Preparation method of Huai Chrysanthemum polysaccharide sulfated derivative and application of Huai Chrysanthemum polysaccharide sulfated derivative in tumor resistance
By isolating and purifying polysaccharides from Huaiju and sulfating modification, sulfated derivatives of Huaiju polysaccharides were prepared, which solved the problem of drug resistance of pancreatic cancer to existing treatment methods, achieved effective inhibition of pancreatic cancer cells and reduced toxicity to normal cells.
Patent Information
- Application Number
- CN202311563531.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-21
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art has the problem of extremely limited treatment options in the treatment of pancreatic cancer, especially because pancreatic cancer is highly resistant to chemoradiotherapy, which leads to the urgent need for new treatment methods.
By isolating and purifying the polysaccharide from Huaiju and sulfate modification by the modified chlorosulphonic acid-pyridine method, a sulfated derivative of Huaiju polysaccharide was prepared for anti-tumor treatment.
The sulfated derivative of Huaiju polysaccharide can effectively inhibit the proliferation of pancreatic cancer cells within the concentration range of 10μg/mL to 1000μg/mL. At the same time, it is less toxic to normal pancreatic epithelial cells and hepatocytes, showing good specificity, high efficiency and low toxicity and safety.
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Figure CN120025465A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant extraction, and in particular to a method for preparing a sulfated derivative of chrysanthemum polysaccharide and an application thereof in anti-tumor treatment. Background Art
[0002] Pancreatic cancer is one of the leading causes of death from all cancers worldwide, with a 5-year survival rate of 26% if localized at diagnosis and only 2% if already metastatic at diagnosis. Currently, surgery, embolization, immunotherapy, chemotherapy, and radiotherapy are the main treatments for the disease. However, pancreatic cancer is highly resistant to chemotherapy and radiotherapy, and treatment options are extremely limited. Therefore, new treatments for pancreatic cancer are urgently needed.
[0003] Finding new anticancer drugs from natural resources is an important way to find more efficient and less toxic drugs for the treatment of pancreatic cancer. Polysaccharides exist in almost all organisms and play different functions in life. Polysaccharides have low toxicity and are gradually becoming a new development direction for anti-tumor drugs. Structural improvement and molecular modification of polysaccharides have become a very important strategy to enhance their biological activity. Sulfation modification is an effective way to enhance or change the biological activity of polysaccharides. Therefore, it is of great significance to researchers and patients to carry out sulfation modification of polysaccharides isolated from natural products to obtain polysaccharide derivatives with strong biological activity and relatively low cytotoxicity.
[0004] As one of the four major Huai medicines, Chrysanthemum morifolium is well-known both at home and abroad. It is a famous local medicinal material in my country. Due to its extremely high medicinal value, it has been included in the herbal books of all dynasties. Traditional Chinese medicine believes that Chrysanthemum morifolium has a sweet and bitter taste and is slightly cold in nature. It has the effects of clearing the liver and improving eyesight, dispersing wind-heat, and clearing away heat and detoxifying. In addition, Chrysanthemum morifolium can also be used as a raw material for food, tea, spices, and cosmetics. Polysaccharides are the main active ingredients of Huaijuhua. Structural modification and transformation of Huaijuhua polysaccharides have great development and utilization value. For example, the earlier study by the inventor team, "Structural Characteristics of Chrysanthemum Polysaccharides and Their Activity on NF-κB and Tumor Cells", disclosed the composition of Huaijuhua polysaccharides CMJA0S1 and CMJA0S2, and specifically reported that Huaijuhua polysaccharides showed good mass concentration dependence at 12.3-1 000μg / mL. Among them, the polysaccharide with the highest inhibition rate on PANC-1 cells was the secondary Huaijuhua water-extracted crude polysaccharide CMJA0. When the mass concentration of the polysaccharide was 1 000μg / mL, its inhibition rate reached 69.3%. The inhibition rate of its uniform polysaccharide CMJA0S2 on PANC-1 cells increased with the increase of mass concentration, reaching up to 66.0%. That is, when Huaijuhua polysaccharide is 1000μg / mL, the strongest inhibition rate is only 70%. The above-mentioned polysaccharides are neutral sugars, and a large amount of acidic sugars are also found during extraction. Therefore, how to utilize these acidic sugars to enhance their tumor-killing activity is a topic that technicians in this field continue to study. Summary of the invention
[0005] In view of the deficiencies in the prior art, the present invention designs a method for preparing a sulfated derivative of chrysanthemum polysaccharide and applies the method to anti-tumor.
[0006] One of the purposes of the present invention is to provide a chrysanthemum polysaccharide, wherein the chrysanthemum polysaccharide has the following structure:
[0007]
[0008] Furthermore, the monosaccharide composition of the chrysanthemum polysaccharide includes mannose, rhamnose, galacturonic acid, galactose and arabinose, and the molar ratios thereof are 2.00-6.00:9.01-13.60:6.00-11.01:10.30-14.60, respectively. The weight average molecular weight of the chrysanthemum polysaccharide is 10-70 kDa, the dispersion coefficient D is 1-2, and the number average molecular weight is 10-80 kDa.
[0009] Furthermore, the sugar residue connection mode of the chrysanthemum polysaccharide includes terminal-linked arabinose, 1,5-linked arabinose, 1,2-linked rhamnose, terminal-linked glucuronic acid, terminal-linked galactose, 1,3,5-linked arabinose, 1,2,4-linked rhamnose, 1,4-linked galactose, 1,4-linked galacturonic acid, 1,3-linked galactose, 1,6-linked galactose, 1,3,4-linked galacturonic acid , 1,3,6-linked galactose, the molar ratio of which is 1.00~4.00:1.00~3.00:6.00~12.00:0.50~2.00:5.00~11.00:1.00~4.00:3.00~9.00:2.00~8.00:3.00~11.00; 1.00~4.00:1.00~7.00:1.00~4.00:1.00~2.00:1.00~6.00.
[0010] The second object of the present invention is to provide a method for preparing the chrysanthemum polysaccharide, comprising the following steps:
[0011] a. Polysaccharide extraction: After defatting, the dried buds of Chrysanthemum morifolium are extracted with boiling water, and then washed with alcohol precipitation to obtain crude polysaccharides;
[0012] b. Polysaccharide purification: The obtained crude polysaccharide is separated and purified by anion exchange column and gel column to obtain the chrysanthemum polysaccharide.
[0013] Specifically, the method comprises the following steps:
[0014] a. Polysaccharide extraction: soak the dried medicinal materials of Huaijuhua in 95% ethanol, soak at room temperature for 5 to 9 days, and dry them for use; add water to the dried medicinal materials at a solid-liquid ratio of 1:10 to 1:30 (g / mL), keep it slightly boiling and decocted for 2 to 4 hours / time, and decoct it 8 to 13 times in total, and then combine the filtrates; concentrate the filtrate to 1 / 10 to 1 / 20 of the original volume, cool it naturally to room temperature, and dialyze the concentrate against running water through cellophane for 2 to 3 days; concentrate the dialyzate to 1 / 5 to 1 / 10 of the original volume, cool it naturally to room temperature, centrifuge it at 4000 to 8000 rpm for 10 to 30 minutes, take the supernatant and add 95% ethanol with a volume of 3 to 6 times the volume of the supernatant while stirring, and let it stand overnight; centrifuge the alcohol precipitate at 4000 to 8000 rpm for 10 to 30 minutes, add water to the precipitate to dissolve it, heat it to evaporate the remaining ethanol, freeze it, and freeze it in a freeze dryer to obtain Huaijuhua crude polysaccharide;
[0015] b. Polysaccharide purification: The crude polysaccharide was separated by DEAE anion exchange, with a sample load range of 6 to 10 g dissolved in 60 to 100 mL of deionized water, eluted with 0, 0.05, 0.1, 0.2, 0.4, and 0.8 M NaCl, and the 0.2 M NaCl elution fraction was collected; it was then purified by gel column, with a sample load range of 100 to 200 mg dissolved in 2.5 to 5 mL of deionized water, and eluted with 0.2 M NaCl to obtain chrysanthemum polysaccharide.
[0016] The third object of the present invention is to provide a sulfated derivative of the chrysanthemum polysaccharide, which is obtained by sulfating the chrysanthemum polysaccharide.
[0017] Furthermore, the degree of substitution of the sulfated derivative of chrysanthemum polysaccharide is 0.71 to 1.51.
[0018] The fourth object of the present invention is to provide a method for preparing the sulfated derivatives of Chrysanthemum morifolium polysaccharide, comprising the following steps:
[0019] 1) At a constant temperature of 60-80°C, anhydrous pyridine is stirred uniformly, and chlorosulfonic acid is added at a ratio of 1-5:1 at a rate of 5-11 seconds / drop to obtain an esterifying agent;
[0020] 2) dissolving the chrysanthemum polysaccharide in formamide at a ratio of 10 to 30:1, adding the esterification agent prepared in step 1) after it is fully dissolved, stirring at a constant temperature of 60 to 80° C. for 2 to 4 hours, and cooling to room temperature;
[0021] 3) Adjust the pH value to 7.0, dialyze against saturated sodium bicarbonate for 24 hours, and then dialyze against deionized water for 48 hours, centrifuge the concentrate at 4000-8000 rpm for 10-30 minutes, take the supernatant, freeze it, and then freeze-dry it in a freeze dryer to obtain the sulfated derivative of chrysanthemum polysaccharide.
[0022] The fifth object of the present invention is to provide the use of sulfated derivatives of chrysanthemum polysaccharide in the preparation of anti-tumor drugs or pharmaceutical compositions and functional products.
[0023] Furthermore, the tumor is pancreatic cancer, and the administration concentration is 10 μg / mL to 1000 μg / mL, and most preferably 20 μg / mL to 740 μg / mL.
[0024] The sixth object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned sulfated derivative of chrysanthemum polysaccharide and pharmaceutically acceptable excipients.
[0025] Working principle and beneficial effects of the present invention:
[0026] The present invention separates and purifies a chrysanthemum polysaccharide from chrysanthemum, and the polysaccharide is modified by an improved chlorosulfonic acid-pyridine method to obtain a sulfated derivative of chrysanthemum polysaccharide. According to research, 10 μg / mL to 1000 μg / mL of sulfated derivative of chrysanthemum polysaccharide can inhibit the proliferation of pancreatic cancer cell lines, and no obvious toxicity is observed to normal pancreatic epithelial cells and hepatocytes. This indicates that the sulfated derivative of chrysanthemum polysaccharide of the present invention has good specificity, high efficiency, low toxicity, and good safety. It can be used to prepare anti-tumor drugs or pharmaceutical compositions and functional products. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a schematic diagram of the infrared spectrum of chrysanthemum polysaccharide;
[0028] Figure 2 This is a schematic diagram of the infrared spectrum of the sulfated derivatives of chrysanthemum polysaccharide;
[0029] Figure 3 This is a schematic diagram of the molecular weight determination results of chrysanthemum polysaccharide;
[0030] Figure 4 This is a schematic diagram of the molecular weight determination results of the sulfated derivatives of chrysanthemum polysaccharide;
[0031] Figure 5 Schematic diagram of monosaccharide composition of chrysanthemum polysaccharide, (A) chrysanthemum polysaccharide, (B) mixed standard;
[0032] Figure 6 Chrysanthemum polysaccharide 13 Schematic diagram of C NMR spectrum;
[0033] Figure 7 Schematic diagram of the effect of chrysanthemum polysaccharide and chrysanthemum polysaccharide sulfated derivatives on the viability of PANC-1 pancreatic cancer cells and HPDE6-C7 normal pancreatic epithelial cells;
[0034] Figure 8 This is a schematic diagram of the effect of sulfated derivatives of chrysanthemum polysaccharide on the cell viability of four pancreatic cancer cell lines: PANC-1, BxPC-3, SW1990 and AsPC-1;
[0035] Fig. 9 Schematic diagram of the effect of sulfated derivatives of chrysanthemum polysaccharide on the viability of HPDE6-C7 normal pancreatic epithelial cells and L02 normal human liver cell lines. DETAILED DESCRIPTION
[0036] The following is further described in detail through specific implementation methods:
[0037] 1. Preparation of Chrysanthemum polysaccharide
[0038] a. Polysaccharide extraction: soak the dried medicinal materials of Huaijuhua in 95% ethanol, soak at room temperature for 5 to 9 days, and dry them for use; add water to the dried medicinal materials at a solid-liquid ratio of 1:10 to 1:30 (g / mL), keep it slightly boiling and decocted for 2 to 4 hours / time, and decoct it 8 to 13 times in total, and then combine the filtrates; concentrate the filtrate to 1 / 10 to 1 / 20 of the original volume, cool it naturally to room temperature, and dialyze the concentrate against running water through cellophane for 2 to 3 days; concentrate the dialyzate to 1 / 5 to 1 / 10 of the original volume, cool it naturally to room temperature, centrifuge it at 4000 to 8000 rpm for 10 to 30 minutes, take the supernatant and add 95% ethanol with a volume of 3 to 6 times the volume of the supernatant while stirring, and let it stand overnight; centrifuge the alcohol precipitate at 4000 to 8000 rpm for 10 to 30 minutes, add water to the precipitate to dissolve it, heat it to evaporate the remaining ethanol, freeze it, and freeze it in a freeze dryer to obtain Huaijuhua crude polysaccharide;
[0039] b. Polysaccharide purification: The crude polysaccharide was separated by DEAE anion exchange, with a sample load range of 6 to 10 g dissolved in 60 to 100 mL of deionized water, eluted with 0, 0.05, 0.1, 0.2, 0.4, and 0.8 M NaCl, and the 0.2 M NaCl elution fraction was collected; it was then purified by gel column, with a sample load range of 100 to 200 mg dissolved in 2.5 to 5 mL of deionized water, and eluted with 0.2 M NaCl to obtain chrysanthemum polysaccharide.
[0040] 2. Preparation of Sulfated Derivatives of Chrysanthemum Polysaccharide
[0041] 1) At a constant temperature of 60-80°C, anhydrous pyridine is stirred uniformly, and chlorosulfonic acid is added at a ratio of 1-5:1 at a rate of 5-11 seconds / drop to obtain an esterifying agent;
[0042] 2) dissolving chrysanthemum polysaccharide in formamide at a ratio of 10 to 30:1, adding the esterification agent prepared in step 1), stirring at a constant temperature of 60 to 80° C. for 2 to 4 hours, and cooling to room temperature;
[0043] 3) Adjust the pH value to 7.0, dialyze against saturated sodium bicarbonate for 24 hours, and then dialyze against deionized water for 48 hours. Centrifuge the concentrate at 4000-8000 rpm for 10-30 minutes, take the supernatant, freeze it, and freeze-dry it in a freeze dryer to obtain the sulfated derivative of chrysanthemum polysaccharide.
[0044] 3. Product Analysis and Structural Identification
[0045] 1. Determination of polysaccharide purity:
[0046] Accurately weigh 2-6 mg of polysaccharide sample, dissolve in 300-600 μL of 0.1 M sodium nitrate, oscillate to fully dissolve the sample, centrifuge at 4000-8000 rpm for 5-10 min, and filter the supernatant through a 0.22 μM aqueous filter membrane for purity determination.
[0047] Polysaccharide-specific gel chromatography columns were connected in series: Shodex KS 804 (8.0 mm × 300 mm, exclusion limit 4 × 105 Da) and Shodex KS 802 (8.0 mm × 300 mm, exclusion limit 1 × 104 Da). Chromatographic conditions: mobile phase was 0.1 M sodium nitrate, flow rate was 0.5 mL / min, injection volume was 10-20 μL, column temperature was 35°C, UV absorption wavelength was 280 nm, differential detector temperature was 35°C, 40-60 min / sample.
[0048] 2. Analysis of polysaccharide monosaccharide composition:
[0049] Complete acid hydrolysis: Weigh 2-4 mg of sample, dissolve in 2 mL of distilled water, vortex and oscillate to dissolve as much as possible (heating or ultrasound can be used), add the sample solution to a heart-shaped bottle (specification 50 / 19), then add 2 mL of 4M trifluoroacetic acid (TFA) and mix well, add the hollow stopper of the heart-shaped bottle, and seal the stopper and the contact port of the heart-shaped bottle with medical rubber paste, and heat and hydrolyze at 110°C for 2-5 hours. After hydrolysis, cool, add methanol and evaporate under reduced pressure for several times to remove TFA. Add 200 μL of distilled water to dissolve the hydrolyzate.
[0050] PMP derivatization process: Take 100μL of the 200μL hydrolyzate in the previous step and add it to a 10mL EP tube. Add 100μL of 0.6M NaOH solution and mix well. Add 200μL of freshly prepared 0.5M PMP, seal the tube and mix well. Heat in a 70℃ water bath for 110min. After the reaction is completed, cool to room temperature, then add 200μL of 0.3M HCl, and then add 400μL of deionized water to make the total volume of the system 1mL.
[0051] Preparation of standard solution: Prepare 9 mg / mL of each monosaccharide with deionized water, take 100 μL of each solution and mix them, then you will get 9 kinds of monosaccharide standard solutions, each with a concentration of 1 mg / mL. When derivatizing, take out 100 μL of the standard mixture for derivatization operation.
[0052] Extraction: Oscillate on an oscillator for 3 minutes, centrifuge at 8000 rpm for 5 minutes, and stand at room temperature for 30 minutes. Keep the upper aqueous phase, repeat the extraction with chloroform three times in the same operation, and filter the upper aqueous phase through a 0.22 μm microporous membrane.
[0053] HPLC analysis: The analytical column was a C18 reverse phase column, the mobile phase was phosphate buffer (the volume ratio of pH 7.0 phosphate buffer to acetonitrile was 83:17), the column temperature was 35°C, the flow rate was 1 mL / min, the ultraviolet absorption wavelength was 245 or 254 nm, the injection volume was 10 μL, and the detection time was 1 h / sample.
[0054] The main stretching vibration absorption peaks of the infrared characteristic spectrum of chrysanthemum polysaccharide Figure 1 The infrared characteristic spectrum of the chrysanthemum polysaccharide is basically the same as that of the chrysanthemum polysaccharide shown in the figure. In the infrared characteristic spectrum of the chrysanthemum polysaccharide, 3304.5cm -1 OH stretching vibration absorption peak, 2924.3cm -1 It is the CH stretching vibration absorption peak, 1405.3~1033.1cm -1 It is the vibration signal of CO and sugar ring. 1598.4cm -1 The presence of an absorption peak indicates that the polysaccharide contains uronic acid. Figure 2 This is the infrared characteristic spectrum of the sulfated derivatives of chrysanthemum polysaccharide. Compared with the original sugar spectrum, at 1218.8cm -1 There is an obvious asymmetric stretching vibration peak of S=O at 805.1cm -1 An obvious symmetrical vibration signal peak of COS appears at .
[0055] The chromatographic columns Shodex KS 804 and KS 802 were connected in series, and the molecular weight results were analyzed by GPC. The purity was determined by HPGPC analysis. Figure 3 As shown: the weight average molecular weight (Mw) of chrysanthemum polysaccharide is 10-70 kDa, the number average molecular weight (Mn) is 10-70 kDa, and the dispersion coefficient D is 1-2; Figure 4 As shown: the weight average molecular weight (Mw) of the sulfated derivatives of chrysanthemum polysaccharide is 15-85 kDa, the number average molecular weight (Mn) is 15-85 kDa, and the dispersion coefficient D is 1-2.
[0056] like Figure 5 As shown, A: Monosaccharide composition analysis of chrysanthemum polysaccharide (Man: mannose; Rha: rhamnose; GlcA: glucuronic acid; GalA: galacturonic acid; Glc: glucose; Gal: galactose; Xyl: xylose; Ara: arabinose). B: The peak order of monosaccharide composition measured by PMP pre-column derivatization of 9 monosaccharide standards;
[0057] The monosaccharide composition analysis by PMP pre-column derivatization method showed that the chrysanthemum polysaccharide contained mannose, rhamnose, galacturonic acid, galactose and arabinose, and the molar ratio was 2.00~6.00:9.01~13.60:6.00~11.01:10.30~14.60.
[0058] 3. Analysis of polysaccharide residue connection patterns
[0059] Take 5-10 mg of the sample of the polysaccharide component of Huaijuhua for methylation analysis. The sample is placed in a 50 mL chicken heart bottle, 2 mL of deionized water is added to dissolve, freeze-dry and place in a drying cabinet overnight (the reaction is guaranteed to be anhydrous), and completely dissolved in 2 ml of anhydrous DMSO the next day. After the polysaccharide sample is completely dissolved (if the sample is not well soluble in DMSO, it can be heated and stirred at a constant temperature of 60-80 ° C overnight to dissolve), add 40-60 mg of ground dry sodium hydroxide powder, and stir the reaction at room temperature for 5-9 minutes. In an ice water bath, slowly add 1 mL of iodomethane (30-40 minutes), remove the ice bath, and react at room temperature in the dark for 30 minutes. After the reaction is completed, add 1 ml of deionized water to the reaction system to quench the reaction. The solution is concentrated under reduced pressure to remove excess iodomethane, dialyzed against deionized water for 24-72 hours, the dialysate is concentrated, frozen and freeze-dried in a freeze dryer. Repeat the above steps 4-5 times. The completely methylated sample was completely acid hydrolyzed, 4 ml of 2M TFA was added and reacted at 110°C for 4 h, and methanol was added 4 to 5 times until there was no irritating sour taste after cooling to room temperature, 2 ml of water and 50 mg of sodium borohydride were added for reduction, and the mixture was sealed and reacted at room temperature for 3 h, and sodium borohydride was added to terminate the reaction after neutralization with 25% acetic acid solution, and methanol was added 4 to 5 times to remove excess acid until there was no irritating sour taste, and the mixture was placed in a 100°C oven for drying for 15 min, and then 3 ml of acetic anhydride was added and placed in a 100°C oven for acetylation, and the reaction was carried out for 1.5 h, and toluene was added 4 to 5 times to remove excess acetic anhydride until there was no irritating odor, and finally 15 mL of chloroform and 15 mL of deionized water (v / v was 1:1) were added to the reaction bottle for extraction, and the organic phase was washed with deionized water 5 times, dried over anhydrous sodium sulfate, and the chloroform was concentrated under reduced pressure to 200 to 600 μL, filtered through a 0.22 μm organic phase filter membrane, and placed in a liquid phase vial. The connection mode of polysaccharides was detected and analyzed by GC-MS (Thermo Fisher ISQ7000).
[0060] The GC-MS analysis of sugar residue connection showed that the polysaccharides from Huaihua Chrysanthemum have the following connection modes: terminal-linked arabinose, 1,5-linked arabinose, 1,2-linked rhamnose, terminal-linked glucuronic acid, terminal-linked galactose, 1,3,5-linked arabinose, 1,2,4-linked rhamnose, 1,4-linked galactose, 1,4-linked galacturonic acid, 1,3-linked galactose, 1,6-linked galactose, 1,3,4-linked galacturonic acid, The molar ratio of 1,3,6-linked galactose is 1.00~4.00: 1.00~3.00: 6.00~12.00: 0.50~2.00: 5.00~11.00: 1.00~4.00: 3.00~9.00: 2.00~8.00: 3.00~11.00; 1.00~4.00: 1.00~7.00: 1.00~4.00: 1.00~2.00: 1.00~6.00, as shown in the following table.
[0061] Table 1 shows the results of sugar residue connection mode of chrysanthemum polysaccharide
[0062]
[0063]
[0064] 4. Determination of polysaccharide sulfation substitution degree:
[0065] Reagent preparation:
[0066] 1) 1 mol / L hydrochloric acid solution: Measure 10 mL of concentrated hydrochloric acid (10 M) into a 100 mL volumetric flask and add deionized water to the mark.
[0067] 2) Barium chloride-gelatin reagent: weigh 1.25g gelatin into a beaker and add 100mL deionized water. Heat in water and keep
[0068] Stir until dissolved, place at room temperature, pour into a 250mL volumetric flask, rinse the residual solution in the beaker with a small amount of deionized water and dilute to the mark. Take 250mL of this solution, add 2.5g of barium chloride, place on a magnetic stirrer and stir until dissolved, then place at 4°C overnight.
[0069] 3) 3% trifluoroacetic acid solution (w / v): weigh 30.0 g of trifluoroacetic acid and dissolve it in 500 mL.
[0070] mL volumetric flask to volume.
[0071] 4) Standard sulfuric acid solution: Accurately weigh 86.7 mg of anhydrous sodium sulfate and dissolve it in 50 mL of 1 mol / L hydrochloric acid solution and stir until dissolved. Place the dissolved solution in a 100 mL volumetric flask and add 1 mol / L hydrochloric acid solution to the mark.
[0072] Standard curve drawing:
[0073] Accurately measure 0, 0.04, 0.08, 0.12, 0.16, and 0.2 mL into a stoppered test tube, and then add 1 mol / L hydrochloric acid solution until the total solution volume is 0.2 mL. Add 3.8 mL of 3% trifluoroacetic acid solution and 1 mL of barium chloride-gelatin solution to each test tube, shake evenly, and let stand at room temperature for 20 minutes. Use a blank tube (add 0 mL of standard solution) as a reference and measure the absorbance value at 360 nm.
[0074] Determination of samples:
[0075] Accurately weigh 4.5 mg of sample, place in a stoppered test tube, add 4.5 mL of 1 mol / L hydrochloric acid solution, and shake the sample.
[0076] Dissolve, seal the tube and hydrolyze in a 100℃ oven for 2.5h. After the reaction is finished, cool to room temperature, take 0.2mL for determination, add 3.8mL 3% trifluoroacetic acid solution and 1mL barium chloride-gelatin solution to the remaining operation standard curve, shake evenly and let stand at room temperature for 20min. Use a blank tube (add 0mL of standard solution) as a reference and measure the absorbance value at 360nm.
[0077] The sulfate substitution degree is calculated as follows:
[0078] DS = M × W% / (96-80 × W%)
[0079] Wherein W% is the content of sulfate groups in the sulfate-derivatized polysaccharide, M is the molecular weight of the sugar residue, and the chrysanthemum polysaccharide is rhamnogalacturonic acid polysaccharide, so M is 162.
[0080] The sulfation degree of the sulfated polysaccharide was determined to be about 1.17.
[0081] 5. Polysaccharide NMR analysis:
[0082] Take 20-40 mg of the polysaccharide sample of Chrysanthemum japonicum, add 0.5 mL of D2O to dissolve it, add 2.5 μL of acetone as the internal standard (δH=2.29 ppm, δC=31.5 ppm), and measure the one-dimensional NMR spectrum at 25°C on a Bruker AVANCE III 500M NMR instrument. The results are as follows: Figure 6As shown. In the 13C NMR spectrum of the chrysanthemum polysaccharide, the terminal carbon signals at δ110-δ108 are C1 signals of T-arabinose, 1,5-arabinose, 1,3,5-arabinose and terminal arabinose, respectively; the terminal carbon signals at δ106-δ104 are C1 signals of 1,3,6-galactose, 1,3-galactose, 1,6-galactose and terminal galactose, respectively; the terminal carbon signals at δ99-δ101 are C1 signals of 1,2-rhamnose and 1,2,4-rhamnose, respectively; the terminal carbon signals at δ92-δ100 are C1 signals of 1,4-galacturonic acid, 1,3,4-galacturonic acid and T-galacturonic acid, respectively; and the signal peak of rhamnose methyl carbon is at δ17.74. From the above results, it can be found that the polysaccharide is RG-1 type polysaccharide.
[0083] The specific structure of chrysanthemum polysaccharide is:
[0084]
[0085] 4. Determination of antitumor activity of sulfated derivatives of chrysanthemum polysaccharide
[0086] Different cell lines were cultured in different culture media. AsPC-1, BxPC-3 and HPDE6-C7 cells were cultured in 1640 medium with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. LO2, SW1990 and PANC-1 cells were cultured in DMEM medium with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. All cell lines were cultured in 5% CO 2 Culture in a 37°C constant temperature incubator. Count the cells that are in good growth condition and plate them in a 96-well plate at a density of 2000 to 5000 cells per well, with 43 to 6 replicates per group. After the cells adhere to the wall (about 8 hours), add the compound solution of the target concentration to treat the cells, and the control group is treated with the carrier solution. After continuing to act in a 37°C constant temperature incubator for 72 hours, add 10μL MTT (5mg / mL) to each well, gently pat evenly, and place it in the incubator for further incubation for 4 hours. Then, aspirate the culture medium and MTT, and add 150μL DMSO to each well. Place the culture plate on a shaker and shake for 15 to 30 minutes until the formazan is completely dissolved. Set the wavelength of the microplate reader to 490nm, measure the absorbance of each well at this wavelength, and set the zero well in the experiment. Calculate the cell viability according to the formula, cell viability = (treatment group OD-zero well OD) / (control group OD-zero well OD) × 100%.
[0087] The MTT experiment was used to detect the cell viability of four pancreatic cancer cells, PANC-1, BxPC-3, SW1990 and AsPC-1, as well as normal pancreatic epithelial cells HPDE6-C7 and normal liver cells LO2 after 72 hours. The results showed that Huaijuhua polysaccharide had no significant inhibitory activity on the proliferation of pancreatic cancer cells. After sulfation modification, it showed significant inhibitory activity on the proliferation of pancreatic cancer cells with less toxic side effects. The results are shown in Figure 7 .Depend on Figure 8 It can be seen that the inhibition rate of 14.44μM (721.5μg / mL) of the sulfated derivative of chrysanthemum polysaccharide on pancreatic cancer cells PANC-1, BxPC-3 and SW1990 reached 40-60% within 72 hours, and the inhibition rate on metastatic pancreatic cancer cell AsPC-1 was as high as 80%. Compared with gemcitabine (GEM), the first-line clinical drug for pancreatic cancer, the inhibition rate of the sulfated derivative of chrysanthemum polysaccharide on pancreatic cancer cell lines at a concentration of 14.44μM is very close to that of 10μM (2.6μg / mL) gemcitabine, indicating that the sulfated derivative of chrysanthemum polysaccharide has a good anti-proliferation effect on pancreatic cancer cells. Fig. 9 It can be seen that even at a high concentration of 14.44 μM, the inhibition rate of the sulfated derivatives of chrysanthemum polysaccharide on normal pancreatic epithelial cells HPDE6-C7 and normal hepatocytes LO2 is still less than 20%, which is far lower than the inhibition rate of gemcitabine. This indicates that the sulfated derivatives of chrysanthemum polysaccharide are less toxic to normal pancreatic epithelial cells and hepatocytes.
[0088] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A kind of chrysanthemum polysaccharide, in, The chrysanthemum polysaccharide has the following structure:
2. The chrysanthemum polysaccharide according to claim 1, Features: The monosaccharide composition includes mannose, rhamnose, galacturonic acid, galactose and arabinose, and the molar ratios thereof are 2.00-6.00:9.01-13.60:6.00-11.01:10.30-14.60 respectively; the weight average molecular weight of the chrysanthemum polysaccharide is 10-70 kDa, and the dispersion coefficient D is 1-2.
3. The chrysanthemum polysaccharide according to claim 1, Features: The sugar residues are linked in a manner that includes terminally linked arabinose, 1,5-linked arabinose, 1,2-linked rhamnose, terminally linked glucuronic acid, terminally linked galactose, 1,3,5-linked arabinose, 1,2,4-linked rhamnose, 1,4-linked galactose, 1,4-linked galacturonic acid, 1,3-linked galactose, 1,6-linked galactose, 1,3,4-linked galacturonic acid, 1,3,6 -linked galactose, the molar ratio is 1.00~4.00:1.00~3.00:6.00~12.00:0.50~2.00:5.00~11.00:1.00~4.00:3.00~9.00:2.00~8.00:3.00~11.00; 1.00~4.00:1.00~7.00:1.00~4.00:1.00~2.00:1.00~6.
00.
4. The method for preparing chrysanthemum polysaccharide according to any one of claims 1 to 3, Features: The following steps are involved: a. Polysaccharide extraction: After defatting, the dried buds of Chrysanthemum morifolium are extracted with boiling water, and then freeze-dried by alcohol precipitation to obtain crude polysaccharides; b. Polysaccharide purification: The obtained crude polysaccharide is separated and purified by anion exchange column and gel column to obtain the chrysanthemum polysaccharide.
5. A sulfated derivative of chrysanthemum polysaccharide, Features: The invention is obtained by sulfating the chrysanthemum polysaccharide described in any one of claims 1 to 3.
6. The sulfated derivative of chrysanthemum polysaccharide according to claim 5, Features: The degree of substitution by sulfation is 0.71 to 1.
51.
7. The method for preparing the sulfated derivatives of Chrysanthemum morifolium polysaccharide according to claim 6, It is characterized in that The following steps are involved: 1) At a constant temperature of 60-80°C, anhydrous pyridine is stirred uniformly, and chlorosulfonic acid is added at a ratio of 1-5:1 at a rate of 5-11 seconds / drop to obtain an esterifying agent; 2) dissolving the chrysanthemum polysaccharide in formamide at a ratio of 10 to 30:1, adding the esterification agent prepared in step 1) after it is fully dissolved, stirring at a constant temperature of 60 to 80° C. for 2 to 4 hours, and cooling to room temperature; 3) Adjust the pH value to 7.0, dialyze against saturated sodium bicarbonate for 24 hours, and then dialyze against deionized water for 48 hours, centrifuge the concentrate at 4000-8000 rpm for 10-30 minutes, take the supernatant, freeze it, and then freeze-dry it in a freeze dryer to obtain the sulfated derivative of chrysanthemum polysaccharide.
8. Use of the sulfated derivatives of Chrysanthemum flos polysaccharides according to claim 6 in the preparation of anti-tumor drugs or pharmaceutical compositions and functional products.
9. The use according to claim 8, Features: The tumor is pancreatic cancer, and the administration concentration is 10 μg / mL to 1000 μg / mL.
10. A pharmaceutical composition comprising the sulfated derivative of Chrysanthemum flos polysaccharide according to claim 5, and pharmaceutically acceptable excipients.