Tianfu peanut 18 leaf polysaccharide and its preparation method and application

Abstract

The present invention discloses a polysaccharide (AHL-P) from the leaf of Tianfu peanut 18, and its preparation method and application. AHL-P is composed of (1→4)-glucose, (1→4)-xylose, (1→4,6)-glucose, →1)-glucose, (1→4)-galactose, and (1→4)-arabinose, wherein the molar ratio of glucose:xylose:galactose:arabinose is 7:2:1:1, and the molar ratio of (1→4)-glucose, (1→4,6)-glucose, →1)-glucose is 3:2:2. The preparation process of the polysaccharide includes the steps of hot water extraction, alcohol precipitation, protein removal, purification, etc., and the operation is simple and convenient. The present invention discloses the structure of AHL-P and studies the immunomodulatory activity of the polysaccharide, providing a new idea and technical basis for further research and development of Tianfu peanut 18 leaf polysaccharide products.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant polysaccharide applications, and in particular relates to a polysaccharide from the leaves of Tianfu peanut 18, and a preparation method and application thereof. Background Art

[0002] Peanuts (Arachis hypogaea), also known as groundnuts and longevity nuts, are the mature seeds of the leguminous plant Arachis hypogaea. They are known for their tonifying, longevity-promoting, anti-aging, and beauty-enhancing properties, earning them the nickname "longevity nuts." Peanuts are a major oilseed crop worldwide, second only to rapeseed in terms of cultivated area. China is a major peanut producer, with an annual output of approximately 15 million tons, ranking first in the world.

[0003] Peanut polysaccharides are the second most abundant nutritional component of peanuts, after protein. Polysaccharides contain more complex biological information than proteins and nucleic acids. Research on peanut polysaccharides has been on the rise in recent years. Like other plant polysaccharides, peanut polysaccharides possess both primary and higher-order structures. Because the number of monosaccharides is greater than the number of amino acids that make up proteins, and because they also have more linkage sites, determining the structure of these branched heteropolysaccharides is much more challenging than determining the structure of proteins. Therefore, in-depth research into the primary and higher-order structures of peanut polysaccharides is crucial for their development and application. Numerous methods have been studied for extracting peanut polysaccharides from peanuts, including hot water extraction, acid-base extraction, enzymatic extraction, and assisted extraction techniques. Each method has its own advantages and disadvantages, resulting in polysaccharide extraction yields ranging from 5.6% to 13.78%. Song et al. (Song Y, Du BJ, Zhou Y, et al. Optimization of extraction process by response surface methodology and preliminary structural analysis of polysaccharides from defatted peanut (Arachis hypogaea) cakes [J]. Carbohydrate research, 2011 (346): 305-310) used ethanol to extract polysaccharides from peanuts, and obtained the polysaccharide component DPCP-1 after separation and purification. The structure of the polysaccharide was analyzed by FT-IR technology, and the spectrum showed that the polysaccharide had the highest peaks at 1023, 1079, and 1154 cm -1 There are strong absorption peaks near 1415 and 1239 cm, indicating the presence of pyranose ring; -1 The absorption peaks near 1730 cm indicate the presence of CO and OH structures; -1 The absorption peaks near 3368 and 2931 cm indicate the absence of uronic acid; -1The presence of an absorption peak near the polysaccharide indicates that the polysaccharide has a typical polysaccharide structure. The monosaccharide composition of peanut polysaccharide is relatively complex, containing rhamnose, fucose, arabinose, xylose, glucose and galactose. Structural analysis shows that its monosaccharide is an α-pyranose ring with an acetylamino modification.

[0004] Plant polysaccharides have a variety of biological activities, including immune regulation, anti-tumor, blood pressure reduction, blood lipid reduction, anti-radiation, antibacterial, antiviral and other health effects. The activity of plant polysaccharides is mainly to utilize their similar structures to construct immune regulation mechanisms to produce immune activity. At present, the research on peanut polysaccharides has only reported on liver protection, blood sugar reduction and anti-oxidation. Yao Xiufen et al. (Protective effect of peanut crude polysaccharides on acute liver damage caused by carbon tetrachloride and alcohol in mice [J]. Food Science, 2011 (32): 261-265) studied the protective effect of peanut crude polysaccharides extracted from peanuts on acute liver damage caused by carbon tetrachloride (CCl4) and alcohol in mice. The results showed that the polysaccharides at doses of 50, 100 and 200 mg / kg could significantly inhibit the increase of malondialdehyde content, liver index, alanine aminotransferase and aspartate aminotransferase activities caused by acute liver damage caused by CCl4, effectively inhibit the decrease of superoxide dismutase activity in the liver, and improve the degree of liver tissue damage in mice to varying degrees. This shows that peanut polysaccharides can increase the activity of superoxide dismutase in the body, promptly remove free radicals produced by CCl4 and alcohol metabolism in the body, avoid the accumulation of reactive oxygen free radicals in the body, reduce the generation of lipid peroxidation product malondialdehyde, stabilize cell membranes, and play a positive role in resisting lipid peroxidation damage. Liu Hui et al. (Study on Enzymatic Extraction of Peanut Polysaccharides and Their Antioxidant Activity [J]. Oil and Fats Engineering, 2012 (12): 54-56) used peanuts as raw materials, extracted peanut polysaccharides by enzymatic method, and studied their antioxidant activity. The results showed that peanut polysaccharides had a strong effect on ·OH, ·O 2- The free radicals showed strong scavenging ability, with IC50 values ​​of 0.81 mg / mL and 0.17 mg / mL respectively. This may be because the presence of peanut polysaccharides inhibited some free radicals and directly scavenged some free radicals, thus having good antioxidant properties. Yang Wei et al. (Study on the Hypoglycemic Activity of Water-Extracted Peanut Meal Polysaccharides [J]. Food Industry Science and Technology, 2010 (12): 330-332) studied the hypoglycemic effect of peanut polysaccharides on diabetic mice induced by alloxan and preliminarily explored the mechanism of action of the hypoglycemic activity. The results showed that different doses of peanut polysaccharides could reduce the blood sugar levels of mice, and a concentration of 200 mg / kg could significantly reduce the blood sugar levels of diabetic mice. Different doses of polysaccharides could increase the immune capacity of diabetic mice and alleviate weight loss. Therefore, it can be seen that peanut polysaccharides have the effect of lowering blood sugar and improving immunity to a certain extent.

[0005] At present, there are no reports on the fine structure of Tianfu peanut 18 leaf polysaccharide and its application in immunomodulatory activity. Summary of the Invention

[0006] To fill the gap in research and develop the polysaccharide from the leaves of Tianfu peanut 18, the inventors conducted a long-term study on the polysaccharide from the leaves of Tianfu peanut 18 for the first time. They conducted extensive experiments specifically on the structure, extraction process, and physiological activity of the polysaccharide, laying a solid foundation for the application of the polysaccharide from Tianfu peanut 18.

[0007] In a first aspect, the present invention provides a polysaccharide (AHL-P) from the leaf of Tianfu peanut 18. The polysaccharide is composed of (1→4)-glucose, (1→4)-xylose, (1→4,6)-glucose, (→1)-glucose, (1→4)-galactose, and (1→4)-arabinose, wherein the molar ratio of glucose:xylose:galactose:arabinose is 7:2:1:1, and the molar ratio of the three glucose residues (1→4)-glucose, (1→4,6)-glucose, and (→1)-glucose is 3:2:2.

[0008] Preferably, the chemical structural formula of the polysaccharide is as follows:

[0009]

[0010] Wherein, n is an integer, 3≤n≤10.

[0011] Preferably, the weight average molecular weight of the polysaccharide is 6000-20000 Da, more preferably 12837 Da.

[0012] In a second aspect, the present invention also provides a method for preparing the above-mentioned Tianfu peanut 18 leaf polysaccharide (AHL-P), the specific preparation process comprising the following steps:

[0013] 1. Take 18 leaves of Tianfu peanut solid powder, extract it with hot water, and sequentially subject the obtained water extract to alcohol precipitation, drying, and protein removal to obtain crude polysaccharide.

[0014] 2. The crude polysaccharide obtained in step 1 is eluted by ion exchange column chromatography, and the eluate is collected.

[0015] 3. The collected eluate is dialyzed and concentrated using a dialysis bag, and then the liquid in the dialysis bag is freeze-dried to obtain the polysaccharide.

[0016] Preferably, in step 1, the temperature of hot water extraction is 80-100°C (such as 80°C, 83°C, 85°C, 88°C, 90°C, 93°C, 95°C, 98°C, 100°C), more preferably 98°C.

[0017] Preferably, in step 1, the material-liquid ratio (W / V, mg / mL) of Tianfu peanut 18 leaf fruiting body powder to hot water is 1:1-10 (such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 17, 1:8, 1:9, 1:10), and more preferably 1:3.

[0018] Preferably, in step 1, the number of hot water extractions is 1-5 times (such as 1 time, 2 times, 3 times, 4 times, 5 times), more preferably 4 times.

[0019] Preferably, in step 1, each hot water extraction time is 1-10 hours (such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours), more preferably 8 hours.

[0020] In a specific embodiment of the present invention, when the alcohol precipitation operation is performed in step 1, the volume ratio of ethanol to the aqueous extract concentrate is 1-10:1 (such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1), preferably 4:1.

[0021] In a specific embodiment of the present invention, the method for removing protein in step 1 is selected from the group consisting of the Sevag method, the trifluorotrichloroethane method, and the trichloroacetic acid method, and more preferably the Sevag method.

[0022] In one embodiment of the present invention, the filler of the ion exchange column chromatography in step 2 is DEAE-cellulose, such as DEAE-52 or DEAE-32, preferably DEAE-52.

[0023] Preferably, a gradient elution method is used in step 2. The eluent is a NaCl solution with a concentration of 0.05-0.3 mol / L, such as 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, or 0.3 mol / L.

[0024] In a specific embodiment of the present invention, the molecular weight cut-off of the dialysis bag in step 3 is 5000-10000 Da (such as 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, 10000 Da), preferably 7000 Da.

[0025] 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), more preferably 2 days.

[0026] In a third aspect, the present invention also provides applications of the Tianfu peanut 18 leaf polysaccharide (AHL-P), such as developing the polysaccharide into products that enhance immunity, active ingredients for cell culture, cell culture reagents, and the like.

[0027] Preferably, the product is a medicine, a health product or a food.

[0028] Preferably, AHL-P can be used alone or in combination with other active ingredients.

[0029] The beneficial effects of the present invention are as follows:

[0030] 1. The present invention determines the molecular weight, monosaccharide composition, chemical structure, etc. of Tianfu peanut 18 leaf polysaccharide (AHL-P), providing a technical basis for further research and development of Tianfu peanut 18 leaf polysaccharide products.

[0031] 2. The present invention studies the immunomodulatory activity of Tianfu peanut 18 leaf polysaccharide (AHL-P), provides a technical basis for the development of products (such as medicines, foods or health products) that enhance the body's immunity, and improves the application value of Tianfu peanut 18.

[0032] 3. The present invention also provides a method for extracting polysaccharides (AHL-P) from Tianfu peanut 18 leaves, providing a new technical idea for the extraction and research of other plant polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown is the HPGPC spectrum of AHL-P;

[0034] Figure 2 Shown is the infrared spectrum of AHL-P;

[0035] Figure 3 Shown is AHL-P 1 H NMR spectrum;

[0036] Figure 4 Shown is AHL-P 13 C NMR spectrum;

[0037] Figure 5 Shown is AHL-P 1 H- 1 H-COSY spectrum;

[0038] Figure 6 Shown is the HMQC spectrum of AHL-P;

[0039] Figure 7 Shown is the HMBC spectrum of AHL-P;

[0040] Figure 8 Shown is the chemical structure of AHL-P;

[0041] Figure 9 Shown are the experimental results of the effect of AHL-P on B cell proliferation;

[0042] Figure 10 Shown are the experimental results of the effect of AHL-P on the proliferation of RAW264.7 cells. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely illustrative and do not limit the scope of the present invention.

[0044] It should be noted that unless otherwise defined, the scientific and technical terms used in the present invention have the common meanings in the technical field.

[0045] In the present invention, “Tianfu peanut 18 (Pleurotus cornucopiae)” refers to a fungus of the subphylum Basidiomycota, class Hymenomycetes, order Agaricales, family Pleurotaceae, genus Pleurotus, which comprises a fruiting body and a mycelium.

[0046] Example 1 Isolation and Extraction of Polysaccharide AHL-P from Tianfu Peanut 18 Leaves

[0047] 1. Isolation and extraction of polysaccharide AHL-P from Tianfu peanut 18 leaves

[0048] 1.1 Extraction of crude polysaccharides from Tianfu peanut 18 leaves

[0049] Weigh 600g of dried Tianfu peanut 18 leaf fruiting bodies and crush them into fruiting body powder. Then, according to a material-liquid ratio of 1:3, add the Tianfu peanut 18 leaf fruiting bodies and distilled water to a beaker, incubate in a 98°C water bath for 6 hours, centrifuge the extracted mixture at 5000r / min for 10 minutes, collect the supernatant and centrifuge again, repeat three times, and finally concentrate all the supernatant to 200mL. Then mix the concentrate with 800mL of anhydrous ethanol, let it stand and precipitate, collect the precipitate and dry it at 40-50°C. Then use the Sevag method to remove the protein in the dried sample to obtain Tianfu peanut 18 leaf crude polysaccharide.

[0050] 1.2. Separation and purification of crude polysaccharides from Tianfu peanut 18 leaves by DEAE-cellulose column chromatography

[0051] Accurately weigh 50g of DEAE cellulose and dissolve it in 1L of ultrapure water. Stir thoroughly until no cellulose particles are visible. Stop stirring. Let stand for 24 hours. Discard the supernatant and soak the cellulose in 0.5mol / L NaOH for 6 hours. Wash with ultrapure water until neutral. Discard the supernatant and soak in 0.5mol / L HCl for 6 hours. Wash with distilled water until neutral. Discard the supernatant and soak in 0.5mol / L NaOH for 6 hours. Wash with distilled water until neutral. Let stand until ready to use.

[0052] The activated DEAE-52 cellulose was loaded onto the column and then equilibrated with distilled water for 24 hours. The crude polysaccharide from the leaves of Tianfu peanut 18 prepared in step 1.1 was then added to 200 mL of pure water and mixed evenly. The mixture was centrifuged at 12000 r / min for 10 minutes, 5 mL of the supernatant was added to the DEAE-52 cellulose column, and eluted with different concentrations of NaCl solution (0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L) as the mobile phase. The polysaccharide was determined by the sulfuric acid-phenol method. The eluate was collected and concentrated to 5 mL.

[0053] The eluted concentrate was dialyzed using a dialysis bag (Mw≥7kDa) for 48 hours, and then centrifuged at 12000r / min for 10 minutes. After centrifugation, the precipitate was collected and freeze-dried to obtain Tianfu peanut 18 leaf polysaccharide, which was named AHL-P.

[0054] 2. Structural identification of polysaccharide AHL-P from Tianfu peanut 18 leaves

[0055] The structure of Tianfu peanut 18 leaf polysaccharide (AHL-P) was analyzed using acid hydrolysis, methylation analysis, high performance gel permeation chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance technology.

[0056] 2.1 Determination of molecular weight

[0057] 10 mg of Tianfu peanut 18 leaf polysaccharide AHL-P sample was accurately weighed, dissolved in 1 mL ddH2O, ultrasonicated for 5 min, and then subjected to HPGPC analysis. Figure 1 ) showed that the weight average molecular weight of 18 leaf polysaccharide AHL-P was 12837 Da.

[0058] 2.2 Fourier transform infrared spectroscopy analysis of polysaccharide AHL-P from Tianfu peanut 18 leaves

[0059] 2 mg of Tianfu peanut 18 leaf polysaccharide AHL-P sample was accurately weighed, mixed with KBr, ground, and tableted. The sample was then analyzed by Fourier transform infrared spectrometry at 4000 cm -1 -400cm -1 Scan within the range. FTIR spectrum (see Figure 2 ) shows that the wave number is 3431.036cm -1 、2930.688cm -1 、1636.342cm -1 、1401.732cm -1 、1153.556cm -1 、1084.932cm-1 and 1026.867cm -1 There are typical polysaccharide absorption peaks in the following places. The Fourier transform infrared spectrum of polysaccharide AHL-P in Tianfu peanut 18 leaves shows that: 3431.036cm -1 The OH stretching vibration peak is at 2930.688 cm -1 The -CH2 stretching vibration peak is at 1636.342 cm -1 The C=O stretching vibration peak is at 1401.732 cm -1 The peak of CH in-plane bending vibration is 1153.556 cm -1 1084.932cm -1 and 1026.867cm -1 The CO stretching vibration peak is at 671.568 cm -1 The peak at is the in-plane bending vibration peak of =CH. The Fourier transform infrared spectrum results show that AHL-P has typical polysaccharide structural characteristics.

[0060] 2.3 Nuclear Magnetic Resonance Analysis of AHL-P Polysaccharide from Tianfu Peanut 18 Leaves

[0061] Accurately weigh 50 mg of 18 leaf polysaccharide AHL-P sample, 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. The results are shown in Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 and Figure 7 .

[0062] AHL-P 1 H NMR spectrum results (see Figure 3 ) showed that AHL-P has six anomeric hydrogen signals with chemical shifts of δ5.29, δ5.23, δ5.10, δ4.96, δ4.84, and δ4.53, respectively, with an integration ratio of 3:2:2:2:1:1. The signals between δ3.0-4.2 ppm are assigned to the hydrogen signals of C2-C6 in the sugar residues.

[0063] AHL-P 13 C NMR spectrum results (see Figure 4 ) showed that AHL-P has a total of 6 anomeric carbon signals with chemical shifts of δ107.49, δ99.74, δ99.52, δ98.57, δ95.75 and δ91.87. The signals between δ60-80 ppm are assigned to the carbon signals of C2-C6 in the sugar residue.

[0064] AHL-P 1 H- 1 H-COSY spectrum results (see Figure 5) indicates the coupling relationship between adjacent hydrogen nuclei. Signals A (δ5.29 / δ3.46), B (δ5.23 / δ3.47), C (δ5.10 / δ3.42), D (δ4.96 / δ4.01), E (δ4.84 / δ3.46), and F (δ4.53 / δ3.15) are attributed to the coupling signals between H1 and H2 of (1→4)-glucose, (1→4,6)-glucose, (→1)-glucose, (1→4)-xylose, (1→4)-galactose, and (1→4)-arabinose, respectively.

[0065] The chemical shift results of all hydrogen atoms are shown in Table 1.

[0066] Table 1 Chemical shifts of H atoms in AHL-P

[0067]

[0068] The HMQC spectrum results of AHL-P (see Figure 6 ) indicates the close-range 1 H and 13 The coupling relationship between C. Signals A (H1 / C1δ5.29 / δ99.52), B (H1 / C1δ5.23 / δ99.74), C (H1 / C1δ5.10 / δ91.87), D (H1 / C1δ4.96 / δ107.49), E (H1 / C1δ4.84 / δ98.57) and F (H1 / C1δ4.53 / δ95.75) are attributed to the resonance coupling signals of H1 and C1 on (1→4)-glucose, (1→4,6)-glucose, (→1)-glucose, (1→4)-xylose, (1→4)-galactose and (1→4)-arabinose residues, respectively.

[0069] The HMBC spectrum results of AHL-P (see Figure 7 ) indicates the remote related 1 H and 13 The coupling relationship between C. Signals δ5.29 / δ73.07, δ5.10 / δ69.84, δ4.96 / δ76.76 and δ4.84 / δ72.76 are attributed to the resonance coupling signals between H1 and C3 of (1→4)-glucose, (→1)-glucose, (1→4)-xylose and (1→4)-galactose, respectively. The signal δ3.61 / δ72.61 is attributed to the resonance coupling signal between H5 and C3 of (1→4,6)-glucose. The signal δ3.29 / δ73.07 is attributed to the resonance coupling signal between H4 and C2 of the group (1→4)-arabinose.

[0070] The chemical shift results of all carbon atoms are shown in Table 2.

[0071] Table 2 Chemical shifts of C atoms in AHL-P

[0072]

[0073] 2.4 GC-MS analysis of polysaccharide AHL-P from Tianfu peanut 18 leaves after methylation and silanization derivatization

[0074] Accurately weigh 20 mg of AHL-P sample, add it to a sealed beaker, then add 2 mL of DMSO (dimethyl sulfoxide) to the sealed beaker. Gently shake the beaker to fully dissolve the AHL-P. Then, add 200 mg of NaOH until the NaOH just dissolves. Place the beaker on a shaker and shake at room temperature for 1 hour. Then, add 1.5 mL of iodomethane and react for 1 hour in the dark. Terminate the reaction by adding water. Extract the product with chloroform and dry it to obtain the methylated polysaccharide.

[0075] The methylated polysaccharide was hydrolyzed with trifluoroacetic acid (TFA). After complete hydrolysis, the product was washed three times with water to obtain a completely methylated acid hydrolyzate. The hydrolyzate was then reacted with 2 mL of hexamethyldisilazane, 1 mL of trimethylchlorosilane, and 2 mL of anhydrous pyridine. The reaction was continued in a 50°C water bath for 20 minutes. The product was centrifuged in a low-temperature high-speed centrifuge at 12,000 rpm / min at 4°C for 10 minutes, and the precipitate was discarded. The supernatant was filtered through a 0.22 μm filter, and the supernatant was used for GC-MS analysis. The results are shown in Table 3.

[0076] Table 3: AHL-P methylation results analysis

[0077]

[0078] The results in Table 3 show that AHL-P has a pyranose ring and is composed of glucose (Glc), xylose (Xyl), galactose (Gal), and arabinose (Arab). Its primary structure is a polysaccharide with (1→4,6)-D-glucose and (1→4)-glucose as the backbone, (1→4)-xylose, (1→4)-arabinose, and (1→4)-galactose as the branches, and (1→1)-glucose as the terminal sugar.

[0079] The chemical structure of AHL-P was obtained based on the results of experiments 2.1-2.4 (see Figure 7 and Figure 8 ).

[0080] Example 2: Study on the immunomodulatory activity of Tianfu peanut 18 leaf polysaccharide AHL-P

[0081] 1.1 Effect of AHL-P on B cell proliferation

[0082] The B cells were cultured in vitro to the logarithmic growth phase, and then counted using a cell counting plate. The B cell culture medium was then diluted with new culture medium to 1×10 5 / mL. 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, the experimental groups were added with different mass concentrations of AHL-P solution (final mass concentration was 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL), the positive control was added with 100 μL of LPS solution (final mass concentration was 5 μg / mL), and the blank group was added with 100 μL of cell culture medium. After culturing in a CO2 incubator for 24 hours, 5 μl of CCK-8 solution was added to each well, and the plates were placed in a CO2 incubator for 3 hours. The absorbance value at a wavelength of 450 nm was then detected using an enzyme-labeled instrument, and the measurement results were recorded and analyzed (see Figure 9 ).

[0083] By Figure 9 As can be seen, compared with the blank group (CK), the LPS group significantly (P < 0.01) promoted B cell proliferation, with a proliferation rate of 124.46%. At different concentrations of AHL-P, all experimental groups significantly (P < 0.01) promoted B cell proliferation. When the AHL-P concentration was 10 μg / mL, the effect of AHL-P on B cell proliferation was most pronounced, with a maximum proliferation rate of 104.39%. This indicates that AHL-P can effectively promote B cell proliferation.

[0084] 1.2 Effect of AHL-P on RAW264.7 cell proliferation

[0085] The specific experimental process is the same as 1.1. The only difference is that the B cells in 1.1 are replaced with RAW264.7 cells. The experimental results are shown in Figure 10 .

[0086] Depend on Figure 10 As can be seen, compared with the blank group (CK), the LPS group significantly (P < 0.05) promoted the proliferation of RAW264.7 cells, with a proliferation rate of 55.55%. At different concentrations of AHL-P, all experimental groups significantly (P < 0.01) promoted the proliferation of RAW264.7 cells. When the AHL-P concentration was 10 μg / mL, the proliferation effect of AHL-P on RAW264.7 cells was the most significant, with a maximum proliferation rate of 32.25%. This indicates that AHL-P can effectively promote the proliferation of RAW264.7 cells.

[0087] The results of experiments 1.1 and 1.2 indicate that AHL-P, a polysaccharide from the leaves of Tianfu peanut 18, can effectively enhance the body's immune system. To further enhance the value of AHL-P, the polysaccharide could be used in the development of food or health products.

[0088] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or directly or indirectly applied to other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Application of Tianfu peanut 18 leaf polysaccharide in the preparation of medicines, health products or foods for enhancing immunity, characterized by: The polysaccharide is composed of (1→4)-glucose, (1→4)-xylose, (1→4,6)-glucose, (→1)-glucose, (1→4)-galactose, and (1→4)-arabinose, wherein the molar ratio of glucose:xylose:galactose:arabinose is 7:2:1:1, and the molar ratio of (1→4)-glucose, (1→4,6)-glucose, and (→1)-glucose is 3:2:2; the structural formula of the polysaccharide is: The weight average molecular weight of the polysaccharide is 8000-20000 Da.

2. The use according to claim 1, characterized in that The preparation process of Tianfu peanut 18 leaf polysaccharide comprises the following steps: Take 18 leaves of Tianfu peanut powder, extract it with hot water, and then precipitate it with alcohol, dry it, and remove the protein to obtain crude polysaccharide. Passing the crude polysaccharide through ion exchange column chromatography, eluting, and collecting the eluate; The eluate is dialyzed and concentrated using a dialysis bag, and then the liquid in the dialysis bag is freeze-dried to obtain the polysaccharide.

3. The use according to claim 2, characterized in that: The temperature of the hot water is 80-100° C., the material-liquid ratio of the Tianfu peanut 18 leaf powder to the hot water is 1:1-10, the hot water extraction times are 1-5 times, and the extraction time for each time is 1-10 hours; The volume ratio of ethanol to the water extract concentrate in the alcohol precipitation is 1-10:

1.

4. The use according to claim 2, characterized in that: The filler of the ion exchange column chromatography is DEAE-cellulose.

5. The use according to claim 4, characterized in that: The DEAE-cellulose is DEAE-52 or DEAE-32.

6. The use according to claim 2, characterized in that: The elution is gradient elution, the eluent is NaCl solution, and the NaCl concentration is 0.05-0.3 mol / L.

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