Hydrogen peroxide / ascorbic acid assisted gynura divaricata polysaccharide extraction method

Abstract

The invention belongs to the field of biochemistry, and relates to a method for extracting gynura divaricata polysaccharide by using a hydrogen peroxide / ascorbic acid system. Comprising the following steps: adding deionized water, hydrogen peroxide and ascorbic acid into degreased gynura divaricata powder, and stirring and extracting in an oil bath; centrifuging and concentrating the obtained reaction solution, and carrying out alcohol precipitation on the obtained concentrated solution; and redissolving the obtained precipitate by using deionized water, dialyzing by using a dialysis bag with the molecular weight cut-off of 3500 Da, and then concentrating and freeze-drying to obtain the gynura divaricata crude polysaccharide GDP-H. The molecular weight of the gynura divaricata crude polysaccharide GDP-H is greatly reduced, and the biological activity is also remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry and relates to a method for extracting Glehnia littoralis polysaccharide by utilizing a hydrogen peroxide / ascorbic acid system. Background Art

[0002] Gynura divaricate (L.) DC., also known as white-backed Panax notoginseng and moon grass, is a plant of the genus Panax notoginseng in the Asteraceae family. It is distributed in Guangdong, Zhejiang and other places. It is a traditional Chinese medicinal material, widely used to improve blood circulation, stop bleeding, clear away heat and detoxify, and enhance immunity. Studies have shown that one of the main active ingredients of Gynura divaricate is polysaccharide, which has significant biological activity, including antioxidant, blood pressure lowering, blood sugar lowering and anti-inflammatory effects. Because Gynura divaricate polysaccharide is natural and has no toxic side effects, it has become one of the important directions of the food and pharmaceutical industry and a hot spot for research.

[0003] The commonly used extraction methods for polysaccharides of Glechoma longituba mainly include hot water extraction, acid-base hydrolysis and enzymolysis. The hot water extraction method usually uses water and Glechoma longituba powder to mix and extract at high temperature. This method is simple and easy, but the yield is low, and the extracted polysaccharide has a large molecular weight, poor water solubility and relatively low biological activity. The acid-base hydrolysis method destroys the cell wall structure and releases polysaccharides through the hydrolysis of acid or alkali. Although this method can improve the extraction rate of polysaccharides, the operation is complicated, the treatment conditions are harsh, and the structure and biological activity of polysaccharides may be damaged. Although the enzymolysis method can selectively degrade the cell wall and release polysaccharides, it is expensive and the operating conditions are also complicated. Although these commonly used methods are effective, they still have low extraction efficiency, cumbersome process, high operating cost, and insufficient protection of polysaccharide structure and biological activity. Therefore, it is necessary to further improve the extraction method of Glechoma longituba polysaccharides. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method, which can improve the extraction rate and biological activity of Glechoma longituba polysaccharide.

[0005] In order to solve the above technical problems, the present invention provides a hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method, comprising the following steps:

[0006] S1. Add deionized water to defatted Glechoma longituba powder to form a mixed solution. Add hydrogen peroxide (H2O2) to the mixed solution under light-proof conditions. 2 O 2 ) and ascorbic acid (Vc) to form a reaction system, placing the reaction system in a 90-100° C. oil bath and stirring for 3±0.5 h to obtain a reaction solution;

[0007] The molar ratio of hydrogen peroxide to ascorbic acid is 1:1±0.1;

[0008] Defatted Glechoma longituba powder: deionized water = 1g: 30±10mL solid-liquid ratio;

[0009] In the reaction system, H 2 O 2 The concentration of is 5~25mmol / L, and the concentration of Vc is 5~25mmol / L;

[0010] S2, cooling the reaction solution obtained in step S1 to room temperature and then centrifuging (8000-10000 rpm, 15-20 min), concentrating the supernatant obtained by centrifugation (concentrating at 50° C.-60° C.) to 30%-35% of the original volume to obtain a concentrated solution;

[0011] Add anhydrous ethanol to the concentrate, stir evenly, place at 0-5°C for alcohol precipitation for 24±2h, centrifuge (8000-10000rpm, 15-20min), and obtain a precipitate; the volume ratio of the concentrate to the anhydrous ethanol is 1:3.5-4.5;

[0012] The precipitate was re-dissolved in deionized water, and dialyzed in deionized water for 72±2 h using a dialysis bag with a molecular weight cut-off of 3500 Da to obtain a retentate (the liquid retained in the dialysis bag);

[0013] The retentate was concentrated (concentrated at 50°C to 60°C) to 20% to 25% of the original volume, and freeze-dried (freeze-dried at -40°C to -60°C for 22 to 26 hours) to obtain crude polysaccharide GDP-H (H 2 O 2 / Vc-assisted extraction of crude polysaccharides from Glehnia littoralis).

[0014] Note: During the dialysis process, replace the deionized water every 3 to 6 hours.

[0015] As an improvement of the hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method of the present invention:

[0016] In the re-dissolution in step S2, the volume of deionized water is 0.8 to 1.2 times the volume of the concentrated solution.

[0017] As a further improvement of the hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method of the present invention:

[0018] Defatted Glechoma longituba powder: deionized = 1g: 31mL water-to-liquid ratio;

[0019] In the reaction system: H 2 O 2 The concentration of is 21mmol / L, the concentration of Vc is 21mmol / L;

[0020] The reaction system was placed in a 94° C. oil bath and stirred for 3 h to obtain a reaction solution.

[0021] As a further improvement of the hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method of the present invention, in S2:

[0022] The reaction solution obtained in step S1 is cooled and centrifuged, and the supernatant obtained by centrifugation is concentrated to

[0023] 1 / 3 of the original volume to obtain the concentrated solution;

[0024] Add anhydrous ethanol to the concentrate, stir evenly, place at 4°C for 24 hours, and then centrifuge to obtain a precipitate; the volume ratio of the concentrate to anhydrous ethanol is 1:4;

[0025] The obtained precipitate was re-dissolved in deionized water and dialyzed in deionized water for 72 h using a dialysis bag with a molecular weight cut-off of 3500 Da to obtain a retentate (the liquid retained in the dialysis bag); the volume ratio of deionized water: concentrate = 0.8:1;

[0026] The retentate was concentrated to 1 / 4 of the original volume and freeze-dried to obtain crude polysaccharide of Gnaphalium chinense (GDP-H).

[0027] As a further improvement of the hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method of the present invention, the crude polysaccharide (GDP-H) of Gnaphalium chinense obtained in step S2 is separated and purified, comprising the following steps:

[0028] ① Rough classification:

[0029] Prepare a 20±0.5 mg / mL polysaccharide solution of crude polysaccharide (GDP-H) from Glehnia littoralis with deionized water, and centrifuge 4.8-5.2 mL of the polysaccharide solution to obtain a supernatant;

[0030] The supernatant was evenly added to the surface of the filler of the glass chromatography column, and eluted with deionized water and gradient concentration NaCl solution as eluents in turn through a DEAE Cellulose-52 chromatography column (column specifications: 60 cm × Φ2.6 cm, effective length: 50 cm), with an elution rate of 1 mL / min;

[0031] The sugar-containing eluates corresponding to 0.1 mol / L and 0.3 mol / L NaCl solutions were collected respectively, and two preliminary purified polysaccharide fractions of Glechoma longituba polysaccharide were obtained after concentration, dialysis and freeze-drying.

[0032] Note: The gradient concentration of NaCl solution is 0.1, 0.3, 0.5, 0.7, 0.9 mol / L NaCl solution;

[0033] When it is found that the detected eluate does not contain polysaccharides, the next eluent is replaced for elution; the automatic collector collects the eluate at a speed of 10 min / tube.

[0034] ②Purification:

[0035] The two preliminarily purified polysaccharide components obtained in step ① are respectively subjected to the following operations:

[0036] Weigh the preliminarily purified polysaccharide component and prepare it into a 10 mg / mL polysaccharide component solution with deionized water, take 4.8-5.2 mL of the polysaccharide component solution and centrifuge to obtain a supernatant;

[0037] The supernatant was filtered through a 0.45 μm filter membrane, and evenly loaded into a Sephadex G-100 gel column (the column size was 60 cm × Φ1.6 cm, and the effective volume of the column was 50 cm × Φ1.6 cm), and eluted with deionized water at a flow rate of 0.25 mL / min; the eluate was collected;

[0038] The eluate was concentrated and freeze-dried to a constant weight to obtain two purified polysaccharide fractions, GDP1-H and GDP3-H.

[0039] Note: The phenol-sulfuric acid method is used to detect the ultraviolet absorbance of the eluate of each group at 490nm, and an elution curve is drawn. When it is found that the eluate detected does not contain polysaccharides, the elution is stopped.

[0040] The present invention also provides the use of crude polysaccharide GDP-H of Glechoma longituba and purified polysaccharide components GDP1-H and GDP3-H as functional ingredients, wherein the functionality includes antioxidant, anti-aging activity and intestinal flora regulating activity.

[0041] The present invention adopts hydrogen peroxide / ascorbic acid auxiliary method to extract polysaccharide from Glehnia littoralis, and its molecular weight is greatly reduced. Low molecular weight polysaccharide is not only more soluble, but also improves the contact area and reaction speed with free radicals, and its structure is more open, surface active groups are exposed, and the antioxidant activity in vitro and in vivo is enhanced, effectively protecting cells from oxidative damage, reducing cell apoptosis, thereby helping to slow down the aging process. In addition, low molecular weight polysaccharide is more easily absorbed and utilized by the human body, and polysaccharide can be used as prebiotics to selectively promote the growth and reproduction of beneficial bacteria (such as lactic acid bacteria, bifidobacteria, etc.), and improve the balance of intestinal microecology.

[0042] Compared with the prior art, the technical advantages of the present invention are:

[0043] 1. Compared with the crude polysaccharide (GDP-W) of Glehnia littoralis obtained by the traditional hot water extraction method, the extraction rate of the crude polysaccharide (GDP-H) of Glehnia littoralis obtained by the present invention is significantly increased; and compared with the hot water extraction method, no additional equipment is required.

[0044] 2. Compared with the crude polysaccharide of Ganoderma lucidum (GDP-W, molecular weight 6874 kDa) obtained by the traditional hot water extraction method, the crude polysaccharide of Ganoderma lucidum (GDP-H, molecular weight 15.75 kDa) obtained by the present invention has a greatly reduced molecular weight and significantly improved biological activity. GDP-H and its two purified components have significant antioxidant, anti-aging and intestinal flora regulating effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0046] In the figure: GDP-W is water-extracted Glehnia littoralis polysaccharide, GDP represents crude Glehnia littoralis polysaccharide (GDP-H), GDP1 represents the purified polysaccharide component GDP1-H, and GDP3 represents the purified polysaccharide component GDP3-H.

[0047] Figure 1 is the elution curve;

[0048] Figure 2 It is a comparison of the DPPH radical scavenging rates of polysaccharide and other samples at different concentrations;

[0049] Figure 3 It is a comparison of ABTS free radical scavenging rates of polysaccharide and other samples at different concentrations;

[0050] Figure 4 It is a comparison of the OH radical scavenging rates of polysaccharide and other samples at different concentrations;

[0051] Figure 2 to Figure 4 Middle: Ascorbic acid (Vc) as a positive control;

[0052] Figure 5 Comparison of the effects of polysaccharide samples at different concentrations on the survival rate of N2 nematodes; M9 buffer was used as the blank control (Control);

[0053] Figure 6 The effect of polysaccharide and other samples on the life span of N2 nematodes; the NGM culture plate without drugs was used as the blank control (Control);

[0054] Figure 7 The effect of polysaccharide and other samples on lipofuscin in N2 nematodes; the NGM culture plate without drugs was used as the blank control (Control);

[0055] Figure 8 is the effect of polysaccharide and other samples on the activity of ROS, SOD, CAT, and MDA in N2 nematodes;

[0056] Figure 8 middle:

[0057] A: Effects of samples on ROS in N2 nematodes; B: Effects of samples on SOD activity in N2 nematodes; C: Effects of samples on CAT activity in N2 nematodes; D: Effects of samples on MDA activity in N2 nematodes;

[0058] Fig. 9 It is the analytical determination of short-chain fatty acids in in vitro fermentation of polysaccharide and other samples at the same concentration;

[0059] Fig.10 The effects of polysaccharides and other samples on intestinal flora;

[0060] Fig.10 middle:

[0061] A: Diversity index bar graph; B: Venn diagram at OUT level; Figures C and D: Relative abundance of intestinal flora at phylum and genus levels; E: PCoA analysis graph;

[0062] Fig.11 Analysis of the species composition of intestinal flora and its association with SCFAs;

[0063] Fig.11 middle:

[0064] A: Genus level community heat map; B: LEfSe multi-level species hierarchy tree and significantly enriched bacterial community histogram; C: Environmental factor Heatmap;

[0065] Figures 9 to 11 Middle: YCFA basal medium was used as blank control; G1 was YCFA (blank) group, G2 was fructooligosaccharide (FOS) group, G3 was GDP group, G4 was GDP1 group, and G5 was GDP3 group. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0067] In the present invention, the stirring speed is generally set to 800-1000 rpm.

[0068] The defatted white cabbage powder is obtained by subjecting the white cabbage powder to conventional defatting pretreatment. The defatting pretreatment comprises the following steps: taking an appropriate amount of white cabbage powder (water content ≤5%, passed through a 100-mesh sieve), adding anhydrous ethanol at a solid-liquid ratio of 1:20 (g / mL, w / v), condensing and refluxing at 90°C for 2h, and suction filtering. The obtained filter cake repeats the above steps of adding anhydrous ethanol-condensing and refluxing-suction filtering until the filtrate is colorless, thereby removing impurities such as lipids, flavonoids and pigments. The filter cake obtained by the last suction filtration is dried at 60°C to a constant weight to obtain the defatted white cabbage powder.

[0069] Example 1-1: A hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method, comprising the following steps:

[0070] S1. Add deionized water to the defatted Ganoderma lucidum powder at a solid-liquid ratio of 1:31 (g / mL, w / v), stir and mix to form a mixed solution;

[0071] Under light-proof conditions, hydrogen peroxide and ascorbic acid are added to the mixed solution to form a reaction system; the molar ratio of hydrogen peroxide to ascorbic acid is 1:1, and the concentrations of hydrogen peroxide and ascorbic acid in the reaction system are both 21 mmol / L;

[0072] The reaction system was placed in a 94° C. oil bath and stirred for 3 h to obtain a reaction solution.

[0073] S2, cooling the reaction solution obtained in step S1 to room temperature and centrifuging (10000 rpm, 15 min), taking the supernatant and concentrating it at 60° C. to 1 / 3 of the original volume to obtain a concentrated solution;

[0074] Add anhydrous ethanol to the concentrate, stir evenly, place in a 4°C refrigerator for alcohol precipitation for 24 hours, and then centrifuge (10000 rpm, 15 min) to obtain a precipitate; wherein the volume ratio of the concentrate to anhydrous ethanol is 1:4;

[0075] The obtained precipitate was re-dissolved with deionized water, the volume of deionized water being 0.8 times that of the concentrate, and then placed in a dialysis bag with a molecular weight cut-off of 3500 Da and dialyzed in deionized water for 72 h to obtain a retentate (the liquid retained in the dialysis bag);

[0076] Note: During dialysis, replace deionized water every 3 to 6 hours;

[0077] The retentate was concentrated to 1 / 4 of the original volume at 60°C, and freeze-dried at -50°C for 24 hours to obtain Glechoma longituba crude polysaccharide (GDP-H). The extraction rate of Glechoma longituba crude polysaccharide was 21.05%.

[0078] Extraction rate = (mass of crude G. chinensis polysaccharide after freeze-drying / mass of defatted G. chinensis powder) × 100%.

[0079] Embodiment 1-2, relative to embodiment 1-1, makes the following changes:

[0080] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:25, and the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 25 mmol / L; the reaction system was placed in a 95° C. oil bath and stirred for extraction for 3 h; the rest was the same as Example 1-1.

[0081] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 16.35%.

[0082] Embodiment 1-3, with respect to embodiment 1-1, makes the following changes:

[0083] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:35, and the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 15 mmol / L; the reaction system was placed in a 95°C oil bath and stirred for extraction for 3 hours, and the rest was the same as Example 1-1.

[0084] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 16.77%.

[0085] Embodiment 1-4, with respect to embodiment 1-1, makes the following changes:

[0086] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:25, and the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 20 mmol / L; the reaction system was placed in a 100° C. oil bath and stirred for extraction for 3 h, and the rest was the same as Example 1-1.

[0087] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 17.43%.

[0088] Embodiment 1-5, with respect to embodiment 1-1, makes the following changes:

[0089] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:30, and the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 15 mmol / L; the reaction system was placed in a 100°C oil bath and stirred for extraction for 3 hours, and the rest was the same as Example 1-1.

[0090] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 16.82%.

[0091] Embodiment 1-6, with respect to embodiment 1-1, makes the following changes:

[0092] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:35, and the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 20 mmol / L; the reaction system was placed in a 100° C. oil bath and stirred for extraction for 3 h, and the rest was the same as Example 1-1.

[0093] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 18.21%.

[0094] Embodiment 1-7, with respect to embodiment 1-1, makes the following changes:

[0095] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:30; the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 25 mmol / L; the reaction system was placed in a 90° C. oil bath and stirred for extraction for 3 h, and the rest was the same as Example 1-1.

[0096] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 18.1%.

[0097] Embodiment 1-8, with respect to embodiment 1-1, makes the following changes:

[0098] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:35; the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 20 mmol / L; the reaction system was placed in a 90° C. oil bath and stirred for extraction for 3 h, and the rest was the same as Example 1-1.

[0099] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 19.2%.

[0100] Embodiment 1-9, with respect to embodiment 1-1, makes the following changes:

[0101] The solid-liquid ratio of the defatted white cabbage powder and deionized water in step S1 of Example 1-1 was adjusted to 1:30; the concentrations of hydrogen peroxide and ascorbic acid in the reaction system were both 20 mmol / L; the reaction system was placed in a 95° C. oil bath and stirred for extraction for 3 h, and the rest was the same as Example 1-1.

[0102] The crude polysaccharide of Glehnia littoralis (GDP-H) was obtained, and the extraction rate was 20.91%.

[0103] Comparative Example 1: In Example 1-1, "adding hydrogen peroxide and ascorbic acid" was changed to "not adding hydrogen peroxide and ascorbic acid", that is, the amount of hydrogen peroxide and ascorbic acid was 0, and the rest was the same as in Example 1-1, and water-extracted Ginkgo biloba crude polysaccharide (GDP-W) was obtained, and the extraction rate was 12.32%.

[0104] Embodiment 2:

[0105] ①. Accurately weigh 200 mg of the crude polysaccharide (GDP-H) of Glechoma longituba obtained in Example 1-1, add deionized water to prepare a 20 mg / mL crude polysaccharide solution of Glechoma longituba, take 5 mL of the crude polysaccharide solution of Glechoma longituba and centrifuge (8000 rpm, 10 min) to collect the supernatant, and use a rubber-tipped dropper to slowly and evenly add the supernatant to the filler surface of the glass chromatography column, pass through a cellulose DEAE-52 chromatography column (column specifications are 60 cm×Φ2.6 cm, and the effective length is 50 cm), and use deionized water and NaCl solutions of different concentrations (0.1, 0.3, 0.5, 0.7, 0.9 mol / L) to elute in turn. The elution rate of the sample is controlled to be 1 mL / min, and the speed of the automatic collector is 10 min / tube, and the eluate corresponding to each eluent is collected. After the collection is completed, the elution of each group is tested for ultraviolet absorbance at 490 nm using the phenol-sulfuric acid method, and an elution curve is drawn, such as Figure 1 .

[0106] Note: The ultraviolet absorption value of the eluent at 490nm is detected by the sulfuric acid-phenol method to determine whether it contains polysaccharides. When the detected eluent is found to contain no polysaccharides, the next eluent is replaced for elution.

[0107] Since the sugar content in the eluents of 0.5 mol / L, 0.7, and 0.9 mol / L NaCl was low, no further study was conducted. Since the GDP0-H component of the deionized water eluent was very easy to absorb moisture during storage and was unstable, it was not collected later.

[0108] The two sugar-containing eluates obtained by eluting with 0.1 mol / L and 0.3 mol / L NaCl solutions as eluents were evaporated and concentrated to 1 / 10 of the original volume at 60°C, and dialyzed in deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 3500Da to achieve the purpose of desalination (the deionized water was replaced every 3 to 6 hours during this period). The retentate (the liquid retained in the dialysis bag) was then freeze-dried to constant weight at -50 to -60°C, thereby obtaining two preliminary purified polysaccharide components of Gynura polysaccharide.

[0109] ②, the two preliminary purified polysaccharide components were subjected to the following steps respectively:

[0110] Weigh the preliminarily purified polysaccharide component, prepare a 10 mg / mL polysaccharide component solution with deionized water, take 5 mL of the polysaccharide component solution and centrifuge (8000 rpm, 10 min), collect the supernatant and filter it through a 0.45 μm filter membrane, and evenly add the sample to a dextran Sephadex G-100 gel column glass chromatography column (the specification of the chromatography column is 60 cm×Φ1.6 cm, and the effective length is 50 cm); adjust the deionized water to elute at a flow rate of 0.25 mL / min, and collect the eluate (collect 1 tube every 20 min). Use the phenol-sulfuric acid method to detect the ultraviolet absorbance value of the eluate at 490 nm, and draw an elution curve. When it is found that the detected eluate does not contain polysaccharides, stop eluting.

[0111] The eluates containing polysaccharides were combined and concentrated by evaporation at 60°C to 1 / 10 of the original volume, and freeze-dried at -50°C to -60°C to constant weight, thereby obtaining two purified polysaccharide components, GDP1-H and GDP3-H.

[0112] Experiment 1: Determination of molecular weight and content of polysaccharides and other components:

[0113] The molecular weights of the crude polysaccharide of Ganoderma lucidum (GDP-H) obtained in Example 1-1, the crude polysaccharide of Ganoderma lucidum (GDP-W) extracted from Comparative Example 1, and the purified polysaccharide components GDP1-H and GDP3-H obtained in Example 2 were measured; the results are shown in Table 1 below;

[0114] The contents of polysaccharides and other components of GDP-W, GDP1-H and GDP3-H were also determined, and the results are shown in Table 2:

[0115] Table 1. Molecular weight comparison

[0116]

[0117] Table 2. Comparison of the composition of crude polysaccharides GDP-H1-1, GDP1-H2-1, and GDP3-H2-1

[0118]

[0119]

[0120] Note: Man, GlcA, Rha, GalA, Glc, Gal, Xyl, Ara and Fuc represent mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose and fucose, respectively.

[0121] Experiment 2: Antioxidant activity

[0122] Taking ascorbic acid (Vc) as a positive control, the crude polysaccharide of Ganoderma lucidum (GDP-H) obtained in Example 1-1, the water-extracted crude polysaccharide of Ganoderma lucidum (GDP-W) obtained in Comparative Example 1, and the purified polysaccharide components GDP1-H and GDP3-H obtained in Example 2 were tested for DPPH free radical scavenging ability, ABTS scavenging ability, and ·OH scavenging ability, respectively.

[0123] The DPPH free radical scavenging experiment was carried out using the method reported in the literature (KW LI, J WANG, ZZ HUANG, et al. Solid structure and antioxidant activity in vitro of angelica sinensis polysaccharide-Ce(IV)[J]. Chemistry&Biodiversity, 2023, 20(3): e202200813). The results are as follows Figure 2 .Depend on Figure 2 It can be seen that the free radical scavenging ability of GDP-H is much higher than that of GDP-W, GDP1-H and GDP3-H. When the concentration reaches 4 mg / mL, the free radical scavenging ability of GDP is 95.24%, which is close to the free radical scavenging ability of VC (97.00%).

[0124] The ABTS free radical scavenging experiment was carried out using the method reported in the literature (KW WANG, XY SHENG, XJ CHEN, et al. Characterization and antioxidant activities of polysaccharide extracted from benincasa var how [J]. Food Science and Technology, 2022, 42: e88421.). The results are as follows Figure 3 .

[0125] Depend on Figure 3 It can be seen that when the concentration reaches 5 mg / mL, the free radical scavenging ability of GDP-H is 92.96%, which is close to the free radical scavenging ability of VC (98.01%).

[0126] The OH radical scavenging experiment was carried out using the method reported in the literature (H TIAN, HF LIU, WK SONG, et al. Polysaccharide from caulerpalentillifera: extraction optimization with response surface methodology, structure and antioxidant activities [J]. Natural Product Research, 2021, 35(20): 3417-3425.). The results are as follows: Figure 4 .Depend on Figure 4 It can be seen that the hydroxyl radical scavenging ability of GDP3-H is slightly stronger than that of GDP-W, GDP-H and GDP1-H.

[0127] The comparison of antioxidant activity is shown in Table 3 below.

[0128] Table 3. Comparison of antioxidant activity of GDP-W, GDP-H, GDP1-H, and GDP3-H

[0129]

[0130]

[0131] Experiment 3: Toxicity test of crude polysaccharide from Glehnia littoralis to N2 nematodes:

[0132] The method reported in the literature (YH QIN, FF CHEN, ZZ TANG, et al. Ligusticum chuanxiong hortas a medicinal and edible plant foods: Antioxidant, anti-aging and neuroprotective properties in caenorhabditis elegans [J] Frontiers in Pharmacology, 2022, 13: 1043434.) was adopted, and M9 buffer was used as a blank control (Control). The crude polysaccharide of Glehnia littoralis (GDP-H) obtained in Example 1-1, the water-extracted crude polysaccharide of Glehnia littoralis (GDP-W) obtained in Comparative Example 1, and the purified polysaccharide components GDP1-H and GDP3-H obtained in Example 2 were respectively subjected to toxicity tests on L4 stage Caenorhabditis elegans N2.

[0133] The culture method of Caenorhabditis elegans N2 can refer to the following Experiment 4.

[0134] The results are as follows Figure 5 and Table 4. Therefore, it can be seen that after 48 h of acute toxicity experiment, the survival rate of nematodes in each group was above 85%, so in subsequent experiments 4 to 6, GDP-W, GDP-H, GDP1-H and GDP3-H were all selected at concentrations of 0.5 mg / mL, 1 mg / mL and 2 mg / mL for experiments.

[0135] Table 4. Effects of GDP-W, GDP-H, GDP1-H, and GDP3-H on the lifespan of N2 nematodes

[0136]

[0137] Experiment 4: Effects on the life span of N2 nematodes:

[0138] The effects of the crude Ganoderma lucidum polysaccharide (GDP-H) obtained in Example 1-1, the water-extracted Ganoderma lucidum crude polysaccharide (GDP-W) obtained in Comparative Example 1, and the purified polysaccharide components GDP1-H and GDP3-H obtained in Example 2 on the lifespan of N2 nematodes when used as drugs were detected; NGM culture medium without drugs was used as a blank control (Control).

[0139] The specific experimental methods are as follows:

[0140] ① Preparation of reagents

[0141] LB liquid medium: 10 g sodium chloride, 5 g yeast powder, 10 g tryptone, add deionized water to 1000 mL. Autoclave at 121°C for 15 min.

[0142] LB solid medium: Add 15g of agar powder to LB liquid medium. Autoclave at 121℃ for 15min, pour into a plate and cool for later use.

[0143] M9 buffer: 0.025 g anhydrous magnesium sulfate, 0.6 g sodium dihydrogen phosphate, 0.5 g sodium chloride and 0.3 g potassium dihydrogen phosphate, add deionized water to 100 mL. Autoclave at 121°C for 15 min.

[0144] Nematode growth medium (NGM medium): 0.005 g cholesterol, 17 g agar powder, 2.5 g tryptone, 3 g sodium chloride, 0.11 g calcium chloride, 0.12 g magnesium sulfate, 3.4 g potassium dihydrogen phosphate, add deionized water to 1000 mL. Autoclave at 121°C for 15 min, pour into a plate and cool for later use.

[0145] Drug-containing NGM medium: Pipette 100 μL of the sample solution and add it to the NGM plate coated with E. coli OP50, and let it stand until the medium absorbs the drug. Prepare it one day in advance and store it in a 4°C refrigerator for later use.

[0146] Nematode freezing solution: 1.36 g potassium dihydrogen phosphate, 1.17 g sodium chloride and 60 mL glycerol, add deionized water to make up to 100 mL. Autoclave at 121°C for 15 min.

[0147] ②Cultivation of E. coli OP50

[0148] Activation: Thaw the E.coli OP50 strain stored in a -80℃ refrigerator quickly in a 37℃ warm water bath, take 100μL of E.coil OP50 strain and add it to 10mL LB liquid culture medium, and culture it in a 37℃ constant temperature shaker.

[0149] Single cloning: E. coli OP50 was inoculated on LB solid medium by streak method and cultured at 37℃ for 12h.

[0150] Expanded culture: Pick a single colony after monoclone and add it to LB liquid culture medium and mix well. Culture it in a constant temperature shaker at 37℃ for 12h, take small tubes and transfer them to a 4℃ refrigerator for later use.

[0151] Spread NGM plates: Take 100 μL of the cultured bacterial solution and spread it on a blank NGM medium. Be careful not to cover the area within 1 to 2 mm of the edge to prevent nematodes from crawling to the edge and affecting the count.

[0152] Cryopreservation: Mix the bacterial solution with glycerol (concentration 40%) in a 1:1 ratio and store in a -80℃ freezer.

[0153] ③Cultivation of N2 nematodes

[0154] Recovery: Thaw the nematodes stored at -80°C at room temperature, centrifuge, wash the nematodes twice with PBS, move the nematodes to NGM plates coated with E. coli OP50, culture at 20°C, and observe the recovery the next day.

[0155] Culture: Nematodes were transferred to NGM plates coated with E.coil OP50 and cultured in an inverted sealed container at 20°C.

[0156] Passaging: Cut a small piece of the agar plate with nematodes, transfer it to an NGM plate coated with E. coli OP50, and culture it at a constant temperature of 20℃.

[0157] Cryopreservation: When the culture plate contains a large number of nematodes in the L1 and L2 stages, culture them in a starved state for 48 hours, wash the worms twice with M9 buffer, centrifuge to retain the precipitate, add nematode freezing solution, mix well, preserve with sealing film, and freeze in a -80℃ refrigerator.

[0158] Synchronization: Synchronization of nematodes is performed using the time-limited egg-laying method. Nematodes at the peak of egg-laying are transferred to a blank NGM plate, and the nematodes are picked out after 2 hours, trying to ensure that the nematodes hatched in the plate are at the same stage. Thus, the corresponding N2 nematodes at the L4 stage are obtained after synchronization.

[0159] ④. Transfer the synchronized N2 nematodes in the L4 stage to culture plates containing different concentrations of GDP-W (0.5 mg / mL, 1 mg / mL and 2 mg / mL), GDP-H (0.5 mg / mL, 1 mg / mL and 2 mg / mL), GDP1-H (0.5 mg / mL, 1 mg / mL and 2 mg / mL) and GDP3-H (0.5 mg / mL, 1 mg / mL and 2 mg / mL). Use the NGM culture plates without drugs as blank controls. There are 60 nematodes in each group and culture at a constant temperature of 20°C. Transfer the nematodes to new culture plates containing drugs every day. The day of drug administration is recorded as day 0, and the survival rate of the nematodes is recorded. Draw a lifespan curve. Figure 6 It can be seen that after being treated with different concentrations of GDP-W, GDP-H, GDP1-H and GDP3-H, the lifespan of N2 nematodes was extended to a certain extent compared with the blank control group. Among them, 2mg / mL GDP-H and GDP3-H had a better effect on extending the lifespan of nematodes, and the average lifespan was extended by 19.17% and 14.07% respectively compared with the blank group.

[0160] Experiment 5: Accumulation of lipofuscin in N2 nematodes

[0161] The accumulation of lipofuscin in N2 nematodes was detected when the crude polysaccharide of Ganoderma lucidum (GDP-H) obtained in Example 1-1, the water-extracted crude polysaccharide of Ganoderma lucidum (GDP-W) obtained in Comparative Example 1, and the purified polysaccharide components GDP1-H and GDP3-H obtained in Example 2 were used as drugs; NGM culture medium without drugs was used as a blank control (Control).

[0162] The synchronized N2 nematodes at the L4 stage (same as the method in Experiment 4) were transferred to culture plates containing different concentrations of GDP-W (0.5mg / mL, 1mg / mL and 2mg / mL), GDP-H (0.5mg / mL, 1mg / mL and 2mg / mL), GDP1-H (0.5mg / mL, 1mg / mL and 2mg / mL) and GDP3-H (0.5mg / mL, 1mg / mL and 2mg / mL). The NGM culture plates without drugs were used as blank controls. There were 30 nematodes in each group and they were cultured at a constant temperature of 20°C. After 5 days, 3 to 5 nematodes were placed on the agarose gel and paralyzed with 3 to 5 drops of imidazole for a few minutes. The nematodes were rinsed with saline (0.9% sodium chloride solution). The accumulation of lipofuscin in the N2 nematodes was recorded under a fluorescent inverted microscope and the relative fluorescence value was calculated using Image software. Figure 7 It can be seen that the relative average optical density values ​​of lipofuscin fluorescence in nematodes treated with GDP-W, GDP-H, GDP1-H and GDP3-H show that the accumulation of lipofuscin in nematodes in the GDP-W, GDP-H, GDP1-H and GDP3-H treatment groups is lower than that in the blank group, indicating that the GDP-W, GDP-H, GDP1-H and GDP3-H treatment groups all inhibit the accumulation of lipofuscin in nematodes. Among them, the effect of GDP-H at a concentration of 2 mg / mL is the most obvious, which is reduced by 43.23% compared with the blank group.

[0163] Experiment 6

[0164] The drug-free NGM culture plate was used as a blank control (Control), the plate only containing paraquat (Paraquat) was used as a model group, and the crude polysaccharide of Ganoderma lucidum (GDP-H) obtained in Example 1-1, the water-extracted crude polysaccharide of Ganoderma lucidum (GDP-W) obtained in Comparative Example 1, and the purified polysaccharide components GDP1-H and GDP3-H obtained in Example 2 were added with paraquat as the experimental group.

[0165] The synchronized N2 nematodes at the L4 stage (prepared in the same way as in Experiment 4) were transferred to culture plates containing different concentrations of GDP-W (0.5 mg / mL, 1 mg / mL and 2 mg / mL), GDP-H (0.5 mg / mL, 1 mg / mL and 2 mg / mL), GDP1-H (0.5 mg / mL, 1 mg / mL and 2 mg / mL) and GDP3-H (0.5 mg / mL, 1 mg / mL and 2 mg / mL), with 600 ± 20 nematodes in each group, cultured at 20°C. After 5 days of culture, the experiment set up a blank group, a model group and an experimental group. The model group and the experimental group were respectively stressed with 100 μmol / L paraquat for 12 hours. The nematodes were rinsed with PBS solution and transferred to a centrifuge tube. The nematodes were allowed to settle naturally, and the precipitate was retained and the supernatant was discarded. This was repeated 2 to 3 times. The nematodes were crushed using a cell disruptor (200 W, 15 min), followed by centrifugation (4000 rpm, 2 min), and the supernatant was collected.

[0166] ROS determination in N2 nematodes: 96 black-well plates were used for plating, 50 μL of supernatant and 50 μL of DCFH-DA probe (100 μM) were added to each well, and incubated for 1 h. The fluorescence intensity of each well was measured by an ELISA instrument, and the inhibition rate of the fluorescence intensity of the nematode body by the polysaccharide of Glechoma longituba was calculated according to the instructions of the ROS kit (Nanjing Jiancheng Bioengineering Institute).

[0167] MDA, SOD, CAT activity test: The supernatant was tested according to the instructions of MDA, SOD, CAT kit (Nanjing Jiancheng Bioengineering Institute). Figure 8A shows that when the concentration of polysaccharide addition reaches 2 mg / mL, the ROS content in nematodes treated with GDP-W, GDP-H, GDP1-H and GDP3-H decreased by 23.90%, 39.78%, 17.79% and 24.39% respectively compared with the model group. This shows that GDP-H has a better ability to reduce ROS levels. Figure 8 From B and C, it can be seen that when the concentration of polysaccharide added reaches 2 mg / mL, GDP-H has a better ability to enhance the activity of SOD and CAT. Figure 8 It can be seen from D that when the concentration of polysaccharide addition reached 2 mg / mL, GDP-H showed a better inhibitory effect on MDA.

[0168] Experiment 7

[0169] The method reported in the literature (Zhou Jiamin. Purification of extracellular polysaccharides of Lactobacillus rhamnosus and study on lipid-lowering and intestinal flora regulation activity [D]; Zhejiang Gongshang University, 2021.) was adopted, YCFA basal medium was used as blank control, and oligofructose (FOS), crude polysaccharide of Ganoderma lucidum (GDP-H) obtained in Example 1-1, and purified polysaccharide components GDP1-H and GDP3-H obtained in Example 2 were used as experimental groups for analysis and determination of short-chain fatty acids. Fig. 9 It can be seen that from the perspective of individual SCFAs, acetic acid accounted for the highest proportion in each experimental group, among which the acetic acid concentrations in groups G2, G3, G4 and G5 were all over 30.00 mM, significantly higher than those in group G1 (11.81 ± 1.01 mM). In addition, the propionic acid content in each experimental group was also significantly higher than that in the blank group.

[0170] Experiment 8

[0171] Intestinal microbial analysis was performed through simulated in vitro fermentation of Polysaccharides from Brassica rapa in the human intestine.

[0172] YCFA basal medium: 2 g sodium bicarbonate, 10 μL vitamin K, 0.04 g potassium dihydrogen phosphate, 2.0 g peptone, 0.04 g potassium dihydrogen phosphate, 0.025 g heme, 0.1 g sodium chloride, 0.5 g cysteine ​​hydrochloride, 2.0 g yeast extract, 0.01 g magnesium sulfate heptahydrate, 0.01 g calcium chloride, 2 mL Tween-80, 0.5 g bile salts, 4 mL resazurin solution (prepared into a 0.25 g / L solution first), dissolve in distilled water to 1 L, and adjust the pH to 7 with 0.1 mol / L HCl.

[0173] Blank group culture medium: YCFA basal culture medium was used as the blank control group, recorded as G1.

[0174] Experimental group culture medium: Accurately weigh equal amounts of fructooligosaccharide (FOS), GDP-H, GDP1-H and GDP3-H, respectively, add them into YCFA basal medium to dissolve (8 mg / mL), record them as G2 group, G3 group, G4 group and G5 group, sterilize them at 121℃ for 30 min, cool them to room temperature and place them in the anaerobic workstation.

[0175] In this experiment, stool samples were collected from 6 volunteers, including 3 males and 3 females. All volunteers had no intestinal diseases, had a normal diet, regular bowel movements, and had not taken antibiotics for at least 3 months before sampling.

[0176] Weigh equal amounts of feces from volunteers, mix them, and immediately add them into 100 mL of sterilized PBS to prepare a mixed feces solution with a final concentration of 10% (w / v). Centrifuge (500 r / min, 5 min) to obtain the supernatant.

[0177] The human feces supernatant was placed in an anaerobic workstation and quickly inoculated into groups G1, G2, G3, G4 and G5. 1 mL (10%) of feces mixed suspension was inoculated into every 10 mL of culture medium and fermented in an anaerobic workstation at 37°C for 24 hours. After 24 hours, the mixture was centrifuged (10,000 rpm, 10 min) to obtain the precipitate.

[0178] The precipitate was subjected to microbial diversity sequencing, which was performed by Beijing Novogene Technology Co., Ltd. The PCR amplification primer was 341F_806R (forward primer: CCTAYGGGRBGCASCAG, reverse primer: GGACTACHVGGGTWTCTAAT).

[0179] The operational taxonomic units (OTUs) were analyzed using bioinformatics methods, and the similarity was 97%. Fig.10 A is the Coverage, Simpson, Chao and Shannon indexes, which are often used to reflect the α diversity of species. Compared with the blank group, the species richness and diversity of the intestinal flora in the four experimental groups decreased. The Venn diagram can more intuitively show the similarity and overlap of OTU composition between samples. Fig.10 From B, we can see that the five groups of samples overlapped 96 OUTs. The blank group retained 285 OUTs, while the G2, G3, G4, and G5 groups retained 237, 224, 275, and 263 OUTs, respectively, which were all lower than those of the blank group. The trend was consistent with the results of α diversity analysis. Fig.10As shown in C, at the phylum level, the microbial communities of the samples in each group were mainly composed of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Compared with the blank group (G1 group), the relative abundance of Firmicutes and Bacteroidetes in groups G2, G3, G4, and G5 increased, which is consistent with the trend of short-chain fatty acid content mentioned above. Specifically, the relative abundance of Firmicutes was 54.50%, 40.87%, 41.12%, and 42.68%, respectively (30.21% in group G1), and the relative abundance of Bacteroidetes was 14.17%, 22.16%, 9.07%, and 13.59%, respectively (3.92% in group G1). The abundance of Actinobacteria also increased significantly in each group. On the other hand, compared with the G1 group, the relative abundance of Proteobacteria in each group was significantly reduced, and the relative abundance of Proteobacteria in the four groups was 16.50%, 34.29%, 34.23%, and 37.00%, respectively (53.43% in the G1 group). Fig.10 From D, we can see that at the genus level, each group is mainly composed of Megasphaera, Escherichia coli Shigella, Bacteroides, Bifidobacterium, etc. Compared with the blank group (G1 group), the relative abundance of Escherichia coli Shigella in groups G2, G3 and G4 decreased significantly, by 37.5%, 28% and 48%, respectively. Compared with group G1, group G3 was able to increase the relative abundance of Bifidobacterium (21.72%). In addition, the relative abundance of Bacteroides and Lactobacillus in the experimental group also increased. Fig.10 From E, we can see that the samples of the blank group are basically concentrated in one area, and the samples of the sample group are basically concentrated in one area. This shows that compared with the normal group, the intestinal microorganisms of the experimental group have changed significantly.

[0180] The community heat map also analyzes the intestinal microbiome at the genus level, using colors to represent the relative abundance of different microorganisms, which can visually show the differences in microbial species composition in the samples. Fig.11 The results of A showed that after 24 hours of in vitro fermentation, the intestinal flora composition of the experimental group and the blank group changed significantly. Specifically, compared with the blank group (G1 group), the relative abundance of beneficial bacteria in the experimental groups (G2, G3, G4 and G5), such as Bacteroides, Bifidobacterium, Lactobacillus, Prevotella, and Collinsella, increased, while the relative abundance of harmful bacteria such as Escherichia coli, Shigella, Morganella, Phascolarctobacterium, Enterobacter, and Peneproteus decreased. Fig.11B is the histogram of significantly enriched bacteria in LEfSe of the experimental group. At the genus level, the blank group was significantly enriched with: Enterobacter, Escherichia coli-Shigella; at the family level, Morganellaceae and others were enriched. After GDP fermentation, the experimental group showed significant enrichment of probiotics Lactobacillusaceae, Lactobacillus, Bifidobacteraceae, Bifidobacterium, Bacteroidetes, Bacteroides, and Macrosphaera; GDP1 and GDP3 also promoted the growth of Bifidobacteriales and Bifidobacterium of Bifidobacteriaceae. The heat map of the association analysis between intestinal flora and short-chain fatty acids is an important tool for exploring the relationship between intestinal microorganisms and metabolism in in vitro fermentation studies. Fig.11 It can be seen from the C that the intestinal flora has a low correlation with isobutyric acid and isovaleric acid at the genus level, while the correlation with valeric acid, acetic acid, butyric acid and propionic acid is more significant. The correlation between total short-chain fatty acids and acetic acid is similar. The addition of polysaccharides can be used by the above species to generate short-chain fatty acids, thus having a positive impact on human intestinal health.

[0181] Finally, it should be noted that the above are only some specific embodiments of the present invention. The present invention is not limited to these embodiments, and there may be many other variations. Any variation that can be directly derived or associated with the content disclosed by a person skilled in the art should be included in the protection scope of the present invention.

Claims

1. Hydrogen peroxide / ascorbic acid assisted polysaccharide extraction method, characterized in that The following steps are involved: S1. Deionized water is added to defatted Glechoma longituba powder to form a mixed solution. Under light-proof conditions, hydrogen peroxide and ascorbic acid are added to the mixed solution to form a reaction system. The reaction system is placed in an oil bath at 90 to 100° C. and stirred for extraction for 3±0.5 h to obtain a reaction solution. The molar ratio of hydrogen peroxide to ascorbic acid is 1:1±0.1; Defatted Glechoma longituba powder: deionized water = 1g: 30±10mL solid-liquid ratio; In the reaction system, the concentration of H2O2 is 5-25 mmol / L, and the concentration of Vc is 5-25 mmol / L; S2, cooling the reaction solution obtained in step S1 to room temperature and then centrifuging, and concentrating the supernatant obtained by centrifugation to 30% to 35% of the original volume to obtain a concentrated solution; Add anhydrous ethanol to the concentrate, stir evenly, place at 0-5°C for alcohol precipitation for 24±2h, and centrifuge to obtain a precipitate; the volume ratio of the concentrate to anhydrous ethanol is 1:3.5-4.5; The precipitate was redissolved in deionized water and dialyzed in deionized water for 72±2 h using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain a retentate; The retentate is concentrated to 20% to 25% of the original volume, and freeze-dried to obtain crude Glehnia littoralis polysaccharide.

2. The hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method according to claim 1, characterized in that: In the re-dissolution in step S2, the volume of deionized water is 0.8 to 1.2 times the volume of the concentrated solution.

3. The hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method according to claim 1 or 2, characterized in that: Defatted Glechoma longituba powder: deionized = 1g: 31mL water-to-liquid ratio; In the reaction system: the concentration of H2O2 is 21mmol / L, the concentration of Vc is 21mmol / L; The reaction system was placed in a 94° C. oil bath and stirred for 3 h to obtain a reaction solution.

4. The hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method according to claim 3, characterized in that In S2: The reaction solution obtained in step S1 is cooled and centrifuged, and the supernatant obtained by centrifugation is concentrated to 1 / 3 of the original volume to obtain a concentrated solution; Add anhydrous ethanol to the concentrate, stir evenly, place at 4°C for 24 hours, and then centrifuge to obtain a precipitate; the volume ratio of the concentrate to anhydrous ethanol is 1:4; The obtained precipitate was re-dissolved in deionized water and dialyzed in deionized water for 72 h using a dialysis bag with a molecular weight cut-off of 3500 Da to obtain a retentate; Deionized water: concentrate = 0.8:1 volume ratio; The retentate was concentrated to 1 / 4 of the original volume and freeze-dried to obtain crude polysaccharide of Glechoma longituba.

5. The hydrogen peroxide / ascorbic acid-assisted polysaccharide extraction method according to any one of claims 1 to 4, characterized in that: The crude polysaccharide of Glehnia littoralis obtained in step S2 is separated and purified, comprising the following steps: ① Rough classification: Take the crude polysaccharide of Glechoma longituba and prepare it into a polysaccharide solution of 20±0.5 mg / mL with deionized water, take 4.8-5.2 mL of the polysaccharide solution and centrifuge to obtain the supernatant; The supernatant was evenly added to the surface of the filler of the glass chromatography column, and eluted with deionized water and gradient concentration NaCl solution as eluents in turn through the DEAE Cellulose-52 chromatography column at a rate of 1 mL / min; The sugar-containing eluates corresponding to 0.1 mol / L and 0.3 mol / L NaCl solutions were collected respectively, and two preliminary purified polysaccharide fractions of Glechoma longituba polysaccharide were obtained after concentration, dialysis and freeze-drying. ②Purification: The two preliminarily purified polysaccharide components obtained in step ① are respectively subjected to the following operations: Weigh the preliminarily purified polysaccharide component and prepare it into a 10 mg / mL polysaccharide component solution with deionized water, take 4.8-5.2 mL of the polysaccharide component solution and centrifuge to obtain a supernatant; The supernatant was filtered through a 0.45 μm filter membrane, evenly loaded into a Sephadex G-100 gel column, eluted with deionized water at a flow rate of 0.25 mL / min, and the eluate was collected; The eluate was concentrated and freeze-dried to obtain two purified polysaccharide fractions, GDP1-H and GDP3-H.

6. Use of crude polysaccharide GDP-H from Glechoma longituba and purified polysaccharide components GDP1-H and GDP3-H, characterized in that: As a functional ingredient, the functionality includes antioxidant, anti-aging activity and intestinal flora regulating activity.

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