Sulfated polygonatum sibiricum polysaccharide, a preparation method thereof and application thereof in preparation of a medicine for treating liver cancer

Abstract

This invention belongs to the field of antitumor drugs, disclosing a sulfated Polygonatum sibiricum polysaccharide, its preparation method, and its application in the preparation of drugs for treating liver cancer. The invention uses dried Polygonatum sibiricum rhizome as raw material, purifies it to obtain Polygonatum sibiricum polysaccharide, and then uses a sulfur trioxide-pyridine complex as a sulfation reagent to perform sulfation modification, thus preparing sulfated Polygonatum sibiricum polysaccharide. Sulfated Polygonatum sibiricum polysaccharide significantly improves water solubility, optimizes molecular spatial conformation and charge distribution, and induces cancer cell apoptosis by damaging mitochondrial membrane potential, increasing intracellular ROS levels, and downregulating the expression of key phosphorylated proteins in the PI3K / Akt / mTOR signaling pathway. In animal experiments, it effectively inhibited tumor growth without causing significant systemic toxicity in mice, demonstrating good in vivo biocompatibility. Therefore, it has important application value.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drugs, specifically relating to a sulfated Polygonatum polysaccharide, its preparation method, and its application in the preparation of drugs for treating liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC), originating from hepatocytes, accounts for approximately 90% of primary liver cancers and is the leading histological type, exhibiting the most severe malignant symptoms. Due to the lack of early symptoms in HCC, most patients are diagnosed at an advanced stage. Advanced HCC is typically treated with a combination of surgical resection, arterial chemoembolization, radiofrequency ablation, broad-spectrum tyrosine kinase inhibitors, immunotherapy, and anti-angiogenic therapy. However, liver cancer exhibits significant resistance to these traditional treatments, with a high likelihood of disease recurrence and no significant prolongation of patient survival. Furthermore, the high incidence of severe drug-related adverse events and intolerance to toxicity in patients with liver failure pose significant obstacles to achieving optimal efficacy with traditional cytotoxic chemotherapy drugs. With years of research into HCC, many advanced treatment methods have been continuously discovered.

[0003] Polysaccharides are high molecular weight compounds composed of ten or more monosaccharides linked by glycosidic bonds. As one of the main active ingredients of natural plants, they have relatively few toxic side effects and possess numerous pharmacological activities, such as anti-tumor, hypoglycemic, antioxidant, and immunomodulatory effects. They have become an important candidate resource for the discovery of novel and safe anti-tumor drugs and functional foods.

[0004] Polygonatum is a perennial herb belonging to the genus Polygonatum in the family Liliaceae. Its rhizome has the effects of tonifying the kidneys and replenishing essence, enhancing immunity, regulating lipids, lowering blood sugar, and lowering blood pressure. Polygonatum has been used for over two thousand years and is a well-known medicinal herb in traditional Chinese medicine. Its fleshy rhizome has the effects of tonifying qi and nourishing yin, strengthening the spleen and moistening the lungs, and tonifying the kidneys and promoting body fluids. Polysaccharides are one of its main active ingredients. Studies have shown that Polygonatum polysaccharides have various effects such as anti-oxidation, anti-diabetic, anti-tumor, anti-fatigue, anti-inflammatory, lipid-lowering, immune-enhancing, and cardiovascular-improving effects. However, the relatively low efficacy of natural medicines limits its application. Summary of the Invention

[0005] The purpose of this invention is to provide a sulfated Polygonatum polysaccharide and its preparation method. The sulfated Polygonatum polysaccharide, through sulfation chemical modification, significantly improves its water solubility and enhances its biological activity. It can effectively intervene in the PI3K / Akt / mTOR signaling pathway, inhibit the proliferation of liver cancer cells, and promote cell apoptosis, thus exhibiting good anti-liver cancer effects.

[0006] Another objective of this invention is to provide an application of sulfated Polygonatum polysaccharide in the preparation of drugs for treating liver cancer.

[0007] The specific technical solution of this invention is as follows:

[0008] This invention provides a method for preparing sulfated Polygonatum polysaccharide, the method comprising the following steps:

[0009] 1) Disperse Polygonatum polysaccharide powder in a solvent and react it with a sulfation reagent;

[0010] 2) After the sulfation reaction in step 1) is completed, the reaction product is cooled to room temperature, and then neutralized, dialyzed, concentrated and freeze-dried to obtain crude sulfated Polygonatum polysaccharide derivatives (s-PSPs).

[0011] 3) The crude sulfated Polygonatum polysaccharide derivative was eluted, concentrated and freeze-dried to obtain sulfated Polygonatum polysaccharide (s-PSP).

[0012] In step 1), the liquid-to-solid ratio of the Polygonatum polysaccharide powder to the solvent is 8-12 mg / mL.

[0013] In step 1), the solvent is one or more of pyridine or n-butanol.

[0014] In step 1), the sulfation reagent is one or more of sulfur trioxide-pyridine complex, chlorosulfonic acid, or concentrated sulfuric acid.

[0015] In step 1), the sulfation reaction is carried out at a temperature of 70-90°C for 1-3 hours.

[0016] In step 1), the mass ratio of the Polygonatum polysaccharide powder to the sulfation reagent is 1:2~4.

[0017] In step 2), the dialysis time is 48-72 hours and the molecular weight cutoff is 1000 Da.

[0018] In step 3), the elution is performed using a Sephadex G-25 gel chromatography column with deionized water.

[0019] This invention provides a sulfated Polygonatum polysaccharide prepared using the above-described preparation method.

[0020] The degree of sulfation substitution of the sulfated Polygonatum polysaccharide is 1.2-1.45.

[0021] This invention provides an application of the sulfated Polygonatum polysaccharide, which can inhibit the PI3K / Akt / mTOR pathway, thereby exerting an anti-proliferative effect on liver cancer cells, and can be used to prepare drugs for treating liver cancer.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] Sulfation significantly optimized the physicochemical properties and bioactivity of Polygonatum polysaccharides, successfully preparing sulfated Polygonatum polysaccharide (s-PSP) with enhanced anti-hepatocellular carcinoma effects. This provides important theoretical and experimental support for the high-value development of Polygonatum polysaccharides, the research and development of anti-tumor functional foods and candidate drugs. Sulfation significantly improved water solubility, reduced molecular weight, and maintained the integrity of the basic sugar chain structure, overcoming the limitations of poor water solubility and insufficient activity of natural n-PSP. In vitro experiments showed that s-PSP significantly inhibited the proliferation of HepG2 hepatocellular carcinoma cells compared to n-PSP, and significantly reduced tumor volume. Mechanistic studies revealed that s-PSP induces cancer cell apoptosis by damaging mitochondrial membrane potential and increasing intracellular ROS levels, thereby inhibiting the PI3K / Akt / mTOR signaling pathway and resulting in significant necrosis and increased apoptosis in tumor tissue. Furthermore, in vivo administration of s-PSP had no significant adverse effects on mouse body weight or organ indices, demonstrating good biosafety and low toxicity. Attached Figure Description

[0024] Figure 1 A is a schematic diagram of the n-PSP and s-PSP extraction process; B is the n-PSP elution curve; C is the Sephadex G-100 elution curve; D is the UV spectrum of n-PSP; E is the HPGPC spectrum of n-PSP; F is the molecular weight distribution spectrum of n-PSP; and G is the monosaccharide composition analysis of n-PSP.

[0025] Figure 2 In the image, A is the GC chromatogram of PMAAs obtained by methylation derivatization of n-PSP, and B and F are the mass spectrometry fragment images corresponding to each sugar residue;

[0026] Figure 3 Here is the NMR spectrum of n-PSP, where Figure 3 In the spectrum, A is the 1H spectrum, B is the 13C spectrum, C is the DEPT-135 spectrum, D is the HSQC spectrum, E is the COSY spectrum, and F is the HMBC spectrum.

[0027] Figure 4 The prediction structure for n-PSP;

[0028] Figure 5 In the figure, A is the elution curve of s-PSP, B is the elution curve of Sephadex G-25, and C is the ultraviolet spectrum of s-PSP.

[0029] Figure 6 The NMR spectrum of s-PSP is shown below. Figure 6 In the image, A is the 13C spectrum and B is the DEPT-135 spectrum.

[0030] Figure 7In the diagram, A is the overall XPS spectrum of n-PSP and s-PSP, B is the S2p peak diagram of n-PSP and s-PSP, C is the C1s peak diagram of n-PSP, D is the O1s peak diagram of n-PSP, E is the C1s peak diagram of s-PSP, and F is the O1s peak diagram of s-PSP.

[0031] Figure 8 The effects of n-PSP and s-PSP on HepG2 cell proliferation were detected using the CCK8 assay. Figure 8 In the first case, A was treated with n-PSP and s-PSP for 24 hours, and B was treated with n-PSP and s-PSP for 48 hours.

[0032] Figure 9 Graphs showing the quantitative analysis of wound healing and cell migration rates;

[0033] Figure 10 Image A shows flow cytometry analysis of apoptosis using Annexin V-FITC / PI staining; image B shows mitochondrial membrane potential assessment using JC-1 staining; image C shows intracellular ROS levels measured using DCFH-DA staining; image D shows representative Western blot images of PI3K, Akt, mTOR, and their phosphorylated proteins in tumor cells; image E shows quantitative analysis of PI3K and its phosphorylated protein expression levels; image F shows quantitative analysis of Akt and its phosphorylated protein expression levels; and image G shows quantitative analysis of mTOR and its phosphorylated protein expression levels.

[0034] Figure 11 A is a schematic diagram of mouse model establishment and treatment; B is a picture of mouse tumor; C is a picture of tumor section with HE staining; D is a picture of mouse weight change; E is a picture of mouse tumor volume change.

[0035] Figure 12 TUNEL staining image of tumor tissue;

[0036] Figure 13 This is an elemental result analysis diagram for Example 1. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0039] Comparative Example 1

[0040] A method for extracting Polygonatum polysaccharide powder includes the following steps:

[0041] 1) Disperse the Polygonatum rhizome powder in water at a solid-liquid ratio of 50 mg / mL, and extract the Polygonatum rhizome powder at 90℃ for 120 min each time, repeating 3 times to obtain the supernatant after extraction.

[0042] 2) The supernatant after extraction was concentrated to 20% of its original volume by rotary evaporator under reduced pressure at 60 °C to obtain a concentrate. Anhydrous ethanol with a volume of 4 times that of the concentrate was added to the concentrate, and the mixture was precipitated at 4 °C for 12 h. The precipitate was collected by centrifugation.

[0043] 3) The precipitate was dissolved in distilled water and deproteinized using Sevag reagent with a volume ratio of chloroform and butanol of 4:1. Then, it was decolorized using macroporous adsorption resin D101. The resulting aqueous fraction was dialyzed in distilled water at 4°C for 72 h. The dialysate was freeze-dried at -80°C for 72 h to obtain crude Polygonatum polysaccharide powder.

[0044] 4) Dissolve 200 mg of crude Polygonatum polysaccharide powder in 40 mL of deionized water, filter through a 0.22 μm microporous membrane, and load the sample onto a DEAE-52 anion exchange chromatography column. Elute with a gradient of deionized water and 0.1, 0.2, 0.3, 0.4 and 0.5 mol / L NaCl solutions at a flow rate of 0.8 mL / min. Collect the elution products for 5 min per tube.

[0045] 5) The eluent was concentrated to 20% of its original volume by a rotary evaporator under reduced pressure at 60°C. It was then dialyzed in deionized water for 48 hours using a 1000 Da dialysis bag, with the water changed every 4 hours. Finally, it was freeze-dried at -80°C for 72 hours to obtain preliminarily purified Polygonatum polysaccharide.

[0046] 6) Weigh 40 mg of preliminarily purified Polygonatum polysaccharide, dissolve it in 4 mL of deionized water, filter it through a 0.22 μm microporous membrane, and load it onto a Sephadex G-75 gel column. Elute isocratically with deionized water at a flow rate of 0.8 mL / min, collecting the eluent for 5 min per tube. Detect the eluent fractions using HPGPC. Combine the fractions with the same retention time and symmetrical peak shape, concentrate them, and freeze-dry them to obtain Polygonatum polysaccharide powder, labeled as n-PSP.

[0047] Example 1

[0048] A method for preparing sulfated Polygonatum polysaccharide includes the following steps:

[0049] 1) Stir at room temperature, completely dissolve 100 mg of n-PSP prepared in Comparative Example 1 in 10 mL of anhydrous pyridine, add 300 mg of sulfur trioxide-pyridine complex, and stir the mixture at 80°C for 2 h to carry out sulfation reaction;

[0050] 2) After the reaction was completed, the reaction mixture was cooled to room temperature by an ice-water bath and neutralized by adding 1M NaOH solution. The resulting solution was dialyzed for 72 hours using a 1000 Da dialysis bag, concentrated, and freeze-dried to obtain crude sulfated Polygonatum polysaccharide derivative.

[0051] 3) The s-PSPs powder was eluted with deionized water using a Sephadex G-25 gel chromatography column. The main fraction was collected, concentrated, and lyophilized to obtain sulfated Polygonatum polysaccharide, which was labeled as s-PSP.

[0052] Test Example 1

[0053] UV analysis:

[0054] Weigh n-PSP and s-PSP using a balance, and prepare 1 mg / mL solutions with deionized water. Preheat the UV-Vis spectrophotometer for 30 minutes, and perform a full-wavelength UV scan in the range of 190–900 nm. Zero the meter with distilled water, record the absorbance values, and determine the characteristic absorption peaks. The test results for n-PSP are as follows: Figure 1 As shown in Figure D, the UV-Vis absorption spectrum of n-PSP shows no absorption at 260 and 280 nm, indicating that n-PSP contains little or no nucleic acid and protein; the test results for s-PSP are as follows... Figure 5 As shown in Figure C, the UV absorption spectrum of s-PSP has a typical absorption peak at 200 nm, indicating that the basic structure of the modified polysaccharide has not changed. After adding the sulfate group to s-PSP, a new absorption peak appeared near 260 nm, indicating that it was caused by the n→π* transition induced by S=O.

[0055] Molecular weight analysis:

[0056] The molecular weight of n-PSP was determined by gel permeation chromatography-differential refractive index-multi-angle light scattering. The sample was dissolved in a 0.1M NaNO3 aqueous solution containing 0.02wt% NaN3 to a final concentration of 1mg / mL, and then filtered through a 0.45μm filter before being analyzed.

[0057] The chromatographic system used was a gel chromatography-differential chromatography-multi-angle laser light scattering system. The liquid chromatography system was a Thermo U3000, the differential detector was a Wyatt technology Optilab T-rEX, and the laser light scattering detector was a Wyatt technology DAWN HELEOS II. The specific chromatographic column and elution conditions were as follows: 300×8 mm gel size exclusion columns Ohpak SB-805 HQ and Ohpak SB-803 HQ were used in series, the column temperature was 45℃, the injection volume was 100 μL, the mobile phase was 0.1 M NaNO3 aqueous solution containing 0.02 wt% NaN3, the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min. The chromatographic data were processed using ASTRA 6.1 software.

[0058] The molecular weight of s-PSP was determined by high performance gel permeation chromatography. 5 mg of the sample from Example 1 was weighed and added to 1 mL of 0.05 M sodium chloride solution to prepare a 5 mg / mL test sample solution. The test sample solution was centrifuged at 8000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm microporous membrane. The sample was transferred to a 2 mL injection bottle and analyzed using high performance gel permeation chromatography column chromatography.

[0059] The n-PSP test results described in Comparative Example 1 are as follows: Figure 1 As shown in Figures E, F, and G, n-PSP in Figure E exhibits a symmetrical single peak with a retention time of 10.581 min and a purity of 99%, with a stable baseline, indicating the homogeneity of the purified n-PSP; Figure 1 As shown in Figure F, the SEC-MALLS-RI system analysis results show that the weight-average molecular weight and number-average molecular weight of n-PSP are 5041 and 4479 Da, respectively, and the polydispersity index is 1.125, proving that n-PSP is a polysaccharide with a narrow molecular weight distribution.

[0060] The test results of S-PSP are as follows Figure 5 As shown in Figure A, the average molecular weight of s-PSP is 1980 Daltons, the number average molecular weight is 1435 Daltons, and the polydispersity index is calculated to be 1.38.

[0061] Monosaccharide composition analysis of n-PSP:

[0062] Weigh 5 mg of n-PSP sample, add 1 mL of 2 M trifluoroacetic acid solution, and hydrolyze at 60 or 121 °C for 2 h. Dry under nitrogen, wash 2-3 times with methanol, dry under nitrogen again, dissolve in sterile water to obtain the hydrolyzed monosaccharide, transfer to a chromatographic vial for analysis, and use Thermo ICS 5000. +The ion chromatography system is equipped with a 150×3.0mm, 10μm CarboPac™ PA20 liquid chromatography column. The n-PSP monosaccharide components are analyzed and detected using an electrochemical detector. The injection volume is controlled at 5μL, the mobile phase A is H2O, the mobile phase B is 0.1M NaOH, and the mobile phase C is 0.1M NaOH and 0.2M NaAc. The flow rate is 0.5mL / min, and the column temperature is 30℃.

[0063] Using monosaccharides as standards, the GC-MS ion chromatograms of n-PSP, monosaccharides hydrolyzed at 60℃, and monosaccharides hydrolyzed at 121℃ were compared. Among these, n-PSP... Figure 1 The α-line of G, a monosaccharide hydrolyzed at 60℃, such as Figure 1 The b line of G, monosaccharides hydrolyzed at 121℃, such as Figure 1 The c-line of G showed that n-PSP is mainly composed of fructose and glucose, with a molar ratio of 93.25:6.75.

[0064] Methylation analysis:

[0065] n-PSP was methylated in anhydrous DMSO using NaOH and CH3I. The methylated polysaccharide was then hydrolyzed with 2 M TFA at 100°C for 4 hours. The product was reduced with NaBH4 and then acetylated with acetic anhydride at 100°C for 2.5 hours to obtain partially methylated sugar alcohol acetyl ester derivatives (PMAAs).

[0066] PMAAs were extracted with dichloromethane and analyzed by gas chromatography-mass spectrometry. The test results are as follows: Figure 2 As shown in AF, n-PSP, after being methylated, hydrolyzed, reduced, and acetylated by iodomethane, becomes... Figure 2 As shown in Figure A, five key peaks appeared in the GC chromatogram. The monosaccharide composition results indicate that n-PSP consists of the furanose form of fructose and the pyranose form of glucose. Therefore, after analysis of the MS fragment, as shown... Figure 3As shown in the BF diagram, each peak was identified; the prominent peaks at 21.87 and 22.07 min were inferred to be 2,5-di-O-acetyl-1,3,4,6-tetra-O-methylmannitol / glucitol, indicating the presence of terminal fructose; furthermore, the high-response peaks at 26.26 and 26.36 min were 1,2,5-tri-O-acetyl-3,4,6-tri-O-methylmannitol / glucitol, indicating the presence of fructose linked at positions 1 and 2. Additionally, peaks at 26.05 and 30.34 min were also observed. The relative intensity peaks at min 27.29 were 2,5,6-tri-O-acetyl-1,3,4-tri-O-methylmannitol / glucitol and 1,2,5,6-tetra-O-acetyl-3,4-di-O-methylmannitol / glucitol, indicating the presence of fructose linked at positions 2,6 and 1,2,6, respectively. The small peak at min 27.29 was inferred to be 1,5,6-tri-O-acetyl-2,3,4-tri-O-methylglucitol, indicating the presence of trace amounts of glucose linked at position 1,6.

[0067] Nuclear magnetic resonance analysis:

[0068] 40 mg of the samples from Example 1 and Comparative Example 1 were accurately weighed and dissolved in 0.5 mL of 99.9% deuterium water. The 1H and 13C NMR spectra and two-dimensional NMR spectra were recorded at 600 MHz using a JEOL JNM-ECZ600R / S1 spectrometer (Japan). The data were analyzed using MestReNova 6.1.0-6224 software.

[0069] The test results of Comparative Example 1 are as follows Figure 3 As shown in Figure AF, the 1H NMR spectrum indicates that the proton signal is concentrated in the δ 3.3–5.5 range. Figure 3 As shown in Figure A, the 13C NMR spectrum signal is concentrated in the δ 55-110 region, with no peak signal between δ 150-220, indicating that n-PSP does not contain uronic acid such as... Figure 3 As shown in B.

[0070] Four strong signals at δ 106.11, 106.57, 106.71, and 106.88 were found in the anomeric carbon region, but they disappeared in the DEPT-135 spectrum. Figure 2 As shown in C, this indicates that all four anomers are quaternary carbons. Furthermore, no hydrogen-related signals were observed in the HSQC spectrum. Figure 2As shown in Figure D, based on the monosaccharide composition and methylation results, the anomeric carbon signals δ 106.57, 106.88, 106.11, and 106.71 can be inferred to be the C-2 signals of β-D-Fruf-(2→, →6)-β-D-Fruf-(2→, →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→) residues, and are respectively labeled as residues A, B, C, and D. Simultaneously, the signals δ 65.27 and 66.82 in the DEPT-135 spectrum are inverted peaks, identified as the C-1 and C-6 signals of fructose. Since C-1 is correlated with the HSQC at 3.78 ppm, 3.78 ppm is considered the H-1 signal; similarly, since C-6 is correlated with the HSQC at 3.71 ppm, 3.71 ppm is considered the H-6 signal. From H-6 to 4.18... ppm such as Figure 2 The COSY correlation shown in E indicates that 4.18 ppm is considered the H-5 signal. From H-6 to 71.80 ppm, as shown... Figure 2 The HMBC correlation shown in Figure F, along with the COSY correlation from 4.18 ppm to 3.92 ppm and the HSQC correlation from 3.92 ppm to 71.80 ppm, indicate that 71.80 ppm and 3.92 ppm are C-4 and H-4 signals, respectively. 4.15 ppm shows a COSY correlation with H-4 and an HMBC correlation with C-1, indicating that 4.15 ppm is an H-3 signal. Therefore, based on the HSQC correlation between 4.15 ppm and 77.09 ppm, 77.09 ppm is a C-3 signal.

[0071] Sugar residue E: →6)-α-D-Glcp-(1→, given that the anodic carbon signal of residue E is 95.08 ppm and the anodic carbon chemical shift is less than 100 ppm, consistent with the chemical shift range of a typical α-glycosidic bond, residue E is determined to be a glucose residue with an α configuration. The H-1 chemical shift of sugar residue E, determined by 1H NMR, is δ 5.36 ppm. The H-2 shift of residue E was determined by the cross peaks δ 5.36 / 3.51 ppm in the COSY spectrum. Following the same method, the H-3 to H-6 signals can be determined sequentially using the COSY spectrum, i.e., the H-3 to H-6 chemical shifts of sugar residue E are assigned to δ 3.97 ppm, δ 3.65 ppm, δ 3.83 ppm, and δ 4.06 ppm, respectively. H-2 has a signal at 73.80 ppm in the HSQC spectrum, therefore 73.80... ppm is the C-2 signal. H-3 and 3.65ppm have a COSY correlation signal, and H-3 and 69.82ppm have an HMBC correlation signal, thus determining that 3.65ppm is the H-4 signal, 69.82ppm is the C-4 signal, H-4 and 72.48ppm have an HMBC correlation signal, thus determining that 72.48ppm is the C-5 signal, and H-5 and 66.50ppm have an HMBC correlation signal, thus determining that 66.50ppm is the C-6 signal.

[0072] In summary, the structure of Comparative Example 1 was determined to be a branched fructan, with its main chain composed of →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, β-D-Fruf-(2→, and →6)-α-D-Glcp-(1→) residues, among which →1)-β-D-Fruf-(2→) linkages are the most common. Branching units were identified as terminal β-D-Fruf-(2→ and →2)-β-D-Fruf-(6→), which are linked at the C-6 position of the →1,6)-β-D-Fruf-(2→) residues in the main chain; the predicted structure of n-PSP is as follows. Figure 4 As shown.

[0073] The test results of the s-PSP prepared in Example 1 are as follows: Figure 6 As shown in Figures A and B, the s-PSP's 13 The C NMR signal is concentrated in the δ 55–110 region, with a strong signal observed in the δ 90–110 region of the anomeric carbon, but this signal disappears in the DEPT-135 spectrum. This indicates that the anomeric carbon is a quaternary carbon, exhibiting chemical shift characteristics of the carbon on the fructose ring. Compared to the DEPT-135 spectrum of n-PSP, ... Figure 3 B, Figure 6The B s-PSP spectrum showed a characteristic carbon signal specific to sulfation in the region δ≈68–75 ppm. This signal corresponds to the carbon after the polysaccharide backbone hydroxyl group (–OH) is replaced by a sulfate group (–OSO3⁻). Due to the strong electron-withdrawing effect of –OSO3⁻, the electron cloud density of the carbon nucleus decreases, the deshielding effect is enhanced, and the chemical shift shifts to a lower field, which is a direct indicator of the successful introduction of the sulfate group. At the same time, the splitting and intensity changes of the backbone carbon signal also reflect the change in the spatial conformation of the polysaccharide after the introduction of the sulfate group, which is indirect evidence of successful modification. The C-6 carbon signal shifted from δ≈66.82 ppm in n-PSP to δ≈68.81 ppm in s-PSP, confirming that the C-6 hydroxyl group has undergone sulfation substitution.

[0074] XPS Analysis:

[0075] After compressing 5 mg of sample into a tablet, attach it to the sample tray and place the sample into the sample chamber of the Thermo Scientific K-Alpha XPS instrument. The pressure in the sample chamber should be less than 2.0 × 10⁻⁶. -7 At mbar, the sample is sent into the analysis chamber with a spot size of 400 μm, an operating voltage of 12 kV, and a filament current of 6 mA. The full spectrum scan pass energy is 150 eV with a step size of 1 eV, and the narrow spectrum scan pass energy is 50 eV with a step size of 0.1 eV.

[0076] Test results are as follows Figure 7 As shown in AF, from Figure 7 As can be seen from A, n-PSP does not contain the S element, while s-PSP contains selected elements, namely O1s at 532.38 eV, C1s at 284.88 eV, and S2p at 168.48 eV; Figure 7 The BE image shows the magnified XPS spectrum. Figure 7 The peak at 168.48 eV in B is a characteristic peak of S2p, indicating that the sulfur atom has a valence state of S. 6+ The peak at 531.58 eV originates from the O1s group in the hydroxyl group. Figure 7 As shown in D, n-PSP may contain four types of carbon atoms, such as Figure 7 As shown in Figure C: the peak at 287.48 eV originates from the C1s of the carbonyl group, the peak at 286.18 eV originates from the C1s of the CO bond, and the peaks at 284.88 eV and 283.38 eV are attributed to the C1s of the C-C or CH bonds. n-PSPs may contain three types of oxygen atoms, such as... Figure 7 As shown in Figure D: the peak at 532.38 eV originates from the O1s of the carbonyl group, and the peak at 531.58 eV originates from the O1s of the CO bond. After sulfation, the carbon signal at 284.88 eV is significantly weakened and shifts to 285.28 eV, indicating a COS carbon signal. Figure 7 As shown in E. Therefore, it can be deduced that -SO3 - It exists in s-PSP.

[0077] Test Example 2

[0078] The effects of n-PSP and s-PSP on HepG2 cell proliferation were detected using the CCK8 assay.

[0079] The specific procedure is as follows: HepG2 cells with good growth and a confluence rate of 90% were digested with trypsin and then prepared into 5×10⁶ cells using complete culture medium. 4 Single-cell suspensions of cells / mL were seeded into 96-well plates and cultured at 37°C and 5% CO2 for 24 h. After cell adhesion, the supernatant was discarded, and 100 μL of serum-free MEM medium was added to prepare polysaccharide solutions with mass concentrations of 0, 100, 200, 300, 400, and 500 μg / mL of BSA, n-PSP, and s-PSP. Each group was replicated in 5 groups. The cells were incubated at 37°C and 5% CO2 for 24 h or 48 h. After discarding the supernatant, 10 μL of Solarbio CCK8 reagent was added to each well and the cells were cultured for another 2 h. The absorbance (OD) value at 450 nm was measured using a microplate reader, and the average value was used to plot a growth curve.

[0080] Experimental results are as follows Figure 8 As shown, at a dose of 500 μg / mL, after treatment with sulfated Polygonatum polysaccharide for 24 h and 48 h, the survival rate of HepG2 liver cancer cells decreased to approximately 50% and 40%, respectively, both significantly lower than that of the unmodified Polygonatum polysaccharide group at the same time point; while the inhibition rate of the unmodified polysaccharide was less than 20% at both 24 h and 48 h, indicating that sulfated modification can significantly enhance the in vitro proliferative inhibitory activity of Polygonatum polysaccharide against liver cancer cells, and this effect is significantly time- and concentration-dependent.

[0081] Annexin V FITC / PI double staining method for detecting cell apoptosis:

[0082] 5×10 per hole 5 Two HepG2 cells were seeded into each well of a 6-well plate (2 mL per well). Cells were treated with sulfated Polygonatum sibiricum polysaccharide at concentrations of 50, 100, and 200 μg / mL for 24 h. Corresponding negative and blank wells were prepared. After treatment, the cells were washed 2-3 times with PBS, digested with trypsin (without EDTA), and the remaining cells were collected in centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was carefully aspirated to obtain a cell pellet. 500 μL of 1× Binding Buffer was added to the cell pellet, and the cells were gently pipetted, vortexed, and resuspended. Cells were counted and prepared to a 1×10⁻⁶ plate. 5100 μL of a high-density cell suspension was mixed with a buffer, followed by 5 μL of Annexin V-FITC. The mixture was incubated in the dark at room temperature for 15 min. 5 μL of PI was added 5 min before detection. The mixture was filtered through a 300-mesh sieve and then analyzed by flow cytometry.

[0083] The results are as follows Figure 10 As shown in Figure A, the compound significantly induced apoptosis in HepG2 cells. The apoptosis-inducing effect was significantly enhanced at medium and high concentrations of the compound.

[0084] Mitochondrial membrane potential analysis (MMP):

[0085] HepG2 cells in logarithmic growth phase were selected, with a cell density of 1×10⁻⁶. 5 Cells were seeded at a density of 100 cells / mL in 6-well plates and incubated overnight in a CO2 incubator until the cells were stable. After treating the cells with sulfated Polygonatum sibiricum polysaccharide at concentrations of 50, 100, and 200 μg / mL for 24 h, the cells were collected, centrifuged at 1000 rpm for 5 min, and washed three times with PBS. 500 µL of pre-prepared JC-1 staining working solution was added to each tube and gently shaken to ensure the probe fully contacts the cells. The cells were incubated in the dark for 20 min. Finally, the cells were washed twice with JC-1 staining buffer, and 0.2 mL of anti-fluorescence quencher was added. The fluorescence intensity was quantitatively measured by flow cytometry, and the data were processed using FlowJo.

[0086] Experimental results are as follows Figure 10 As shown in Figure B, s-PSP increases the green fluorescence produced by HepG2 cells, meaning that s-PSP can induce early apoptosis of HepG2 cells in a concentration-dependent manner.

[0087] Experiment to detect reactive oxygen species (ROS) levels:

[0088] HepG2 cells in logarithmic growth phase and in good condition were used at a dose of 2×10⁻⁶. 5 Cells were seeded at the desired density in 6-well plates and allowed to grow overnight to stabilize. The target compound solution at the desired concentration was then added and incubated for 24 h. Cells were collected in 1.5 mL EP tubes, pre-chilled, and centrifuged for 5 minutes. 1 mL of diluted DCFH-DA solution was added to each tube, and the cells were thoroughly resuspended. The tubes were incubated in the dark for 20 minutes, gently inverting every 4 minutes to ensure adequate contact between DCFH-DA and the cells. After incubation, the cells were centrifuged, the solution discarded, and the cells were resuspended in basal medium, washed three times, and sieved. ROS levels in the cells were then analyzed by flow cytometry.

[0089] Experimental results are as follows Figure 10As shown in C, s-PSP can induce upregulation of ROS levels to some extent, which suggests that it may cause HepG2 cell death through a ROS-dependent pathway.

[0090] Wound healing experiment:

[0091] HepG2 cells were seeded into six-well plates and cultured to 90% confluence. Straight scratches were made with a 20 μL pipette tip, washed three times with serum-free DMEM, and then added with complete culture medium and incubated for another period. Microscopic observation and photography were performed at 0 h, 24 h and 48 h of culture. The healing rate was calculated using ImageJ to measure the width of the cells and to assess their migration ability.

[0092] Experimental results are as follows Figure 9 As shown, after 48 h of intervention with 100 μg / mL s-PSP, the scratch gap increased by 35.20% compared with the control group, and the cell density decreased, indicating that s-PSP treatment inhibited cancer cell migration in a dose-dependent and time-dependent manner, revealing the potential of s-PSP to inhibit cancer cell metastasis.

[0093] Western blot analysis

[0094] Total protein was extracted from HepG2 cells using pre-chilled RIPA lysis buffer, supplemented with benzyl sulfonyl fluoride (PMSF) and a phosphatase inhibitor. Protein concentration was quantified using a BCA assay kit. Samples containing 15–30 μg of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in TBST buffer containing 5% skim milk at room temperature for 1 hour. Primary antibodies against PI3K, phosphorylated PI3K, Akt, phosphorylated Akt, mTOR, phosphorylated mTOR, and β-actin were then added, and the membranes were incubated overnight at 4°C. After washing with TBST buffer, the membranes were incubated with enzyme-labeled secondary antibody at room temperature for 1 hour. Protein signals were acquired using an Amersham ImageQuant™ 800 Western blot imaging system, and protein expression levels were quantified using ImageJ software.

[0095] Sulfation Substitution Degree Test

[0096] The degree of sulfation substitution (DS) of the s-PSP was determined by field emission scanning electron microscopy. The s-PSP described in Example 1 was fixed on an aluminum support by a conductive carbon ribbon and coated with a thin gold layer to prevent charging. The atomic percentages of carbon, oxygen, sodium, and sulfur were quantified, and DS was calculated based on the atomic percentages of sulfur and carbon, using the following formula:

[0097] DS = At ​​%S / At %C × 6;

[0098] In the formula, At %S is the atomic percentage of sulfur, At %C is the atomic percentage of carbon, and monosaccharide analysis shows that Example 1 is composed of glucose and fructose. Glucose and fructose are hexoses, so the coefficient in the above formula is taken as 6.

[0099] Test results are as follows Figure 13 As shown, the degree of sulfation substitution in Example 1 is calculated to be 1.39 using the above formula.

[0100] Test Example 3

[0101] Animal experiments:

[0102] Four-week-old SPF-grade male BALB / c-NU nude mice, weighing 15±2 g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All animals were housed under pathogen-free conditions, with the environment controlled at 20-25℃ and relative humidity at 50±5%. Prior to the experiment, all mice were allowed one week to acclimatize to the new environment. Throughout the experiment, they were allowed free access to tap water and fed standard pelleted feed, with a light / dark cycle every 12 hours. All experimental procedures strictly followed the "Guidelines for the Care and Use of Laboratory Animals" and were approved by the Animal Ethics Committee of Wannan Medical College.

[0103] After a 7-day acclimatization period, all mice except the control group received a subcutaneous injection of HepG2 cells into the right axilla, at a dose of 5 × 10⁻⁶ cells. 5 Establish a HepG2 nude mouse tumor-bearing model using one mouse per animal, until the tumor volume reaches approximately 100 mm. 3 Forty-eight mice were randomly divided into six groups of eight each: 1) Control group: injected with saline, without HepG2 cells; 2) Model group: injected with saline and HepG2 cells; 3) 5-fluorouracil (5-Fu) group: injected with 25 mg / kg / day 5-Fu; 4) s-PSP-low dose group (s-PSP-L) group: injected with 100 mg / kg / day s-PSP; 5) s-PSP-medium dose group (s-PSP-M) group: injected with 200 mg / kg / day s-PSP; (6) s-PSP-high dose group (s-PSP-H) group: injected with 400 mg / kg / day s-PSP.

[0104] Throughout the experiment, tumor size was observed and measured every two days using calipers. After the last administration, tumors were weighed and euthanized by cervical dislocation. Tumors, spleens, and livers were carefully collected and weighed immediately for further study. The formula used to calculate tumor volume is as follows: Tumor volume (V) = (Length × Width) 2 ) / 2. The immune organ index is expressed as the ratio of organ weight to body weight.

[0105] Histological observation of tumor tissue was performed by fixing mouse tumor tissue in 10% neutral formaldehyde solution and dehydrating it with graded ethanol solutions. After paraffin embedding, 4µm sections were taken, stained with hematoxylin and eosin, and observed under a light microscope.

[0106] After dewaxing, rehydration, and permeabilization with proteinase K, tumor tissue was fluorescently labeled with TdT enzyme, nuclei were stained with DAPI, and after mounting, the positive rate was randomly counted in three fields under a fluorescence microscope. The positive rate formula is as follows:

[0107] Tunel positivity rate % = (Tunel positive cells / total cells) × 100%.

[0108] Depend on Figure 11 As shown in the tumor images of group B, the tumor volume and weight in the Model group were significantly higher than those in all treatment groups; the quantitative statistical results are as follows. Figure 11 As shown in Figure E, after 15 days of intervention, the tumor volume of each s-PSP dose group decreased in a dose-dependent manner. The tumor volume of the s-PSP-H group was significantly lower than that of the Model group, and the tumor inhibition effect was close to that of the positive control 5-Fu group, suggesting that s-PSP can effectively inhibit the growth of tumors in vivo.

[0109] Hematoxylin and eosin (H&E) staining uses the basic dye hematoxylin and the acidic dye eosin. Chromatin in the cell nucleus turns purple under the influence of hematoxylin, nucleic acids in the cytoplasm turn blue, while components in the cytoplasm and extracellular matrix turn red under the influence of eosin. Therefore, H&E staining was chosen to determine the inhibitory effect and cytotoxicity of 5-fluorouracil and s-PSP on liver cancer cells. Figure 11 As shown in Figure C, the model group mice had a larger number of tumor cells with relatively intact morphology and structure, and the cells were arranged more tightly. The cell nuclei were uniformly stained blue by the dye. After treatment with 5-fluorouracil and s-PSP, the solid tumor cells became loosely arranged, exhibited varying degrees of necrosis, and their cell structures were damaged. The 5-FU group and the high-dose group mice showed more apoptotic cells in their tumor cells. With increasing s-PSP concentration, the damage to solid tumor cells became more severe, with the cells becoming sparsely arranged, and the nuclei showing nuclear condensation and nuclear fragmentation. This indicates that the degree of damage to solid tumor cells caused by s-PSP may be dose-dependent.

[0110] The results of mouse weight changes are shown in Figure 11D: The weight of mice in the Model group showed a slow upward trend as the tumor progressed, while the weight of mice in the 5-Fu group showed a significant decrease, which suggests the toxic effects of chemotherapy drugs; the weight changes of each s-PSP dose group were not significantly different from those in the Control group, indicating that s-PSP effectively inhibited tumors without causing significant systemic toxicity to mice, and has good in vivo biosafety.

[0111] The TUNEL-DAPI double staining method was used to detect cell apoptosis in tumor tissues of each group. Green fluorescence represented TUNEL-positive apoptotic cells, and blue fluorescence represented DAPI-stained cell nuclei. Results showed that only a small amount of scattered green fluorescence was observed in the Model group tumor tissue, indicating a low proportion of apoptotic cells. The green fluorescence signal of each s-PSP dose group gradually increased with increasing dosage, with a significant increase in the number of TUNEL-positive cells and a more dense fluorescence distribution in the s-PSP-H group. The 5-Fu group also showed a strong green fluorescence signal. Figure 12 As shown, the above results indicate that s-PSP can induce tumor cell apoptosis in HCC-bearing mice in a dose-dependent manner, and its pro-apoptotic effect is similar to that of the positive control drug 5-Fu.

[0112] In summary, s-PSP can inhibit tumor growth in HCC-bearing mice in a dose-dependent manner in vivo, induce pathological damage in tumor tissue, and has good biosafety, making it a potential low-toxicity natural active ingredient for liver cancer treatment.

[0113] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0114] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing sulfated polygonatum polysaccharide, characterized in that, The preparation method includes the following steps: 1) Disperse Polygonatum polysaccharide powder in a solvent and react it with a sulfation reagent; 2) After the sulfation reaction in step 1) is completed, the reaction product is cooled to room temperature, and then neutralized, dialyzed, concentrated and freeze-dried to obtain crude sulfated Polygonatum polysaccharide derivative; 3) The crude sulfated Polygonatum polysaccharide derivative was eluted, concentrated and freeze-dried to obtain sulfated Polygonatum polysaccharide.

2. The method of claim 1, wherein the sulfated polygonatum polysaccharide is prepared by the following steps of: In step 1), the solvent is one or more of pyridine or n-butanol. ​ 3. The method for preparing sulfated polygonatum polysaccharide according to claim 1 or 2, characterized in that, In step 1), the liquid-to-solid ratio of the Polygonatum polysaccharide powder to the solvent is 8-12 mg / mL.

4. The method of claim 1, wherein the sulfated polygonatum polysaccharide is prepared by the following steps of: The sulfation reaction described in step 1) is carried out at a temperature of 70-90°C for 1-3 hours. ​ 5. The method of claim 1, wherein the sulfated polygonatum polysaccharide is prepared by the following steps of: In step 1), the sulfation reagent is one or more of sulfur trioxide-pyridine complex, chlorosulfonic acid, or concentrated sulfuric acid. ​ 6. The method for preparing sulfated Polygonatum polysaccharide according to claim 1 or 5, characterized in that, The mass ratio of Polygonatum polysaccharide powder to sulfation reagent in step 1) is 1:2~4.

7. The method for preparing sulfated Polygonatum polysaccharide according to claim 1, characterized in that, The dialysis time described in step 2) is 48-72 hours, and the molecular weight cutoff is 1000 Da.

8. The method for preparing sulfated Polygonatum polysaccharide according to claim 1, characterized in that, In step 3), the elution is performed using a Sephadex G-25 gel chromatography column with deionized water.

9. A sulfated Polygonatum polysaccharide prepared by the preparation method according to any one of claims 1-8, characterized in that, The degree of sulfation substitution of the sulfated Polygonatum polysaccharide is 1.2-1.

45.

10. The use of sulfated Polygonatum polysaccharide as described in claim 9 in the preparation of a drug for treating liver cancer.

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