Tianfu peanut 26-leaf polysaccharide as well as preparation method and application thereof

Abstract

The invention provides Tianfu peanut 26-leaf polysaccharide (AHL-P2) as well as a preparation method and application thereof. The AHL-P2 is composed of glucose, galactose and xylose, and the molar ratio of glucose residues to galactose residues to xylose residues is 6: 4: 3; the main chain of the AHL-P2 comprises a (1-> 6)-galactose residue, a (1-> 2, 6)-galactose residue, a (1-> 3, 6)-glucose residue, a (1-> 4, 6)-glucose residue and a (1-> 4)-glucose residue; and respectively linking-> 1)-xylose residue and-> 2)-glucose residue. The AHL-P2 is obtained through the steps of extraction and purification, has a good effect in the aspects of immunoregulation and tumor resistance, provides a technical support for developing products for enhancing the immunity of the organism, such as drugs, functional foods and health care products, and greatly widens the application field.

Description

Technical Field

[0001] The present invention relates to the technical field of plant leaf polysaccharide application, and in particular to a Tianfu peanut 26 leaf polysaccharide and a preparation method and application thereof. Background Art

[0002] The scientific name of peanut (Arachis hypogaea L) is groundnut. It belongs to the genus Arachis in the subfamily Fabaceae, and is one of the important oil crops and cash crops. my country's annual peanut production ranks first in the world, and the planting area ranks second. Whether in industrial structure adjustment or international competition, peanuts occupy an important position in agricultural products. "Tianfu Peanut" is one of the main peanut variety series in my country, and is the main variety cultivated in the peanut production area of ​​the Sichuan Basin. The current main varieties are Tianfu No. 18, Tianfu No. 24, Tianfu No. 26 and Tianfu No. 30, and the pearl bean varieties Tianfu No. 22, Tianfu No. 32, Tianfu No. 35 and Tianfu No. 11; Tianfu No. 33 and Tianfu No. 36 are the latest high-oleic acid varieties cultivated and are being demonstrated and promoted. Among them, Tianfu No. 11 has the characteristics of moderate growth period, wide adaptability, good taste, strong disease resistance and fertilizer resistance and lodging resistance; Tianfu No. 22 has the characteristics of high kernel rate, high oil content, good traits and strong disease resistance and stress resistance; Tianfu No. 24 has the characteristics of lodging resistance, strong cold resistance and strong seed dormancy; Tianfu No. 26 has the characteristics of early maturity, high oil content, high yield, lodging resistance, strong cold resistance, strong seed dormancy and strong disease resistance.

[0003] At present, the main peanut products are peanut kernels, peanut shells, peanut stems, peanut roots and peanut leaves. Peanut kernels are the main products, which can be used for oil extraction or directly used. It has been found that the biological activities of peanuts include anti-cancer, anti-inflammatory, antibacterial, immune regulation and cardiovascular protection. Peanut shells are byproducts, which contain valuable chemical components, such as flavonoids, resveratrol and other functional components; peanut shell flavonoids play an important role in immune regulation, digestive system, nervous system and other aspects. Du Lei et al. determined the antioxidant activity of flavonoid extracts in peanut shells by comparing their scavenging effects on hydroxyl free radicals and DPPH free radicals. Resveratrol has the functions of anti-tumor, immune regulation, lowering blood lipids, reducing serum lipids and accelerating liver metabolic activities. Therefore, resveratrol is a high-quality material for making health products. A small amount of peanut roots are used as fuel, and most of them are used as the best raw materials for extracting resveratrol. Qualitative and quantitative analysis of the functional components of peanut stems and leaves found that they are rich in polysaccharides, but due to process limitations, the extraction rate and antioxidant activity of polysaccharides are low.

[0004] Studies have shown that peanut polysaccharides have antioxidant, liver protection, and blood sugar lowering effects. Peanut polysaccharides are polar molecules that are soluble in water and are usually extracted from defatted peanut meal using water, acid-base, and enzyme methods. However, there are few reports on the structural analysis, immune activity, and anti-tumor activity of peanut leaf polysaccharides. Summary of the invention

[0005] The invention overcomes the defects in the prior art and provides a polysaccharide from the leaf of Tianfu peanut 26 and a preparation method and application thereof.

[0006] The first aspect of the present invention provides a polysaccharide from the leaves of peanut 26 (AHL-P2), which is a heteropolysaccharide composed of glucose, galactose and xylose, wherein the molar ratio of glucose, galactose and xylose residues is 6:4:3.

[0007] Specifically, the main chain in the chemical structure of the polysaccharide includes (1→6)-galactose residues, (1→2,6)-galactose residues, (1→3,6)-glucose residues, (1→4,6)-glucose residues, and (1→4)-glucose residues.

[0008] Preferably, the main chain is connected to →1)-xylose residues and / or →2)-glucose residues, respectively.

[0009] Further preferably, the chemical structure of AHL-P2 includes (1→2,6)-galactose residues connected to (1)-xylose residues, (1→3,6)-glucose residues connected to (1)-xylose residues, or (1→4,6)-glucose residues connected to (2)-glucose residues.

[0010] Preferably, the weight average molecular weight of the polysaccharide is 4000-20000 Da.

[0011] In a specific embodiment of the present invention, the weight average molecular weight of the polysaccharide is 8476 Da.

[0012] Preferably, the chemical structural formula of the polysaccharide is as follows:

[0013]

[0014] Wherein, n is an integer of 1-20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably, an integer of 2-15, and more preferably, an integer of 3-10.

[0015] Among them, Glu is glucose, Gal is galactose, and Xyl is xylose.

[0016] The second aspect of the present invention provides a composition, which comprises the polysaccharide described in the first aspect and a pharmaceutically acceptable excipient.

[0017] Preferably, the pharmaceutically acceptable excipients are selected from: one or more of pH regulators, excipients, stabilizers, surfactants, solubilizers, cosolvents, antioxidants, buffers, suspending agents, emulsifiers, flavoring agents, thickeners, wetting agents, fillers, adhesives, lubricants, disintegrants, coating materials, penetration enhancers, plasticizers, antibacterial agents and preservatives.

[0018] Preferably, in the composition, the polysaccharide described in the first aspect can be used alone or in combination with other types of active ingredients.

[0019] Preferably, the composition can be administered by any suitable route, such as enteral administration (e.g., oral, rectal) or parenteral administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, respiratory tract administration, etc.).

[0020] Preferably, the composition can be in any suitable dosage form, such as a dosage form for gastrointestinal administration or parenteral administration, such as any one of tablets, powders, emulsions, injections (preferably solution injections), suspensions, capsules, pills, granules, powder injections, lozenges, sprays, aerosols, powder mists, lotions, ointments, pastes, patches, eye drops, nasal drops, suppositories, effervescent tablets, pills, gels and films.

[0021] The third aspect of the present invention provides a method for preparing polysaccharides from the leaves of Tianfu peanut 26, the preparation method comprising extraction of crude polysaccharides and purification of the crude polysaccharides.

[0022] Preferably, the crude polysaccharide extraction method is selected from one or more of hot water extraction, water extraction and alcohol precipitation, alkali extraction, enzymatic extraction, microwave-assisted method and ultrasonic method, preferably water extraction and alcohol precipitation.

[0023] Preferably, the crude polysaccharide purification method is selected from one or more of the following: stepwise precipitation method, salting-out method, metal complex method, fiber column chromatography method, quaternary ammonium salt precipitation method, gel column chromatography method and electrophoresis method, preferably fiber column chromatography method.

[0024] The specific steps of the preparation method of Tianfu peanut 26 leaf polysaccharide are as follows:

[0025] (1) extracting the powder of peanut leaves from Tianfu 26 plants with hot water, concentrating the water extract, and precipitating with alcohol to obtain crude polysaccharides;

[0026] (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate;

[0027] (3) The eluate obtained in step (2) is dialyzed using a dialysis bag, concentrated, and dried to obtain the polysaccharide.

[0028] Preferably, in step (1), the extraction temperature can be 85-95°C (such as 85°C, 86°C, 88°C, 90°C, 92°C, 93°C, 95°C), and more preferably 92°C.

[0029] Preferably, in step (1), the mass ratio (W / V, mg / mL) of Tianfu peanut 26 leaf powder to water is 1:1-1:10 (such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10), and more preferably 1:4.

[0030] Preferably, in step (1), the extraction is performed 1-5 times (e.g., 1, 2, 3, 4, 5 times).

[0031] Preferably, in step (1), each extraction time is 1-10 hours (such as 1 hour, 3 hours, 6 hours, 8 hours, 10 hours).

[0032] In a specific embodiment of the present invention, the extraction is performed three times, and each extraction time is 8 hours.

[0033] Preferably, in the alcohol precipitation step of step (1), the volume ratio of alcohol to the concentrated aqueous extract is 1-10:1 (such as 1:1, 3:1, 4:1, 5:1, 8:1, 10:1), and more preferably 4:1;

[0034] Preferably, in the alcohol precipitation step (1), the alcohol reagent is selected from one of methanol, ethanol, isopropanol and n-butanol, and more preferably ethanol.

[0035] Preferably, step (1) further comprises: removing impurities and drying the crude polysaccharide.

[0036] Preferably, the impurity removal is to remove protein from crude polysaccharide.

[0037] Preferably, the method for removing protein is selected from the group consisting of the Sevag method, the trifluorotrichloroethane method, and the trichloroacetic acid method, and more preferably the Sevag method.

[0038] Preferably, the commonly used solvents in the Sevag method are chloroform and alcohol reagents, and the alcohol reagent is selected from methanol, ethanol, isopropanol, n-butanol, and amyl alcohol, and is more preferably n-butanol or amyl alcohol.

[0039] Preferably, the volume ratio of crude polysaccharide solution, chloroform and n-butanol in the Sevag method is (15-35):(1-10):(0.5-3), for example, 15:1:0.5, 15:5:0.5, 15:10:0.5, 15:15:0.5, 20:1:0.7, 20:5:1, 20:10:1.5, 25:5:1, 25:10:1.5, 30:5:1, 30:10:1.5 or 35:10:3, etc.

[0040] Preferably, the drying method described in step (1) is selected from one or a combination of two or more of vacuum drying, freeze drying, air flow drying, spray drying, microwave drying and natural drying.

[0041] In a specific embodiment of the present invention, the ion exchange column in step (2) can be a cellulose column, and its filler is DEAE-cellulose, such as DEAE-52 or DEAE-32, preferably DEAE-52.

[0042] Preferably, the elution method in step (2) is gradient elution. The eluent used for elution is NaCl solution, and its concentration is 0.01-1.0 mol / L, such as 0.01 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 1.0 mol / L.

[0043] Preferably, in step (3), the molecular weight cutoff of the dialysis bag is 5000-10000Da (such as 5000Da, 6000Da, 7000Da, 8000Da, 9000Da, 10000Da), and more preferably 7000Da.

[0044] Preferably, in step (3), the dialysis time is 2-4 days (such as 2 days, 2.5 days, 3 days, 3.5 days, 4 days), and more preferably 2 days.

[0045] Preferably, the drying method described in step (3) is selected from one or a combination of two or more of vacuum drying, freeze drying, air flow drying, spray drying, microwave drying and natural drying, and freeze drying is more preferred.

[0046] The fourth aspect of the present invention provides the use of Tianfu peanut 26 leaf polysaccharide in the preparation of products that enhance immunity and anti-tumor activity.

[0047] Preferably, the product is food, health care product or medicine.

[0048] In some embodiments of the present invention, the product is an anti-tumor drug.

[0049] In other embodiments of the present invention, the product is a health product for enhancing immunity.

[0050] Preferably, the concentration of Tianfu peanut 26 leaf polysaccharide in the product is 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL and 20 μg / mL.

[0051] Preferably, the tumor disease includes but is not limited to gastric cancer, ascites cancer, non-small cell lung cancer, liver cancer, nasopharyngeal carcinoma, breast cancer, cervical cancer, esophageal cancer, colon cancer, laryngeal cancer, malignant melanoma, prostate cancer, kidney cancer, bladder cancer, lung adenocarcinoma, and ovarian cancer.

[0052] The beneficial effects of the present invention are:

[0053] 1. The present invention separates and purifies Tianfu peanut leaves to obtain Tianfu peanut 26 leaf polysaccharide (AHL-P2), analyzes and identifies its molecular weight, monosaccharide composition, chemical structure, etc., and determines its weight average molecular weight and structural composition.

[0054] 2. Tianfu peanut 26 leaf polysaccharide (AHL-P2) has significant immunomodulatory activity, especially at a concentration of 10μg / mL, the proliferation rate of RAW 264.7 cells is the highest; especially at a concentration of 20μg / mL, the proliferation rate of T cells and B cells is the highest. Such results show that AHL-P2 plays a more powerful role in resisting pathogen invasion and removing foreign bodies from the body. AHL-P2 also has good anti-tumor efficacy and significant anti-tumor activity, especially at a concentration of 10μg / mL, the inhibition rate of MFC cells and S180 cells is the highest. In summary, the outstanding characteristics of AHL-P2 provide technical support for the development of products such as medicines, functional foods, and health products that enhance the body's immunity, greatly broaden the application field, deeply integrate into the biopharmaceutical and health industries, and bring significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0056] Figure 1 Shown is the GPC spectrum of AHL-P2.

[0057] Figure 2 Shown is the infrared spectrum of AHL-P2.

[0058] Figure 3 AHL-P2 is shown 1 H NMR spectrum.

[0059] Figure 4 AHL-P2 is shown 13 C NMR spectrum.

[0060] Figure 5 AHL-P2 is shown 1 H- 1 H-COSY spectrum.

[0061] Figure 6 Shown is the HMQC spectrum of AHL-P2.

[0062] Figure 7 Shown is the HMBC spectrum of AHL-P2.

[0063] Figure 8 The chemical structure of AHL-P2 is shown, wherein A is the structural formula of AHL-P2 and B is the simplified structural formula of AHL-P2.

[0064] Fig.9A Shown are cell diagrams of the effect of AHL-P2 on B cell proliferation, wherein 1A and 1B are blank groups, 1A is a local enlarged view of 1B, 2A and 2B are 1.25 μg / mL AHL-P2 groups, 2A is a local enlarged view of 2B, 3A and 3B are 2.5 μg / mL AHL-P2 groups, 3A is a local enlarged view of 3B, 4A and 4B are 5 μg / mL AHL-P2 groups, 4A is a local enlarged view of 4B, 5A and 5B are 10 μg / mL AHL-P2 groups, 5A is a local enlarged view of 5B, 6A and 6B are 20 μg / mL AHL-P2 groups, 6A is a local enlarged view of 6B, 7A and 7B are LPS groups, 7A is a local enlarged view of 7B.

[0065] Fig. 9B Shown are the experimental results of the effect of AHL-P2 on B cell proliferation, where *** represents P<0.001 and **** represents P<0.0001.

[0066] Fig. 10A The cell diagrams shown show the effect of AHL-P2 on T cell proliferation, wherein 1A and 1B are blank groups, 1A is a local enlarged view of 1B, 2A and 2B are 1.25 μg / mL AHL-P2 groups, 2A is a local enlarged view of 2B, 3A and 3B are 2.5 μg / mL AHL-P2 groups, 3A is a local enlarged view of 3B, 4A and 4B are 5 μg / mL AHL-P2 groups, 4A is a local enlarged view of 4B, 5A and 5B are 10 μg / mL AHL-P2 groups, 5A is a local enlarged view of 5B, 6A and 6B are 20 μg / mL AHL-P2 groups, 6A is a local enlarged view of 6B, 7A and 7B are LPS groups, 7A is a local enlarged view of 7B.

[0067] Fig. 10BShown are the experimental results of the effect of AHL-P2 on T cell proliferation, where ** represents P < 0.01.

[0068] Fig.11A Shown are cell diagrams of the effect of AHL-P2 on the proliferation of RAW264.7 cells, wherein 1A and 1B are blank groups, 1A is a local enlarged view of 1B, 2A and 2B are 1.25 μg / mL AHL-P2 groups, 2A is a local enlarged view of 2B, 3A and 3B are 2.5 μg / mL AHL-P2 groups, 3A is a local enlarged view of 3B, 4A and 4B are 5 μg / mL AHL-P2 groups, 4A is a local enlarged view of 4B, 5A and 5B are 10 μg / mL AHL-P2 groups, 5A is a local enlarged view of 5B, 6A and 6B are 20 μg / mL AHL-P2 groups, 6A is a local enlarged view of 6B, 7A and 7B are LPS groups, 7A is a local enlarged view of 7B.

[0069] Fig. 11B Shown are the experimental results of the effect of AHL-P2 on the proliferation of RAW264.7 cells, where *** represents P<0.001 and **** represents P<0.0001.

[0070] Fig.12 Shown are the cell images of the inhibitory effect of AHL-P2 on the proliferation of MFC cells, where 1A and 1B are blank groups, 1A is a local enlarged image of 1B, 2A and 2B are 1.25 μg / mL AHL-P2 groups, 2A is a local enlarged image of 2B, 3A and 3B are 2.5 μg / mL AHL-P2 groups, 3A is a local enlarged image of 3B, 4A and 4B are 5 μg / mL AHL-P2 groups, 4A is a local enlarged image of 4B, 5A and 5B are 10 μg / mL AHL-P2 groups, 5A is a local enlarged image of 5B, 6A and 6B are MAN groups, 6A is a local enlarged image of 6B.

[0071] Fig.13A Shown are cell images of the inhibitory proliferation effect of AHL-P2 on S180 cells, where 1A and 1B are blank groups, 1A is a local enlarged view of 1B, 2A and 2B are 1.25 μg / mL AHL-P2 groups, 2A is a local enlarged view of 2B, 3A and 3B are 2.5 μg / mL AHL-P2 groups, 3A is a local enlarged view of 3B, 4A and 4B are 5 μg / mL AHL-P2 groups, 4A is a local enlarged view of 4B, 5A and 5B are 10 μg / mL AHL-P2 groups, 5A is a local enlarged view of 5B, 6A and 6B are MAN groups, 6A is a local enlarged view of 6B.

[0072] Fig. 13BThe results show the experimental effect of AHL-P2 on the proliferation inhibition of S180 cells, where * represents P<0.05 and ** represents P<0.01. DETAILED DESCRIPTION

[0073] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0074] In the present invention, "Tianfu peanut leaves (Arachis Hypogaea leaves)" refers to the by-products of the Tianfu peanut series varieties cultivated by the Nanchong Academy of Agricultural Sciences in Sichuan Province.

[0075] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0076] Example 1 Isolation and Extraction of Polysaccharide (AHL-P2) from Peanut 26 Leaves

[0077] 1. Isolation and extraction of polysaccharide (AHL-P2) from Tianfu peanut 26 leaves

[0078] 1.1 Extraction of crude polysaccharides from Tianfu peanut 26 leaves by water extraction and alcohol precipitation

[0079] Weigh 200g of dried Tianfu peanut 26 leaves and crush them, add the crushed Tianfu peanut 26 leaves and distilled water in a beaker at a ratio of 1:4, place in a 92°C water bath for 8 hours, collect the supernatant and concentrate, repeat 3 times, and finally concentrate all the supernatant to 200mL. Add four times the volume of anhydrous ethanol to precipitate, collect and dry the precipitate, remove the protein in the extract, and obtain Tianfu peanut 26 leaf crude polysaccharide.

[0080] 1.2 Isolation and purification of crude polysaccharides from Tianfu peanut 26 leaves by DEAE-52 cellulose column chromatography

[0081] Accurately weigh 50g DEAE-52 cellulose and dissolve it in 1L ultrapure water. Stir thoroughly. Stop stirring if no cellulose particles are visible to the naked eye. Let stand for 24 hours, discard the supernatant and set aside. Prepare 0.5mol / L NaOH, soak the cellulose for 6 hours, wash with ultrapure water until neutral, discard the supernatant, add 0.5mol / L HCl and soak for 6 hours, wash with distilled water until neutral, discard the supernatant; add 0.5mol / L NaOH and soak again for 6 hours, wash with distilled water until neutral, and let stand for use.

[0082] After the activated cellulose is loaded into the column, the crude polysaccharide can be separated and purified after balancing the column with distilled water for 24 hours. The crude polysaccharide is dissolved in distilled water. According to the characteristics that proteins are denatured in organic solvents such as chloroform and are insoluble in water, the ratio of crude polysaccharide aqueous solution, chloroform and n-butanol is adjusted to 25:5:1. The mixture is shaken vigorously for 30 minutes, and the protein and chloroform-n-butanol form a gel. Then centrifuge, collect the supernatant (5mL) and add it to the DEAE cellulose column, and add different concentrations of NaCl (0.01mol / L, 0.05mol / L, 0.1mol / L) for elution. The polysaccharide is determined by the sulfuric acid-phenol method. The eluate is concentrated to 5mL and the sample is purified on the cellulose column. Dialysis is performed with a dialysis bag (Mw≥7kDa) for 48 hours, the polysaccharide solution in the dialysis bag is taken out, centrifuged, the supernatant is collected, and freeze-dried to obtain Tianfu peanut 26 leaf polysaccharide, named AHL-P2.

[0083] 2. Structural identification of polysaccharide (AHL-P2) from the leaves of Tianfu peanut 26

[0084] The structure of AHL-P2 was elucidated using acid hydrolysis, methylation analysis, high performance gel permeation chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance technology.

[0085] 2.1 Determination of molecular weight

[0086] 10 mg of AHL-P2 sample was dissolved in 1 mL of ddH2O, sonicated for 5 min, and subjected to GPC analysis.

[0087] The GPC spectrum of AHL-P2 is shown in Figure 1 As shown, the weight average molecular weight of AHL-P2 is 8476 Da.

[0088] 2.2 IR spectrum analysis of AHL-P2

[0089] 2 mg of AHL-P2 was mixed with KBr and pressed into a pellet, and the infrared spectrophotometer was used to scan the range of 4000 cm-1-400 cm-1.

[0090] The primary structure of AHL-P2 was characterized by Fourier transform infrared spectroscopy. Figure 2 As shown, in the characteristic peak area (4000~1250cm -1 ), 3379.96cm -1 The strong broad peak at 2942.66cm is the OH stretching vibration peak of the sugar molecule, indicating the existence of hydrogen bonds within or between polysaccharide molecules; -1 The CH stretching vibration peak of methylene is at 1681.83cm -1 The peak at 1406.91cm is the asymmetric stretching vibration peak of C=O;-1 The peak of CH in-plane bending vibration is at 1250-500 cm -1 ), 1214.20cm -1 、1139.19cm -1 and 1077.75cm -1 The peak at 804.036 cm is the stretching vibration peak of the pyranose ring CO. -1 、725.034cm -1 The peak at is the in-plane bending vibration peak of =CH. The FT-IR data of AHL-P2 show that AHL-P2 has the characteristic absorption peak of polysaccharides and contains pyranose ring in its structure.

[0091] 2.3 NMR analysis of AHL-P2

[0092] Take 50 mg of AHL-P2 and dissolve it in 0.6 mL of heavy water (D2O), put it into a nuclear magnetic resonance tube, and detect it on a nuclear magnetic resonance instrument.

[0093] AHL-P2 1 H NMR results are as follows Figure 3 The results showed that AHL-P2 had seven anomeric hydrogen signals, namely: δ5.11, δ5.04, δ4.95, δ4.93, δ4.87, δ4.40, δ4.33, with an integral ratio of 0.58: 0.23: 0.61: 0.46: 0.17: 0.43: 0.24. The signals between δ3.0 and 4.2 were assigned to the hydrogen signals of C2-C6 in the sugar residues.

[0094] AHL-P2 13 C NMR results are as follows Figure 4 As shown, AHL-P2 has seven anomeric carbon signals at δ109.39, δ107.43, δ106.23, δ103.27, δ103.10, δ99.33, and δ97.98. The signals between δ60 and 80 are assigned to the carbon signals of C2-C6 in the sugar residues.

[0095] AHL-P2 1 H- 1 H-COSY spectrum Figure 5As shown, the coupling relationship between adjacent hydrogen nuclei can be identified. Signals A (δ5.11 / δ4.11), B (H1 / H2: δ5.04 / 4.08), C (H1 / H2: δ4.95 / 4.03), D (H1 / H2: δ4.93 / 4.03), E (H1 / H2: δ4.87 / 3.76), F (H1 / H2: δ4.40 / 3.57), G (H1 / H2: δ4.33 / 3.47) correspond to the sugar residues 1→4-glucose (A), →2-glucose (B), →1-xylose (C), 1→2,6-galactose (D), 1→3,6-glucose (E), 1→6-galactose (F), 1→4,6-glucose (G) groups on H1 and H2 signals. According to 1 H- 1 H COSY spectrum, the chemical shifts of H2, H3, H4, H5 and H6 of group A are δ4.11, δ4.11, δ4.00, δ4.08 and δ3.85, respectively.

[0096] All hydrogen chemical shifts are summarized in Table 1.

[0097] Table 1 AHL-P2 1 Chemical shift of H

[0098]

[0099] The HMQC spectrum results of AHL-P2 (see Figure 6 ) indicates the close-range 1 H and 13 The coupling relationship between C. Signals A (H1 / C1: δ5.11 / δ109.39), B (H1 / C1: δ5.04 / δ106.23), C (H1 / C1: δ4.95 / δ107.43), D (H1 / C1: δ4.93 / δ97.98), E (H1 / C1: δ4.87 / δ99.33), F (H1 / C1: δ4.40 / δ103.10), and G (H1 / C1: δ4.33 / δ103.27) are respectively attributed to the resonance signals of H1 and C1 on the groups 1→4-glucose (A), →2-glucose (B), →1-xylose (C), 1→2,6-galactose (D), 1→3,6-glucose (E), 1→6-galactose (F), and 1→4,6-glucose (G).

[0100] The HMBC spectrum results of AHL-P2 (see Figure 7 ) indicates the remote related 1 H and 13The signals δ5.11 / δ82.57, δ5.04 / δ75.47, δ4.95 / δ61.13, δ4.93 / δ63.52 and δ4.87 / δ70.80 are respectively assigned to the resonance coupling signals between H1 and C3 of the signals (1→4)-glucose (A), (→2)-glucose (B), (→1)-xylose (C), (1→2,6)-galactose (D) and (1→3,6)-glucose (E); the signals δ3.64 / δ67.66 and δ3.95 / δ76.85 are respectively assigned to the resonance coupling signals between H4 and C2 of the groups (1→6)-galactose (F) and (1→4,6)-glucose (G).

[0101] The chemical shifts of all carbons are summarized in Table 2.

[0102] Table 2 AHL-P2 13 Chemical shift of C

[0103]

[0104] 2.4 GC-MS analysis after methylation and silylation of AHL-P2

[0105] Weigh 20 mg of AHL-P2 sample, seal the beaker, add 2 mL of DMSO (dimethyl sulfoxide) to the sealed beaker, and gently shake the beaker to fully dissolve AHL-P2. Then add 200 mg of NaOH until NaOH just does not dissolve and place it in a shaker to shake at room temperature for 1 hour. After the shaking is completed, add 1.5 mL of iodomethane, react in the dark for 1 hour, and add water to terminate the reaction after the reaction. Extract the product with chloroform and dry it to obtain methylated polysaccharides. After the methylated polysaccharide is completely hydrolyzed with TFA, wash it with water three times to obtain a methylated completely acid hydrolyzed product.

[0106] The above sample was fully reacted with 2 mL of hexamethyldisilazane, 1 mL of trimethylchlorosilane and 2 mL of anhydrous pyridine, and incubated in a water bath at 50°C for 20 min. The sample was centrifuged at 12000 rpm / min and 4°C for 10 min in a low-temperature high-speed centrifuge. The precipitate was discarded and filtered with a 0.22 μm filter. The upper layer of the solution was used for GC-MS analysis.

[0107] The methylation results are shown in Table 3 , indicating that the main repeating structural unit of AHL-P2 is composed of 1,4-linked glucose and 1,4-linked arabinose residues linked alternately.

[0108] Table 3 Analysis of AHL-P2 methylation results

[0109]

[0110] The chemical structure of AHL-P2 was obtained based on the results of experiments 2.1-2.4 (see Figure 8 ).

[0111] Example 2 Determination of immunomodulatory activity of Tianfu peanut 26 leaf polysaccharide (AHL-P2)

[0112] 1. Reagents

[0113] CCK-8 kit, RPIM1640, FBS, DMSO, double antibodies, etc. are all commercially available products.

[0114] 2. Instruments

[0115] Microplate reader; cell culture incubator.

[0116] 3. Methods

[0117] 3.1 Effect of AHL-P2 on the proliferation of immune cells (B cells, T cells and RAW264.7 cells)

[0118] The effect of AHL-P2 on the proliferation of T cells, B cells and RAW264.7 cells was determined by the cell counting kit (CCK-8) method.

[0119] T cells, B cells and RAW264.7 cells were cultured in vitro to the logarithmic growth phase. After counting with a cell counter, the cell suspension was diluted to 1×105 / mL with a new culture medium. The cell suspension was added to a 96-well plate, 100 μL per well, and the 96-well plate was placed in a CO2 incubator for 24 hours. After 24 hours, different mass concentrations of AHL-P2 solution (final mass concentration of 1.25, 2.5, 5, 10, 20 μg / mL) were added to the experimental group, 100 μL LPS solution (final mass concentration of 5 μg / mL) was added to the positive control, and 100 μL cell culture medium was added to the blank group. After 24 hours of culture in a CO2 incubator, 5 μl of CCK-8 was added to each well, and the absorbance value (450 nm) was detected on a microplate reader and an image was taken after being placed in a CO2 incubator for 3 hours.

[0120] 3.2 Effect of AHL-P2 on the proliferation of tumor cells (MFC cells, S180 cells)

[0121] The CCK-8 method was used to detect the inhibitory effect of polysaccharides on MFC and S180 cells. The experimental groups were added with different mass concentrations of AHL-P2 solution (final mass concentration of 1.25, 2.5, 5, 10 μg / mL), and the positive control group (MAN group) was added with an equal volume of mannan peptide solution (MAN) with a final concentration of 5 μg / mL, and the rest was the same as 3.1.

[0122] 4. Results

[0123] 4.1 Effect of AHL-P2 on B cell proliferation

[0124] The results are as follows Fig.9A -B shows that compared with the blank group, the LPS group can significantly (P<0.0001) promote the proliferation of B cells, with a proliferation rate of 89.02%; when the final concentration of AHL-P2 is in the range of 1.25-20μg / mL, it can significantly promote the proliferation of B cells; and when the final concentration of AHL-P2 is 20μg / mL (P<0.0001), the effect of AHL-P2 on the proliferation of B cells is most obvious, with the maximum proliferation rate reaching 94.74%.

[0125] 4.2 Effect of AHL-P2 on T cell proliferation

[0126] The results are as follows Fig. 10A -B, compared with the blank group, the LPS group can significantly (P < 0.01) promote the proliferation of T cells. When the final concentration of AHL-P2 is in the range of 1.25-20 μg / mL, it can significantly promote the proliferation of T cells. When the final concentration of AHL-P2 is 20 μg / mL (P < 0.01), the effect of AHL-P2 on the proliferation of T cells is most obvious, and the maximum proliferation rate reaches 59.68%.

[0127] 4.3 Effect of AHL-P2 on the proliferation of RAW264.7 cells

[0128] The results are as follows Fig.11A -B, compared with the blank group, the LPS group could significantly (P<0.0001) promote the proliferation of RAW264.7 cells; when the final concentration of AHL-P2 was in the range of 1.25-20 μg / mL, it could significantly promote the proliferation of RAW264.7 cells; when the final concentration of AHL-P2 was 10 μg / mL (P<0.001), the proliferation effect of AHL-P2 on RAW264.7 cells was most obvious, and the maximum proliferation rate reached 85.99%.

[0129] 4.4 Inhibitory effect of AHL-P2 on proliferation of MFC cells

[0130] The results are as follows Fig.12 As shown in the figure, compared with the blank group, the MAN group could significantly (P<0.0001) inhibit the proliferation of MFC cells; when the final concentration of AHL-P2 was in the range of 1.25-10 μg / mL, it could significantly inhibit the proliferation of MFC cells; and when the final concentration of AHL-P2 was 10 μg / mL (P<0.0001), the inhibitory effect of AHL-P2 on MFC cells was the most obvious, with the maximum inhibition rate reaching 32.63%

[0131] 4.5 Effect of AHL-P2 on the proliferation inhibition of S180 cells

[0132] The results are as follows Fig.13A -B, compared with the blank group, the MAN group could significantly (P<0.01) inhibit the proliferation of S180 cells; when the final concentration of AHL-P2 was 2.5-10μg / mL, it could significantly inhibit the proliferation of S180 cells; and when the final concentration of AHL-P2 was 10μg / mL (P<0.01), the inhibitory effect of AHL-P2 on S180 cells was the most obvious, with the maximum inhibition rate reaching 22.12%.

[0133] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0134] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A polysaccharide, characterized in that The polysaccharide is composed of glucose, galactose and xylose, wherein the molar ratio of glucose, galactose and xylose residues is 6:4:3; Preferably, the main chain in the chemical structure of the polysaccharide includes (1→6)-galactose residues, (1→2,6)-galactose residues, (1→3,6)-glucose residues, (1→4,6)-glucose residues, and (1→4)-glucose residues; the main chain is respectively connected to (1→1)-xylose residues and / or (2)-glucose residues.

2. The polysaccharide according to claim 1, characterized in that The polysaccharide has the following structure: Wherein, n is an integer from 1 to 20; Preferably, n is an integer of 2-15, and more preferably, an integer of 3-10.

3. The polysaccharide according to claim 1, characterized in that The weight average molecular weight of the polysaccharide is 4000-20000 Da.

4. The polysaccharide according to claim 1, characterized in that The polysaccharide is Tianfu peanut 26 leaf polysaccharide, and its weight average molecular weight is 8476Da.

5. A composition, characterized in that The composition comprises the polysaccharide according to any one of claims 1 to 4, and pharmaceutically acceptable excipients.

6. The method for preparing the polysaccharide according to claim 1, characterized in that: The preparation method of the polysaccharide comprises the extraction of crude polysaccharide and the purification of crude polysaccharide.

7. The preparation method according to claim 6, characterized in that: The specific steps of the preparation method are as follows: (1) extracting the powder of peanut leaves from Tianfu 26 plants with hot water, concentrating the water extract, and precipitating with alcohol to obtain crude polysaccharides; (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate; (3) The eluate obtained in step (2) is dialyzed using a dialysis bag, concentrated, and dried to obtain the polysaccharide.

8. The preparation method according to claim 7, characterized in that: In the step (1), the mass ratio of Tianfu peanut 26 leaf powder to water is 1:1-1:10, preferably 1:4; Preferably, in step (1), the extraction is performed 1 to 5 times; Preferably, in step (1), each extraction time is 1-10 hours; Preferably, step (1) further comprises: removing impurities and drying the crude polysaccharide.

9. The preparation method according to claim 7, characterized in that: The filler of the ion exchange column chromatography in step (2) is DEAE-cellulose, such as DEAE-52 or DEAE-32, preferably DEAE-52; Preferably, the elution method in step (2) is gradient elution; the eluent used for elution is NaCl solution with a concentration of 0.01-1.0 mol / L.

10. Use of the polysaccharide according to any one of claims 1 to 4 in the preparation of a product for enhancing immunity and anti-tumor activity.

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