A luffin polysaccharide, and a preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical compounds, and particularly relates to a platycodon grandiflorum polysaccharide as well as a preparation method and application thereof. The platycodon grandiflorum polysaccharide is named as PGP-1. The platycodon grandiflorum polysaccharide PGP-1 is simply and efficiently extracted from the platycodon grandiflorum root through an improved extraction process. Pharmacological activity shows that the PGP-1 has significant immunoregulatory activity and inhibits cancer cell proliferation, which indicates that the PGP-1 can be applied to preparation of immunoregulatory drugs or antitumor drugs, and provides a new choice for development of immunoregulatory drugs and antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical compounds, and particularly relates to a platycodon grandiflorum polysaccharide as well as 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 animals and plants and the cell walls of microorganisms in nature, and are one of the four basic substances constituting life activities. In recent years, with the researches in cell biology and molecular biology, it is found that polysaccharides have various pharmacological activities, including anti-tumor, immune system enhancement, anti-inflammatory, anticoagulation, anti-virus, anti-aging and blood sugar reduction. 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 is a perennial herb that can withstand cold. Its roots are used as a traditional Chinese medicine, and have the effects of relieving cough, clearing the lungs and draining pus. In addition to medicinal use, platycodon grandiflorum is often pickled into salted vegetables or pickles for eating. Platycodon grandiflorum 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, have not been fully developed and utilized. As a 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 resources.

[0004] Platycodon grandiflorum polysaccharide belongs to inulin-type polysaccharide. There are many methods for extracting, separating and purifying platycodon grandiflorum 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. In the prior art, there are problems such as low extraction rate of platycodon grandiflorum polysaccharide, long time consumption, low purity of preparation, low activity of polysaccharide and complicated preparation process. Extracting platycodon grandiflorum polysaccharide with specific biological activity has positive significance for the resource utilization of platycodon grandiflorum, and also has important significance for the industrialization development of platycodon grandiflorum polysaccharide. SUMMARY

[0005] In order to solve the above technical problems, the present application first provides a platycodon grandiflorum polysaccharide, which is named PGP-1, and the structural formula is shown as formula A:

[0006] A,

[0007] In the formula, m+n=9, and m and n are both greater than 0.

[0008] The present application further provides a preparation method of the platycodon grandiflorum polysaccharide, comprising the following steps:

[0009] S1. After the dried root of Platycodon grandiflorum is ground, extracted, the extract is centrifuged, rotary evaporated, alcohol precipitated, deproteinized and freeze-dried to obtain a crude polysaccharide extract;

[0010] S2. The crude polysaccharide extract is dissolved into a solution of 15 mg / mL, and purified by DEAE-52 cellulose ion exchange column;

[0011] S3. The purified component is dialyzed by using a filter membrane of 1000 Da, and the concentrated solution after dialysis is collected, dried to obtain the desired polysaccharide PGP-1 of Platycodon grandiflorum.

[0012] Preferably, the specific method of step S1 is as follows:

[0013] S11. After the root of Platycodon grandiflorum is ground, it is soaked in acetone at room temperature for 1.5 days, and then dried at 45℃ to obtain pretreated Platycodon grandiflorum powder;

[0014] S12. The Platycodon grandiflorum powder is mixed with pure water at a ratio of 1g: (10-20) mL, and extracted at a temperature of 60-100℃ for 60-120 min, filtered, and the extraction is repeated for 1-3 times, the filtrates are combined, centrifuged and rotary evaporated;

[0015] 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;

[0016] S14. Sevage reagent is added to the polysaccharide extract at a volume ratio of 5:1, stirred, and after standing and layering, the third layer solution at the bottom is taken, rotary evaporated at 50℃ to remove the Sevage reagent, and the concentrated solution is freeze-dried to obtain a crude polysaccharide extract.

[0017] Preferably, the Platycodon grandiflorum powder is mixed with pure water at a ratio of 1g: 15 mL, extracted at a temperature of 75℃ for 100 min, filtered, and the extraction is repeated for 1-3 times, the filtrates are combined, centrifuged and rotary evaporated.

[0018] The application also provides the use of the polysaccharide of Platycodon grandiflorum as described above in the preparation of a medicine with immunomodulatory effect.

[0019] Preferably, the medicine is used for activating and / or enhancing the immune function of macrophages, for example, the medicine can improve the viability of macrophages, improve the release of pro-inflammatory factors, and improve the phagocytic ability of macrophages.

[0020] The application also provides the use of the polysaccharide of Platycodon grandiflorum as described above in the preparation of an anti-tumor medicine.

[0021] Preferably, the medicine induces tumor cell apoptosis and inhibits tumor cell proliferation.

[0022] Preferably, the tumor is liver cancer or lung cancer.

[0023] The application finally provides an immunomodulatory or antitumor medicine comprising a pharmaceutically effective dose of the above-mentioned platycodon grandiflorum polysaccharide and a pharmaceutically acceptable carrier.

[0024] Preferably, the platycodon grandiflorum polysaccharide accounts for 60-90% of the total mass or total volume of the medicine.

[0025] 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 medicine.

[0026] Preferably, the pharmaceutically acceptable carrier comprises one or more of excipients, stabilizers, antioxidants, coloring agents, diluents, and 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.

[0027] Preferably, the medicine is any one of an injection, a tablet, a granule, a pill, a capsule, a suspension, or an emulsion.

[0028] The application has the following beneficial effects:

[0029] 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 immunomodulatory 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 immunomodulatory drugs or antitumor drugs, and provides a new choice for the development of immunomodulatory drugs and antitumor drugs.

[0030] 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 immunomodulatory activity and antitumor activity, thereby widening the application field of platycodon grandiflorum polysaccharide.

[0031] 3. The present application is simple and convenient to operate, has mild extraction conditions, low energy consumption, and small pollution, is easy to realize industrialization, and meets the needs of modern environmental protection and low-carbon economy. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 As single factor test results, A in the figure is the effect of extraction time on the extraction rate of jiegeng crude polysaccharide, B is the effect of extraction temperature on the extraction rate of jiegeng crude polysaccharide, C is the effect of solid-liquid ratio on the extraction rate of jiegeng crude polysaccharide, and D is the effect of extraction times on the extraction rate of jiegeng crude polysaccharide.

[0033] Figure 2 A is the HPGPC chart of PGP-1, B is the HPAEC-PAD chart of PGP-1, C is the UV chart of PGP-1, and D is the IR chart of PGP-1.

[0034] Figure 3 A is the H NMR chart of PGP-1, B is the C NMR chart of PGP-1, and C is the DEPT135 chart of PGP-1. 1 H- 1 H spectrum, B is 13 C NMR spectrum, C is DEPT135 spectrum.

[0035] Figure 4 A is the H NMR chart of PGP-1, B is the C NMR chart of PGP-1, and C is the DEPT135 chart of PGP-1. 1 H- 1 H COSY spectrum, B is the HSQC spectrum.

[0036] Figure 5 A is the HMBC spectrum, and B is the ROESY spectrum.

[0037] Figure 6 A is the effect of PGP-1 on the activity of RAW264.7 cells, B is the effect of PGP-1 on the NO release amount of RAW264.7 cells, C is the effect of PGP-1 on the IL-6 release amount of RAW264.7 cells, D is the effect of PGP-1 on the TNF-α release amount of RAW264.7 cells, and E is the effect of PGP-1 on the phagocytic activity of RAW264.7 cells.

[0038] Figure 7 A is the effect of OGP on the NO release amount of RAW264.7 cells, B is the effect of ACP on the NO release amount of RAW264.7 cells, and C is the effect of DDP on the NO release amount of RAW264.7 cells.

[0039] Figure 8 The results of anti-tumor activity of PGP-1 pure product. In the figure, A is the effect of PGP-1 on the viability of HepG2 liver cancer cells, and B is the effect of PGP-1 on the viability of A549 lung cancer cells.

[0040] Figure 9 The results of anti-tumor activity of OGP, ACP and DDP which have been reported to have immunomodulatory activity. In the figure, A-C are the effects of OGP, ACP and DDP on the viability of A549 lung cancer cells, respectively, and D-F are the effects of OGP, ACP and DDP on the viability of HepG2 liver cancer cells, respectively.

[0041] Figure 10 Flow cytometry graphs of PGP-1 on the apoptosis of HepG2 liver cancer cells and A549 lung cancer cells. In the figure, A is the flow cytometry graph of PGP-1 on the apoptosis of HepG2 liver cancer cells, and B is the flow cytometry graph of PGP-1 on the apoptosis of A549 lung cancer cells.

[0042] Figure 11 The effects of PGP-1 on the apoptosis of HepG2 liver cancer cells and A549 lung cancer cells. In the figure, A is the quantitative result graph of PGP-1 on the apoptosis of HepG2 liver cancer cells, and B is the quantitative result graph of PGP-1 on the apoptosis of A549 lung cancer cells.

[0043] Figures 6-11 Compared with the normal group, P<0.05, P<0.01, P < 0.001, P <0.0001. DETAILED DESCRIPTION

[0044] Unless otherwise specified, the terms used herein have meanings commonly understood by those skilled in the art.

[0045] The technical solutions of the present application will be described in more detail below in conjunction with examples.

[0046] In this application, room temperature refers to 25±5℃, and the Platycodon grandiflorum (Jacq.) A. DC. used in the examples is a common commercially available product, and the Platycodon grandiflorum used in the examples is produced in Bozhou City, Anhui Province. The chemical reagents used in the examples have a purity of preferably analytical grade. Platycodon grandiflorus

[0047] Example 1

[0048] Optimization of extraction conditions

[0049] 1. Single factor experiment 1. Single factor experiment

[0050] 1)Effect of extraction time on extraction rate of crude polysaccharide from Jiegeng

[0051] Precisely weigh 2 g of the pretreated Jiegeng powder obtained in step S11, and extract for 30 min, 60 min, 90 min, 120 min and 150 min, respectively, under the conditions of a solid-liquid ratio of 20 mL / g and an extraction temperature of 80°C. After cooling to room temperature, remove the Jiegeng powder residue with gauze, repeatedly extract the residue for 3 times, centrifuge at 4500 rpm / min for 10 min, collect the supernatant, and concentrate to an appropriate concentration using a rotary evaporator. Stir 95% ethanol into the obtained supernatant to adjust the final alcohol concentration in the polysaccharide extract to 75%, and then place it in a 4°C refrigerator to be refrigerated overnight. Centrifuge at 4500 rpm / min for 10 min, discard the supernatant, and freeze-dry the obtained precipitate to a constant weight to obtain crude polysaccharide from Jiegeng. Calculate the extraction rate to determine the optimal extraction time.

[0052] The results are shown in Table 2. Figure 1 Table 2: Effect of extraction time on extraction rate of crude polysaccharide from Jiegeng

[0053] 2)Effect of extraction temperature on extraction rate of crude polysaccharide from Jiegeng

[0054] Precisely weigh 2 g of the pretreated Jiegeng powder obtained in step S11, and extract for 30 min, 60 min, 90 min, 120 min and 150 min, respectively, under the conditions of a solid-liquid ratio of 20 mL / g and an extraction temperature of 80°C. After cooling to room temperature, remove the Jiegeng powder residue with gauze, repeatedly extract the residue for 3 times, centrifuge at 4500 rpm / min for 10 min, collect the supernatant, and concentrate to an appropriate concentration using a rotary evaporator. Stir 95% ethanol into the obtained supernatant to adjust the final alcohol concentration in the polysaccharide extract to 75%, and then place it in a 4°C refrigerator to be refrigerated overnight. Centrifuge at 4500 rpm / min for 10 min, discard the supernatant, and freeze-dry the obtained precipitate to a constant weight to obtain crude polysaccharide from Jiegeng. Calculate the extraction rate to determine the optimal extraction time.

[0055] The results are shown in Table 3. Figure 1 Table 3: Effect of extraction temperature on extraction rate of crude polysaccharide from Jiegeng

[0056] 3)Effect of solid-liquid ratio on extraction rate of crude polysaccharide from Jiegeng

[0057] 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.

[0058] 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.

[0059] 4) Effect of extraction times on the extraction rate of lycorus root crude polysaccharide

[0060] 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.

[0061] 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.

[0062] 2, Response surface experiment

[0063] 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.

[0064] Table 1. Factor level coding table

[0065] ;

[0066] Table 2. Four variable response surface design matrix expressed in coded units and response value of jiegeng crude polysaccharide extraction rate

[0067] ;

[0068] 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 multivariate regression analysis as follows:

[0069] 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

[0070] 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.

[0071] According to the response surface results, the jiegeng crude polysaccharide extraction conditions 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.

[0072] Example 2

[0073] 1. Extraction of jiegeng polysaccharide PGP-1

[0074] Jiegeng polysaccharide PGP-1 has the following general structure:

[0075] ;

[0076] In the formula, m+n=9, and m and n are both >0.

[0077] The preparation method of the lycianthes polysaccharide PGP-1 comprises the following steps:

[0078] S11. The dried lycianthes root is crushed 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 lycianthes powder;

[0079] S12. 2000 g of the pretreated lycianthes powder is mixed with 30 L of purified water, the temperature is adjusted to 75°C, and the mixture is immersed in a water bath for 100 min, and then filtered with gauze. The residue is extracted for another 2 times. The filtrates are combined, centrifuged, and the supernatant is concentrated to a suitable concentration by a rotary evaporator at 50°C.

[0080] S13. After concentration, the supernatant is alcohol precipitated, 95% ethanol is added under stirring to adjust the final alcohol concentration in the polysaccharide extract to 75%, and the mixture is sealed with plastic wrap and placed in a refrigerator at 4°C overnight. After centrifugation, the polysaccharide precipitate is dissolved in an appropriate amount of water, and concentrated to a concentration of 15-20 mg / mL without alcohol taste by a rotary evaporator at 50°C. The polysaccharide extract is obtained.

[0081] S14. 1000 mL of the polysaccharide extract is added with 200 mL of Sevage reagent (chloroform:n-butanol=4:1 v / v) in a separatory funnel, mixed thoroughly, and then allowed to stand to separate into layers. The lower two layers (organic solvent and protein layer) are discarded, and the third layer at the bottom is taken out. The residual organic solvent is removed by a rotary evaporator at 50°C, and the volume of the concentrated solution is adjusted. The Sevage reagent is removed, and the concentrated solution is freeze-dried to obtain the crude polysaccharide PGP with an extraction rate of about 19%.

[0082] S2. The protein-free PGP is completely dissolved in distilled water by magnetic stirring to a concentration of 15 mg / mL, and then loaded onto a DEAE-52 cellulose ion exchange column. When the sample liquid surface almost coincides with the gel surface, ultrapure water is added to flush the column, and the same fractions are combined and concentrated for the next step of purification.

[0083] S3. The fractions after DEAE column purification are concentrated by rotary evaporation, and then dialyzed using a dialysis bag with a molecular weight cut-off of 1000 Da and ultrapure water. After concentration, the dialysis bag is freeze-dried to obtain the uniform and symmetrical polysaccharide fraction PGP-1.

[0084] The PGP-1 product obtained by the above preparation has the following properties: white powder, yield of 21.77%, and purity of 99%.

[0085] Example 3

[0086] Identification of PGP-1

[0087] 1. Determination of molecular weight

[0088] 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.

[0089] 2. Monosaccharide composition determination

[0090] Polysaccharide hydrolysis: Take a clean chromatographic bottle, weigh 2 mg of polysaccharide sample, add 1 mL of 3 M TFA acid solution, heat at 60°C for 3 hours. Blow dry with nitrogen. Add 99.99% methanol to wash, and then blow dry, repeat the methanol washing 2-3 times. Add an appropriate amount of pure water to dissolve, filter and transfer into a chromatographic bottle for detection.

[0091] Standard preparation: Accurately weigh the required standard for this project, add water to prepare a 10 mg / mL standard solution master single standard, then take an appropriate amount of standard master single standard to prepare a standard mixed standard with a maximum target concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL, and prepare a series of standard solutions according to the concentration gradient required for detection, filter and transfer into a chromatographic bottle for detection.

[0092] Instrument parameters: Thermo ICS 5000+ ion chromatography system was used, and an electrochemical detector was used to analyze and detect the 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.

[0093] According to Figure 2 A, 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.

[0094] 3. Methylation experiment

[0095] 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°C for 4 h after adding 1 mL 2 M TFA, after the reaction is completed, spin out TFA by adding methanol multiple times. 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°C for 4 h, then spin dry, chloroform extraction, then wash with pure water 3 times, take the chloroform layer to analyze by GCMS-QP2010 SE gas chromatography mass spectrometer (Shimadzu). The results are shown in Table 3.

[0096] Table 3. Methylation analysis of PGP-1

[0097] ;

[0098] 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.

[0099] 4、UV analysis

[0100] 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).

[0101] The results are shown in Table 3. Methylation analysis of PGP-1 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.

[0102] 5、IR analysis

[0103] 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 .

[0104] Results are shown in Table 1. Figure 2 The peaks in the region of 1000-800 cm -1 The bands at 937, 879 and 817 cm -1 indicate the presence of β-configuration of fructose in PGP-1.

[0105] 6. NMR analysis

[0106] 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 spectrometer (Bruker) was used to analyze the sample at 25 °C. 1 H, 13 C, DEPT-135, COSY, HSQC, HMBC, ROESY information. Results are shown in Table 2. Figures 3-5

[0107] Finally, it can be determined that the structure of the compound PGP-1 prepared in Example 1 is as follows:

[0108] ;

[0109] wherein m+n=9, and m and n are both >0.

[0110] Example 4

[0111] Immunomodulatory activity of PGP-1

[0112] 1. Experimental materials

[0113] RAW264.7 macrophages were purchased from China Typical Culture Collection Center; Ophiopogon japonicus polysaccharide (OGP), Asparagus cochinchinensis polysaccharide (ACP) and Dendrobium polysaccharide (DDP) reported to have immunomodulatory activity were used as control groups in the NO release experiment. Ophiopogon japonicus polysaccharide (OGP) was extracted according to the method described in the second page of the second part of the literature

[0114] Comparative studies on the immunoregulatory effects of three polysaccharides using high content imaging system; Asparagus cochinchinensis polysaccharide (ACP) was extracted according to the method described in the second page of the literature Asparagus cochinchinensis polysaccharide: structure, immunomodulatory activity and in vitro antioxidant activity; Dendrobium polysaccharide (DDP) was extracted according to the method described in the second page of the literature

[0115] ​A novel polysaccharide from Dendrobium devonianum serves as a TLR4 agonist for activating macrophages》second page second part of the method extraction obtained.

[0116] Three literature specific information:

[0117] 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.

[0118] ②Mu D, Saren Gao, Bao Liang, et al. Structure, immunomodulatory activity and in vitro antioxidant activity of asparagus polysaccharide [J]. Chinese Journal of Food Science, 2022, 22(8): 51-60. DOI: 10.16429 / j.1009-7848.2022.08.006.

[0119] 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.

[0120] Instrument reagent, see Table 4.

[0121] Table 4 Instrument reagent

[0122] .

[0123] 2. Cell culture, subculture, cryopreservation and recovery

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 3. Cell viability detection

[0128] 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). Meanwhile, the LPS (1 μg / mL) group was used as a positive control. 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 0 μg / mL group was used as a control.

[0129] 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 proliferative activity on RAW264.7 cells.

[0130] 4. NO, immune factor release, and phagocytic capacity detection

[0131] 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.

[0132] 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.

[0133] 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.

[0134] Example 5

[0135] PGP-1's antitumor activity

[0136] Human liver cancer cells HepG2 and human lung cancer cells A549 were purchased from the American Type Culture Collection (ATCC) in the United States.

[0137] CytoFLEX S flow cytometer (Beckman, USA), and the other reagents and instruments are the same as those in Table 4.

[0138] 1. Cell viability detection

[0139] 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 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 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 at 450 nm wavelength using a multifunctional microplate reader.

[0140] See 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.

[0141] According to Figure 9 A, B, C, Ophiopogon, Asparagus, and Dendrobium all have similar inhibitory effects on A549 cell proliferation, 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 Dendrobium homopolysaccharide being the most effective, but PGP-1 had a stronger inhibitory effect on HepG2 cell proliferation.

[0142] 2. Apoptosis detection

[0143] After treating HepG2 and A549 cells (5 x 10 5 After 72 hours of treatment with PGP-1, the harvested cells were fixed in 70% ethanol in PBS at -20°C for 7 hours. Annexin V / PI assay was used to determine whether PGP-1 induced apoptosis in cancer cells. See 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 HepG-2 cell apoptosis reached 36.88%, and the percentage of A549 cell apoptosis reached 36.08%.

[0144] 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.

[0145] 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.

[0146] 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. The use of a polysaccharide in a balloon in the preparation of an anti-tumor drug, characterized in that, The tumor is liver cancer or lung cancer, and the luffah polysaccharide is named PGP-1, and the structural formula is shown in formula (A): （A）， In the formula, m+n=9, m and n are both greater than 0, and the molecular weight is less than 3 kDa.

2. Use according to claim 1, wherein The preparation method of the luffah polysaccharide PGP-1 comprises the following steps: S1. After the luffah root is dried, it is ground, extracted, centrifuged, rotary evaporated, alcohol precipitated, deproteinated and freeze-dried to obtain a crude polysaccharide extract; S2. The crude polysaccharide extract is dissolved into a solution of 15 mg / mL, and is purified by a DEAE-52 cellulose ion exchange column; S3. The purified component is dialyzed by using a 1000 Da filter membrane, and the concentrated solution after dialysis is collected, dried to obtain the required luffah polysaccharide PGP-1.

3. Use according to claim 2, wherein the compound is ###0002### The specific method of step S1 is as follows: S11. After the luffah root is ground, it is soaked in acetone at room temperature for 1.5 days, and then dried at 45℃ to obtain pretreated luffah powder; S12. The luffah powder is mixed with pure water at a ratio of 1g: (10-20) mL, and extracted at a temperature of 60-100℃ for 60-120 min, filtered, and extracted repeatedly for 1-3 times, and the filtrate is 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 liquid 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 and Sevage reagent 5:1, the Sevage reagent is added to the polysaccharide extract, stirred, and after standing and layering, the third layer solution at the bottom is taken, and the Sevage reagent is removed by rotary evaporation at 50℃, and the concentrated solution is freeze-dried to obtain a crude polysaccharide extract.

4. The use according to claim 3, wherein the compound is ###0002### In the step S12, the luffah powder is mixed with pure water at a ratio of 1g: 15 mL, extracted at a temperature of 75℃ for 100 min, filtered, and extracted repeatedly for 1-3 times, and the filtrate is combined, centrifuged and rotary evaporated.

5. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The luffah polysaccharide as claimed in claim 1 and a pharmaceutically acceptable carrier.

6. The medicament according to claim 5, wherein The luffah polysaccharide accounts for 60-90% of the total mass or total volume of the medicine.

7. The medicament according to claim 5, wherein The pharmaceutically acceptable carrier comprises one or several carriers with the functions of excipients, stabilizers, antioxidants, colorants, diluents and sustained-release agents.

8. The medicament according to claim 5, wherein The medicine is any one of injection, tablet, granule, pill, capsule, suspension or emulsion.

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