New application of codonopsis pilosula seeds or extracts thereof, and codonopsis pilosula seed polysaccharide as well as preparation method and application thereof

Abstract

The invention provides application of codonopsis pilosula seeds or extracts thereof in preparation of drugs with an immune regulation effect or a blood sugar reduction effect or health-care food beneficial to immunity enhancement. The invention provides a codonopsis pilosula seed crude polysaccharide, a purified polysaccharide, a preparation method and application. The polysaccharide is extracted based on the Codonopsis pilosula seeds, structural analysis and biological activity research of the Codonopsis pilosula seed polysaccharide are carried out, and a certain theoretical basis is provided for development and utilization of the Codonopsis pilosula seeds. According to the method, comprehensive development and utilization of codonopsis pilosula resources are facilitated, and the economic value and the product added value of codonopsis pilosula are improved. Meanwhile, the research can expand the industrial chain of codonopsis pilosula to a certain extent, and has an important promotion effect on increasing the income of medicinal herb farmers and promoting the local medicinal material industry and economic development.

Description

Technical Field

[0001] The present invention relates to the use of Codonopsis pilosula seeds or their extracts in the preparation of drugs with immunomodulatory effects or health foods helpful for enhancing immunity, and a polysaccharide from Codonopsis pilosula seeds. Background Art

[0002] Codonopsis pilosula is the dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) L. T. Shen, and Codonopsis tangshen Oliv. of the family Campanulaceae, and is one of the bulk Chinese medicinal materials in China. Modern pharmacological studies have shown that Codonopsis pilosula has pharmacological effects such as anti - inflammation, improving immunity, and anti - aging.

[0003] The non - medicinal parts refer to the plant organs or tissues that are not used as the main medicinal parts during the use of medicinal plants in traditional Chinese medicine. There have been relevant reports on the reuse of non - medicinal parts such as stems, leaves, rhizome tips, and fibrous roots generated during the planting, harvesting, and processing of Codonopsis pilosula, which has expanded the application channels of Codonopsis pilosula resources. However, the research on the development and utilization of Codonopsis pilosula seed resources is still shallow. The yield of Codonopsis pilosula seeds per mu is 15 - 20 kg, and only 4 - 5 kg of seeds are needed per mu when sowing directly. Due to the poor storage property of Codonopsis pilosula seeds, the germination rate drops below 50% after 1 - year storage at room temperature and less than 10% after 2 - year storage. Therefore, every year, there are Codonopsis pilosula seeds that are not planted or sold, which are either left idle or directly discarded, causing a waste of Codonopsis pilosula resources to a certain extent. Codonopsis pilosula polysaccharide is one of the main active components of Codonopsis pilosula and has various biological activities such as antioxidant, immunity - enhancing, and anti - inflammatory. At present, there have been many reports on polysaccharides from non - medicinal parts of Codonopsis pilosula. For example, the acidic polysaccharides contained in the stems, leaves, rhizome tips, and fibrous roots of Codonopsis pilosula var. modesta and the stems and leaves of Codonopsis pilosula and Codonopsis tangshen all have certain antioxidant activities. However, there has not yet been a systematic study on Codonopsis pilosula seed polysaccharide. Summary of the Invention

[0004] The present invention provides a new use of Codonopsis pilosula seeds or their extracts. The present invention also provides a crude polysaccharide and a purified polysaccharide from Codonopsis pilosula seeds, as well as their preparation methods and uses.

[0005] The present invention provides the use of Codonopsis pilosula seeds or their extracts in the preparation of drugs with immunomodulatory effects or hypoglycemic effects or health foods helpful for enhancing immunity.

[0006] Among them, the Codonopsis pilosula extract is Codonopsis pilosula seed polysaccharide.

[0007] The present invention provides a crude polysaccharide from Codonopsis pilosula seeds, which is obtained by extracting the crude polysaccharide from Codonopsis pilosula seeds by the water extraction and alcohol precipitation method and purifying it by the Sevag method. It contains Ara, Gal, Xyl, GalA, Rha, Man, and their mass ratio is 45.6:18.9:11.9:9.3:7.7:6.7;

[0008] Among them, the contents of total sugar, protein, and uronic acid are 27.61±0.64%, 14.68±0.17%, and 15.03±0.33% respectively; the content of uronic acid is 14.65708%-15.26203%; the average particle size is 450.91nm, and the particle size is distributed in the range of 0-2000nm.

[0009] The present invention also provides a preparation method of the crude polysaccharide from Codonopsis pilosula seeds described in claim 3, which includes the steps:

[0010] a. Pretreatment of Codonopsis pilosula seeds: Weigh Codonopsis pilosula powder and soak it in 95% ethanol overnight. Heat and reflux for defatting the next day, filter, combine the filter residues, and dry them to obtain defatted powder of Codonopsis pilosula seeds;

[0011] b. Extraction of crude polysaccharide (CPSP-0): Weigh the defatted powder and mix it with water, heat and reflux for water extraction, filter and collect the filtrate, concentrate it under reduced pressure, slowly add 95% ethanol to the concentrated solution, stir evenly, place it at 4°C, centrifuge at a speed of 4000rpm / min, wash the precipitate with acetone, ether, and absolute ethanol twice respectively, and dry it at 60°C to obtain the crude polysaccharide CPSP-0 (Codonopsis pilosula seedspolysaccharides) in the form of white powder;

[0012] c. Preliminary purification of CPSP-0: Weigh the dried product and dissolve it completely in water. Add Sevag reagent (chloroform: n-butanol = 4:1, v / v) to the dissolved solution, shake it with a mixer at 5000rpm / min for 5min, centrifuge at 4000rpm / min for 10min, discard the lower layer of protein and organic mixed emulsion in the centrifuge tube, and recover the upper layer solution; repeat the above process 6-7 times until no obvious white protein precipitate precipitates; concentrate the upper layer of polysaccharide solution under reduced pressure, load it into a dialysis bag with a molecular weight cut-off of 3000Da, dialyze it with running water for 72h, and freeze-dry to obtain the crude polysaccharide CPSP of Codonopsis pilosula seeds.

[0013] The present invention provides a purified polysaccharide from Codonopsis pilosula seeds, which is a neutral polysaccharide CPSP-1, acidic polysaccharides: CPSP-2, CPSP-3, CPSP-4, CPSP-5 separated from the crude polysaccharide of Codonopsis pilosula seeds;

[0014] Among them, CPSP-1 is mainly composed of Ara, Gal, Man, Xyl, Glc and Rha in a ratio of 52.0:28.6:7.8:5.3:4.9; the contents of total sugar, protein and uronic acid are 23.35±0.25%, 8.25±0.07%, 2.01±0.12% respectively;

[0015] The contents of total sugar, protein and uronic acid in CPSP-2 are 23.44±0.67%, 3.13±0.20%, 6.55±0.17% respectively;

[0016] CPSP-3 is mainly composed of Ara, Gal, Xyl, Glc, GlcA, Man, GalA, Rha in a ratio of 42.5:26.4:22.1:2.8:2.4:1.8:1.7:0.4; the contents of total sugar, protein and uronic acid are 32.87±0.87%, 5.14±0.11%, 8.62±0.10% respectively;

[0017] CPSP-4 is mainly composed of Ara, GalA, Gal, Xyl, Rha, GlcA, Glc, Man in a ratio of 37.7:19.7:17.4:13.7:5.9:2.6:2.0:1.0; the contents of total sugar, protein and uronic acid are 21.74±0.87%, 11.28±0.33%, 24.22±0.13% respectively; the average particle size is 1155.75nm;

[0018] CPSP-5 is mainly composed of Ara, Rha, GalA, Xyl, Gal, Man in a ratio of 28.5:24.5:22.4:12.1:10.6:1.9; the molecular weight of CPSP-5 is 12389Da; the contents of total sugar, protein and uronic acid are 10.76±0.22%, 6.09±0.64%, 36.71±0.18% respectively; the average particle size is 1438.87nm.

[0019] The present invention also provides a preparation method of the purified polysaccharide from Codonopsis pilosula seeds, which separates the crude polysaccharide from Codonopsis pilosula seeds by anion exchange column chromatography and purifies it by gel filtration column chromatography.

[0020] It includes the following steps:

[0021] a. Swelling and activation of the packing: DEAE-52 is soaked in deionized water and the water is changed every 2h; soaked in 0.5M sodium hydroxide solution for 1h and washed with water until neutral; soaked in 0.5M hydrochloric acid solution for 1h and washed with water until neutral.

[0022] b. Column packing: The packing material is added to a 0.5 M sodium hydroxide solution. The base solution is added to the bottom of the column. After gently stirring the packing material, it is added to the chromatography column (with a specification of Ф5.5 cm × 50 cm) in a swirling manner. Stir from bottom to top to make the packing material evenly deposit at the bottom of the column. Stop packing when it reaches 5 cm from the upper end of the chromatography column. Wash the packing material with water until it is neutral and compact it overnight.

[0023] c. Sample loading: Take the crude polysaccharide from Codonopsis pilosula seeds and dissolve it in water. Filter it through a 0.45 μm water-based filter. Turn on the constant flow pump to lower the liquid level until it is flush with the packing material. Load the sample in a swirling manner. Turn off the pump when the liquid level is flush with the packing material and allow for adsorption equilibrium for 0.5 h.

[0024] c. Elution: Wash the neutral polysaccharide with water, and elute the acidic polysaccharide with 0.05 M, 0.1 M, 0.2 M, and 0.4 M NaCl solutions respectively. The elution flow rate is 3 mL / min, and collect 200 tubes at 10 mL / tube. Detect the polysaccharide every 3 tubes using the phenol-sulfuric acid method. Plot the elution curve with the absorbance as the vertical axis and the elution tube number as the horizontal axis. Collect the eluate, concentrate it under reduced pressure at 60 °C, dialyze it against running water for 48 h, and freeze-dry to obtain the various fractionated components of CPSP.

[0025] d. Swelling: The Sephadex G-150 dextran gel packing material is swollen with pure water.

[0026] e. Column packing: Add pure water to the bottom of the column, turn on the pump, gently stir the packing material and add it to the chromatography column (with a specification of Φ1.6 cm × 90 cm) in a swirling manner. Stop packing when it reaches 5 cm from the upper end of the chromatography column. Wash it with water for 10 column volumes and then compact it overnight.

[0027] f. Sample loading: Weigh each fractionated component and dissolve it in pure water. Filter it through a 0.45 μm water-based filter. Turn on the constant flow pump to lower the liquid level until it is flush with the packing material. Load the sample in a swirling manner. Turn off the pump when the liquid level is flush with the packing material and allow for adsorption equilibrium for 0.5 h. The flow rate is 0.4 mL / min.

[0028] g. Elution: Use water as the eluent, the elution flow rate is 0.4 mL / min, collect 15 tubes at 10 mL / tube, detect the polysaccharide in each tube using the phenol-sulfuric acid method. Plot the elution curve with the absorbance as the vertical axis and the elution tube number as the horizontal axis. Collect the eluate according to each elution curve, concentrate it under reduced pressure at 60 °C, dialyze it against running water for 48 h, and freeze-dry to obtain the fractionated and purified components of CPSP.

[0029] The present invention provides the use of the above-mentioned crude polysaccharide from Codonopsis pilosula seeds and the purified polysaccharide from Codonopsis pilosula seeds in the preparation of drugs or health foods that are helpful for antioxidation.

[0030] The present invention provides the use of the above-mentioned crude polysaccharide from Codonopsis pilosula seeds and the purified polysaccharide from Codonopsis pilosula seeds in the preparation of drugs or health foods with immunomodulatory effects.

[0031] The present invention provides the use of the crude polysaccharide from Codonopsis pilosula seeds and the purified polysaccharide from Codonopsis pilosula seeds in the preparation of a drug with hypoglycemic effect or a health food helpful for maintaining a healthy blood glucose level.

[0032] Based on the extraction of polysaccharides from Codonopsis pilosula seeds, the present invention conducts structural analysis and bioactivity research on the polysaccharides from Codonopsis pilosula seeds, providing a certain theoretical basis for the development and utilization of Codonopsis pilosula seeds. The present invention helps to realize the comprehensive development and utilization of Codonopsis pilosula resources, enhancing the economic value and product added value of Codonopsis pilosula. At the same time, this research can, to a certain extent, expand the industrial chain of Codonopsis pilosula, playing an important promoting role in increasing the income of medicinal herb farmers and promoting the local medicinal herb industry and economic development. Description of the Drawings

[0033] Figure 1 Flow chart for the extraction of crude polysaccharide from Codonopsis pilosula seeds;

[0034] Figure 2 Standard curve A. Glucose B. Bovine serum albumin C. Galacturonic acid;

[0035] Figure 3 HPLC chromatogram of the monosaccharide composition of CPSP-0;

[0036] Figure 4 HPLC chromatogram of the monosaccharide composition of CPSP;

[0037] Figure 5 Infrared spectrum of CPSP-0;

[0038] Figure 6 Infrared spectrum of CPSP;

[0039] Figure 7 Particle size analysis diagram of CPSP-0 and CPSP;

[0040] Figure 8 Ultraviolet absorption spectrum of CPSP-0 and CPSP;

[0041] Figure 9 Standard curve of polysaccharide molecular weight;

[0042] Figure 10 Gradient separation and elution curve of CPSP;

[0043] Figure 11 Purification elution curve of the fractionated polysaccharide from Codonopsis pilosula seeds;

[0044] Figure 12 HPGPC chromatogram (A) and ultraviolet absorption spectrum (B) of CPSP-1;

[0045] Figure 13HPLC chromatogram of the monosaccharide composition of CPSP-1;

[0046] Figure 14 Infrared spectrum of CPSP-1;

[0047] Figure 15 Particle size distribution map (A) and triple helix structure analysis (B) of CPSP-1;

[0048] Figure 16 HPGPC chromatogram (A) and ultraviolet absorption spectrum (B) of CPSP-3;

[0049] Figure 17 HPLC chromatogram of the monosaccharide composition of CPSP-3;

[0050] Figure 18 Infrared spectrum of CPSP-3;

[0051] Figure 19 Particle size distribution map (A) and triple helix structure analysis (B) of CPSP-3;

[0052] Figure 20 HPGPC chromatogram (A) and ultraviolet absorption spectrum (B) of CPSP-4;

[0053] Figure 21 HPLC chromatogram of the monosaccharide composition of CPSP-4;

[0054] Figure 22 Infrared spectrum of CPSP-4;

[0055] Figure 23 Particle size distribution map (A) and triple helix structure analysis (B) of CPSP-4;

[0056] Figure 24 HPGPC chromatogram (A) and ultraviolet absorption spectrum (B) of CPSP-5;

[0057] Figure 25 HPLC chromatogram of the monosaccharide composition of CPSP-5;

[0058] Figure 26 Infrared spectrum of CPSP-5;

[0059] Figure 27 Particle size distribution map (A) and triple helix structure analysis (B) of CPSP-5;

[0060] Figure 28 DPPH free radical scavenging activity;

[0061] Figure 29 ABTS free radical scavenging activity;

[0062] Figure 30 OH radical scavenging activity;

[0063] Figure 31 O2- radical scavenging activity;

[0064] Figure 32 Reducing power;

[0065] Figure 33 α-amylase (A) and α-glucosidase (B) inhibitory activities;

[0066] Figure 34 Toxic effects on RAW 264.7 cells at different concentrations (100, 200, 400 μg / mL);

[0067] Figure 35 Effect on NO secretion by RAW 264.7 cells;

[0068] Figure 36 Schematic diagram of animal experiment design;

[0069] Figure 37 Changes in the mass (A), thymus index (B), and spleen (C) index of mice;

[0070] Figure 38 Pathological changes in spleen tissues of each group (20X);

[0071] Figure 39 Dilution curve A.sobs index B.Shannon index;

[0072] Figure 40 Alpha diversity analysis A.Ace index B.Chao index C.Shannon index D.Simpson index;

[0073] Figure 41 Dysbiosis (A) and PCoA analysis (B);

[0074] Figure 42 Community composition analysis A.phylum level B.genus level. Specific implementation mode

[0075] Example 1 Extraction and structural analysis of crude polysaccharides from Codonopsis pilosula seeds of the present invention

[0076] 1 Materials and methods

[0077] 1.1 Main experimental materials

[0078] In September 2022, Codonopsis pilosula seeds were purchased from Yongxin Township, Jingyuan County, Gansu Province. The surface dust and residual stems and leaves were washed away with running water, dried and crushed. The powder was passed through a 60-mesh sieve and stored at 4°C.

[0079] 1.2 Experimental methods

[0080] 1.2.1 Extraction of crude polysaccharides from Codonopsis pilosula seeds

[0081] The crude polysaccharides were prepared by water extraction and ethanol precipitation method, and were preliminarily purified by Sevag method. The extraction process is as Figure 1 shown.

[0082] (1) Pretreatment of Codonopsis pilosula seeds: Weigh a certain amount of powder and soak it overnight with 95% ethanol at a mass-to-liquid ratio of 1:6. Reflux and degrease it three times by heating the next day, each time for 1.5 h. Filter with a filter cloth, combine the filter residues, and dry them to obtain defatted powder of Codonopsis pilosula seeds.

[0083] (2) Extraction of crude polysaccharides (CPSP-0): Weigh a certain amount of defatted powder and extract it by heating and refluxing with water at a mass-to-liquid ratio of 1:10 three times, each time for 2 h. Filter with a filter cloth and collect the filtrate. Concentrate it under reduced pressure at 60 °C to 1 / 4 of the initial volume. Slowly add 4 times the volume of 95% ethanol to the concentrated solution, stir evenly, place it at 4 °C for 12 h, centrifuge it at 4000 rpm / min for 10 min. Wash the precipitate with acetone, ether, and absolute ethanol twice respectively, and dry it at 60 °C to obtain white powdery crude polysaccharides CPSP-0 (Codonopsis pilosula seeds polysaccharides) of Codonopsis pilosula seeds. Weigh it and calculate the yield.

[0084] (3) Preliminary purification of CPSP-0: Weigh the dried product and dissolve it completely in water. Add 10 mL of Sevag reagent (chloroform: n-butanol = 4:1, v / v) to 40 mL of the dissolution solution (10 mg / mL). Vibration for 5 min with a mixer at 5000 rpm / min, centrifuge at 4000 rpm / min for 10 min, discard the lower layer of protein and organic mixed emulsion in the centrifuge tube, and recover the upper layer solution. Repeat the above process 6 - 7 times until no obvious white protein precipitate precipitates, indicating that the protein is basically removed. Concentrate the upper layer of polysaccharide solution under reduced pressure to a certain volume, put it into a dialysis bag (3000 Da), dialyze it with running water for 72 h, and freeze-dry it to obtain crude polysaccharides CPSP (Codonopsis pilosula seeds polysaccharides) of Codonopsis pilosula seeds. Weigh it and calculate the yield.

[0085] 1.2.2 Determination of the main chemical composition of crude polysaccharides from Codonopsis pilosula seeds

[0086] 1.2.2.1 Determination of total sugar content

[0087] The total sugar content was determined by the phenol-sulfuric acid method.

[0088] (1) Solution preparation: ① 5% phenol: Weigh 2.5 g of phenol (re-distilled) and dissolve it in water, then make up the volume to 50 mL in a brown volumetric flask. ② 1 mg / mL glucose standard solution: Weigh 10 mg of glucose standard and dissolve it in water, then make up the volume to 10 mL in a volumetric flask.

[0089] (2) Standard curve preparation: Pipette 1 mL of the standard solution into a 5 mL volumetric flask and make up the volume. Then pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL of the diluted solution into test tubes respectively, add water to make up to 1 mL. Add 1 mL of 5% phenol to each tube, shake well. Slowly add 5 mL of concentrated sulfuric acid in an ice-water bath, shake well. React in a boiling water bath for 15 min, then cool in an ice-water bath. Measure the absorbance at 490 nm. Each group is measured in parallel three times and the average value is taken. Plot the standard curve with the standard glucose mass concentration on the x-axis and the absorbance on the y-axis.

[0090] 1.2.2.2 Determination of protein content

[0091] The Coomassie brilliant blue method is used to determine the protein content.

[0092] (1) Solution preparation: ① Coomassie brilliant blue G 250: Weigh 100 mg of G 250 and dissolve it in 50 mL of 95% ethanol, then add 100 mL of 85% phosphoric acid and make up the volume to 1 L in a brown volumetric flask. ② 1 mg / mL BSA standard solution: Weigh 10 mg of BSA and dissolve it in water, then make up the volume to 10 mL in a volumetric flask.

[0093] (2) Standard curve preparation: Pipette 1 mL of the protein standard solution into a 5 mL volumetric flask and make up the volume. Then pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL of the diluted solution into test tubes respectively, add water to make up to 1 mL. Add 5 mL of G-250 solution to each tube, shake well. React at 25 °C for 15 min. Measure the absorbance at 595 nm. Each group is measured in parallel three times and the average value is taken. Plot the standard curve with the standard protein mass concentration on the x-axis and the absorbance on the y-axis.

[0094] 1.2.2.3 Determination of uronic acid content

[0095] The m-hydroxybiphenyl method is used to determine the uronic acid content.

[0096] (1) Solution preparation: ① 0.15% m-hydroxybiphenyl: Weigh 0.15 g of m-hydroxybiphenyl and dissolve it in 0.5% NaOH solution, then make up the volume to 100 mL in a brown volumetric flask with the alkaline solution. ② 1.5 mg / mL sodium tetraborate / sulfuric acid solution: Weigh 0.478 g of sodium tetraborate and dissolve it in sulfuric acid, then make up the volume to 100 mL in a volumetric flask with sulfuric acid. ③ 1 mg / mL galacturonic acid standard solution: Weigh 10 mg of galacturonic acid standard and dissolve it in water, then make up the volume to 10 mL in a volumetric flask.

[0097] (2) Preparation of standard curve: Pipette 1 mL of the standard solution into a 10 mL volumetric flask and make up to the mark. Take 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL of the made-up solution into test tubes respectively, add water to make up to 1 mL, slowly add 5 mL of sodium tetraborate / sulfuric acid solution in an ice-water bath, shake well, react in a boiling water bath for 15 min, cool in an ice-water bath, add 100 μL of 0.15% m-hydroxybiphenyl solution, shake well for color development, measure the absorbance at a wavelength of 520 nm within 5 min, measure in parallel three times for each group, take the average value, use the mass concentration of standard uronic acid as the abscissa and the absorbance as the ordinate to draw the standard curve.

[0098] 1.2.3 Determination of monosaccharide composition of crude polysaccharide from Codonopsis pilosula seeds

[0099] The monosaccharide composition of the crude polysaccharide from Codonopsis pilosula seeds was determined by pre-column derivatization with PMP and HPLC method.

[0100] (1) Derivatization of mixed monosaccharide standards: ① Preparation of mixed monosaccharide standard solution: Prepare standard solutions of Glc, Man, Ara, Rha, Gal, GalA, Rib, GlcA, Fuc, Xyl at 5 mg / mL respectively, and then pipette 200 μL of each standard solution and mix for standby. ② PMP derivatization: Pipette 400 μL of the mixed standard solution, 400 μL of 0.3 mo1 / L sodium hydroxide solution, 500 μL of ultrapure water and 800 μL of 0.5 M PMP-methanol solution respectively, vortex and mix well, react under sealed water bath conditions at 70 °C for 1 h, cool to room temperature, add 400 μL of 0.3 M hydrochloric acid to neutralize the reaction solution, add 2 mL of chloroform to extract the residual PMP in the reaction solution, extract 3 times, collect the upper aqueous phase and filter through a 0.22 μm organic microporous membrane, and take samples for testing.

[0101] (2) Acid hydrolysis and derivatization of polysaccharide from Codonopsis pilosula seeds: ① Prepare a 5 mg / mL crude polysaccharide solution, pipette 2 mL of the polysaccharide solution into a 25 mL three-way valve reaction flask, add 4.0 mL of 4.0 M trifluoroacetic acid and a magnetic stirrer, seal with insulating tape, fill nitrogen into the reaction flask with a water bath nitrogen blower, replace the air in the bottle with a circulating water pump, carry out acid hydrolysis at 120 °C in an oil bath for 5 h, rotary evaporate the hydrolysis solution under reduced pressure at 55 °C, repeat adding 3 mL of methanol multiple times to remove the residual trifluoroacetic acid (until the methanol solution is neutral), and finally add 1.5 mL of ultrapure water to dissolve to obtain the hydrolysis product. ② PMP derivatization: The operation is the same as that of the mixed monosaccharide standards.

[0102] (3) HPLC determination conditions: The equipment is Agilent 1260 infinity Ⅱ; diode array detector (detection wavelength 250 nm); WondaSil C 18Superb chromatographic column (4.5×250 mm, 5 μm); column temperature 30 °C; injection volume 20 μL; mobile phase is a mixture of 0.05 M phosphate buffer (pH 6.7) and chromatographic grade acetonitrile (v / v, 83:17); flow rate is 1 mL / min.

[0103] 1.2.4 Infrared spectrum analysis of crude polysaccharides from Codonopsis pilosula seeds

[0104] The IR absorption spectrum of the crude polysaccharide was determined by the potassium bromide tablet method. First, potassium bromide was dried at 120 °C for 4 h and placed in a desiccator for later use. Then, 2.0 mg of polysaccharide and 300 mg of KBr were weighed and mixed, ground into fine powder with an agate mortar, and finally, the spectrum in the range of 450 - 4000 cm -1 was scanned using an FT-IR instrument.

[0105] 1.2.5 Particle size of crude polysaccharides from Codonopsis pilosula seeds

[0106] A 5 mg / ml crude polysaccharide solution was prepared, and the particle size was measured at room temperature.

[0107] 1.2.6 Ultraviolet spectrum analysis of crude polysaccharides from Codonopsis pilosula seeds

[0108] A 0.1 mg / mL crude polysaccharide solution was prepared, and the ultraviolet absorption spectrum of the solution was scanned in the range of 200 - 600 nm of ultraviolet-visible light wavelength.

[0109] 1.2.7 Data processing

[0110] Experimental data were expressed as mean ± standard deviation, and Origin 2022 software was used for plotting.

[0111] 2 Results

[0112] 2.1 Yield of crude polysaccharides from Codonopsis pilosula seeds

[0113] (1) Defatting rate: 4600 g of Codonopsis pilosula seed powder was defatted with 95% ethanol, and 3285 g of defatted powder was obtained after drying. The defatting rate was 28.59%. (2) Yield of CPSP-0: 2800 g of defatted powder was subjected to water extraction and ethanol precipitation, and 142.62 g of white powdery CPSP-0 was obtained after drying. The yield was 5.09%. (3) Yield of CPSP: 120.2 g of CPSP-0 was deproteinized by the Sevag method, and 25.27 g of light yellow powdery CPSP was obtained. The yield was 21.02%.

[0114] 2.2 Determination of the main chemical composition of crude polysaccharides from Codonopsis pilosula seeds

[0115] Preparation of standard curves for glucose, bovine serum albumin, and galactose. The glucose standard curve is as Figure 2As shown in A, the curve regression equation is Y = 0.0107x + 0.0126 (R 2 = 0.9997), and the linear relationship is good in the concentration range of 10.1 - 60.6 μg / mL. The standard curve of bovine serum albumin is as Figure 2 shown in B. The regression equation of the curve is Y = 0.00545x + 0.094 (R 2 = 0.9993), and the linear relationship is good in the concentration range of 20.15 - 120.9 μg / mL. The standard curve of galacturonic acid is as Figure 2 shown in C. The regression equation of the curve is Y = 0.113x + 0.0194 (R 2 = 0.9997), and the linear relationship is good in the concentration range of 10.4 - 52 μg / mL.

[0116] Main chemical compositions of crude polysaccharides from Codonopsis pilosula seeds. Within the linear ranges of the three standard curves, the main chemical compositions of CPSP - 0 and CPSP were determined. The contents of total sugar, protein, and uronic acid in CPSP - 0 were 11.89 ± 0.24%, 21.30 ± 0.10%, and 5.45 ± 0.05% respectively. The contents of total sugar, protein, and uronic acid in CPSP were 27.61 ± 0.64%, 14.68 ± 0.17%, and 15.03 ± 0.33% respectively. After removing protein from CPSP - 0 by Sevag method, the protein content of CPSP decreased, while the contents of total sugar and uronic acid increased significantly, and the uronic acid content reached 15.03%.

[0117] 2.3 Analysis of monosaccharide composition of crude polysaccharides from Codonopsis pilosula seeds

[0118] The results of monosaccharide composition of CPSP - 0 are as Figure 3 shown. CPSP - 0 is composed of Ara, Gal, Man, Xyl, GalA, Glc, Rha, GlcA, Rib in a ratio of 39.3:17.4:17.0:12.7:5.7:3.9:2.0:1.2:0.7. The results of monosaccharide composition of CPSP are as Figure 4 shown. CPSP is composed of Ara, Gal, Xyl, GalA, Rha, Man in a ratio of 45.6:18.9:11.9:9.3:7.7:6.7.

[0119] 2.4 FT - IR analysis of crude polysaccharides from Codonopsis pilosula seeds

[0120] The FT - IR results of CPSP - 0 are as Figure 5 shown. The strong absorption peak at 3419.25 cm -1 is attributed to the stretching vibration of O - H. The absorption peak near 2925.28 cm -1 is for the stretching vibration of C - H, and the absorption peak at 2855.67 cm-1 The absorption peak at 1743.27 cm -1 is a secondary peak for the absorption peak, indicating the presence of methylene. The absorption peak at 1640.26 cm -1 is attributed to the stretching vibration of C=O, indicating that CPSP-0 contains methyl ester carboxyl and may contain a small amount of uric acid. The absorption peak at 1538.18 cm -1 is attributed to the stretching vibration of C=O in the amide group. The absorption peak at 1403.86 cm

[91] is attributed to the amide II band, indicating that CPSP-0 contains protein -1 The absorption peak at 1101.69 cm -1 is the bending vibration of C-O in the stretching mode in the form of pyranose. The wave number is 618.57 cm -1 The absorption peak is the out-of-plane bending vibration of C-H.

[0121] The FT-IR results of CPSP are as Figure 6 shown. The absorption peaks near 3417.58 cm-1 and 2925.90 cm-1 are attributed to the stretching vibrations of O-H and C-H respectively. After removing the protein, the secondary peak of CPSP at 2850 cm -1 disappears. The absorption peak at 1640.73 cm -1 is attributed to the stretching vibration of C=O in the amide group. The absorption peak at 1550.64 cm -1 The absorption peak near 1406 cm -1 is a characteristic absorption peak caused by the bending vibration of C-H. The absorption peak at 1242.91 cm -1 is the bending vibration of O-H in the carboxyl group and the asymmetric vibration of C-O-C. The absorption peak at 1097.96 cm -1 is the absorption peak for the stretching vibration of C-O. The absorption peak at 895.01 cm -1 is the characteristic peak of α-type glycoside between sugar units.

[0122] 2.5 Particle size of crude polysaccharide from Codonopsis pilosula seeds

[0123] The particle size of CPSP-0 is as Figure 7 shown in A. Its average particle size is 417.88 nm, and the particle size is distributed around 200 - 1000 nm. The particle size of CPSP is as Figure 7 shown in B. Its average particle size is 450.91 nm, and the particle size is distributed in the range of 0 - 2000 nm, with a wider distribution range than CPSP-0.

[0124] 2.6 Ultraviolet spectral analysis of crude polysaccharide from Codonopsis pilosula seeds

[0125] The ultraviolet absorption spectra of CPSP-0 and CPSP are asFigure 8 As shown in Figure 8 , CPSP-0 and CPSP have no obvious absorption peaks at 260 nm and 280 nm, indicating that they contain almost no nucleic acid and protein. However, chemical analysis shows that the protein contents of the two are 21.30% and 14.68% respectively, which are different experimental results from instrumental analysis.

[0126] 3 Discussion

[0127] In this invention, 95% ethanol is used to remove the fat-soluble components of Codonopsis pilosula seeds, and the defatting rate is 28.59%. The crude polysaccharide CPSP-0 of Codonopsis pilosula seeds is obtained by hot water extraction method, and the extraction rate is 5.09%. The contents of total sugar, protein and uronic acid in CPSP-0 are 11.89%, 21.30% and 5.45% respectively, containing more protein. After removing protein from CPSP-0 by Sevag method, CPSP is obtained, and the yield is 21.02%; the contents of total sugar and uronic acid double and increase to 27.61% and 15.03% respectively, and still contain 14.68 ± 0.17% protein; it shows that the Sevag effect is mild, which can increase the contents of total sugar and uronic acid, but fails to remove all the protein; CPSP-0 and CPSP have complex monosaccharide compositions, among which the proportion of arabinose is the highest, which may be the reason for their low total sugar content. The particle sizes of CPSP-0 and CPSP are similar, but the particle size of CPSP is larger, and it may be easy to form aggregates with uneven sizes in water, which is related to its higher proportion of galacturonic acid. The infrared scanning results show that both CPSP-0 and CPSP have characteristic absorption peaks of polysaccharides.

[0128] 4 Summary

[0129] The crude polysaccharide CPSP-0 of Codonopsis pilosula seeds is prepared by the method of water extraction and alcohol precipitation, and CPSP is obtained by removing protein by Sevag method. Both of them have infrared characteristic peaks of polysaccharides, are mainly composed of arabinose, contain a certain amount of protein and uronic acid, and may be acidic polysaccharides.

[0130] Example 2 Isolation, Purification and Structural Analysis of Polysaccharides from Codonopsis pilosula Seeds

[0131] 1 Materials and Methods

[0132] 1.1 Main Experimental Materials and Reagents

[0133] The main experimental material is the crude polysaccharide (CPSP) of Codonopsis pilosula seeds. Congo red reagent (batch number 0FDER51N) is purchased from Anhui Zesheng Technology Co., Ltd., and the rest of the reagents are the same as those in Table 1-2.

[0134] 1.2 Experimental Methods

[0135] 1.2.1 Isolation of Polysaccharides from Codonopsis pilosula Seeds

[0136] Anion exchange column chromatography was used. Based on the principle that acidic polysaccharides are adsorbed by DEAE cellulose-52 while neutral polysaccharides are not adsorbed when the solution pH is 6, CPSP was separated.

[0137] (1) Swelling and activation of the packing material: DEAE-52 was soaked in deionized water for 12 h, with water changed every 2 h; soaked in 0.5 M sodium hydroxide solution for 1 h, washed with water until neutral; soaked in 0.5 M hydrochloric acid solution for 1 h, washed with water until neutral.

[0138] (2) Column packing: The packing material was added with 0.5 M sodium hydroxide solution, and a small amount of alkali solution was also added to the bottom of the column. After gently stirring the packing material, it was added to the chromatography column (with a specification of Ф5.5 cm × 50 cm) in a circular flow manner. Stir from bottom to top to make the packing material evenly deposited at the bottom of the column. Stop packing when it reaches 5 cm from the upper end of the chromatography column, wash the packing material with water until neutral, and compact it overnight. It should be ensured that there are no faults and no bubbles inside the packing material.

[0139] (3) Sample loading: 2 g of CPSP was dissolved in 30 mL of pure water, filtered through a 0.45 μm water-based filter. Turn on the constant flow pump to lower the liquid level to be flush with the packing material, load the sample in a circular flow manner, turn off the pump when the liquid level is flush with the packing material, and perform adsorption equilibrium for 0.5 h.

[0140] (4) Elution: Neutral polysaccharides were eluted with water, and acidic polysaccharides were eluted with 0.05 M, 0.1 M, 0.2 M, and 0.4 M NaCl solutions respectively. The elution flow rate was 3 mL / min, and 200 tubes were collected at 10 mL / tube. The phenol-sulfuric acid method was used to detect polysaccharides every 3 tubes. An elution curve was plotted with the absorbance as the ordinate and the elution tube number as the abscissa. Refer to each section of the elution curve to collect the eluate, concentrate it under reduced pressure at 60 °C, dialyze it with running water for 48 h, and freeze-dry to obtain each fractionated component of CPSP.

[0141] 1.2.2 Purification of Codonopsis pilosula seed polysaccharides

[0142] Gel filtration column chromatography was used. Based on the principle that polysaccharides with large molecular weights elute from the chromatography column first and polysaccharides with small molecular weights elute later, each fractionated component of CPSP was purified.

[0143] (1) Swelling: The Sephadex G-150 dextran gel packing material was swollen with 20 times the volume of pure water for 48 h, and the water was changed several times during this period.

[0144] (2) Column packing: An appropriate amount of pure water was added to the bottom of the column, the pump was turned on, and after gently stirring the packing material, it was added to the chromatography column (with a specification of Φ1.6 cm × 90 cm) in a circular flow manner. Stop packing when it reaches 5 cm from the upper end of the chromatography column, wash with water for 10 column volumes, and compact it overnight. It should be ensured that there are no faults and no bubbles inside the packing material.

[0145] (3) Sample addition: Weigh 30 mg of the fractionated component and dissolve it in 3 mL of pure water. Filter through a 0.45 μm water-based filter. Turn on the constant flow pump to lower the liquid level until it is flush with the packing material. Add the sample in a circular motion. Turn off the pump when the liquid level is flush with the packing material, and allow for 0.5 h of adsorption equilibrium. The flow rate is 0.4 mL / min.

[0146] (4) Elution: Use water as the eluent. The elution flow rate is 0.4 mL / min, and collect 15 tubes at 10 mL / tube. Detect the polysaccharide in each tube using the phenol-sulfuric acid method. Plot the elution curve with the absorbance as the ordinate and the elution tube number as the abscissa. Collect the eluate according to each section of the elution curve, concentrate it under reduced pressure at 60 °C, dialyze it against running water for 48 h, and freeze-dry to obtain the fractionated and purified component of CPSP.

[0147] 1.2.3 Determination of the main chemical composition of Codonopsis pilosula seed polysaccharide: The same as in Example 1.

[0148] 1.2.4 Determination of the molecular weight of Codonopsis pilosula seed polysaccharide

[0149] The molecular weight (Molecular weight, Mw) of Codonopsis pilosula seed polysaccharide was determined by HPGPC method.

[0150] (1) Determination conditions: The equipment is Agilent 1260 infinity Ⅱ GPC; unit pump; the chromatographic column is Agilent PLaquagel-OH SEC MIXED-H (8 μm, 300×7.5 mm); differential refractive index detector; column temperature 40 °C; mobile phase is 0.1 M NaNO3; injection volume is 20 μL; flow rate is 1 mL / min.

[0151] (2) Fitting the standard curve: Prepare polysaccharide standard solutions with molecular weights of 6.3, 9.8, 22, 49.4, 107, 216, 348, and 739 kD, respectively, with a concentration of 1 mg / mL for each. Measure according to the above experimental conditions. Use the retention time as the abscissa and the logarithm of the molecular weight (lg Mw) as the ordinate to obtain the fitted standard curve. The fitting result is as Figure 9 shown, and the regression equation is Y = 12.786 - 1.0234x (R 2 = 0.9961). (3) Determination of the molecular weight of the polysaccharide sample: Prepare a 1 mg / mL solution of the purified polysaccharide sample of Codonopsis pilosula seeds. After determination by HPGPC, calculate the molecular weight of the sample according to the retention time.

[0152] 1.2.5 Ultraviolet spectrum analysis of Codonopsis pilosula seed polysaccharide, the same as in Example 1.

[0153] 1.2.6 Determination of the monosaccharide composition of Codonopsis pilosula seed polysaccharide, the same as in Example 1.

[0154] 1.2.7 Infrared spectrum analysis of Codonopsis pilosula seed polysaccharide, the same as in Example 1.

[0155] 1.2.8 Particle size of Codonopsis pilosula seed polysaccharide, the same as in Example 1.

[0156] 1.2.9 Determination of the triple-helix structure of Codonopsis pilosula seed polysaccharide

[0157] Under the condition of increasing solution alkalinity (0 - 0.6 M NaOH), the maximum absorption wavelength of the reaction solution of Congo red and polysaccharide with a triple-helix conformation will first show a red shift, then a blue shift, and finally tend to be stable. Based on this, it is judged whether the polysaccharide has a triple-helix structure.

[0158] (1) Solution preparation: ① Weigh 10 mg of polysaccharide sample and dissolve it in water, and make up the volume to 10 mL with water in a volumetric flask. ② 80 μM Congo red solution: Weigh 28 mg of Congo red and dissolve it in water, and make up the volume to 500 mL with water in a volumetric flask, and store it in the dark. ③ 1 M NaOH solution: Weigh 4 g of sodium hydroxide and dissolve it in water, and make up the volume to 100 mL with water in a volumetric flask.

[0159] (2) UV scanning: Take 7 centrifuge tubes in the dark, add 1 mL of polysaccharide solution and 1 mL of Congo red solution in sequence, gently shake and mix well, then add 0, 0.23, 0.5, 0.86, 1.35, 2, 3 mL of NaOH solution in sequence, shake evenly, and let it stand in the dark at room temperature for 10 min. Using water as the blank and the Congo red solution without adding the sample as the control, perform UV scanning at 400 - 600 nm. Taking the NaOH concentration as the abscissa and the maximum absorption wavelength as the ordinate, record the change of the maximum absorption wavelength of the polysaccharide sample at different NaOH concentrations.

[0160] 1.2.10 Statistical methods

[0161] Experimental data are expressed as mean ± standard deviation, and Origin 2022 software is used for plotting.

[0162] 2 Results

[0163] 2.1 Isolation of Codonopsis pilosula seed polysaccharide

[0164] A total of 24 g of crude Codonopsis pilosula seed polysaccharide (CPSP) was separated by DEAE cellulose-52 anion exchange column. Figure 10It is an elution curve. After eluting with water and NaCl solutions of different gradients, 5 elution peaks were separated. The component eluted with pure water was denoted as CPSP-1, and the collection tube numbers were 52 - 101; the component eluted with 0.05 M NaCl was denoted as CPSP-2, and the collection tube numbers were 273 - 310; the component eluted with 0.1 M NaCl was denoted as CPSP-3, and the collection tube numbers were 473 - 510; the component eluted with 0.2 M NaCl was denoted as CPSP-4, and the collection tube numbers were 673 - 755; the component eluted with 0.4 M NaCl was denoted as CPSP-5, and the collection tube numbers were 876 - 919. The corresponding eluates of each component were collected respectively. After concentration, dialysis, and freeze-drying, five components of Codonopsis pilosula seed polysaccharide CPSP were preliminarily separated, and their yields were 2.29%, 1.10%, 4.60%, 9.10%, and 13.12% (calculated based on the dry weight of CPSP) in sequence. The masses of the obtained dry components were 549.6 mg, 264 mg, 1104 mg, 2184 mg, and 3149 mg respectively. Among them, the yield of CPSP-5 component was the highest, and the yield of CPSP-4 component was the second highest.

[0165] 2.2 Purification of Codonopsis pilosula seed polysaccharide

[0166] The components separated by DEAE cellulose-52 anion exchange column were purified by Sephadex G-150 dextran gel chromatography column with pure water elution. From Figure 11 it can be seen that after eluting the 5 polysaccharide components with pure water, 4 highly pure single purified components were obtained. The collection tube numbers of CPSP-1 were 1 - 15; the collection tube numbers of CPSP-2 were 2 - 11; the collection tube numbers of CPSP-3 were 3 - 8; the collection tube numbers of CPSP-4 were 3 - 8; the collection tube numbers of CPSP-5 were 3 - 10. After reduced pressure concentration, dialysis, and freeze-drying, the purified components of Codonopsis pilosula seed polysaccharide were obtained, and the yields were 91.70%, 28.22%, 66.46%, 83.33%, and 77.49% respectively.

[0167] 2.3 Main chemical composition of Codonopsis pilosula seed polysaccharide

[0168] The total sugar, protein, and uronic acid contents of CPSP-1 were 23.35±0.25%, 8.25±0.07%, and 2.01±0.12% respectively; those of CPSP-2 were 23.44±0.67%, 3.13±0.20%, and 6.55±0.17% respectively; those of CPSP-3 were 32.87±0.87%, 5.14±0.11%, and 8.62±0.10% respectively; those of CPSP-4 were 21.74±0.87%, 11.28±0.33%, and 24.22±0.13 respectively; those of CPSP-5 were 10.76±0.22%, 6.09±0.64%, and 36.71±0.18% respectively. The protein contents of the 5 purified polysaccharides were all lower than that of CPSP (14.68±0.17%), indicating that DEAE-52 cellulose column chromatography had a certain protein removal effect. All 5 purified components contained a certain amount of uronic acid. As the NaCl concentration in the separation eluent increased, the eluted components contained more uronic acid. The contents of CPSP-4 and CPSP-5 were 24.22% and 36.71% respectively. Since the yield of the CPSP-2 component was relatively low, subsequent structural analysis and biological activity research were not carried out.

[0169] 2.4 Structural analysis of the purified polysaccharide CPSP-1 from Codonopsis pilosula seeds

[0170] 2.4.1 Molecular weight determination and ultraviolet spectrum analysis

[0171] The results of the molecular weight determination of CPSP-1 are as Figure 12 shown in A. CPSP-1 presented two absorption peaks, with molecular weights of 176270 Da and 7647 Da respectively, and the proportions were 35.29% and 64.07%, indicating that CPSP-1 was not a homogeneous polysaccharide.

[0172] The results of the ultraviolet absorption spectrum of CPSP-1 are as Figure 12 shown in B. CPSP-1 had no obvious absorption peaks at 260 nm and 280 nm, indicating that it contained almost no nucleic acids and free proteins.

[0173] 2.4.2 Monosaccharide composition analysis

[0174] The results of the monosaccharide composition of CPSP-1 are as Figure 13 shown. CPSP-1 was composed of Ara, Gal, Man, Xyl, Glc, and Rha in a ratio of 52.0:28.6:7.8:5.3:4.9.

[0175] 2.4.3 Infrared spectrum analysis

[0176] The FT-IR results of CPSP-1 are as Figure 14 shown. 3419.41 cm -1The strong absorption peak nearby is the stretching vibration of O-H. 2930.59 cm -1 The absorption peak nearby is the stretching vibration of C-H ] 。1652.63 cm -1 and 1539.78 cm -1 The absorption peaks at 1652.63 cm and 1539.78 cm are attributed to the stretching vibration of C=O in the amide group. 1412.11 cm -1 The absorption peak at 1412.11 cm is for the bending vibration of C-H. 1078.54 cm -1 、1039.44 cm -1 The absorption peaks nearby indicate that CPSP-1 may have a pyran ring. 864.68 cm -1 The absorption peak at 864.68 cm indicates the presence of α-type glycosidic bonds.

[0177] 2.4.4 Particle Size and Triple Helix Structure

[0178] The particle size of CPSP-1 is as Figure 15 shown in A. The average particle size of CPSP-1 is 2122.30 nm, with a wide and extensive distribution, indicating that it is easy to form large aggregates in water.

[0179] The determination results of the triple helix structure of CPSP-1 are as Figure 15 shown in B. The change trend of the maximum absorption wavelength of CPSP-1 is similar to that of the congo red control, indicating that it does not contain a triple helix structure.

[0180] 2.5 Structural Analysis of the Purified Polysaccharide CPSP-3 from Codonopsis pilosula Seeds

[0181] 2.5.1 Molecular Weight Determination and UV Spectral Analysis

[0182] The determination results of the molecular weight of CPSP-3 are as Figure 16 shown in A. CPSP-3 shows two absorption peaks as a whole, with molecular weights of 1885979 Da and 22190 Da respectively, accounting for 66.98% and 33.02%. This indicates that CPSP-3 is not a homogeneous polysaccharide.

[0183] The UV absorption spectral results of CPSP-3 are as Figure 16 shown in B. CPSP-3 has no obvious absorption peaks at 260 nm and 280 nm, indicating that it contains almost no nucleic acids and proteins.

[0184] 2.5.2 Monosaccharide Composition Analysis

[0185] The monosaccharide composition results of CPSP-3 are as Figure 17As shown, CPSP-3 is composed of Ara, Gal, Xyl, Glc, GlcA, Man, GalA, and Rha in a ratio of 42.5:26.4:22.1:2.8:2.4:1.8:1.7:0.4.

[0186] 2.5.3 Infrared Spectroscopy Analysis

[0187] The FT-IR results of CPSP-3 are as Figure 18 shown. The strong absorption peak near 3420.57 cm -1 is attributed to the stretching vibration of O-H. The absorption peak near 2930.19 cm -1 is for the stretching vibration of C-H. The absorption peak at 1643.82 cm -1 is for the bending vibration of O-H. The absorption peaks at 1412.24 cm -1 and 1385.31 cm -1 are for the bending vibration of C-H. The absorption peaks at 1074.78 cm -1 and 1041.64 cm -1 are for the C-O stretching vibration in the pyranose ring. The absorption peak at 864.38 cm -1 is attributed to the α-type glycosidic bond. The absorption peak at 778.49 cm -1 is for the symmetric stretching vibration of C-O-C in the pyran ring.

[0188] 2.5.4 Particle Size and Triple Helix Structure

[0189] The particle size of CPSP-3 is as Figure 19 shown in A. The average particle size of CPSP-3 is 1052.32 nm.

[0190] The determination results of the triple helix structure of CPSP-3 Figure 19 are shown in B. As the concentration of sodium hydroxide increases, the maximum absorption wavelength of the mixture of CPSP-3 and congo red does not show a significant red shift. Therefore, it is judged that CPSP-3 does not contain a triple helix structure.

[0191] 2.6 Structural Analysis of the Purified Polysaccharide CPSP-4 from Codonopsis pilosula Seeds

[0192] 2.6.1 Molecular Weight Determination and Ultraviolet Spectroscopy Analysis

[0193] The molecular weight determination results of CPSP-4 are as Figure 20 shown in A. CPSP-4 shows two absorption peaks, with molecular weights of 2056454 Da and 12929 Da, respectively, and the proportions are 10.6% and 89.40%, indicating that CPSP-4 is not a homogeneous polysaccharide.

[0194] The ultraviolet absorption spectrum of CPSP-4 is as Figure 20As shown in Figure B, CPSP-4 has no obvious absorption peaks at 260 nm and 280 nm, indicating that it contains almost no nucleic acids and proteins.

[0195] 2.6.2 Monosaccharide composition analysis

[0196] The monosaccharide composition results of CPSP-4 are as Figure 21 shown. CPSP-4 is composed of Ara, GalA, Gal, Xyl, Rha, GlcA, Glc, and Man in a ratio of 37.7:19.7:17.4:13.7:5.9:2.6:2.0:1.0.

[0197] 2.6.3 Infrared spectroscopy analysis

[0198] The FT-IR results of CPSP-4 are as Figure 22 shown. The strong absorption peak near 3410.48 cm -1 is attributed to the stretching vibration of O-H. The absorption peak near 2930.73 cm -1 is the stretching vibration of C-H. The absorption peaks at 1740.08 cm -1 and 1420.09 cm -1 are the C=O stretching vibrations in the carboxyl group, indicating that CPSP-4 contains galacturonic acid. The absorption peak at 1640.33 cm -1 is attributed to the stretching vibration of C=O in the amide group. The absorption peak at 1243.73 cm -1 is the deformation vibration of O-H, which indicates the presence of rhamnose. The absorption peaks at 1091.31 cm -1 , 1042.64 cm -1 and 770.52 cm -1 are the characteristic peaks of pyranose. The absorption peaks at 866.28 cm -1 and 817.02 cm -1 are attributed to the α-glycosidic bond configuration. The absorption peak at 614.44 cm -1 is attributed to the symmetric stretching vibration of the furan ring.

[0199] 2.6.4 Particle size and triple helix structure

[0200] The particle size of CPSP-4 is as Figure 23 shown in Figure A. The average particle size of CPSP-4 is 1155.75 nm.

[0201] The determination of the triple helix structure of CPSP-4 is as Figure 23 shown in Figure B. As the concentration of sodium hydroxide increases, the change trend of the maximum absorption wavelength of CPSP-4 and the congo red control is similar, and it is judged that it does not contain a triple helix structure.

[0202] 2.7 Structural Analysis of Codonopsis pilosula Seed Purified Polysaccharide CPSP-5

[0203] 2.7.1 Molecular Weight Determination and Ultraviolet Spectrum Analysis

[0204] The molecular weight determination results of CPSP-5 are as Figure 24 shown in Figure A. CPSP-5 presents two absorption peaks with molecular weights of 206,870 Da and 12,389 Da respectively, accounting for 4.31% and 95.69%. The purity of the second absorption peak is relatively high, indicating that CPSP-5 is relatively homogeneous.

[0205] The ultraviolet absorption spectrum results of CPSP-5 are as Figure 24 shown in Figure B. CPSP-5 has no obvious absorption peaks at 260 nm and 280 nm, indicating that it contains almost no nucleic acids and proteins.

[0206] 2.7.2 Monosaccharide Composition Analysis

[0207] The monosaccharide composition results of CPSP-5 are as Figure 25 shown. CPSP-5 is composed of Ara, Rha, GalA, Xyl, Gal, and Man in a ratio of 28.5:24.5:22.4:12.1:10.6:1.9.

[0208] 2.7.3 Infrared Spectrum Analysis

[0209] The FT-IR results of CPSP-5 are as Figure 26 shown. The strong absorption peak near 3418.84 cm -1 is attributed to the stretching vibration of O-H. The absorption peak near 2923.51 cm -1 is the stretching vibration of C-H. The absorption peak at 1740.08 cm -1 is the C=O stretching vibration in the carboxyl group, indicating that CPSP-5 contains galacturonic acid. The absorption peak at 1629.36 cm -1 is the symmetric stretching vibration of carboxyl C=O. The absorption peak at 1419.29 cm -1 is the bending vibration of C-H. The absorption peak at 1244.12 cm -1 is the bending vibration of C-H, which indicates the presence of uronic acid in CPSP-5. The absorption peaks near 1097 - 1041 cm -1 are the characteristic peaks of the pyranose ring. The absorption peak at 894.46 cm -1 is attributed to the β-glycosidic bond. The absorption peak at 862.28 cm -1 is the characteristic absorption peak of the a-type pyranose. The absorption peak at 832.92 cm -1 is the characteristic absorption peak of the α-type glycosidic bond. The absorption peak at 770.41 cm -1 is the characteristic absorption peak of the pyranose.

[0210] 2.7.4 Particle Size and Triple Helix Structure

[0211] The particle size of CPSP-5 is as Figure 27 shown in A. The average particle size of CPSP-5 is 1438.87 nm.

[0212] The determination result of the triple helix structure of CPSP-5 is as Figure 27 shown in B. With the increase of sodium hydroxide concentration, the maximum absorption wavelength of the mixed solution of CPSP-5 and congo red did not show a significant red shift, and the change trend was similar to that of the congo red control. It was judged that CPSP-5 did not contain a triple helix structure.

[0213] 3 Summary

[0214] Five purified components were isolated from the crude polysaccharide of Codonopsis pilosula seeds: CPSP-1, CPSP-2, CPSP-3, CPSP-4, CPSP-5. The yield of CPSP-2 was relatively low, so no subsequent research was carried out. The remaining four components had infrared characteristic absorption peaks of polysaccharides, were mainly composed of arabinose, and the uronic acid content increased in turn. Among them, CPSP-5 was a homogeneous polysaccharide with relatively high purity.

[0215] The beneficial effects of the present invention are demonstrated by the following ergonomics tests.

[0216] Experimental Example 1 In vitro Biological Activity of Polysaccharides from Codonopsis pilosula Seeds

[0217] 1 Materials

[0218] 1.1 Main Experimental Materials

[0219] The crude polysaccharides of Codonopsis pilosula seeds in the first part (CPSP-0, CPSP) and the purified polysaccharide components in the second part (CPSP-1, CPSP-3, CPSP-4, CPSP-5).

[0220] 1.2 Experimental Methods

[0221] 1.2.1 In vitro Antioxidant Activity of Polysaccharides from Codonopsis pilosula Seeds

[0222] 1.2.1.1 DPPH Free Radical Scavenging Activity

[0223] The purple solution of DPPH free radical has a strong absorption at 517 nm. The antioxidant scavenges the free radical and makes the solution fade, and the degree of fading is related to the scavenging degree. Using L-ascorbic acid (VC) as a control, it was determined with reference to the method of Liu et al. and appropriately modified.

[0224] (1) Solution preparation: Weigh 0.9858 mg of DPPH, dissolve it in absolute ethanol, and make up the volume to 25 mL to obtain a 0.1 mM DPPH solution, which is stored in the dark at 4 °C.

[0225] (2) Scavenging reaction: Add 400 μL of DPPH solution to 200 μL of polysaccharide solutions with different concentrations (0.5, 1, 2, 3, 4, 5 mg / mL), mix well by shaking, react in the dark at room temperature for 30 min, zero with pure water, and measure the absorbance at 517 nm. Calculate the DPPH radical scavenging rate according to the following formula:

[0226]

[0227] A1, A2, and A3 are the absorbances of the radical scavenging group (polysaccharide, VC), the sample background absorption group (pure water instead of DPPH solution), and the blank group (pure water instead of the sample), respectively.

[0228] 1.2.1.2 ABTS radical scavenging activity

[0229] The green ABTS+· solution formed by the reaction of ABTS and potassium persulfate (K2S2O8) has a strong absorption at 734 nm. Antioxidants scavenging free radicals can fade the solution, and the degree of fading is related to the scavenging degree. Using VC as a control, it was measured with reference to the method of Erel et al. and appropriately modified.

[0230] (1) Solution preparation: Weigh 40.6 mg of ABTS diammonium salt, dissolve it in water, and make up the volume to 10 mL to obtain a 7.4 mM ABTS diammonium salt solution. Weigh 7 mg of K2S2O8, dissolve it in water, and make up the volume to 10 mL to obtain a 2.6 mM K2S2O8 solution. Take equal volumes of the ABTS diammonium salt and K2S2O8 solutions, mix them, and let them stand at room temperature in the dark for 12 h. Dilute with water by 10 - 20 times until the absorbance is 0.70 ± 0.02 to obtain the ABTS+· solution, which is stored in the dark at 4 °C.

[0231] (2) Scavenging reaction: Add 800 μL of ABTS+· solution to 200 μL of polysaccharide solutions with different concentrations (0.5, 1, 2, 3, 4, 5 mg / mL), mix well by shaking, react in the dark at room temperature for 6 min, measure the absorbance at 734 nm, and zero with pure water. Calculate the ABTS+· radical scavenging rate according to the following formula:

[0232]

[0233] A1, A2, and A3 are the absorbances of the radical scavenging group (polysaccharide, VC), the sample background absorption group (pure water instead of ABTS radical solution), and the blank group (pure water instead of the sample), respectively.

[0234] 1.2.1.3 OH radical scavenging activity

[0235] The purple solution formed by the reaction of ·OH generated by the Fenton reaction with salicylic acid has a strong absorption at 510 nm. The antioxidant scavenges free radicals and causes the solution to fade. The degree of fading is related to the scavenging degree. Using VC as a control, it was measured with reference to the method of Zhao Honghong et al. and appropriately modified.

[0236] (1) Solution preparation: ① Weigh 62.15 mg of salicylic acid, dissolve it with absolute ethanol and make up the volume to 50 mL to obtain a 9 mM salicylic acid solution. ② Weigh 125 mg of FeSO4·7H2O, dissolve it with water and make up the volume to 50 mL to obtain a 9 mM ferrous sulfate solution. ③ Weigh 249.35 mg of 6% H2O2, make up the volume to 50 mL with water to obtain an 8.8 mM hydrogen peroxide solution. All the above solutions are stored in the dark at 4 °C.

[0237] (2) Scavenging reaction: Add 200 μL of FeSO4, H2O2, and salicylic acid solutions to 200 μL of polysaccharide solutions with different concentrations (0.5, 1, 2, 3, 4, 5 mg / mL) respectively, shake well, react in the dark at 37 °C for 30 min, measure the absorbance at 560 nm, and zero with pure water. Calculate the OH free radical scavenging rate according to the following formula:

[0238]

[0239] A1, A2, and A3 are the absorbances of the free radical scavenging group (polysaccharide, VC), the sample background absorption group (pure water instead of FeSO4, H2O2, salicylic acid), and the blank group (pure water instead of the sample) respectively.

[0240] 1.2.1.4 O2 - Free radical scavenging activity

[0241] The blue ·O2 generated by the NADH-PMS-NBT system - The solution has a strong absorption at 560 nm. The antioxidant can scavenge free radicals and make the system clear. The degree of clarity is related to the scavenging degree. Using VC as a control, it was measured with reference to the method of Bi et al. and appropriately modified

[132] .

[0242] (1) Solution preparation: ① Weigh 12.25 mg of NBT, dissolve it with water and make up the volume to 50 mL to obtain a 0.30 mM NBT solution. ② Weigh 33.2 mg of NADH, dissolve it with water and make up the volume to 50 mL to obtain a 0.936 mM NADH solution. ③ Weigh 1.85 mg of PMS, dissolve it with water and make up the volume to 50 mL to obtain a 0.12 mM PMS solution. All the above solutions are stored in the dark at 4 °C.

[0243] (2) Scavenging reaction: Add 200 μL of NBT, NADH, and PMS solutions to 200 μL of polysaccharide solutions with different concentrations (0.5, 1, 2, 3, 4, 5 mg / mL) respectively, mix well by shaking, react in the dark at 25 °C for 5 min, measure the absorbance at 560 nm, and zero with pure water. Calculate the O2 - Free radical scavenging rate:

[0244]

[0245] A1, A2, and A3 are the absorbances of the free radical scavenging group (polysaccharide, VC), the sample background absorption group (pure water instead of NBT, NADH, PMS), and the blank group (pure water instead of polysaccharide) respectively.

[0246] 1.2.1.5 Determination of reducing power

[0247] After the antioxidant reacts with potassium ferricyanide to form Fe 2+ and reacts with ferric chloride, the reaction solution has a strong absorption at 700 nm, and the degree of absorption is related to the strength of the antioxidant. Using VC as a control, it was determined with reference to Li's method and appropriately modified.

[0248] (1) Solution preparation: ① Weigh 1.0 g of potassium ferricyanide, dissolve it in water and make up the volume to 100 mL to obtain a 1.0% potassium ferricyanide solution. ② Weigh 3.58 g of Na2HPO4·12H2O, dissolve it in water and make up the volume to 50 mL, weigh 3.12 g of NaH2PO4·2H2O, dissolve it in water and make up the volume to 100 mL; Pipette 50 mL of Na2HPO4 and 83.35 mL of NaH2PO4 and mix (3:5) to obtain a 0.2 M PBS solution (pH 6.6). ③ Weigh 10 g of TCA, dissolve it in water to 100 mL to obtain a 10% TCA solution. ④ Weigh 50 mg of FeCl3, dissolve it in water and make up the volume to 50 mL to obtain a 0.1% FeCl3 solution. The above solutions are all stored in the dark at 4 °C.

[0249] (2) Reduction reaction: Add 500 μL of potassium ferricyanide and PBS solution to 200 μL of polysaccharide solutions with different concentrations (0.5, 1, 2, 3, 4, 5 mg / mL) respectively, mix well by shaking, react in a water bath at 50 °C for 20 min, add 500 μL of TCA solution to terminate the reaction after cooling, and finally add 100 μL of FeCl3 solution, zero with pure water, and measure the absorbance at 700 nm. Calculate the reducing power according to the following formula:

[0250]

[0251] A1 and A0 are the absorbances of the reduction group (polysaccharide, VC) and the blank group (pure water instead of polysaccharide) respectively.

[0252] 1.2.2 In vitro hypoglycemic activity of Codonopsis pilosula seeds polysaccharide

[0253] 1.2.2.1 Determination of α-amylase inhibitory activity

[0254] The reducing sugar produced by α-amylase catalyzing starch can reduce DNS to form a red-brown substance, which has absorption at 540 nm, and the inhibitor can inhibit this process. Using acarbose as a control, it was determined by referring to the method of Deng et al. and making appropriate modifications

[133] 。

[0255] (1) Solution preparation: ① 0.1M phosphate buffer PBS (pH 6.9): Weigh 8.95 g of Na2HPO4·12H2O, dissolve it in water and make up the volume to 250 mL. Weigh 3.9 g of NaH2PO4·2H2O, dissolve it in water and make up the volume to 250 mL; Pipette 220 mL of Na2HPO4 and 180 mL of NaH2PO4, mix them and add 156 mg of NaCl. ② 10 mg / mL soluble starch solution: Weigh 0.5 g of starch, dissolve it with boiling PBS, boil for 20 min, cool and make up the volume to 50 mL. ③ 1 u / mL α-amylase solution: Weigh 10 mg of α-amylase (4000 u / g) and dissolve it in 400 mL of PBS. ④ 3,5-dinitrosalicylic acid: Weigh 3.25 g of DNS, dissolve it in a small amount of water, then transfer it to a 500 mL brown volumetric flask containing 162.5 mL of 2M NaOH, add 22.5 g of glycerol, shake well, make up the volume to 500 mL with water, and use it after one week

[0256] (2) Inhibitory reaction: Add 200 μL of α-amylase solution, 400 μL of PBS to 200 μL of polysaccharide solutions with different concentrations (0.5, 1, 2, 3, 4, 5 mg / mL) respectively, react at 37 °C for 10 min, then add 200 μL of starch solution, react at 37 °C for 5 min, add 2 mL of DNS, boil for 5 min for color development, cool in an ice-water bath, add 5 mL of water, mix well and measure the absorbance at 540 nm. Calculate the inhibitory ability according to the following formula:

[0257]

[0258] A1, A2 and A3 are the absorbances of the inhibition group (polysaccharide, acarbose), the sample background absorption group (PBS instead of α-amylase) and the blank group (pure water instead of polysaccharide) respectively

[0259] 1.2.2.2 Determination of α-glucosidase inhibitory activity

[0260] p-Nitrophenol produced by the hydrolysis of pNPG by α-glucosidase has light absorption at a wavelength of 405 nm. The addition of enzyme inhibitors can reduce the formation of p-nitrophenol. Using acarbose as a control, it was measured according to the method of Peng et al. with appropriate modifications.

[0261] (1) Solution preparation: ① 0.1M PBS (pH 6.8): Weigh 3.58 g of Na2HPO4·12H2O, dissolve it in water and make up the volume to 100 mL. Weigh 1.56 g of NaH2PO4·2H2O, dissolve it in water and make up the volume to 100 mL; Pipette 51 mL of Na2HPO4 and 49 mL of NaH2PO4 and mix them to obtain. ② 0.2M Na2CO3: Weigh 2.1198 g of anhydrous sodium carbonate, dissolve it in water and make up the volume to 100 mL. ③ 0.04 u / mL α-glucosidase: Weigh 2 mg of α-glucosidase (50 u / mg), dissolve it in 5 ml of PBS to obtain a stock solution of 20 u / mL, and then pipette 100 μL of the stock solution and make up the volume to 50 mL with PBS. ④ 0.5 mM PNPG: Weigh 7.53 mg of PNPG, dissolve it in PBS and make up the volume to 50 mL.

[0262] (2) Inhibition reaction: Add 200 μL of α-glucosidase solution to 200 μL of polysaccharide solutions with different concentrations (0.5, 1, 2, 3, 4, 5 mg / mL), mix well by shaking, react at 37 °C for 10 min, then add 100 μL of PNPG, react at 37 °C for 30 min, then add 400 μL of sodium carbonate to terminate the reaction, let it stand at room temperature for 5 min, zero with pure water, and measure the absorbance at 405 nm. Calculate the reducing ability according to the following formula:

[0263]

[0264] A1, A2, and A3 are the absorbances of the inhibition group (polysaccharide, acarbose), the sample background absorption group (PBS instead of α-glucosidase), and the blank group (pure water instead of polysaccharide), respectively.

[0265] 1.2.3 In vitro immunomodulatory activity of Codonopsis pilosula seed polysaccharide

[0266] 1.2.3.1 RAW 264.7 cell culture

[0267] (1) Cell resuscitation: After thawing the cryopreserved cells in a 37 °C water bath for 1 min, pipette 1 mL of the cell suspension into a 10 mL centrifuge tube, centrifuge at 1000 rpm / min for 5 min, discard the supernatant, add 8 mL of complete medium (89% DMEM basal medium, 10% FBS, 1% double antibody) to resuspend, gently blow and mix well, then transfer to a cell culture dish (diameter 10 cm), shake well and culture for 24 h (37 °C, 5% CO2).

[0268] (2) Cell passage: Discard the supernatant in the culture dish, gently wash twice with PBS, resuspend with 2 mL of medium, mix well and divide into 2 culture dishes, then add 7 mL of medium to each dish and culture for 24 h. The third-generation cells were used for subsequent experiments.

[0269] (3) Cell cryopreservation: After washing the cells with PBS, add 2 mL of cell cryopreservation solution (90% FBS, 10% DMSO), pipette and mix well, then aliquot into 2 cryotubes, place in a cell programmable cryopreservation box, incubate at -80 °C overnight, and transfer to liquid nitrogen the next day.

[0270] 1.2.3.2 Cytotoxicity assay

[0271] Determined by the MTT method. Except for the blank group, inoculate 2×10 4 cells per well in a 96-well plate, and the volume of each well is 100 μL. After culturing for 24 h, discard the supernatant. The blank group and the zero-adjustment group add complete medium, the positive control group adds 2.5 μg / mL LPS solution (prepared with complete medium), and the sample group adds 100, 200, 400 μg / mL polysaccharide solution (prepared with complete medium). Each group has 5 replicates and the volume of each well is 100 μL. After culturing for 24 h, add 10 μL of MTT (5 mg / mL, prepared with PBS) to the four groups of wells in the dark. After culturing for 4 h, carefully remove the medium, add 150 μL of DMSO to each well and shake for 10 min, measure the absorbance at 490 nm, and calculate the proliferation activity according to the following formula:

[0272]

[0273] A1, A2, and A3 are the absorbances of the treatment groups (positive control, experiment), the zero-adjustment group, and the blank group, respectively.

[0274] 1.2.3.3 Determination of NO release

[0275] Inoculate 2×10 5 cells per well in a 12-well plate, and the volume of each well is 2 mL. After culturing for 24 h, discard the supernatant. The positive control group adds 2.5 μg / mL LPS solution, the sample group adds 100, 200, 400 μg / mL polysaccharide solution, and the blank group adds complete medium. Each group has 3 replicates and 2 mL per well. After culturing for 24 h, measure the NO content according to the method of the NO kit.

[0276] 1.3 Data processing

[0277] Experimental data are expressed as mean ± standard deviation, and plotted and analyzed using Origin 2022 software and Graphpad Prism 8.0 software.

[0278] 2 Results

[0279] 2.1 In vitro antioxidant activity of Codonopsis pilosula seed polysaccharides

[0280] (1) Results of DPPH radical scavenging: As Figure 28 shown, the scavenging activity of Codonopsis pilosula seed polysaccharides on DPPH radicals increased with the increase of concentration. Among them, CPSP, CPSP-0 and CPSP-5 were in the top three and the highest scavenging rates all exceeded 60%. Their IC 50 values were 2.123, 1.327, and 1.331 mg / mL respectively.

[0281] (2) Results of ABTS radical scavenging: All 6 polysaccharide components had high scavenging effects on ABTS radicals ( Figure 29 ). At a concentration of 3 mg / mL, the scavenging rates all exceeded 90%. The IC 50 values of CPSP-0, CPSP, CPSP-1, CPSP-3, CPSP-4, and CPSP-5 were 0.358, 0.727, 0.501, 1.025, 0.875, and 0.799 mg / mL respectively. From the IC 50 values, CPSP-0, CPSP-1, and CPSP-5 were in the top three in terms of scavenging activity.

[0282] (3) Results of OH radical scavenging: As Figure 30 shown, the scavenging rates of most polysaccharide components on OH radicals were relatively low. CPSP-0 showed a good scavenging rate (up to 83.79% at most), followed by CPSP-5 (up to 46.81% at most). Their IC 50 values were 2.272 and 6.03 mg / mL.

[0283] (4) Results of O2 - radical scavenging: As Figure 31 shown, each component of Codonopsis pilosula seed polysaccharides had a certain scavenging ability on O2 - radicals. Among them, CPSP-0 had the best scavenging activity, with the highest inhibition rate of 71.09%. Its IC 50 value was 3.623 mg / mL.

[0284] (5) Determination of reducing power: As Figure 32 shown, all 6 polysaccharide components had high reducing power. Among them, the reducing power of CPSP-0 at a concentration of 5 mg / mL was close to that of VC. Its IC 50 value was 0.331 mg / mL.

[0285] 2.2 In vitro hypoglycemic activity of Codonopsis pilosula seed polysaccharides

[0286] At concentrations of 0.5 - 5 mg / mL, the 6 polysaccharide components had activation and inhibition effects on α-amylase activity of no more than 20% respectively (Figure 33 A); however, they have a good inhibitory effect on α-glucosidase activity ( Figure 33 B), and the inhibition rates all exceed 50%. Among them, CPSP-4, CPSP-5, and CPSP-1 rank in the top three, and the IC 50 values are 1.893, 2.282, and 3.015 mg / mL respectively.

[0287] 2.3 Immunomodulatory activity of Codonopsis pilosula seed polysaccharide in vitro

[0288] 2.3.1 Cytotoxicity assay

[0289] The cytotoxic effect of Codonopsis pilosula seed polysaccharide on RAW 264.7 cells is as Figure 34 shown. The 6 polysaccharide components have different degrees of inhibitory effects on cell activity at different concentrations; however, the CPSP-5 component does not significantly affect cell activity at 100 and 200 μg / mL.

[0290] 2.3.2 NO release amount

[0291] The results of the effect of Codonopsis pilosula seed polysaccharide on NO secretion by RAW 264.7 cells are as Figure 35 shown. The 6 components can promote NO release by cells at different concentrations.

[0292] 3 Summary

[0293] This chapter studied the in vitro antioxidant, hypoglycemic, and immunomodulatory activities of crude polysaccharides (CPSP-0, CPSP) and their purified components (CPSP-1, CPSP-3, CPSP-4, CPSP-5) of Codonopsis pilosula seeds. The results showed that the 6 components have different degrees of antioxidant capacity and inhibitory activity against α-glucosidase. Among them, CPSP-0 and CPSP-5 have better antioxidant activity, and CPSP-4 and CPSP-5 have the best hypoglycemic activity. In addition, CPSP-5 does not affect the activity of RAW 264.7 cells at a certain concentration and promotes NO secretion by cells. The above results indicate that CPSP-5 has good biological activity.

[0294] Experimental Example 2 In vivo immunomodulatory activity of CPSP-5

[0295] 1 Materials and methods

[0296] 1.1 Main experimental materials

[0297] Codonopsis pilosula seed polysaccharide CPSP-5. SPF-grade 5-week-old male Balb / C mice, weighing 20±2 g, were provided and raised by the Experimental Animal Center of Zunyi Medical University. The animal production license of this center is SCXK(Qian)2021-0002. This experiment complied with the review and approval requirements of the Experimental Animal Ethics Committee of Zunyi Medical University (No. ZMU21-2412-011).

[0298] 1.2 Experimental methods

[0299] 1.2.1 Mouse modeling and sample collection

[0300] The mice were cultured at a temperature of 23±2 °C and a humidity of 50%±5%, with 12 hours of light and dark alternating and free access to food and water. Sixty mice were randomly divided into 6 groups: normal control group (NC), model group (CTX), positive control group (LMS), low, medium, and high-dose polysaccharide groups (LCPSP-5, MCPSP-5, HCPSP-5). After 3 days of adaptive culture, on the 4th to 6th days, the mice in the NG group were intraperitoneally injected with normal saline, and the mice in the remaining groups were injected with 80 mg / kg of cyclophosphamide to construct an immunosuppressive model. On the 7th to 13th days, the mice in the NG group and the CTX group were gavaged with pure water, and the mice in the LMS group, LCPSP-5 group, MCPSP-5 group, and HCPSP-5 group were gavaged with 40 mg / kg of LMS, 100 mg / kg of CPSP-5, 200 mg / kg of CPSP-5, and 400 mg / kg of CPSP-5, respectively. After fasting for 12 h, on the 14th day, the feces, serum, spleen, and thymus of the mice were collected and stored at -80 °C. The animal experiment design is as Figure 36 shown.

[0301] 1.2.2 Changes in mouse body weight, thymus index, and spleen index

[0302] The body weight of the mice was recorded daily, and the status of the mice was observed. After the thymus and spleen of the mice were taken out, they were rinsed with normal saline, blotted dry with filter paper, and weighed. The index changes were calculated according to the following formula:

[0303]

[0304] m1 is the thymus / spleen mass, and m2 is the mouse weight.

[0305] 1.2.3 Morphological analysis of the spleen

[0306] The spleens of the mice in the NC group, CTX group, LMS group, and MCPSP-5 group were fixed with 10% formaldehyde and sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for hematoxylin-eosin (HE) staining.

[0307] 1.2.4 Intestinal flora analysis

[0308] Fecal pellets of mice in the NC group, CTX group, LMS group, and MCPSP-5 group were collected and placed in sterile centrifuge tubes, and then sent to Shanghai Majorbio Co., Ltd. under dry ice environment for high-throughput DNA sequencing analysis. The general method is as follows. According to The total genomic DNA of the microbial community was extracted by the method of soil DNAKit (Omega Biotek, Norcross, GA, USA) kit. The V3-V4 variable region of the 16S rRNA gene was amplified by PCR using the upstream primer 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and the downstream primer 806R (5’-GGACTAC HVGGGTWTCTAAT-3’). The PCR products were recovered by 2% agarose gel and purified using a DNA gel recovery and purification kit. After purification, quantification was performed using Qubit 4.0 (Thermo, USA), and the purified amplicons were sequenced on the Illumina Nextseq2000 platform.

[0309] 2 Results

[0310] 2.1 Changes in mouse body weight, thymus index, and spleen index

[0311] The change in body weight is as shown in Figure 37 A. During the adaptation period from day 1 to day 3, the body weights of mice in each group generally showed an increasing trend, and a decreasing trend after modeling; on day 12, compared with the NC group, the body weight of mice in the CTX group showed a significant difference, and there was no difference in the other groups. The changes in thymus index and spleen index are as shown in Figure 37 B, Figure 37 C. The thymus index and spleen index of the CTX group were significantly decreased compared with the NC group (P<0.05), and there was no significant difference between the LMS group, CPSP-5 group and the NC group. This indicates that LMS and CPSP-5 can regulate the body weight, thymus index and spleen index of mice to a certain extent and improve the physiological conditions of mice caused by CTX.

[0312] 2.2 Morphological analysis of the spleen

[0313] The spleen is the largest peripheral lymph organ in the human body and also the main site of immune response. Its morphological changes can directly reflect the immune state. The results of HE staining are as shown in Figure 38 . The spleen of mice in the NC group developed well, with normal distribution of red pulp and white pulp and clear boundaries ( Figure 38 A). Compared with the NC group, the splenocytes of mice in the CTX group were arranged loosely and the red pulp was greatly reduced ( Figure 38 B, red arrow), indicating that CTX significantly damaged the normal structure of the spleen. The white pulp T lymphocytes in the spleen of mice in the LMS group were arranged tightly and the number increased ( Figure 38C (black arrow), suggesting that LMS may enhance immune function by promoting the proliferation of T lymphocytes. Although the number of red pulp and white pulp in the mice of the MCPSP-5 polysaccharide group was lower than that of the NC group, it was higher than that of the CTX group, indicating that CPSP-5 may alleviate the spleen injury caused by CTX by protecting the spleen structure ( Figure 38 D).

[0314] 2.3 Intestinal flora analysis

[0315] ASV taxonomic analysis. A total of 18 fecal samples, 6 in each of the NC group, CTX group, and MCPSP-5 group, were sequenced, and a total of 1,093,828 raw single-end reads were obtained; after denoising and chimera removal of the original sequence reads, 58,526 effective high-resolution sequences and 7223 exact sequence variants ASVs were obtained. Figure 39 is the dilution curve of the intestinal microbiota at the ASVs level. The abscissa is the number of sequencing Reads, and the ordinate is the Sobs (i.e., observed species) index (39A), Shannon index ( Figure 39 B). As the number of sequences increased, the dilution curves of all samples tended to saturate, indicating that the sequencing data was reasonable and could be used for subsequent bioinformatics analysis.

[0316] Alpha diversity analysis. ACE ( Figure 40 A), Chao ( Figure 40 B) are indices for estimating species richness; Shannon ( Figure 40 C), Simpson ( Figure 40 D) are indices for measuring species diversity. Compared with the NC group, there were no significant differences in the ACE, Chao, Shannon, and Simpson indices of the CTX group and MCPSP-5 group, indicating that short-term administration of cyclophosphamide and MCPSP-5 did not affect the Alpha diversity of the intestinal microbiota.

[0317] Dysbiosis and Beta diversity analysis. Figure 41 A is the dysbiosis index map at the ASV taxonomic level. The ordinate is the intestinal flora dysbiosis index (MDI), and the larger the MDI value, the greater the degree of flora disorder. Taking the NC group as the healthy group, the intestinal flora of the mice in the CTX group and MCPSP-5 group were significantly dysbiotic (P<0.01), but the MDI value of the MCPSP-5 group was lower than that of the CTX group, indicating that CPSP could alleviate intestinal flora dysbiosis. The principal coordinate analysis (PCoA) based on the abund_jaccard distance algorithm is as Figure 41As shown in Figure B, there were significant differences in the microbial community structures among different treatment groups (CTX, NC, MCPSP5) (P = 0.038); the distribution of the CTX group was more concentrated on PC1 and PC2, and the microbial community structure was more consistent; while the distributions of the NC group and the MCPSP5 group in the figure were more dispersed, and the variation in the microbial community structure was larger.

[0318] For the community composition analysis, the microbial community composition and relative abundance of fecal samples were analyzed at the phylum and genus levels respectively. Figure 42 Figure A shows the distribution at the phylum level of microorganisms. In all groups, the relative abundances of Bacteroidota and Firmicutes were relatively high, which were the main components of the gut microbiota and were crucial for maintaining gut health. Compared with the NC group, the relative abundance of Bacteroidota in the CTX group decreased; compared with the CTX group, the relative abundance of Bacteroidota in the MCPSP-5 group increased. The ratio of Firmicutes / Bacteroidota (F / B) was used to evaluate the balance of the gut microbiota. The F / B value of the CTX group increased, causing gut microbiota imbalance, while CPSP-5 could reverse this trend to a certain extent.

[0319] Figure 42 Figure B shows the distribution at the genus level of microorganisms. In all groups, norank_f__Muribaculaceae, Lachnospiraceae_NK4A136_group, and Lactobacillus were the dominant bacterial species. Compared with the NC group, the relative abundance of norank_f__Muribaculaceae in the CTX group decreased, while the relative abundances of Lachnospiraceae_NK4A136_group and Lactobacillus increased. Compared with the CTX group, the relative abundance of norank_f__Muribaculaceae in the MCPSP-5 group increased.

[0320] 3 Summary

[0321] In this chapter, through a CTX-induced immunosuppressed mouse model, the in vivo immunomodulatory activity of Codonopsis pilosula seed polysaccharide CPSP-5 was explored. The results showed that CPSP-5 could alleviate the weight loss caused by CTX, protect the structure and function of the immune organ (spleen), and improve gut microbiota dysbiosis by regulating the composition of the gut microbiota, and had immunomodulatory activity.

[0322] This article took Codonopsis pilosula seed polysaccharide as the research object, first explored its structure and biological activities such as antioxidant and hypoglycemic effects, and then combined with gut microbiomics to investigate the effects of the purified polysaccharide fraction on immunosuppressed mice. The specific conclusions are summarized as follows:

[0323] (1) Codonopsis pilosula seeds were defatted, subjected to water extraction and alcohol precipitation, and deproteinized by Sevag to obtain crude polysaccharides CPSP-0 and CPSP respectively. They contain a certain amount of protein and complex monosaccharide compositions and exhibit certain biological activities. Among them, CPSP-0 has higher antioxidant activity.

[0324] (2) After CPSP was separated and purified by DEAE-50 and Sephadex G-150, purified polysaccharides CPSP-1, CPSP-2, CPSP-3, CPSP-4 and CPSP-5 were obtained. Among them, CPSP-2 was not subjected to subsequent research due to low yield. CPSP-1, CPSP-3, and CPSP-4 are heterogeneous polysaccharides but have certain antioxidant and enzyme inhibitory activities.

[0325] (3) CPSP-5 is a polysaccharide with high purity and high activity, with a molecular weight of 12389 Da, composed of Ara, Rha, GalA, Xyl, Gal, and Man in a ratio of 28.5:24.5:22.4:12.1:10.6:1.9, and has good antioxidant and α-glucosidase inhibitory activities. In addition, CPSP-5 does not significantly affect the activity of RAW264.7 at a certain concentration, promotes its release of NO, and enhances cellular immunity. In vivo animal experiments showed that CPSP-5 can improve the changes in body weight, thymus index, and spleen index of immunosuppressed mice, regulate the composition of intestinal flora, and play an immunomodulatory role.

Claims

1. Use of Codonopsis pilosula seeds or extracts thereof in the preparation of drugs having immunomodulatory or hypoglycemic effects or health foods that help enhance immunity.

2. The use according to claim 1, characterized in that: The codonopsis extract is codonopsis seed polysaccharide.

3. A crude polysaccharide from Codonopsis pilosula seeds, characterized in that: It uses water extraction and alcohol precipitation method to extract crude polysaccharide from Codonopsis pilosula seeds and Sevag method to purify it, which contains Ara, Gal, Xyl, GalA, Rha, Man, and the mass ratio is 45.6:18.9:11.9:9.3:7.7:6.7; Among them, the contents of total sugar, protein and uronic acid were 27.61±0.64%, 14.68±0.17% and 15.03±0.33% respectively; the content of uronic acid was 14.65708%-15.26203%; the average particle size was 450.91nm, and the particle size was distributed in the range of 0 to 2000nm.

4. A method for preparing crude polysaccharide from Codonopsis pilosula seeds according to claim 3, characterized in that: It includes the steps: a. Pretreatment of Codonopsis seeds: Weigh Codonopsis powder and soak it in 95% ethanol overnight, heat it under reflux to defat it the next day, filter it, combine the filter residue, and dry it to obtain Codonopsis seed defatted powder; b. Extraction of crude polysaccharide (CPSP-0): weigh defatted powder and mix with water, heat and reflux water extraction, filter and collect the filtrate, concentrate under reduced pressure, slowly add 95% ethanol to the concentrate, stir evenly, place at 4°C, centrifuge at a speed of 4000rpm / min, wash the precipitate with acetone, ether and anhydrous ethanol twice in sequence, and dry at 60°C to obtain white powdery crude polysaccharide CPSP-0 (Codonopsis pilosula seedspolysaccharides); c. Preliminary purification of CPSP-0: weigh the dry product and add water to completely dissolve it, add Sevag reagent (chloroform: n-butanol = 4:1, v / v) to the dissolved solution, use a 5000 rpm / min mixer to shake for 5 minutes, centrifuge at 4000 rpm / min for 10 minutes, discard the lower layer of protein and organic mixed emulsion in the centrifuge tube, and recover the upper layer solution; repeat the above process 6 to 7 times until no obvious white protein precipitates; the upper layer polysaccharide solution is concentrated under reduced pressure, loaded into a dialysis bag 3000Da, dialyzed with running water for 72 hours, and freeze-dried to obtain the crude polysaccharide CPSP of Codonopsis pilosula seeds.

5. A purified polysaccharide from Codonopsis pilosula seeds, characterized in that: It is the neutral polysaccharide CPSP-1, acidic polysaccharide: CPSP-2, CPSP-3, CPSP-4, CPSP-5 obtained by separating the crude polysaccharide of Codonopsis pilosula seed as claimed in claim 3; Among them, CPSP-1 was mainly composed of Ara, Gal, Man, Xyl, Glc, and Rha in the ratio of 52.0:28.6:7.8:5.3:4.9; the total sugar, protein, and uronic acid contents were 23.35±0.25%, 8.25±0.07%, and 2.01±0.12%, respectively; The total sugar, protein, and uronic acid contents of CPSP-2 were 23.44±0.67%, 3.13±0.20%, and 6.55±0.17%, respectively; CPSP-3 was mainly composed of Ara, Gal, Xyl, Glc, GlcA, Man, GalA, and Rha in the ratio of 42.5:26.4:22.1:2.8:2.4:1.8:1.7:0.4; the contents of total sugar, protein, and uronic acid were 32.87±0.87%, 5.14±0.11%, and 8.62±0.10%, respectively; CPSP-4 was mainly composed of Ara, GalA, Gal, Xyl, Rha, GlcA, Glc, and Man in a ratio of 37.7:19.7:17.4:13.7:5.9:2.6:2.0:1.0; the contents of total sugar, protein, and uronic acid were 21.74±0.87%, 11.28±0.33%, and 24.22±0.13%, respectively; the average particle size was 1155.75nm; CPSP-5 is mainly composed of Ara, Rha, GalA, Xyl, Gal, and Man in the ratio of 28.5:24.5:22.4:12.1:10.6:1.9; the molecular weight of CPSP-5 is 12389Da; the contents of total sugar, protein, and uronic acid are 10.76±0.22%, 6.09±0.64%, and 36.71±0.18%, respectively; the average particle size is 1438.87nm.

6. The method for preparing the purified polysaccharide from Codonopsis pilosula seeds according to claim 5, characterized in that: The method adopts anion exchange column chromatography to separate and gel filtration column chromatography to purify the codonopsis seed crude polysaccharide described in claim 3.

7. The method for preparing purified polysaccharide from Codonopsis pilosula seeds according to claim 6, characterized in that: It includes the following steps: a. Swelling and activation of filler: Soak DEAE-52 in deionized water, changing the water every 2 hours; soak in 0.5M sodium hydroxide solution for 1 hour, wash with water until neutral; soak in 0.5M hydrochloric acid solution for 1 hour, wash with water until neutral; b. Column loading: Add 0.5M sodium hydroxide solution to the filler, add alkali solution to the bottom of the column, gently stir the filler and drain it into the chromatography column (specification: Ф5.5cm×50cm) in a circular manner, stir from bottom to top to make the filler evenly deposit at the bottom of the column, stop loading the column when it is deposited 5cm from the top of the chromatography column, wash the filler with water until it is neutral, and compact it overnight; c. Sample addition: Dissolve the crude polysaccharide from Codonopsis pilosula seeds in water, filter with a 0.45 μm water filter, turn on the constant flow pump to lower the liquid level to the same level as the filler, add the sample in a circular motion, turn off the pump when the liquid level is level with the filler, and allow the adsorption equilibrium to last for 0.5 h; c. Elution: neutral polysaccharides were eluted with water, and acidic polysaccharides were eluted with 0.05M, 0.1M, 0.2M and 0.4M NaCl solutions respectively; the elution flow rate was 3mL / min, 10mL / tube was used to collect 200 tubes; polysaccharides were detected every 3 tubes using the phenol-sulfuric acid method, and the elution curve was drawn with absorbance as the ordinate and the number of eluted tubes as the abscissa; The eluate was collected, concentrated under reduced pressure at 60°C, dialyzed against running water for 48 h, and freeze-dried to obtain the fractionated components of CPSP; d. Swelling: Sephadex G-150 dextran gel filler is expanded by adding pure water; e. Column loading: Add pure water to the bottom of the column, turn on the pump, gently stir the filler and add it to the chromatography column (specification: Φ1.6cm×90cm) in a circular manner, and stop loading the column when it is deposited 5cm from the upper end of the chromatography column. Rinse with water for 10 column volumes and compact overnight; f. Sample addition: Weigh each graded component and dissolve it in pure water, filter it with a 0.45μm water filter, turn on the constant flow pump to make the liquid level drop to the same level as the filler, add the sample in a circular manner, turn off the pump when the liquid level is flush with the filler, and allow the adsorption equilibrium to last for 0.5h; the flow rate is 0.4mL / min; g. Elution: Use water as eluent, the elution flow rate is 0.4 mL / min, collect 15 tubes at 10 mL / tube, use phenol-sulfuric acid method to detect polysaccharides in each tube, draw elution curve with absorbance as ordinate and number of elution tubes as abscissa, collect eluent according to each elution curve, concentrate under reduced pressure at 60°C, and dialyze in running water for 48 h, freeze-dried to obtain the fractionated purified fractions of CPSP.

8. Use of the crude polysaccharide from Codonopsis pilosula seeds according to claim 3 or the purified polysaccharide from Codonopsis pilosula seeds according to claim 5 in the preparation of drugs or health foods that contribute to anti-oxidation.

9. Use of the crude polysaccharide from Codonopsis pilosula seeds according to claim 3 or the purified polysaccharide from Codonopsis pilosula seeds according to claim 5 in the preparation of medicines or health foods having immunomodulatory effects.

10. Use of the crude polysaccharide from Codonopsis pilosula seeds according to claim 3 or the purified polysaccharide from Codonopsis pilosula seeds according to claim 5 in the preparation of a drug having a blood sugar lowering effect or a health food that helps maintain a healthy blood sugar level.

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