Preparation method and application of pectic polysaccharide degradation product of overground part of radix tetrastigme

Abstract

The invention relates to the field of traditional Chinese medicines, and discloses a preparation method and application of a pectic polysaccharide degradation product of an overground part of radix tetrastigme. The preparation method comprises the following steps: 1) extracting radix tetrastigme crude polysaccharide in the overground part of radix tetrastigme; 2) dialyzing the radix tetrastigme crude polysaccharide, and performing gradient elution through a cellulose column to obtain an eluent; (3) eluting with gel column water to obtain radix tetrastigme pectic polysaccharide; (4) carrying out enzymolysis on the radix tetrastigme pectic polysaccharide by using pectinase; and eluting with a gel column to obtain a degradation product of the tetrastigma hemsleyanum pectic polysaccharide. Compared with a product before enzymolysis, the pectic polysaccharide degradation product of the aboveground part of the radix tetrastigme, which is prepared by the method disclosed by the invention, has more advantages in the aspect of immune activation effect, and is beneficial to the development and application of the aboveground part of the radix tetrastigme in the fields of foods and medicines.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and particularly relates to a preparation method and application of a degradation product of pectin polysaccharide from the aerial part of Tetrastigma hemsleyanum Diels et Gilg. Background Art

[0002] Tetrastigma hemsleyanum Diels et Gilg is a rare medicinal plant unique to China, with antipyretic, antioxidant, antibacterial, immunomodulatory, antiviral and anti-inflammatory activities, and is commonly used to treat diseases such as infantile high fever, pneumonia, asthma, rheumatism and hepatitis (CN118615315A, CN117815258A), and has high research value. The above-mentioned effects of Tetrastigma hemsleyanum Diels et Gilg are attributed to its rich bioactive components, such as flavonoids, polysaccharides, phenolic acids and alkaloids. Tetrastigma hemsleyanum Diels et Gilg contains rich polysaccharides, which have anti-inflammatory, anti-tumor, immunomodulatory and other effects, and are safe and non-toxic. However, although extensive studies have been conducted on the biological activities of Tetrastigma hemsleyanum Diels et Gilg, its structural characteristics have not been fully elucidated.

[0003] Traditionally, the tuberous roots of Tetrastigma hemsleyanum Diels et Gilg are its main medicinal parts. In recent years, it has been found that the aerial part of Tetrastigma hemsleyanum Diels et Gilg also has high medicinal value. Extracting polysaccharides from the aerial part of Tetrastigma hemsleyanum Diels et Gilg can make full use of resources, reduce waste, and improve social and economic benefits. In recent years, water-soluble polysaccharides from Tetrastigma hemsleyanum Diels et Gilg have become a research hotspot, and these polysaccharides have attracted much attention due to their unique separation sources, molecular weights and monosaccharide compositions. Among them, pectin polysaccharides are mainly divided into homogalacturonan (HG), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) according to their compositional differences. However, there is no report on the research of pectin polysaccharides from Tetrastigma hemsleyanum Diels et Gilg at present, and pectin polysaccharides have complex structures, their structural characterization is difficult, and their active regions are difficult to elucidate. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a preparation method and application of a degradation product of pectin polysaccharide from the aerial part of Tetrastigma hemsleyanum Diels et Gilg. The present invention for the first time extracts a degradation product of pectin polysaccharide from the aerial part of Tetrastigma hemsleyanum Diels et Gilg, which has more advantages in terms of immune activation efficacy compared with the pectin polysaccharide of Tetrastigma hemsleyanum Diels et Gilg before enzymatic hydrolysis, and is conducive to the development and application of the aerial part of Tetrastigma hemsleyanum Diels et Gilg in the food and pharmaceutical fields.

[0005] The specific technical solution of the present invention is as follows: In the first aspect, the present invention provides a preparation method of a degradation product of pectin polysaccharide from the aerial part of Tetrastigma hemsleyanum Diels et Gilg, which comprises the following steps: 1) Extract the crude polysaccharide of Tetrastigma hemsleyanum Diels et Gilg from the aerial part of Tetrastigma hemsleyanum Diels et Gilg.

[0006] 2) Dialyze the crude polysaccharide of Tetrastigma hemsleyanum Diels et Gilg, and perform gradient elution through a cellulose column to obtain an eluate containing STHP4.

[0007] 3) Elute with water through a gel column to obtain the pectin polysaccharide STHP4 of Tetrastigma hemsleyanum Diels et Gilg.

[0008] 4) Use pectinase to enzymatically hydrolyze the pectin polysaccharide of Tetrastigma hemsleyanum Diels et Gilg., and elute the enzymatic hydrolysis product through a gel column to obtain the pectin polysaccharide degradation products STHP4-1 and STHP4-2 of Tetrastigma hemsleyanum Diels et Gilg.

[0009] As described in the background art section, there are currently few studies on the pectin polysaccharide of Tetrastigma hemsleyanum Diels et Gilg., and pectin polysaccharides have a complex structure, making their structural characterization difficult and their active regions difficult to elucidate. For this reason, the present invention for the first time extracts and isolates a pectin polysaccharide STHP4 highly rich in homogalacturonan (HG) from the aerial parts of Tetrastigma hemsleyanum Diels et Gilg.; different from the usual research on plant polysaccharide extracts in the prior art, the present invention further obtains two components STHP4-1 and STHP4-2 by enzymatically hydrolyzing STHP4. By evaluating the structural characterization and immune activation effects of STHP4 and its degradation products, their main active regions are revealed, which is beneficial to the development and application of the aerial parts of Tetrastigma hemsleyanum Diels et Gilg. in the food and pharmaceutical fields.

[0010] Specifically, the pectin polysaccharide of Tetrastigma hemsleyanum Diels et Gilg. and its degradation products prepared by the method of the present invention have no obvious cytotoxicity, and can promote the secretion of NO, IL-1β, TNF-α and IL-6 in RAW 264.7 cells, and among them, one of the degradation products, STHP4-1, has better activity. It shows that the pectin polysaccharide of Tetrastigma hemsleyanum Diels et Gilg. and its degradation products have good immune activation effects, and are safe and non-toxic, and can be used for the product development of immune activators.

[0011] Preferably, in step 4), the enzymatic hydrolysis temperature is 40-45 °C and the time is 40-50 h.

[0012] More preferably, step 4) specifically includes: dissolving the pectin polysaccharide of Tetrastigma hemsleyanum Diels et Gilg. in an acetate buffer solution at 4-6 mg / mL, adding 0.8-1.2 mg of pectinase to react at 40-45 °C; adding 0.4-0.6 mg and 0.8-1.2 mg of pectinase respectively at 22-26 h and 34-38 h of the reaction; after a total enzymatic hydrolysis of 40-50 h, inactivate the pectinase, centrifuge, filter, and centrifuge again, and elute the supernatant with water through a gel column to obtain the pectin polysaccharide degradation product of Tetrastigma hemsleyanum Diels et Gilg.

[0013] Different enzymatic hydrolysis conditions have a significant impact on the composition and immune activation efficacy of the enzymatic hydrolysis products. The present invention finds that the pectin polysaccharide degradation product of Tetrastigma hemsleyanum Diels et Gilg. obtained by the above optimal enzymatic hydrolysis process has particularly prominent immune activation efficacy, and the effect is better than that of the pectin polysaccharide of Tetrastigma hemsleyanum Diels et Gilg. before enzymatic hydrolysis.

[0014] Preferably, step 1) specifically includes: extracting the dried and pulverized above-ground parts of Tetrastigma hemsleyanum with ethanol aqueous solution A, and performing suction filtration; drying the filter residue and then extracting it with water, centrifuging, taking the supernatant and performing suction filtration, and concentrating it to a viscous state; adding ethanol aqueous solution B and stirring for extraction; sealing and standing still, centrifuging, redissolving the precipitate with water, evaporating ethanol, placing the concentrated solution in an environment of -90 to -70 °C for more than 12 h, and obtaining crude polysaccharide of Tetrastigma hemsleyanum through freeze-drying.

[0015] Preferably, in step 1), the above-ground parts of Tetrastigma hemsleyanum are leaves and / or stems.

[0016] Preferably, in step 1), the ethanol concentration in the ethanol aqueous solution A is 75 - 85 vol%, the temperature is 75 - 85 °C; the solid-liquid ratio for extraction is 1:13 - 17.

[0017] Preferably, in step 1), the temperature for water extraction is 90 - 98 °C.

[0018] Preferably, in step 1), the rotation speed for centrifugation is 3000 - 5000 rpm, and the time is 3 - 7 min.

[0019] Preferably, in step 1), the temperature for the first concentration is 55 - 65 °C; the temperature for the second concentration is 35 - 45 °C.

[0020] Preferably, in step 1), the ethanol concentration in the ethanol aqueous solution B is 90 - 98 vol%, and the solid-liquid ratio for extraction ≤ 1:3.

[0021] Preferably, in step 1), the temperature for sealing and standing still is 1 - 6 °C, and the time is 8 - 12 h.

[0022] Preferably, in step 1), the temperature for evaporating ethanol is 35 - 45 °C.

[0023] Preferably, in step 2), the cut-off molecular weight of the dialysis bag used for dialysis is 2.0 - 3.5 kDa, and the dialysis time is 60 - 80 h.

[0024] Preferably, in step 2), the filler of the cellulose column is DEAE-52 cellulose.

[0025] Preferably, in step 2), gradient elution is performed successively with water, 0.08 - 0.12, 0.18 - 0.22, 0.28 - 0.32, 0.38 - 0.42 mol / L NaCl solutions, the flow rate is 0.8 - 1.2 cm / min, and the last obtained eluate is taken.

[0026] Preferably, step 3) specifically includes: concentrating the eluate at 55-65 °C, desalting by dialysis (cut-off pore size 2.5-3.5 kDa), and freeze-drying to obtain preliminarily purified Polygonum multiflorum Thunb. polysaccharide; preparing an aqueous solution of Polygonum multiflorum Thunb. polysaccharide with a concentration of 6-10 mg / mL, loading the sample onto a Sephadex 200 gel column at a flow rate of 0.2-0.4 cm / min, and the volume of each sample loading being 5-10% of the column bed volume; eluting with water at a flow rate of 0.2-0.4 cm / min, and freeze-drying the eluted fractions to obtain purified Polygonum multiflorum Thunb. pectin polysaccharide.

[0027] Preferably, in step 4): the pH of the acetate buffer solution is 4.3-4.7.

[0028] Preferably, in step 4): the model of the gel column is Sephadex 200.

[0029] In the second aspect, the present invention provides the Polygonum multiflorum Thunb. pectin polysaccharide obtained in step 3) of the above preparation method, whose molecular weight distribution is 2 kDa-10000 kDa; the molecular weight is concentrated at 119 kDa; there are a total of 18 partially methylated alditol acetate derivatives (PMAAs): t-Araf, 1,5-Araf, 1,3,5-Araf, 1,2-Rhap, 1,3,4-Rhap, 1,2,4-Rha, t-Galp, 1,2-Galp, 1,6-Galp, 1,3,6-Galp, t-GalAp, 1,4-GalAp, 1,3,4-GalAp, 1,3-Glcp, 1,6-Glcp, t-GlcAp, 1,4-GlcAp, 1,2,3-Manp; among them, GalAp accounts for 55-65%; the monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and their molar ratio is (7-8):(4.5-5.5):(9-11):(51-53):(0.3-0.4):(14-15):(9-10).

[0030] In the third aspect, the present invention provides the degradation product of the Polygonum multiflorum Thunb. pectin polysaccharide obtained by the above preparation method, wherein: The average molecular weight of the STHP4-1 is 9.9 kDa-251 kDa, including mannose, rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, and their molar ratio is (29-31):(11-13):(17-19):(5.5-6.5):(20-22):(11-13); The average molecular weight of the said STHP4-2 is 1.387 kDa, which is mainly composed of free monosaccharides, including galacturonic acid, galactose, arabinose and glucose, and their molar ratios are respectively (75-77):(0.8-0.9):(2.5-3.5):(18-20).

[0031] In the fourth aspect, the present invention provides the application of the tinospora sagittata pectin polysaccharide or the degradation product of the tinospora sagittata pectin polysaccharide obtained by the above preparation method in the preparation of an immune activator.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention first extracts pectin polysaccharide and its degradation product from the aerial part of tinospora sagittata through a specific process. The pectin polysaccharide and its degradation product of tinospora sagittata have no obvious cytotoxicity, and can promote the secretion of NO, IL-1β, TNF-α and IL-6 in RAW 264.7 cells, and among them, one of the degradation products, STHP4-1, has better activity. It shows that the pectin polysaccharide and degradation product of tinospora sagittata have good immune activation effects, are safe and non-toxic, and can be used for the product development of immune activators. Description of the Drawings

[0033] Figure 1 It is the separation, purification, monosaccharide composition and molecular weight analysis of STHP4. (A) Elution curve of polysaccharide THP4 eluted with 0.3 mol / L Nacl from tinospora sagittata on a Superdex 200 gel chromatography column (1.5×100 cm); (B) Molecular weight analysis of STHP4. (C) Monosaccharide composition analysis: a) Standard; b) STHP4.

[0034] Figure 2 It is the elution curve of the pectinase hydrolysis product of STHP4 on a Superdex 200 gel chromatography column (1.5×100 cm).

[0035] Figure 3 It is the molecular weight and monosaccharide composition analysis of the degradation products STHP4-1 and STHP4-2 of STHP4. (A) Molecular weight analysis: a) STHP4-1; b) STHP4-2; c) Standard; (B) Monosaccharide composition analysis: a) Standard; b) STHP4-1; c) STHP4-2; (C) Free monosaccharide composition analysis: a) Standard; b) STHP4-2; (D) Molar proportion of the monosaccharide composition of STHP4, STHP4-1 and STHP4-2.

[0036] Figure 4 It is the NMR spectrum of STHP4. (A) 1H; (B) 13C; (C) DEPT-135.

[0037] Figure 5 It is the NMR spectrum of STHP4. (D) HSQC

[0038] Figure 6 It is the NMR spectrum of STHP4. (E) TOCSY.

[0039] Figure 7 It is the NMR spectrum of STHP4. (F) COSY.

[0040] Figure 8 It is the NMR spectrum of STHP4. (G) HMBC; (H) NOESY.

[0041] Figure 9 It is the comparison diagram of 1H spectra of STHP4 (a) and STHP4-1 (b) at 4.25 - 5.50 ppm.

[0042] Figure 10 It is the speculated chemical structure diagram of STHP4.

[0043] Figure 11 It is the immunological activity analysis of STHP4, STHP4-1 and STHP4-2. (A) Effect on cell viability; (B) Effect on NO release; (C) Expression of inflammatory factor mRNAs in RAW 264.7 cells: a) IL-6; b) TNFα; c) IL-1β (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05). Detailed implementation mode

[0044] The present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification, but the implementation modes of the present invention are not limited thereto. The experimental raw materials and reagents involved in the following embodiments can all be obtained from commercial channels. For the experimental methods without specific conditions noted in the embodiments, they are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0045] General embodiment A preparation method of the degraded product of the pectin polysaccharide from the aerial part of Tetrastigma hemsleyanum Diels et Gilg, which comprises the following steps: 1) Extract the crude polysaccharide of Tetrastigma hemsleyanum Diels et Gilg from the aerial part.

[0046] In some preferred embodiments, step 1) specifically includes: extracting the dried and crushed aerial part of Tetrastigma hemsleyanum Diels et Gilg with ethanol aqueous solution A, and performing suction filtration; after drying the filter residue, extracting it with water, centrifuging, taking the supernatant and performing suction filtration, and concentrating it to a viscous state; adding ethanol aqueous solution B and stirring for extraction; sealing and standing still, centrifuging, dissolving the precipitate with water, evaporating ethanol, placing the concentrated solution in an environment of -90 to -70 °C for more than 12 h, and obtaining the crude polysaccharide of Tetrastigma hemsleyanum Diels et Gilg through freeze-drying.

[0047] In some more preferred embodiments, the aerial part of Tetrastigma hemsleyanum Diels et Gilg is leaves and / or stems.

[0048] In some more preferred embodiments, the concentration of ethanol in the aqueous ethanol solution A is 75 - 85 vol%, the temperature is 75 - 85 °C; the solid-liquid ratio for extraction is 1:13 - 17.

[0049] In some more preferred embodiments, the temperature for water extraction is 90 - 98 °C.

[0050] In some more preferred embodiments, the rotation speed for centrifugation is 3000 - 5000 rpm, and the time is 3 - 7 min.

[0051] In some more preferred embodiments, the temperature for the first concentration is 55 - 65 °C; the temperature for the second concentration is 35 - 45 °C.

[0052] In some more preferred embodiments, the concentration of ethanol in the aqueous ethanol solution B is 90 - 98 vol%, and the solid-liquid ratio for extraction is ≤1:3.

[0053] In some more preferred embodiments, the temperature for sealed static placement is 1 - 6 °C, and the time is 8 - 12 h.

[0054] In some more preferred embodiments, the temperature for evaporating ethanol is 35 - 45 °C.

[0055] 2) Dialyze the crude polysaccharide of Tetrastigma hemsleyanum, and perform gradient elution through a cellulose column to obtain an eluate containing STHP4.

[0056] In some preferred embodiments, the cut-off molecular weight of the dialysis bag used for dialysis is 2.0 - 3.5 kDa, and the dialysis time is 60 - 80 h.

[0057] In some preferred embodiments, the packing material of the cellulose column is DEAE - 52 cellulose.

[0058] In some preferred embodiments, perform gradient elution successively with water, 0.08 - 0.12, 0.18 - 0.22, 0.28 - 0.32, 0.38 - 0.42 mol / L NaCl solutions, the flow rate is 0.8 - 1.2 cm / min, and take the eluate obtained in the last time.

[0059] 3) Perform water elution through a gel column to obtain the pectin polysaccharide STHP4 of Tetrastigma hemsleyanum.

[0060] In some preferred embodiments, step 3) specifically includes: concentrating the eluate at 55 - 65 °C, desalting by dialysis (cut-off pore size 2.0 - 3.5 kDa), and freeze-drying to obtain preliminarily purified Polygonum multiflorum Thunb. polysaccharide; preparing an aqueous solution of Polygonum multiflorum Thunb. polysaccharide with a concentration of 6 - 10 mg / mL, loading it onto a Sephadex 200 gel column at a flow rate of 0.2 - 0.4 cm / min, and the volume of each loading being 5 - 10% of the column bed volume; eluting with water at a flow rate of 0.2 - 0.4 cm / min, and freeze-drying the eluted fractions to obtain purified Polygonum multiflorum Thunb. pectin polysaccharide.

[0061] In some preferred embodiments, the molecular weight distribution of Polygonum multiflorum Thunb. pectin polysaccharide is in the range of 2 kDa - 10000 kDa: the molecular weight is concentrated at 119 kDa; there are a total of 18 partially methylated alditol acetate derivatives (PMAAs): t-Araf, 1,5-Araf, 1,3,5-Araf, 1,2-Rhap, 1,3,4-Rhap, 1,2,4-Rha, t-Galp, 1,2-Galp, 1,6-Galp, 1,3,6-Galp, t-GalAp, 1,4-GalAp, 1,3,4-GalAp, 1,3-Glcp, 1,6-Glcp, t-GlcAp, 1,4-GlcAp, 1,2,3-Manp; among them, GalAp accounts for 55 - 65%; the monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose, and their molar ratio is (7 - 8):(4.5 - 5.5):(9 - 11):(51 - 53):(0.3 - 0.4):(14 - 15):(9 - 10).

[0062] 4) Enzymatically hydrolyze Polygonum multiflorum Thunb. pectin polysaccharide with pectinase, the enzymatic hydrolysis temperature is 40 - 45 °C, and the time is 40 - 50 h; the enzymatic hydrolysis product is eluted through a gel column to obtain Polygonum multiflorum Thunb. pectin polysaccharide degradation products STHP4-1 and STHP4-2.

[0063] In some more preferred embodiments, step 4) specifically includes: dissolving Polygonum multiflorum Thunb. pectin polysaccharide in acetate buffer solution at 4 - 6 mg / mL, adding 0.8 - 1.2 mg of pectinase to react at 40 - 45 °C; adding 0.4 - 0.6 mg and 0.8 - 1.2 mg of pectinase respectively at 22 - 26 h and 34 - 38 h of the reaction; after a total enzymatic hydrolysis of 40 - 50 h, inactivate the pectinase, centrifuge, filter, and centrifuge again, and the supernatant is eluted through a gel column with water to obtain Polygonum multiflorum Thunb. pectin polysaccharide degradation products.

[0064] In some more preferred embodiments, the pH of the acetate buffer solution is 4.3 - 4.7.

[0065] In some more preferred embodiments, the model of the gel column is Sephadex 200.

[0066] In some preferred embodiments, the average molecular weight of the STHP4-1 is from 9.9 kDa to 251 kDa, and it includes mannose, rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, and the molar ratio thereof is (29-31):(11-13):(17-19):(5.5-6.5):(20-22):(11-13).

[0067] In some preferred embodiments, the average molecular weight of the STHP4-2 is 1.387 kDa, and it is mainly composed of free monosaccharides, including galacturonic acid, galactose, arabinose and glucose, and the molar ratio thereof is respectively (75-77):(0.8-0.9):(2.5-3.5):(18-20).

[0068] Specific examples and comparative examples Example 1: Preparation and degradation of the pectin polysaccharide from the aerial part of Tetrastigma hemsleyanum 1.1 Experimental reagents and instruments Chemical reagents: 95% ethanol, concentrated sulfuric acid (98%), phenol, carbazole, trifluoroacetic acid, NaBD4, DMSO, acetic acid, dichloromethane, methyl iodide, acetic anhydride, methanol, hydrochloric acid, chloroform, sodium hydroxide, tetramethylsilane (TMS), deuterated water (D2O), etc. DEAE-52 cellulose (Shanghai Yuanye Bio-Technology Co., Ltd.), Superdex 200 glucan gel (Beijing Weishi Bohui Chromatography Technology Co., Ltd.).

[0069] Kit: Brandford protein concentration assay kit (Beyotime P0006).

[0070] Experimental instruments: constant-current peristaltic pump (Lange Constant Current Pump Co., Ltd.), 1.5×30 cm glass chromatography column, 2.6×50 cm glass chromatography column (Shanghai Lianta Instruments Co., Ltd.), HC-0210-10 medium-pressure chromatography column (Beijing Ruida Henghui Technology Development Co., Ltd.), BS-100N automatic fraction collector (Shanghai Huxi Analytical Instrument Factory Co., Ltd.), rotary evaporator (Shanghai Shensheng Technology Co., Ltd.), LDZ5-2 low-speed balancing centrifuge (Beijing Jingli Centrifuge Co., Ltd.), SHZ-D(III) circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd.), Freezone 4.5L freeze dryer (Labconco, USA), 3 kDa dialysis bag (Shanghai Yuanye Biotechnology Co., Ltd.), high-performance liquid chromatography system (equipped with OHpak SB-805 HQ, SB-804HQ, SB-803HQ gel columns) (Shodex, Japan), Waters2410 refractive index detector (Waters, USA), Ultimate 3000 high-performance liquid chromatography system (Thermo Fisher Scientific, USA).

[0071] ICS 5000+ high-performance liquid chromatography system (equipped with Dionex TM CarboPac TM PA-20 anion exchange column) (Thermo Fisher, USA), Agilent 7890A-5977B gas chromatography system (equipped with Agilent BPX70 chromatographic column) (Agilent Technologies, USA), various types of pipettes (Eppendorf, Germany).

[0072] Experimental equipment: water bath, round-bottom flask, Buchner funnel, filter paper, glass rod, beaker.

[0073] 1.2 Experimental methods 1.2.1 Extraction of Tetrastigma hemsleyanum crude polysaccharide Take the dried above-ground leaves and stems of Tetrastigma hemsleyanum, crush them, weigh 100 g, extract twice with 80% (v / v) ethanol aqueous solution (temperature is 80 °C), and filter by suction. The filter residue is dried in a ventilated place at room temperature, and then extracted twice with pure water at 95 °C. Centrifuge at 4000 rpm for 5 min, take the supernatant and filter by suction. Concentrate to a viscous state with a rotary evaporator at a temperature of 60 °C. Slowly add more than 3 times 95% ethanol, and stir rapidly with a magnetic stirrer (slow first and then fast). Seal and leave overnight at 4 °C. Centrifuge at 4000 rpm for 5 min, collect the precipitate, redissolve it with pure water, and evaporate the ethanol with a rotary evaporator at a temperature set at 40 °C. Freeze the concentrated solution at -80 °C for more than 12 h, and then freeze-dry to obtain Tetrastigma hemsleyanum crude polysaccharide.

[0074] 1.2.2 Isolation and Purification of Tetrastigma hemsleyanum Diels et Gilg Crude Polysaccharide 1.2.2.1 Dialysis Dissolve the crude polysaccharide in pure water and dialyze it in pure water for 72 h (cut-off pore size 3 kDa). Take out the liquid in the dialysis bag and store it in a refrigerator at 4°C.

[0075] 1.2.2.2 Purification by Anion Exchange Chromatography Column Use DEAE-52 cellulose as the packing material for the anion exchange chromatography column. Use a constant flow pump to determine the flow rate. Pass deionized water through the column for 3 - 5 column bed volumes to reach the equilibrium state (linear flow rate 1.5 cm / min). Take an appropriate amount of the deionized aqueous solution of the dialyzed Tetrastigma hemsleyanum Diels et Gilg crude polysaccharide for sample loading (linear flow rate 0.25 cm / min), and elute it with a gradient of deionized water, 0.1, 0.2, 0.3, 0.4 mol / L NaCl solutions (linear flow rate 1 cm / min). Use an automatic fraction collector to collect the eluted fractions into test tubes. Detect the eluted fractions by the sulfuric acid-phenol method. Take the fraction eluted with 0.3 mol / L NaCl, concentrate it by rotary evaporation under reduced pressure (temperature 60°C), dialyze to remove salts (cut-off pore size 3 kDa), and freeze-dry to obtain the preliminarily purified Tetrastigma hemsleyanum Diels et Gilg polysaccharide THP4.

[0076] 1.2.2.3 Purification by Gel Chromatography Column Use Superdex 200 as the packing material for the gel chromatography column. Use a constant flow pump to determine the flow rate. Pass deionized water through the column for 3 - 5 column bed volumes to reach the equilibrium state (linear flow rate 0.3 cm / min). Take the deionized aqueous solution of the preliminarily purified polysaccharide THP4 (concentration 8 mg / mL, linear flow rate 0.3 cm / min) for sample loading. To achieve good separation effect, the sample loading volume each time is 5 - 10% of the column bed volume. Elute with deionized water (linear flow rate 0.3 cm / min), and use an automatic fraction collector to collect the fractions into test tubes. Detect the eluted fractions by the sulfuric acid-phenol method. Take the eluted fractions and freeze-dry to obtain the purified Tetrastigma hemsleyanum Diels et Gilg polysaccharide STHP4.

[0077] 1.2.3 Degradation of Tetrastigma hemsleyanum Diels et Gilg Polysaccharide STHP4 Dissolve 30 mg of STHP4 in 6 mL of acetate buffer solution (pH 4.5, 0.1 M), and add 1 mg of pectinase at 42°C and react for 24 h. Then add 0.5 mg and 1 mg of pectinase at 24 h and 36 h of reaction respectively. After the reaction is completed, treat the residual enzyme protein at 100°C for 10 min to inactivate it, and then remove it by centrifugation and filtration. Centrifuge the enzymolysis solution at 3500 rpm for 5 min and collect the supernatant. Further separate it by Superdex 200 gel permeation chromatography.

[0078] 1.2.4 Molecular Weight Detection of Tetrastigma hemsleyanum Diels et Gilg Polysaccharide STHP4 and Its Degradation Products The molecular weight of the purified polysaccharide from *Tetrastigma hemsleyanum* was detected using a Waters high performance liquid chromatography system equipped with three OHpak SB-803HQ, SB-804HQ, and SB-805HQ chromatographic columns (8×300 mm). 5 mg of the polysaccharide from *Tetrastigma hemsleyanum* was completely dissolved in 1 mL of 0.05 mol / L NaCl solution (concentration: 5 mg / mL). Before injection, the sample was filtered through a 0.22 μm filter membrane. The injection volume was 30 μL each time. 0.05 mol / L NaCl solution was used as the mobile phase, with a flow rate of 0.65 mL / min and a column temperature of 40 °C. The molecular weight of the polysaccharide from *Tetrastigma hemsleyanum* was determined by comparing the retention time with the standard curve of dextran with different molecular weights (molecular weight range: 0.1 - 670 kDa).

[0079] 1.2.5 Detection of monosaccharide composition of the polysaccharide STHP4 from *Tetrastigma hemsleyanum* and its degradation products The analysis of the monosaccharide composition was performed using the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization method. 5 mg of the polysaccharide from *Tetrastigma hemsleyanum* was hydrolyzed with 1 mL of 2 mol / L trifluoroacetic acid (TFA) at 121 °C for 2 h, then dried under nitrogen and washed with methanol to remove the residual TFA. This process was repeated 2 - 3 times. 0.2 mL of the monosaccharide standard or sample solution was taken, 0.5 mL of 0.5 mol / L PMP methanol solution and 0.2 mL of 0.5 mol / L sodium hydroxide solution were added. After mixing, the reaction was carried out at 70 °C for 1 h. Finally, 0.2 mL of 0.5 mol / L hydrochloric acid was added to neutralize the sodium hydroxide, and then 1 mL of chloroform was added to remove the excess PMP. The aqueous phase was carefully separated, made up to 1 mL with water, and the organic phase was discarded. After the sample was filtered through a 0.22 μm filter membrane, it was analyzed using a high performance liquid chromatography system. The chromatographic column was a ZORBAX Eclipse XDB-C18 column, equipped with an ultraviolet detector, and the detection temperature was 30 °C. The injection volume was 10 μL, and the mobile phase consisted of phosphate buffer solution A (pH 6.8) and acetonitrile B, with a volume ratio of A to B of 83:17 and a flow rate of 0.8 mL / min.

[0080] 1.2.6 Methylation analysis of the polysaccharide STHP4 from *Tetrastigma hemsleyanum* and its degradation products Dissolve 10 mg of the sample in 1 mL of distilled water, add 1 mL of carbodiimide solution (100 mg / mL), react for 2 h, then add 1 mL of imidazole solution (2 mol / L) and 1 mL of NaBD4 solution (30 mg / mL), and continue to react for 3 h. After the reaction is terminated, add 100 μL of glacial acetic acid, then perform dialysis for 48 h and freeze-dry. Next, dissolve the sample in 500 μL of dimethyl sulfoxide (DMSO), add 1 mg of sodium hydroxide for methylation reaction for 30 min, and then add 50 μL of methyl iodide (CH3I) and react for 1 h. Then add 1 mL of water and 2 mL of dichloromethane, mix well and centrifuge, discard the aqueous phase, and repeat the water washing 3 times. Collect the dichloromethane phase and evaporate to dryness. Add 100 μL of trifluoroacetic acid (TFA, 2 mol / L) and react at 121 °C for 90 min, then evaporate to dryness. Add 50 μL of ammonia water (2 mol / L) and 50 μL of NaBD4 solution (1 mol / L), mix and react at room temperature for 2.5 h. After the reaction is terminated, add 20 μL of acetic acid and blow dry with nitrogen. Wash with 250 μL of methanol and then blow dry with nitrogen again. Add 250 μL of acetic anhydride, mix and react at 100 °C for 2.5 h, add 1 mL of water and let stand for 10 min. Finally, add 500 μL of dichloromethane, mix and centrifuge, discard the aqueous phase, and repeat the water washing 3 times. Take the lower dichloromethane phase and detect it by gas chromatography-mass spectrometry (GC-MS, Agilent 7890A-5977B, Agilent Technologies, USA). The chromatographic column is HP-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm). The temperature programming is as follows: the initial column temperature is maintained at 50 °C for 1 min, then heated from 50 °C to 130 °C (heating rate is 3 °C / min), and finally maintained at 230 °C for 2 min. Mass spectrometry parameters: full scan mode, scanning mass range is 30 - 600 m / z.

[0081] 1.2.7 NMR spectrum analysis of Tetrastigma hemsleyanum polysaccharide STHP4 and its degradation products Dissolve the sample (20 - 30 mg) in 0.5 mL of D2O, and then transfer it to a 5 mm NMR tube for testing.

[0082] Measure one-dimensional nuclear magnetic 1 1H-NMR, 13 13C-NMR, DEPT-135 and two-dimensional nuclear magnetic COSY, HSQC, HMBC, NOESY and TOCSY spectra. Data processing is analyzed using MestReNova 12.0 software (Mestrelab Research S.L).

[0083] 1.3 Experimental results 1.3.1 Yield of crude polysaccharide from Tetrastigma hemsleyanum Light yellow flaky polysaccharides were extracted, with a yield of 13% (dry weight ratio of polysaccharides from Tetrastigma hemsleyanum / Tetrastigma hemsleyanum powder).

[0084] 1.3.2 Preparation of purified polysaccharide STHP4 from Tetrastigma hemsleyanum The yield of polysaccharide THP4 from Tetrastigma hemsleyanum preliminarily purified by anion exchange chromatography column was 13.68% (dry weight ratio of preliminarily purified polysaccharides from Tetrastigma hemsleyanum / crude polysaccharides from Tetrastigma hemsleyanum), and its appearance was light yellow flocculent. The yield of polysaccharide STHP4 from Tetrastigma hemsleyanum purified by gel column was 62.5%, and its appearance was white flocculent.

[0085] 1.3.3 Degradation of STHP4 Pectinase (EC 3.2.1.15) was used to degrade STHP4, and the degradation products were purified by gel column Superdex200. STHP4 could be degraded into two major components, named STHP4-1 and STHP4-2( Figure 2 ).

[0086] 1.3.4 Molecular weights of STHP4, STHP4-1 and STHP4-2 The average molecular weight of STHP4 was measured to be 119 kDa; STHP4-1 was mainly composed of four polysaccharides, with average molecular weights of 251 kDa, 85 kDa, 14 kDa and 9.9 kDa respectively; the average molecular weight of STHP4-2 was 1.387 kDa( Figure 1 and Figure 3 ).

[0087] 1.3.5 Monosaccharide compositions of STHP4, STHP4-1 and STHP4-2 The monosaccharide composition of STHP4 was mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and their molar ratio was 7.52:4.87:10.71:52.82:0.36:14.48:9.24. Among them, galacturonic acid accounted for more than 50%. The monosaccharide composition of STHP4-1 included mannose, rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, and their molar ratio was 30.22:12.43:18.19:5.96:21.18:12.02. STHP4-2 was mainly composed of free monosaccharides, including galacturonic acid, galactose, arabinose and glucose, and their molar ratios were 76.47:0.86:3.07:19.60 respectively. See details in( Figure 1 , Figure 3 , Table 3).

[0088] 1.3.6 Chemical composition identification of STHP4, STHP4-1 and STHP4-2 The sugar contents in STHP4, STHP4-1 and STHP4-2 were measured by the sulfuric acid-phenol method and were 56.05%, 53.55% and 21.1% respectively. The uronic acid contents in STHP4, STHP4-1 and STHP4-2 were measured by the sulfuric acid-carbazole method and were 82.81%, 57.97% and 17.63%. The protein contents in STHP4 were measured by the Bradford method and were 0.69%, 1.37% and 0.43% (Table 3).

[0089] 1.3.7 Methylation analysis of STHP4, STHP4-1 and STHP4-2 STHP4 had a total of 18 partially methylated alditol acetate derivatives (PMAAs): t-Araf, 1,5-Araf, 1,3,5-Araf, 1,2-Rhap, 1,3,4-Rhap, 1,2,4-Rhap, t-Galp, 1,2-Galp, 1,6-Galp, 1,3,6-Galp, t-GalAp, 1,4-GalAp, 1,3,4-GalAp, 1,3-Glcp, 1,6-Glcp, t-GlcAp, 1,4-GlcAp, 1,2,3-Manp. Among them, GalAp accounted for 61.56% (Table 1). The methylation analysis results of STHP4-1 and STHP4-2 are shown in detail in Table 3 and Table 4.

[0090] 1.3.8 Nuclear magnetic resonance analysis of STHP4, STHP4-1 and STHP4-2 The nuclear magnetic resonance analysis of STHP4, STHP4-1 and STHP4-2 is shown in detail in Figures 4 - 9 and Table 5. Combining the structural analysis of the degradation products STHP4-1 and STHP4-2 of STHP4, the possible structure of STHP4 deduced is shown in detail in Figure 10 .

[0091] Table 1: Methylation analysis of STHP4 Table 2: Chemical composition analysis of STHP4, STHP4-1 and STHP4-2 A: Oligosaccharides in STHP4-2; B: Free monosaccharides in STHP4-2.

[0092] Table 3: Methylation analysis of STHP4-1 Table 4: Methylation analysis of STHP4-2 Table 5: 1 1H and 13 13C chemical shifts (ppm) in the NMR spectrum of STHP4 in D2O Example 2: Immunostimulatory effects of Tetrastigma hemsleyanum pectin polysaccharide STHP4 and its degradation products 2.1 Experimental materials 2.1.1 Experimental cells and culture Mouse macrophages (RAW 264.7) were cultured in DMEM (Gibco, USA) at 37 °C and 5% CO2, supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin and streptomycin.

[0093] 2.1.2 Experimental reagents and instruments Experimental reagents: Lipopolysaccharide (LPS, Sigma-Aldrich, Cat.039M4004V). CCK-8 (BioSharp, Cat.68096017). DMSO (Amresco, Cat.V900090).

[0094] Kits: NO detection kit (Beyotime Biotechnology Co., Ltd., batch number: 022421210608); RNA extraction kit (Vazyme, R711); HiScript II QRT SuperMix kit (Vazyme, R223-01); Cham QSYBR qPCR Master Mix kit (Vazyme, Q331-03).

[0095] Experimental instruments: Epoch2 microplate reader (BioTek, USA); CytoFLEX flow cytometer (Beckman, USA); Direct-Q8 ultrapure water integrator (Millipore, USA). StepOnePlus Real-TimePCR instrument (Applied Biosystems, USA); 7500 PCR instrument (Bio-Rad, USA); Heraeus Multifuge X1R high-speed refrigerated centrifuge (Thermo fisher, USA); Nanodrop 2000 (Thermofisher, USA).

[0096] 2.2 Experimental methods 2.2.1 Effects of Tetrastigma hemsleyanum pectin polysaccharide STHP4 and its degradation products on the viability of RAW 264.7 cells The MTT method was used to detect the effects of STHP4, a pectic polysaccharide from Tetrastigma hemsleyanum Diels et Gilg, and its degradation products STHP4-1 and STHP4-2 on the viability of RAW 264.7 cells. RAW 264.7 cells in the logarithmic growth phase were inoculated at 1×10 4 / well into a 96-well plate, 100 μL / well, and cultured in an incubator at 37 °C and 5% CO2 for 12 h. In the drug-added groups, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL of STHP4, STHP4-1, and STHP4-2 were added respectively. The blank control group was added with phosphate buffer PBS and cultured in the incubator. 100 ng / mL LPS was used as the positive control. After 24 h, 10 μL of CCK-8 was added to each well, carefully mixed, incubated at 37 °C for 2 h, and the absorbance was measured at 450 nm.

[0097] Cell survival rate (%) = [(mean OD value of the drug-treated group - OD value of the blank well) / (OD of the blank control group - OD of the blank well)] × 100% 2.2.2 Effects of STHP4, a pectic polysaccharide from Tetrastigma hemsleyanum Diels et Gilg, and its degradation products on NO release According to the above experimental grouping in 2.2.1, the cell supernatant was taken for detection 24 h after adding the drugs. The specific operation was carried out according to the kit: Take out Griess Reagent I and II and restore to room temperature. Dilute the standard product with the solution used for the sample. Add the standard product and the sample into a 96-well plate at 50 μL / well. Add room temperature Griess Reagent I to each well at 50 μL / well. Add room temperature Griess Reagent II to each well at 50 μL / well. Detect the OD value at 540 nm. Calculate the concentration of NO in each experimental group according to the standard product curve.

[0098] 2.2.3 Effects of STHP4 and STHP4-1 on the expression of cytokines IL-6, TNFα, and IL-1β RAW 264.7 cells were inoculated at 5×10 5 / well into a 6-well culture plate. In the drug-treated groups, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL of STHP4 and STHP4-1 were added respectively. The positive control group was added with 100 ng / mL LPS and acted for 24 h. Total RNA was extracted using an RNA extraction kit. Reverse transcribed into cDNA using HiScript II QRT SuperMix for qPCR. Fluorescent quantitative PCR was carried out using Cham Q SYBR qPCR Master Mix, and 2 -ΔΔCtThe relative expression levels of IL-6, TNFα, and IL-1β mRNA were calculated by the method. The primer sequences of IL-6, TNFα, and IL-1β are shown in Table 6.

[0099] Table 6: Primer sequences of IL-6, TNFα, and IL-1β 2.3 Experimental results 2.3.1 Tetrastigma hemsleyanum pectin polysaccharide STHP4 and its degradation products have no obvious effect on the growth of RAW264.7 cells The CCK-8 method was used to detect the effects of STHP4, STHP4-1, and STHP4-2 on the growth of RAW 264.7 cells. The results showed that in the concentration range of 2.5 to 100 μg / mL, these three polysaccharides had no obvious effect on the growth of RAW 264.7 cells ( Figure 11 A).

[0100] 2.3.2 STHP4 and STHP4-1 significantly promote the release of NO in RAW 264.7 cells The effects of STHP4, STHP4-1, and STHP4-2 on the release of NO in RAW 264.7 cells were analyzed by a NO detection kit. STHP4-1 could significantly promote the release of NO at a low concentration of 10 μg / mL, STHP4 could significantly promote the release of NO at a high concentration of 100 μg / mL, while STHP4-2 had no obvious effect ( Figure 11 B). These results indicate that STHP4-1 may be the active region of STHP4.

[0101] 2.3.3 STHP4 and STHP4-1 significantly promote the release of inflammatory factors in RAW 264.7 cells RT-PCR was used to detect the expression of inflammatory factors IL-6, TNFα, and IL-1β by STHP4 and STHP4-1. As Figure 7 shown in C, both STHP4 and STHP4-1 could significantly promote the mRNA expression of IL-6, TNFα, and IL-1β.

[0102] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And, the experimental methods described herein are conventional methods unless otherwise specified; the reagents, biological materials, and equipment described herein can be obtained from commercial sources unless otherwise specified.

[0103] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of the degradation product of pectin polysaccharide from the aboveground part of Tetrastigma hemsleyanum, which is characterized in that It includes: 1) Extract the crude polysaccharide of Tetrastigma hemsleyanum from its above-ground parts; 2) Dialyze the crude polysaccharide of Tetrastigma hemsleyanum, and perform gradient elution through a cellulose column to obtain an eluate; 3) Wash and elute through a gel column to obtain the pectin polysaccharide of Tetrastigma hemsleyanum; 4) Use pectinase to enzymatically hydrolyze the pectin polysaccharide of Tetrastigma hemsleyanum, with the enzymatic hydrolysis temperature being 40 - 45 °C and the time being 40 - 50 h; after elution through a gel column, obtain the degradation product of the pectin polysaccharide of Tetrastigma hemsleyanum.

2. The preparation method according to claim 1, characterized in that: Step 1) specifically includes: Extract the dried and pulverized above-ground parts of Tetrastigma hemsleyanum with aqueous ethanol solution A, and perform suction filtration; after drying the filter residue, extract it with water, centrifuge, take the supernatant for suction filtration, and concentrate it to a viscous state; add aqueous ethanol solution B for stirring extraction; seal and let it stand still, centrifuge, dissolve the precipitate with water, evaporate ethanol, place the concentrated solution in an environment of -90~-70 °C for more than 12 h, and obtain the crude polysaccharide of Tetrastigma hemsleyanum through freeze-drying.

3. The preparation method according to claim 2, characterized in that: In step 1), the above-ground parts of Tetrastigma hemsleyanum are leaves and / or stems; in the aqueous ethanol solution A, the ethanol concentration is 75 - 85 vol%, and the temperature is 75 - 85 °C; the solid-liquid ratio for extraction is 1:13 - 17; the temperature for water extraction is 90 - 98 °C; the rotation speed for centrifugation is 3000 - 5000 rpm, and the time is 3 - 7 min; the temperature for the first concentration is 55 - 65 °C; the temperature for the second concentration is 35 - 45 °C; in the aqueous ethanol solution B, the ethanol concentration is 90 - 98 vol%, and the solid-liquid ratio for extraction ≤ 1:3; the temperature for sealing and standing still is 1 - 6 °C, and the time is 8 - 12 h; the temperature for evaporating ethanol is 35 - 45 °C.

4. The preparation method according to claim 2, wherein: In step 2), the cut-off molecular weight of the dialysis bag used for dialysis is 2.0 - 3.5 kDa, and the dialysis time is 60 - 80 h; the packing material of the cellulose column is DEAE - 52 cellulose; Perform gradient elution successively with water, 0.08 - 0.12, 0.18 - 0.22, 0.28 - 0.32, 0.38 - 0.42 mol / L NaCl solutions, with a flow rate of 0.8 - 1.2 cm / min, and take the eluate obtained in the last time.

5. The preparation method according to claim 1, characterized in that: Step 3) specifically includes: Concentrate the eluate at 55 - 65 °C, dialyze to desalt, and perform freeze-drying to obtain the preliminarily purified polysaccharide of Tetrastigma hemsleyanum; prepare an aqueous solution of the polysaccharide of Tetrastigma hemsleyanum with a concentration of 6 - 10 mg / mL, use a gel column Sephadex 200, load the sample at a flow rate of 0.2 - 0.4 cm / min, and the volume of each sample loading is 5 - 10% of the column bed volume; elute with water at a flow rate of 0.2 - 0.4 cm / min, and perform freeze-drying on the eluted fractions to obtain the purified pectin polysaccharide of Tetrastigma hemsleyanum.

6. The preparation method according to claim 1, wherein: Step 4) specifically includes: Dissolve the pectin polysaccharide of Tetrastigma hemsleyanum in an acetate buffer solution at 4 - 6 mg / mL, add 0.8 - 1.2 mg pectinase for reaction at 40 - 45 °C; supplement 0.4 - 0.6 mg and 0.8 - 1.2 mg pectinase respectively at 22 - 26 h and 34 - 38 h of the reaction; after a total enzymatic hydrolysis of 40 - 50 h, inactivate the pectinase, centrifuge, filter, centrifuge again, and elute the supernatant through a gel column with water to obtain the degradation product of the pectin polysaccharide of Tetrastigma hemsleyanum.

7. The preparation method according to claim 6, characterized in that: In step 4): The pH of the acetate buffer solution is 4.3 - 4.7; The model of the gel column is Sephadex 200.

8. The tetrastigma hemsleyanum pectin polysaccharide obtained in step 3) of the preparation method according to any one of claims 1 - 7, characterized in that: The molecular weight distribution is from 2 kDa to 10000 kDa; There are 18 partially methylated alditol acetate derivatives: t-Ara f , 1,5-Ara f , 1,3,5-Ara f , 1,2-Rha p , 1,3,4-Rha p , 1,2,4-Rha ， t-Gal p , 1,2-Gal p , 1,6-Gal p , 1,3,6-Gal p , t-GalA p , 1,4-GalA p , 1,3,4-GalA p , 1,3-Glc p , 1,6-Glc p , t-GlcA p , 1,4-GlcA p , 1,2,3-Man p ; among them, GalA p accounts for 55 - 65%; The monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molar ratio thereof is (7 - 8):(4.5 - 5.5):(9 - 11):(51 - 53):(0.3 - 0.4):(14 - 15):(9 - 10).

9. The degradation product of tetrastigma hemsleyanum pectin polysaccharide obtained by the preparation method according to any one of claims 1-7, characterized in that: The average molecular weight is from 9.9 kDa to 251 kDa, including mannose, rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, and the molar ratio thereof is (29 - 31):(11 - 13):(17 - 19):(5.5 - 6.5):(20 - 22):(11 - 13).

10. Use of the tetrastigma hemsleyanum pectin polysaccharide or the degradation product of the tetrastigma hemsleyanum pectin polysaccharide obtained by the preparation method according to any one of claims 1 - 7 in the preparation of an immune activator.

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