Extraction and purification method of green tea cell wall polysaccharide with blood sugar lowering activity and green tea cell wall polysaccharide obtained

Abstract

The present application relates to a kind of green tea cell wall polysaccharide with hypoglycemic activity extraction and purification method and the green tea cell wall polysaccharide obtained, and five different tea polysaccharides are extracted by sequential extraction method using water, CDTA, saturated sodium carbonate and 1M NaOH and 4M NaOH solution, and the best hypoglycemic activity of green tea cell wall polysaccharide is screened by sugar metabolism enzyme activity inhibition, and further purified by gel column chromatography.Compared with prior art, the whole operation process of the present application has relatively mild reaction condition, can greatly retain the activity of green tea cell wall polysaccharide and reduce the loss of polysaccharide component to the maximum, has the advantages of high extraction rate, low energy consumption, small solvent consumption, simple operation and low economic cost, etc., lays a foundation for the study of the physicochemical properties and biological activity of green tea cell wall polysaccharide, and has important significance for the utilization of green tea cell wall polysaccharide in food, medical care and cosmetics and other fields.

Description

Technical Field

[0001] This invention relates to the field of tea polysaccharide extraction technology, and in particular to a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity, and the obtained green tea cell wall polysaccharides. Background Technology

[0002] Plant cell walls are primarily composed of cellulose, hemicellulose, and pectin, along with proteins and lignin bound to the cell wall. The components of the cell wall are cross-linked by ions and covalent bonds to form a robust cell wall that resists physical penetration. Cellulose is the main load-bearing component of the cell wall, consisting of 30 to 36 glucose chains linked by β-1,4 hydrogen bonds, forming an insoluble and inelastic crystalline material. Cellulose is interconnected with high-molecular-weight hemicellulose molecules (usually xylo-glucan or arabinoxylan), which are linked to the surface of cellulose microfibrils and embedded in the pectin matrix via hydrogen bonds. Pectin is the collective term for the galacturonic acid-rich portion of the cell wall. Pectin is typically rhamnogalacturonic acid I, composed of alternating rhamnosine and galacturonic acid backbones and various side groups, primarily galactose and arabinose. Pectin interacts with or binds to (hemi)cellulose via hydrogen bonds or covalent bonds. Single extraction methods cannot be used to study the polysaccharides of various components in the cell wall; therefore, sequential extraction is a suitable alternative method for studying the actual state of plant cell wall polysaccharides and intracellular polysaccharides. Sequential extraction involves using different chemical reagents stepwise to extract various components from the cell wall. Currently, sequential extraction is widely used in the study of cell wall polysaccharides from different plant materials. Sequential extraction typically uses several chemical reagents, such as water, chelating agents, acids, sodium carbonate, and alkalis. Chelating agents (CDTA, EDTA, etc.) can disrupt the calcium bridges in the cell wall that are cross-linked with pectin polysaccharides, releasing the pectin polysaccharides. Sodium carbonate and dilute alkalis can be used to extract pectin polysaccharides that are loosely or tightly bound to hemicellulose or cellulose via covalent linkages. The soluble portion of concentrated alkali is rich in xylose and arabinose side chains. The analytical results of cell wall polysaccharide components obtained by sequential extraction are of great significance for understanding the structure and function of plant cell walls. These studies help reveal the differences and interactions of cell wall polysaccharides in different plant materials, providing important evidence for the effective utilization and application of plant resources.

[0003] Chemical characterization of tea cell wall polysaccharides includes monosaccharide composition, molecular weight (Mw), monosaccharide sequence, glycosidic bond position, degree of branching, configuration, and overall molecular conformation. To date, more than 120 tea polysaccharides have been extracted and isolated from various types of tea. Tea polysaccharides are mainly composed of 2-10 monosaccharides, including glucose, rhamnose, arabinose, mannose, ribose, xylose, galactose, fucose, galacturonic acid, and glucuronic acid, with average Mw ranging from 1.02 to 4940 kDa. Due to the influence of tea species, tea processing techniques, and polysaccharide isolation methods, the structure of tea polysaccharides is extremely complex. Previous structural studies of tea polysaccharides have mainly focused on water-soluble polysaccharides, rather than cell wall polysaccharides; further research and refinement in this area are urgently needed. CN104059160A discloses a Hericium erinaceus cell wall polysaccharide and its preparation method. The Hericium erinaceus cell wall polysaccharide contains water-soluble and alkali-soluble cell wall polysaccharides, which are prepared by removing intracellular polysaccharides, pulverizing and extracting with water, alkali extraction with 0.1-1M NaOH solution and alcohol precipitation. However, this method cannot retain the activity of green tea cell wall polysaccharides to a large extent or minimize the loss of polysaccharide components, and cannot obtain cell wall polysaccharides with different chemical compositions and structural characteristics.

[0004] Tea polysaccharides (TPS) from tea have been widely used in traditional Chinese and Japanese medicine for the treatment of diabetes. Due to the increasing global incidence of diabetes, the hypoglycemic effect of TPS has attracted widespread attention. TPS exhibits a strong inhibitory effect on enzymes related to glucose metabolism in the human body, and its hypoglycemic activity is concentration-dependent, providing a reference for the rational dosage of this potential drug. These findings expand the potential applications of natural plant polysaccharides in the treatment of diabetes and provide important clues and research directions for the development of new diabetes treatments. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity, and the resulting green tea cell wall polysaccharides, in order to solve the above-mentioned problems. This method shortens the extraction cycle and reduces economic costs, obtaining green tea cell wall polysaccharides with high purity and strong activity. It can prepare tea cell wall polysaccharides with different structural characteristics and hypoglycemic activities, providing a reference for the application of green tea cell wall polysaccharides, expanding the deep processing technology of green tea, increasing the output value of green tea, and having important significance in the fields of food, health products, medicine and health, and cosmetics.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity. The method includes: sequentially extracting green tea with water, cyclohexanediaminetetraacetic acid (CDTA), sodium carbonate, and sodium hydroxide solutions of different concentrations, followed by impurity removal, dialysis, and freeze-drying to obtain cell wall polysaccharides with hypoglycemic activity.

[0008] Furthermore, the method includes the following steps:

[0009] (1) After grinding green tea leaves, pass them through a mesh sieve to obtain tea powder. Add ultrapure water and extract at 37 ℃ for 2 h. Then centrifuge to collect the supernatant. Repeat the above steps to extract the precipitate until no sugar content is detected in the supernatant. Combine the supernatants and dialyze. The supernatant is the extract. Keep the tea residue and dry it to obtain dried tea residue for subsequent steps. Add ethanol and let stand at 4 ℃ overnight. Then centrifuge to collect the precipitate. Redissolve the precipitate in water and centrifuge to collect the supernatant. Repeat 3 times. Combine the supernatants and freeze dry to obtain water-extracted tea polysaccharides (W-TPS).

[0010] (2) The dried tea residue obtained in step (1) was added to 70% ethanol, shaken on a shaker, and the solution was centrifuged to collect the precipitate. The precipitate was washed repeatedly with 70% ethanol 3-5 times and washed once with anhydrous ethanol. The precipitate was then placed in a fume hood to dry, resulting in alcohol-insoluble tea residue. The alcohol-insoluble tea residue was added to Tris-maleic acid buffer, 2 mmol / L CaCl2 and amylase were added, and the mixture was shaken on a shaker to obtain tea residue with starch removed. The tea residue with starch removed was washed 3-5 times and centrifuged to collect the precipitate and dried. The dried tea residue with starch removed was added to cyclohexanediaminetetraacetic acid solution, shaken on a shaker, centrifuged, and the supernatant was dialyzed to obtain the precipitate and extract. The precipitate was washed 3-5 times and dried to obtain tea residue after CDTA extraction. After dialysis, ethanol was added, and the mixture was allowed to stand overnight at 4 ℃. The precipitate was then centrifuged to collect the precipitate. The precipitate was reconstituted with water, centrifuged, and the supernatant was collected. This process was repeated three times. The supernatants were combined and freeze-dried to obtain CDTA-extracted tea polysaccharides (C-TPS).

[0011] (3) Add the tea residue obtained from step (2) after CDTA extraction to a saturated sodium carbonate solution, and add 25 mmol / L NaBH4 to the solution. Shake the solution, centrifuge, and dialyze the supernatant to obtain the precipitate and extract. Wash the precipitate 3-5 times and dry it to obtain the tea residue after saturated sodium carbonate extraction. Add ethanol, let stand overnight at 4 ℃, and then centrifuge to collect the precipitate. Redissolve the precipitate in water, centrifuge to collect the supernatant, repeat 3 times, combine the supernatants, and freeze-dry to obtain sodium carbonate-extracted tea polysaccharides (N-TPS).

[0012] (4) Add the tea residue obtained from step (3) after extraction with saturated sodium carbonate to 1 mol / L NaOH solution, and add 25 mmol / L NaBH4 to the solution. Shake the mixture, centrifuge, and dialyze the supernatant to obtain the precipitate and extract. Wash the precipitate 3-5 times and dry it to obtain the tea residue extracted with 1 mol / L NaOH solution. Add ethanol, let stand overnight at 4 ℃, and centrifuge to collect the precipitate. Redissolve the precipitate in water, centrifuge to collect the supernatant, repeat 3 times, combine the supernatants, and freeze-dry to obtain the polysaccharides extracted with 1 mol / L NaOH solution (1 M NaOH-extracted tea polysaccharides, 1 M-TPS).

[0013] (5) Add the tea residue obtained in step (4) after extraction with 1 mol / L NaOH solution to 4 mol / L NaOH solution, and add 25 mmol / L NaBH4 to the solution. Shake the mixture, centrifuge, and dialyze the supernatant to obtain the precipitate and extract. After rotary evaporation and concentration, add ethanol, let stand overnight at 4 ℃, and centrifuge to obtain the precipitate. Redissolve the precipitate in water, centrifuge to obtain the supernatant, repeat 3 times, combine the supernatants, and freeze dry to obtain the polysaccharides extracted with 4 mol / L NaOH solution (4 M NaOH-extracted tea polysaccharides, 4 M-TPS).

[0014] (6) Protein removal: Trichloroacetic acid was added to the water-extracted polysaccharide obtained in step (1), the CDTA-extracted polysaccharide obtained in step (2), the sodium carbonate-extracted polysaccharide obtained in step (3), the 1 mol / L NaOH solution-extracted polysaccharide obtained in step (4), and the 4 mol / L NaOH solution-extracted polysaccharide obtained in step (5). The precipitate was removed by centrifugation, and the supernatant was taken to obtain a protein-free polysaccharide solution. The protein-free polysaccharide solution was adjusted to pH 7 and then dialyzed. Then it was freeze-dried to obtain the protein-free water-extracted polysaccharide, the protein-free CDTA-extracted polysaccharide, the protein-free sodium carbonate-extracted polysaccharide, the protein-free 1 mol / L NaOH solution-extracted polysaccharide, and the protein-free 4 mol / L NaOH solution-extracted polysaccharide, respectively.

[0015] Furthermore, the specific process of step (1) is as follows:

[0016] Green tea leaves were ground and passed through an 80-mesh sieve to obtain tea powder. The tea powder was added to ultrapure water at a ratio of 1:20 and extracted at 37 ℃ for 2 h. The supernatant was then collected by centrifugation. The precipitate was extracted again by repeating the above steps until no sugar content was detected in the supernatant. The supernatants were combined and dialyzed for 72 h using a 7000 Da dialysis bag. The supernatant was used as the extract. The tea residue was retained and dried to obtain dried tea residue for subsequent steps. Ethanol was added at a volume ratio of extract to ethanol of 1:3 and allowed to stand overnight at 4 ℃. The precipitate was then collected by centrifugation. The precipitate was reconstituted with water and centrifuged to collect the supernatant. This process was repeated 3 times. The supernatants were combined and freeze-dried to obtain water-extracted tea polysaccharides (W-TPS).

[0017] Furthermore, the specific process of step (2) is as follows:

[0018] The dried tea residue obtained in step (1) was added to 70% ethanol at a material-to-liquid ratio of 1:10, and shaken on a shaker at room temperature for 12 h. The solution was centrifuged to collect the precipitate. The precipitate was washed repeatedly with 70% ethanol 3-5 times and washed once with anhydrous ethanol to remove alcohol-soluble substances. The precipitate was then placed in a fume hood to air dry, resulting in alcohol-insoluble tea residue. The alcohol-insoluble tea residue was added to Tris-maleic acid buffer at a material-to-liquid ratio of 1:10. The concentration of the Tris-maleic acid buffer was 20 mmol / L, and the pH of the Tris-maleic acid buffer was 6.9 (20 mmol / L, pH=6.9). 2 mmol / L CaCl2 and amylase were added. The ratio of amylase to alcohol-insoluble tea residue was 20 mg amylase / 5 g tea residue. The mixture was shaken on a shaker at 37 ℃ for 4 hours. To remove starch, obtain starch-free tea residue. Wash the starch-free tea residue 3-5 times, centrifuge, collect the precipitate, and dry it in a fume hood. Add the dried starch-free tea residue to a cyclohexanediaminetetraacetic acid (CDTA) solution (CDTA solution, solvent: pure water) at a material-to-liquid ratio of 1:10. The concentration of the CDTA solution is 50 mmol / L, and the pH of the CDTA solution is 6.5 (50 mmol / L, pH=6.5). Extract by shaking at 37 °C for 12 h. Then centrifuge and dialyze the supernatant using a 7000 Da dialysis bag for 72 h to obtain the precipitate and extract. Wash the precipitate 3-5 times and dry it in a fume hood to obtain the CDTA-extracted tea residue. After dialysis, add ethanol at a ratio of extract:ethanol = 1:3, let stand overnight at 4 °C, and then centrifuge to collect the precipitate. The precipitate was reconstituted with water and centrifuged to obtain the supernatant. This process was repeated three times. The supernatants were combined and freeze-dried to obtain CDTA-extracted tea polysaccharides (C-TPS).

[0019] Furthermore, the specific process of step (3) is as follows:

[0020] The tea residue obtained from step (2) after CDTA extraction was added to a saturated sodium carbonate solution at a ratio of 1:10, and 25 mmol / L NaBH4 was added to the solution to protect the polysaccharide. The mixture was shaken at 37 °C for 12 h. After centrifugation, the supernatant was dialyzed for 72 h using a 7000 Da dialysis bag to obtain the precipitate and extract. The precipitate was washed 3-5 times and then dried to obtain the tea residue after saturated sodium carbonate extraction. Ethanol was added at a volume ratio of extract to ethanol of 1:3, and the mixture was allowed to stand overnight at 4 °C. The precipitate was then centrifuged to obtain the precipitate. The precipitate was reconstituted with water and centrifuged to obtain the supernatant. This process was repeated 3 times. The supernatants were combined and freeze-dried to obtain the sodium carbonate-extracted tea polysaccharides (N-TPS).

[0021] Furthermore, the specific process of step (4) is as follows:

[0022] The tea residue obtained from step (3) after extraction with saturated sodium carbonate was added to 1 mol / L NaOH solution at a ratio of 1:10, and 25 mmol / L NaBH4 was added to the solution to protect the polysaccharides. The mixture was shaken at 37 °C for 12 h. After centrifugation, the supernatant was dialyzed for 72 h using a 7000 Da dialysis bag (7000 Da, pH=6-7) to obtain the precipitate and extract. The precipitate was washed 3-5 times and then dried to obtain the tea residue extracted with 1 mol / L NaOH solution. Ethanol was added at a volume ratio of extract to ethanol of 1:3, and the mixture was allowed to stand overnight at 4 °C. The precipitate was collected by centrifugation. The precipitate was reconstituted with water and centrifuged to obtain the supernatant. This process was repeated 3 times. The supernatants were combined and freeze-dried to obtain the polysaccharides extracted with 1 mol / L NaOH solution (1 M NaOH-extracted tea polysaccharides, 1 M-TPS).

[0023] Furthermore, the specific process of step (5) is as follows:

[0024] The tea residue obtained in step (4) after extraction with 1 mol / L NaOH solution was added to 4 mol / L NaOH solution at a material-to-liquid ratio of 1:10, and 25 mmol / L NaBH4 was added to the solution. The mixture was shaken at 37 °C for 12 h. After centrifugation, the supernatant was dialyzed for 72 h using a 7000 Da dialysis bag (7000 Da, pH=6-7) to obtain the precipitate and extract. After rotary evaporation and concentration, ethanol was added at a volume ratio of 1:3 between the water extract and ethanol. The mixture was allowed to stand overnight at 4 °C and centrifuged to obtain the precipitate. The precipitate was reconstituted with water and centrifuged to obtain the supernatant. This process was repeated 3 times. The supernatants were combined and freeze-dried to obtain the polysaccharides extracted with 4 mol / L NaOH solution (4 M NaOH-extracted tea polysaccharides, 4 M-TPS).

[0025] Further, in step (6), trichloroacetic acid is added until the solution system contains 10% by mass of trichloroacetic acid; in step (6), dialysis is performed for 72 h using a dialysis bag with a mass of 3500 Da.

[0026] Furthermore, all the unspecified centrifugation conditions mentioned above were 5000 rpm for 10 min.

[0027] Furthermore, all the freeze-drying conditions not mentioned above are -20°C for 48 hours.

[0028] The obtained green tea cell wall polysaccharides were obtained using the above-described extraction and purification methods. The green tea cell wall polysaccharides with hypoglycemic activity include one or more of the following: polysaccharides extracted with CDTA after protein removal, polysaccharides extracted with sodium carbonate after protein removal, polysaccharides extracted with 1 mol / L NaOH solution after protein removal, and polysaccharides extracted with 4 mol / L NaOH solution after protein removal.

[0029] Furthermore, the total sugar content of the polysaccharide extracted from the 4 mol / L NaOH solution after protein removal is 631.12 mg / g; the uronic acid content of the polysaccharide extracted from the CDTA solution after protein removal is 260.75 mg / g; and the molecular weight of the water-extracted polysaccharide after protein removal reaches 2.55 × 10⁻⁶. 5 Da; The polysaccharide extracted from the 1 mol / L NaOH solution after protein removal has a molecular weight of 8.95 × 10⁻⁶. 3 The polysaccharide extracted from CDTA after protein removal has a galacturonic acid content of 49.83%; the polysaccharide extracted from sodium carbonate after protein removal has a glucosamine content of 45.32%; and the polysaccharide extracted from 1 mol / L NaOH solution after protein removal has a glucuronic acid content of 50.54%.

[0030] Furthermore, water-extractable polysaccharides were obtained through water extraction, and after starch removal, alcohol extraction, and impurity removal, alcohol-insoluble tea residue was obtained. Cell wall polysaccharides were then obtained through sequential extraction with CDTA, saturated sodium carbonate, 1 M NaOH, and 4 M NaOH.

[0031] Furthermore, after protein removal, CDTA showed the highest extraction rate, reaching 1.93%; the polysaccharide (cell wall polysaccharide) extracted with 4 mol / L NaOH solution had the highest total sugar content, at 631.12 mg / g; the water-extracted polysaccharide had the highest uronic acid content, at 265.81 mg / g; and the water-extracted polysaccharide had the highest molecular weight, at 2.55 × 10⁻⁶. 5 Da, while the polysaccharide extracted from 1 mol / L NaOH solution had the lowest molecular weight, at 8.95 × 10⁻⁶. 3 Da.

[0032] Further analysis of monosaccharide composition revealed significant differences in the monosaccharide composition of polysaccharides extracted using different solvents. C-TPS exhibited the highest galacturonic acid content at 49.83%, N-TPS the highest glucosamine content at 45.32%, and 1 M-TPS the highest glucuronic acid content at 50.54%. Compositional analysis correlated these components with different polysaccharide components found in the tea cell walls. The CDTA-extracted portion was pectin, while the sodium carbonate and 1 M NaOH-extracted portions were likely pectin and hemicellulose complexes. The 4 M NaOH-extracted polysaccharide was primarily composed of hemicellulose and cellulose. Infrared spectroscopy showed similar peak shapes for each polysaccharide, but the water-extracted and CDTA-extracted polysaccharides showed characteristic peaks for uronic acid, and the characteristic peaks for pyranose were stronger. Scanning electron microscopy (SEM) revealed significant morphological differences among the polysaccharides. The SEM images show the differences in structure and morphology among the various polysaccharides, indicating that the structure of polysaccharides is closely related to different extraction methods, polysaccharide composition, and molecular weight.

[0033] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects:

[0034] 1) The present invention proposes a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity and the obtained green tea cell wall polysaccharides. The reaction conditions of the entire operation process are relatively mild, which can retain the activity of green tea cell wall polysaccharides to the greatest extent and minimize the loss of polysaccharide components. It has the advantages of high extraction rate, low energy consumption, low solvent consumption, simple operation and low economic cost. It lays the foundation for studying the physicochemical properties and biological activities of green tea cell wall polysaccharides and is of great significance for the utilization of green tea cell wall polysaccharides in the fields of food, medical care and cosmetics.

[0035] 2) The present invention proposes a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity and the obtained green tea cell wall polysaccharides. Through sequential extraction, polysaccharides with different polysaccharide compositions and molecular weights can be extracted in each step. The polysaccharides extracted sequentially by CDTA, sodium carbonate and sodium hydroxide solutions of different concentrations (1M and 4M) showed the most significant hypoglycemic activity in vitro.

[0036] 3) The present invention proposes a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity, and the obtained green tea cell wall polysaccharides. Sequential extraction yielded five polysaccharides with different structures and compositions (water-extracted polysaccharides, CDTA-extracted polysaccharides, sodium carbonate-extracted polysaccharides, 1M sodium hydroxide-extracted polysaccharides, and 4M sodium hydroxide-extracted polysaccharides). The CDTA-extracted, sodium carbonate-extracted, 1M sodium hydroxide-extracted, and 4M sodium hydroxide-extracted polysaccharides are all cell wall polysaccharides. The five polysaccharides have different structures, with the 4M sodium hydroxide-extracted polysaccharide fragment being more complete and thinner. All obtained polysaccharides exhibit inhibitory effects on α-glucosidase, with the 4M sodium hydroxide-extracted polysaccharide showing an inhibition rate of 51.1%. This method can maximize the preservation of the activity of green tea cell wall polysaccharides and minimize the loss of polysaccharide components. The polysaccharides extracted from purified 4M sodium hydroxide include neutral sugars, uronic acids, and reducing sugars. They exhibit resistance to degradation in the digestive environment of the human mouth and esophagus, allowing them to safely reach the stomach and intestines, particularly interacting with gut microbiota. This may further regulate carbohydrate metabolism by modulating the gut microbiota. Attached Figure Description

[0037] Figure 1 This is a schematic flowchart of the extraction and purification method for green tea cell wall polysaccharides with hypoglycemic activity according to the present invention.

[0038] Figure 2 Gel permeation chromatograms of polysaccharides extracted from green tea and cell wall polysaccharides: W-TPS (A), C-TPS (B), N-TPS (C), 1M-TPS (D), 4M-TPS (E), p4M-TPS (F) (Note: The numbers in the figure indicate the retention time at the peak, and 38.5 min is the mobile phase peak).

[0039] Figure 3Ion chromatograms of cell wall polysaccharides mentioned in standard monosaccharides and Yichang fried green tea (diluted 25 times) (where: (1) Fucose, (2) Rhamnose, (3) Arabinose, (4) Glucosamine, (5) Galactose, (6) Glucose, (7) Xylose, (8) Fructose, (9) Mannose, (10) GalA, (11) GluA).

[0040] Figure 4 Infrared spectra of polysaccharides extracted from green tea and cell wall polysaccharides (A); SEM images of polysaccharides extracted from Yichang roasted green tea and cell wall polysaccharides on α-glucosidase (B); and W-TPS (C), C-TPS (D), N-TPS (E), 1 M-TPS (F), and 4 M-TPS (G) (magnification: 250×, scale: 100 μm).

[0041] Figure 5 Elution curves for the purification of 4M-TPS using a DEAE-Sepharose FF column. Detailed Implementation

[0042] The present invention will now be described in detail with reference to specific embodiments, but this is by no means a limitation thereof. Any preparation methods, materials, structures, or compositional ratios not explicitly described in this technical solution are considered common technical features disclosed in the prior art.

[0043] The green tea mentioned is Yichang pan-fried green tea.

[0044] Example

[0045] like Figure 1 As shown, this embodiment provides a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity. The method includes: sequentially extracting green tea with water, cyclohexanediaminetetraacetic acid (CDTA), sodium carbonate, and sodium hydroxide solutions of different concentrations; followed by impurity removal, dialysis, and freeze-drying to obtain the cell wall polysaccharides with hypoglycemic activity. The method specifically includes the following steps:

[0046] (1) Green tea leaves are ground and passed through an 80-mesh sieve to obtain tea powder. The tea powder is added to ultrapure water at a ratio of 1:20 and water-extracted at 37 ℃ for 2 h. Then, the supernatant is collected by centrifugation. The precipitate is extracted by repeating the above steps until no sugar content is detected in the supernatant. The supernatants are combined and dialyzed for 72 h using a 7000 Da dialysis bag. The supernatant is the extract. The tea residue is retained and dried to obtain dried tea residue for subsequent steps. Ethanol is added at a volume ratio of extract:ethanol = 1:3 and allowed to stand overnight at 4 ℃. Then, the precipitate is collected by centrifugation. The precipitate is reconstituted with water and centrifuged to collect the supernatant. This process is repeated 3 times. The supernatants are combined and freeze-dried to obtain water-extracted tea polysaccharides (W-TPS).

[0047] (2) The dried tea residue obtained in step (1) was added to 70% ethanol at a ratio of 1:10 and shaken on a shaker at room temperature (shaking speed of 100-200 rpm, the same below) for 12 h. The solution was centrifuged to collect the precipitate. The precipitate was washed repeatedly with 70% ethanol 3-5 times and washed once with anhydrous ethanol to remove alcohol-soluble substances. The precipitate was then placed in a fume hood to dry, resulting in alcohol-insoluble tea residue. The alcohol-insoluble tea residue was added to Tris-maleic acid buffer at a ratio of 1:10. The concentration of the Tris-maleic acid buffer was 20 mmol / L and the pH of the Tris-maleic acid buffer was 6.9 (20 mmol / L, pH=6.9). 2 mmol / L CaCl2 and amylase were added. The ratio of amylase to alcohol-insoluble tea residue was 20 mg amylase / 5 g tea residue (20 mg amylase / 5 g tea residue). The mixture was shaken on a shaker at 37 ℃ for 4 h. To remove starch, tea residue was obtained. The residue was washed 3-5 times, centrifuged, and the precipitate was dried in a fume hood. The dried residue was then added to a cyclohexanediaminetetraacetic acid (CTA) solution at a ratio of 1:10 (50 mmol / L, pH 6.5). The mixture was extracted at 37 °C with shaking for 12 h. The residue was then centrifuged, and the supernatant was dialyzed for 72 h using a 7000 Da dialysis bag (7000 Da). The precipitate and extract were obtained. The precipitate was washed 3-5 times and dried in a fume hood to obtain CDTA-extracted tea residue. After dialysis, ethanol was added at a ratio of extract to ethanol of 1:3, and the mixture was allowed to stand overnight at 4 °C. The precipitate was then centrifuged. The precipitate was reconstituted with water and centrifuged to obtain the supernatant. This process was repeated three times. The supernatants were combined and freeze-dried to obtain CDTA-extracted tea polysaccharides (C-TPS).

[0048] (3) The tea residue obtained from step (2) after CDTA extraction was added to a saturated sodium carbonate solution at a ratio of 1:10, and 25 mmol / L NaBH4 was added to the solution to protect the polysaccharide. The mixture was shaken at 37 °C for 12 h. After centrifugation, the supernatant was dialyzed for 72 h using a 7000 Da dialysis bag (7000 Da, pH=6-7) to obtain the precipitate and extract. The precipitate was washed 3-5 times and then dried to obtain the tea residue after saturated sodium carbonate extraction. Ethanol was added at a volume ratio of extract:ethanol = 1:3, and the mixture was allowed to stand overnight at 4 °C. The precipitate was then centrifuged to obtain the precipitate. The precipitate was reconstituted with water and centrifuged to obtain the supernatant. This process was repeated 3 times. The supernatants were combined and freeze-dried to obtain the sodium carbonate-extracted tea polysaccharides (N-TPS).

[0049] (4) Add the tea residue obtained from step (3) after extraction with saturated sodium carbonate to 1 mol / L NaOH solution at a ratio of 1:10, and add 25 mmol / L NaBH4 to the solution to protect the polysaccharide. Shake and extract for 12 h at 37 ℃. After centrifugation, dialyze the supernatant using a 7000 Da dialysis bag for 72 h (7000 Da, pH=6-7) to obtain the precipitate and extract. Wash the precipitate 3-5 times and dry it to obtain the tea residue extracted with 1 mol / L NaOH solution. Add ethanol at a volume ratio of extract:ethanol = 1:3, let stand overnight at 4 ℃, and collect the precipitate by centrifugation. Redissolve the precipitate in water and centrifuge to obtain the supernatant. Repeat 3 times, combine the supernatants, and freeze-dry to obtain the polysaccharide extracted with 1 mol / L NaOH solution (1 M NaOH-extracted tea polysaccharides, 1 M-TPS).

[0050] (5) The tea residue obtained in step (4) after extraction with 1 mol / L NaOH solution was added to 4 mol / L NaOH solution at a ratio of 1:10, and 25 mmol / L NaBH4 was added to the solution. The mixture was shaken at 37 °C for 12 h. After centrifugation, the supernatant was dialyzed for 72 h using a 7000 Da dialysis bag (7000 Da, pH=6-7) to obtain precipitate and extract. After rotary evaporation and concentration, ethanol was added at a volume ratio of 1:3 between the water extract and ethanol. The mixture was allowed to stand overnight at 4 °C and centrifuged to obtain precipitate. The precipitate was reconstituted with water and centrifuged to obtain supernatant. This process was repeated 3 times. The supernatants were combined and freeze-dried to obtain polysaccharides extracted with 4 mol / L NaOH solution (4 M NaOH-extracted tea polysaccharides, 4 M-TPS).

[0051] (6) Protein removal: Trichloroacetic acid was added to the water-extracted polysaccharide obtained in step (1), the CDTA-extracted polysaccharide obtained in step (2), the sodium carbonate-extracted polysaccharide obtained in step (3), the 1 mol / L NaOH solution-extracted polysaccharide obtained in step (4), and the 4 mol / L NaOH solution-extracted polysaccharide obtained in step (5) until the solution contained 10% by mass of trichloroacetic acid. The precipitate was removed by centrifugation, and the supernatant was taken to obtain a protein-removed polysaccharide solution. The protein-removed polysaccharide solution was adjusted to pH 7 and dialyzed for 72 h using a 3500 Da dialysis bag. Then, it was lyophilized to obtain the protein-removed water-extracted polysaccharide, the protein-removed CDTA-extracted polysaccharide, the protein-removed sodium carbonate-extracted polysaccharide, the protein-removed 1 mol / L NaOH solution-extracted polysaccharide, and the protein-removed 4 mol / L NaOH solution-extracted polysaccharide, respectively.

[0052] This embodiment provides a green tea cell wall polysaccharide with hypoglycemic activity, obtained by the above-described extraction and purification method. The green tea cell wall polysaccharide with hypoglycemic activity includes one or more of the following: polysaccharide extracted by CDTA after protein removal, polysaccharide extracted by sodium carbonate after protein removal, polysaccharide extracted by 1 mol / L NaOH solution after protein removal, and polysaccharide extracted by 4 mol / L NaOH solution after protein removal.

[0053] The following analyses were performed on the polysaccharides extracted from water after protein removal, polysaccharides extracted from CDTA after protein removal, polysaccharides extracted from sodium carbonate after protein removal, polysaccharides extracted from 1 mol / L NaOH solution after protein removal, and polysaccharides extracted from 4 mol / L NaOH solution after protein removal.

[0054] 1. Determination of basic components

[0055] Basic component analysis: Total sugar determination: SN / T 4260-2015

[0056] Glucuronic acid content determination: determined by the m-hydroxybiphenyl method.

[0057] Determination of polyphenol content: Refer to the spectrophotometer method in GB / T 8313-2018.

[0058] Determination of soluble protein content: GB5009.5-2016.

[0059] 2. Molecular weight determination

[0060] The relative molecular weights of water-extracted polysaccharides and four types of tea cell wall polysaccharides (dihydrogenated from CDTA, sodium carbonate, 1 mol / L NaOH, and 4 mol / L NaOH) after protein removal were determined using high-performance gel permeation chromatography (HPPGPC). Samples were dissolved in ultrapure water to a concentration of 2 mg / mL. One mL of sample was centrifuged for 10 min (10000 r / min) and filtered through a 0.22 μm filter. 30 μL of sample was injected into the HPPGPC system. The mobile phase was 0.02 mol / L phosphate buffer (pH 6.8), and the elution columns were Shodex SB-805HQ (8 mm × 300 mm) and Shodex SB-804HQ (8 mm × 300 mm). Column and detector temperatures were controlled at 25 °C and 30 °C, respectively, with a flow rate of 0.3 mL / min.

[0061] Different molecular weight dextran standards (T3, T6, T10, T100, T500, and T1000) were prepared into 2 mg / mL solutions, filtered through a 0.22 μm filter membrane, and injected sequentially. Chromatographic results were recorded. A standard curve was plotted with retention time tR (min) on the x-axis and the logarithm of the weight-average molecular weight of dextran (Mw) (lgMw) on the y-axis. Based on the retention times of the polysaccharide samples and the standard curve, the molecular weight of the polysaccharide samples was calculated.

[0062] 3. Determination of monosaccharide composition

[0063] Ion chromatography was used to qualitatively and quantitatively determine the monosaccharide composition of water-extracted polysaccharides after protein removal and four types of tea cell wall polysaccharides after protein removal (polysaccharides extracted from CDTA after protein removal, polysaccharides extracted from sodium carbonate after protein removal, polysaccharides extracted from 1 mol / L NaOH solution after protein removal, and polysaccharides extracted from 4 mol / L NaOH solution after protein removal).

[0064] (1) Sample pretreatment:

[0065] Take 2 mg of sample, add 3 mL of 2 mol / L trifluoroacetic acid, and hydrolyze in an oven at 110 ℃ for 4 h. After hydrolysis, place in a nitrogen evaporator to dry (temperature <45 ℃). After drying, add 3 mL of methanol solution, and repeat the methanol-nitrogen evaporation operation 3 times to remove TFA. Dissolve the hydrolyzed sample in 2 mL of ultrapure water, centrifuge for 5 min (5000 r / min), and retain the supernatant.

[0066] Testing conditions:

[0067] Analysis was performed using a Dionex ICS 5000+ system at a flow rate of 0.45 mL / min. A 2 mmol / L sodium hydroxide solution was used as the eluent. The analysis time was 40 min, and the injection volume was 30 μL. Monosaccharide standards (including glucose, arabinose, fructose, glucuronic acid, galactose, xylose, galacturonic acid, rhamnose, glucosamine, mannose, and fucose) were processed according to the above sample pretreatment method. The peak times and peak areas of the standards were compared with those of the polysaccharide samples to determine the types of monosaccharides and the molar concentration of each monosaccharide.

[0068] Infrared spectral scanning

[0069] Qualitative analysis of five tea polysaccharides obtained by sequential extraction (water-extracted polysaccharides after protein removal and four tea cell wall polysaccharides after protein removal: polysaccharides extracted with CDTA after protein removal, polysaccharides extracted with sodium carbonate after protein removal, polysaccharides extracted with 1 mol / L NaOH solution after protein removal, and polysaccharides extracted with 4 mol / L NaOH solution after protein removal) was performed using Fourier transform infrared spectroscopy. A small amount of dried polysaccharide sample was ground with KBr into a fine powder using an agate mortar and pestle. The powder was then pressed into transparent sheets using a tablet press and subsequently tested on a Fourier transform infrared spectrometer. The spectral scanning range was 4000-400 cm⁻¹. -1 .

[0070] 5. Scanning electron microscope

[0071] A small amount of dried test sample (water-extracted polysaccharide after protein removal and four types of tea cell wall polysaccharides after protein removal: polysaccharide extracted from CDTA after protein removal, polysaccharide extracted from sodium carbonate after protein removal, polysaccharide extracted from 1 mol / L NaOH solution after protein removal, and polysaccharide extracted from 4 mol / L NaOH solution after protein removal) was evenly spread and adhered to the conductive adhesive on the sample stage. The sample was then placed in a vacuum sputtering instrument to deposit a layer of gold as a conductive film on its surface. Subsequently, the morphology and structure of the sample were observed using a scanning electron microscope, and images were acquired and analyzed at an appropriate electron microscope magnification.

[0072] 6. Determination of α-glucosidase inhibition rate activity

[0073] The α-glucosidase inhibitory activity of tea polysaccharides (water-extracted polysaccharides after protein removal and four types of tea cell wall polysaccharides after protein removal: polysaccharides extracted with CDTA after protein removal, polysaccharides extracted with sodium carbonate after protein removal, polysaccharides extracted with 1 mol / L NaOH solution after protein removal, and polysaccharides extracted with 4 mol / L NaOH solution after protein removal) was determined using a colorimetric method with an enzyme-linked immunosorbent assay (ELISA) reader. The experimental groups were: blank group Ab (without test solution and enzyme solution), control group Ac (without test solution), test solution group As, and test solution blank group Asb (without enzyme solution). Nitrophenyl-α-D-glucopyranoside (PNPG) was used as the chromogenic substrate, and the entire experiment was performed in 96-well plates. In the experiment, according to different groups, 60 μL of PBS phosphate buffer (67 mmol / L, pH=6.8), 20 μL of α-glucosidase solution (0.25 U / mL), and 20 μL of different concentrations of test sample solutions (the concentration gradient of tea polysaccharide test solution was set to 0.1, 1, 10, 100, and 1000 μg / mL) were added to the plate sequentially. After low-speed shaking and homogenization, the plate was heated in a 37 ℃ water bath for 10 min. Then, 20 µL of saturated PNPG solution was added to each well as the reaction substrate to initiate the enzyme reaction. After low-speed shaking and homogenization, the plate was heated in a 37 ℃ water bath for 20 min to allow the reaction to proceed fully. After the reaction was completed, 80 μL of Na2CO3 solution (0.2 mol / L) was quickly added to the system to finally catalyze and stop the reaction. After low-speed shaking and homogenization, the absorbance of each well was measured using a microplate reader at a wavelength of 405 nm. Finally, the inhibition rate of the sample against α-glucosidase was calculated according to the following formula:

[0074] Formula (3-1)

[0075] 7. Determination of α-amylase inhibition rate activity

[0076] The α-amylase inhibitory activity of tea polysaccharides (water-extracted polysaccharides after protein removal and four types of tea cell wall polysaccharides after protein removal: polysaccharides extracted with CDTA after protein removal, polysaccharides extracted with sodium carbonate after protein removal, polysaccharides extracted with 1 mol / L NaOH solution after protein removal, and polysaccharides extracted with 4 mol / L NaOH solution after protein removal) was determined using the 3,5-dinitrosalicylic acid method. The experimental groups were: blank group Ab (without test solution and enzyme solution), control group Ac (without test solution), test solution group As, and test solution blank group Asb (without enzyme solution). Starch solution was used as the chromogenic substrate in this experiment. In the experiment, 67 mmol / L phosphate buffer (pH=6.8) was added sequentially to each stoppered graduated test tube. Then, 50 μL of different concentrations of the test sample solution (the concentration gradient of tea polysaccharide test solution was set to 0.05, 0.1, 0.5, 1, and 5 mg / mL) and 50 μL of 2.5 U / mL α-amylase (dissolved in PBS buffer, pH=6.8) were added. The mixtures were vortexed and incubated at 37.5 ℃ for 10 min. Next, 100 μL of 1% starch solution (dissolved in PBS buffer, pH=6.8) was added, mixed, and incubated at 37.5 ℃ for 10 min. After the reaction was complete, 200 μL of DNS solution was added and incubated in a boiling water bath for 5 min. The reaction was then quickly cooled to room temperature to terminate the reaction. After thorough shaking, the absorbance was measured using a UV spectrophotometer. Using PBS buffer as a blank, the absorbance was measured at 540 nm after dilution. Finally, the inhibition rate of the sample against α-amylase was calculated using the following formula:

[0077]

[0078] 8. Determination of sucrase inhibition rate activity

[0079] The sucrase inhibitory activity of tea polysaccharide samples was determined according to a previously described method for determining α-amylase inhibitory activity. The experimental groups were: blank group Ab (without test solution and enzyme solution), control group Ac (without test solution), test solution group As, and test solution blank group Asb (without enzyme solution). Sucrose solution was used as the substrate. In each stoppered graduated test tube, 67 mmol / L PBS buffer (pH=6.8) was added sequentially, followed by 50 μL of different concentrations of the test sample solution (tea polysaccharide test solution concentration gradient set to 0.05, 0.1, 0.5, 1, 5 mg / mL) and 50 μL of 10 U / mL sucrase (dissolved in PBS buffer, pH=6). The mixture was vortexed and incubated at 37.5 ℃ for 10 min. Then, 100 μL of 0.5% sucrose solution (dissolved in PBS buffer, pH=6) was added, mixed, and incubated at 37.5 ℃ for 10 min. After the reaction was complete, 200 μL of DNS solution was added and the mixture was reacted in a boiling water bath for 5 min. The reaction was then quickly terminated by cooling the mixture to room temperature. After thorough shaking, the absorbance was measured using a UV spectrophotometer. PBS buffer was used as a blank, and the absorbance was measured at 540 nm after dilution. Finally, the inhibition rate of the sample against sucrase was calculated using the following formula:

[0080]

[0081] Isolation and purification of cell wall polysaccharides from Yichang pan-fried green tea

[0082] Crude polysaccharide samples obtained through in vitro screening for glycemic enzyme inhibition activity were dissolved in phosphate-buffered saline (PBS) (pH=6.8, 0.02 mol / L) to a polysaccharide concentration of 50 mg / mL. After filtration through a 0.22 μm filter, the solution was washed onto a gel column packed with DEAE-Sepharose Fast Flow gel packing until equilibrium was reached, and then loaded onto the column. The polysaccharide solution was slowly and evenly added dropwise to an anion exchange gel column (2.5 cm × 60 cm). After stabilization, the flow was turned on, and the column was eluted sequentially with PBS buffer solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl. The concentration gradient was changed after elution until no sugar was detected. The eluent was collected using an automated collector (2 mL / min, 2 min / tube). The total sugar content of the collected liquid was determined using the phenol-sulfuric acid method. The elution curve was plotted with the OD value at 490 nm as the ordinate and the number of tubes as the abscissa. The eluents from the single peaks were combined, concentrated, and dialyzed for 72 h using a 3500 Da dialysis bag. The resulting polysaccharide sample was then lyophilized.

[0083] 10. Simulated in vitro digestion experiment

[0084] Preparation of simulated saliva: Accurately weigh 15.1 mmol KCl, 1.5 mmol CaCl2, 0.15 mmol MgCl2, 3.7 mmol KH2PO4, 0.06 mmol (NH4)2CO3, 13.6 mmol NaHCO3 and 1.1 mmol HCl, dissolve and mix them evenly with ultrapure water, and bring the volume to 1 L to obtain simulated saliva.

[0085] Preparation of simulated gastric juice: Accurately weigh 6.9 mmol KCl, 0.15 mmol CaCl2, 0.12 mmol MgCl2, 0.9 mmol KH2PO4, 0.5 mmol (NH4)2CO3, 25.0 mmol NaHCO3, 47.2 mmol NaCl and 15.6 mmol HCl, dissolve and mix them evenly with ultrapure water, and bring the volume to 1 L to obtain simulated gastric juice.

[0086] Preparation of simulated intestinal fluid: Accurately weigh 6.8 mmol KCl, 0.6 mmol CaCl2, 0.33 mmol MgCl2, 0.8 mmol KH2PO4, 85.0 mmol NaHCO3, 38.4 mmol NaCl and 8.4 mmol HCl, dissolve and mix them evenly with ultrapure water, and bring the volume to 1 L to obtain simulated intestinal fluid.

[0087] First, 100.0 mL of sample solution (20.0 mg / mL), 15 mg α-amylase (1000 U / mg), and 100.0 mL of simulated saliva were mixed and incubated at 37 °C (pH = 7.0) for 1 h to simulate salivary digestion. Then, 150.0 mL of salivary digestion solution, 0.2 g pepsin (3000 U / g), and 150.0 mL of simulated gastric juice were mixed and rapidly adjusted to pH = 3.0 to trigger simulated gastric digestion, and incubated at 37 °C for 4 h to simulate gastric juice digestion. Finally, 180.0 mL of gastric juice digested sample, 1.47 g bile salts, 1.8 g pancreatin (4000 U / g), and 180.0 mL of simulated intestinal juice were mixed and rapidly adjusted to pH = 7.0 to trigger simulated intestinal digestion, and incubated at 37 °C for 4 h to simulate intestinal juice digestion. After the simulated digestion was completed, the samples were collected for further analysis. The collected samples were first used to determine the reducing sugar content, and then treated with ethanol, dialyzed (3.0 kDa), and freeze-dried in sequence before being used for subsequent experimental determinations.

[0088] 11. Analysis of sugar content before and after digestion

[0089] Basic component analysis: Determination of total sugar content: SN / T 4260-2015

[0090] Determination of uronic acid content: The m-hydroxybiphenyl method was used for determination.

[0091] Formula for calculating digestibility:

[0092]

[0093] The test results are as follows:

[0094] 1. Yichang pan-fried green tea water mentions cell wall polysaccharide extraction rate

[0095] Table 1. Extraction rate of cell wall polysaccharides from Yichang pan-fried green tea (%)

[0096]

[0097] Note: Different superscript letters indicate significant differences between groups. p< (0.05), and perform intragroup significance analysis for each row separately.

[0098] As shown in Table 1, cell wall polysaccharides from Yichang pan-fried green tea were extracted using a sequential extraction method. Initial extraction revealed significant differences in polysaccharide yields among different solvents. Water extraction and CDTA extraction yielded relatively high yields (1.98% and 2.24%, respectively), while saturated Na2CO3 extraction yielded a low yield of only 0.61%. Component analysis showed that, except for the polysaccharide fraction extracted by CDTA, the extracted polysaccharides all contained high protein content. Therefore, protein removal was performed on the initial polysaccharide samples. Protein removal results showed that the water extraction fraction had a high initial polysaccharide extraction rate, but after protein removal, the extraction rate decreased to 1.48%, indicating significant sample loss during the removal process. The polysaccharide content extracted by CDTA remained as high as 1.93% even after protein removal. Similarly, the polysaccharide fractions extracted by saturated Na2CO3, 1 M NaOH, and 4 M NaOH showed less protein loss after removal, indicating that some soluble proteins were extracted during the water extraction of tea. Overall, water extraction, CDTA extraction, and 4M NaOH extraction yielded relatively high polysaccharide extraction rates.

[0099] 2. The basic components of Yichang pan-fried green tea water include cell wall polysaccharides.

[0100] Table 2. Basic composition of cell wall polysaccharides in Yichang pan-fried green tea (mg / g)

[0101]

[0102] Note: Different superscript letters indicate significant differences between groups. p<(0.05), and each column was analyzed separately for significant differences within the group.

[0103] The composition of polysaccharide fractions obtained by sequential extraction with different solvents showed significant differences. The total sugar content of cell wall polysaccharides extracted with water and 4 M NaOH was relatively high, at 577.13 mg / g and 631.12 mg / g, respectively, while the polysaccharide fraction extracted with sodium carbonate had a lower total sugar content of 192.71 mg / g. Simultaneously, the polysaccharides extracted with water and CDTA contained high uronic acid contents, at 265.81 mg / g and 260.75 mg / g, respectively. The polyphenol content of the polysaccharide fractions obtained by different extraction methods varied greatly. The polysaccharide fraction extracted with sodium carbonate contained a relatively high polyphenol content of 83.46 mg / g, while the fraction extracted with CDTA contained almost no polyphenols, indicating that chelating agents may disrupt the binding of polysaccharides and polyphenols in the CDTA-extracted polysaccharide.

[0104] 3. Molecular weight determination

[0105] The weight-average molecular weight of five tea polysaccharides obtained by sequential extraction was determined by gel permeation chromatography. The test results are as follows: Figure 2 As shown, different molecular weight dextran standards (T3, T6, T10, T100, T500, and T1000) were injected sequentially, and chromatograms were collected to obtain the retention time tR for each molecular weight dextran standard. A standard curve was plotted with retention time tR (min) on the x-axis and the logarithm of the weight-average molecular weight of dextran Mw (lgMw) on the y-axis. The standard linear equation was obtained:

[0106] (R) 2 =0.993)

[0107] The weight-average molecular weight of the polysaccharide samples was calculated based on the retention time and molecular weight standard curve. The molecular weight of W-TPS was found to be 2.55 × 10⁻⁶. 5 The molecular weight of Da,C-TPS is 5.8 × 10⁻⁶. 4 The molecular weight of Da,N-TPS is 1.18 × 10⁻⁶. 4 The molecular weight of Da, 1M-TPS is 8.95 × 10⁻⁶. 3 The molecular weight of Da,4 M-TPS is 1.35 × 10⁻⁶. 5 As can be seen from the above results, the water-extracted polysaccharide has the largest molecular weight. During the sequential extraction process, the molecular weight of the polysaccharide shows a decreasing trend, indicating that the chelating agent and alkali will gradually hydrolyze and release the large molecular polysaccharide during the extraction process.

[0108] 4. Monosaccharide composition analysis

[0109] The ion chromatograms obtained by determining the monosaccharide composition of the five polysaccharide components are shown below. Figure 3 The monosaccharide composition results are shown in Table 3. W-TPS consists of rhamnose, arabinose, glucosamine, glucose, xylose, galacturonic acid, and glucuronic acid, with a molar ratio of 1.00:0.31:1.29:0.68:0.14:2.47:0.15. Arabinose, glucosamine, and galacturonic acid are the main sugar units in the W-TPS structure. The arabinose mainly originates from the decomposition and release of lignocellulose in the tea cell walls during water extraction. The high galactose content may be due to the transformation of other monosaccharide residues into galactose residues during alkali extraction.

[0110] C-TPS is composed of rhamnose, arabinose, glucosamine, glucose, mannose, galacturonic acid, and glucuronic acid in a molar ratio of 1.00:1.03:0.85:0.31:0.37:3.86:0.33, with rhamnose, arabinose, glucosamine, and galacturonic acid being the main monosaccharides. C-TPS has the highest galacturonic acid content (49.83%). Galacturonic acid in tea is an oxidation product of galactose, possibly originating from the decomposition of pectin and other polysaccharides in tea.

[0111] N-TPS is composed of rhamnose, arabinose, glucosamine, glucose, galacturonic acid, and glucuronic acid in a molar ratio of 1.00:0.67:2.02:0.29:0.13:0.36, with rhamnose, arabinose, and glucosamine being the main monosaccharides. N-TPS has the highest glucosamine content (45.32%). Its main form in the polysaccharides of tea cell walls is chitosan, a major component in the formation of cellulose and hemicellulose. Under alkaline conditions, the hydrogen bonds between hemicellulose and cellulose break due to cell wall swelling, demonstrating that sodium carbonate effectively disrupts the structure of tea cell walls during extraction, dissolving the polysaccharides.

[0112] 1M-TPS is composed of rhamnose, arabinose, glucosamine, galactose, galacturonic acid, and glucuronic acid in a molar ratio of 1.00:1.73:1.33:18.65:5.02:9.17, with galactose, galacturonic acid, and glucuronic acid being the main monosaccharides. 1M-TPS has the highest glucuronic acid content (50.54%), which acts as a building block of pectin and hemicellulose in tea cell walls. It forms complex three-dimensional structures through covalent bonds with other monosaccharides and polysaccharides, playing a role in maintaining cell wall structure and function. It is effectively extracted and released during alkaline extraction. Galactose was only detected in 1M-TPS, and it is mainly found in cellulose and hemicellulose of the cell walls, indicating that the main components of the cell wall polysaccharide fraction extracted by alkaline extraction are cellulose and hemicellulose.

[0113] 4 M-TPS is composed of rhamnose, arabinose, glucosamine, glucose, xylose, fructose, galacturonic acid, and glucuronic acid, with a molar ratio of 1.00:4.00:4.10:3.11:0.98:1.03:2.04:0.34. Arabinose, glucosamine, and glucose are the main monosaccharides. Fructose was detected for the first time among these five polysaccharides and is a major component of pectin. These polysaccharides are linked by α-1,4- and α-1,2-glycosidic bonds, indicating that strong alkali can effectively disrupt the glycosidic bonds between pectin molecules in the cell wall, dissolving and releasing them. The monosaccharide composition of tea polysaccharides is affected by the extraction process; the extraction method is the main reason for the differences in monosaccharide composition among the aforementioned polysaccharides.

[0114] Among them: (1) Fucose, (2) Rhamnose, (3) Arabinose, (4) Glucosamine, (5) Galactose, (6) Glucose, (7) Xylose, (8) Fructose, (9) Mannose, (10) GalA, (11) GluA.

[0115] Table 3. Composition of cell wall polysaccharides and monosaccharides in Yichang pan-fried green tea (mol%)

[0116]

[0117] Note: - indicates not detected.

[0118] 5. Infrared spectroscopy analysis

[0119] Infrared spectroscopy test results as follows Figure 4 A. All five polysaccharide samples exhibited similar absorption peaks across multiple wavenumber ranges, with a peak at 3440 cm⁻¹. -1 up to 3400 cm -1 The peaks in this wavenumber range represent the stretching vibrations of the hydroxyl group (-OH). 2930 cm⁻¹ -1 The peak at 1630 cm⁻¹ represents the stretching vibration of methyl (-CH₃) or methylene (-CH₂), indicating the presence of alkanes in the polysaccharide sample. -1 The peak at 1400 cm⁻¹ represents the stretching vibration of a non-resonant double bond (C=C), indicating the presence of esterified carboxyl groups or unsaturated bonds in the polysaccharide component. -1 The peak at 1100 cm⁻¹ represents the bending vibration of the CH bond, which may originate from the carbon-hydrogen bonds in the polysaccharide molecule. -1 -1010 cm -1The peaks at [value] represent the stretching vibrations of the sugar ring, and these peaks are attributed to the pyran ring in the polysaccharide molecule. [Peaks less than 1000 cm⁻¹] -1 The peak at 660 cm⁻¹ typically represents the out-of-plane vibration of CH in olefins or aromatics. -1 -600 cm -1 The peaks at these points typically represent bending vibrations of the sugar ring, and these peaks also indicate an association with the pyran ring structure in the polysaccharide molecule. Figure 4 Polysaccharide fractions extracted by water extraction, CDTA, and sodium carbonate were at 1742 cm⁻¹ -1 and 1630 cm -1 Double peaks were observed at 1740 cm. -1 The peak at 1630 cm⁻¹ typically corresponds to the C=O vibration of the methyl esterified carboxyl group (COOR), while the peak at 1630 cm⁻¹ corresponds to the C=O vibration of the methyl esterified carboxyl group (COOR). -1 The peak at 915 and 830 cm⁻¹ typically corresponds to the C=O vibration of the ionic carboxyl group (COO-), and the presence of this peak indicates the presence of a uronic acid structure in the sample; peaks at 915 and 830 cm⁻¹ were observed in the polysaccharide fraction extracted by CDTA. -1 The presence of absorption peaks indicates that the polysaccharides exist in the form of α-D-pyran; the water-extracted polysaccharides, CDTA-extracted polysaccharides, and sodium carbonate extracts showed absorption peaks at 1100, 1070, and 1010 cm⁻¹. -1 The presence of multiple strong absorption peaks indicates the presence of a pyran ring structure in the polysaccharide molecule.

[0120] 6. Scanning Electron Microscope (SEM)

[0121] Five polysaccharide components obtained by sequential extraction were analyzed using scanning electron microscopy (SEM). Figure 4 As shown in Figure C, the observation results indicate that the water-extracted polysaccharide (W-TPS) exhibits a network-like flocculated state, while the polysaccharide extracted from the cell wall using chelating agents and alkalis mainly presents a granular and layered morphology, suggesting that the water-extracted polysaccharide components have a relatively intact structure during the extraction process. The polysaccharide extracted using cyclohexanediaminetetraacetic acid (CDTA) (C-TPS) shows a loose and fine powder structure. Figure 4 (D) This indicates that the chelating agent effectively disrupted the cross-links between macromolecular polysaccharide structures in the tea cell wall, causing them to fragment into polysaccharide fragments. The polysaccharides extracted using sodium carbonate (Na₂CO₃) (N-TPS), 1M sodium hydroxide (1M-TPS), and 4M sodium hydroxide (4M-TPS) all exhibited a lamellar structure. Notably, the polysaccharides extracted using sodium carbonate exhibited fine fragmentation and elongated branching shapes. Figure 4 E), indicating that sodium carbonate can extract highly branched pectin polysaccharides from tea cell walls. The polysaccharides extracted from 1M and 4M sodium hydroxide have similar morphologies, but the polysaccharide fragments extracted from 1M sodium hydroxide are relatively smaller and thicker (E). Figure 4F), while the polysaccharide fragments extracted with 4M sodium hydroxide were more complete and thinner ( Figure 4 G). These SEM observations indicate that the polysaccharide structure is closely related to different extraction methods, polysaccharide composition, and molecular weight.

[0122] 7. Inhibition rate of enzymes related to glucose metabolism

[0123] like Figure 4 As shown in B, all five tea polysaccharides exhibited inhibitory effects on α-glucosidase, and this inhibitory effect was concentration-dependent. The inhibitory effect on α-glucosidase increased with increasing polysaccharide concentration. At low concentrations (0.1 and 1 μg / mL), these tea polysaccharides showed the least inhibitory effect on α-glucosidase. When the concentration reached 10 μg / mL, the tea polysaccharides began to inhibit α-glucosidase activity, with 4M-TPS showing the most significant inhibitory effect (23.7%). At higher concentrations, especially at 1000 μg / mL, both 1M-TPS and 4M-TPS showed strong inhibitory effects on α-glucosidase, with 4M-TPS achieving an inhibition rate of 51.1%. Notably, the inhibitory effects of these five tea polysaccharides on α-amylase and sucrase were negligible. Based on the inhibitory rates of various tea polysaccharides on enzymes related to glucose metabolism, 4M-TPS exhibited excellent hypoglycemic activity.

[0124] 8. Separation and purification of 4M-TPS

[0125] In the purification of tea polysaccharides, gradient elution with salt solutions of varying concentrations is frequently used as the mobile phase to exchange and elute polysaccharides bound to the column packing material. This method is popular because the interaction between ions in the salt solution and polysaccharide molecules is enhanced, thereby increasing their charged properties. Changes in salt solution concentration lead to changes in ionic strength, which in turn affects the properties of the displaced polysaccharides. Elution in a neutral solution yields neutral polysaccharides, while elution with increasing salt concentration yields polysaccharides with increased acidity. This study further purified 4M-TPS (which exhibited the most significant hypoglycemic activity in vitro). Figure 5 When phosphate-buffered saline (PBS) was used as the elution buffer, a large amount of tea polysaccharides were eluted. However, subsequent elution with a concentration gradient of sodium chloride solution yielded very little polysaccharide. Therefore, the fractions eluted and collected under PBS conditions were selected, combined, concentrated, and freeze-dried to obtain purified tea polysaccharides, named p4M-TPS. Furthermore, the elution curves showed that the polysaccharide component extracted with 4M sodium hydroxide was mainly composed of neutral sugars.

[0126] 9. Chemical composition, molecular weight, and in vitro digestion of p4M-TPS

[0127] The chemical composition of p4M-TPS was determined to contain 553.44 mg / g of neutral sugars, 104.22 mg / g of uronic acid, and 391.32 mg / g of reducing sugars (Table 4). When determining the total sugar content using the phenol-sulfuric acid method with glucose as a standard, the results may be biased due to differences in the degree of reaction between monosaccharides. This method is also used to determine neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. When glucose is used as a standard, the measured total sugar content is closer to the neutral sugar content. Therefore, the results from the phenol-sulfuric acid method and the m-hydroxybiphenyl method should be combined to determine the total sugar content of p4M-TPS, which is 657.66 mg / g. The soluble protein content of p4M-TPS is 14.26 mg / g, and no polyphenols were detected, indicating that the DEAE-Sepharose Fast Flow ion exchange column has strong separation and purification capabilities. Figure 2 As shown in Figure F, the molecular weight of p4M-TPS was determined by gel permeation chromatography. The results showed a single and symmetrical elution peak, indicating the homogeneity of the p4M-TPS composition. The molecular weight of p4M-TPS was 2.42 × 10⁻⁶. 5 Da.

[0128] Table 4. Chemical composition and content (mg / g) of p4 M-TPS

[0129]

[0130] Note: - indicates not detected.

[0131] In vitro digestion experiments using p4M-TPS showed that the content of neutral sugars and uronic acids in tea polysaccharides decreased after simulated digestion. The changes in uronic acid content before and after digestion were significant (Table 5), similar to the digestibility characteristics of Fu brick tea polysaccharides. This phenomenon indicates that tea polysaccharides possess resistance to degradation in the digestive environment of the human oral cavity and esophagus, allowing them to safely reach the stomach and intestines, particularly interacting with intestinal microorganisms. This may further regulate carbohydrate metabolism by modulating the gut microbiota.

[0132] Table 5. Changes in sugar content (mg / g) before and after p4 M-TPS digestion.

[0133]

[0134] Note: - indicates not detected.

[0135] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for extracting and purifying green tea cell wall polysaccharides having blood glucose-lowering activity, characterized by, The method comprises: Green tea is sequentially extracted by water, cyclohexanediaminetetraacetic acid, sodium carbonate and different concentrations of sodium hydroxide solution, and then impurities are removed, dialysis and freeze-drying are performed to obtain cell wall polysaccharide with hypoglycemic activity; The method comprises the following steps: (1) Green tea leaves are ground and passed through a mesh screen to obtain tea powder, and then ultrapure water is added, and water extraction is performed at 37 DEG C for 2 hours, and then centrifugation is performed to obtain supernatant, and the precipitate is repeatedly extracted by the above steps until no sugar content is detected in the supernatant, and then the supernatant is combined and dialyzed, the supernatant is an extraction solution, and the tea residue is retained and dried to obtain dried tea residue for subsequent steps; ethanol is added, and then the solution is placed at 4 DEG C overnight, and then centrifugation is performed to collect the precipitate; the precipitate is redissolved in water, and then centrifugation is performed to obtain supernatant, and the above steps are repeated for 3 times, and then the supernatant is combined and freeze-dried to obtain water-extracted polysaccharide; (2) The dried tea residue obtained in step (1) is added with 70% ethanol, and then the solution is shaken on a shaking table, and then centrifugation is performed to collect the precipitate, and then the precipitate is repeatedly washed with 70% ethanol for 3-5 times, and then the precipitate is washed with anhydrous ethanol once, and then the precipitate is placed in a fume hood to dry to obtain alcohol-insoluble tea residue; the alcohol-insoluble tea residue is added with Tris-maleic acid buffer solution, and then 2mmol / L CaCl2 and amylase are added, and then the solution is shaken on a shaking table to obtain tea residue from which starch is removed, and then the tea residue from which starch is removed is washed for 3-5 times, and then centrifugation is performed to obtain precipitate which is dried, and then the dried tea residue from which starch is removed is added with cyclohexanediaminetetraacetic acid solution, and then the solution is shaken on a shaking table, and then centrifugation is performed, and then the supernatant is dialyzed to obtain precipitate and extraction solution, and then the precipitate is washed for 3-5 times, and then the precipitate is dried to obtain tea residue extracted by CDTA; after dialysis is completed, ethanol is added, and then the solution is placed at 4 DEG C overnight, and then centrifugation is performed to obtain precipitate, and then the precipitate is redissolved in water, and then centrifugation is performed to obtain supernatant, and the above steps are repeated for 3 times, and then the supernatant is combined and freeze-dried to obtain CDTA-extracted polysaccharide; (3) The tea residue extracted by CDTA obtained in step (2) is added with saturated sodium carbonate solution, and then 25mmol / L NaBH4 is added in the solution, and then the solution is shaken on a shaking table, and then centrifugation is performed, and then the supernatant is dialyzed to obtain precipitate and extraction solution, and then the precipitate is washed for 3-5 times, and then the precipitate is dried to obtain tea residue extracted by saturated sodium carbonate; ethanol is added, and then the solution is placed at 4 DEG C overnight, and then centrifugation is performed to obtain precipitate; the precipitate is redissolved in water, and then centrifugation is performed to obtain supernatant, and the above steps are repeated for 3 times, and then the supernatant is combined and freeze-dried to obtain sodium carbonate-extracted polysaccharide; (4) The tea residue extracted by saturated sodium carbonate obtained in step (3) is added with 1mol / L NaOH solution, and then 25mmol / L NaBH4 is added in the solution, and then the solution is shaken on a shaking table, and then centrifugation is performed, and then the supernatant is dialyzed to obtain precipitate and extraction solution, and then the precipitate is washed for 3-5 times, and then the precipitate is dried to obtain tea residue extracted by 1mol / L NaOH solution; ethanol is added, and then the solution is placed at 4 DEG C overnight, and then centrifugation is performed to obtain precipitate; the precipitate is redissolved in water, and then centrifugation is performed to obtain supernatant, and the above steps are repeated for 3 times, and then the supernatant is combined and freeze-dried to obtain 1mol / L NaOH solution-extracted polysaccharide; (5) the tea residue obtained after extraction by 1 mol / L NaOH solution in step (4) is added into 4 mol / L NaOH solution, 25 mmol / L NaBH4 is added into the solution, and the solution is shaken and centrifuged, and the supernatant is dialyzed to obtain a precipitate and an extraction solution; after being concentrated by rotary evaporation, ethanol is added, and the solution is placed at 4℃ overnight, and then centrifuged to obtain a precipitate; the precipitate is redissolved in water, and the supernatant is obtained by centrifugation, and the operation is repeated for three times, the supernatants are combined, and the combined supernatant is freeze-dried to obtain polysaccharides extracted by 4 mol / L NaOH solution; The green tea is preferably Yichang pan-fried green tea.

2. The method of claim 1, wherein the green tea cell wall polysaccharide having a blood sugar-lowering activity is extracted and purified, and the method is characterized by, The specific process of step (1) is as follows: After the green tea leaves are ground, the tea powder is obtained by passing through an 80-mesh screen, the tea powder is added into ultrapure water at a ratio of 1:20, and the tea powder is extracted in water at 37℃ for 2 hours, and then the supernatant is obtained by centrifugation, the precipitate is repeatedly extracted by the above steps until no sugar is detected in the supernatant, the supernatants are combined, and the combined supernatant is dialyzed by using a dialysis bag with a molecular weight of 7000 Da for 72 hours, the supernatant is the extraction solution, the tea residue is reserved and dried to obtain dried tea residue, and the dried tea residue is used in subsequent steps; ethanol is added into the extraction solution at a volume ratio of 1:3, the solution is placed at 4℃ overnight, and then the precipitate is collected by centrifugation; the precipitate is redissolved in water, and the supernatant is obtained by centrifugation, and the operation is repeated for three times, the supernatants are combined, and the combined supernatant is freeze-dried to obtain water-extracted polysaccharides.

3. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 1, characterized in that, The specific process of step (2) is as follows: The dried tea residue obtained in step (1) is added into 70% ethanol at a ratio of 1:10, the solution is shaken at room temperature for 12 hours, the precipitate is collected by centrifugation, the precipitate is repeatedly washed with 70% ethanol for 3-5 times, and then washed once with anhydrous ethanol, and then the precipitate is placed in a fume hood to dry, to obtain alcohol-insoluble tea residue; the alcohol-insoluble tea residue is added into Tris-maleic acid buffer at a ratio of 1:10, the concentration of the Tris-maleic acid buffer is 20 mmol / L, the pH of the Tris-maleic acid buffer is 6.9, 2 mmol / L CaCl2 and amylase are added, the ratio of amylase to alcohol-insoluble tea residue is 20 mg amylase / 5 g tea residue, the solution is shaken at 37℃ for 4 hours to remove starch, to obtain starch-removed tea residue, the starch-removed tea residue is washed for 3-5 times, and then the precipitate is collected by centrifugation and placed in a fume hood to dry, the dried starch-removed tea residue is added into a solution of cyclohexanediamine tetraacetic acid at a ratio of 1:10, the concentration of the solution of cyclohexanediamine tetraacetic acid is 50 mmol / L, the pH of the solution of cyclohexanediamine tetraacetic acid is 6.5, the solution is shaken at 37℃ for 12 hours, and then the supernatant is obtained by centrifugation and dialyzed by using a dialysis bag with a molecular weight of 7000 Da for 72 hours, to obtain a precipitate and an extraction solution, the precipitate is washed for 3-5 times, and then placed in a fume hood to dry, to obtain tea residue extracted by CDTA; after dialysis, ethanol is added into the extraction solution at a ratio of 1:3, the solution is placed at 4℃ overnight, and then the precipitate is collected by centrifugation, the precipitate is redissolved in water, and the supernatant is obtained by centrifugation, and the operation is repeated for three times, the supernatants are combined, and the combined supernatant is freeze-dried to obtain CDTA-extracted polysaccharides.

4. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 1, characterized in that, The specific process of step (3) is as follows: The tea residue after CDTA extraction obtained in step (2) was added into saturated sodium carbonate solution at a ratio of 1:10 of solid to liquid, and 25 mmol / L of NaBH4 was added into the solution to protect polysaccharides, and extraction was carried out at 37°C for 12 hours under shaking table. After centrifugation, the supernatant was dialyzed in a dialysis bag with a molecular weight of 7000 Da for 72 hours to obtain a precipitate and an extraction solution. The precipitate was washed for 3-5 times and then dried to obtain tea residue after saturated sodium carbonate extraction. The extraction solution: ethanol was added at a volume ratio of 1:3, and the mixture was placed at 4°C overnight, and then centrifuged to obtain a precipitate. The precipitate was redissolved in water and centrifuged to obtain a supernatant, which was repeated for 3 times. The supernatants were combined and freeze-dried to obtain polysaccharides extracted by sodium carbonate.

5. The method of claim 1, wherein the green tea cell wall polysaccharide having a blood sugar-lowering activity is extracted and purified, and the method is characterized by, The specific process of step (4) is as follows: The tea residue after saturated sodium carbonate extraction obtained in step (3) was added into 1 mol / L NaOH solution at a ratio of 1:10 of solid to liquid, and 25 mmol / L of NaBH4 was added into the solution to protect polysaccharides, and extraction was carried out at 37°C for 12 hours under shaking table. After centrifugation, the supernatant was dialyzed in a dialysis bag with a molecular weight of 7000 Da for 72 hours to obtain a precipitate and an extraction solution. The precipitate was washed for 3-5 times and then dried to obtain tea residue after 1 mol / L NaOH solution extraction. Ethanol was added at a volume ratio of 1:3 of extraction solution to ethanol, and the mixture was placed at 4°C overnight, and then centrifuged to obtain a precipitate. The precipitate was redissolved in water and centrifuged to obtain a supernatant, which was repeated for 3 times. The supernatants were combined and freeze-dried to obtain polysaccharides extracted by 1 mol / L NaOH solution.

6. The method of claim 1, wherein the green tea cell wall polysaccharide having a blood sugar-lowering activity is extracted and purified, and the method is characterized by, The specific process of step (5) is as follows: The tea residue after 1 mol / L NaOH solution extraction obtained in step (4) was added into 4 mol / L NaOH solution at a ratio of 1:10 of solid to liquid, and 25 mmol / L of NaBH4 was added into the solution, and extraction was carried out at 37°C for 12 hours under shaking table. After centrifugation, the supernatant was dialyzed in a dialysis bag with a molecular weight of 7000 Da for 72 hours to obtain a precipitate and an extraction solution. The extraction solution was concentrated by rotary evaporation, and ethanol was added at a volume ratio of 1:3 of water extraction solution to ethanol, and the mixture was placed at 4°C overnight, and then centrifuged to obtain a precipitate. The precipitate was redissolved in water and centrifuged to obtain a supernatant, which was repeated for 3 times. The supernatants were combined and freeze-dried to obtain polysaccharides extracted by 4 mol / L NaOH solution.

7. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 1, characterized in that, The following step (6) is performed after step (5): (6) Protein removal: trichloroacetic acid was added to the water-extracted polysaccharides obtained in step (1), the CDTA-extracted polysaccharides obtained in step (2), the sodium carbonate-extracted polysaccharides obtained in step (3), the 1 mol / L NaOH solution-extracted polysaccharides obtained in step (4), and the 4 mol / L NaOH solution-extracted polysaccharides obtained in step (5), respectively. The precipitate was removed by centrifugation, and the supernatant was obtained to obtain polysaccharide solutions after protein removal. The polysaccharide solutions after protein removal were adjusted to pH 7, dialyzed, and then freeze-dried to obtain water-extracted polysaccharides after protein removal, CDTA-extracted polysaccharides after protein removal, sodium carbonate-extracted polysaccharides after protein removal, 1 mol / L NaOH solution-extracted polysaccharides after protein removal, and 4 mol / L NaOH solution-extracted polysaccharides after protein removal, respectively. In step (6), trichloroacetic acid is added to the solution system containing 10% mass concentration of trichloroacetic acid; In step (6), a dialysis bag with a molecular weight of 3500 Da is used for dialysis for 72 h; In step (6), the freeze-drying condition is -20℃ for 48 h.

8. The green tea cell wall polysaccharide with hypoglycemic activity prepared by the extraction and purification method according to any one of claims 1-7, characterized in that, The green tea cell wall polysaccharide with hypoglycemic activity comprises one or more of the following: polysaccharide extracted by CDTA after removing protein, polysaccharide extracted by sodium carbonate after removing protein, polysaccharide extracted by 1 mol / L NaOH solution after removing protein, and polysaccharide extracted by 4 mol / L NaOH solution after removing protein.

9. The green tea cell wall polysaccharide having blood glucose lowering activity according to claim 8, wherein, The total sugar content of the polysaccharide extracted by 4 mol / L NaOH solution after removing protein is 631.12 mg / g; The uronic acid content of the polysaccharide extracted by CDTA after removing protein is 260.75 mg / g; The molecular weight of polysaccharide extracted by the 1 mol / L NaOH solution after removing protein reaches 8.95 x 10 3 Da; The galacturonic acid content of the polysaccharide extracted by CDTA after removing protein reaches 49.83%; The glucosamine content of the polysaccharide extracted by sodium carbonate after removing protein reaches 45.32%; The glucuronidase content of the polysaccharide extracted by 1 mol / L NaOH solution after removing protein reaches 50.54%.

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