Thunberg fritillary bulb polysaccharide as well as extraction and purification method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to thunberg fritillary bulb polysaccharide as well as an extraction and purification method and application thereof. The extraction and purification method comprises water extraction, alcohol precipitation and two times of purification, the extraction and purification method is stable in process and good in repeatability, and a polysaccharide component with high uniformity can be efficiently obtained from the thunberg fritillary bulb. In addition, the intestinal flora structure of a human body can be remarkably regulated, specifically, the capacity of the flora for totally producing short-chain fatty acids (SCFAs, such as acetic acid, propionic acid and butyric acid) is improved, the relative abundance of beneficial bacteria (such as bifidobacteria and bacteroides) is increased, and the abundance of potential harmful bacteria is reduced; in-vitro proliferation of common probiotics (such as bifidobacterium adolescentis, bacteroides multiflora and the like) can be remarkably promoted, it is indicated that the Zhejiang fritillaria thunbergii has excellent prebiotic potential, and a new way is provided for deep development and high-value utilization of the Zhejiang fritillaria thunbergii.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a polysaccharide from Fritillaria thunbergii, its extraction and purification method, and its applications. Background Technology

[0002] Fritillaria thunbergii is a traditional Chinese medicine with the effects of clearing heat and resolving phlegm, relieving cough and dissipating nodules. Modern research shows that the polysaccharides in Fritillaria thunbergii have various biological activities such as immunomodulation, antioxidation, and antitumor activity.

[0003] However, current research on Fritillaria thunbergii polysaccharides mainly focuses on crude extracts or primary purified products. For example, CN120617092A discloses a whitening gel based on biological polysaccharides and its preparation method. The preparation method of Fritillaria thunbergii polysaccharides includes the following steps: weighing crude powder of Fritillaria thunbergii, mixing with deionized water, ultrasonically extracting, filtering, concentrating the filtrate, adding ethanol and stirring thoroughly, allowing to stand, centrifuging, and freeze-drying the precipitate to obtain Fritillaria thunbergii polysaccharides.

[0004] For example, CN103483459A discloses an ultrasonic-assisted extraction process for Fritillaria thunbergii polysaccharide and a health product containing Fritillaria thunbergii polysaccharide, including the following steps: (1) degreasing; (2) water extraction, taking dried Fritillaria thunbergii, crushing it, and extracting polysaccharide according to the ultrasonic-assisted extraction process, that is, the extraction conditions are: material-liquid ratio of 1:15, ultrasonic power of 120W, extraction temperature of 80℃, and extraction time of 1.5h; the filter residue is extracted at least three times, the filtrate is combined to obtain Fritillaria thunbergii polysaccharide water extract; (3) alcohol precipitation; (4) deproteinization to obtain purified Fritillaria thunbergii polysaccharide. However, this technical solution does not specify whether the extracted polysaccharide component is a single component. It can be seen that the extraction of Fritillaria thunbergii polysaccharide in the prior art is still limited to crude extract or primary purified product.

[0005] The gut microbiota is closely related to human health, and dysbiosis is associated with various chronic diseases. Regulating the gut microbiota through dietary components or medications has become a research hotspot. Polysaccharides, as prebiotics, can selectively promote the growth of beneficial bacteria and inhibit harmful bacteria, making them ideal gut microbiota regulators. However, the regulatory effects of Fritillaria thunbergii polysaccharides on the gut microecology using existing technologies have not been systematically reported.

[0006] Based on the current state of the technology, the extraction of Fritillaria thunbergii polysaccharides in the existing technology is still limited to crude extracts or primary purified products, and there are still technical problems that need to be solved, such as directions for further exploration of the application of Fritillaria thunbergii polysaccharides. Summary of the Invention

[0007] To solve the above-mentioned technical problems, the present invention provides a method for extracting and purifying polysaccharides from Fritillaria thunbergii, the method comprising the following steps:

[0008] Step 1, Water extraction and alcohol precipitation: Mix Fritillaria thunbergii powder with distilled water and extract to obtain an extract. After concentration, add ethanol to the concentrate, let it stand to precipitate, and collect the precipitate to obtain crude Fritillaria thunbergii polysaccharide. Step 2, First purification: Dissolve the crude polysaccharide of Fritillaria thunbergii from Step 1 in deionized water, load the sample onto an ion exchange column, and elute with deionized water and sodium chloride solution in sequence. Monitor the sugar content online and collect the main peak elution fraction. Step 3, Secondary purification: The main peak eluent obtained in Step 2 is dialyzed and desalted using a semi-permeable membrane, and then freeze-dried to obtain the Fritillaria thunbergii polysaccharide (abbreviated as FTP-1).

[0009] Furthermore, the particle size of the Fritillaria thunbergii powder mentioned in step 1 is 45-65 mesh.

[0010] Furthermore, the particle size of the Fritillaria thunbergii powder mentioned in step 1 is preferably 60 mesh; When the powder of Fritillaria thunbergii is too fine, filtration / centrifugation is extremely difficult, the filtrate is turbid, and subsequent protein removal and purification become much more difficult; when the powder of Fritillaria thunbergii is too coarse, the polysaccharides inside the Fritillaria thunbergii are difficult to release, resulting in a large waste of raw materials.

[0011] Furthermore, the ratio of Fritillaria thunbergii powder to distilled water in step 1 is 1:(10-30), with units of g / mL.

[0012] Furthermore, the extraction temperature in step 1 is 80-100℃, and the extraction time is 1-3 h.

[0013] Furthermore, the extraction in step 1 is performed 1-3 times, and the extracts need to be combined after multiple extractions.

[0014] Furthermore, the concentration described in step 1 is a rotary evaporation concentration, with a temperature of 40-55℃ and a rotation speed of 30-150 rpm. To prevent boiling over during the initial concentration, a temperature of 40℃ and a rotation speed of 30 rpm are used for rotary evaporation. After stabilization, the temperature and rotation speed are appropriately increased to improve the concentration efficiency.

[0015] Furthermore, the volume ratio of the concentrate to ethanol in step 1 is 1:(4-5).

[0016] Furthermore, the settling temperature in step 1 is 2-4℃, and the settling time is 12-16h.

[0017] Furthermore, the preferred temperature for standing in step 1 is 4°C, and the preferred time is 12 hours.

[0018] Furthermore, the drying temperature in step 1 is -60 to -80°C, and the drying time is 10-12 hours.

[0019] Furthermore, the drying temperature in step 1 is preferably -80°C, and the drying time is preferably 12 hours.

[0020] Furthermore, in step 2, the ratio of the crude polysaccharide from Fritillaria thunbergii dissolved in deionized water is 1:(40-50), with units of g / mL.

[0021] Furthermore, the ion exchange column mentioned in step 2 is a DEAE-52 cellulose ion exchange column.

[0022] Furthermore, the concentration of the sodium chloride solution in step 2 is 0.2-0.8M.

[0023] Furthermore, the elution in step 2 is a linear gradient elution, wherein deionized water is used for elution from 0 to 40 min, and sodium chloride solution with a concentration of 0.2-0.8 M is used for elution from 40 to 280 min.

[0024] Furthermore, the concentrations of the sodium chloride solution are 0.2 M, 0.4 M, 0.6 M, and 0.8 M, respectively.

[0025] Furthermore, the flow rate of the linear gradient elution is 3-5 mL / min.

[0026] Furthermore, the method for online monitoring of sugar content in step 2 is either the phenol-sulfuric acid method or the sulfuric acid-carbazole method.

[0027] Furthermore, the molecular weight cutoff of the semipermeable membrane described in step 3 is 3500-14000 Da.

[0028] Furthermore, the molecular weight cutoff of the semipermeable membrane in step 3 is preferably 3500 Da or 8000-14000 Da.

[0029] Furthermore, the freeze-drying temperature in step 3 is -60 to -80°C under vacuum for 12-24 hours.

[0030] The present invention also provides a Fritillaria thunbergii polysaccharide, which is obtained by extraction and purification using the above-mentioned extraction and purification method for Fritillaria thunbergii polysaccharide.

[0031] Furthermore, the weight-average molecular weight (Mw) of the Fritillaria thunbergii polysaccharide is 11-12 kDa, comprising glucose (Glc), galactose (Gal), and mannose (Man), with mass percentages of (97-98) wt%, (1-2) wt%, and (0.5-1) wt%, respectively.

[0032] The present invention also provides a product for regulating intestinal flora, the product comprising the above-mentioned Fritillaria thunbergii polysaccharide.

[0033] Furthermore, the intestinal flora includes, but is not limited to, species of the genera *Bifidobacterium* and *Bacteroides*.

[0034] Furthermore, the product is used to regulate the gut microbiota of mammals.

[0035] Furthermore, the product is one of the following: a drug, a food, or a food additive.

[0036] The beneficial effects of this invention are as follows: 1. This invention provides a method for extracting and purifying polysaccharides from Fritillaria thunbergii. The process is stable and reproducible, efficiently obtaining a highly homogeneous polysaccharide component from Fritillaria thunbergii. The high homogeneity of the component means that the weight-average molecular weight (Mw) of the Fritillaria thunbergii polysaccharide is 11-12 kDa, comprising glucose (Glc), galactose (Gal), and mannose (Man), with mass percentages of (97-98) wt%, (1-2) wt%, and (0.5-1) wt%, respectively. 2. The extraction and purification process is highly efficient and stable, with excellent repeatability and operability: The three-step process constructed in this invention, consisting of water extraction and alcohol precipitation, primary purification using a DEAE-52 cellulose ion exchange column, and secondary purification via semi-permeable membrane dialysis and desalting, has clearly defined parameters and a standardized operating procedure. By adjusting key parameters such as the material-liquid ratio, elution concentration, and dialysis molecular weight cutoff, efficient extraction and precise purification of Fritillaria thunbergii polysaccharides can be achieved. The entire process requires no special or complex equipment, and each step can be repeated. The target polysaccharide component can be stably obtained in different embodiments, with an extraction rate of 18%-22%. This solves the technical problems of existing technologies where Fritillaria thunbergii polysaccharide extraction only yields crude extracts or primary purified products, has poor process stability, and unsatisfactory purification effects, making it suitable for large-scale industrial production. 3. Obtaining highly homogeneous and structurally distinct pure Fritillaria thunbergii polysaccharide, filling a technological gap: The Fritillaria thunbergii polysaccharide (FTP-1) purified by this process has a stable weight-average molecular weight of 11-12 kDa. The monosaccharide composition is mainly glucose (97-98 wt%), supplemented by galactose (1-2 wt%) and mannose (0.5-0.7 wt%). The component ratio is uniform and the structural characteristics are clear. High-performance gel permeation chromatography shows a single symmetrical peak without interference from impurities. Compared with crude polysaccharides with unclear components in existing technologies, the polysaccharide product of this invention has high purity and stable physicochemical properties, laying a material foundation for its bioactivity research and industrial application, and filling the technological gap in the purification and preparation of highly homogeneous Fritillaria thunbergii polysaccharides. 4. Significantly regulates intestinal flora structure, achieving a dual effect of enriching beneficial bacteria and inhibiting harmful bacteria: The Fritillaria thunbergii polysaccharide of this invention has a precise intestinal flora regulation effect. Verified by an in vitro fermentation model, it can significantly alter the community structure of the human intestinal flora: directionally increasing the relative abundance of beneficial bacteria such as Bifidobacterium and Bacteroides, while effectively reducing the abundance of potentially harmful or conditionally pathogenic bacteria such as Escherichia coli; it has a specific screening effect on the microbial community, shaping a healthy flora structure with short-chain fatty acid-producing bacteria as the core, solving the problem that the intestinal microecological regulation effect of Fritillaria thunbergii polysaccharide has not been systematically reported in existing technologies, and providing a novel natural active substance for the intervention of intestinal flora imbalance; 5. Enhancing the short-chain fatty acid synthesis capacity of gut microbiota and strengthening intestinal metabolic health: Fritillaria thunbergii polysaccharide significantly improves the total production of short-chain fatty acids (SCFAs) by gut microbiota. Compared with the blank control group, the production of core short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, as well as total SCFAs, in its fermentation broth increased significantly (p<0.01). Short-chain fatty acids, as key beneficial metabolites of gut microbiota, can regulate intestinal barrier function, immune function, and energy metabolism. This invention's polysaccharide enhances the synthesis level of SCFAs in gut microbiota, thereby regulating intestinal health at the metabolic level, demonstrating significant biological activity and a clear mechanism of action. 6. Significantly promotes the in vitro proliferation of probiotics and possesses excellent natural prebiotic potential: Verification through probiotic growth curve testing shows that the Fritillaria thunbergii polysaccharide of this invention has no growth inhibitory effect on common intestinal probiotics such as Bifidobacterium adolescentis, Bacteroides multiforme, Bacteroides ovalis, and Parabacterium chrysogenum. On the contrary, it can significantly accelerate the growth rate of probiotics, enabling them to enter the stationary phase earlier, and the final biomass (OD) is also higher. 600 Overall, the levels were higher than the control group, demonstrating a clear probiotic-promoting effect on gut symbiotic probiotics. Compared to traditional chemical prebiotics or other plant polysaccharides, the polysaccharides of this invention are of natural plant origin, exhibiting high safety and significant probiotic effects, making them a novel and excellent candidate for natural prebiotics. 7. Expanding the application dimensions of Fritillaria thunbergii to achieve in-depth development and high-value utilization of traditional Chinese medicine: As a traditional Chinese medicine, Fritillaria thunbergii's current applications are mostly concentrated on its traditional effects of clearing heat and resolving phlegm, relieving cough and dissipating nodules. Research on its polysaccharide components is also limited to basic activities such as immune regulation and antioxidation. This invention is the first to discover the intestinal flora regulation and prebiotic functions of Fritillaria thunbergii polysaccharides, expanding the application field of Fritillaria thunbergii from traditional Chinese medicine preparations to intestinal health-related pharmaceuticals, health foods, food additives, and other fields. This achieves in-depth development and high-value utilization of traditional Chinese medicine, enhances the industrial added value of Fritillaria thunbergii, and provides new technical ideas and application directions for the diversified development of traditional Chinese medicine resources. 8. High biocompatibility and wide range of applications: The Fritillaria thunbergii polysaccharide of this invention is extracted from the bulb of the natural Chinese medicinal herb Fritillaria thunbergii. The purification process uses mild solvents such as water and ethanol and physical purification methods throughout, leaving no toxic or harmful reagent residues. In vitro experiments have not found any toxic side effects on probiotics or normal intestinal flora, demonstrating high biocompatibility. Based on its clear intestinal flora regulation function and prebiotic potential, it can be widely used in various intestinal health regulation products, including pharmaceuticals, functional health foods, special dietary foods, and food additives, with rich application scenarios and broad industrial prospects. Attached Figure Description

[0037] Figure 1 Figure showing the extraction, purification, and compositional analysis of polysaccharides from Fritillaria thunbergii. Figure 2 A graph showing changes in gut microbiota at different levels; Figure 3 A graph showing the changes in the content of different types of short-chain fatty acids in the fermentation broth after the microbial community metabolizes polysaccharides. Figure 4 A graph showing the effect of Fritillaria thunbergii polysaccharide on the α and β diversity of human gut microbiota. Detailed Implementation

[0038] Example 1 This embodiment provides a polysaccharide from Fritillaria thunbergii and a method for its extraction and purification. The method includes the following steps: Step 1, Water Extraction and Alcohol Precipitation: Mix 500g of powdered Fritillaria thunbergii bulb (particle size between 55-60 mesh) with 5L of distilled water and reflux at 95℃ for 2h to obtain a first extract. Repeat the above extraction steps with the residue to obtain a second extract. Combine the two extracts, concentrate them, and then rotary evaporate them. To prevent bumping during the initial concentration, use a temperature of 40℃ and a speed of 30 rpm for rotary evaporation. After stabilization, appropriately increase the temperature to 50℃ and the speed to 70 rpm to improve the concentration efficiency. Concentrate to 500mL. While stirring, add 4 times the volume of anhydrous ethanol to the concentrate, place it in a 4℃ refrigerator and let it stand for 12h to precipitate. Centrifuge (8000rpm, 15min) to collect the precipitate, and then wash it with anhydrous ethanol, acetone, and ether respectively. Vacuum dry at -60℃ for 12h to obtain 35g of crude Fritillaria thunbergii polysaccharide. Step 2, First Purification: Dissolve 2g of crude Fritillaria thunbergii polysaccharide from Step 1 in 100 mL of deionized water, centrifuge to remove insoluble matter, and load the sample onto a DEAE-52 cellulose column (2.6cm × 40cm) pre-equilibrated with deionized water. Elute sequentially with deionized water, 0.2M, 0.4M, 0.6M, and 0.8M NaCl solutions at a flow rate of 3-5 mL / min. Collect 5 mL from each tube, and determine the sugar content of each tube using the phenol-sulfuric acid method. Plot the elution curve, as shown below. Figure 1Figure A shows the purification results of Fritillaria thunbergii polysaccharide using DEAE-52 cellulose column, with the main peak eluted fraction collected. Where the horizontal axis represents the collection tube number, and the vertical axis on the left represents OD. 490 (The absorbance value for polysaccharide content determination using the phenol-sulfuric acid method reflects the polysaccharide concentration). The right-hand vertical axis represents the concentration gradient of NaCl in the eluent, specifically: 1. Elution phase and polysaccharide distribution Initial stage (Tube 0-40): NaCl concentration is 0 (water elution), and the first obvious OD appears. 490 The peak indicates that the component is a neutral polysaccharide, which does not bind to the DEAE-52 anion exchange column and can be washed away by water.

[0039] NaCl gradient elution stage (Tube 40-140): The NaCl concentration is gradually increased from 0 to 0.8 mol / L. Multiple small ODs appear as the salt concentration increases. 490 The presence of peaks indicates the presence of multiple acidic polysaccharide components. These components carry a negative charge and can only be eluted by competing with salt ions for binding sites. Different components are eluted sequentially at different salt concentrations due to differences in charge.

[0040] 2. Purification Results and Conclusions Polysaccharide composition diversity: The crude polysaccharide of Fritillaria thunbergii contains at least one major polysaccharide component, and the charge and molecular weight of different components are different.

[0041] Separation results: DEAE-52 column chromatography successfully separated polysaccharide components with different charges. The eluents from the corresponding collection tubes can be combined to obtain multiple polysaccharide components with higher homogeneity.

[0042] Content characteristics: The neutral polysaccharide eluted by water is the most abundant component (first peak OD). 490 The highest concentration was close to 0.9, while the content of acidic polysaccharide components was relatively low and their distribution was scattered.

[0043] Step 3, Secondary Purification: The main peak eluent obtained in Step 2 was placed into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed with deionized water for 48 hours, with the water changed every 8 hours, and then freeze-dried at -80℃ for 12 hours to obtain a white flocculent pure product of Fritillaria thunbergii polysaccharide (abbreviated as FTP-1), with an extraction rate of 19%.

[0044] The Fritillaria thunbergii polysaccharide (FTP-1) prepared in this embodiment was analyzed using high-performance gel permeation chromatography (HPGPC) coupled with multi-angle laser light scattering (MALLS) and differential detector (RI). The results showed a single symmetrical peak, as... Figure 1Figures B (showing the molecular weight determination results of purified Fritillaria thunbergii polysaccharide) and C (showing the monosaccharide composition analysis results of purified Fritillaria thunbergii polysaccharide) indicate that it is a homogeneous component with a weight-average molecular weight (Mw) of 11.86 kDa. like Figure 1 In Figure B: The horizontal axis of this graph represents retention time, and the vertical axis on the left represents peak intensity. This result is a graph showing the molecular weight determination of the neutral polysaccharide component of purified Fritillaria thunbergii. The HPLC elution peaks of the purified Fritillaria thunbergii are relatively simple, with a retention time of approximately 14.309 min, indicating that it is a low molecular weight oligosaccharide.

[0045] like Figure 1 Figure C: This figure shows the monosaccharide composition analysis results of purified Fritillaria thunbergii polysaccharides. The lower part of Figure C shows the HPLC detection results of a mixed standard of nine monosaccharides: mannose (Man), rhamnose (Rha), glucuronic acid (GlcUA), galacturonic acid (GalUA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc). The upper part of Figure C shows the monosaccharide composition results of the purified Fritillaria thunbergii neutral polysaccharides, with glucose (Glc), galactose (Gal), and mannose (Man) as the main components.

[0046] The detailed analysis of the HPLC detection results is as follows: Mannose (Man): A distinct chromatographic peak appears at a retention time of approximately 20 min, indicating it is one of the polysaccharide components; Glucose (Glc): The strongest chromatographic peak appears at a retention time of approximately 40 min, indicating it is the main monosaccharide component of this polysaccharide; Galactose (Gal): A distinct chromatographic peak appears at a retention time of approximately 45 min, indicating it has a relatively high content.

[0047] In summary, the purified Fritillaria thunbergii polysaccharide is a heteropolysaccharide, with glucose (Glc) as the main monosaccharide component, and also containing galactose (Gal) and mannose (Man). The ratio of glucose (Glc): galactose (Gal): mannose (Man) is 97.99 wt%: 1.35 wt%: 0.66 wt%.

[0048] Example 2 This embodiment provides a polysaccharide from Fritillaria thunbergii and a method for its extraction and purification. The method includes the following steps: Step 1, Water Extraction and Alcohol Precipitation: Mix 500g of powdered Fritillaria thunbergii bulb (particle size between 55-60 mesh) with 15L of distilled water and reflux at 80℃ for 3h to obtain a first extract. Repeat the above extraction steps with the residue to obtain a second extract. Combine the two extracts, concentrate them, and then rotary evaporate them. To prevent boiling over during the initial concentration, use a temperature of 40℃ and a speed of 30 rpm for rotary evaporation. After stabilization, appropriately increase the temperature to 50℃ and the speed to 70 rpm to improve the concentration efficiency. Concentrate to 500mL. While stirring, add 5 times the volume of anhydrous ethanol to the concentrate, place it in a 2℃ refrigerator and let it stand for 16h to precipitate. Centrifuge (8000rpm, 15min) to collect the precipitate, and then wash it with anhydrous ethanol, acetone, and ether respectively. Vacuum dry at -80℃ for 12h to obtain 32g of crude Fritillaria thunbergii polysaccharide. Step 2, First purification: Dissolve 2g of crude polysaccharide from Fritillaria thunbergii in 80 mL of deionized water, centrifuge to remove insoluble matter, and load the sample onto a DEAE-52 cellulose column (2.6cm×40cm) pre-equilibrated with deionized water. Elute in stages with deionized water, 0.2M, 0.4M, 0.6M, and 0.8M NaCl solutions at a flow rate of 3-5 mL / min. Collect 5 mL from each tube, and determine the sugar content of each tube using the phenol-sulfuric acid method. Plot the elution curve and collect the main peak elution fraction. Step 3, Secondary Purification: The main peak eluent obtained in Step 2 was placed into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed with deionized water for 48 hours, with the water changed every 8 hours, and then freeze-dried at -60℃ for 24 hours to obtain pure white flocculent Fritillaria thunbergii polysaccharide (abbreviated as FTP-1), with an extraction rate of 21%.

[0049] The Fritillaria thunbergii polysaccharide (FTP-1) prepared in this embodiment was analyzed using high-performance gel permeation chromatography (HPGPC) coupled with multi-angle laser light scattering (MALLS) and differential detector (RI). The results showed a single symmetrical peak, indicating that it is a homogeneous component, and the weight-average molecular weight (Mw) was determined to be 11.74 kDa. In this embodiment, the main components of Fritillaria thunbergii polysaccharide are glucose (Glc), galactose (Gal), and mannose (Man). The ratio of glucose (Glc):galactose (Gal):mannose (Man) is 97.64 wt%: 1.44 wt%: 0.92 wt%.

[0050] Example 3 This embodiment provides a polysaccharide from Fritillaria thunbergii and a method for its extraction and purification. The method includes the following steps: Step 1, Water Extraction and Alcohol Precipitation: Mix 500g of powdered Fritillaria thunbergii bulb (particle size between 55-60 mesh) with 10L of distilled water and reflux at 100℃ for 1h to obtain a first extract. Repeat the above extraction steps with the residue to obtain a second extract. Repeat the above extraction steps with the residue to obtain a third extract. Combine the three extracts, concentrate them, and then rotary evaporate them. To prevent boiling over during the initial concentration, use a temperature of 40℃ and a speed of 30 rpm for rotary evaporation. After stabilization, appropriately increase the temperature to 50℃ and the speed to 70 rpm to improve the concentration efficiency. Concentrate to 500mL. Add 5 times the volume of anhydrous ethanol to the concentrate while stirring. Place it in a 2℃ refrigerator and let it stand for 16h to precipitate. Centrifuge (8000 rpm, 15min) to collect the precipitate, and then wash it with anhydrous ethanol, acetone, and ether respectively. Dry it under vacuum at -80℃ for 12h to obtain 33g of crude Fritillaria thunbergii polysaccharide. Step 2, First purification: Dissolve 2g of crude polysaccharide from Fritillaria thunbergii in 90 mL of deionized water, centrifuge to remove insoluble matter, and load the sample onto a DEAE-52 cellulose column (2.6cm×40cm) pre-equilibrated with deionized water. Elute in stages with deionized water, 0.2M, 0.4M, 0.6M, and 0.8M NaCl solutions at a flow rate of 3-5 mL / min. Collect 5 mL from each tube, and determine the sugar content of each tube using the phenol-sulfuric acid method. Plot the elution curve and collect the main peak elution fraction. Step 3, Secondary Purification: The main peak eluent obtained in Step 2 was placed into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed with deionized water for 48 hours, with the water changed every 8 hours, and then freeze-dried at -80℃ for 18 hours to obtain a pure white flocculent Fritillaria thunbergii polysaccharide (abbreviated as FTP-1) with an extraction rate of 22%.

[0051] The Fritillaria thunbergii polysaccharide (FTP-1) prepared in this embodiment was analyzed using high-performance gel permeation chromatography (HPGPC) coupled with multi-angle laser light scattering (MALLS) and differential detector (RI). The results showed a single symmetrical peak, indicating that it is a homogeneous component, and the weight-average molecular weight (Mw) was determined to be 11.93 kDa. In this embodiment, the main components of Fritillaria thunbergii polysaccharide are glucose (Glc), galactose (Gal), and mannose (Man). The ratio of glucose (Glc):galactose (Gal):mannose (Man) is 97.81 wt%: 1.27 wt%: 0.92 wt%.

[0052] Example 4 This embodiment provides a polysaccharide from Fritillaria thunbergii and a method for its extraction and purification. The method includes the following steps: Step 1, Water Extraction and Alcohol Precipitation: Mix 500g of powdered Fritillaria thunbergii bulb (particle size between 45-50 mesh) with 10L of distilled water and reflux at 100℃ for 1h to obtain a first extract. Repeat the above extraction steps with the residue to obtain a second extract. Repeat the above extraction steps with the residue to obtain a third extract. Combine the three extracts, concentrate them, and then rotary evaporate them. To prevent bumping during the initial concentration, use a temperature of 40℃ and a speed of 30 rpm for rotary evaporation. After stabilization, appropriately increase the temperature to 50℃ and the speed to 70 rpm to improve the concentration efficiency. Concentrate to 500mL. Add 5 times the volume of anhydrous ethanol to the concentrate while stirring. Place it in a refrigerator at 2℃ and let it stand for 16h to precipitate. Centrifuge (8000rpm, 15min) to collect the precipitate, and then wash it with anhydrous ethanol, acetone, and ether respectively. Dry it under vacuum at -80℃ for 12h to obtain 29g of crude Fritillaria thunbergii polysaccharide. Step 2, First purification: Dissolve 2g of crude polysaccharide from Fritillaria thunbergii in 90 mL of deionized water, centrifuge to remove insoluble matter, and load the sample onto a DEAE-52 cellulose column (2.6cm×40cm) pre-equilibrated with deionized water. Elute in stages with deionized water, 0.2M, 0.4M, 0.6M, and 0.8M NaCl solutions at a flow rate of 3-5 mL / min. Collect 5 mL from each tube, and determine the sugar content of each tube using the phenol-sulfuric acid method. Plot the elution curve and collect the main peak elution fraction. Step 3, Secondary Purification: The main peak eluent obtained in Step 2 was placed into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed with deionized water for 48 hours, with the water changed every 8 hours, and then freeze-dried at -80℃ for 18 hours to obtain a white flocculent pure product of Fritillaria thunbergii polysaccharide (abbreviated as FTP-1), with an extraction rate of 18%.

[0053] The Fritillaria thunbergii polysaccharide (FTP-1) prepared in this embodiment was analyzed using high-performance gel permeation chromatography (HPGPC) coupled with multi-angle laser light scattering (MALLS) and differential detector (RI). The results showed a single symmetrical peak, indicating that it is a homogeneous component, and the weight-average molecular weight (Mw) was determined to be 11.93 kDa. In this embodiment, the main components of Fritillaria thunbergii polysaccharide are glucose (Glc), galactose (Gal), and mannose (Man). The ratio of glucose (Glc):galactose (Gal):mannose (Man) is 97.25 wt%: 1.97 wt%: 0.78 wt%.

[0054] Example 5 This embodiment provides a polysaccharide from Fritillaria thunbergii and a method for its extraction and purification. The method includes the following steps: Step 1, Water Extraction and Alcohol Precipitation: Mix 500g of powdered Fritillaria thunbergii bulb (particle size between 60-65 mesh) with 5L of distilled water and reflux at 100℃ for 1h to obtain a first extract. Repeat the above extraction steps with the residue to obtain a second extract. Repeat the above extraction steps with the residue to obtain a third extract. Combine the three extracts, concentrate them, and then rotary evaporate them. To prevent boiling over during the initial concentration, use a temperature of 40℃ and a speed of 30 rpm for rotary evaporation. After stabilization, appropriately increase the temperature to 50℃ and the speed to 70 rpm to improve the concentration efficiency. Concentrate to 500mL. While stirring, add 5 times the volume of anhydrous ethanol to the concentrate, place it in a 2℃ refrigerator and let it stand for 16h to precipitate. Centrifuge (8000 rpm, 15min) to collect the precipitate, and then wash it with anhydrous ethanol, acetone, and ether respectively. Vacuum dry at -80℃ for 12h to obtain 33g of crude Fritillaria thunbergii polysaccharide. Step 2, First purification: Dissolve 2g of crude polysaccharide from Fritillaria thunbergii in 90 mL of deionized water, centrifuge to remove insoluble matter, and load the sample onto a DEAE-52 cellulose column (2.6cm×40cm) pre-equilibrated with deionized water. Elute in stages with deionized water, 0.2M, 0.4M, 0.6M, and 0.8M NaCl solutions at a flow rate of 3-5 mL / min. Collect 5 mL from each tube, and determine the sugar content of each tube using the phenol-sulfuric acid method. Plot the elution curve and collect the main peak elution fraction. Step 3, Secondary Purification: The main peak eluent obtained in Step 2 was placed into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed with deionized water for 48 hours, with the water changed every 8 hours, and then freeze-dried at -80℃ for 18 hours to obtain a pure white flocculent Fritillaria thunbergii polysaccharide (FTP-1) with an extraction rate of 19%.

[0055] The Fritillaria thunbergii polysaccharide (FTP-1) prepared in this embodiment was analyzed using high-performance gel permeation chromatography (HPGPC) coupled with multi-angle laser light scattering (MALLS) and differential detector (RI). The results showed a single symmetrical peak, indicating that it is a homogeneous component, and the weight-average molecular weight (Mw) was determined to be 11.93 kDa. In this embodiment, the main components of Fritillaria thunbergii polysaccharide are glucose (Glc), galactose (Gal), and mannose (Man). The ratio of glucose (Glc):galactose (Gal):mannose (Man) is 97.74 wt%: 1.92 wt%: 0.34 wt%.

[0056] Example 6 This example illustrates the effect of Fritillaria thunbergii polysaccharide (FTP-1) prepared in Example 1 on the growth of probiotics, and specifically includes the following steps: Step 1: Strain Activation: *Bifidobacterium adolescentis*, *Bacteroides thetaiotaomicron*, *Bacteroides ovatus*, and *Parabacteroides goldsteinii* were streaked onto their respective solid media for resuscitation, and then inoculated into liquid media for subculturing to the logarithmic growth phase. *Bacteroides* strains were primarily activated using GAM solid and liquid media, while *Bifidobacterium* was primarily activated using YPD liquid and solid media.

[0057] Step 2, Growth Curve Determination: The activated bacterial strain was inoculated at a rate of 2% into liquid culture medium containing 1% FTP-1 (experimental group) or without FTP-1 (control group); it was then incubated under anaerobic conditions at 37℃. Samples were taken every 6-12 hours, and the optical density (OD) at 600nm was measured using a microplate reader. 600 )value.

[0058] The results are as follows Figure 2 As shown, the AB plot is a stacked bar chart of the relative abundance of microbial communities at the phylum and genus levels, used to show the differences in community composition at the phylum level between Ctrl (control group) and CB (treatment group) samples. The X-axis represents the sample number (Ctrl1-5 are control group, CB1-6 are treatment group), and the Y-axis represents the relative abundance (%), indicating the proportion of a certain phylum in the total community of the sample. Different colors represent different bacteria.

[0059] Therefore, community composition analysis at the phylum and genus levels showed that the treatment with Fritillaria thunbergii polysaccharide significantly altered the microbial community structure: in the control group, Proteobacteria was the dominant phylum and Escherichia was the dominant genus, resulting in a balanced and diverse community structure; after treatment with Fritillaria thunbergii polysaccharide, the community shifted, with Proteobacteria (phylum level) and Escherichia (genus level) becoming extremely enriched at the phylum and genus levels, respectively, becoming the absolutely dominant groups, while Acidobacteria, Planctomycetes (phylum level) and most non-Escherichia genera were significantly suppressed. This indicates that the treatment with Fritillaria thunbergii polysaccharide has a strong screening effect on the microbial community, ultimately shaping a tolerant community with Proteobacteria and Escherichia as its core.

[0060] at the same time, Figure 2C and F in the graph represent: C is the growth curve of *Bifidobacterium adolescentis* in *Fritillaria thunbergii*; D is the growth curve of *Parabacteroides goldsteinii* in *Fritillaria thunbergii*; E is the growth curve of *Bacteroides thetaiotaomicron* in *Fritillaria thunbergii*; and F is the growth curve of *Bacteroides ovatus* in *Fritillaria thunbergii*. This paper presents the in vitro growth curves of four beneficial intestinal bacteria strains—Bifidobacterium adolescentis, Bacteroides thetaiotaomicron, Bacteroides ovatus, and Parabacteroides goldsteinii—treated with Fritillaria thunbergii polysaccharide (CB). Growth curve analysis showed that, compared with the control group, CB treatment had no growth inhibitory effect on any of the tested strains; on the contrary, it generally exhibited a positive effect of promoting growth or tolerance: the growth rate of each strain was significantly accelerated under CB treatment, they entered the stationary phase earlier, and the final biomass (OD) was higher. 600 The overall levels were higher than those in the control group. These results indicate that the purified Fritillaria thunbergii polysaccharide (CB) isolated in this study has a clear probiotic effect on these gut symbiotic probiotics and possesses potential application value as a novel prebiotic.

[0061] Example 7 This embodiment describes an in vitro experiment on regulating intestinal flora using the Fritillaria thunbergii polysaccharide (FTP-1) prepared in Example 1. The specific steps include: Step 1, Preparation of fermentation substrate: A 10 mg / mL solution of Fritillaria thunbergii polysaccharide was prepared using sterile phosphate buffered saline (PBS) solution as the experimental group, and an equal volume of PBS solution was used as the blank control group. Step 2, preparation of fecal inoculum: Fresh fecal samples were collected from 3 healthy volunteers and homogenized into a 5-10% homogenate using pre-reduced phosphate buffer solution (PBS solution) under anaerobic conditions. The filtrate was then collected after filtration. Step 3, In vitro fermentation: In the anaerobic workstation, the fermentation basal medium (which mainly includes 2 g / L peptone, 2 g / L yeast extract, 0.1 g / L NaCl, 0.01 g / L K₂HPO₄, 0.04 g / L KH₂PO₄, and 0.01 g / L MgSO₄) is prepared. 7H2O, 0.01 g / L CaCl2 2H2O, 2 g / L NaHCO3, 2 mL Tween-80, 0.05 g / L hemin, 10 μL vitamin K1, 0.5 g / L L-cysteine, 0.5 g bile salts, and 0.01 g resazurin were mixed with the experimental or control group substrates at a ratio of 1:10 and fermented anaerobically at 37°C for 24 h. Step 4, Detection and Analysis: Samples were taken at the fermentation endpoint, and the content of short-chain fatty acids (acetic acid, propionic acid, butyric acid, etc.) in the fermentation broth was determined by gas chromatography (GC). The results are as follows: Figure 3 The figure shows the changes in the content of different types of short-chain fatty acids in the fermentation broth after the microbial community metabolizes polysaccharides. The total SCFAs yield in the experimental group (CB group) was significantly higher than that in the control group (Ctrl group). The specific analysis is as follows: Figure 3 This study demonstrates the changes in short-chain fatty acid (SCFA) content in the fermentation broth after fermentation with Fritillaria thunbergii polysaccharide. Compared with the control group, the CB group showed significantly higher levels of all detected SCFA components (acetic acid, propionic acid, 2...). Methylpropionic acid, butyric acid, 3 The concentrations of methylbutyric acid, valeric acid, and hexanoic acid were all significantly increased (p<0.01); the total SCFAs production increased approximately 5-fold, with the total concentration in the CB group being approximately 160 μg / mL, while the control group was only about 30 μg / mL. Combined with the aforementioned results showing that the treatment with Fritillaria thunbergii polysaccharide significantly enriched short-chain fatty acid-producing bacteria such as Bifidobacterium and Bacteroides, this suggests that Fritillaria thunbergii polysaccharide selectively promotes the growth of SCFA-producing strains and enhances the metabolic function of the gut microbiota, thereby significantly increasing the level of SCFAs synthesis. These results further confirm that Fritillaria thunbergii polysaccharide has a clear probiotic effect, and can exert its intestinal health regulatory function by regulating gut microbiota metabolism and increasing the production of beneficial metabolites.

[0062] Meanwhile, total bacterial DNA was extracted from the fermentation broth, and the bacterial community structure was analyzed by 16S rRNA gene high-throughput sequencing. The results showed that compared with the control group (Ctrl group), the relative abundance of Bifidobacterium and Bacteroides was significantly increased in the experimental group (CB group), while the abundance of conditionally pathogenic bacteria such as Escherichia coli decreased.

[0063] Simultaneously, routine diversity analyses of changes in human gut microbiota α and β were performed, and the results were as follows: Figure 4 As shown.

[0064] Figure 4Figure A shows the dilution curves. The horizontal X-axis represents the number of sequencing reads sampled (representing sequencing depth), and the vertical Y-axis represents species richness (usually referring to the number of observed OTUs / ASVs, i.e., the total number of detected "species"). Each curve represents an independent sample, showing the trend of the number of species detected in that sample as sequencing depth increases. All curves exhibit a typical trend of "rapid rise → slowing growth → approaching a plateau".

[0065] Therefore, when none of the curves show a "continuous steep rise", it indicates that there is no serious problem of insufficient sequencing depth, and the results of subsequent diversity analysis (such as α diversity and β diversity) based on these data are reliable.

[0066] Figure 4 Figure B in the figure shows the results of β-diversity analysis, used to illustrate the similarity / difference in microbial community structure among different samples. PC1 (66.12%) is the first principal component, explaining 66.12% of the inter-sample variation, and is the most significant factor contributing to community differences. PC2 (17.7%) is the second principal component, explaining 17.7% of the variation, and is a secondary source of difference. All samples in the figure are clearly divided into two independent clusters, each enclosed by an ellipse (typically a 95% confidence interval).

[0067] Together, these two dimensions explained 83.82% of the total variation, indicating that they effectively reflected the community differences between samples, and the results are reliable. The complete separation and lack of overlap between the two clusters in each sample indicated a highly significant difference in the microbial community structure between the two groups.

[0068] Figure 4The CF plot shows the results of α-diversity analysis, used to describe the species richness and evenness within a single sample. The Chao1 index (Figure C) estimates the species richness of the community (including unobserved rare species); a higher value indicates a greater total number of species. Observed species (Figure D) represents the number of species actually observed (i.e., the number of OTUs / ASVs detected in sequencing), directly reflecting the detectable species richness. The Shannon index (Figure E) comprehensively reflects species richness and evenness; a higher value indicates higher community diversity (more species and even distribution). The Simpson index (Figure F) reflects the dominance / diversity of the community (the value here is close to 1; a higher value indicates higher diversity and a lower proportion of dominant species). The α-diversity of the microbial community in the Ctrl group was significantly higher than that in the CB group (including the Fritillaria thunbergii polysaccharide group). In terms of species richness (Chao1, Observed species): the Ctrl group had more species, while the CB group had fewer species. In terms of community diversity and evenness (Shannon, Simpson): the Ctrl group community was more complex and even, while the CB group showed decreased diversity, possibly indicating a concentration of dominant species.

[0069] Therefore, compared with the control group (Ctrl), the CB-treated group showed a significant decrease in microbial community α-diversity. Specifically, the Chao1 index (p<0.05) and the number of observed species (p<0.05) decreased significantly, indicating that CB treatment led to a decrease in community species richness; the Shannon index (p<0.01) and the Simpson index (p<0.01) also decreased significantly, indicating that CB treatment resulted in decreased community diversity, reduced species evenness, and an increased proportion of dominant species.

[0070] It should be understood that the present invention is not limited to what has been described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of the invention is limited only by the appended claims.

Claims

1. A method for extracting and purifying polysaccharides from Fritillaria thunbergii, characterized in that, The method includes the following steps: Step 1, Water extraction and alcohol precipitation: Mix Fritillaria thunbergii powder with distilled water and extract to obtain an extract. After concentration, add ethanol to the concentrate, let it stand to precipitate, and collect the precipitate to obtain crude Fritillaria thunbergii polysaccharide. Step 2, First purification: Dissolve the crude polysaccharide of Fritillaria thunbergii from Step 1 in deionized water, load the sample onto an ion exchange column, and elute with deionized water and sodium chloride solution in sequence. Monitor the sugar content online and collect the main peak elution fraction. Step 3, Secondary purification: The main peak eluent obtained in Step 2 is dialyzed and desalted using a semi-permeable membrane, and then freeze-dried to obtain the Fritillaria thunbergii polysaccharide.

2. The method for extracting and purifying Fritillaria thunbergii polysaccharide according to claim 1, characterized in that, The ratio of Fritillaria thunbergii powder to distilled water in step 1 is 1:(10-30), with units of g / mL.

3. The method for extracting and purifying Fritillaria thunbergii polysaccharide according to claim 1, characterized in that, The volume ratio of the concentrate to ethanol in step 1 is 1:(4-5).

4. The method for extracting and purifying Fritillaria thunbergii polysaccharide according to claim 1, characterized in that, The ion exchange column mentioned in step 2 is a DEAE-52 cellulose ion exchange column.

5. The method for extracting and purifying Fritillaria thunbergii polysaccharide according to claim 1, characterized in that, The concentration of the sodium chloride solution in step 2 is 0.2-0.8M.

6. The method for extracting and purifying Fritillaria thunbergii polysaccharide according to claim 1, characterized in that, The method for online monitoring of sugar content mentioned in step 2 is either the phenol-sulfuric acid method or the sulfuric acid-carbazole method.

7. The method for extracting and purifying Fritillaria thunbergii polysaccharide according to claim 1, characterized in that, The semipermeable membrane described in step 3 has a molecular weight cutoff of 3500-14000 Da.

8. A polysaccharide from Fritillaria thunbergii, characterized in that, The Fritillaria thunbergii polysaccharide was obtained by extraction and purification using the extraction and purification method of Fritillaria thunbergii polysaccharide according to any one of claims 1-7.

9. The Fritillaria thunbergii polysaccharide according to claim 8, characterized in that, The weight-average molecular weight of the Fritillaria thunbergii polysaccharide is 11-12 kDa, and it includes glucose, galactose and mannose, with mass percentages of (97-98) wt%, (1-2) wt%, and (0.5-1) wt%, respectively.

10. A product for regulating intestinal flora, characterized in that, The product includes the Fritillaria thunbergii polysaccharide as described in any one of claims 8-9.

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