White cork polysaccharide, its preparation method and use for preparing drugs for treating colorectal cancer

Abstract

The application discloses white gualou polysaccharide, a preparation method thereof and an application of the white gualou polysaccharide in preparing a colorectal cancer treatment drug; the white gualou crude polysaccharide is extracted by a hot water extraction method, and three new polysaccharides are separated and purified from the white gualou by a combined purification process of DEAE-52 cellulose ion exchange chromatography and dextran gel G-200 gel permeation chromatography; the molecular weight, monosaccharide composition, glycosidic bond connection mode, triple helix structure and micro morphology of the white gualou polysaccharide are systematically characterized; the three polysaccharides all show concentration-dependent anti-colorectal cancer activity on an AB zebra fish tumor model constructed by DiI fluorescently labeled HCT116 human colorectal cancer cells; and the application provides a new candidate molecule and application basis for the development of a colorectal cancer treatment drug and related functional food.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry, specifically relating to the isolation, purification, structural characterization, and application in the preparation of anti-colorectal cancer drugs from the traditional Chinese medicine *Ampelopsis japonica*. This invention establishes methods for the preparation and structural analysis of *Ampelopsis japonica* polysaccharides and clarifies their anti-colorectal cancer activity. Background Technology

[0002] While commonly used chemotherapy drugs such as 5-fluorouracil can inhibit tumor cell proliferation and delay disease progression to some extent, long-term use easily induces drug resistance in tumor cells and is accompanied by significant toxic side effects such as bone marrow suppression and gastrointestinal reactions. Therefore, the development of safe and effective anti-colorectal cancer drugs is of great clinical significance.

[0003] Naturally derived polysaccharides are an important source of antitumor active ingredients due to their non-toxicity and good biocompatibility. Studies have shown that the antitumor activity of polysaccharides is closely related to their structural characteristics, including molecular weight distribution, monosaccharide composition, glycosidic bond linkage, and degree of branching. Obtaining high-purity, homogeneous polysaccharides is fundamental for structural characterization and activity studies. However, the separation and purification process of polysaccharides is complex, requiring repeated optimization of extraction and purification conditions to remove impurities and obtain structurally stable polysaccharides with acceptable purity.

[0004] Ampelopsis japonica is a traditional Chinese medicine with effects such as clearing heat and detoxifying, promoting wound healing and tissue regeneration. Modern pharmacological studies suggest that its extracts have certain anti-tumor potential. However, research on the systematic isolation and purification of Ampelopsis japonica polysaccharides, polysaccharide preparation, and their anti-colorectal cancer activity is still lacking.

[0005] Zebrafish models, due to their high homology with the human genome (approximately 84% of human pathogenic genes can be found in zebrafish homologs) and advantages such as rapid reproduction, low breeding costs, and short experimental cycles, have been widely used for rapid screening and activity evaluation of antitumor drugs. Constructing a tumor model by injecting Dil-labeled human colorectal cancer HCT116 cells into the yolk sac of zebrafish can effectively simulate the development and progression of human colorectal cancer, providing an intuitive and efficient in vivo screening platform for evaluating the antitumor efficacy of candidate drugs.

[0006] This invention, through optimizing extraction and separation process parameters, has for the first time purified three novel polysaccharides from *Ampelopsis japonica*. The structures were characterized using a combination of techniques including NMR, IR, and GC-MS. Their antitumor activity was evaluated in a zebrafish colorectal cancer model and compared with the positive control drug 5-fluorouracil, providing a material basis and technical support for the development of novel anti-colorectal cancer drugs. Summary of the Invention

[0007] To address the current technical problems of imperfect separation and purification processes for Gynostemma pentaphyllum polysaccharides, lack of research on pure polysaccharide preparation and fine structure, and unclear anti-colorectal cancer activity, this invention provides three types of Gynostemma pentaphyllum polysaccharides, their preparation methods, and their applications in the preparation of anti-colorectal cancer drugs.

[0008] This invention employs hot water extraction to extract crude polysaccharides from *Ampelopsis japonica*. A combined purification process, combining DEAE-52 cellulose ion exchange chromatography with Sephadex G-200 gel permeation chromatography, was used to isolate and purify three novel polysaccharides from *Ampelopsis japonica* for the first time. Subsequently, their structures were identified using infrared spectroscopy (IR), nuclear magnetic resonance (NMR), high-performance gel permeation chromatography (HPGPC), monosaccharide composition analysis, and methylation-GC-MS analysis. Their anti-colorectal cancer activity was also evaluated, laying the foundation for the application of *Ampelopsis japonica* in the development of anti-colorectal cancer drugs.

[0009] The technical solution of the present invention is as follows:

[0010] Polysaccharides from Gynostemma pentaphyllum: AJP-1a, AJP-2a, AJP-3a;

[0011] AJP-1a has a relative molecular weight of 281 kDa and is composed of →1)-D-Glcp, →3)-D-Glcp-(1→, →4)-D-Glcp-(1→, →6)-D-Glcp-(1→, →3,4)-D-Glcp-(1→, →2,4)-D-Glcp-(1→ and →4,6)-D-Glcp-(1→). It is a homogeneous dextran with a glucose content of 100%.

[0012] AJP-2a has a relative molecular weight of 1435 kDa and is composed of →4)-D-Arap-(1→, →1)-D-Glcp, →4)-D-Glcp-(1→, →6)-D-Glcp-(1→, →3,4)-D-Galp-(1→ and →4,6)-D-Glcp-(1→). It contains 96.65% glucose, 1.68% galactose, and 1.66% arabinose.

[0013] The relative molecular weight of AJP-3a is 251.1 kDa, derived from -(1→)-L-Araf, -(1→)-L-Arap, →4)-L-Arap-(1→,→1)-D-GlcpA,→2)-D-Galp-(1→,→2)-L-Arap-(1→,→4)-D-GlcpA-(1→,→3)-D-Galp-(1→,→6)-D-Ma Composed of np-(1→,→6)-D-Galp-(1→,→2,3)-D-Manp-(1→,→3,4)-D-Manp-(1→,→3,6)-D-GlcpA-(1→ and→3,4,6)-D-Manp-(1→), with a galactose content of 22.79%, mannose content of 20.54%, arabinose content of 42.35%, and glucuronic acid content of 14.3%.

[0014] The present invention uses UV, FT-IR, HPLC, HPGPC, GC-MS, 1D-NMR, 2D-NMR, FESEM and AFM techniques to characterize the structures of three types of white peony polysaccharides.

[0015] Surface microstructure analysis revealed that AJP-1a exhibits irregular pores and wrinkles, with obvious unevenness and cracks, presenting a complex network or porous morphology; AJP-2a has obvious fibrous or filamentous structures, which may be formed by the aggregation of polysaccharide molecular chains; AJP-3a presents an irregular cluster-like, coiled and folded sheet-like structure, with polysaccharide chains intertwined and stacked, and an uneven surface, exhibiting a flexible and dense aggregation morphology; furthermore, AJP-3a has a triple helix structure, while AJP-1a and AJP-2a do not have a triple helix structure.

[0016] The preparation method of the polysaccharide from *Ampelopsis japonica* described in this invention is as follows:

[0017] S1. Take the raw medicinal material of Bai Lian, crush it, and then extract it with water and precipitate it with alcohol to collect the polysaccharide precipitate;

[0018] The specific operation is as follows: Take the raw medicinal material of Bai Lian, crush it and pass it through a 50-mesh sieve. Mix the medicinal powder with water at a material-to-liquid ratio of 1:15-30 (g / mL), heat to 95-105℃ and extract for 1.5-2.5h. Filter, and repeat the extraction 1-2 times with the filter residue. Combine the extracts, concentrate under reduced pressure to 1 / 5-1 / 3 of the original volume, centrifuge, take the supernatant, add anhydrous ethanol to the system until the ethanol volume fraction is 70-90%, precipitate with alcohol at 4℃ for 8-16h, and collect the polysaccharide precipitate by centrifugation.

[0019] S2. The polysaccharide precipitate collected in S1 is redissolved in water, and the resulting solution is subjected to deproteinization, decolorization, dialysis and freeze-drying in sequence to obtain crude polysaccharide AJP.

[0020] The specific procedure is as follows: Dissolve the polysaccharide precipitate again in water, and adjust the pH of the resulting solution to 8-9 (using 0.5 mol / L...). Adjust with NaOH solution), heat to 80-100℃, add CaCl2 solid to a final concentration of 45-55 g / L, boil for 25-35 min, cool to room temperature, filter, adjust the pH of the filtrate to 6.8-7.2 (using 0.1 mol / L hydrochloric acid), add anhydrous ethanol to 75-90% ethanol volume fraction, place at 4℃ for 8-16 h, centrifuge, collect the precipitate, dissolve in water and repeat the above operation until the protein content is less than 5%, completing the deproteinization process; redissolve the deproteinized precipitate in water, add activated carbon (preferably 1-3% of the solution mass), stir at 45-55℃ for 35-45 min for decolorization, then centrifuge to remove the activated carbon, completing the decolorization process; transfer the decolorized solution to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze at 3-5℃ for 24-72 h, changing the distilled water every 6-8 h, then freeze-dry to obtain crude polysaccharide AJP;

[0021] S3. The crude polysaccharide AJP obtained in S2 was initially separated by DEAE-52 anion exchange chromatography. Gradient elution was performed using 0-0.5 mol / L NaCl aqueous solution as the eluent. The elution was monitored by the phenol-sulfuric acid method and the elution curve was plotted. The eluents of each component were combined according to the elution curve, and then concentrated under reduced pressure, dialyzed and freeze-dried to obtain polysaccharides AJP-1, AJP-2 and AJP-3.

[0022] The specific operation is as follows: Crude polysaccharide AJP is dissolved in distilled water to prepare a crude polysaccharide AJP solution of 5-15 mg / mL. After filtration through a 0.45 μm aqueous microporous membrane, the solution is added to a chromatographic column (26×300 mm) packed with DEAE-52 packing material for separation. The sample loading volume is 5-15 mL. Gradient elution is performed using NaCl aqueous solutions of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L as eluents, with a flow rate of 1-2 mL / min. The elution curve is monitored using the phenol-sulfuric acid method and a elution curve is plotted. The eluents of each component are combined according to the elution curve, concentrated under reduced pressure, dialyzed at 3-5℃ for 24-72 h, and freeze-dried to obtain polysaccharides AJP-1, AJP-2, and AJP-3.

[0023] The phenol-sulfuric acid method is as follows: Take 1.0 mL of the eluent sample, add 1.0 mL of distilled water, then add 1.0 mL of 6% phenol, quickly add 5.0 mL of concentrated sulfuric acid, mix thoroughly, let stand for 30 min, and measure the absorbance at 490 nm.

[0024] Before use, the DEAE-52 packing material undergoes the following treatment: Distilled water is added to the new DEAE-52 packing material, stirred thoroughly, and soaked for 2.5-3.5 hours to remove surface impurities. The mixture is then filtered under reduced pressure, followed by soaking in a 0.4-0.6 mol / L HCl solution for 1.5-2.5 hours, followed by filtration under reduced pressure, washing with distilled water until neutral, and then further soaking in a 0.4-0.6 mol / L NaOH solution for 1.5-2.5 hours, followed by filtration under reduced pressure, and washing with distilled water until neutral. The treated packing material is then soaked in a 15-25% ethanol solution and stored at 3-5°C for long-term preservation.

[0025] Used DEAE-52 packing material can be regenerated and reused. The regeneration method is as follows: First, rinse the used DEAE-52 packing material with saturated NaCl solution to remove impurities adsorbed in the packing material. Then, rinse with distilled water. Subsequently, soak it in 0.4-0.6 mol / L HCl solution and 0.4-0.6 mol / L NaOH solution for 1.5-2.5 hours respectively. Filter under reduced pressure, wash with distilled water until neutral, and store in 15-25% ethanol solution at 3-5℃.

[0026] S4. The polysaccharides AJP-1, AJP-2, and AJP-3 obtained in S3 were purified using dextran gel G-200. Elution was performed using 0.15-0.25 mol / L NaCl aqueous solution as eluent. The elution was monitored using the phenol-sulfuric acid method, and the elution curves were plotted. The eluents of each component were combined according to the elution curves, and the polysaccharides AJP-1a, AJP-2a, and AJP-3a were obtained by vacuum concentration, dialysis, and freeze-drying.

[0027] The specific operation is as follows: Polysaccharides AJP-1, AJP-2, and AJP-3 were purified using a dextran gel G-200 column (26×600mm) with a loading concentration of 5-15 mg / mL and a loading volume of 5-20 mL. Elution was performed using a 0.15-0.25 mol / L NaCl aqueous solution as the eluent at a flow rate of 0.2-0.8 mL / min. The elution was monitored using the phenol-sulfuric acid method, and the elution curves were plotted. The eluents corresponding to single symmetrical peaks were combined according to the elution curves, concentrated under reduced pressure, dialyzed at 3-5℃ for 24-72 h, and freeze-dried to obtain the polysaccharides AJP-1a, AJP-2a, and AJP-3a of *Ampelopsis japonica*.

[0028] Before use, dextran gel G-200 is treated as follows: at room temperature, the dextran gel G-200 dry powder is soaked in distilled water for 24-72 hours, and stirred continuously to ensure that the gel swells fully. The floating gel fragments on the upper layer are removed and it is ready for use.

[0029] The polysaccharides from *Ampelopsis japonica* described in this invention can be used to prepare anti-colorectal cancer drugs. AB strain zebrafish were used as an in vitro evaluation model for colorectal cancer. A tumor model was constructed by injecting a suspension of DiI-labeled HCT116 colorectal cancer cells into the yolk sac of zebrafish. A blank control group, a positive control group, and a polysaccharide experimental group were set up. After incubation, ImageJ software was used to quantify fluorescence-related indicators in the tumor region, and statistical analysis was combined to evaluate the antitumor activity of the three *Ampelopsis japonica* polysaccharides.

[0030] The polysaccharide obtained by the present invention has novel and unique structural features and significant anti-colorectal cancer activity. It can be used to prepare new drugs for the treatment of colorectal cancer, or as an intermediate for anti-colorectal cancer drugs or as a plant raw material for functional foods.

[0031] Compared with the prior art, the present invention has the following characteristics:

[0032] 1) This invention is the first to develop a process for separating and purifying polysaccharides from Gynostemma pentaphyllum, and provides a suitable method for separating polysaccharides from Gynostemma pentaphyllum.

[0033] 2) This invention is the first to use a DEAE-52 anion exchange column combined with a dextran gel G-200 gel permeation chromatography column to perform multiple separation and purification of Gynostemma pentaphyllum polysaccharides, obtaining structurally uniform Gynostemma pentaphyllum polysaccharides AJP-1a, AJP-2a, and AJP-3a.

[0034] 3) This invention is the first to conduct cold field scanning electron microscopy (FESEM) experiments on polysaccharides of AJP-1a and found that AJP-2a has a loose and porous network structure, AJP-2a has an irregular cluster-like curled and folded shape, and AJP-3a exhibits a dense and wrinkled sheet-like structure. Each of the three has a characteristic microscopic morphology.

[0035] 4) This invention is the first to conduct atomic force microscopy (AFM) experiments on polysaccharides of Gynostemma pentaphyllum and found that these three polysaccharides have different degrees of branching and aggregation characteristics. The results are consistent with the detection results of GC-MS, NMR and FESEM.

[0036] 5) This invention is the first to conduct a Congo red test on the polysaccharide of Gynostemma pentaphyllum and found that AJP-3a has a triple helix structure.

[0037] 6) This invention is the first to report that the polysaccharides AJP-1a, AJP-2a, and AJP-3a of Amygdalin can effectively inhibit the growth of colorectal cancer cells in zebrafish and have good anti-colorectal cancer activity. Attached Figure Description

[0038] Figure 1 Flowchart for the extraction, separation, and purification of polysaccharides from Gynostemma pentaphyllum.

[0039] Figure 2Elution curves of Gynostemma pentaphyllum polysaccharides: A: Elution curve of crude Gynostemma pentaphyllum polysaccharide on DEAE-cellulose-52 column; B: Elution curve of AJP-1 on Sephadex G-200 column; C: Elution curve of AJP-2 on Sephadex G-200 column; D: Elution curve of AJP-3 on Sephadex G-200 column.

[0040] Figure 3 A: Structural analysis diagrams of AJP-1a, AJP-2a, and AJP-3a; B: HPGPC spectra of AJP-1a, AJP-2a, and AJP-3a; C: Monosaccharide composition spectra of AJP-1a, AJP-2a, and AJP-3a; D: Infrared spectra of AJP-1a, AJP-2a, and AJP-3a.

[0041] Figure 4 Results of Congo Red experiments with AJP-1a, AJP-2a, and AJP-3a.

[0042] Figure 5 FESEM images of AJP-1a, AJP-2a, and AJP-3a.

[0043] Figure 6 AFM diagram of AJP-1a.

[0044] Figure 7 AFM diagram of AJP-2a.

[0045] Figure 8 AFM diagram of AJP-3a.

[0046] Figure 9 GC-MS spectra of methylation of AJP-1a, AJP-2a, and AJP-3a.

[0047] Figure 10 AJP-1a 1 H-NMR spectrum.

[0048] Figure 11 AJP-1a 13 C-NMR spectrum.

[0049] Figure 12 HSQC spectrum of AJP-1a.

[0050] Figure 13 COSY spectrum of AJP-1a.

[0051] Figure 14 HMBC spectrum of AJP-1a.

[0052] Figure 15 AJP-2a1 H-NMR spectrum.

[0053] Figure 16 AJP-2a 13 C-NMR spectrum.

[0054] Figure 17 HSQC spectrum of AJP-2a.

[0055] Figure 18 COSY spectrum of AJP-2a.

[0056] Figure 19 HMBC spectrum of AJP-2a.

[0057] Figure 20 AJP-3a 1 H-NMR spectrum.

[0058] Figure 21 AJP-3a 13 C-NMR spectrum.

[0059] Figure 22 HSQC spectrum of AJP-3a.

[0060] Figure 23 COSY spectrum of AJP-3a.

[0061] Figure 24 HMBC spectrum of AJP-3a.

[0062] Figure 25 The structure of AJP-1a.

[0063] Figure 26 The structure of AJP-2a.

[0064] Figure 27 The structure of AJP-3a.

[0065] Figure 28 Fluorescence microscopy images of the inhibitory effects of AJP-1a, AJP-2a, and AJP-3a on the growth of HCT116 tumor cells in zebrafish.

[0066] Figure 29 Effects of AJP-1a, AJP-2a, and AJP-3a on zebrafish tumor models. Detailed Implementation

[0067] The present invention will now be described in detail and clearly through specific embodiments and in conjunction with the accompanying drawings. Obviously, the scope of protection of the present invention is not limited thereto.

[0068] In the following embodiments,

[0069] The medicinal material, Bai Lian, comes from Hebei Kangyiqiang Pharmaceutical Co., Ltd.

[0070] DEAE-52 anion exchange column packing was purchased from Whatman, UK.

[0071] The dextran gel G-200 column was purchased from Beijing Ruida Heng Hui Technology Development Co., Ltd.

[0072] This invention utilizes a novel extraction, separation, and purification method to obtain three polysaccharides with distinct basic structures from *Ampelopsis japonica*, and provides comprehensive and systematic structural characterization of these polysaccharides. Given the potential antitumor activity of *Ampelopsis japonica*, the anti-colorectal cancer activity of these three polysaccharides was evaluated by constructing a zebrafish tumor model of DiI-labeled HCT116 colorectal cancer cells, laying the foundation for the development of therapeutic drugs for colorectal cancer.

[0073] Example 1: Extraction, separation and purification of polysaccharides from Gynostemma pentaphyllum

[0074] Dried *Bletilla striata* tubers were pulverized using a pulverizer and passed through a 50-mesh sieve to obtain *Bletilla striata* powder. Distilled water was added at a material-to-liquid ratio of 1:20 (g / mL), and the mixture was stirred until homogeneous. The powder was transferred to a round-bottom flask and extracted under reflux at 100℃ for 2 h. This extraction was repeated twice, with centrifugation after cooling in between. The two extracts were combined and concentrated under reduced pressure to 1 / 5 of the original volume. The mixture was centrifuged (3000 rpm, 10 min), and the supernatant was collected. Anhydrous ethanol was slowly added until the ethanol volume fraction reached 80%, and the mixture was allowed to stand overnight at 4℃. After ethanol precipitation, the mixture was centrifuged (6000 rpm, 10 min), and the polysaccharide precipitate was collected. The precipitate was redissolved in distilled water at a ratio of 1:20 (g / mL) to obtain a crude extract of *Bletilla striata* polysaccharides.

[0075] Take 200 mL of the crude extract, adjust the pH to 8-9 with 0.5 mol / L NaOH, heat to 85℃, add CaCl2 solid to a final concentration of 50 g / L, boil for 30 min, cool, and filter. Adjust the pH of the filtrate to 6.8-7.2 with 0.1 mol / L hydrochloric acid, add anhydrous ethanol to 80% ethanol volume, let stand overnight at 4℃, then centrifuge (6000 rpm, 10 min) to collect the precipitate. Rehydrate the precipitate and repeat the above deproteinization process 3 times until the protein content is less than 5%. Add 2.5% activated carbon to the deproteinized polysaccharide solution, stir and decolorize at 50℃ for 40 min, centrifuge (10000 rpm, 10 min), repeat 2-3 times until the activated carbon is completely removed. Transfer the deproteinized and decolorized polysaccharide solution to a dialysis bag (molecular weight cutoff 3500 Da), dialyze at 4℃ for 48 h, changing the dialysate every 8 h to remove small molecule impurities. After dialysis, the solution was freeze-dried to obtain crude polysaccharide AJP from *Ampelopsis japonica*.

[0076] The crude polysaccharide AJP from *Ampelopsis japonica* was dissolved in distilled water to prepare a 10 mg / mL solution. After filtration through a 0.45 μm aqueous filter membrane, 10 mL of the solution was loaded onto a DEAE-52 cellulose column (26 × 300 mm). Gradient elution was performed sequentially using 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions as mobile phases at a flow rate of 1.5 mL / min, collecting 5 mL from each tube. The polysaccharide content in each tube was monitored using the phenol-sulfuric acid method (1.0 mL of polysaccharide sample was mixed with 1.0 mL of distilled water and 1.0 mL of 6% phenol, then 5.0 mL of concentrated sulfuric acid was rapidly added, mixed thoroughly, allowed to stand for 30 min, and the absorbance was measured at 490 nm). Elution curves were plotted. Based on the curves, the major components were combined, concentrated under reduced pressure, dialyzed (4℃, 48 h, molecular weight cutoff 3500 Da), and freeze-dried to obtain polysaccharides AJP-1, AJP-2, and AJP-3, respectively.

[0077] The three polysaccharides were further purified using a dextran gel G-200 column (26×600 mm) with 0.2 mol / L NaCl solution as eluent, a flow rate of 0.5 mL / min, a loading concentration of 10 mg / mL, and a loading volume of 10 mL, with 7 mL collected per tube. Elution curves were monitored and plotted using the phenol-sulfuric acid method. Components corresponding to single symmetrical peaks were combined, concentrated under reduced pressure, dialyzed (4℃, 48 h, molecular weight cutoff 3500 Da), and freeze-dried to obtain the polysaccharides AJP-1a, AJP-2a, and AJP-3a.

[0078] Figure 1 This study demonstrates a complete process for extracting, separating, and purifying polysaccharides from the raw medicinal material of *Bletilla striata*. The yield of crude polysaccharides from *Bletilla striata* was 9.6% relative to the dried medicinal material. Three main components (AJP-1, AJP-2, and AJP-3) were obtained by DEAE-52 ion-exchange chromatography, followed by further purification using a dextran gel G-200 to yield three polysaccharides: AJP-1a, AJP-2a, and AJP-3a. HPGPC analysis showed that all three exhibited a single symmetrical peak, indicating good homogeneity and different molecular weights, suitable for subsequent monosaccharide composition, FT-IR, GC-MS methylation analysis, NMR analysis, and surface microstructure studies.

[0079] Example 2: Structural characterization of Gynostemma pentaphyllum polysaccharides

[0080] 1) Molecular weight determination (HPGPC)

[0081] The purity and relative molecular mass of polysaccharides AJP-1a, AJP-2a, and AJP-3a from *Gynostemma pentaphyllum* were determined by high-performance gel permeation chromatography (HPGPC). The chromatographic system was a Waters e2695 HPLC system (Waters Corporation), equipped with a TSKgel G3000PWxl column (7.8 mm × 300 mm, 7 μm, Sigma-Aldrich) and an ELSD detector. The mobile phase was pure water, the flow rate was 0.5 mL / min, the column temperature was 40 °C, the injection volume was 10 μL, and the run time was 30 min.

[0082] like Figure 3 As shown in Figure A, the gel chromatograms of AJP-1a, AJP-2a, and AJP-3a all exhibit a single symmetrical peak, indicating that the three polysaccharides possess good homogeneity. Based on the retention times of each polysaccharide and the dextran standard curve (Lg(MW) = –0.3292 × tR + 9.0539, R...),... 2 = 0.9903), the relative molecular masses of AJP-1a, AJP-2a and AJP-3a were calculated to be 281 kDa, 1435 kDa and 251.1 kDa, respectively.

[0083] (2) Fourier transform infrared spectroscopy (FT-IR) analysis

[0084] Take 2 mg of polysaccharide sample, mix it with 200 mg of KBr, compress it into a tablet, and use a Fourier transform infrared spectrometer (Thermo Scientific Nicolet iS50, USA) to observe it at 400–4000 cm⁻¹. -1 Infrared spectra were collected within the range.

[0085] Figure 3 C represents the infrared spectra of AJP-1a, AJP-2a, and AJP-3a. All three are located at 3269 cm⁻¹. -1 All peaks exhibit strong and broad absorption, attributed to the O–H stretching vibrations between polysaccharide molecules; 2914 cm⁻¹ -1 The absorption peak at that point corresponds to the stretching vibrations of C–H (including methylene and methine) on the sugar ring, reflecting the alkyl skeleton characteristics of the sugar ring.

[0086] At 1800–1400 cm -1 In this region, only AJP-3a is at 1740 cm. -1 A distinct absorption peak appears at 1604 cm⁻¹, which is attributed to the C=O stretching vibration of the carboxyl group (–COOH) in glucuronic acid, indicating the presence of a glucuronic acid component. Meanwhile, AJP-3a shows an absorption peak at 1604 cm⁻¹. -1 It exhibits a sharper peak shape at 1404 cm. -1Absorption peaks appear at these locations, corresponding to the –COO group after deprotonation of the carboxyl group. - The asymmetric and symmetric stretching vibrations are observed, while AJP-1a and AJP-2a show no characteristic absorption in this region, consistent with the fact that neither contains glucuronic acid. At 1200–800 cm⁻¹ -1 The vibrational region of the sugar ring and glycosidic bond, 1141 cm -1 The characteristic absorption peaks of α-pyranose are observed here, with AJP-1a and AJP-2a showing distinct peak shapes, reaching 920 cm⁻¹. -1 With 830 cm -1 The strong absorption of both indicates that they contain an α-D-glucopyranose structure. In contrast, AJP-3a has weaker absorption in this region, consistent with its mixed vibrational characteristics of multiple pyranose rings, including mannose and galactose.

[0087] (3) Monosaccharide composition analysis (HPLC)

[0088] The monosaccharide composition of AJP-1a, AJP-2a and AJP-3a was determined by pre-column derivatization combined with high performance liquid chromatography using PMP.

[0089] Accurately weigh 2 mg of each dried polysaccharide sample and place them in a digestion tube. After purging with nitrogen, add 4 mL of 3 mol / L trifluoroacetic acid (TFA) and hydrolyze in an oil bath at 100℃ for 8 h to completely hydrolyze the polysaccharides into monosaccharides. After cooling, evaporate the TFA, dissolve in 5 mL of methanol, and dry under reduced pressure. Repeat three times to remove residual acid. Dissolve the residue in 1 mL of pure water, centrifuge (10000 rpm, 10 min), and take 100 μL of the supernatant. Add 100 μL of 0.3 mol / L NaOH solution and 120 μL of 0.5 mol / L PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution sequentially, and react in a water bath at 70℃ for 1 h for derivatization. After cooling the reaction solution, add 100 μL of 0.3 mol / L HCl for neutralization, then add an equal volume of chloroform for vortex extraction. After centrifugation, collect the upper aqueous phase, filter through a 0.22 μm microporous membrane, and perform HPLC analysis.

[0090] 10 mg each of monosaccharide standards (rhamnose, xylose, galactose, arabinose, glucose, mannose, galacturonic acid, and glucuronic acid) were dissolved in 10 mL of pure water, centrifuged, and 100 μL of the supernatant was taken and subjected to PMP derivatization and sample processing under the same conditions as above, as a control.

[0091] Chromatographic analysis was performed using an Agilent 1260 high-performance liquid chromatograph equipped with a Kramasil 100-5-C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was acetonitrile-0.1 mol / L phosphate buffer (17:83, pH 6.8), with isocratic elution, a flow rate of 1.0 mL / min, a column temperature of 30 °C, a UV detection wavelength of 250 nm, an injection volume of 10 μL, and a run time of 70 min.

[0092] like Figure 3 As shown in Table B and Table 1, there are significant differences in the monosaccharide composition of AJP-1a, AJP-2a, and AJP-3a. AJP-1a contains only glucose and is a homogeneous dextran; AJP-2a is a heteropolysaccharide with glucose as its core, composed of glucose (96.65%), a small amount of galactose (1.68%), and arabinose (1.66%); AJP-3a does not contain glucose and mainly contains arabinose (42.35%), galactose (22.79%), mannose (20.54%), and glucuronic acid (14.3%), belonging to a complex heteropolysaccharide.

[0093] The above monosaccharide composition results are consistent with the column chromatography behavior: AJP-1a and AJP-2a can be eluted with pure water and low concentration of sodium chloride, while AJP-3a requires a higher concentration of salt solution for elution, which is related to its presence of acidic components such as glucuronic acid.

[0094] Table 1 Monosaccharide composition of AJP-1a, AJP-2a and AJP-3a of Gynostemma pentaphyllum polysaccharides

[0095]

[0096] (4) Analysis of the triple helix structure of Congo red

[0097] Prepare aqueous solutions of AJP-1a, AJP-2a, and AJP-3a with a concentration of 1 mg / mL, respectively. Take 2 mL of each solution and add 2 mL of 50 μmol / L Congo red solution and 1 mL of NaOH solution of different concentrations, respectively, to achieve final NaOH concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. After mixing, scan the wavelength range of 200-800 nm and record the maximum absorption wavelength for each system. Use pure water as a blank control and plot a curve with NaOH concentration on the x-axis and maximum absorption wavelength on the y-axis. Simultaneously, use deionized water instead of polysaccharide solution to prepare a control solution with Congo red and NaOH using the same method, and measure and plot the control curve.

[0098] The Congo red assay is a commonly used method for detecting whether polysaccharides possess a triple helix structure. Congo red can form a complex with polysaccharides that have a triple helix structure, causing a red shift in the maximum absorption wavelength compared to Congo red itself. For example... Figure 4 As shown, after AJP-1a and AJP-2a bound to Congo red, no significant red shift was observed in the maximum absorption wavelength at various NaOH concentrations, and their trends were basically consistent with those of the control solution, indicating that neither of them possesses a triple helix structure. However, after AJP-3a bound to Congo red, a significant red shift in the maximum absorption wavelength was observed at various NaOH concentration gradients, and the magnitude of the red shift conformed to the characteristic pattern of triple helix polysaccharides binding to Congo red, indicating that AJP-3a possesses a triple helix structure.

[0099] (5) Cold field emission scanning electron microscopy (FESEM) analysis

[0100] The microstructure of polysaccharides was observed using cold field emission scanning electron microscopy (CFET). Small amounts of lyophilized AJP-1a, AJP-2a, and AJP-3a samples were taken using antistatic tweezers and evenly dispersed on a sample stage coated with conductive adhesive. The sample stage was gently shaken, and loosely adhered samples were blown away with a syringe. The sample stage was then subjected to gold sputtering in an ion sputtering apparatus to enhance conductivity, and subsequently transferred to an S-8020 CFET (Hitachi, Japan) for observation. Images were acquired at magnifications of 1.0 k×, 5.0 k×, and 10.0 k× in high vacuum mode with an accelerating voltage of 5.0 kV.

[0101] FESEM can be used to observe the morphology of polysaccharides, and their microstructure is closely related to their biological activity. For example... Figure 5 As shown, AJP-1a exhibits a loose, interwoven network structure with abundant and highly interconnected surface pores. This may be attributed to its status as a single dextran, where moderate interactions between sugar chains result in a more relaxed spatial conformation, which is beneficial for improving water solubility. AJP-2a has a rough surface and exhibits a dense, clustered aggregate morphology with small and fewer surface pores, suggesting a higher molecular weight, greater branching, and tighter interactions between sugar chains, making it prone to forming a condensed structure. AJP-3a, on the other hand, displays obvious filamentous cross-linking and coiled morphology. Under high magnification, the highly coiled characteristics of individual filaments are visible. This structure is closely related to the formation of a triple helix conformation, consistent with the results of the Congo red experiment confirming that AJP-3a possesses a triple helix structure. The anti-colorectal cancer activities of three polysaccharides from *Ampelopsis japonica* are related to their microstructural characteristics: the loose network structure of AJP-1a increases the contact area with tumor cells; the dense cluster structure of AJP-2a may affect its activity by regulating the dissolution rate; and the coiled filamentous cross-linked structure and triple helix conformation of AJP-3a together constitute the structural basis for its significant antitumor activity. Previous studies have shown that polysaccharides with triple helix structures generally possess strong immunomodulatory and antitumor activities.

[0102] (6) Atomic force microscopy (AFM) analysis

[0103] Lyophilized AJP-1a, AJP-2a, and AJP-3a samples were dissolved in deionized water to prepare solutions with a concentration of 5 μg / mL. A suitable amount was dropped onto freshly peeled mica sheets and dried overnight at 25–30 °C. Observation was performed using a Dimension Edge atomic force microscope (Bruck, Germany) in tapping mode, and images were acquired using NanoScope software.

[0104] Atomic force microscopy (AFM) is an important tool for observing the morphology of polysaccharide molecules. To directly observe the conformational characteristics of AJP-1a, AJP-2a, and AJP-3a, imaging analysis was performed on each polysaccharide sample at a concentration of 5 μg / mL. Figure 6 , Figure 7 , Figure 8 As shown, AJP-1a mainly exhibits fine granular or short-chain structures, which are cross-linked to form a relatively loose network porous morphology. In contrast, AJP-2a shows stronger intermolecular interactions at the same concentration, forming larger and thicker clusters or spherical aggregates, exhibiting a more dense aggregation state. AJP-3a shows a mixed aggregation state, with both small-sized dispersed structures and obvious large-sized aggregates in the image, accompanied by localized sparse and unevenly distributed areas. All three polysaccharides exhibit a peak-shaped aggregation morphology, with heights significantly higher than the theoretical height of a single polysaccharide chain (0.1–1 nm), indicating molecular aggregation and suggesting branched structures, with differences in the degree of branching and branch length among the polysaccharides. Observation revealed that these structures are intertwined, forming aggregates of various morphologies, with some areas exhibiting a network structure. The above observations are basically consistent with the scanning electron microscopy images.

[0105] (7) GC-MS analysis of methylation

[0106] To clarify the glycosidic bond linkages of AJP-1a, AJP-2a, and AJP-3a, methylation combined with GC-MS was used for analysis. Because AJP-3a contains glucuronic acid, it requires pretreatment with glucuronic acid reduction, while AJP-1a and AJP-2a can be directly methylated. The specific steps are as follows:

[0107] AJP-1a, AJP-2a, and AJP-3a were pulverized separately in a ball mill. 3 mg of each sample was accurately weighed into an EP tube, freeze-dried for 5 h, and then transferred to a desiccator for overnight drying. NaOH was ground into a fine powder, vacuum-dried at 80℃ for 8 h, and then transferred to a desiccator for overnight drying.

[0108] AJP-3a uronic acid reduction pretreatment: Weigh 20 mg of AJP-3a and dissolve it in 10 mL of deionized water. Add 200 mg of N-cyclohexyl-N'-[2-(N-methylmorpholine)ethyl]carbodiimide p-toluenesulfonate. Under magnetic stirring, add 0.01 mol / L HCl dropwise to adjust the pH to 4.75 and maintain the reaction for 3 h. Add 20 mL of 2 mol / L sodium borohydride solution dropwise, adjust the pH to 7.00 with 4 mol / L HCl, continue stirring, add a few drops of isopropanol to defoam, and continue the reaction at room temperature for 12 h. Transfer the reaction solution to a dialysis bag (molecular weight cutoff 500 Da) and dialyze for 24 h. Concentrate under reduced pressure to 10 mL and freeze-dry. Repeat the above reduction reaction 2–3 times to ensure complete reduction of uronic acid. Take the reduced sample and proceed with the methylation reaction in the subsequent steps.

[0109] Dry AJP-1a, AJP-2a samples, and the reduced AJP-3a sample were placed in dry 10 mL round-bottom flasks, sealed, and purged with nitrogen to fully dissolve the polysaccharides. 500 μL of ultradry dimethyl sulfoxide was added, and the mixture was stirred until completely dissolved. 5 mg of dry NaOH powder was added, and the mixture was stirred overnight to allow the hydroxyl groups on the sugar chains to react fully with the NaOH. 100 μL of iodomethane was added to the reaction solution, and the reaction was terminated by adding 1 mL of water after 2 h. After the methylation reaction was complete, 2 mL of dichloromethane was added for extraction. After vortexing and centrifugation (3000 rpm, 10 min), the lower organic phase was collected. 3 mL of pure water was added to wash the organic phase, and after vortexing and centrifugation, the aqueous phase was discarded. This washing process was repeated three times, and the dichloromethane layer was evaporated to dryness. The methylation products were detected using Fourier transform infrared spectroscopy (FTIR). If 3500 cm⁻¹... - The absence of characteristic absorption peaks for hydroxyl groups near the ¹ indicates complete methylation; if methylation is incomplete, repeat the methylation steps described above. Add 2 mL of 2 mol / L trifluoroacetic acid to the fully methylated product, purge with nitrogen, and then reflux in an oil bath at 110 °C for 8 h. After cooling to room temperature, evaporate to dryness, dissolve in 1–2 mL of methanol, and evaporate to dryness again. Repeat this process three times to remove residual trifluoroacetic acid. Add 200 μL of 2 mol / L ammonia and 200 μL of 1 mol / L sodium borodeuteride to the hydrolysis product, mix well, and react under nitrogen protection for 24 h. Stop the reaction by adding 20 μL of acetic acid, evaporate to dryness, wash twice with 1 mL of methanol, and repeatedly evaporate to dryness. Add 250 μL of acetic anhydride to the reaction system, mix well, and react in an oil bath at 100℃ for 2 h. Cool to room temperature, add 1 mL of water and let stand for 10 min, then add dichloromethane for vortex extraction, centrifuge (3000 rpm, 10 min), discard the aqueous phase, and repeat the washing with water three times. Take the dichloromethane layer, add anhydrous sodium sulfate and dry to obtain partially methylated aldose acetates (PMAAs), filter through a 0.22 μm microporous membrane and perform GC-MS analysis.

[0110] GC-MS analysis conditions: Agilent HP5-ms column (30 m × 0.32 mm, 0.25 μm); injection port temperature 250℃; interface temperature 250℃; carrier gas: high-purity helium, flow rate 1.5 mL / min; temperature program: initial temperature 140℃ held for 2 min, then increased to 230℃ at 3℃ / min and held for 3 min; injection volume 1 μL, split ratio 10:1; mass spectrometer detector: EI ionization source (70 eV), scan range m / z 30–600.

[0111] GC-MS spectra of methylation of AJP-1a, AJP-2a, and AJP-3a are shown below. Figure 9 As shown, the types of sugar residues and their relative molar ratios are listed in Table 2. The results indicate that AJP-1a is mainly composed of →1)-D-Glcp, →3)-D-Glcp-(1→, →4)-D-Glcp-(1→, →6)-D-Glcp-(1→, →3,4)-D-Glcp-(1→, →2,4)-D-Glcp-(1→ and →4,6)-D-Glcp-(1→). AJP-2a mainly includes →4)-D-Arap-(1→, →1)-D-Glcp, →4)-D-Glcp-(1→, →6)-D-Glcp-(1→, →3,4)-D-Galp-(1→ and →4,6)-D-Glcp-(1→). AJP-3 The glycosidic bond linkage of α is quite complex, including various linkage modes such as -(1→)-L-Araf, -(1→)-L-Arap, →4)-L-Arap-(1→、→1)-D-GlcpA, →2)-D-Galp-(1→、→2)-L-Arap-(1→、→4)-D-GlcpA-(1→、→3)-D-Galp-(1→、→6)-D-Manp-(1→、→6)-D-Galp-(1→、→2,3)-D-Manp-(1→、→3,4)-D-Manp-(1→、→3,6)-D-GlcpA-(1→ and →3,4,6)-D-Manp-(1→).

[0112] Table 2. Methylation analysis results of three polysaccharide monomers from Gynostemma pentaphyllum.

[0113]

[0114]

[0115] (8) NMR analysis

[0116] Weigh 50 mg of each of the following polysaccharides from *Ampelopsis japonica* (AJP-1a, AJP-2a, and AJP-3a): dissolve each in 1 mL of heavy water, freeze-dry, and repeat the heavy water exchange process twice. The freeze-dried samples are then dried overnight in a freeze dryer. Finally, dissolve each sample in 0.6 mL of heavy water, and after complete dissolution, transfer the solution to an NMR tube, filling it to a height of 3-4 cm. Allow the solution to stand at room temperature before testing. The test temperature is 298 K, and the test items include one-dimensional... 1 H NMR and 13 C NMR, 2D COSY, HSQC, HMBC.

[0117] By combining the monosaccharide composition and methylation analysis results of *Ampelopsis japonica* polysaccharides with one-dimensional and two-dimensional NMR results, the basic structures of *Ampelopsis japonica* polysaccharides AJP-1a, AJP-2a, and AJP-3a were elucidated. The anomeric hydrogen and anomeric carbon signals of AJP-1a were mainly concentrated between δ 4.8–5.5 ppm and δ 98–101 ppm; the anomeric hydrogen and anomeric carbon signals of AJP-2a were mainly concentrated between δ 4.5–5.5 ppm and δ 92–100 ppm. 13 Based on the C NMR, HSQC and COSY spectra results, and referring to the methylation results, monosaccharide composition and relevant references, it is inferred that sugar residue A of AJP-1a is α-D-Glcp-(1→), sugar residue B is →4)-α-D-Glcp-(1→), sugar residue C is →3,4)-α-D-Glcp-(1→), sugar residue D is →2,4)-α-D-Glcp-(1→), and sugar residue E is →4,6)-α-D-Glcp-(1→). AJP-2a has sugar residue A as α-D-Glcp-(1→), sugar residue B as →4)-α-D-Glcp-(1→), sugar residue C as →6)-α-D-Glcp-(1→), sugar residue D as →3,4)-α-D-Galp-(1→), and sugar residue E as →4,6)-α-D-Glcp-(1→). The sugar residue A of AJP-3a is α-L-Araf-(1→), sugar residue B is α-L-Arap-(1→), sugar residue C is →4)-β-L-Arap-(1→), sugar residue D is α-D-GlcpA-(1→), sugar residue E is →2)-β-D-Galp-(1→, sugar residue F is →3)-β-D-Galp-(1→, sugar residue G is →6)-α-D-Manp-(1→, sugar residue H is →2,3)-α-D-Manp-(1→, sugar residue I is →3,6)-α-D-GlcpA-(1→). 1 H and 13 C chemical shifts are shown in Table 3.

[0118] Table 3 AJP-1a, AJP-2a, AJP-3a13 C / 1 H NMR chemical shift

[0119]

[0120] Based on the sugar residues in the sample 13 C and 1 H chemical shifts were used to characterize the linkage sequence between sugar residues using HMBC maps.

[0121] In AJP-1a, the H-1 (δ5.25) / C-1 (δ99.9) of terminal residue A is coupled with the C-3 (δ76.61) / H-3 (δ3.94) of residue C and the C-6 (δ69.2) / H-6 (δ3.74) of residue E, respectively; the H-1 (δ5.31) / C-1 (δ100.09) of branch point residue B is coupled with the C-6 (δ69.2) / H-6 (δ3.74) of residue E, the C-2 (δ76.82) / H-2 (δ3.77) of residue D, and the C-4 (δ77.6) / H-4 (δ3.77) of residue C, respectively; the H-1 (δ5.34) / C-1 (δ98.5), H-4 (δ3.59) / C-4 (δ76.84), and H-6 of residue E are coupled sequentially. (δ3.74) / C-6 (δ69.2) also forms a bidirectional coupling with C-4 (δ76.65) / H-4 (δ3.56), C-1 (δ100.09) / H-1 (δ5.31) of residue B and C-1 (δ99.9) / H-1 (δ5.25) of residue A; H-3 (δ3.94) / C-3 (δ76.61) of residue C and H-4 (δ3.79) / C-4 (δ78.01) of residue D are also coupled with C-1 (δ100.09) / H-1 (δ5.31) of residue B. Based on the analysis of one-dimensional and two-dimensional NMR information and methylation results, it is inferred that the main chain of AJP-1a is mainly composed of →4)-α-D-Glcp-(1→), and the side chains are composed of →3,4)-α-D-Glcp-(1→), →2,4)-α-D-Glcp-(1→) and →4,6)-α-D-Glcp-(1→). The C-1 position is connected to the O-3 position of sugar residue C and the O-6 position of sugar residue E in the main chain, respectively, and sugar residue B serves as a branch point connecting the residues.

[0122] In AJP-2a, HMBC spectra revealed coupling signals between H-1 (δ 4.88) of sugar residue C and C-4 (δ 76.85) of sugar residue B; H-1 (δ 5.13) of sugar residue D and C-4 (δ 76.85) of sugar residue B; H-1 (δ 5.34) of sugar residue E and C-4 (δ 76.85) of sugar residue B; and H-1 (δ 5.25) of sugar residue A and C-4 (δ 76.85) of sugar residue B. This indicates that the anomeric carbons of sugar residues C, D, E, and A are all attached to the hydroxyl group at the C-4 position of sugar residue B. Simultaneously, H-1 (δ 5.25) of sugar residue A and C-6 (δ 76.85) of sugar residue C are also coupled. The presence of a coupling signal at δ 69.54 indicates that the anomeric carbon of sugar residue A is also attached to the hydroxyl group at the C-6 position of sugar residue C; the H-1 (δ 4.88) of sugar residue C is coupled with the C-6 (δ 69.43) of sugar residue E, and with the C-4 (δ 74.09) of sugar residue D, indicating that the anomeric carbon of sugar residue C is simultaneously attached to the hydroxyl group at the C-6 position of sugar residue E and the hydroxyl group at the C-4 position of sugar residue D. Therefore, based on the combined one-dimensional and two-dimensional NMR spectra and methylation results, the main chain of AJP-2a is mainly composed of →4)-α-D-Glcp-(1→), →4,6)-α-D-Glcp-(1→) and →3,4)-α-D-Galp-(1→), while the side chains are composed of α-D-Glcp-(1→) and →6)-α-D-Glcp-(1→), which are connected to the O-4 position of sugar residue B, the O-6 position of sugar residue C, and the O-4 position of sugar residue D in the main chain, respectively.

[0123] In AJP-3a, NMR results show that the main chain is composed of sugar residues linked by multiple glycosidic bonds, such as →3)-α-D-Manp-(1→3)-α-D-GlcpA-(1→、→3)-β-D-Galp-(1→3)-α-D-GlcpA-(1→、→4)-β-L-Arap-(1→3)-α-D-GlcpA-(1→、→4)-α-D-GlcpA-(1→4)-β-L-Arap-(1→、→3 ...3)-α-D-GlcpA-(1→4)-β-L-Arap-(1→、→3)-α-D-GlcpA-(1→3)-α-D-GlcpA-(1→3)-α-D-GlcpA-(1→4)-β-L-Arap-(1→、→3)-α-D-GlcpA- The core branch residues are →3,6)-α-D-GlcpA-(1→), which can connect to →3)-β-D-Galp-(1→, →4)-β-L-Arap-(1→, →2)-β-D-Galp-(1→) at different sites, forming a complex main chain backbone. Compared with AJP-1a and AJP-2a, the polysaccharide chain structure of AJP-3a is more complex, with more types of sugar residues and denser branch connections. Its complex multi-branched structure also provides a structural basis for its bioactivity research.

[0124] Example 3: Anti-colorectal cancer activity of white peony polysaccharide

[0125] DiI fluorescently labeled HCT116 colorectal cancer cell zebrafish tumor model: Normally developed 48 hpf (hours post-fertilization) AB strain zebrafish embryos were selected and placed in 48-well cell culture plates, divided into 11 groups (n=10 embryos / group), and subjected to 72 h of drug intervention. The groups were set as follows: blank control group (Control, E3 water), positive control group (5-fluorouracil, 5-FU, 40 μg / mL), AJP-1a treatment group (50, 100, 200 μg / mL), AJP-2a treatment group (25, 50, 100 μg / mL), and AJP-3a treatment group (12.5, 25, 50 μg / mL). 500 μL of the corresponding treatment solution was added to each well, and the plates were incubated at 28.5℃, with the treatment solution replaced with fresh solution every 24 h.

[0126] After incubation, fluorescence images of tumor cells in zebrafish were observed and captured using a motorized inverted fluorescence microscope (NIB950-FL, Nexcope). The fluorescence area and intensity of the tumor region were quantitatively detected using morphometric analysis software (ImageJ, Bethesda, USA). The fluorescence signal quantification results reflected the tumor cell growth. All data are expressed as mean ± standard error (SEM). One-way ANOVA and Tukey's multiple comparison test were used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0127] Using zebrafish as a model organism, the inhibitory effects of AJP-1a, AJP-2a, and AJP-3a on colorectal cancer were evaluated. The results of tumor fluorescence intensity and area in zebrafish after 72 h are as follows: Figure 28 , Figure 29 As shown in the figure, compared with the blank control group, the fluorescence signal of the positive control 5-FU group was significantly reduced (P<0.001), indicating that the polysaccharide of *Ampelopsis japonica* has significant antitumor activity.

[0128] All three types of *Ampelopsis japonica* polysaccharides exhibited concentration-dependent inhibition of HCT116 tumor cell growth in zebrafish: AJP-3a showed the best anti-colorectal cancer activity, exhibiting significant tumor-suppressing effects even at a low concentration of 12.5 μg / mL. At 25 μg / mL, its tumor-suppressing effect was comparable to that of the positive control drug 5-FU, and at 50 μg / mL, the inhibitory effect was further enhanced and significantly superior to 5-FU. AJP-1a required medium to high concentrations of 100 μg / mL and 200 μg / mL to show significant tumor-suppressing activity, and the inhibitory effect slightly increased with increasing concentration. AJP-2a showed some tumor-suppressing effects at concentrations of 25 μg / mL and 50 μg / mL, and at a concentration of 100 μg / mL, the tumor-suppressing activity was significantly enhanced and approached the level of the positive control drug.

[0129] The differences in activity are closely related to the structural characteristics of the polysaccharides: AJP-3a possesses a unique triple helix structure and filamentous cross-linked microstructure. The triple helix structure is the key structural basis for the antitumor activity of polysaccharides, enhancing their interaction with tumor cells; therefore, it exhibits the lowest effective tumor-inhibiting concentration and the strongest activity. AJP-1a is a linear dextran with a simple structure and fewer sites of interaction with tumor cells, thus requiring higher concentrations (100 μg / mL and above) to show significant activity. AJP-2a is mainly composed of glucose, with moderate branching and structural complexity; it can inhibit tumor growth at concentrations of 25 μg / mL and 50 μg / mL, and its tumor-inhibiting effect at a concentration of 100 μg / mL is close to that of a positive control drug. In summary, the anti-colorectal cancer activity of *Ampelopsis japonica* polysaccharides is highly correlated with their molecular structure, among which AJP-3a has the potential to be developed into a colorectal cancer therapeutic drug or functional food.

Claims

1. A polysaccharide from *Ampelopsis japonica*, characterized in that, The polysaccharides from *Ampelopsis japonica* are selected from: AJP-1a, AJP-2a, and AJP-3a; AJP-1a has a relative molecular weight of 281 kDa and is composed of →1)-D-Glcp, →3)-D-Glcp-(1→, →4)-D-Glcp-(1→, →6)-D-Glcp-(1→, →3,4)-D-Glcp-(1→, →2,4)-D-Glcp-(1→ and →4,6)-D-Glcp-(1→). It is a homogeneous dextran with a glucose content of 100%. AJP-2a has a relative molecular weight of 1435 kDa and is composed of →4)-D-Arap-(1→, →1)-D-Glcp, →4)-D-Glcp-(1→, →6)-D-Glcp-(1→, →3,4)-D-Galp-(1→ and →4,6)-D-Glcp-(1→). It contains 96.65% glucose, 1.68% galactose, and 1.66% arabinose. The relative molecular weight of AJP-3a is 251.1 kDa, derived from -(1→)-L-Araf, -(1→)-L-Arap, →4)-L-Arap-(1→,→1)-D-GlcpA,→2)-D-Galp-(1→,→2)-L-Arap-(1→,→4)-D-GlcpA-(1→,→3)-D-Galp-(1→,→6)-D-Ma Composed of np-(1→,→6)-D-Galp-(1→,→2,3)-D-Manp-(1→,→3,4)-D-Manp-(1→,→3,6)-D-GlcpA-(1→ and→3,4,6)-D-Manp-(1→), with a galactose content of 22.79%, mannose content of 20.54%, arabinose content of 42.35%, and glucuronic acid content of 14.3%.

2. The method for preparing *Ampelopsis japonica* polysaccharide as described in claim 1, characterized in that, The preparation method is as follows: S1. Take the raw medicinal material of Bai Lian, crush it, and then extract it with water and precipitate it with alcohol to collect the polysaccharide precipitate; S2. The polysaccharide precipitate collected in S1 is redissolved in water, and the resulting solution is subjected to deproteinization, decolorization, dialysis and freeze-drying in sequence to obtain crude polysaccharide AJP. S3. The crude polysaccharide AJP obtained in S2 was initially separated by DEAE-52 anion exchange chromatography. Gradient elution was performed using 0-0.5 mol / L NaCl aqueous solution as the eluent. The elution was monitored by the phenol-sulfuric acid method and the elution curve was plotted. The eluents of each component were combined according to the elution curve, and then concentrated under reduced pressure, dialyzed and freeze-dried to obtain polysaccharides AJP-1, AJP-2 and AJP-3. S4. The polysaccharides AJP-1, AJP-2, and AJP-3 obtained in S3 were purified using dextran gel G-200. Elution was performed using 0.15-0.25 mol / L NaCl aqueous solution as the eluent. The elution was monitored using the phenol-sulfuric acid method, and the elution curves were plotted. The eluents of each component were combined according to the elution curves, and then concentrated under reduced pressure, dialyzed, and freeze-dried to obtain the polysaccharides AJP-1a, AJP-2a, and AJP-3a of *Ampelopsis japonica*.

3. The method for preparing *Ampelopsis japonica* polysaccharide as described in claim 2, characterized in that, The S1 procedure is as follows: Take the raw medicinal material of *Bai Lian*, pulverize it and pass it through a 50-mesh sieve. Mix the medicinal powder with water at a material-to-liquid ratio of 1:15-30, heat to 95-105℃ and extract for 1.5-2.5 hours. Filter, and repeat the extraction 1-2 times with the filter residue. Combine the extracts, concentrate under reduced pressure to 1 / 5-1 / 3 of the original volume, centrifuge, take the supernatant, add anhydrous ethanol to the system until the ethanol volume fraction is 70-90%, precipitate at 4℃ for 8-16 hours, and collect the polysaccharide precipitate by centrifugation.

4. The method for preparing the polysaccharide from *Ampelopsis japonica* as described in claim 2, characterized in that, The S2 procedure is as follows: Dissolve the polysaccharide precipitate again in water. Adjust the pH of the resulting solution to 8-9. Heat to 80-100℃, add CaCl2 solid to a final concentration of 45-55 g / L, boil for 25-35 min, cool to room temperature, filter, adjust the pH of the filtrate to 6.8-7.2, add anhydrous ethanol to a volume fraction of 75-90%, place at 4℃ for 8-16 h, centrifuge, collect the precipitate, dissolve in water, and repeat the above operation until the protein content is below 5%, completing the deproteinization process. Dissolve the deproteinized precipitate again in water, add activated carbon, stir at 45-55℃ for 35-45 min to decolorize, then centrifuge to remove the activated carbon, completing the decolorization process. Transfer the decolorized solution to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze at 3-5℃ for 24-72 h, changing the distilled water every 6-8 h, then freeze-dry to obtain crude polysaccharide AJP.

5. The method for preparing the polysaccharide from *Ampelopsis japonica* as described in claim 2, characterized in that, The S3 procedure is as follows: Crude polysaccharide AJP is dissolved in distilled water to prepare a crude polysaccharide AJP solution of 5-15 mg / mL. After filtration through a 0.45 μm aqueous microporous membrane, the solution is added to a chromatographic column packed with DEAE-52 packing material for separation. The sample loading volume is 5-15 mL. Gradient elution is performed using NaCl aqueous solutions of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L as eluents, with a flow rate of 1-2 mL / min. The elution curve is monitored using the phenol-sulfuric acid method, and the elution curves are plotted. The eluents of each component are combined according to the elution curves, concentrated under reduced pressure, dialyzed at 3-5℃ for 24-72 h, and freeze-dried to obtain polysaccharides AJP-1, AJP-2, and AJP-3.

6. The method for preparing the polysaccharide from *Ampelopsis japonica* as described in claim 2, characterized in that, The S4 procedure is as follows: Polysaccharides AJP-1, AJP-2, and AJP-3 are purified using a dextran gel G-200 column, with a loading concentration of 5-15 mg / mL and a loading volume of 5-20 mL. Elution is performed using a 0.15-0.25 mol / L NaCl aqueous solution as the eluent at a flow rate of 0.2-0.8 mL / min. The elution is monitored using the phenol-sulfuric acid method, and the elution curves are plotted. The eluents corresponding to single symmetrical peaks are combined according to the elution curves, concentrated under reduced pressure, dialyzed at 3-5℃ for 24-72 h, and freeze-dried to obtain the polysaccharides AJP-1a, AJP-2a, and AJP-3a of *Ampelopsis japonica*.

7. The use of the polysaccharide from *Ampelopsis japonica* as described in claim 1 in the preparation of anti-colorectal cancer drugs.

Images