Method for extracting and purifying green tea cell wall polysaccharide with hypoglycemic activity and obtained green tea cell wall polysaccharide

Abstract

The invention relates to an extraction and purification method of green tea cell wall polysaccharide with hypoglycemic activity and the obtained green tea cell wall polysaccharide. The green tea is extracted by using water, CDTA, saturated sodium carbonate, 1M NaOH solution and 4M NaOH solution through a sequential extraction method to obtain five different tea polysaccharides; the green tea cell wall polysaccharide with the best hypoglycemic activity is screened out through activity inhibition of glycometabolism enzyme, and is further purified by gel column chromatography. Compared with the prior art, the method has the advantages that the reaction conditions in the whole operation process are mild, the activity of the green tea cell wall polysaccharide can be reserved to the greatest extent, the loss of polysaccharide components is reduced to the greatest extent, the extraction rate is high, the energy consumption is low, the solvent dosage is small, the operation is simple, and the economic cost is low; a foundation is laid for researching the physicochemical property and biological activity of the green tea cell wall polysaccharide, and the method has important significance on utilization of the green tea cell wall polysaccharide in the fields of food, medical care, cosmetics and the like.

Description

Technical Field

[0001] The invention relates to the technical field of tea polysaccharide extraction, and in particular to a method for extracting and purifying green tea cell wall polysaccharide with blood sugar lowering activity and the obtained green tea cell wall polysaccharide. Background Art

[0002] Plant cell walls are composed primarily of cellulose, hemicellulose, and pectin, as well as proteins and lignin bound to the cell wall. The components of the cell wall are cross-linked into a network through ionic and covalent bonds, forming a tough cell wall that resists physical penetration. Cellulose, the main load-bearing component of the cell wall, consists 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 (usually xyloglucan or arabinoxylan) molecules, which are hydrogen-bonded to the surface of the cellulose microfibrils and embedded in a pectin matrix. Pectin is a general term for the galacturonic acid-rich part of the cell wall. Pectin is usually rhamnosegalacturonic acid I, which consists of an alternating rhamnose and galacturonic acid backbone and various side groups, mainly galactose and arabinose. Pectin interacts or binds with (hemi)cellulose through hydrogen bonds or covalent bonds. A single extraction method cannot study the polysaccharides of each component in the cell wall. Therefore, the sequential extraction method is a suitable alternative method for studying the actual conditions of plant cell wall polysaccharides and intracellular polysaccharides. The sequential extraction method extracts various components in the cell wall by gradually using different chemical reagents. At present, the sequential extraction method has been widely used in the study of cell wall polysaccharides in different plant materials. Sequential extraction usually uses several chemical reagents, such as water, chelating agents, acids, sodium carbonate and alkali. Chelating agents (CDTA, EDTA, etc.) can destroy the calcium bridges cross-linked with pectin polysaccharides in the cell wall and release pectin polysaccharides. Sodium carbonate and dilute alkali can be used to extract pectin polysaccharides that are loosely or tightly bound to hemicellulose or cellulose through covalent bonding. The concentrated alkali soluble part is rich in xyloglucan and arabinose side chains. The analysis 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 to reveal the differences and interactions of cell wall polysaccharides in different plant materials and provide an important basis for the effective utilization and application of plant resources.

[0003] The chemical characterization of tea cell wall polysaccharides includes monosaccharide composition, molecular weight (Mw), monosaccharide sequence, location of glycosidic bonds, degree of branching, configuration and conformation of the entire molecule. To date, more than 120 tea polysaccharides have been extracted and separated 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 an average Mw ranging from 1.02 to 4940 kDa. Due to the influence of factors such as tea tree species, tea processing technology and tea polysaccharide separation 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. This aspect of research urgently needs further supplementation and improvement. CN104059160A discloses a Hericium erinaceus cell wall polysaccharide and a preparation method thereof. The Hericium erinaceus cell wall polysaccharide comprises water-soluble and alkali-soluble cell wall polysaccharides and is prepared by removing intracellular polysaccharides, crushing and water extraction, alkali extraction using a 0.1-1M NaOH solution and alcohol precipitation. However, this method cannot retain the activity of green tea cell wall polysaccharides to a great extent and minimize the loss of polysaccharide components, and cannot produce cell wall polysaccharides with different chemical compositions and structural characteristics.

[0004] Tea polysaccharides (TPS) in tea are widely used in the treatment of diabetes in traditional Chinese and Japanese medicine. As the incidence of diabetes continues to increase worldwide, the hypoglycemic effect of TPS has attracted widespread attention. TPS has a strong inhibitory effect on the enzyme system related to sugar metabolism in the human body and its hypoglycemic activity is concentration-dependent, which provides a reference for its reasonable dosage as a potential drug. These findings expand the potential application scope of natural plant polysaccharides in the treatment of diabetes and provide important clues and research directions for the development of new diabetes treatment drugs. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and to provide a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity and the obtained green tea cell wall polysaccharides, thereby shortening the extraction cycle and reducing the economic cost, obtaining green tea cell wall polysaccharides with high purity and strong activity, and preparing tea cell wall polysaccharides with different structural characteristics and hypoglycemic activity, providing a reference for the application of green tea cell wall polysaccharides, expanding the deep processing technology of green tea, and increasing the output value of green tea, which is of great significance in the fields of food, health products, medicine and health, and cosmetics.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity, the method comprising: sequentially extracting green tea with water, cyclohexanediaminetetraacetic acid (CDTA), sodium carbonate and sodium hydroxide solutions of different concentrations, and then undergoing steps such as impurity removal, dialysis and freeze drying to obtain cell wall polysaccharides with hypoglycemic activity.

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

[0009] (1) Grind the green tea leaves and sieve them to obtain tea powder, add ultrapure water, extract at 37°C for 2h, then centrifuge to obtain the supernatant, repeat the above steps with the precipitate until the sugar content in the supernatant is no longer detected, combine the supernatants for dialysis, the supernatant is the extract, retain the tea residue and dry it to obtain dried tea residue for subsequent steps; add ethanol, let it stand at 4°C overnight, then centrifuge to collect the precipitate; redissolve the precipitate with water and centrifuge to obtain the supernatant, repeat 3 times, combine the supernatants, and obtain water-extracted tea polysaccharides (W-TPS) after freeze-drying.

[0010] (2) Add 70% ethanol to the dried tea residue obtained in step (1), shake on a shaker, centrifuge the solution to collect the precipitate, wash the precipitate repeatedly with 70% ethanol for 3-5 times, wash it once with anhydrous ethanol, and then place the precipitate in a fume hood to dry to obtain alcohol-insoluble tea residue; add Tris-maleic acid buffer, 2mmol / L CaCl2 and amylase to the alcohol-insoluble tea residue, shake on a shaker to obtain tea residue from which starch has been removed, wash the tea residue from which starch has been removed for 3-5 times, centrifuge to obtain the precipitate, dry it, add cyclohexanediaminetetraacetic acid solution to the dried tea residue from which starch has been removed, shake on a shaker, centrifuge, and dialyze the supernatant to obtain a precipitate and an extract, wash the precipitate for 3-5 times, and then dry it to obtain tea residue extracted with CDTA; after the dialysis is completed, add ethanol, let it stand at 4°C overnight, and then centrifuge to obtain the precipitate. The precipitate was re-dissolved in 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).

[0011] (3) Add the tea residue obtained in step (2) after CDTA extraction to a saturated sodium carbonate solution, add 25 mmol / L NaBH4 to the solution, shake on a shaker, centrifuge and dialyze the supernatant to obtain a precipitate and an extract, wash the precipitate 3-5 times and then dry it to obtain the tea residue extracted with saturated sodium carbonate; add ethanol, let it stand at 4°C overnight, and then centrifuge to obtain the precipitate; redissolve the precipitate with water and centrifuge to obtain the supernatant, repeat this process 3 times, combine the supernatants, and freeze-dry to obtain sodium carbonate-extracted polysaccharides (Na2CO3-extracted tea polysaccharides, N-TPS).

[0012] (4) adding 1 mol / L NaOH solution to the tea residues extracted with saturated sodium carbonate obtained in step (3), and adding 25 mmol / L NaBH4 to the solution, shaking on a shaker, and dialyzing the supernatant after centrifugation to obtain a precipitate and an extract, washing the precipitate 3-5 times, and drying it to obtain tea residues extracted with 1 mol / L NaOH solution; adding ethanol, standing at 4°C overnight, and collecting the precipitate by centrifugation; re-dissolving the precipitate with water, and then centrifuging to obtain the supernatant, repeating this process 3 times, combining the supernatants, and freeze-drying to obtain polysaccharides extracted with 1 mol / L NaOH solution (1M NaOH-extracted tea polysaccharides, 1M-TPS).

[0013] (5) Adding the tea residue obtained in step (4) after extraction with 1 mol / L NaOH solution to 4 mol / L NaOH solution, and adding 25 mmol / L NaBH4 to the solution, shaking on a shaker, and dialyzing the supernatant after centrifugation to obtain a precipitate and an extract; adding ethanol after rotary evaporation concentration, standing at 4°C overnight, and centrifuging to obtain a precipitate; re-dissolving the precipitate with water and centrifuging to obtain the supernatant, repeating 3 times, combining the supernatants, and freeze-drying to obtain polysaccharides extracted with 4 mol / L NaOH solution (4M NaOH-extracted tea polysaccharides, 4M-TPS).

[0014] (6) Protein removal: trichloroacetic acid is 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), respectively, and the precipitate is removed by centrifugation, and the supernatant is collected to obtain a protein-removed polysaccharide solution. The protein-removed polysaccharide solution is 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.

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

[0016] The green tea leaves are ground and passed through an 80-mesh sieve to obtain tea powder, and ultrapure water is added to the tea powder at a solid-liquid ratio of 1:20, and the mixture is extracted with water at 37°C for 2 hours, followed by centrifugation to obtain the supernatant, and the precipitate is extracted repeatedly with the above steps until the sugar content is no longer detected in the supernatant, and the supernatants are combined and dialyzed for 72 hours using a 7000Da dialysis bag, the supernatant being the extract, and 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 the mixture is allowed to stand at 4°C overnight, followed by centrifugation to collect the precipitate; the precipitate is redissolved with water and then centrifuged to obtain the supernatant, and the process is repeated 3 times, the supernatants are combined, and water-extracted tea polysaccharides (W-TPS) are obtained after freeze-drying.

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

[0018] The dried tea residue obtained in step (1) is added with 70% ethanol at a solid-liquid ratio of 1:10, and the mixture is shaken on a shaker at room temperature for 12 hours. The solution is centrifuged to collect a precipitate, and the precipitate is repeatedly washed with 70% ethanol for 3-5 times, and washed once with anhydrous ethanol to remove alcohol-soluble substances. The precipitate is then 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 at a solid-liquid ratio of 1:10, wherein the concentration of the Tris-maleic acid buffer is 20 mmol / L, and the pH of the Tris-maleic acid buffer is 6.9 (20 mmol / L, pH=6.9), and 2 mmol / L CaCl2 and amylase, amylase and alcohol-insoluble tea residue in a ratio of 20 mg amylase / 5 g tea residue (20 mg amylase / 5 g tea residue), shake on a shaker at 37°C for 4 hours to remove starch, obtain tea residue with starch removed, wash the tea residue with starch removed 3-5 times, centrifuge to obtain a precipitate, place it in a fume hood to dry, add cyclohexanediaminetetraacetic acid solution (CDTA solution, the solvent is pure water) to the dried tea residue with starch removed at a ratio of 1:10 between solid and liquid, and the concentration of the cyclohexanediaminetetraacetic acid solution is 50mmol / L, the pH of the cyclohexanediaminetetraacetic acid solution is 6.5 (50mmol / L, pH=6.5), shake extraction at 37°C for 12h, then centrifuge and dialyze the supernatant for 72h using a 7000Da dialysis bag to obtain a precipitate and an extract, wash the precipitate 3-5 times, place it in a fume hood to dry, and obtain tea residue extracted by CDTA; after dialysis, add ethanol at a ratio of extract: ethanol = 1:3, let it stand at 4°C overnight, and then centrifuge to obtain the precipitate. The precipitate is re-dissolved in water and centrifuged to obtain the supernatant, repeated 3 times, the supernatant is combined, and the CDTA-extracted tea polysaccharides (C-TPS) are obtained after freeze-drying.

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

[0020] The tea residue obtained in step (2) after CDTA extraction is added to a saturated sodium carbonate solution at a material-liquid ratio of 1:10, and 25 mmol / L NaBH4 is added to the solution to protect the polysaccharide, and the extraction is performed on a shaking table at 37°C for 12 hours. After centrifugation, the supernatant is dialyzed using a 7000Da dialysis bag for 72 hours to obtain a precipitate and an extract, and the precipitate is washed 3-5 times and then dried to obtain the tea residue after saturated sodium carbonate extraction; ethanol is added at a volume ratio of extract: ethanol = 1:3, and the mixture is allowed to stand at 4°C overnight, and then centrifuged to obtain the precipitate; the precipitate is redissolved with water and then centrifuged to obtain the supernatant, and the process is repeated 3 times. The supernatants are combined and freeze-dried to obtain sodium carbonate-extracted polysaccharides (Na2CO3-extracted tea polysaccharides, N-TPS).

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

[0022] The tea residue obtained in step (3) after extraction with saturated sodium carbonate was added with 1 mol / L NaOH solution at a material-liquid ratio of 1:10, and 25 mmol / L NaBH4 was added to the solution to protect the polysaccharide, and the extraction was performed on a shaking table at 37°C for 12 hours. After centrifugation, the supernatant was dialyzed for 72 hours using a 7000Da dialysis bag (7000DA, pH=6-7) to obtain a precipitate and an extract, and 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: ethanol = 1:3, and the mixture was allowed to stand at 4°C overnight, and the precipitate was collected by centrifugation; the precipitate was redissolved with water and then centrifuged to obtain the supernatant, and the process was repeated 3 times. The supernatants were combined and freeze-dried to obtain polysaccharides extracted with 1 mol / L NaOH solution (1M NaOH-extracted tea polysaccharides, 1M-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 into 4 mol / L NaOH solution at a material-liquid ratio of 1:10, and 25 mmol / L NaBH4 was added into the solution, and the mixture was shaken and extracted at 37°C for 12 h. After centrifugation, the supernatant was dialyzed for 72 h using a 7000Da dialysis bag (7000DA, pH=6-7) to obtain a precipitate and an extract. After rotary evaporation and concentration, ethanol was added at a volume ratio of 1:3 between the water extract and ethanol, and the mixture was allowed to stand at 4°C overnight, and the precipitate was obtained by centrifugation. The precipitate was redissolved with water, and the supernatant was obtained by centrifugation. The process was repeated 3 times, and the supernatants were combined. After freeze-drying, polysaccharides extracted with 4 mol / L NaOH solution (4MNaOH-extracted tea polysaccharides, 4M-TPS) were obtained.

[0025] Furthermore, in step (6), trichloroacetic acid is added to the solution system to contain 10% mass concentration of trichloroacetic acid; in step (6), dialysis is performed for 72 hours using a 3500Da dialysis bag.

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

[0027] Furthermore, all the above-mentioned lyophilization conditions not specified were -20°C and 48h.

[0028] The obtained green tea cell wall polysaccharide is obtained by the above-mentioned extraction and purification method. The green tea cell wall polysaccharide with hypoglycemic activity includes one or more of the polysaccharide extracted by CDTA after protein removal, the polysaccharide extracted by sodium carbonate after protein removal, the polysaccharide extracted by 1 mol / L NaOH solution after protein removal, and the polysaccharide extracted by 4 mol / L NaOH solution after protein removal.

[0029] Furthermore, the total sugar content of the polysaccharide extracted by 4 mol / L NaOH solution after protein removal is 631.12 mg / g; the uronic acid of the polysaccharide extracted by CDTA after protein removal is 265.81 mg / g; the molecular weight of the water-extracted polysaccharide after protein removal reaches 2.55×10 5 The molecular weight of the polysaccharide extracted by sodium carbonate after protein removal reaches 8.95×10 3 Da; the galacturonic acid content of the polysaccharide extracted by CDTA after the protein removal reaches 49.83%; the glucosamine content of the polysaccharide extracted by sodium carbonate after the protein removal reaches 45.32%; the glucuronic acid content of the polysaccharide extracted by 1 mol / L NaOH solution after the protein removal reaches 50.54%.

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

[0031] Furthermore, after protein removal, the extraction rate of CDTA was the highest, reaching 1.93%; the total sugar content of polysaccharides (cell wall polysaccharides) extracted by 4 mol / L NaOH solution was the highest, at 631.12 mg / g; the uronic acid content of polysaccharides extracted by CDTA was the highest, at 265.81 mg / g; the molecular weight of water-extracted polysaccharides was the highest, at 2.55×10 5 Da, while the molecular weight of polysaccharide extracted by sodium carbonate was the lowest, which was 8.95×10 3 Da.

[0032] Furthermore, the monosaccharide composition results showed that there were significant differences in the monosaccharide composition of polysaccharides extracted by different solvents. C-TPS had the highest galacturonic acid content of 49.83%, N-TPS had the highest glucosamine content of 45.32%, and 1M-TPS had the highest glucuronic acid content of 50.54%. Through composition analysis, each part corresponded to different polysaccharide components in the tea cell wall. The CDTA extraction part was pectin, the sodium carbonate and 1MNaOH extraction parts might be pectin and hemicellulose complexes, and the 4MNaOH extraction polysaccharides were mainly composed of hemicellulose and cellulose. The infrared spectrum results showed that the polysaccharides had similar peak shapes, except that the characteristic peaks of uronic acid appeared in the water-extracted polysaccharides and CDTA-extracted polysaccharides, and the characteristic peaks of pyranose were stronger. The scanning electron microscopy results showed that the morphologies of the polysaccharides were quite different. The electron microscopy observation results showed the differences between the structures and morphologies of different polysaccharides, which indicated that the structure of polysaccharides was closely related to different extraction methods, the composition of polysaccharides and the molecular weight.

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

[0034] 1) The present invention provides 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, and the activity of the green tea cell wall polysaccharides can be retained to a great extent and the loss of polysaccharide components can be reduced to a maximum extent. It has the advantages of high extraction rate, low energy consumption, small amount of solvent, simple operation and low economic cost, etc., which lays a 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 sequentially extracted with CDTA, sodium carbonate and sodium hydroxide solutions of different concentrations (1M and 4M) show 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, which are sequentially extracted to obtain 5 polysaccharides with different structures and components (polysaccharides extracted with water, polysaccharides extracted with CDTA, polysaccharides extracted with sodium carbonate, polysaccharides extracted with 1M sodium hydroxide, and polysaccharides extracted with 4M sodium hydroxide). The polysaccharides extracted with CDTA, the polysaccharides extracted with sodium carbonate, the polysaccharides extracted with 1M sodium hydroxide, and the polysaccharides extracted with 4M sodium hydroxide are all cell wall polysaccharides, and the structures of the 5 polysaccharides are different. Among them, the polysaccharide fragments extracted with 4M sodium hydroxide are more complete and thinner. The obtained polysaccharides all have an inhibitory effect on α-glucosidase, and the inhibition rate of the polysaccharides extracted with 4M sodium hydroxide reaches 51.1%, which can retain the activity of green tea cell wall polysaccharides to a great extent and minimize the loss of polysaccharide components. The purified 4M sodium hydroxide-extracted polysaccharides include neutral sugars, uronic acids, and reducing sugars, which are resistant to decomposition in the digestive environment of the human mouth and esophagus and can safely reach the stomach and intestines, especially interacting with intestinal microorganisms, which may further regulate carbohydrate metabolism by regulating the intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure is a schematic diagram of the process of extracting and purifying the green tea cell wall polysaccharide with hypoglycemic activity according to the present invention.

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

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

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

[0041] Figure 5 This is the elution curve of 4M-TPS separated and purified by DEAE-Sepharose FF column. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with specific embodiments, but is by no means intended to limit the present invention. Features such as preparation methods, materials, structures or composition ratios not explicitly described in this technical solution are considered to be common technical features disclosed in the prior art.

[0043] The green tea is Yichang stir-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 for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity comprises: sequentially extracting green tea with water, cyclohexanediaminetetraacetic acid (CDTA), sodium carbonate and sodium hydroxide solutions of different concentrations, and then removing impurities, dialysis and freeze-drying to obtain cell wall polysaccharides with hypoglycemic activity. The method specifically comprises the following steps:

[0046] (1) After grinding green tea leaves, pass through an 80-mesh sieve to obtain tea powder, add ultrapure water at a solid-liquid ratio of 1:20 to the tea powder, extract at 37°C for 2 hours, then centrifuge to obtain the supernatant, repeat the above steps for the precipitate to extract until the sugar content in the supernatant is no longer detected, combine the supernatants and use a 7000Da dialysis bag for 72 hours, the supernatant is the extract, retain the tea residue and dry it to obtain dried tea residue for subsequent steps; add ethanol at a volume ratio of extract: ethanol = 1:3, let stand at 4°C overnight, then centrifuge to collect the precipitate; redissolve the precipitate with water and centrifuge to obtain the supernatant, repeat 3 times, combine the supernatants, and obtain water-extracted tea polysaccharides (W-TPS) after freeze-drying.

[0047] (2) adding 70% ethanol to the dried tea residue obtained in step (1) at a solid-liquid ratio of 1:10, shaking on a shaker at room temperature (shaking rate of 100-200 rpm, the same below) for 12 h, centrifuging the solution to collect the precipitate, washing the precipitate repeatedly with 70% ethanol for 3-5 times, and washing it once with anhydrous ethanol to remove alcohol-soluble substances, and then placing the precipitate in a fume hood to dry to obtain alcohol-insoluble tea residue; adding Tris-maleic acid buffer to the alcohol-insoluble tea residue at a solid-liquid ratio of 1:10, wherein the concentration of the Tris-maleic acid buffer is 20 mmol / L, and the pH of the Tris-maleic acid buffer is 6.9 (20 mmol / L, pH=6.9), adding 2 mmol / L CaCl2 and amylase, amylase and alcohol-insoluble tea residue in a ratio of 20 mg amylase / 5 g tea residue (20 mg amylase / 5 g tea residue), shake on a shaker at 37°C for 4 hours to remove starch, obtain tea residue with starch removed, wash the tea residue with starch removed 3-5 times, centrifuge to obtain a precipitate, place it in a fume hood to dry, add cyclohexanediaminetetraacetic acid solution after drying at a solid-liquid ratio of 1:10, the concentration of the cyclohexanediaminetetraacetic acid solution is 50 mmol / L, the The pH of the cyclohexanediaminetetraacetic acid solution was 6.5 (50mmol / L, pH=6.5). The extraction was performed on a shaking table at 37°C for 12h, followed by centrifugation and the supernatant was dialyzed for 72h using a 7000Da dialysis bag (7000Da) to obtain a precipitate and an extract. The precipitate was washed 3-5 times and placed in a fume hood to dry to obtain tea residue extracted with CDTA. After dialysis, ethanol was added at a ratio of extract: ethanol = 1:3, and the mixture was allowed to stand at 4°C overnight, and then centrifuged to obtain the precipitate. The precipitate was re-dissolved in water and centrifuged to obtain the supernatant. This was repeated 3 times, and the supernatants were combined and freeze-dried to obtain CDTA-extracted teapolysaccharides (C-TPS).

[0048] (3) The tea residue obtained in step (2) after CDTA extraction is added to a saturated sodium carbonate solution at a material-liquid ratio of 1:10, and 25 mmol / L NaBH4 is added to the solution to protect the polysaccharide, and the extraction is performed on a shaking table at 37°C for 12 hours. After centrifugation, the supernatant is dialyzed using a 7000Da dialysis bag for 72 hours (7000DA, pH=6-7) to obtain a precipitate and an extract, and the precipitate is washed 3-5 times and then dried to obtain the tea residue after saturated sodium carbonate extraction; ethanol is added at a volume ratio of extract: ethanol = 1:3, and the mixture is allowed to stand at 4°C overnight, and then centrifuged to obtain the precipitate; the precipitate is redissolved in water and then centrifuged to obtain the supernatant, and the process is repeated 3 times. The supernatants are combined and freeze-dried to obtain sodium carbonate-extracted polysaccharides (Na2CO3-extracted tea polysaccharides, N-TPS).

[0049] (4) The tea residue obtained in step (3) after extraction with saturated sodium carbonate was added with 1 mol / L NaOH solution at a material-liquid ratio of 1:10, and 25 mmol / L NaBH4 was added to the solution to protect the polysaccharide, and the extraction was performed on a shaking table at 37°C for 12 h. After centrifugation, the supernatant was dialyzed for 72 h using a 7000Da dialysis bag (7000DA, pH=6-7) to obtain a precipitate and an extract, and 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: ethanol = 1:3, and the mixture was allowed to stand at 4°C overnight, and the precipitate was collected by centrifugation; the precipitate was redissolved with water and centrifuged to obtain the supernatant, and the process was repeated 3 times. The supernatants were combined and freeze-dried to obtain polysaccharides extracted with 1 mol / L NaOH solution (1M NaOH-extracted tea polysaccharides, 1M-TPS).

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

[0051] (6) Protein removal: trichloroacetic acid is 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-extracted polysaccharide obtained in step (4), and the 4 mol / L NaOH-extracted polysaccharide obtained in step (5) respectively until the solution system contains 10% trichloroacetic acid by mass, the precipitate is removed by centrifugation, and the supernatant is collected to obtain a protein-removed polysaccharide solution. The protein-removed polysaccharide solution is adjusted to pH 7, dialyzed for 72 h using a 3500Da dialysis bag, and then freeze-dried to obtain the water-extracted polysaccharide after protein removal, the CDTA-extracted polysaccharide after protein removal, the sodium carbonate-extracted polysaccharide after protein removal, the 1 mol / L NaOH-extracted polysaccharide after protein removal, and the 4 mol / L NaOH-extracted polysaccharide after protein removal, respectively.

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

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

[0054] 1. Determination of basic ingredients

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

[0056] Determination of uronic acid content: m-hydroxybiphenyl method was used.

[0057] Determination of polyphenol content: refer to the spectrophotometer method in GBT8313-2018.

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

[0059] 2. Molecular weight determination

[0060] The relative molecular weights of water-extracted polysaccharides after protein removal and four tea cell wall polysaccharides after protein removal (polysaccharides extracted by CDTA after protein removal, polysaccharides extracted by sodium carbonate after protein removal, polysaccharides extracted by 1 mol / L NaOH solution after protein removal, and polysaccharides extracted by 4 mol / L NaOH solution after protein removal) were determined by high performance gel permeation chromatography: the samples were dissolved in ultrapure water and prepared to a concentration of 2 mg / mL. After taking 1 mL of the sample, centrifuged for 10 min (10000 r / min) and passed through a 0.22 μm filter membrane. 30 μL of the sample was injected into the HPGPC 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). The column temperature and detector temperature were controlled at 25 ° C and 30 ° C, respectively, and the flow rate was 0.3 mL / min.

[0061] Dextran standards of different molecular weights (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 to record the chromatogram results. A standard curve was drawn with retention time tR (min) as the horizontal axis and the logarithm (lgMw) of the weight average molecular weight (Mw) of dextran as the vertical axis. The molecular weight of the polysaccharide sample was calculated based on the retention time of the polysaccharide sample and the standard curve.

[0062] 3. Determination of monosaccharide composition

[0063] Ion chromatography was used to qualitatively and quantitatively detect the monosaccharide composition in 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 place in a 110°C oven for hydrolysis for 4 hours. After hydrolysis, place in a nitrogen blower to dry (temperature <45°C). After drying, add 3 mL of methanol solution, and repeat the methanol-nitrogen blow operation 3 times to remove TFA. Dissolve the hydrolyzed sample in 2 mL of ultrapure water, centrifuge for 5 minutes (5000 r / min), and retain the supernatant.

[0066] (2) Testing conditions:

[0067] The Dionex ICS 5000+ system was used for analysis, and the flow rate was set to 0.45 mL / min. Sodium hydroxide with a concentration of 2 mmol / L 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 treated according to the above sample pretreatment method, and the peak time and peak area of ​​the standards were compared with the measured polysaccharide samples to determine the type of monosaccharide and determine the molar concentration of each monosaccharide.

[0068] 4. Infrared spectrum scanning

[0069] The five tea polysaccharides obtained by sequential extraction method (water-extracted polysaccharides after protein removal and four tea cell wall polysaccharides after protein removal (polysaccharides extracted by CDTA after protein removal, polysaccharides extracted by sodium carbonate after protein removal, polysaccharides extracted by 1 mol / L NaOH solution after protein removal, and polysaccharides extracted by 4 mol / L NaOH solution after protein removal)) were qualitatively analyzed by Fourier transform infrared spectroscopy: a small amount of dry polysaccharide sample was ground evenly with KBr into fine powder using an agate mortar, placed in a tablet press and pressed into a transparent thin sheet, and then placed on a Fourier transform infrared spectrometer for sample testing. The spectral scanning range was: 4000-400cm -1 .

[0070] 5. Scanning electron microscope

[0071] Take a small amount of dry test samples (water-extracted polysaccharides after protein removal and four 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, polysaccharides extracted from 4 mol / L NaOH solution after protein removal)), evenly spread them on the conductive glue on the sample table, and then place the samples in a vacuum spray coating instrument to coat a layer of gold on the surface as a conductive film. Subsequently, the morphology and structure of the samples were observed using a scanning electron microscope, and images were collected at appropriate electron microscope magnifications for analysis.

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

[0073] The α-glucosidase inhibitory activity of tea polysaccharides (water-extracted polysaccharides after protein removal and four 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)) was determined by colorimetric method using an enzyme marker. The experimental groups were divided into blank group Ab (no test solution and enzyme solution), control group Ac (no test solution), test solution group As, and test solution blank group Asb (no enzyme solution). This experimental method uses nitrophenyl-α-D-pyranoglucoside (PNPG) as a colorimetric substrate, and the entire experimental process is carried out in a 96-well plate. In the experiment, 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 sample solution of different concentrations were added to the plate in sequence according to different groups (the concentration gradient of tea polysaccharide test solution was set to 0.1, 1, 10, 100, 1000 μg / mL). After low-speed oscillation, the plate was placed in a 37°C water bath for 10 min. Then, 20 μL of PNPG saturated solution was added to each well as a reaction substrate to start the enzyme reaction. After low-speed oscillation, the plate was placed in a 37°C water bath for 20 min for full reaction. After the reaction, 80 μL of Na2CO3 solution (0.2 mol / L) was quickly added to the system to terminate the catalytic reaction. After low-speed oscillation, the absorbance of each well was measured on the microplate reader, and the measurement wavelength was 405 nm. Finally, the inhibition rate of the sample on α-glucosidase was calculated according to the following formula:

[0074] I(%)={[(Ac-Ab)-(As-Asb)] / (Ac-Ab)}×100%

[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 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)) was determined by 3,5-dinitrosalicylic acid method. The experimental groups were blank group Ab (no test solution and enzyme solution), control group Ac (no test solution), test solution group As, and test solution blank group Asb (no enzyme solution). The experimental method uses starch solution as a chromogenic substrate. In the experiment, phosphate buffer (67mmol / L, pH=6.8) was added to each stoppered graduated test tube in turn, followed by 50μL of the sample solution to be tested (the concentration gradient of the tea polysaccharide test solution was set to 0.05, 0.1, 0.5, 1, 5mg / mL) and 50μL of 2.5U / mL α-amylase (dissolved in PBS buffer, pH=6.8), the above mixture was mixed with a vortex oscillator and placed at 37.5℃ for 10min, then 100μL of 1% starch solution (dissolved in PBS buffer, pH=6.8) was added and mixed and placed at 37.5℃ for 10min. After the reaction was completed, 200μL of DNS solution was added and reacted under boiling water bath conditions for 5min, and then quickly placed in cold water to cool to room temperature to terminate the reaction. After oscillation, the absorbance was measured using a UV spectrophotometer, with PBS buffer as a blank, and the OD value was measured at 540nm after dilution. Finally, the inhibition rate of the sample on α-amylase was calculated according to the following formula:

[0077] I(%)={[(Ac-Ab)-(As-Asb)] / (Ac-Ab)}×100%

[0078] 8. Determination of sucrase inhibition activity

[0079] The sucrase inhibitory activity of tea polysaccharide samples was determined according to the method described previously for determining the inhibitory activity of α-amylase. The experimental groups were divided into blank group Ab (no test solution and enzyme solution), control group Ac (no test solution), test solution group As, and test solution blank group Asb (no enzyme solution). The experimental method used sucrose solution as the substrate. In the experiment, PBS buffer (67mmol / L, pH=6.8) was added to each stoppered graduated test tube in turn, followed by adding 50μL of the test sample solution of different concentrations (the concentration gradient of the tea polysaccharide test solution was set to 0.05, 0.1, 0.5, 1, and 5mg / mL) and 10U / mL sucrase (dissolved in PBS buffer, pH=6) 50μL, the above mixed solution was mixed using a vortex oscillator and placed at 37.5℃ for 10min, and then 100μL of 0.5% sucrose solution (dissolved in PBS buffer, pH=6) was added and mixed and placed at 37.5℃ for 10min. After the reaction is completed, add 200 μL DNS solution and react in a boiling water bath for 5 minutes, then quickly place in cold water and cool to room temperature to terminate the reaction. After oscillation, use a UV spectrophotometer to measure the absorbance, use PBS buffer as a blank, and measure the OD value at 540 nm after dilution. Finally, calculate the sample's inhibition rate of sucrase according to the following formula:

[0080] I(%)-{[(Ac-Ab)-(As-Asb)] / (Ac-Ab)}×100%

[0081] 9. Isolation and purification of cell wall polysaccharides from Yichang stir-fried green tea

[0082] The crude polysaccharide sample obtained by in vitro hypoglycemic enzyme inhibition activity screening was dissolved in phosphate buffer solution PBS (pH = 6.8, 0.02mol / L) until the polysaccharide solution concentration was 50mg / mL, and after filtering through a 0.22μm filter membrane, the gel column equipped with DEAE-Sepharose Fast Flow gel filler was rinsed to equilibrium and then loaded with the sample, and the polysaccharide solution was slowly and evenly added to the anion gel column (2.5cm×60cm) with a dropper, and the liquid flow switch was turned on after stabilization, and the PBS buffer solution containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5mol / L NaCl was used in turn for gradient elution, and the concentration gradient was switched after elution until no sugar was detected. The experiment used an automatic collector to collect the eluate (2mL / min, 2min / tube), and the total sugar content of the collected liquid was determined using the phenol-sulfuric acid method, and the elution curve was drawn with its OD value at 490nm as the ordinate and the number of tubes as the abscissa. The eluates under a single peak were combined, concentrated, and dialyzed for 72 h using a 3500 Da dialysis bag. The polysaccharide sample was obtained after freeze-drying.

[0083] 10. Simulated in vitro digestion experiment

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

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

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

[0087] First, 100.0 mL of sample solution (20.0 mg / mL), 15 mg of 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 the salivary digestion process. Subsequently, 150.0 mL of salivary digestion solution, 0.2 g of pepsin (3000 U / g) and 150.0 mL of simulated gastric juice were mixed and quickly adjusted to pH = 3.0 to trigger simulated gastric digestion, and incubated at 37°C for 4 h to simulate the gastric juice digestion process. Finally, 180.0 mL of gastric juice digestion sample, 1.47 g of bile salts, 1.8 g of pancreatin (4000 U / g) and 180.0 mL of simulated intestinal juice were mixed and quickly adjusted to pH = 7.0 to trigger simulated intestinal digestion. Incubated at 37°C for 4 h to simulate the intestinal juice digestion process. After the simulated digestion, 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 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] Digestibility calculation formula:

[0092]

[0093] The above test results are as follows:

[0094] 1. Yichang Chaoqing Green Tea Water and Cell Wall Polysaccharide Extraction Rate

[0095] Table 1 Extraction rate of cell wall polysaccharides from Yichang Chaoqing green tea (%)

[0096]

[0097] Note: Different superscript letters represent significant differences among the groups (p<0.05), and each row was analyzed for significant differences within the group.

[0098] As shown in Table 1, the cell wall polysaccharides of Yichang stir-fried green tea were extracted by sequential extraction. After preliminary extraction, it was found that the yields of polysaccharides extracted by different solvents were very different. Among them, the water extraction and CDTA extraction yields were higher, 1.98% and 2.24% respectively, while the saturated Na2CO3 extraction yield was lower, only 0.61%. After component determination, it was found that except for the polysaccharide components extracted by CDTA, the extracted polysaccharides all contained high protein content, so the initial polysaccharide samples were deproteinized. The results of deproteinization showed that the extraction rate of polysaccharides in the water extraction part was higher during the initial extraction, while after deproteinization, the extraction rate of polysaccharides in the water extraction dropped to 1.48%, and the sample loss was large during the deproteinization process. The polysaccharide content extracted by CDTA was still as high as 1.93% after deproteinization. Similarly, the polysaccharide parts extracted by saturated Na2CO3, 1M NaOH and 4M NaOH had less loss after deproteinization, which indicates that some soluble proteins have been extracted in the water extraction of tea leaves. Overall, water extraction, CDTA extraction and 4M NaOH have higher extraction rates for polysaccharides.

[0099] 2. Yichang Chaoqing Green Tea mentioned the basic components of cell wall polysaccharides

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

[0101]

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

[0103] The compositions of polysaccharide fractions extracted by different solvents by sequential extraction method were significantly different. The total sugar content of cell wall polysaccharides extracted by water and 4M NaOH was higher, 577.13 mg / g and 631.12 mg / g, respectively, while the polysaccharide fraction extracted by sodium carbonate had a lower total sugar content of 192.71 mg / g. At the same time, the polysaccharides extracted by water and CDTA contained higher uronic acid content, 265.81 mg / g and 260.75 mg / g, respectively. The polyphenol content in the polysaccharide fractions obtained by different extraction methods was very different. The polysaccharide fraction extracted by sodium carbonate contained a higher polyphenol content of 83.46 mg / g, while the fraction extracted by CDTA contained almost no polyphenol, indicating that chelating agents may destroy the binding of polysaccharides and polyphenols in the polysaccharides extracted by CDTA.

[0104] 3. Molecular weight determination

[0105] The weight average molecular weight of the 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 in sequence, and the chromatograms were collected to obtain the retention time tR of various molecular weight dextran standards. The standard curve was drawn with the retention time tR (min) as the abscissa and the logarithm of the weight average molecular weight Mw of dextran (lgMw) as the ordinate. The standard linear equation was obtained:

[0106] lgMw=-0.2347tR+12.074(R 2 =0.993)

[0107] The weight average molecular weight of the polysaccharide sample was calculated based on the retention time of the polysaccharide sample and the molecular weight standard curve. The molecular weight of W-TPS was 2.55×10 5 Da, molecular weight of C-TPS 5.8×10 4 Da, molecular weight of N-TPS 1.18×10 4 Da, molecular weight of 1M-TPS 8.95×10 3 The molecular weight of 4M-TPS is 1.35×10 5 Da, from the above results, it can be seen that the molecular weight of water-extracted polysaccharides is the largest. During the sequential extraction process, the molecular weight of polysaccharides shows a downward trend, indicating that the chelating agent and alkali will gradually hydrolyze and release the large molecular weight polysaccharides 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 in Figure 2. Figure 3The monosaccharide composition results are shown in Table 3. W-TPS is composed 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 structure of W-TPS. Arabinose mainly comes from the decomposition and release of lignocellulose in the cell wall of tea leaves during water extraction. The high content of galactose may be due to the transformation of the residues of other monosaccharides into galactose residues during the alkali extraction process.

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

[0111] N-TPS is composed of rhamnose, arabinose, glucosamine, glucose, galacturonic acid and glucuronic acid, with a molar ratio of 1.00:0.67:2.02:0.29:0.13:0.36, of which rhamnose, arabinose and glucosamine are the main monosaccharides. N-TPS has the highest glucosamine content (45.32%), and its main form in the polysaccharide of tea cell wall is chitosan, which is the main component of cellulose and hemicellulose. Under alkaline conditions, the hydrogen bonds between hemicellulose and cellulose will break due to cell wall swelling, proving that sodium carbonate can effectively destroy the structure of tea cell wall and dissolve polysaccharides during the extraction process.

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

[0113] 4M-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, of which arabinose, glucosamine and glucose are the main monosaccharides. Fructose was detected for the first time in these five polysaccharides and is the main component of pectin. They are connected by α-1,4- and α-1,2-glycosidic bonds, indicating that strong alkali can fully destroy the glycosidic bonds between pectins in the cell wall and dissolve and release them. The monosaccharide composition of tea polysaccharides is affected by the extraction process, and the extraction method is the main reason for the difference in monosaccharide composition between the above-mentioned different 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 Yichang Chaoqing green tea water mentioned cell wall polysaccharide monosaccharide composition (mol%)

[0116]

[0117] Note: -: indicates not measured.

[0118] 5. Infrared spectroscopy analysis

[0119] Infrared spectrum test results are as follows Figure 4 A. The five polysaccharide samples have similar absorption peaks in multiple wavenumber ranges, -1 To 3400cm -1 The peaks in this wavenumber range represent the stretching vibration of hydroxyl (-OH). -1 The peak at 1630cm 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 represents the stretching vibration of the non-resonant double bond (C=C), indicating the presence of esterified carboxyl groups or unsaturated bonds in the polysaccharide component. -1 The peak at 1100cm represents the bending vibration of the CH bond, which may come from the carbon-hydrogen bond in the polysaccharide molecule. -1 -1010cm -1 The peaks at represent the stretching vibrations of the sugar rings, which are attributed to the pyran rings in the polysaccharide molecules.-1 The peak at 660 cm usually represents the out-of-plane vibration of CH of olefins or aromatics. -1 -600cm -1 The peaks at usually represent bending vibrations of the sugar rings, and these peaks also indicate association with the pyran ring structure in the polysaccharide molecules. Figure 4 The polysaccharide components extracted by water, CDTA and sodium carbonate were at 1742 cm -1 and 1630cm -1 There are double peaks observed at 1740cm -1 The peak at 1630 cm-1 is usually associated with the C=O vibration of the methyl esterified carboxyl group (COOR), while the peak at 1630 cm-1 is usually associated with the C=O vibration of the methyl esterified carboxyl group (COOR). -1 The peak at 915 and 830 cm-1 is usually corresponding to the C=O vibration of the ionic carboxyl group (COO-). The presence of this peak indicates that the sample contains uronic acid structure; -1 The absorption peaks showed that the polysaccharide existed in the form of α-D-pyranose; the water-extracted polysaccharide, CDTA-extracted polysaccharide and sodium carbonate-extracted polysaccharide showed absorption peaks at 1100, 1070 and 1010 cm -1 Multiple strong absorption peaks appear at the bottom, indicating the presence of pyran ring structure in the polysaccharide molecule.

[0120] 6. Scanning Electron Microscope (SEM)

[0121] The five polysaccharide components obtained by the sequential extraction method were analyzed using scanning electron microscopy (SEM). Figure 4 As shown in Figure C, the observation results show that the water-extracted polysaccharide (W-TPS) presents a flocculated state with a network interweaving, while the polysaccharide extracted from the cell wall using chelating agents and alkali mainly presents granular and lamellar morphologies, which indicates that the structure of the water-extracted polysaccharide component is relatively intact during the extraction process. The polysaccharide (C-TPS) extracted using cyclohexanediaminetetraacetic acid (CDTA) shows a loose and fine powdery structure ( Figure 4 D), which indicates that the chelating agent effectively destroyed the cross-links between the macromolecular polysaccharide structures in the tea cell wall, causing them to break into polysaccharide fragments. The polysaccharides extracted using sodium carbonate (Na2CO3) (N-TPS), 1M sodium hydroxide (1M-TPS), and 4M sodium hydroxide (4M-TPS) all showed a flaky structure. It is worth noting that the polysaccharides extracted with sodium carbonate showed fine fragments and elongated branched shapes ( Figure 4 E), which indicates that sodium carbonate can extract highly branched pectin polysaccharides from tea cell walls. The polysaccharides extracted by 1M sodium hydroxide and 4M sodium hydroxide have similar morphologies, but the polysaccharide fragments extracted by 1M sodium hydroxide are relatively smaller and thicker ( Figure 4 F), while the polysaccharide fragments extracted with 4M sodium hydroxide were more complete and thinner ( Figure 4G). These SEM observations showed that the polysaccharide structures were closely related to the different extraction methods, polysaccharide compositions, and molecular weights.

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

[0123] like Figure 4 As shown in Figure B, all five tea polysaccharides have an inhibitory effect on α-glucosidase, and the inhibitory effect is concentration-dependent. As the concentration of polysaccharides increases, the inhibitory effect on α-glucosidase increases. At low concentrations (0.1 and 1 μg / mL), these tea polysaccharides have the least inhibitory effect on α-glucosidase. When the concentration reaches 10 μg / mL, tea polysaccharides begin to inhibit the activity of α-glucosidase, among which 4M-TPS has the most obvious inhibitory effect (23.7%). At higher concentrations, especially at 1000 μg / mL, 1M-TPS and 4M-TPS both show strong inhibitory effects on α-glucosidase, among which the inhibition rate of 4M-TPS reaches 51.1%. It is worth noting that the inhibitory effects of these five tea polysaccharides on α-amylase and sucrase are negligible. Based on the inhibition rate of various tea polysaccharides on enzymes related to glucose metabolism, 4M-TPS has excellent hypoglycemic activity.

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

[0125] In the purification process of tea polysaccharides, gradient elution with salt solutions of different concentrations is often used as the mobile phase to exchange and elute the polysaccharides bound to the filler in the chromatographic column. This method is popular because the interaction between the ions in the salt solution and the polysaccharide molecules is enhanced, thereby increasing their charged properties. Changes in the concentration of the salt solution lead to changes in the ionic strength, which in turn affects the properties of the replaced polysaccharides. Elution in a neutral solution can obtain neutral polysaccharides, while elution with a salt solution can obtain polysaccharides with increased acidity as the salt concentration increases. This study further purified 4M-TPS ( Figure 5 ). When phosphate buffered saline (PBS) was used as the eluent, a large amount of tea polysaccharides were eluted. However, when a sodium chloride solution with a concentration gradient was subsequently used for elution, the amount of polysaccharide elution detected was very small. Therefore, the components eluted and collected under PBS conditions were selected, combined, concentrated and freeze-dried to obtain purified tea polysaccharides, named p4M-TPS. In addition, the elution curve 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] By measuring the chemical composition of p4M-TPS, it was found that the contents of neutral sugars, uronic acid, and reducing sugars were 553.44 mg / g, 104.22 mg / g, and 391.32 mg / g, respectively (Table 4). When the phenol-sulfuric acid method is used to determine the total sugar content with glucose as the standard, the results may be biased due to the different degrees of reaction between monosaccharides. This method is also used to determine neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. When glucose is used as the standard, the measured total sugar content is closer to the content of neutral sugars. Therefore, the results of 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 are detected, indicating that the DEAE-Sepharose Fast Flow ion exchange column has a powerful separation and purification effect. As Figure 2 As shown in Figure F, the molecular weight of p4M-TPS was measured by gel permeation chromatography, and the result showed a single and symmetrical elution peak, indicating the uniformity of the p4M-TPS composition. The molecular weight of p4M-TPS is 2.42×10 5 Da.

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

[0129]

[0130] Note: -: indicates not measured.

[0131] The in vitro digestion experiment of p4M-TPS showed that the neutral sugar and uronic acid contents of tea polysaccharides decreased after simulated digestion. The uronic acid content changed significantly before and after digestion (Table 5), which is similar to the digestion characteristics of Fuzhuan tea polysaccharides. This phenomenon shows that tea polysaccharides are resistant to decomposition in the digestive environment of the human mouth and esophagus, and can safely reach the stomach and intestines, especially interacting with intestinal microorganisms, which may further regulate carbohydrate metabolism by regulating intestinal flora.

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

[0133]

[0134] Note: -: indicates not measured.

[0135] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. A method for extracting and purifying green tea cell wall polysaccharides having hypoglycemic activity, characterized in that: The method comprises: Green tea is sequentially extracted with water, cyclohexanediaminetetraacetic acid, sodium carbonate and sodium hydroxide solutions of different concentrations, and then subjected to impurity removal, dialysis and freeze-drying to obtain cell wall polysaccharides with hypoglycemic activity.

2. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 1, characterized in that: The method comprises the following steps: (1) Grinding green tea leaves and sieving to obtain tea powder, adding ultrapure water, extracting at 37°C for 2h, then centrifuging to obtain the supernatant, repeating the above steps with the precipitate until the sugar content in the supernatant is no longer detected, combining the supernatants for dialysis, the supernatant is the extract, retaining the tea residue and drying to obtain dried tea residue for subsequent steps; adding ethanol, standing at 4°C overnight, then centrifuging to collect the precipitate; re-dissolving the precipitate with water and centrifuging to obtain the supernatant, repeating 3 times, combining the supernatants, and freeze-drying to obtain water-extracted polysaccharides; (2) Add 70% ethanol to the dried tea residue obtained in step (1), shake on a shaker, centrifuge the solution to collect the precipitate, wash the precipitate repeatedly with 70% ethanol 3-5 times, wash once with anhydrous ethanol, and then place the precipitate in a fume hood to dry to obtain alcohol-insoluble tea residue; add Tris-maleic acid buffer, add 2mmol / L CaCl2 and amylase, shake on a shaker to obtain tea residue after starch removal, wash the tea residue after starch removal 3-5 times, centrifuge to obtain the precipitate and dry, add cyclohexanediaminetetraacetic acid solution after drying, shake on a shaker, centrifuge and dialyze the supernatant to obtain a precipitate and an extract, wash the precipitate 3-5 times, dry it, and obtain tea residue after CDTA extraction; after dialysis, add ethanol, let it stand at 4°C overnight, and then centrifuge to obtain the precipitate. Redissolve the precipitate with water and centrifuge to obtain the supernatant, repeat 3 times, combine the supernatants, and freeze-dry to obtain the polysaccharide extracted by CDTA; (3) adding the tea residue obtained in step (2) after CDTA extraction to a saturated sodium carbonate solution, and adding 25 mmol / L NaBH4 to the solution, shaking on a shaker, centrifuging and dialyzing the supernatant to obtain a precipitate and an extract, washing the precipitate 3-5 times, and drying to obtain the tea residue extracted with saturated sodium carbonate; adding ethanol, standing at 4°C overnight, and then centrifuging to obtain the precipitate; re-dissolving the precipitate with water and centrifuging to obtain the supernatant, repeating 3 times, combining the supernatants, and freeze-drying to obtain the polysaccharide extracted with sodium carbonate; (4) adding a 1 mol / L NaOH solution to the tea residue obtained in step (3) after extraction with saturated sodium carbonate, and adding 25 mmol / L NaBH4 to the solution, shaking on a shaker, centrifuging, dialyzing the supernatant to obtain a precipitate and an extract, washing the precipitate 3-5 times, and drying to obtain the tea residue extracted with a 1 mol / L NaOH solution; adding ethanol, standing at 4°C overnight, and centrifuging to collect the precipitate; re-dissolving the precipitate with water, and centrifuging to obtain the supernatant, repeating 3 times, combining the supernatants, and freeze-drying to obtain a polysaccharide extracted with a 1 mol / L NaOH solution; (5) Adding 4 mol / L NaOH solution to the tea residue extracted with 1 mol / L NaOH solution obtained in step (4), and adding 25 mmol / L NaBH4 to the solution, shaking on a shaker, and dialyzing the supernatant after centrifugation to obtain a precipitate and an extract; adding ethanol after rotary evaporation concentration, standing at 4°C overnight, and centrifuging to obtain a precipitate; re-dissolving the precipitate with water and centrifuging to obtain the supernatant, repeating 3 times, combining the supernatants, and freeze-drying to obtain a polysaccharide extracted with 4 mol / L NaOH solution.

3. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 2, characterized in that: The specific process of step (1) is as follows: The green tea leaves are ground and passed through an 80-mesh sieve to obtain tea powder, and ultrapure water is added to the tea powder at a solid-liquid ratio of 1:20, and the mixture is extracted at 37°C for 2 hours, followed by centrifugation to obtain the supernatant, and the precipitate is extracted repeatedly with the above steps until the sugar content is no longer detected in the supernatant, and the supernatants are combined and dialyzed for 72 hours using a 7000Da dialysis bag, the supernatant being the extract, and 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 the mixture is allowed to stand at 4°C overnight, followed by centrifugation to collect the precipitate; the precipitate is redissolved with water and then centrifuged to obtain the supernatant, and the process is repeated 3 times, the supernatants are combined, and the water-extracted polysaccharides are obtained after freeze-drying.

4. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 2, characterized in that: The specific process of step (2) is as follows: The dried tea residue obtained in step (1) is added with 70% ethanol at a solid-liquid ratio of 1:10, and the mixture is shaken on a shaker at room temperature for 12 hours. The solution is centrifuged to collect the precipitate, and the precipitate is repeatedly washed with 70% ethanol for 3-5 times, and washed once with anhydrous ethanol. The precipitate is then 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 at a solid-liquid ratio of 1:10, wherein the concentration of the Tris-maleic acid buffer is 20 mmol / L, and the pH of the Tris-maleic acid buffer is 6.9, and 2 mmol / L The ratio of CaCl2 and amylase, amylase and alcohol-insoluble tea residue is 20 mg amylase / 5 g tea residue, shake on a shaker at 37°C for 4 hours to remove starch, obtain tea residue with starch removed, wash the tea residue with starch removed 3-5 times, centrifuge to obtain the precipitate and place it in a fume hood to dry, add cyclohexanediaminetetraacetic acid solution to the dried tea residue with starch removed at a solid-liquid ratio of 1:10, the concentration of the cyclohexanediaminetetraacetic acid solution is 50 mmol / L, the pH of the cyclohexanediaminetetraacetic acid solution is 6.5, shake on a shaker at 37°C for 12 hours, then centrifuge and dialyze the supernatant for 72 hours using a 7000Da dialysis bag to obtain a precipitate and an extract, wash the precipitate 3-5 times, place it in a fume hood to dry, and obtain tea residue extracted with CDTA; after the dialysis is completed, add ethanol at a ratio of extract: ethanol = 1:3, let it stand overnight at 4°C, and then centrifuge to obtain the precipitate. The precipitate was re-dissolved in water and centrifuged to obtain the supernatant. This process was repeated three times. The supernatants were combined and freeze-dried to obtain the polysaccharide extracted by CDTA.

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

6. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 2, characterized in that: The specific process of step (4) is as follows: The tea residue obtained in step (3) after extraction with saturated sodium carbonate is added with 1 mol / L NaOH solution at a material-liquid ratio of 1:10, and 25 mmol / L NaBH4 is added to the solution to protect the polysaccharide, and the extraction is performed on a shaking table at 37°C for 12 h. After centrifugation, the supernatant is dialyzed using a 7000Da dialysis bag for 72 h to obtain a precipitate and an extract, and the precipitate is washed 3-5 times and then dried to obtain the tea residue extracted with 1 mol / L NaOH solution; ethanol is added at a volume ratio of extract: ethanol = 1:3, and the mixture is allowed to stand at 4°C overnight, and the precipitate is collected by centrifugation; the precipitate is redissolved with water and then centrifuged to obtain the supernatant, and the process is repeated 3 times. The supernatants are combined and freeze-dried to obtain the polysaccharide extracted with 1 mol / L NaOH solution.

7. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 2, characterized in that: The specific process of step (5) is as follows: The tea residue obtained in step (4) after extraction with 1 mol / L NaOH solution is added into 4 mol / L NaOH solution at a material-liquid ratio of 1:10, and 25 mmol / L NaBH4 is added into the solution, and the mixture is extracted by shaking on a shaking table at 37°C for 12 h. After centrifugation, the supernatant is dialyzed using a 7000Da dialysis bag for 72 h to obtain a precipitate and an extract; after rotary evaporation and concentration, ethanol is added at a volume ratio of 1:3 between the water extract and ethanol, and the mixture is allowed to stand at 4°C overnight, and the precipitate is obtained by centrifugation; the precipitate is redissolved with water, and the supernatant is obtained by centrifugation, and the mixture is repeated 3 times. The supernatants are combined, and the polysaccharide extracted with 4 mol / L NaOH solution is obtained after freeze-drying.

8. The method for extracting and purifying green tea cell wall polysaccharides with hypoglycemic activity according to claim 2, characterized in that: After step (5), perform the following step (6): (6) Protein removal: adding trichloroacetic acid 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-extracted polysaccharide obtained in step (4), and the 4 mol / L NaOH-extracted polysaccharide obtained in step (5), respectively, centrifuging to remove the precipitate, taking the supernatant to obtain a protein-removed polysaccharide solution, adjusting the pH of the protein-removed polysaccharide solution to 7, dialyzing, and then freeze-drying to obtain the water-extracted polysaccharide after protein removal, the CDTA-extracted polysaccharide after protein removal, the sodium carbonate-extracted polysaccharide after protein removal, the 1 mol / L NaOH-extracted polysaccharide after protein removal, and the 4 mol / L NaOH-extracted polysaccharide after protein removal, respectively; In step (6), trichloroacetic acid is added to the solution system to contain 10% by mass concentration of trichloroacetic acid; In step (6), dialysis was performed for 72 h using a 3500 Da dialysis bag; In step (6), the freeze-drying conditions are -20°C, 48h.

9. The green tea cell wall polysaccharide with hypoglycemic activity prepared by the extraction and purification method according to any one of claims 1 to 8, characterized in that: 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.

10. The green tea cell wall polysaccharide with hypoglycemic activity according to claim 9, characterized in that: The total sugar content of the polysaccharide extracted with the 4 mol / L NaOH solution after protein removal is 631.12 mg / g; The uronic acid content of the polysaccharide extracted by CDTA after protein removal is 265.81 mg / g; The molecular weight of the polysaccharide extracted by sodium carbonate after protein removal reaches 8.95×10 3 Da; The galacturonic acid content of the polysaccharide extracted by CDTA after protein removal reaches 49.83%; The glucosamine content of the polysaccharide extracted by sodium carbonate after protein removal reaches 45.32%; The glucuronic acid content of the polysaccharide extracted with the 1 mol / L NaOH solution after protein removal reaches 50.54%.

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