Search

CN-122011233-A - Ampelopsis polysaccharide, preparation method thereof and application of Ampelopsis polysaccharide in preparation of colorectal cancer therapeutic drugs

CN122011233ACN 122011233 ACN122011233 ACN 122011233ACN-122011233-A

Abstract

The invention discloses Japanese ampelopsis polysaccharide and a preparation method thereof as well as application of the Japanese ampelopsis polysaccharide in preparing colorectal cancer treatment medicaments, wherein the Japanese ampelopsis polysaccharide is extracted by adopting a hot water extraction method, three novel polysaccharides are firstly separated and purified from Japanese ampelopsis by a combined purification process of combining DEAE-52 cellulose ion exchange chromatography and sephadex G-200 gel permeation chromatography, the Japanese ampelopsis polysaccharide is systematically characterized in molecular weight, monosaccharide composition, glycosidic bond connection mode, triple helix structure, microscopic morphology and the like, and the three polysaccharides show concentration-dependent anti-colorectal cancer activity on an AB-zebra fish tumor model constructed by DiI fluorescent labeled HCT116 human colorectal cancer cells, and the Japanese ampelopsis polysaccharide provides a novel candidate molecule and application basis for development of colorectal cancer treatment medicaments and related functional foods.

Inventors

  • TAO YI
  • LUO WEI

Assignees

  • 浙江工业大学

Dates

Publication Date
20260512
Application Date
20260414

Claims (7)

  1. 1. The ampelopsis polysaccharide is characterized in that the ampelopsis polysaccharide is selected from AJP-1a, AJP-2a and AJP-3a; AJP-1a has a relative molecular weight of 281kDa 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 → and has a glucose content of 100% as a uniform glucan; AJP-2a has a relative molecular weight of 1435kDa, consisting 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 → 96.65% glucose content, 1.68% galactose content, 1.66% arabinose content; AJP-3a has a relative molecular weight of 251.1kDa and consists of -(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 → galactose content of 22.79%, mannose content of 20.54%, arabinose content of 42.35% and glucuronic acid content of 14.3%.
  2. 2. The method for preparing ampelopsis polysaccharide according to claim 1, wherein the method comprises the following steps: s1, taking Japanese ampelopsis raw medicinal materials, crushing, extracting with water, precipitating with alcohol, and collecting polysaccharide precipitate; s2, re-dissolving the polysaccharide precipitate collected in the step S1 in water, and sequentially deproteinizing, decolorizing, dialyzing and freeze-drying the obtained solution to obtain crude polysaccharide AJP; S3, performing primary separation on crude polysaccharide AJP obtained in the step S2 by adopting DEAE-52 anion exchange chromatography, performing gradient elution by taking 0-0.5mol/L NaCl aqueous solution as an eluent, monitoring by using a phenol-sulfuric acid method, drawing an outflow curve, merging eluents of all components according to the outflow curve, and performing reduced pressure concentration, dialysis and freeze drying to obtain polysaccharide AJP-1, polysaccharide AJP-2 and polysaccharide AJP-3; S4, purifying polysaccharide AJP-1, AJP-2 and AJP-3 obtained in the step S3 by using sephadex G-200, eluting by using 0.15-0.25mol/L NaCl aqueous solution as an eluent, monitoring by using a phenol-sulfuric acid method, drawing an outflow curve, merging eluents of all components according to the outflow curve, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain Japanese ampelopsis polysaccharide AJP-1a, AJP-2a and AJP-3a respectively.
  3. 3. The preparation method of the ampelopsis polysaccharide according to claim 2, wherein S1 is characterized in that the preparation method comprises the following steps of taking the raw ampelopsis medicinal material, crushing the raw ampelopsis medicinal material, sieving the crushed raw ampelopsis medicinal material with a 50-mesh sieve, mixing medicinal material powder with water according to a feed liquid ratio of 1:15-30, heating the mixture to 95-105 ℃ to extract 1.5-2.5h, filtering the mixture, repeatedly extracting filter residues for 1-2 times, combining the extracting solutions, concentrating the extracting solutions under reduced pressure to 1/5-1/3 of the original volume, centrifuging the extracting solutions, taking supernatant, adding absolute ethyl alcohol until the volume fraction of the system ethyl alcohol is 70-90%, precipitating the system ethyl alcohol at 4 ℃ for 8-16h, and centrifuging the extracting polysaccharide precipitate.
  4. 4. The method for preparing Japanese ampelopsis polysaccharide according to claim 2, wherein S2 comprises the steps of dissolving polysaccharide precipitate in water again, regulating pH of the obtained solution to 8-9, heating to 80-100 ℃, adding CaCl 2 solid to a final concentration of 45-55g/L, boiling for 25-35min, cooling to room temperature, filtering, regulating pH of filtrate to 6.8-7.2, adding absolute ethanol to 75-90% of ethanol volume, standing at 4 ℃ for 8-16h, centrifuging, collecting precipitate, adding water to dissolve until protein content is lower than 5%, finishing deproteinization treatment, redissolving the deproteinized precipitate with water, adding active carbon, stirring at 45-55 ℃ for decoloration for 35-45min, centrifuging to remove active carbon, finishing decoloration treatment, transferring the decolored solution into a dialysis bag with molecular weight of 3500Da, dialyzing at 3-5 ℃ for 24-72h, replacing distilled water every 6-8h, and freeze-drying to obtain crude polysaccharide AJP.
  5. 5. The method for preparing Japanese ampelopsis polysaccharide according to claim 2, wherein S3 comprises the steps of dissolving crude polysaccharide AJP in distilled water to prepare a crude polysaccharide AJP solution of 5-15mg/mL, filtering with a 0.45 μm water-based microporous membrane, adding to a chromatographic column filled with DEAE-52 for separation, loading 5-15mL, gradient eluting with 0, 0.1, 0.2, 0.3, 0.4 and 0.5mol/L NaCl aqueous solution as eluent at a flow rate of 1-2mL/min, monitoring with phenol-sulfuric acid method, drawing an outflow curve, combining the eluents of the components according to the outflow curve, concentrating under reduced pressure, dialyzing at 3-5 ℃ for 24-72h, and freeze-drying to obtain polysaccharides AJP-1, AJP-2 and AJP-3.
  6. 6. The preparation method of Japanese ampelopsis polysaccharide according to claim 2, wherein S4 is characterized in that polysaccharide AJP-1, AJP-2 and AJP-3 are purified by using a sephadex G-200 chromatographic column, the loading concentration is 5-15mg/mL, the loading amount is 5-20mL, naCl aqueous solution with the flow rate of 0.15-0.25mol/L is used as an eluent, the flow rate is 0.2-0.8mL/min, the monitoring is carried out by using a phenol-sulfuric acid method, an outflow curve is drawn, the eluents corresponding to single symmetrical peaks are combined according to the outflow curve, and the eluents are concentrated under reduced pressure, dialyzed for 24-72h at 3-5 ℃ and freeze-dried to obtain Japanese ampelopsis polysaccharide AJP-1a, AJP-2a and AJP-3a respectively.
  7. 7. Use of the ampelopsis polysaccharide according to claim 1 for preparing an anti-colorectal cancer drug.

Description

Ampelopsis polysaccharide, preparation method thereof and application of Ampelopsis polysaccharide in preparation of colorectal cancer therapeutic drugs Technical Field The invention belongs to the technical field of natural pharmaceutical chemistry, and particularly relates to separation and purification, structural characterization and application of three novel polysaccharides in a traditional Chinese medicine Japanese ampelopsis herb in preparation of anti-colorectal cancer drugs. The invention establishes a preparation and structure analysis method of Japanese ampelopsis polysaccharide, and confirms the anti-colorectal cancer activity. Background At present, the clinical common chemotherapeutics such as 5-fluorouracil can inhibit the proliferation of tumor cells and delay the disease progression to a certain extent, but the long-term use of the chemotherapeutics is easy to induce the drug resistance of the tumor cells, and the chemotherapeutics are accompanied with obvious toxic and side effects such as bone marrow suppression, digestive tract reaction and the like. Therefore, the research and development of safe and efficient anti-colorectal cancer drugs has important clinical significance. The polysaccharide from natural sources has the advantages of no toxicity, good biocompatibility and the like, and is an important source of antitumor active ingredients. Researches show that the antitumor activity of polysaccharide is closely related to the structural characteristics of polysaccharide, including molecular weight distribution, monosaccharide composition, glycosidic bond connection mode, branching degree and the like, and the obtained high-purity uniform polysaccharide is the basis for developing structural characterization and activity researches. However, the polysaccharide separation and purification process is complex, and impurities can be removed by repeatedly optimizing the extraction and purification conditions, so that the polysaccharide with stable structure and qualified purity can be obtained. Ampelopsis japonica is a traditional Chinese medicinal material, has the effects of clearing heat and removing toxicity, healing sore and promoting tissue regeneration, and modern pharmacological research suggests that the extract has a certain antitumor potential. However, the current research on systematic separation and purification of ampelopsis polysaccharide, polysaccharide preparation and anti-colorectal cancer activity is still blank. The zebra fish model has the advantages of rapid propagation, low breeding cost, short experimental period and the like because of high homology with human genome (about 84% of human pathogenic genes can find homologous genes in zebra fish), and has been widely applied to rapid screening and activity evaluation of antitumor drugs. Dil-marked human colorectal cancer HCT116 cells are injected into the zebra fish yolk sac to construct a tumor model, so that the occurrence and development processes of human colorectal cancer can be effectively simulated, and an intuitive and efficient in-vivo screening platform is provided for evaluating the anti-tumor effect of candidate drugs. According to the invention, three novel polysaccharides are purified from Japanese ampelopsis for the first time by optimizing extraction and separation process parameters, structural characterization is performed by comprehensively applying NMR, IR, GC-MS and other technical means, the antitumor activity of the novel polysaccharides is evaluated in a zebra fish colorectal cancer model, and the novel polysaccharides are compared with a positive drug 5-fluorouracil, so that a material basis and technical support are provided for research and development of novel anti-colorectal cancer drugs. Disclosure of Invention Aiming at the technical problems that the existing Japanese ampelopsis polysaccharide system separation and purification technology is imperfect, the pure polysaccharide preparation and the fine structure research are deficient, and the anti-colorectal cancer activity is not clear, the invention provides three Japanese ampelopsis polysaccharides, the preparation method thereof and the application of preparing anti-colorectal cancer drugs. The invention adopts a hot water extraction method to extract the Ampelopsis japonica crude polysaccharide, and three novel polysaccharides are obtained by separating and purifying the Ampelopsis japonica crude polysaccharide for the first time through a combined purification process of combining DEAE-52 cellulose ion exchange chromatography and Sephadex G-200 (Sephadex G-200) gel permeation chromatography. And then adopting methods such as infrared spectrum (IR), nuclear Magnetic Resonance (NMR), high Performance Gel Permeation Chromatography (HPGPC), monosaccharide composition analysis, methylation-GC-MS analysis and the like to carry out structural identification, and evaluating the colorectal cancer resistance activity of the Japanese ampelo