Platycodon grandiflorum polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical compounds, and particularly relates to platycodon grandiflorum polysaccharide as well as a preparation method and application thereof. The platycodon grandiflorum polysaccharide is named as PGP-1. According to the application, the platycodon grandiflorum polysaccharide PGP-1 is simply and efficiently extracted from platycodon grandiflorum roots through an improved extraction process, and pharmacological activity shows that the PGP-1 has remarkable immunomodulatory activity and a cancer cell proliferation inhibition effect, which prompts that the PGP-1 can be applied to preparation of immunomodulatory drugs or anti-tumor drugs, and has broad application prospects. And a new choice is provided for developing immunomodulatory drugs and anti-tumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical compounds, and particularly relates to a platycodon grandiflorum JACQ polysaccharide, a preparation method and application thereof. BACKGROUND

[0002] Polysaccharides are sugar chains combined by glycosidic bonds, and are a class of sugar substances with complex molecular structures. Polysaccharides widely exist in the cell membranes of plants and animals and microbial cell walls in nature, and are one of the four basic substances constituting life activities. In recent years, with the research of cell biology and molecular biology, it is found that polysaccharides have various pharmacological activities, including anti-tumor, immune system enhancement, anti-inflammatory, anticoagulation, antiviral, anti-aging and blood glucose lowering effects. Scholars have isolated more than one hundred polysaccharide compounds from nature, among which polysaccharides extracted from plants are particularly important. Due to the characteristics of various functions, few side effects and high safety, they have attracted the attention of scholars, and have become a research hotspot in the medical and food health industries.

[0003] Platycodon grandiflorum JACQ is a cold-resistant perennial herb, and its roots are used as a traditional Chinese medicinal material, which has the effects of relieving cough, clearing lung and pus discharge. In addition to medicinal use, platycodon grandiflorum JACQ is often pickled into salted vegetables or pickles for eating. Platycodon grandiflorum JACQ contains rich natural products and bioactive substances, such as amino acids, vitamins, flavonoids, saponins and the like. Polysaccharides, as one of the beneficial ingredients in platycodon grandiflorum JACQ, have not been fully developed and utilized. As a kind of bioactive substance with low toxicity and high safety, it has great market potential, and if it can be reasonably developed, it will be helpful for the utilization of platycodon grandiflorum JACQ resources.

[0004] Platycodon grandiflorum JACQ polysaccharide belongs to inulin-type polysaccharide. There are many methods for extracting, separating and purifying platycodon grandiflorum JACQ polysaccharide, and the types and structures of polysaccharides obtained by different preparation methods are not the same. Different structures show great differences in biological activities. There are problems such as low extraction rate, long time consumption, low purity, low polysaccharide activity and complicated preparation process in the prior art. Extraction of platycodon grandiflorum JACQ polysaccharide with specific biological activity has positive significance for the resource utilization of platycodon grandiflorum JACQ, and also has important significance for the industrialization development of platycodon grandiflorum JACQ polysaccharide. SUMMARY

[0005] In order to solve the above technical problems, the present application first provides a platycodon grandiflorum JACQ polysaccharide, which is named PGP-1, and the structural formula is shown as formula A: A, In the formula, m+n=9, and m and n are both >0.

[0006] The present application further provides a preparation method of the platycodon grandiflorum JACQ polysaccharide, comprising the following steps: S1. After the platycodon grandiflorum JACQ roots are dried, they are ground, extracted, centrifuged, rotary evaporated, alcohol precipitated, deproteinized and freeze-dried to obtain a crude polysaccharide extract; S2. Dissolve the polysaccharide crude extract into a solution of 15 mg / mL, and purify through a DEAE-52 cellulose ion exchange column; S3. Perform dialysis on the purified component using a 1000 Da filter membrane, collect the concentrated solution after dialysis, dry, and obtain the desired polysaccharide PGP-1 of Platycodon grandiflorum.

[0007] Preferably, the specific method of step S1 is as follows: S11. Grind the roots of Platycodon grandiflorum, soak the ground roots in acetone at room temperature for 1.5 days, and then dry at 45°C to obtain pretreated Platycodon grandiflorum powder; S12. Mix the Platycodon grandiflorum powder with purified water at a ratio of 1 g : (10-20) mL, and extract at a temperature of 60-100°C for 60-120 min, filter, repeat the extraction 1-3 times, combine the filtrates, centrifuge, and rotary evaporate; S13. Add 95% ethanol to the rotary evaporated liquid, adjust the final concentration of ethanol in the liquid to 75%, and centrifuge after overnight standing at 4°C. Dissolve the precipitate in water, and rotary evaporate at 50°C until the ethanol is evaporated to obtain a polysaccharide extract; S14. Add Sevage reagent to the polysaccharide extract at a volume ratio of 5:1, stir, and after standing and layering, take the third layer of solution at the bottom, rotary evaporate at 50°C to remove the Sevage reagent, and freeze-dry the concentrated solution to obtain a polysaccharide crude extract.

[0008] Preferably, mix the Platycodon grandiflorum powder with purified water at a ratio of 1 g : 15 mL, extract at a temperature of 75°C for 100 min, filter, repeat the extraction 1-3 times, combine the filtrates, centrifuge, and rotary evaporate.

[0009] The application also provides the use of the polysaccharide of Platycodon grandiflorum as described above in the preparation of a medicament having an immunomodulatory effect.

[0010] Preferably, the medicament is used to activate and / or enhance the immune function of macrophages, for example, the medicament can improve the viability of macrophages, improve the release of pro-inflammatory factors, and improve the phagocytic ability of macrophages.

[0011] The application also provides the use of the polysaccharide of Platycodon grandiflorum as described above in the preparation of an antitumor medicament.

[0012] Preferably, the medicament inhibits the proliferation of tumor cells by inducing apoptosis of tumor cells.

[0013] Preferably, the tumor is liver cancer or lung cancer.

[0014] The application further provides an immunoregulatory or antitumor drug, comprising a pharmaceutically effective dose of the above-mentioned platycodon grandiflorum polysaccharide and a pharmaceutically acceptable carrier.

[0015] Preferably, the platycodon grandiflorum polysaccharide accounts for 60-90% of the total mass or total volume of the drug.

[0016] Preferably, the platycodon grandiflorum polysaccharide accounts for 60%, 62%, 65%, 70%, 72%, 75%, 78%, 80%, 85%, 88% or 90% of the total mass or total volume of the drug.

[0017] Preferably, the pharmaceutically acceptable carrier comprises one or more of excipients, stabilizers, antioxidants, coloring agents, diluents, sustained-release agents, such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose and the like.

[0018] Preferably, the drug is any one of an injection, a tablet, a granule, a pill, a capsule, a suspension or an emulsion.

[0019] The application has the following advantages: 1. The application can simply and efficiently obtain a novel platycodon grandiflorum polysaccharide PGP-1 through an improved extraction process. Pharmacological activity tests show that PGP-1 has significant immunoregulatory activity. After RAW264.7 macrophages are treated with 25-200 μg / mL of PGP-1 for 48 h, the cell viability increases in a concentration-dependent manner, and the release of active factors such as NO and TNF-α is significantly increased. The phagocytic activity of RAW264.7 macrophages is also significantly enhanced. After HepG2 liver cancer cells and A549 lung cancer cells are treated with 800 μg / mL of PGP-1 for 72 h, the cell viability can be effectively inhibited, and the inhibition rate reaches 50%, without affecting the viability of human normal liver cells and lung fibroblasts. The platycodon grandiflorum polysaccharide PGP-1 provided by the application can be applied to the preparation of immunoregulatory drugs or antitumor drugs, and provides a new choice for the development of immunoregulatory drugs and antitumor drugs.

[0020] 2. The platycodon grandiflorum polysaccharide obtained by the application has high purity and a molecular weight of less than 3 kDa, is a new type of small molecular polysaccharide, and has certain immunoregulatory activity and antitumor activity, thereby widening the application field of platycodon grandiflorum polysaccharide.

[0021] 3. The application has the advantages of simple and convenient operation, mild extraction conditions, low energy consumption, small pollution, easy industrialization, and compliance with the requirements of modern environmental protection and low-carbon economy. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1The results are from a single-factor experiment. In the figure, A represents the effect of extraction time on the extraction rate of crude polysaccharide from Platycodon grandiflorus, B represents the effect of extraction temperature on the extraction rate of crude polysaccharide from Platycodon grandiflorus, C represents the effect of material-liquid ratio on the extraction rate of crude polysaccharide from Platycodon grandiflorus, and D represents the effect of extraction times on the extraction rate of crude polysaccharide from Platycodon grandiflorus.

[0023] Figure 2 Characterization of pure PGP-1: A is the HPGPC image of PGP-1, B is the HPAEC-PAD image of PGP-1, C is the UV image of PGP-1, and D is the IR image of PGP-1.

[0024] Figure 3 This is the NMR spectrum of pure PGP-1, where A in the spectrum represents... 1 H- 1 H spectrum, B is... 13 C10 NMR spectrum, where C10 represents DEPT135 spectrum.

[0025] Figure 4 This is the NMR spectrum of pure PGP-1, where A in the spectrum represents... 1 H- 1 The H COSY spectrum is shown, and B in the figure is the HSQC spectrum.

[0026] Figure 5 The image shows the NMR spectrum of pure PGP-1. In the image, A is the HMBC spectrum and B is the ROESY spectrum.

[0027] Figure 6 The results show the immunomodulatory activity of pure PGP-1. In the figure, A represents the effect of PGP-1 on the viability of RAW264.7 cells, B represents the effect of PGP-1 on the NO release of RAW264.7 cells, C represents the effect of PGP-1 on the IL-6 release of RAW264.7 cells, D represents the effect of PGP-1 on the TNF-α release of RAW264.7 cells, and E represents the effect of PGP-1 on the phagocytic activity of RAW264.7 cells.

[0028] Figure 7 The results show the immunomodulatory activities of homogenized polysaccharides from Ophiopogon japonicus, Asparagus cochinchinensis, and Dendrobium nobile, which have been reported to possess immunomodulatory activities. In the figure, A represents the effect of Ophiopogon japonicus polysaccharide (OGP) on NO release from RAW264.7 cells, B represents the effect of Asparagus cochinchinensis polysaccharide (ACP) on NO release from RAW264.7 cells, and C represents the effect of Dendrobium nobile polysaccharide (DDP) on NO release from RAW264.7 cells.

[0029] Figure 8 The results show the antitumor activity of pure PGP-1. In the figure, A represents the effect of PGP-1 on the viability of HepG2 liver cancer cells, and B represents the effect of PGP-1 on the viability of A549 lung cancer cells.

[0030] Figure 9The results show the antitumor activities of homogenized polysaccharides from Ophiopogon japonicus, Asparagus cochinchinensis, and Dendrobium nobile, which have been reported to have immunomodulatory activities. In the figure, AC represents the effects of Ophiopogon japonicus polysaccharide (OGP), Asparagus cochinchinensis polysaccharide (ACP), and Dendrobium nobile polysaccharide (DDP) on the viability of A549 lung cancer cells, and DF represents the effects of Ophiopogon japonicus polysaccharide (OGP), Asparagus cochinchinensis polysaccharide (ACP), and Dendrobium nobile polysaccharide (DDP) on the viability of HepG2 liver cancer cells, respectively.

[0031] Figure 10 The figures show flow cytometry images of PGP-1 inducing apoptosis in HepG2 liver cancer cells and A549 lung cancer cells. In the figure, A is the flow cytometry image of PGP-1 inducing apoptosis in HepG2 liver cancer cells, and B is the flow cytometry image of PGP-1 inducing apoptosis in A549 lung cancer cells.

[0032] Figure 11 The figure shows the effect of PGP-1 on apoptosis of HepG2 liver cancer cells and A549 lung cancer cells. Figure A shows the quantitative results of PGP-1 on apoptosis of HepG2 liver cancer cells, and figure B shows the quantitative results of PGP-1 on apoptosis of A549 lung cancer cells.

[0033] Figures 6-11 In comparison with the normal group, P<0.05, P<0.01, P < 0.001, P < 0.0001. Detailed Implementation

[0034] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art.

[0035] The technical solution of the present invention will be described in more detail below with reference to the embodiments.

[0036] In this application, room temperature refers to 25±5℃, and Platycodon grandiflorus ( Platycodon grandiflorus (Jacq.) A. DC.) is a commercially available product. The Platycodon grandiflorus used in the examples is produced in Bozhou City, Anhui Province. The chemical reagents used in the examples are preferably of analytical grade.

[0037] Example 1 Optimization of extraction conditions

[0038] 1. Single-factor experiment

[0039] 1) Effect of extraction time on the extraction rate of crude polysaccharides from Platycodon grandiflorus

[0040] The pretreated lycoris powder 2 g obtained in step S11 was precisely weighed, and was extracted at a solid-liquid ratio of 20 mL / g and an extraction temperature of 80°C for 30 min, 60 min, 90 min, 120 min and 150 min, respectively. After cooling to room temperature, the lycoris powder residue was removed with gauze, the filtrate was repeatedly extracted for 3 times, and then centrifuged at 4500 rpm / min for 10 min. The supernatant was collected and concentrated to an appropriate concentration using a rotary evaporator. 95% ethanol was added to the obtained supernatant under stirring to adjust the final alcohol concentration in the polysaccharide extract to 75%, and then placed in a 4°C refrigerator for cold storage overnight. After centrifugation at 4500 rpm / min for 10 min, the supernatant was discarded, and the obtained precipitate was freeze-dried to a constant weight to obtain lycoris crude polysaccharide. The extraction rate was calculated to determine the optimal extraction time.

[0041] The results are shown in Table 2. Figure 1 In Table 2, when the extraction time reached 90 min, the lycoris polysaccharide extraction rate reached a peak, so the extraction time of 60 min, 90 min and 120 min was selected for the subsequent response surface experiment.

[0042] 2) Effect of extraction temperature on the extraction rate of lycoris crude polysaccharide

[0043] The pretreated lycoris powder 2 g obtained in step S11 was precisely weighed, and was extracted at a solid-liquid ratio of 20 mL / g and an extraction temperature of 80°C for 30 min, 60 min, 90 min, 120 min and 150 min, respectively. After cooling to room temperature, the lycoris powder residue was removed with gauze, the filtrate was repeatedly extracted for 3 times, and then centrifuged at 4500 rpm / min for 10 min. The supernatant was collected and concentrated to an appropriate concentration using a rotary evaporator. 95% ethanol was added to the obtained supernatant under stirring to adjust the final alcohol concentration in the polysaccharide extract to 75%, and then placed in a 4°C refrigerator for cold storage overnight. After centrifugation at 4500 rpm / min for 10 min, the supernatant was discarded, and the obtained precipitate was freeze-dried to a constant weight to obtain lycoris crude polysaccharide. The extraction rate was calculated to determine the optimal extraction time.

[0044] The results are shown in Table 2. Figure 1 In Table 2, when the extraction time reached 90 min, the lycoris polysaccharide extraction rate reached a peak, so the extraction time of 60 min, 90 min and 120 min was selected for the subsequent response surface experiment.

[0045] 3) Effect of solid-liquid ratio on the extraction rate of lycoris crude polysaccharide

[0046] The pretreated lycorus root powder obtained in step S11 was precisely weighed 2 g, and extracted at 80 ℃ for 90 min at a liquid-solid ratio of 10 mL / g, 15 mL / g, 20 mL / g, 25 mL / g, and 30 mL / g, respectively. After cooling to room temperature, the lycorus root powder residue was removed with gauze, and the filtrate was repeatedly extracted for 3 times. The mixture was centrifuged at 4500 rpm / min for 10 min, and the supernatant was collected and concentrated to an appropriate concentration using a rotary evaporator. 95% ethanol was added to the obtained supernatant to adjust the final alcohol concentration in the polysaccharide extract to 80%, and the mixture was stored in a 4 ℃ refrigerator overnight. The mixture was centrifuged at 4500 rpm / min for 10 min, and the supernatant was discarded. The obtained precipitate was freeze-dried to a constant weight to obtain lycorus root crude polysaccharide, and the extraction rate was calculated to determine the optimal liquid-solid ratio.

[0047] The results are shown in Table D. Figure 1 The extraction rate of lycorus root polysaccharide reached a peak when the liquid-solid ratio was 10 mL / g, and then showed a downward trend. Therefore, the liquid-solid ratio of 10 mL / g, 15 mL / g, and 20 mL / g was selected for the subsequent response surface experiment.

[0048] 4) Effect of extraction times on the extraction rate of lycorus root crude polysaccharide

[0049] The pretreated lycorus root powder obtained in step S11 was precisely weighed 2 g, and extracted at 80 ℃ for 90 min at a liquid-solid ratio of 20 mL / g. After cooling to room temperature, the lycorus root powder residue was removed with gauze, and the filtrate was repeatedly extracted for 1 time, 2 times, 3 times, 4 times, and 5 times, respectively. The mixture was centrifuged at 4500 rpm / min for 10 min, and the supernatant was collected and concentrated to an appropriate concentration using a rotary evaporator. 95% ethanol was added to the obtained supernatant to adjust the final alcohol concentration in the polysaccharide extract to 80%, and the mixture was stored in a 4 ℃ refrigerator overnight. The mixture was centrifuged at 4500 rpm / min for 10 min, and the supernatant was discarded. The obtained precipitate was freeze-dried to a constant weight to obtain lycorus root crude polysaccharide, and the extraction rate was calculated to determine the optimal extraction times.

[0050] The results are shown in Table D. Figure 1 The extraction rate of lycorus root polysaccharide reached a peak when the liquid-solid ratio was 10 mL / g, and then showed a downward trend. Therefore, the liquid-solid ratio of 10 mL / g, 15 mL / g, and 20 mL / g was selected for the subsequent response surface experiment.

[0051] 2, Response surface experiment

[0052] According to the single factor experiment results, the corresponding response surface analysis was carried out using factor A time, factor B temperature, factor C liquid material ratio and factor D times. The factor level coding table is shown in Table 1, and the experimental results are shown in Table 2. The data was processed and analyzed using Design-Expert program (version 10.0.1). Using the Box-Behnken design of 29 runs, a second-order polynomial model was obtained. According to the model, the yield of jiegeng polysaccharide was analyzed and predicted, and the influence of each single factor on the yield of polysaccharide was analyzed to obtain the optimal extraction conditions.

[0053] Table 1. Factor level coding table ; Table 2. Four variable response surface design matrix expressed in coded units and response value of jiegeng crude polysaccharide extraction rate ; The data in Table 2 was fitted with a quadratic polynomial, and a polynomial equation for predicting the PGP extraction rate (Y) was obtained by multiple regression analysis as follows: Y = 18.07 + 1.32A - 1.47B + 0.35C + 0.72D - 0.50AB + 0.095AC + 0.58AD+ 1.76BC + 0.11BD - 0.83CD - 1.86 A 2 - 3.81 B 2 - 1.01 C 2 - 4.69 D 2 Where Y is the predicted value of jiegeng crude polysaccharide PGP extraction rate; A, B, C and D are the coded values of the tested time, temperature, liquid material ratio and extraction times, respectively.

[0054] According to the response surface results, the extraction conditions of jiegeng crude polysaccharide were determined as follows: liquid material ratio of 15 mL / g, extraction temperature of 75℃, extraction time of 100 min, and the filter residue was repeatedly extracted for 2 times.

[0055] Example 2

[0056] 1. Extraction of jiegeng polysaccharide PGP-1

[0057] The jiegeng polysaccharide PGP-1 has the following general structure: ; In the formula, m+n=9, and m and n are both >0.

[0058] The preparation method of the jiegeng polysaccharide PGP-1 comprises the following steps: S11. The dried root of Jiegeng was ground and passed through a 60 mesh sieve, then soaked in acetone at room temperature for 1.5 days and dried at 45°C to obtain pretreated Jiegeng powder; S12. 2000 g of the pretreated Jiegeng powder was mixed with 30 L of purified water, the temperature was adjusted to 75°C, and the mixture was extracted in a water bath for 100 min, then filtered with gauze. The residue was extracted twice more. The filtrates were combined, centrifuged, and the supernatant was concentrated to the appropriate concentration using a rotary evaporator at 50°C.

[0059] S13. After concentration, the supernatant was alcohol precipitated by adding 95% ethanol under stirring to adjust the final alcohol concentration in the polysaccharide extract to 75%, and then the mixture was placed in a refrigerator at 4°C overnight. The polysaccharide precipitate was dissolved in an appropriate amount of water, and the solution was concentrated to a concentration of 15-20 mg / mL without alcohol taste using a rotary evaporator at 50°C. The polysaccharide extract was obtained.

[0060] S14. 1000 mL of the polysaccharide extract was added to 200 mL of Sevage reagent (chloroform:n-butanol = 4:1 v / v) in a separatory funnel, mixed thoroughly, and then allowed to stand until the layers were separated. The lower two layers (organic solvent and protein layer) were discarded, and the third layer at the bottom was collected. The residual organic solvent was removed using a rotary evaporator at 50°C, and the volume of the solution was concentrated to an appropriate amount. The Sevage reagent was removed, and the concentrated solution was freeze-dried to obtain crude polysaccharide PGP with an extraction rate of about 19%.

[0061] S2. The protein-free PGP was dissolved in distilled water to a concentration of 15 mg / mL using a magnetic stirrer, and then loaded onto a DEAE-52 cellulose ion exchange column. When the sample liquid surface was almost aligned with the gel surface, ultrapure water was added to flush the column, and the same fractions were combined and concentrated for the next step of purification.

[0062] S3. The fractions after DEAE column purification were concentrated by rotary evaporation, and then dialyzed using a dialysis bag with a cutoff of 1000 Da using ultrapure water. The dialysis bag was concentrated and freeze-dried to obtain a uniform symmetrical polysaccharide fraction PGP-1.

[0063] The PGP-1 pure product obtained by the above preparation: white powder, yield 21.77%, purity 99%.

[0064] Example 3 Identification of PGP-1

[0065] 1. Molecular weight determination

[0066] The 2 mg / mL standard molecular weight dextran (China Food and Drug Inspection Research) (D0, D1, D2, D3, D4, D5) solution and the 2 mg / mL PGP-1 solution prepared in Example 2 were filtered with a 0.45 μm microporous filter and detected by high performance gel permeation chromatography, using a Shimadzu LC-20A instrument equipped with a Shimadzu ELSD-16 detector. The analysis column was a Shodex Ohpak SB-803 HQ, the mobile phase was ultrapure water, the flow rate was 1 mL / min, and the injection volume was 20 μL. The HPGPC chart of PGP-1 is shown in Figure 2 FIG. 2A.

[0067] 2. Monosaccharide composition determination

[0068] Polysaccharide hydrolysis: a clean chromatographic bottle was taken, 2 mg of polysaccharide sample was weighed, 1 mL of 3 M TFA acid solution was added, and heated at 60°C for 3 hours. Nitrogen was blown to dryness. Add 99.99% methanol to wash, and then dry again. Repeat the methanol washing for 2-3 times. Add an appropriate amount of pure water to dissolve, filter and transfer into the chromatographic bottle for detection.

[0069] Standard preparation: the required standards were accurately weighed, 10 mg / mL standard solution stock was prepared by adding water, then an appropriate amount of standard stock was mixed to prepare a standard mixture with a highest indicator concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL, and a series of standard solutions were prepared according to the concentration gradient, filtered and transferred into the chromatographic bottle for detection.

[0070] Instrument parameters: Thermo ICS 5000+ ion chromatography system was used, and electrochemical detector was used for analysis and detection of monosaccharide components. Dionex™ CarboPac™ PA20 (150 3.0 mm, 10 μm) liquid chromatography column was used, the injection volume was 5 μL. The mobile phase A (H2O), the mobile phase B (0.1 M NaOH), the mobile phase C (0.1 M NaOH, 0.2 M NaAc), the flow rate was 0.5 mL / min, and the column temperature was 30°C. The monosaccharide composition analysis results of PGP-1 are shown in Figure 2 FIG. 2B.

[0071] According to Figure 2 FIG. 2A and B, the molecular weight of PGP-1 is 2.13 kDa, and PGP-1 is composed of fructose and glucose, and the molar ratio is 90.43:9.57.

[0072] 3. Methylation experiment

[0073] Take 10 mg PGP-1 into 2 mL anhydrous DMSO, dissolve, then add 100 mg NaH containing oil clothes, add dry magnetic sub, fill in He protection, stir for 2 h, then slowly inject 2 mL iodomethane, avoid light reaction for 4 h, add 1 mL pure water to terminate the reaction. Add 2 mL chloroform to extract 3 times, combine the chloroform layer, then wash with pure water 3 times, take the chloroform layer to spin dry, add appropriate amount of pure water to freeze dry. Repeat the above steps 3 times, take a small amount of methylation sample for infrared detection, until the 3600-3200 cm -1 peak disappears. Take the methylation sample to hydrolyze at 60℃ for 4 h after adding 1 mL 2 M TFA, after the reaction is completed, add methanol repeatedly to spin out TFA. After hydrolysis, add 1 mL pure water and 20 mg NaBH4 to reduce at room temperature for 6 h, then add excess acetic acid to neutralize the residual NaBH4, spin dry, freeze dry, then add 1 mL acetic anhydride and 1 mL anhydrous pyridine in a sealed tube, acetylate at 100℃ for 4 h, then spin dry, chloroform extraction, then pure water washing 3 times, take the chloroform layer to analyze by GCMS-QP2010 SE gas chromatography mass spectrometer (Shimadzu). The results are shown in Table 3.

[0074] Table 3. Methylation analysis of PGP-1 ; It can be seen that PGP-1 corresponds to four connection modes of t-Fruf, 1,2-Fruf, 1,2,6-Fruf and t-Glcp, and the molar ratio is 2.42:8.52:1.31:1.00.

[0075] 4、UV analysis

[0076] Prepare a uniform PGP-1 into a 1 mg / mL aqueous solution, and analyze the absorption of the sample in the range of 200-400 nm by UV-1800 ultraviolet visible analyzer (Shimadzu).

[0077] The results are shown in Table 3. Figure 2 It can be seen that PGP-1 has no obvious absorption peak at 200-400 nm, indicating that the uniform polysaccharide PGP-1 does not contain protein, nucleic acid and other substances.

[0078] 5、IR analysis

[0079] Mix 1 mg of polysaccharide sample PGP-1 with 100 mg of spectral grade KBr powder and press into a tablet. Use Tensor 27 Fourier transform infrared spectrometer (Bruker) to detect the absorption in the range of 4000-400 cm -1 .

[0080] The results are shown in Table 3.Figure 2 The peaks in the region of 1000-800 cm -1 The peaks in the region of 937, 879 and 817 cm -1 The bands in the region of 937, 879 and 817 cm

[0081] 6、NMR analysis

[0082] PGP-1 was dissolved in 0.5 mL D2O and freeze-dried, after repeating the process three times, the sample was dissolved in D2O and transferred into a NMR tube. Subsequently, the Advance III 800 MHz NMR instrument (Bruker) was used to analyze the sample at 25 °C. 1 H, 13 C, DEPT-135, COSY, HSQC, HMBC, ROESY information. The results are shown in order as follows. Figures 3-5

[0083] Finally, it can be determined that the structure of the compound PGP-1 prepared in Example 1 is as follows: ; In the formula, m+n=9, m and n are both >0.

[0084] Example 4 Immunomodulatory activity of PGP-1

[0085] 1、Experimental materials

[0086] RAW264.7 macrophages were purchased from the China Center for Type Culture Collection; OGP, ACP and DDP, which have been reported to have immunomodulatory activity, were used as control groups in the NO release experiment. Among them, OGP was extracted according to the method described in the second page of the second part of the literature 《Comparative studies on the immunoregulatory effects of three polysaccharides using high content imaging system》; ACP was extracted according to the method described in the second page of the literature 《Structure, immunomodulatory activity and in vitro antioxidant activity of Ophiopogon polysaccharide》; DDP was extracted according to the method described in the literature 《A novel polysaccharide from Dendrobium devonianum ​The method of the second page second part is extracted to obtain.

[0087] Three literature specific information: ①Lv X, Chen D, Yang L, et al. Comparative studies on the immunoregulatory effects of three polysaccharides using high content imaging system [J]. International journal of biological macromolecules, 2016, 86: 28-42. DOI: 10.1016 / j.ijbiomac.2016.01.048. ②Mu D, Saren Gao, Bao Liang, et al. Structure, immunomodulatory activity and in vitro antioxidant activity of aspartic polysaccharide [J]. Chinese Journal of Food Science, 2022, 22(8): 51-60. DOI: 10.16429 / j.1009-7848.2022.08.006. ③Wu Y G, Wang K W, Zhao Z R, et al. A novel polysaccharide from Dendrobium devonianum serves as a TLR4 agonist for activating macrophages[J]. International Journal of Biological Macromolecules, 2019, 133: 564-574.DOI: 10.1016 / j.ijbiomac.2019.04.125. Instrument reagent, see Table 4.

[0088] Table 4 Instrument reagent .

[0089] 2. Cell culture, subculture, freezing and recovery

[0090] RAW264.7 cells were cultured in DMEM medium containing 10% FBS at 37°C in a 5% CO2 incubator. When the cells reached 80-90% density, the original culture medium was discarded, and the cells were washed once with PBS. Then, 1 mL of 0.25% trypsin was added, and the cells were observed under a microscope. When the cells were rounded and detached, 2 mL of complete medium was added, and the cells were blown to completely detach and disperse. Then, the cells were centrifuged at 1000 rpm / min for 5 min, the supernatant was discarded, and 12 mL of complete medium was added. After blowing and mixing, 4 mL of cell suspension was added to a new culture dish for continuous culture.

[0091] The freezing solution was prepared according to the ratio of serum to DMSO of 9:1. After centrifugation, the supernatant was discarded, and 1 mL of freezing solution was added to blow the cells evenly. Then, the cells were transferred to a freezing tube. The freezing tube was placed in a -20°C refrigerator for at least 2 hours, then transferred to a -80°C refrigerator, and finally transferred to a liquid nitrogen tank.

[0092] The cells taken from the liquid nitrogen tank were quickly thawed in a 37°C water bath. Then, 1 mL of freezing solution was transferred to 3 mL of serum-free medium, and the cells were dispersed by gentle blowing. After centrifugation at 1000 rpm / min for 5 min, the supernatant was discarded, and 4 mL of complete culture medium was added for culture in an incubator.

[0093] 3. Cell viability detection

[0094] CCK8 reagent was used to detect the viability of RAW264.7 cells. The cells were seeded into a 96-well plate for 24 hours. Then, the supernatant was replaced with medium containing different concentrations of PGP-1, OGP, ACP, and DDP (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL), respectively. At the same time, the group with the addition of LPS (1 μg / mL) was used as the positive control group. After 48 hours of incubation, a mixture of serum-free medium and CCK8 (medium / CCK8 = 10:1, v / v) was added to each well instead of the supernatant. After 1 hour of incubation, the absorbance of each well was detected using a multifunctional microplate reader at 450 nm wavelength. The group with 0 μg / mL was used as the control group.

[0095] See Figure 6 Compared with the control group, the viability of RAW264.7 cells treated with 25-200 μg / mL PGP-1 for 48 hours increased in a concentration-dependent manner. This indicates that PGP-1 has strong proliferation activity on RAW264.7 cells.

[0096] 4. NO, immune factor release amount, and phagocytic ability detection

[0097] 1) RAW264.7 cells were seeded in 96-well plates (1 × 10⁻⁶ cells per well). 4 Macrophages were placed in wells (number of cells / well) and stimulated for 48 h with PGP-1, OGP, ACP, and DDP (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively). The immune factors, NO release, and phagocytic activity of RAW264.7 macrophages were measured using the corresponding kits according to the instructions.

[0098] See Figure 6 In samples B, C, and D, compared to the control group, PGP-1 significantly induced NO production and increased the levels of TNF-α and IL-6. Figure 6 As shown in Figure E, the effect of PGP-1 on the phagocytosis of RAW264.7 cells was detected by the neutral red uptake method. The phagocytic capacity of RAW264.7 cells increased with increasing PGP-1 concentration.

[0099] 2) RAW264.7 cells were stimulated under the same conditions with the prepared Ophiopogon japonicus homogeneous polysaccharide (OGP), Asparagus cochinchinensis homogeneous polysaccharide (ACP), and Dendrobium nobile homogeneous polysaccharide (DDP) (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL). The NO release was as shown in the attached figure. Figure 7 As shown in Figures A, B, and C, the homogeneous polysaccharides from Ophiopogon japonicus, Asparagus cochinchinensis, and Dendrobium nobile have a similar effect to PGP-1 in promoting NO release in RAW264.7 cells.

[0100] Example 5 PGP-1's antitumor activity

[0101] Human liver cancer cells HepG2 and human lung cancer cells A549 were purchased from the American Type Culture Collection (ATCC) in the United States.

[0102] CytoFLEX S flow cytometer (Beckman, USA), and the other reagents and instruments are the same as those in Table 4.

[0103] 1. Cell viability detection

[0104] CCK8 reagent was used to detect the viability of HepG2 and A549 cells. Cells were seeded into 96-well plates for 24 hours, and then the supernatant was replaced with culture medium containing different concentrations of PGP-1, OGP, ACP, DDP (0 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL). After 72 hours of incubation, a mixture of serum-free culture medium and CCK8 (culture medium / CCK8 = 10:1, v / v) was added to each well instead of the supernatant. After 1 hour of incubation, the absorbance of each well was detected at a wavelength of 450 nm using a multifunctional microplate reader.

[0105] Referring to Figure 8 Compared with the control group, PGP-1 significantly inhibited the proliferation of HepG2 and A549 cells. The inhibition rate of HepG2 liver cancer cells and A549 lung cancer cells reached 50% after 72 h of PGP-1 at 800 μg / mL.

[0106] According to Figure 9 A, B, C, Ophiopogon japonicus, Asparagus cochinchinensis, and Dendrocalamopsis sinensis all had similar inhibition rates on A549 cells as PGP-1, with an inhibition rate close to 50% at 800 μg / mL; as Figure 9 As shown in D, E, and F, all three homopolysaccharides showed proliferation inhibition on HepG2 cells, with Dendrocalamopsis sinensis homopolysaccharide being the most effective. However, PGP-1 had a stronger inhibitory effect on the proliferation of HepG2 cells.

[0107] 2. Apoptosis detection

[0108] After treating HepG2 and A549 cells (5 x 10 5 After 72 hours of treatment with PGP-1, the harvested cells were fixed in PBS containing 70% ethanol at -20°C for 7 hours. Annexin V / PI assay was used to determine whether PGP-1 induced apoptosis in cancer cells. Referring to Figure 10 and Figure 11 After treating cancer cells with PGP-1, the percentage of apoptotic cells showed a concentration-dependent increase. When the concentration was 800 μg / mL, the percentage of cell apoptosis in HepG-2 reached 36.88%, and the percentage of cell apoptosis in A549 reached 36.08%.

[0109] These results suggest that PGP-1 may inhibit cancer cell activity by inducing apoptosis, and has a better effect compared to other homopolysaccharides. Therefore, PGP-1 has research value as a lead compound for developing anti-tumor drugs.

[0110] In conclusion, the PGP-1 can promote the proliferation of RAW264.7 macrophages, improve the phagocytosis of the RAW264.7 macrophages, induce the RAW264.7 macrophages to produce immune factors such as NO, TNF-α and IL-6, and can induce the apoptosis of HepG2 and A549 cells and inhibit the proliferation of tumor cells. It is shown that the PGP-1 has the immunoregulatory activity and the anti-tumor activity, and has the potential to be developed as a lead compound of an immunoadjuvant and an anti-tumor drug.

[0111] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application; although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A luffin polysaccharide, characterized in that, The lycianthes polysaccharide is named as PGP-1, and the structural formula is shown as formula (A): （A）， In the formula, m+n=9, and m and n are both greater than 0.

2. A method of preparing the shatianmian polysaccharide according to claim 1, characterized in that, The method comprises the following steps: S1. After the lycianthes root is dried, it is ground, extracted, centrifuged, rotary evaporated, alcohol precipitated, protein removed, and freeze-dried to obtain a crude polysaccharide extract; S2. The crude polysaccharide extract is dissolved into a solution with a concentration of 15 mg / mL, and then purified by a DEAE-52 cellulose ion exchange column; S3. The purified component is dialyzed by using a filter membrane with a pore size of 1000 Da, and the concentrated solution after dialysis is collected, dried, and the lycianthes polysaccharide PGP-1 is obtained.

3. The method for preparing Platycodon grandiflorus polysaccharide as described in claim 2, characterized in that, The specific method of step S1 is as follows: S11. After the lycianthes root is ground, it is soaked in acetone at room temperature for 1.5 days, and then dried at 45 ℃ to obtain pretreated lycianthes powder; S12. The lycianthes powder is mixed with pure water at a ratio of 1 g to (10-20) mL, and then extracted at a temperature of 60-100 ℃ for 60-120 min, filtered, and extracted repeatedly for 1-3 times, and the filtrates are combined, centrifuged, and rotary evaporated; S13. 95% ethanol is added to the rotary evaporated liquid, the final concentration of ethanol in the liquid is adjusted to 75%, and the mixture is placed at 4 ℃ overnight, centrifuged, and the precipitate is dissolved in water and rotary evaporated at 50 ℃ until the ethanol is evaporated, to obtain a polysaccharide extract; S14. According to the volume ratio of the polysaccharide extract to the Sevage reagent of 5:1, the Sevage reagent is added to the polysaccharide extract, stirred, and then allowed to stand to separate into layers, and the third layer of solution at the bottom is taken, rotary evaporated at 50 ℃ to remove the Sevage reagent, and the concentrated solution is freeze-dried to obtain the crude polysaccharide extract.

4. The method of preparing the shatian polysaccharide according to claim 3, characterized in that, In the step S12, the lycianthes powder is mixed with pure water at a ratio of 1 g to 15 mL, extracted at a temperature of 75 ℃ for 100 min, filtered, and extracted repeatedly for 1-3 times, and the filtrates are combined, centrifuged, and rotary evaporated.

5. The lycianthes polysaccharide of claim 1 is used for preparing a medicine with immunoregulatory effect.

6. The use according to claim 5, wherein the compound is ###0002### The medicine is used for activating and / or enhancing the immune function of macrophages.

7. The lycianthes polysaccharide of claim 1 is used for preparing an anti-tumor medicine.

8. Use according to claim 7, wherein the compound is ###0002### The tumor is liver cancer or lung cancer.

9. An immunomodulatory or antitumor drug, characterized in that, The medicine comprises a pharmaceutically effective dose of the lycianthes polysaccharide of claim 1 and a pharmaceutically acceptable carrier.

10. The medicament according to claim 9, wherein The lycianthes polysaccharide accounts for 60-90% of the total mass or total volume of the medicine.

11. The medicament according to claim 9, wherein The pharmaceutically acceptable carrier comprises one or more of excipients, stabilizers, antioxidants, coloring agents, diluents, and sustained-release agents.

12. The medicament according to claim 9, wherein The medicine is any one of an injection, a tablet, a granule, a pill, a capsule, a suspension, or an emulsion.

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