Method for separating and purifying polysaccharides from jinhuazang tea and application thereof

Abstract

This invention discloses a method and application for the separation and purification of polysaccharides from *Tea laurentii*, belonging to the field of *Tea laurentii* technology. The method includes pulverizing and sieving *Tea laurentii*, extracting with ethanol, centrifuging to collect the precipitate; adjusting the pH of the precipitate with water, adding a compound enzyme for enzymatic hydrolysis, centrifuging to collect the supernatant, vacuum concentrating, and precipitating with alcohol by centrifugation to obtain a crude polysaccharide extract. The extract is then further purified by adding water to adjust the pH, adding papain for enzymatic hydrolysis, heating and holding at a constant temperature, adding a compound solvent, centrifuging to collect the upper aqueous phase, treating with petroleum ether, adsorbing with a compound adsorbent, and dialysis to obtain refined crude polysaccharides. These are then further purified by chromatography, gradient elution, concentration dialysis, and freeze-drying to finally obtain *Tea laurentii* polysaccharides. This invention utilizes a multi-step synergistic purification process involving compound enzymatic hydrolysis, compound solvent removal, compound adsorbent adsorption, and gradient elution to effectively remove proteins, fat-soluble pigments, small molecule impurities, and salts from *Tea laurentii* polysaccharides, significantly improving the purity and uniformity of the product.

Description

Technical Field

[0001] This invention relates to the field of Tibetan tea technology, specifically to a method for separating and purifying polysaccharides from Tibetan tea and its application. Background Technology

[0002] Type 2 diabetes is a globally prevalent chronic metabolic disease. The long-term use of chemically synthesized drugs in its treatment is prone to side effects, making naturally derived hypoglycemic active ingredients a research hotspot. Tea polysaccharides, as natural active substances that are both food and medicine, have shown good potential in regulating glucose metabolism. The polysaccharides formed by the microbial fermentation of golden flower Tibetan tea have unique fermentation and transformation characteristics, which have further development value.

[0003] In the field of preparation of polysaccharides from Tibetan tea, existing extraction and purification processes mostly employ a simple combination of water extraction and alcohol precipitation to remove impurities. This approach is insufficient for the targeted separation of proteins, fat-soluble pigments, and small molecule impurities in polysaccharides, resulting in poor purity and uniformity of the obtained products. Consequently, the hypoglycemic activity is suboptimal, which limits the application of Tibetan tea polysaccharides in hypoglycemic functional products.

[0004] Based on this, the present invention designs a method for separating and purifying polysaccharides from Tibetan tea and its application to solve the above problems. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, this invention provides a method for isolating and purifying polysaccharides from Tibetan tea, comprising the following steps:

[0006] S1: Grind and sieve the golden flower Tibetan tea to obtain golden flower Tibetan tea powder, add ethanol, extract and centrifuge, and collect the precipitate;

[0007] S2: Add water to the precipitate of S1, adjust the pH to 5.0-5.5, add the compound enzyme, and enzymatically hydrolyze to obtain the enzymatic hydrolysate;

[0008] S3: After the enzymatic hydrolysate is extracted, centrifuge and collect the supernatant extract;

[0009] S4: Vacuum concentrate the supernatant extract, cool to obtain concentrate A, precipitate with alcohol and centrifuge, collect the precipitate solid to obtain crude polysaccharide extract;

[0010] S5: Add water to the crude polysaccharide extract and stir. Adjust the pH to 6.0-6.5, add papain, and perform enzymatic hydrolysis. Heat to 78-82℃ and keep warm. After cooling, add the compound solvent, shake and centrifuge, and collect the upper aqueous phase.

[0011] S6: Add petroleum ether to the upper aqueous phase and mix well. Centrifuge to collect the lower aqueous phase. Add composite adsorbent, shake to adsorb, filter to collect the filtrate, dialyze, vacuum concentrate, precipitate with alcohol, centrifuge and dry to obtain refined crude polysaccharide.

[0012] S7: Dissolve the refined crude polysaccharide in water to prepare a polysaccharide mother liquor, centrifuge to obtain the supernatant, load the supernatant onto the sample, elute to obtain the pre-chromatographic solution, and combine the eluents of the main polysaccharide peak;

[0013] S8: Elute the pre-chromatographic solution by loading it onto the sample, and then elute sequentially with NaCl solutions of 0.05-0.1M, 0.1-0.2M, 0.2-0.3M, and 0.3-0.4M.

[0014] S9: Collect all eluents, combine the eluents corresponding to the main peak, concentrate under vacuum to obtain concentrate B, dialyze concentrate B to obtain dialyzed concentrate, freeze dry to obtain golden flower Tibetan tea polysaccharide.

[0015] Furthermore, in S2, the complex enzymes are pectinase, cellulase and papain in a mass ratio of 1.5-2.3:1.4-2.1:0.8-1.2.

[0016] Furthermore, the composite adsorbent is composed of modified diatomaceous earth and macroporous resin AB-8 in a mass ratio of 1.8-2.4:1.

[0017] Furthermore, the modified diatomaceous earth is prepared as follows: take natural diatomaceous earth, add 2-3 times its volume of distilled water, stir at 25-30℃ for 20-30 min, ball mill at 300-400 r / min for 1.5-2 h to obtain diatomaceous earth particles, measure 2-3 times the mass of natural diatomaceous earth with distilled water, add 1-1.5% of methionine by mass of diatomaceous earth at 150-200 r / min at a rate of 1-2 mL / min, continue stirring for 15-20 min to obtain a methionine aqueous solution, add diatomaceous earth particles at 0.5-1.0 g / min to the methionine aqueous solution, increase the stirring rate by 20-50 r / min, disperse at 25-30℃ for 30-40 min to obtain a dispersion;

[0018] Add 0.6-0.8% titanate coupling agent (by weight of natural diatomaceous earth) to the dispersion at a rate of 1-2 mL / min. After the addition is complete, raise the temperature to 60-65℃ at 2-5℃ / min and react at a constant temperature of 150-200 r / min for 1.5-2 h. Cool to room temperature, filter and collect the filter cake. Wash the filter cake with distilled water, using 1-1.5 times the weight of natural diatomaceous earth each time. Stir and wash for 10-15 min, then filter under vacuum. Repeat washing until the pH of the washing solution is 6.5-7.5. Dry the washed filter cake at 60-65℃ and a vacuum of 0.06-0.08 MPa for 4-6 h, then pulverize and pass through a 200-mesh sieve to obtain modified diatomaceous earth.

[0019] Furthermore, the composite solvent is composed of chloroform and n-butanol in a volume ratio of 4-6:1.

[0020] Furthermore, S6 specifically involves: adding an equal volume of petroleum ether to the upper aqueous phase, mixing for 10-15 minutes, centrifuging at 4000-5000 r / min for 5-8 minutes, collecting the lower aqueous phase, adding 12-18% of the volume of the lower aqueous phase to the composite adsorbent, adsorbing at 25-30℃ for 8-12 hours with shaking, collecting the filtrate by filtration, adding the filtrate to a dialysis bag, dialyzing in pure water for 48-60 hours, changing the pure water every 10-12 hours during this period, concentrating under vacuum to 1 / 5-1 / 4 of the original volume, adding 3-4 times the volume of anhydrous ethanol for 6-8 hours of precipitation, collecting the precipitate by centrifugation, drying at 50-60℃ to obtain the refined crude polysaccharide.

[0021] Furthermore, S7 specifically involves: dissolving the refined crude polysaccharide in pure water to prepare a polysaccharide stock solution of 15-30 mg / mL, centrifuging at 9000-10000 r / min for 8-10 min, taking the supernatant, equilibrating the column with pure water until the pH of the eluent is 7.0±0.2, loading the supernatant onto the sample at a flow rate of 2-3 mL / min, eluting with pure water for 2-3 column volumes to obtain the pre-chromatographic solution, and combining the eluents of the main polysaccharide peak.

[0022] Furthermore, S8 specifically involves: passing the pre-chromatographic solution through a pure water equilibrium exchange column until the effluent pH is 7.0 ± 0.2, loading the pre-chromatographic solution at a flow rate of 1-2 mL / min, eluting first with 2-3 column volumes of pure water, and then eluting sequentially with NaCl solutions of 0.05-0.1 M, 0.1-0.2 M, 0.2-0.3 M, and 0.3-0.4 M, maintaining a flow rate of 3-4 mL / min.

[0023] A polysaccharide from *Tea japonica* obtained according to the described separation and purification method has the following structural formula:

[0024] .

[0025] The application of the aforementioned golden flower Tibetan tea polysaccharide in the preparation of a drug for treating type 2 diabetes.

[0026] Compared with the prior art, the beneficial effects of this invention are as follows:

[0027] 1. This invention employs a multi-step synergistic purification process, including compound enzymatic hydrolysis, compound solvent purification, compound adsorbent adsorption, and gradient elution, to effectively remove proteins, fat-soluble pigments, small molecule impurities, and salts from the polysaccharides of *Tea laurentii*, significantly improving the purity and uniformity of the product.

[0028] 2. The polysaccharide of golden flower Tibetan tea prepared by this invention shows good activity in regulating blood glucose levels, improving insulin resistance, and balancing lipid metabolism in type 2 diabetic model animals. At the same time, it can regulate the body's oxidative stress state, inflammatory factor levels, and intestinal microecological metabolism, and has a protective effect on organs such as the liver and kidneys. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0030] Figure 1 This is a scanning electron microscope image of the golden flower Tibetan tea polysaccharide obtained in Example 3 of the present invention;

[0031] Figure 2 The infrared spectrum of the golden flower Tibetan tea polysaccharide obtained in Example 3 of this invention;

[0032] Figure 3 The ultraviolet spectrum of the golden flower Tibetan tea polysaccharide obtained in Example 3 of this invention;

[0033] Figure 4 This is a molecular configuration diagram of the golden flower Tibetan tea polysaccharide obtained in Example 3 of the present invention;

[0034] Figure 5 The absolute molecular weight analysis diagram of the golden flower Tibetan tea polysaccharide obtained in Example 3 of the present invention is shown.

[0035] Figure 6 This is the elution curve of the golden flower Tibetan tea polysaccharide prepared in Example 3 of the present invention using gel chromatography-laser light scattering-differential coupling.

[0036] Figure 7 The X-ray diffraction pattern of the golden flower Tibetan tea polysaccharide obtained in Example 3 of this invention;

[0037] Figure 8 This is a diagram showing the monosaccharide composition analysis of the *Tea japonica* polysaccharide obtained in Example 3 of this invention;

[0038] Figure 9 This is a methylation analysis diagram of the polysaccharide obtained from *Tea ginseng* obtained in Example 3 of the present invention;

[0039] Figure 10 The polysaccharide of *Tea ginseng* obtained in Example 3 of this invention. 1 H spectrum;

[0040] Figure 11 The polysaccharide of *Tea ginseng* obtained in Example 3 of this invention.13 C spectrum;

[0041] Figure 12 This is a COSY diagram of the golden flower Tibetan tea polysaccharide obtained in Example 3 of the present invention;

[0042] Figure 13 The NOESY diagram shows the polysaccharide obtained from *Tea ginseng* obtained in Example 3 of this invention.

[0043] Figure 14 The HSQC diagram of the golden flower Tibetan tea polysaccharide obtained in Example 3 of this invention;

[0044] Figure 15 The above is the HMBC diagram of the golden flower Tibetan tea polysaccharide obtained in Example 3 of this invention;

[0045] Figure 16 The DEPT-135 image is of the polysaccharide obtained from *Tea ginseng* prepared in Example 3 of this invention.

[0046] Figure 17 This is a TOCSY diagram of the polysaccharide obtained from Tibetan tea in Example 3 of the present invention. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0048] Example 1: This example provides a method for isolating and purifying polysaccharides from Tibetan tea, including the following steps:

[0049] S1: Grind the golden flower Tibetan tea into powder, pass it through a 50-mesh sieve to obtain golden flower Tibetan tea powder, weigh 1800g of golden flower Tibetan tea powder, add 5 times the volume of anhydrous ethanol to the golden flower Tibetan tea powder, stir and extract at 25℃ for 1h, centrifuge at 5000r / min for 8min, and collect the precipitate.

[0050] S2: Mix pectinase, cellulase and papain in a mass ratio of 1.5:1.4:0.8 to obtain a compound enzyme. Add 20 times the volume of pure water to the precipitate of S1, adjust the pH to 5.0-5.5, add the compound enzyme at 1.5% of the precipitate mass, and enzymatically hydrolyze at 45℃ for 2 hours to obtain the enzymatic hydrolysate.

[0051] S3: Extract the enzymatic hydrolysate in a 55℃ water bath for 3 hours, sonicating at 100W for 20 minutes every 10 minutes and stirring once every 20 minutes. After extraction, centrifuge at 5000r / min for 8 minutes, collect the supernatant extract, and repeat the above steps (S2-S3) to extract the precipitate residue again. Combine the two extracts.

[0052] S4: The supernatant extract was concentrated to 1 / 10 of its original volume by vacuum rotary evaporation at 45℃ and 0.06MPa. After cooling to room temperature, concentrated solution A was obtained. Anhydrous ethanol was added at a rate of 0.8mL / min at a volume of 3 times that of concentrated solution A. The mixture was precipitated at 4℃ for 8h and centrifuged at 7000r / min for 8min. The precipitated solid was collected to obtain the crude polysaccharide extract.

[0053] S5: Take natural diatomaceous earth, add twice its volume of distilled water, stir at 25℃ for 20 min, and ball mill at 300 r / min for 1.5 h to obtain diatomaceous earth particles. Take twice the amount of distilled water as the mass of natural diatomaceous earth, and add 1% of methionine by the mass of diatomaceous earth at a rate of 1 mL / min at 150 r / min. Continue stirring for 15 min to obtain a methionine aqueous solution. Add diatomaceous earth particles to the methionine aqueous solution at 0.5 g / min, increase the stirring rate by 20 r / min, and disperse at 25℃ for 30 min to obtain a dispersion.

[0054] Add 0.6% titanate coupling agent (311W) of natural diatomaceous earth by mass to the dispersion at a rate of 1 mL / min. After the addition is complete, heat to 60℃ at 2℃ / min and react at a constant temperature of 150 r / min for 1.5 h. Cool to room temperature, filter and collect the filter cake. Wash the filter cake with distilled water, using 1 times the mass of natural diatomaceous earth each time. Stir and wash for 10 min, then filter. Repeat washing until the pH of the washing solution is 6.5-7.5. Dry the washed filter cake at 60℃ and 0.06 MPa vacuum for 4 h, then pulverize and pass through a 200-mesh sieve to obtain modified diatomaceous earth.

[0055] S6: Add 20 times the volume of pure water to the crude polysaccharide extract and stir. Adjust the pH to 6.0-6.5. Add papain at 1% of the mass of the crude polysaccharide extract. Enzymatically hydrolyze at 50℃ for 12h. Heat to 78℃ at 2℃ / min and keep warm for 15min. After naturally cooling to room temperature, add 1 / 5 volume of the composite solvent (composed of chloroform and n-butanol in a volume ratio of 4:1). Shake for 15min. Centrifuge at 5000r / min for 8min. Collect the upper aqueous phase. Repeat 3 times and combine the aqueous phases.

[0056] S7: Add an equal volume of petroleum ether to the upper aqueous phase, mix well for 10 min, centrifuge at 4000 r / min for 5 min, collect the lower aqueous phase, add 12% of the volume of the lower aqueous phase with composite adsorbent (composed of modified diatomaceous earth and macroporous resin AB-8 with a mass ratio of 1.8:1), shake and adsorb at 25℃ for 8 h, filter and collect the filtrate, add the filtrate to a dialysis bag (3000 Da), dialyze in pure water for 48 h, changing the pure water every 10 h during the process, vacuum rotary evaporation to concentrate to 1 / 5 of the original volume, add 3 times the volume of anhydrous ethanol for 6 h, centrifuge to collect the precipitate, dry at 50℃ to obtain refined crude polysaccharide;

[0057] S8: Dissolve the refined crude polysaccharide in pure water to prepare a 15 mg / mL polysaccharide stock solution. Centrifuge at 9000 r / min for 8 min, collect the supernatant, and use a Sephacryl S-400HR separation column (2.6 cm in diameter and 100 cm in height). Equilibrate the column with pure water until the pH of the eluent is 7.0 ± 0.2. Load the supernatant at a flow rate of 2 mL / min and elute with pure water at a rate of 2 column volumes to obtain the pre-chromatographic solution. Determine the total sugar content using the LE-2-097 Total Polysaccharide Content Detection Kit (phenol-sulfuric acid method). Combine the eluents of the main polysaccharide peak.

[0058] S9: Use a DEAE Seplife FF exchange column (2.6 cm in diameter and 40 cm in height). Equilibrate with pure water until the pH of the effluent is 7.0 ± 0.2. Load the pre-chromatographic solution at a flow rate of 1 mL / min. First, elute with 2 column volumes of pure water, and then elute sequentially with 0.05 M, 0.1 M, 0.2 M, and 0.3 M NaCl solutions at a flow rate of 3 mL / min.

[0059] S10: Collect all eluents, combine the eluents corresponding to the main peak, and concentrate them to 1 / 5 of the original volume by vacuum rotary evaporation at 45℃ to obtain concentrate B. Add concentrate B to a dialysis bag (3000 Da) and dialyze in pure water for 36 h to remove salt (NaCl solution eluted in S10). Change the pure water every 8 h during this period to obtain dialysis concentrate. Freeze-dry at -40℃ for 24 h to obtain golden flower Tibetan tea polysaccharide.

[0060] Example 2: This example provides a method for isolating and purifying polysaccharides from Tibetan tea, including the following steps:

[0061] S1: Grind the golden flower Tibetan tea into powder, pass it through a 60-mesh sieve to obtain golden flower Tibetan tea powder, weigh 2000g of golden flower Tibetan tea powder, add 8 times the volume of anhydrous ethanol to the golden flower Tibetan tea powder, stir and extract at 30℃ for 2h, centrifuge at 6000r / min for 10min, and collect the precipitate.

[0062] S2: Mix pectinase, cellulase and papain in a mass ratio of 2.3: 2.1: 1.2 to obtain a compound enzyme. Add 30 times the volume of pure water to the precipitate of S1, adjust the pH to 5.0-5.5, add the compound enzyme at 2.5% of the precipitate mass, and enzymatically hydrolyze at 50℃ for 3 hours to obtain the enzymatic hydrolysate.

[0063] S3: Extract the enzymatic hydrolysate in a 60℃ water bath for 4 hours, sonicating at 150W for 30 minutes every 15 minutes and stirring once every 30 minutes. After extraction, centrifuge at 6000r / min for 10 minutes, collect the supernatant extract, and repeat the above steps (S2-S3) to extract the precipitate residue again. Combine the two extracts.

[0064] S4: The supernatant extract was concentrated to 1 / 8 of its original volume by vacuum rotary evaporation at 55℃ and 0.08MPa. After cooling to room temperature, concentrated solution A was obtained. Anhydrous ethanol was added at a rate of 1.2mL / min at a volume of 4 times that of concentrated solution A. The mixture was precipitated at 8℃ for 12h and centrifuged at 8000r / min for 10min. The precipitated solid was collected to obtain the crude polysaccharide extract.

[0065] S5: Take natural diatomaceous earth, add 3 times its volume of distilled water, stir at 30℃ for 30 min, and ball mill at 400 r / min for 2 h to obtain diatomaceous earth particles. Take 3 times the mass of natural diatomaceous earth of distilled water, add 1.5% of the diatomaceous earth mass of methionine at 2 mL / min at 200 r / min, and continue stirring for 20 min to obtain a methionine aqueous solution. Add diatomaceous earth particles to the methionine aqueous solution at 1.0 g / min, increase the stirring speed by 50 r / min, and disperse at 30℃ for 40 min to obtain a dispersion.

[0066] Add 0.8% titanate coupling agent (311W) of natural diatomaceous earth by weight to the dispersion at a rate of 2 mL / min. After the addition is complete, heat to 65℃ at 5℃ / min and react at a constant temperature of 200 r / min for 2 h. Cool to room temperature, filter and collect the filter cake. Wash the filter cake with distilled water, using 1.5 times the weight of natural diatomaceous earth each time. Stir and wash for 15 min, then filter under vacuum. Repeat washing until the pH of the washing solution is 6.5-7.5. Dry the washed filter cake at 65℃ and 0.08 MPa vacuum for 6 h, then pulverize and pass through a 200-mesh sieve to obtain modified diatomaceous earth.

[0067] S6: Add 30 times the volume of pure water to the crude polysaccharide extract and stir. Adjust the pH to 6.0-6.5. Add papain at 2% of the mass of the crude polysaccharide extract. Enzymatically hydrolyze at 55℃ for 16h. Heat to 82℃ at 4℃ / min and keep warm for 20min. After naturally cooling to room temperature, add 1 / 4 volume of the composite solvent (composed of chloroform and n-butanol in a volume ratio of 6:1). Shake for 20min. Centrifuge at 6000r / min for 10min. Collect the upper aqueous phase. Repeat 4 times and combine the aqueous phases.

[0068] S7: Add an equal volume of petroleum ether to the upper aqueous phase, mix well for 15 min, centrifuge at 5000 r / min for 8 min, collect the lower aqueous phase, add 18% of the volume of the lower aqueous phase to the composite adsorbent (composed of modified diatomaceous earth and macroporous resin AB-8 with a mass ratio of 2.4:1), shake and adsorb at 30℃ for 12 h, filter and collect the filtrate, add the filtrate to a dialysis bag (3000 Da), dialyze in pure water for 60 h, changing the pure water every 12 h during the period, vacuum rotary evaporation to concentrate to 1 / 4 of the original volume, add 4 times the volume of anhydrous ethanol for 8 h, centrifuge to collect the precipitate, dry at 60℃ to obtain the refined crude polysaccharide;

[0069] S8: Dissolve the refined crude polysaccharide in pure water to prepare a 30 mg / mL polysaccharide stock solution. Centrifuge at 10000 r / min for 10 min, collect the supernatant, and use a Sephacryl S-400HR separation column (2.6 cm in diameter and 100 cm in height). Equilibrate the column with pure water until the pH of the effluent is 7.0 ± 0.2. Load the supernatant at a flow rate of 3 mL / min and elute with pure water at a rate of 3 column volumes to obtain the pre-chromatographic solution. Determine the total sugar content by the sulfuric acid-phenol method and combine the eluents of the main polysaccharide peak.

[0070] S9: Use a DEAE Seplife FF exchange column (2.6 cm in diameter and 40 cm in height). Equilibrate with pure water until the pH of the effluent is 7.0 ± 0.2. Load the pre-chromatographic solution at a flow rate of 2 mL / min. First, elute with 3 column volumes of pure water, and then elute sequentially with 0.1 M, 0.2 M, 0.3 M, and 0.4 M NaCl solutions at a flow rate of 4 mL / min.

[0071] S10: Collect all eluents, combine the eluents corresponding to the main peak, and concentrate them to 1 / 4 of the original volume by vacuum rotary evaporation at 55℃ to obtain concentrate B. Add concentrate B to a dialysis bag (3000 Da) and dialyze in pure water for 48 hours to remove salt (NaCl solution eluted in S10). Change the pure water every 10 hours during this period to obtain dialysis concentrate. Freeze-dry at -30℃ for 36 hours to obtain golden flower Tibetan tea polysaccharide.

[0072] Example 3: This example provides a method for isolating and purifying polysaccharides from Tibetan tea, including the following steps:

[0073] S1: Grind the golden flower Tibetan tea into powder, pass it through a 50-mesh sieve to obtain golden flower Tibetan tea powder, weigh 1850g of golden flower Tibetan tea powder, add 6 times the volume of anhydrous ethanol to the golden flower Tibetan tea powder, stir and extract at 28℃ for 2h, centrifuge at 5000r / min for 8min, and collect the precipitate.

[0074] S2: Mix pectinase, cellulase and papain in a mass ratio of 1.9:1.7:1.1 to obtain a compound enzyme. Add 23 times the volume of pure water to the precipitate of S1, adjust the pH to 5.0-5.5, add the compound enzyme at 2.2% of the precipitate mass, and enzymatically hydrolyze at 48℃ for 3 hours to obtain the enzymatic hydrolysate.

[0075] S3: Extract the enzymatic hydrolysate in a 57℃ water bath for 4 hours, sonicating at 120W for 20 minutes every 12 minutes and stirring once every 30 minutes. After extraction, centrifuge at 5400r / min for 10 minutes, collect the supernatant extract, and repeat the above steps (S2-S3) to extract the precipitate residue again. Combine the two extracts.

[0076] S4: The supernatant extract was concentrated to 1 / 10 of its original volume by vacuum rotary evaporation at 52℃ and 0.06MPa. After cooling to room temperature, concentrated solution A was obtained. Anhydrous ethanol was added at a rate of 1mL / min at a volume of 3 times that of concentrated solution A. The mixture was precipitated at 7℃ for 10h and centrifuged at 8000r / min for 10min. The precipitated solid was collected to obtain the crude polysaccharide extract.

[0077] S5: Take natural diatomaceous earth, add 3 times its volume of distilled water, stir at 28℃ for 29 min, and ball mill at 320 r / min for 2 h to obtain diatomaceous earth particles. Take 3 times the mass of natural diatomaceous earth of distilled water, add 1.3% of the diatomaceous earth mass of methionine at 180 r / min at a rate of 2 mL / min, and continue stirring for 19 min to obtain a methionine aqueous solution. Add diatomaceous earth particles to the methionine aqueous solution at 0.7 g / min, increase the stirring rate to 40 r / min, and disperse at 26℃ for 35 min to obtain a dispersion.

[0078] Add 0.6% titanate coupling agent (311W) of natural diatomaceous earth by weight to the dispersion at a rate of 1 mL / min. After the addition is complete, raise the temperature to 62℃ at 4℃ / min and react at a constant temperature of 170 r / min for 1.5 h. Cool to room temperature, filter and collect the filter cake. Wash the filter cake with distilled water, using 1.3 times the weight of natural diatomaceous earth each time. Stir and wash for 12 min, then filter under vacuum. Repeat washing until the pH of the washing solution is 6.5-7.5. Dry the washed filter cake at 62℃ and 0.08 MPa vacuum for 5 h, then pulverize and pass through a 200-mesh sieve to obtain modified diatomaceous earth.

[0079] S6: Add 23 times the volume of pure water to the crude polysaccharide extract and stir. Adjust the pH to 6.0-6.5. Add papain at 1.7% of the mass of the crude polysaccharide extract. Incubate at 52℃ for 14 h. Increase the temperature to 79℃ at 3℃ / min and keep warm for 18 min. After naturally cooling to room temperature, add 1 / 4 volume of the composite solvent (composed of chloroform and n-butanol in a volume ratio of 5:1). Shake for 17 min. Centrifuge at 5200 r / min for 9 min. Collect the upper aqueous phase. Repeat 4 times and combine the aqueous phases.

[0080] S7: Add an equal volume of petroleum ether to the upper aqueous phase, mix well for 12 min, centrifuge at 4400 r / min for 6 min, collect the lower aqueous phase, add 16% of the volume of the lower aqueous phase with composite adsorbent (composed of modified diatomaceous earth and macroporous resin AB-8 with a mass ratio of 2.1:1), shake and adsorb at 26℃ for 11 h, filter and collect the filtrate, add the filtrate to a dialysis bag (3000 Da), dialyze in pure water for 50 h, changing the pure water every 10 h during the period, vacuum rotary evaporation to concentrate to 1 / 5 of the original volume, add 3 times the volume of anhydrous ethanol for 8 h, centrifuge to collect the precipitate, dry at 56℃ to obtain purified crude polysaccharide;

[0081] S8: Dissolve the refined crude polysaccharide in pure water to prepare a polysaccharide stock solution of 24 mg / mL. Centrifuge at 10000 r / min for 8 min, take the supernatant, and use a Sephacryl S-400HR separation column (column diameter 2.6 cm, column height 100 cm). Equilibrate the column with pure water until the pH of the eluent is 7.0 ± 0.2. Load the supernatant at a flow rate of 2 mL / min and elute with pure water for 3 column volumes to obtain the pre-chromatographic solution. Determine the total sugar content by the sulfuric acid-phenol method and combine the eluents of the main polysaccharide peak.

[0082] S9: Use a DEAE Seplife FF exchange column (2.6 cm in diameter and 40 cm in height). Equilibrate with pure water until the pH of the effluent is 7.0 ± 0.2. Load the pre-chromatographic solution at a flow rate of 1-2 mL / min. First, elute with 2 column volumes of pure water, and then elute sequentially with 0.1 M, 0.2 M, 0.3 M, and 0.4 M NaCl solutions at a flow rate of 4 mL / min.

[0083] S10: Collect all eluents, combine the eluents corresponding to the main peak, and concentrate them to 1 / 5 of the original volume by vacuum rotary evaporation at 51℃ to obtain concentrate B. Add concentrate B to a dialysis bag (3000 Da) and dialyze in pure water for 42 h to remove salt (NaCl solution eluted in S10). Change the pure water every 9 h during this period to obtain dialysis concentrate. Freeze-dry at -38℃ for 36 h to obtain golden flower Tibetan tea polysaccharide.

[0084] Comparative Example 1: This comparative example differs from Example 3 in that, in S2, the complex enzyme is replaced with pectinase, cellulase and papain in a mass ratio of 0.5:3.4:2.2.

[0085] Comparative Example 2: This comparative example differs from Example 3 in that the natural diatomaceous earth was not modified, and an equal mass of natural diatomaceous earth was directly selected.

[0086] Comparative Example 3: This comparative example differs from Example 3 in that pre-chromatography was not performed in S8, and the supernatant was directly loaded onto the DEAE seplife FF exchange column.

[0087] Experimental example: Detection of hypoglycemic effect;

[0088] 1. 140 type 2 diabetic rats were randomly divided into 7 groups: DC group (diabetic model group), treatment group (Examples 1-3, Comparative Examples 1-3); and 20 normal rats were used as NC group.

[0089] The treatment group was given 200 mg / kg of Tibetan tea polysaccharide daily by gavage, while the DC and NC groups were given an equal volume of physiological saline daily by gavage. The administration was continued for 6 weeks, and fasting blood glucose was measured once a week during the administration period (results are shown in Table 1). At the end of the 6th week, after the mice were fasted for 12 hours, blood was collected from the orbital cavity, the serum was separated by centrifugation, and stored at -80℃ for later use. The mice were then sacrificed, and the adipose tissue around the liver, kidneys, spleen, and epididymis was dissected, weighed, and the organ index was calculated. The liver tissue was taken to prepare homogenate for the detection of antioxidant indicators.

[0090] 2. Fasting blood glucose was measured weekly. In week 6, glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed, and the area under the curve was calculated. Serum insulin (INS) levels were measured using an ELISA kit, and the insulin resistance index (HOMA-IR) and insulin sensitivity index (HOMA-IS) were calculated.

[0091] 3. Calculate the liver index, kidney index, spleen index, epididymal fat index, and Lee's index:

[0092] Organ Index (%) = (Wet Organ Weight ÷ Fasting Body Weight) × 100%

[0093] Calculate the liver index, kidney index, spleen index, and epididymal fat index using the formulas described above.

[0094] Lee's Index = (Fasting weight) 0.33 (÷body length) × 1000

[0095] Body length is the straight-line distance from the tip of the rat's nose to its anus (in cm), measured with calipers, and fasting weight (g).

[0096] 4. The SOD activity, CAT activity and MDA content in serum were detected using the Abbkine SOD Activity Assay Kit (catalog number KTB1030), the Abbkine CAT Activity Assay Kit (catalog number KTB9040), and the CheKine™ Lipid Peroxidation (Malondialdehyde) Content Detection Kit (catalog number KTB1050), respectively.

[0097] 5. The serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using the triglyceride (TG) assay kit (catalog number A110-1-1), total cholesterol (TCH / T-CHO) assay kit (catalog number A111-1-1), high-density lipoprotein cholesterol (HDL-C) assay kit (catalog number A112-1-1), and low-density lipoprotein cholesterol (LDL-C) assay kit (catalog number A113-1-1) manufactured by Nanjing Jiancheng Biotechnology Research Institute.

[0098] 6. The levels of IL-4, IL-6, and TNF-α in serum were detected using an ELISA kit.

[0099] 7. The activities of AST and ALT in serum were detected using the Aspartate Aminotransferase (AST / GOT) Test Kit (Reiter method) microplate method (Catalog No. C010-2-1) and Alanine Aminotransferase (ALT / GPT) Test Kit (Reiter method) microplate method (Catalog No. C009-2-1) manufactured by Nanjing Jiancheng Biotechnology Research Institute.

[0100] 8. Gas chromatography was used to detect the content of short-chain fatty acids (isovaleric acid, propionic acid, valeric acid, butyric acid, hexanoic acid, acetic acid, isobutyric acid, and 2-methylbutyric acid) in intestinal contents.

[0101] Table 1:

[0102]

[0103] As shown in the table above, the golden flower Tibetan tea polysaccharide prepared in this invention has a significant hypoglycemic effect on type 2 diabetic rats. Before administration, there was no significant difference in fasting blood glucose levels between the DC group and each treatment group, all of which were significantly higher than those in the NC group. After one week of continuous administration, the blood glucose levels in the Examples 1-3 groups showed a significant decrease, and the decrease was significantly greater than that in the Comparative Examples 1-3 groups. As the administration period was extended to 6 weeks, the blood glucose levels in the Examples 1-3 groups steadily decreased to 10.1-11.1 mmol / L, which was significantly lower than that in the DC group, while the blood glucose levels in the Comparative Examples 1-3 groups were 12.1-12.9 mmol / L, showing better results than the Comparative Examples.

[0104] Table 2:

[0105]

[0106] As shown in the table above, the serum insulin content and insulin resistance index of rats in the DC group were significantly higher than those in the NC group, while the insulin sensitivity index was significantly lower, indicating that the model rats had significant insulin resistance. After treatment with golden chrysanthemum tea polysaccharide, the serum insulin content of rats in Examples 1-3 decreased to 4.4-4.6 mIU / L, the insulin resistance index decreased to 1.71-1.75, and the insulin sensitivity index increased to 0.026-0.028. All indicators were significantly better than those in the control groups 1-3.

[0107] Table 3:

[0108]

[0109] As shown in the table above, the liver index, kidney index, spleen index, and Lee's index of rats in the DC group were significantly higher than those in the NC group, indicating that diabetes caused compensatory organ hyperplasia and abnormal body shape. After treatment with golden chrysanthemum tea polysaccharide, the liver index (3.96-4.00%), kidney index (1.00-1.02%), spleen index (0.26-0.28%), and Lee's index (319.9-320.2) of groups 1-3 were all regulated to near the normal range of the NC group, and were significantly better than those of the control groups 1-3.

[0110] Table 4:

[0111]

[0112] As shown in the table above, the serum CAT and SOD activities of rats in the DC group were significantly lower than those in the NC group, while the MDA content was significantly higher, indicating significant oxidative stress damage. After treatment with *Tea japonica* polysaccharide in Examples 1-3, the CAT activity of the groups increased to 169.7-170.3 μmol / min / mL, the SOD activity increased to 93.9-94.3 U / mL, and the MDA content decreased to 22.9-23.2 nmol / mL. All these antioxidant indicators were significantly better than those of the control groups 1-3.

[0113] Table 5:

[0114]

[0115] As shown in the table above, the serum TC, TG, and LDL-C levels in the DC group were significantly higher than those in the NC group, while the HDL-C level was significantly lower, indicating a clear lipid metabolism disorder. After treatment with *Tea japonica* polysaccharide in Examples 1-3, the TC level decreased to 16.72-16.94 mmol / L, the TG level decreased to 2.43-2.61 mmol / L, the LDL-C level decreased to 1.59-1.62 mmol / L, and the HDL-C level increased to 1.03-1.21 mmol / L, demonstrating a significantly better lipid regulation effect than the control groups 1-3.

[0116] Table 6:

[0117]

[0118] As shown in the table above, the serum levels of the anti-inflammatory factor IL-4 in the DC group were significantly lower than those in the NC group, while the levels of the pro-inflammatory factors IL-6 and TNF-α were significantly higher, indicating a significant chronic inflammatory response. After treatment with *Tea japonica* polysaccharide in Examples 1-3, the IL-4 levels increased to 22.98-23.12 pg / mL, the IL-6 levels decreased to 14.18-14.32 pg / mL, and the TNF-α levels decreased to 77.95-78.12 pg / mL, demonstrating a significantly better effect on the balance of inflammatory factors than the control groups 1-3.

[0119] Table 7:

[0120]

[0121] As shown in the table above, the serum ALT and AST activities of rats in the DC group were significantly higher than those in the NC group, indicating impaired liver function. After treatment with golden chrysanthemum tea polysaccharide, the ALT activity in groups 1-3 decreased to 42.98-43.12 nmol / min / mL, and the AST activity decreased to 38.97-39.23 nmol / min / mL. Both indicators were significantly better than those in the control groups 1-3 and close to the normal levels of the NC group.

[0122] Table 8:

[0123]

[0124] As shown in the table above, the contents of most short-chain fatty acids, such as isovaleric acid, propionic acid, butyric acid, and acetic acid, in the intestinal contents of rats in the DC group were significantly lower than those in the NC group, indicating intestinal microecological metabolic disorder. After treatment with *Tea japonica* polysaccharide in Examples 1-3, the contents of various short-chain fatty acids were significantly increased, with acetic acid reaching 798.76-799.23 μg / g, propionic acid reaching 102.59-103.12 μg / g, and butyric acid reaching 61.89-62.08 μg / g. The improvement effects were significantly better than those in Comparative Examples 1-3.

[0125] Characterization example (taking Example 3 as an example);

[0126] 1. Scanning electron microscope;

[0127] Equipment Model: ZEISS Sigma 360; Magnification: 1.00KX; Working Distance: 7.9mm; Accelerating Voltage: 3.00kV; Probe Current: 0.1pA; Signal Mode: SE2 (Secondary Electron Imaging); System Vacuum: 1.87e-06mbar.

[0128] Depend on Figure 1It can be seen that under 1000x magnification (scale bar 20μm), the sample exhibits sheet-like and fragmented structural features. The main body is an irregular sheet shape, with some areas having relatively flat surfaces, but the overall sample has a large number of broken and curled edges. A small amount of fine granular material is attached to the surface, and local pores and depressions are also visible.

[0129] 2. Infrared scanning;

[0130] 5 mg of polysaccharide sample was weighed and mixed with 200 mg of potassium bromide. The mixture was then pressed into 1 mm thick sheets and analyzed using a Nicolet iZ-10 Fourier transform infrared spectrometer with a scanning range of 4000-4000 cm⁻¹. -1 The number of scans was 32, and the resolution was 4.00 cm. -1 ;

[0131] The results are as follows Figure 2 As shown, 3319.1cm -1 This is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. It is located at 2927.16 cm⁻¹. -1 The absorption peak at 1022.33 cm⁻¹ is attributed to the CH stretching vibration. -1 There is an absorption peak at 891 cm⁻¹, which is attributed to the stretching vibration of CO. -1 The nearby signal peaks indicate that it is linked by β-type glycosidic bonds.

[0132] 3. Ultraviolet scanning;

[0133] The polysaccharide sample of Tibetan tea was dissolved in pure water to prepare a 5 mg / mL polysaccharide solution. The polysaccharide solution was scanned and analyzed using a Thermo Fisher Scientific (USA) multi-functional microplate reader. The scanning range was 200 nm starting wavelength, 1000 nm ending wavelength, and 1 nm scanning interval.

[0134] The results are as follows Figure 3 As shown, the blue peak line represents the blank control group (deionized water), and the red peak line represents the polysaccharide solution sample. Compared with the blank control, the polysaccharide solution sample showed no significant absorption in the wavelength range of 200-400nm, indicating that the polysaccharide solution sample contained almost no impurities such as pigments, proteins, and nucleic acids.

[0135] 4. XRD scan;

[0136] The purified polysaccharide sample was dried, pulverized, and passed through a 100-mesh sieve. 20 mg of the sample was weighed onto the stage, pressed and spread evenly, and then tested on the instrument. The power was 1600 W (40 kV × 40 mA), the X-ray source was a copper target Cu-Kα, the wavelength was λ = 0.15406 nm, the scanning range was 5-60° (2θ angle), the step size was 0.02°, and the scanning speed was 4° / min.

[0137] The results are as follows Figure 7 As shown, the figure shows no sharp characteristic diffraction peaks, but rather broadened diffuse peaks, indicating that the sample has an amorphous (non-crystalline) structure and lacks the regular lattice arrangement characteristic of crystals. The absence of characteristic diffraction peaks corresponding to other impurities further confirms the good purification effect of the sample and the absence of residual crystalline impurities.

[0138] 5. Molecular weight analysis;

[0139] The sample was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL. After filtration through a 0.45 μm filter, the sample was analyzed using a gel chromatography-differential-multi-angle laser light scattering system. Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) gel size exclusion columns were connected in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min. The chromatographic data were processed using ASTRA 6.1 software.

[0140] Plot an absolute molecular weight analysis graph with retention time on the x-axis and molar mass on the y-axis (e.g., Figure 5 As shown), a molecular configuration diagram is drawn with molar mass as the x-axis and root mean square radius as the y-axis (e.g.) Figure 4 As shown), a elution curve of gel chromatography-laser light scattering (LS)-differential resonance (RI) was plotted with elution time as the x-axis and signal peak intensity as the y-axis (e.g., Figure 6 (as shown)

[0141] Figure 6 As shown in the figure, two distinct signal peaks (red LS signal and blue RI signal appear simultaneously) are observed, indicating that there are at least two polysaccharide components with different molecular weights in the sample; and the peaks are relatively concentrated and without tails, indicating that the molecular weight distribution of each component is relatively narrow. The results are shown in Table 9.

[0142] Table 9:

[0143]

[0144] The mass fractions in the table above correspond to the proportion of polysaccharide components in the total mass of the sample; the peak range refers to the elution time range of each component peak in the gel chromatogram.

[0145] 6. Monosaccharide composition analysis;

[0146] Weigh 5 mg of polysaccharide sample, add 1 mL of 2 M TFA acid solution, heat at 121 °C for 2 h, purge with nitrogen, dry, add 99.99% methanol to wash, dry again, repeat methanol washing 3 times, add sterile water to dissolve, transfer to chromatographic bottle for analysis.

[0147] The chromatographic system used was a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA). Monosaccharide components were analyzed and detected using an electrochemical detector, employing a Dionex analyzer. TM CarboPac TM PA20 (150*3.0mm, 10μm) HPLC column; injection volume: 5μL; mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc); flow rate: 0.5mL / min; column temperature: 30℃; elution gradient: 0 min A / B / C (95:5:0, V / V), 26 min A / B / C (85:5:10, V / V), 42 min A / B / C (85:5:10, V / V), 42.1 min A / B / C (60:0:40, V / V), 52 min A / B / C (60:40:0, V / V), 52.1 min A / B / C (95:5:0, V / V), 60 min Phase A / Phase B / Phase C (95:5:0, V / V).

[0148] The results are as follows Figure 8 As shown in Table 10, the peak heights of Gal (galactose) and Glc (glucose) are significantly higher, indicating that these two monosaccharides are the main monosaccharide components in the sample; the peak heights of Ara (arabinose) and Xylan (xylose) are lower, belonging to minor monosaccharide components.

[0149] Table 10:

[0150]

[0151] 7. Methylation gas chromatography-mass spectrometry analysis;

[0152] Dissolve 3 mg in 500 μL DMSO, add 1 mg NaOH, incubate for 30 min, add 50 μL iodomethane solution and react for 1 h, add 1 mL water and 2 mL dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the washing with water 3 times.

[0153] The lower dichloromethane phase was removed and dried under nitrogen. 100 μL of 2M TFA was added, and the mixture was reacted at 121 °C for 90 min. The mixture was then evaporated to dryness at 30 °C. 50 μL of 2M ammonia and 50 μL of 1M NaBD4 were added, mixed well, and reacted at room temperature for 2.5 h.

[0154] The reaction was terminated by adding 20 μL of acetic acid, dried under nitrogen, washed twice with 250 μL of methanol, dried under nitrogen, and then 250 μL of acetic anhydride was added. The mixture was vortexed and reacted at 100 °C for 2.5 h.

[0155] Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, wash with water 3 times, take the lower dichloromethane phase, and analyze it by GC-MS.

[0156] The chromatographic system used was an Agilent gas chromatograph (Agilent 6890A; Agilent Technologies, USA), with a BPX70 column (30m × 0.25mm × 0.25μm, SGE, Australia). The injection volume was 1μL, the split ratio was 10:1, the carrier gas was high-purity helium, and the flow rate was 1.5mL / min. The column oven was initially set at 140℃ and held for 2.0min, then programmed to reach 230℃ at a rate of 3℃ / min and held for 3min. The mass spectrometry system used was an Agilent quadrupole mass spectrometer (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The analyte was detected using the EI source in full scan (SCAN) mode, with a mass scan range (m / z) of 50–350.

[0157] The results are as follows Figure 9 As shown in Table 11;

[0158] Table 11:

[0159]

[0160] 8. Nuclear magnetic resonance analysis;

[0161] The purified polysaccharide was fully dissolved in D2O to prepare a polysaccharide solution with a concentration of 40 mg / mL. The dissolved solution was transferred to an NMR tube with an addition volume of 0.5 mL. Quantitative analysis of the target analyte was performed using a Bruker (Germany) 500 MHz NMR spectrometer with a scanning temperature of 25 °C. The liquid probe used was a QXI 1H / 31P / 13C / 15N 5 mm quad-resonance reverse detection probe (Z-gradient, ATM Acc) with the following parameters: signal-to-noise ratio (1H) of 888 and resolution (Hz) of 0.32 (rotating). The BBFO 1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradient, ATM) had the following technical parameters: signal-to-noise ratio (1H) of 798, resolution (Hz) of 0.26 (rotating), signal-to-noise ratio (13C) of 328, and resolution (Hz) of 0.1. The NMR tube is placed in the NMR spectrometer to scan one dimension. 1 H spectrum, 13 C spectrum, DEPT-13, two-dimensional COSY, HSQC, HMBC, NOESY and TOCSY spectra.

[0162] The results are as follows Figure 10-17 As shown, the 1H NMR spectrum of the sample is mainly concentrated between δ 3.0 and 5.5 ppm. Multiple coupled signal peaks were identified in the 4.3-5.4 ppm anodic signal region, indicating the presence of various sugar residues in the sample. The corresponding chemical shifts of the anodic hydrogens are δ 4.46, 4.55, 4.57, 4.87, and 5.32, respectively. Non-anodic hydrogen signals are mainly concentrated in the 3.1-4.2 ppm region. Due to severe overlap, some signals require further analysis using COSY and TOCSY spectra to assign the H2-H6 chemical shifts of each sugar residue. The strong signal peak near δ 4.71 ppm is a solvent peak.

[0163] Multiple signal peaks were identified in the anodic carbon region of the sample. Combining the cross-peaks in the anodic region of the 13C NMR and HSQC spectra, the anodic signals present in the sample were determined to be δ 5.32 / 99.59, 4.55 / 104.34, 4.87 / 98.78, 4.46 / 102.53, and 4.57 / 95.75 ppm, respectively, and denoted as sugar residues A, B, C, D, and E. Combining DEPT-135 and 13C NMR spectra, the signals of methylene groups (δ 60.73, 59.32, 60.46, 69.11 ppm) were obtained. Based on the sample bonding structure (methylation) information and anodic signals, it is inferred that sugar residue A may be →4)-α-D-Glcp-(1→, sugar residue B may be →4)-β-D-Galp-(1→, sugar residue C may be α-D-Glcp-(1→, sugar residue D may be →4,6)-β-D-Galp-(1→, sugar residue E may be →4)-β-D-Manp, and their 1H and 13C chemical shifts are assigned.

[0164] The NMR signal assignment process for major sugar residues is as follows:

[0165] Sugar residue A: The anodic signal δ5.32 / 99.59ppm (H1 / C1) indicates that residue A may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.51ppm) of residue A was determined based on the cross-peak δ5.32 / 3.51ppm, H3 (3.87ppm) of residue A was determined based on the cross-peak δ3.51 / 3.87ppm, H4 (3.57ppm) of residue A was determined based on the cross-peak δ3.87 / 3.57ppm, H5 (3.71ppm) of residue A was determined based on the cross-peak δ3.57 / 3.71ppm, and H6 (3.73, 3.67ppm) of residue A was determined based on the cross-peak δ3.71 / 3.73, 3.67ppm. Thus, the chemical shifts of hydrogen atoms on the complete sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of C1 of residue A was δ99.59ppm, the chemical shift of C2 of residue A was δ71.36ppm, the chemical shift of C3 of residue A was δ73.3ppm, the chemical shift of C4 of residue A was δ76.78ppm, the chemical shift of C5 of residue A was δ71.17ppm, and the chemical shift of C6 of residue A was δ60.73ppm. The chemical shifts of C1 and C4 shifted to the lower field, indicating that the residues were substituted at the O-1 and O-4 positions of the sugar ring. Combined with the methylation analysis results, it was inferred that sugar residue A may be →4)-α-D-Glcp-(1→).

[0166] Sugar residue B: The anodic signal δ4.55 / 104.34ppm (H1 / C1) indicates that residue B may be a β-configuration galactose residue. In the COSY spectrum, H2 (3.18ppm) of residue B was determined based on the cross-peak δ4.55 / 3.18ppm, H3 (3.67ppm) of residue B was determined based on the cross-peak δ3.18 / 3.67ppm, H4 (4.09ppm) of residue B was determined based on the cross-peak δ3.67 / 4.09ppm, H5 (3.56ppm) of residue B was determined based on the cross-peak δ4.09 / 3.56ppm, and H6 (3.64ppm) of residue B was determined based on the cross-peak δ3.56 / 3.64ppm. Thus, the chemical shift of the hydrogen on the sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of C1 of residue B was δ104.34ppm, the chemical shift of C2 of residue B was δ73.84ppm, the chemical shift of C3 of residue B was δ73.3ppm, the chemical shift of C4 of residue B was δ77.64ppm, the chemical shift of C5 of residue B was δ74.50ppm, and the chemical shift of C6 of residue B was δ59.32ppm. Among them, the chemical shifts of C1 and C4 shifted to the lower field, indicating that the residues were substituted at the O-1 and O-4 positions of the sugar ring. Combined with the methylation analysis results, it was inferred that sugar residue B may be →4)-β-D-Galp-(1→).

[0167] Sugar residue C: The anodic signal δ4.87 / 98.78ppm (H1 / C1) indicates that residue C may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.47ppm) of residue C was determined based on the cross-peak δ4.87 / 3.47ppm, H3 (3.59ppm) of residue C was determined based on the cross-peak δ3.47 / 3.59ppm, H4 (3.79ppm) of residue C was determined based on the cross-peak δ3.59 / 3.79ppm, H5 (3.93ppm) of residue C was determined based on the cross-peak δ3.79 / 3.93ppm, and H6 (3.56ppm) of residue C was determined based on the cross-peak δ3.93 / 3.56ppm. Thus, the chemical shift of the hydrogen on the complete sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of C1 was δ98.78ppm, C2 was δ74.5ppm, C3 was δ72.07ppm, C4 was δ71.09ppm, C5 was δ73.12ppm, and C6 was δ60.46ppm. The chemical shift of C1 shifted to a lower field, indicating that the residue was substituted at the O-1 position of the sugar ring. Combined with the methylation analysis results, it was inferred that sugar residue C may be α-D-Glcp-(1→).

[0168] Sugar residue D: The anodic signal δ4.46 / 102.53ppm (H1 / C1) indicates that residue D may be a β-configuration galactose residue. In the COSY spectrum, H2 (3.27ppm) of residue D was determined based on the cross-peak δ4.46 / 3.27ppm, H3 (3.73ppm) of residue D was determined based on the cross-peak δ3.27 / 3.73ppm, H4 (3.86ppm) of residue D was determined based on the cross-peak δ3.73 / 3.86ppm, H5 (3.62ppm) of residue D was determined based on the cross-peak δ3.86 / 3.62ppm, and H6 (3.83ppm) of residue D was determined based on the cross-peak δ3.62 / 3.83ppm. Thus, the chemical shift of the hydrogen on the sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of C1 of residue D was δ102.53ppm, the chemical shift of C2 of residue D was δ72.94ppm, the chemical shift of C3 of residue D was δ73.12ppm, the chemical shift of C4 of residue D was δ76.57ppm, the chemical shift of C5 of residue D was δ74.17ppm, and the chemical shift of C6 of residue D was δ69.11ppm. The chemical shifts of C1, C4, and C6 shifted to the lower field, indicating that the residues were substituted at the O-1, O-4, and O-6 positions of the sugar ring. Combined with the methylation analysis results, it was inferred that sugar residue D may be →4,6)-β-D-Galp-(1→).

[0169] Sugar residue E: The anodic signal δ4.57 / 95.75ppm (H1 / C1) indicates that residue E may be a β-configuration mannose residue. The chemical shift of the carbon atom on the sugar ring was then assigned using the HSQC signal; the C1 chemical shift of residue E was δ95.75ppm. Due to the low content and weak signal of sugar residue E, complete assignment was difficult. Combined with methylation analysis results, it was inferred that sugar residue E may be →4)-β-D-Manp.

[0170] The assignment of all sugar residue shifts can be completed by following the steps above, and the results are shown in Table 12:

[0171] Table 12:

[0172]

[0173] nd is an abbreviation for not detected, meaning it was not detected.

[0174] Based on the chemical shifts of the 13C and 1H of each sugar residue in the sample, the structure and linkage mode of the polysaccharide were analyzed using HMBC and NOESY spectra. According to the HMBC spectrum, there is a cross-peak between H1 and C4 of sugar residue A (δ 5.32 / 76.78 ppm), a cross-peak between H1 and C4 of sugar residue A (δ 5.32 / 77.64 ppm), a cross-peak between C1 and H4 of sugar residue A (δ 99.59 / 3.57 ppm), a cross-peak between H1 and C4 of sugar residue B (δ 4.55 / 76.78 ppm), and a cross-peak between H1 and C4 of sugar residue B. There are cross-peaks at δ4.55 / 77.64ppm, H1 of sugar residue B and C4 of sugar residue D have cross-peaks at δ4.55 / 76.57ppm, C1 of sugar residue B and H4 of sugar residue A have cross-peaks at δ104.34 / 3.57ppm, C1 of sugar residue B and H4 of sugar residue B have cross-peaks at δ104.34 / 4.09ppm, and C1 of sugar residue D and H4 of sugar residue B have cross-peaks at δ102.53 / 4.09ppm. According to the NOESY spectrum, there is a cross peak between H1 and H4 of sugar residue A at δ5.32 / 3.57ppm, a cross peak between H1 and H4 of sugar residue B at δ4.55 / 3.57ppm, a cross peak between H1 and H4 of sugar residue B at δ4.55 / 4.09ppm, and a cross peak between H1 and H6 of sugar residue C at δ4.87 / 3.83ppm.

[0175] Therefore, based on the analysis of one-dimensional and two-dimensional NMR information and methylation results, it is inferred that the polysaccharide is mainly composed of interconnected main chains such as →4)-α-D-Glcp-(1→, →4)-β-D-Galp-(1→ and →4,6)-β-D-Galp-(1→), and the side chains are mainly composed of α-D-Glcp-(1→ connected to the O-6 positions of the sugar residue →4,6)-β-D-Galp-(1→).

[0176] In summary, the structural formula of the *Golden Flower Tibetan Tea* polysaccharide obtained by this invention is as follows:

[0177]

[0178] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for isolating and purifying polysaccharides from Tibetan tea, characterized in that, Includes the following steps: S1: Grind and sieve the golden flower Tibetan tea to obtain golden flower Tibetan tea powder, add ethanol, extract and centrifuge, and collect the precipitate; S2: Add water to the precipitate of S1, adjust the pH to 5.0-5.5, add a compound enzyme, and enzymatically hydrolyze to obtain an enzymatic hydrolysate. The compound enzyme is pectinase, cellulase and papain in a mass ratio of 1.5-2.3:1.4-2.1:0.8-1.

2. S3: Extract the enzymatic hydrolysate in a 55℃ water bath for 3 hours, sonicating once every 10 minutes and stirring once every 20 minutes. After extraction, centrifuge to collect the supernatant extract, and repeat the above steps to extract the precipitate residue. Combine the two extracts. S4: Vacuum concentrate the supernatant extract, cool to obtain concentrate A, add anhydrous ethanol, precipitate at 4℃ for 8 hours, centrifuge, collect the precipitate solid, and obtain crude polysaccharide extract. S5: Add water to the crude polysaccharide extract and stir. Adjust the pH to 6.0-6.

5. Add papain at 1% of the mass of the crude polysaccharide extract for enzymatic hydrolysis. Heat to 78-82℃ and keep warm. After cooling, add the composite solvent, shake and centrifuge, collect the upper aqueous phase, repeat 3 times, and combine the aqueous phases. The composite solvent is composed of chloroform and n-butanol in a volume ratio of 4-6:

1. S6: Add an equal volume of petroleum ether to the upper aqueous phase, mix well for 10-15 min, centrifuge at 4000-5000 r / min for 5-8 min, collect the lower aqueous phase, add 12-18% of the volume of the lower aqueous phase to the composite adsorbent, and adsorb by shaking at 25-30℃ for 8-12 h. Filter and collect the filtrate, add the filtrate to a dialysis bag, and dialyze in pure water for 48-60 h, changing the pure water every 10-12 h during this period. Vacuum rotary evaporation concentrates the filtrate to 1 / 5-1 / 4 of the original volume, adds 3-4 times the volume of anhydrous ethanol for 6-8 h, centrifuges to collect the precipitate, and dries it at 50-60℃ to obtain the refined crude polysaccharide. The composite adsorbent is composed of modified diatomaceous earth and macroporous resin AB-8 in a mass ratio of 1.8-2.4:

1. S7: Dissolve the refined crude polysaccharide in pure water to prepare a polysaccharide stock solution of 15-30 mg / mL. Centrifuge at 9000-10000 r / min for 8-10 min, take the supernatant, and equilibrate the Sephacryl S-400HR separation column with pure water until the pH of the effluent is 7.0±0.

2. Load the supernatant at a flow rate of 2-3 mL / min and elute with pure water for 2-3 column volumes to obtain the pre-chromatographic solution. Combine the eluents of the main polysaccharide peak. S8: Equilibrate the DEAE seplife FF exchange column with pure water until the effluent pH is 7.0±0.

2. Load the pre-chromatographic solution at a flow rate of 1-2 mL / min. First, elute with 2-3 column volumes of pure water, then elute sequentially with a gradient of 0.05-0.1M, 0.1-0.2M, 0.2-0.3M, and 0.3-0.4M NaCl solutions, maintaining a flow rate of 3-4 mL / min. S9: Collect all eluents, combine the eluents corresponding to the main peaks, concentrate under vacuum to obtain concentrate B, dialyze concentrate B in pure water to obtain dialysis concentrate, freeze dry to obtain golden flower Tibetan tea polysaccharide. The modified diatomaceous earth is prepared as follows: Take natural diatomaceous earth, add 2-3 times its volume of distilled water, stir at 25-30℃ for 20-30 min, ball mill at 300-400 r / min for 1.5-2 h to obtain diatomaceous earth particles, measure 2-3 times the mass of natural diatomaceous earth with distilled water, add 1-1.5% of methionine (by mass of diatomaceous earth) at 150-200 r / min at a rate of 1-2 mL / min, continue stirring for 15-20 min to obtain an aqueous solution of methionine, add diatomaceous earth particles at 0.5-1.0 g / min to the aqueous solution of methionine, increase the stirring rate by 20-50 r / min, disperse at 25-30℃ for 30-40 min to obtain a dispersion; Add 0.6-0.8% titanate coupling agent (by weight of natural diatomaceous earth) to the dispersion at a rate of 1-2 mL / min. After the addition is complete, raise the temperature to 60-65℃ at 2-5℃ / min and react at a constant temperature of 150-200 r / min for 1.5-2 h. Cool to room temperature, filter and collect the filter cake. Wash the filter cake with distilled water, using 1-1.5 times the weight of natural diatomaceous earth each time. Stir and wash for 10-15 min, then filter under vacuum. Repeat washing until the pH of the washing solution is 6.5-7.

5. Dry the washed filter cake at 60-65℃ and a vacuum of 0.06-0.08 MPa for 4-6 h, then pulverize and pass through a 200-mesh sieve to obtain modified diatomaceous earth.

2. A polysaccharide from Tibetan tea plant *Tea lycoperdon* prepared by the separation and purification method according to claim 1.

3. The use of the golden flower Tibetan tea polysaccharide as described in claim 2 in the preparation of a drug for treating type 2 diabetes.

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