wrapped in tin foil and preheated after being cleaned with 0.5 mol / L NaOH, water, 5% glacial acetic acid, and ethanol. 1.5 μL of the sample was spotted onto the treated slide using the reverse pipetting method. The slide was dried at 40 °C for 2 min and then equilibrated at 22 °C and 60% RH to form a cured film (cured film: a uniform thin film formed under controlled drying conditions, used for Raman spectroscopy characterization). Spectral acquisition: 785 nm laser, 100% power, exposure 10 s × 3 times, range 200-2000 cm⁻¹. -1Under a microscope, a region with uniform texture inside the deposition ring was selected as the measurement point to improve the reproducibility and stability of the spectrum. 3-5 sites were randomly collected from each sample to truly reflect its Raman peak (coffee ring effect: the ring-shaped deposition phenomenon formed by the migration of solute to the edge during the evaporation of the solution).

[0054] Threshold determination: The Raman spectrum (785 nm laser) of the cured film of the above-mentioned characteristic oligosaccharide reference standard was dynamically monitored. Observations revealed that with increasing self-assembly time, the threshold at 1000 cm⁻¹ increased. -1 Characteristic peaks appear at (e.g.) Figure 11 As shown in Figure C, this peak corresponds to the core structural signal of the β-1,3-glycosidic bond. When the intensity of this peak stabilizes above 50 au, it indicates that effective self-assembly has been completed.

[0055] 2. Comparison of microstructure and Raman spectra of three oligosaccharide-cured films Figure 10 The images show the changes in the microstructure of three oligosaccharide-cured films over time. (A1-3) are acid hydrolysis products of particles; (B1-3) are acid hydrolysis products of medicinal slices; and (C1-3) are enzymatic hydrolysis products of medicinal slices. All images were observed under a 10x objective lens.

[0056] Microscopic imaging of the cured film revealed differences in the self-assembly behavior of the three oligosaccharides (the phenomenon of molecules spontaneously forming ordered aggregate structures): After one day of standing, microscopic imaging of the cured film of the partially acid-hydrolyzed solution of Poria cocos granules showed many spherical aggregates with poor light transmittance, indicating that the oligosaccharides in the Poria cocos granules aggregated disorderedly. After the solution solidified into a film, the adhesion peaks appeared as spherical structures, and the corresponding physical properties were visually displayed. After three days of standing, the number of spherical aggregates decreased, their size decreased, and the light transmittance increased, indicating that the oligosaccharides underwent partial degradation after disordered aggregation, suggesting poor stability. After five days of standing, the number of aggregates decreased again, their size decreased, and the light transmittance increased. Figure 10 (A1-3), the overall structure showed the characteristics of first agglomeration and then degradation, which further confirms that the oligosaccharide in the formulation particles has poor stability.

[0057] After one day of standing, the solidified film of the partially hydrolyzed polysaccharide solution from Poria cocos slices was observed to contain numerous regular, flaky crystals with distinct edges, indicating orderly aggregation of oligosaccharides. After three days, the number of flaky crystals decreased, and the edges became less distinct, suggesting possible degradation of the oligosaccharides. After five days, the flaky crystals transformed into rectangular strips with more distinct edges than after three days. Figure 10 (B1-3) has a weak overall aggregation tendency, which is speculated to be formed by the re-aggregation of some oligosaccharides after degradation and relatively intact ones, manifested as the expansion of plate-like crystals into rectangular strip-like crystals.

[0058] After one day of standing, microscopic imaging of the solidified film of the enzymatic hydrolysis solution of Poria cocos polysaccharides revealed a few three-dimensional crystals with distinct edges, indicating that the enzymatically hydrolyzed oligosaccharides of Poria cocos were orderly aggregated and had good uniformity, and the solidified film of the oligosaccharide solution exhibited three-dimensional crystallization. After three days of standing, the number of three-dimensional square crystals increased, with distinct edges, indicating that the oligosaccharides were again orderly aggregated, exhibiting three-dimensional crystallization, suggesting that the enzymatic hydrolysis products may have self-assembly capabilities. After five days of standing, the crystals disappeared. Figure 10 (C1-3) The overall structure exhibits a pattern of initial crystallization followed by degradation, similar to the characteristics of biocompatible drug delivery carriers with self-assembly capabilities. This suggests that the enzymatic hydrolysis products, due to their weak structural destructiveness, result in relatively intact main chains. While possessing self-assembly capabilities, the relatively poor stability of the corresponding three-dimensional square crystals leads to initial aggregation and crystallization, followed by assembly and increase, and then fragmentation and degradation. The Raman peaks before and after assembly can be used to distinguish the distribution characteristics of branched and main chain Raman peaks. The microscopic aggregation morphology of the three oligosaccharides visually demonstrates the differences in molecular-level structure, corresponding one-to-one with the aggregation tendencies indicated by the molecular weight and particle size distribution of the oligosaccharides.

[0059] Figure 11 These are Raman spectra of three oligosaccharides as a function of storage time. Among them, (A1-3) are acid hydrolysis products of granules; (B1-3) are acid hydrolysis products of medicinal slices; and (C1-3) are enzymatic hydrolysis products of medicinal slices. A, B, and C are parallel experiments.

[0060] The combined technique of "cured film-microscopic imaging-Raman spectroscopy" visualizes the microstructure and correlates it with molecular vibrational characteristics: microscopic imaging shows that it is a spherical aggregate (the medicinal slices are regular crystals), and Raman spectroscopy at 1000 cm⁻¹... -1 The peak intensity decreased; at the same time, during the self-assembly process of the enzymatic hydrolysis products of the medicinal slices, the Raman peak appeared from nowhere (3 days) and then split due to degradation (5 days), further confirming that it is directly related to the integrity of the β-1,3-glycosidic bond.

[0061] Meanwhile, compare the acid hydrolysis products ( Figure 11 As can be seen from A), the granulation process of the formulation will result in a higher peak intensity compared to that of the sliced ​​medicinal materials. Figure 11 (B) decreased. Therefore, the stability strength (≥50 au) at which the enzymatically digested control completed self-assembly was set as the threshold for determining the integrity of the core structural unit. This threshold indicates that the β-1,3-glycosidic bond is a key recognition site for the oral absorption of polysaccharides.

[0062] 3. Specificity verification of characteristic Raman peaks in Poria cocos Partial enzymatic hydrolysis steps: 50 mg of crude polysaccharide from Poria cocos slices or other reference standards were dissolved in 5 mL of PBS-glacial acetic acid buffer (pH 5.0, 50 mmol / L). The mixture was broken up at 80 °C for 10 min and then cooled to 37 °C. β-1,3-endoglucanase was added in two portions, with a total amount of 25 mg and an enzyme / substrate ratio of 0.5 (w / w), each hydrolysis lasting 1.5 h. The hydrolysis process was dynamically monitored using thin-layer chromatography: the developing solvent was n-butanol-water-glacial acetic acid (3:2.5:1), and the chromogenic reagent was diphenylamine-aniline-phosphate. Using maltotriose (Rf≈0.42) as a reference, the optimal hydrolysis time was determined to be 3 h. At the endpoint, 1 mol / L NaOH (20% by volume) was added, and the mixture was inactivated at 130 °C for 3 min. The supernatant was collected by centrifugation to obtain the crude extract of the characteristic oligosaccharide reference standard.

[0063] Figure 12 This is a specificity verification spectrum of the characteristic Raman peak of Poria cocos in the embodiments of the present invention, wherein (A1) Poria cocos slices of different concentrations; (B1) maltodextrin as an excipient; (C1) partially acid hydrolysed products of maltodextrin; (A2) partially acid hydrolysed products of Astragalus membranaceus granules; (B2) partially acid hydrolysed products of Lentinus edodes polysaccharide; (C2) partially acid hydrolysed products of Polyporus umbellatus granules; (A3) partially acid hydrolysed products of Platycodon grandiflorus granules; (B3) partially enzymatic hydrolysed products of Lentinus edodes polysaccharide; (C3) partially enzymatic hydrolysed products of Polyporus umbellatus granules; 1 to 3 in A, B, and C are parallel experiments.

[0064] Specificity verification of characteristic Raman peaks in Poria cocos showed that ( Figure 12 Raman spectra of polysaccharides from different concentrations of Poria cocos slices at 1000 cm⁻¹ -1 The peak appears stably at concentrations of 8 mg / mL and above. Figure 12 (A1), indicating that this peak is concentration-dependent; the acid hydrolysis products of Astragalus and Platycodon grandiflorus formulations with similar oligosaccharide polarity distributions are at 1000-1200 cm⁻¹. -1 Although there are peaks in the region, none have reached 1000 cm. -1 sharp single peak ( Figure 12 A2, Figure 12 (A3); maltodextrin and its acid hydrolysis products at 800-1000 cm -1 There are continuous peaks, 1000-1200 cm. -1 The peaks were adhesion peaks; no peaks were observed at 1000 cm⁻¹. -1 peak( Figure 12 B1 Figure 12 C1); acid and enzymatic hydrolysis products of shiitake polysaccharides at 800, 900, and 1100 cm⁻¹ -1 Peaks appeared in various places, but none reached 1000 cm. -1 peak( Figure 12 B2 Figure 12(B3); Acid and enzymatic hydrolysis products of the Polyporus umbellatus formula granules at 450, 650, 800, 850, 900, and 1050 cm⁻¹ -1 There are no stable peaks at locations such as 1000 cm⁻¹. -1 No sharp single peak ( Figure 12 (C2, C3 of 12). The above results confirm that 1000 cm -1 The spike-like peak is unique to Poria cocos oligosaccharides.

[0065] Example 3. Molecular weight and particle size distribution of the targeted degradation products The supernatant of the acid hydrolysis or enzymatic hydrolysis product was concentrated by centrifugation using a 3kDa ultrafiltration tube. The filtrate was transferred to a 500Da dialysis bag and dialyzed at room temperature for 4 hours. The resulting oligosaccharide was then lyophilized to obtain the target oligosaccharide. A TSKgel GMPWXL column was used with 0.2 mol / L NaCl solution as the mobile phase, a flow rate of 0.4 mL / min, a column temperature of 40℃, a sample concentration of 1.0 mg / mL, and an injection volume of 5 μL. The absolute molecular weight and particle size distribution were determined using a differential refractive index detector coupled with multi-angle laser light scattering.

[0066] Figure 13 These are molecular weight and particle size distribution maps of the directional degradation products. Among them, (A) molecular weight distribution of the directional degradation products of Poria cocos, a1 enzymatic hydrolysis of Poria cocos slices, a2 acid hydrolysis of Poria cocos formula granules, a3 acid hydrolysis of Poria cocos slices; (B) particle size distribution of the acid hydrolysis products of the formula granules; (C) particle size distribution of the acid hydrolysis products of the slices; (D) particle size distribution of the partial acid hydrolysis products of the formula granules; 1-4 in B, C, and D are parallel experiments.

[0067] SEC-MALS analysis shows ( Figure 13 (A) The molecular weight distribution of the acid hydrolysis products of granules is disordered, with peaks sticking together, and the peak at 22.5 min is significantly absent; the molecular weight distribution of the acid hydrolysis products of medicinal slices is clearly layered, with the main peak retained for about 20 min; the molecular weight distribution of the enzymatic hydrolysis products of medicinal slices is relatively uniform, with the main peak retained for about 22.5 min, and the weight-average molecular weight (Mw) is 1328-1454 g / mol. Particle size distribution analysis shows that ( Figure 13 The particle size distribution of the acid-hydrolyzed products (BD) is relatively wide, with the main peak concentrated at 200 nm, showing a tendency to aggregate. The acid-hydrolyzed products of medicinal slices exhibit three distinct particle size peaks at 0.2, 1.5, and 20 nm. The enzymatic hydrolyzed products of medicinal slices have a particle size concentrated at 150 nm and a uniform distribution. These results further confirm that the acid-hydrolyzed products of particles exhibit structural fragment loss and disordered aggregation. The differences in molecular weight and particle size of the above-mentioned directed degradation products in solution provide physical-level expectations for Raman spectroscopy and microscopic imaging analysis.

[0068] Example 4: Comprehensive quality evaluation of Poria cocos formula granule samples 1. Sample Testing: Three batches of Poria cocos granules were analyzed for characteristic degradation sugar spectra according to the method in Example 1. Characteristic peaks were detected in all three batches at RT≈5 min, and the peak height percentage was within the quality control range. 2. Structural Verification: The same batch of samples was analyzed using "cured film-Raman spectroscopy" according to the method in Example 2. The 1000 cm⁻¹ of the three batches of samples showed characteristic peaks. -1 The peak intensities were 65, 72, and 58 au, respectively, all of which were not lower than the threshold (50 au). Overall, all three batches of samples met both criteria and were therefore deemed qualified products.

[0069] 2. Activity verification of cisplatin combination therapy: Groups were set up with single-use groups of oligosaccharide granules and cisplatin at various concentrations, a group of cisplatin (1.21 μg / mL) combined with oligosaccharide granules, and a group of oligosaccharide granules (400 μg / mL) combined with cisplatin. The inhibitory effect of acid hydrolyzed oligosaccharide granules from Poria cocos formula alone and in combination with cisplatin on the proliferation of human liver cancer Huh-7 cells was evaluated using the CCK-8 assay. Cell viability was detected 24 h after intervention with Huh-7 cells.

[0070] Figure 14 This is a graph showing the synergistic effect of the combination of poria cocos formula granules hydrolyzed oligosaccharides and cisplatin on Huh-7 liver cancer cells. (A) Survival rate of Huh-7 cells after 24 hours of treatment with oligosaccharides alone; (B) Survival rate of Huh-7 cells after 24 hours of treatment with cisplatin alone; (C) Survival rate of Huh-7 cells after 24 hours of treatment with a fixed cisplatin concentration (1.21 μg / mL) and different concentrations of oligosaccharides; (D) Survival rate of Huh-7 cells after 24 hours of treatment with a fixed oligosaccharide concentration (400 μg / mL) and different concentrations of cisplatin. "*" indicates P < 0.05 compared to the control group (0 μg / mL), and "****" indicates P < 0.0001 compared to the control group (0 μg / mL).

[0071] Granular acid-hydrolyzed oligosaccharides alone inhibited the half-maximal inhibitory concentration (IC50) of Huh-7 cells. 50 The concentration was 15951 μg / mL (95% confidence interval: 11201-24561 μg / mL, R²=0.8717), and the IC50 concentration of cisplatin alone was 15951 μg / mL (95% confidence interval: 11201-24561 μg / mL, R²=0.8717). 50 The concentration was 2.42 μg / mL (95% confidence interval: 2.23–2.62 μg / mL, R² = 0.9686), and both inhibitors showed concentration-dependent inhibition. Figure 14 (A and B). To further evaluate the combined effect of the two, two combined dosing regimens were designed: ① a fixed cisplatin concentration of 1.21 μg / mL (approximately IC50). 50① Treat Huh-7 cells with 200, 400, and 800 μg / mL granular oligosaccharides for 24 h, respectively; ② Fix the granular oligosaccharide concentration at 400 μg / mL and treat with cisplatin at 0.605, 1.21, and 2.42 μg / mL, respectively, for 24 h.

[0072] When cisplatin was fixed at 1.21 μg / mL, cell viability decreased from 48.1% to 42.8% and then further to 35.33% with increasing granular oligosaccharide concentration (200-800 μg / mL). Figure 14 The concentration-dependent decrease in cisplatin concentration (C) was significantly lower than that of cisplatin alone (67.04%) (one-way ANOVA, Dunnett's post-hoc test, P < 0.001), indicating that granular oligosaccharides could enhance the inhibitory effect of cisplatin. When the granular oligosaccharide concentration was fixed at 400 μg / mL, the survival rate decreased from 49.83% to 42.48% and then to 29.04% with increasing cisplatin concentration (0.605-2.421 μg / mL), significantly lower than the corresponding levels when cisplatin was used alone (87.50%, 67.04%, 54.87%). Figure 14 (B).

[0073] The combination index (CI) for each combination point was calculated using the Chou-Talalay method. The CI values ​​for all combination points ranged from 0.27 to 0.47, all significantly less than 1, indicating a strong synergistic effect between the granular oligosaccharide and cisplatin on Huh-7 cells. The lowest CI values ​​were observed at cisplatin 0.605 μg / mL combined with granular oligosaccharide 400 μg / mL (CI=0.27) and cisplatin 1.21 μg / mL combined with granular oligosaccharide 800 μg / mL (CI=0.27), suggesting a strong synergistic effect at lower cisplatin concentrations or higher oligosaccharide concentrations. Isoequivalence plot analysis showed that the combination points were located below the additive equivalence line, consistent with the CI value conclusions. These results indicate that while the acid-hydrolyzed oligosaccharide from the Poria cocos formula exhibits weak cytotoxicity when used alone, its combination with cisplatin significantly enhances the sensitivity of Huh-7 cells to cisplatin; the CI values ​​are all less than 0.5, suggesting its potential as an adjuvant chemotherapy agent. This invention demonstrates that the Poria cocos formula granules verified by the identification method provided by this invention have the effects of providing cellular immunity and inhibiting tumor cells. In particular, the combination of the formula granules and cisplatin can significantly improve the sensitivity of cells to cisplatin, showing the important value of the method of this invention in verifying the active ingredients and structural integrity of polysaccharide preparations in the selection of tumor treatment drugs.

[0074] 3. DPPH scavenging activity verification: The DPPH method was used. The sample was prepared with 80% methanol and mixed with an equal volume of 0.2 mmol / L DPPH solution. The mixture was reacted at room temperature in the dark for 30 min. The supernatant was centrifuged and the absorbance at 517 nm was measured to calculate the clearance rate.

[0075] Figure 15 Figure 1 shows the in vitro antioxidant activity results of Poria cocos polysaccharides and oligosaccharides. (A) Free radical scavenging rate of crude polysaccharides from Poria cocos slices; (B) Free radical scavenging rate of crude polysaccharides from Poria cocos granules; (C) Free radical scavenging rate of enzymatically hydrolyzed oligosaccharides from Poria cocos slices; (D) Free radical scavenging rate of acid-hydrolyzed oligosaccharides from Poria cocos slices; (E) Free radical scavenging rate of acid-hydrolyzed oligosaccharides from Poria cocos granules; (F) Free radical scavenging rate of vitamin C (Vc) in the positive control. "*" indicates a difference of P < 0.05 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL), "**" indicates a difference of P < 0.01 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL), "***" indicates a difference of P < 0.001 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL), and "****" indicates a difference of P < 0.001 compared to the lowest concentration group (0.25 mg / mL or 3.125 μg / mL). (mg / mL or 3.125 μg / mL), P<0.0001.

[0076] The results of the DPPH free radical scavenging capacity test showed that ( Figure 15 All samples exhibited some antioxidant activity, but most showed no significant dose-dependent effect. The scavenging rate of crude polysaccharides from the medicinal slices ranged from 44.8% to 61.2% within the concentration range of 0.25-3 mg / mL (F(4,20)=2.15, P=0.112). One-way ANOVA showed no clear dose-response relationship (P>0.05). The scavenging rate of crude polysaccharides from the formulated granules ranged from 38.5% to 55.8%, also showing no significant dose-dependent effect (F(4,20)=1.89, P=0.152). The scavenging rate of acid-hydrolyzed oligosaccharides from the medicinal slices ranged from 50.5% to 59.5%, and that from the granules ranged from 48.3% to 57.3%, with neither showing a significant dose-response relationship. It is noteworthy that the scavenging rate of oligosaccharides from the medicinal slices was highest at 0.25 mg / mL (51.2 ± 2.3%), significantly higher than that at 0.5 mg / mL (36.2 ± 1.9%) (Tukey test, P = 0.008). Considering their self-assembly characteristics, it is speculated that at high concentrations, oligosaccharide molecules undergo ordered assembly, leading to a reduction in the exposure of active sites, thus exhibiting stronger activity at lower concentrations. Figure 15 The clearance rate of L-ascorbic acid (Vc) in the positive control group increased from 14.4% to 91.3% within the range of 3.125-50 μg / mL, showing a significant dose-dependent effect (Kruskal-Wallis test, P<0.001), indicating that the detection system was normal. Figure 15 (F).

[0077] Activity evaluation results showed that although the formulated particles had missing structural fragments, their core active units were preserved, which is consistent with the presence of these fragments in the Raman spectrum at 1000 cm⁻¹.-1 The presence and absence of peaks correspond, confirming the rationality of the chemical-physical fingerprint correlation in this invention. It is noteworthy that the 1000 cm⁻¹ peak in the particulate acid hydrolysis product... -1 Although the peak intensity was lower than that of the decoction pieces, its synergistic antitumor activity (CI=0.27-0.47) and antioxidant activity (36.61%-61.54%) were still significant, suggesting that the β-1,3-glycosidic bond fragment reflected by the peak may be the "minimum functional unit" that maintains the key activity, providing experimental basis for subsequent quality control strategies targeting this unit.

[0078] This invention constructs a quality control strategy based on the synergistic effect of characteristic degradation sugar spectra and Raman fingerprinting. Specifically, the study identified characteristic oligosaccharide peaks specific to Poria cocos polysaccharides through partial acid hydrolysis (RT≈5 min). The peak height percentage showed a certain linear relationship with concentration (R²=0.965). Considering the interference of excipients and process fluctuations in actual production, a semi-quantitative quality control range of 4.21%~7.81% (average ±30%) is recommended as the characteristic peak range for Poria cocos formulation granules. Because the structure of the characteristic oligosaccharides is not yet elucidated and there are no commercially available reference standards, no recovery test was conducted. However, the repeatability, precision, linearity, and robustness studies have confirmed the reliability of the method. The Raman spectroscopy was used to determine the peak height at 1000 cm⁻¹. -1 To reflect the core physical fingerprint of β-1,3-glycosidic bond integrity, an intensity ≥50 au was used as the dynamic threshold for structural integrity. Multidimensional evidence shows that although the formulation particles have missing structural fragments, their antioxidant and synergistic antitumor activities are still significant (e.g., DPPH scavenging rate >49%, CI <0.5 when used in combination with cisplatin), confirming that "retention of core structural units" is key to activity. This strengthens the correlation between quality control standards and clinical efficacy, achieving a complete evidence chain for quality control from "component consistency" to "retention of active units." Based on this, this invention constructs a qualitative and quantitative chemical fingerprint (characteristic oligosaccharide peaks) and a physical fingerprint (1000 cm⁻¹)... -1 This strategy employs a collaborative quality control approach that monitors structural integrity and verifies activity in relation to clinical efficacy, utilizing Raman peaks. The core innovation lies in using the β-1,3-endoglucan enzymatic hydrolysis product as the "structural gold standard" to reverse-validate the rationality of acid hydrolysis chromatographic fingerprints and Raman spectral fingerprints. This allows for precise assessment of the β-1,3-glycosidic bond, the core structural unit of Poria cocos polysaccharides, in systems with high excipient interference, without the need for complex standards. This strategy shifts the quality control logic from pursuing "component consistency" to "preservation of active units," proving its feasibility in the Poria cocos-maltodextrin extreme interference model. It provides a methodological reference for the quality control of traditional Chinese medicine polysaccharide preparations under high excipient interference, offering a two-way evaluation approach combining "activity-oriented" and "preservation of characteristic structural units."

[0079] Example 5: Identification of Shiitake Mushroom Formula Granules For shiitake mushroom formulation granules with β-1,3-1,6-glucan as the main active ingredient: Adjustment of S1 (partial acid hydrolysis): Due to slight differences in the structure of shiitake polysaccharides, the acid hydrolysis conditions can be fine-tuned. For example, using 0.2~0.4 mol / L TFA at 70~80℃ for 1~2 hours can produce stable and characteristic oligosaccharide fragments. Establishment of a specific glycosylation profile: Analysis under the same HILIC-FLD conditions will yield a characteristic peak at a specific retention time (e.g., needs to be reconfirmed, such as RT 6.0~6.5 minutes) that is not interfered with by maltodextrin. Application of a general Raman method: Steps 2 and 3 remain essentially unchanged. β-1,3-endoglucanase can also enzymatically hydrolyze shiitake polysaccharides, and the resulting characteristic oligosaccharide reference standard can still be used for "cured membrane-Raman spectroscopy" detection at 1000 cm⁻¹. -1 The Raman peak at the location also reflects the integrity of the core β-1,3-glycosidic bond. Conclusion: By simply adjusting the acid hydrolysis conditions to establish a specific "characteristic degradation sugar spectrum," and combining it with the core "cured film-Raman spectroscopy" structure verification technology of this invention, a synergistic quality control method suitable for shiitake mushroom formulation granules can be constructed. The method of this invention can also be applied to other medicinal materials containing β-glucan.

[0080] It should be noted that in the method of the present invention, the components or steps can obviously be decomposed and / or recombined. These decompositions and / or recombinations should be considered equivalent solutions of the present invention. Furthermore, the steps of performing the above series of processes can naturally be performed in the order described and in chronological order, but are not necessarily required to be performed in chronological order. Some steps can be performed in parallel, overlapping, or independently of each other.

[0081] It should be noted that in the above embodiments, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element. Furthermore, it should be noted that the scope of the methods and apparatuses in the embodiments described above is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0082] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for identifying the active ingredient and structural integrity of a polysaccharide preparation with β-1,3 glycosidic bonds as the main chain, characterized in that, The method includes the following steps: S1: Obtain the crude polysaccharide extract of the sample to be tested, and use an acid solution to perform differential acid hydrolysis on the crude polysaccharide extract based on the difference in glycosidic bond stability to obtain the partial acid hydrolysis product that selectively breaks the β-1,3-glycosidic bonds in the polysaccharide molecule. S2: The partial acid hydrolysis products obtained in S1 are subjected to derivatization based on chromatographic analysis and hydrophilic chromatographic analysis to obtain the characteristic degradation chromatogram of the sample to be tested. S3: Obtain the crude polysaccharide extract of the sample to be tested, and perform enzymatic hydrolysis using a β-1,3-glycosidic bond specific hydrolase to obtain a temperature-controlled β-1,3-glycosidic bond glucosinolate self-assembled mixture. S4: The β-1,3-glucan self-assembled mixture obtained in S3 was used to prepare a cured film, and Raman spectroscopy analysis was performed to obtain a Raman spectrum containing the characteristic peaks of the sample to be detected. S5: Determine the polysaccharide active ingredients and their content in the sample based on whether the characteristic chromatographic peaks of β-1,3-glycosidic bonds are present in the characteristic degradation chromatogram of the sample obtained in S2 and their peak height / area ratio; determine the structural integrity of the polysaccharide active ingredients in the sample based on whether the Raman spectral characteristic peaks of β-1,3-glycosidic bonds are present in the sample obtained in S4.

2. The method according to claim 1, characterized in that, The polysaccharides with β-1,3 glycosidic bonds as the main chain include Poria cocos polysaccharide, Lentinus edodes polysaccharide, Grifola frondosa polysaccharide, Ganoderma lucidum polysaccharide, Trametes versicolor polysaccharide, and Polyporus umbellatus polysaccharide.

3. The method according to claim 2, characterized in that, The acid solution in S1 is a trifluoroacetic acid solution. The reaction conditions for the differentiated acid hydrolysis are set as follows: trifluoroacetic acid solution with a concentration of 0.2 to 0.7 mol / L, acid hydrolysis temperature of 75 to 85°C, and acid hydrolysis time of 1.0 to 2.5 hours. After acid hydrolysis, the product is obtained by rotary evaporation to remove trifluoroacetic acid and then resolution.

4. The method according to claim 3, characterized in that, The derivatization process in S2 is derivatization with ethyl 4-aminobenzoate and sodium cyanoborohydride, the hydrophilic chromatographic analysis is amide column fluorescence chromatography, and the retention time of the characteristic peak of β-1,3 glycosidic bond in the hydrophilic chromatographic analysis is 4.8~6.5 minutes.

5. The method according to claim 4, characterized in that, In step S5, the peak height / area ratio is used as an upper and lower threshold for judging the polysaccharide active ingredients and their content in the sample to be tested, within the range of 4.21% to 7.81%.

6. The method according to claim 2, characterized in that, The β-1,3-glycosidic bond-specific hydrolase in S3 is β-1,3-endoglucanase. The enzymatic hydrolysis reaction conditions are set as follows: enzyme / substrate ratio 0.3~0.7, pH 4.5~5.5, and temperature 35~40℃ for 2~4 hours. After enzymatic hydrolysis, the product is obtained by ultrafiltration, dialysis, and freeze-drying.

7. The method according to claim 6, characterized in that, The cured film in S4 is prepared by mixing the water-soluble product of the enzymatic hydrolysis obtained in S3 with a composite solvent of water-ethanol-0.1% Tween 20, spotting the mixture onto a glass slide, and drying it to form a film.

8. The method according to claim 6, characterized in that, The Raman spectroscopy analysis in S4 uses a 785 nm wavelength laser, with a scanning range of 200 to 2000 cm⁻¹. -1 There are 3 to 5 collection points.

9. The method according to claim 8, characterized in that, In S5, the Raman spectral characteristic peak of the β-1,3-glycosidic bond is determined to be at 1000 cm⁻¹. -1 Peak intensity ≥ 50 au.

10. A system for identifying the active ingredient and structural integrity of polysaccharide preparations with β-1,3 glycosidic bonds as the main chain, using the method described in any one of claims 1-9, characterized in that... The system includes the following modules: (1) Sample processing module, wherein the sample processing module is used to prepare the sample to be tested into a crude polysaccharide extract; (2) Chromatographic analysis module, wherein the chromatographic analysis module is used to derivatize the crude polysaccharide extract and perform chromatographic analysis; (3) Raman spectroscopy analysis module, wherein the Raman spectroscopy analysis module is used to cure the crude polysaccharide extract into a film and perform Raman spectroscopy analysis; (4) Result analysis and output module, which is used to analyze the results output by the chromatography analysis module and the Raman spectroscopy analysis module, and to make a judgment on the polysaccharide active ingredients, content and structural integrity contained in the sample to be tested and output the judgment results.

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