Polygonatum kingianum polysaccharide and application thereof

Abstract

The invention provides polygonatum kingianum polysaccharide and application thereof in a medicine for treating diabetes mellitus, the molecular weight of the polygonatum kingianum polysaccharide is 2365Da, and the polygonatum kingianum polysaccharide is uniform polysaccharide; the polygonatum kingianum polysaccharide is prepared from arabinose, fructose, galactose, glucose and ribose according to the mass ratio of 39.19: 24.28: 2.85: 1.37: 3.38, the polygonatum kingianum polysaccharide is simple in preparation method, low in cost and free of pollution, and the obtained polysaccharide is high in purity; an in-vitro lipid-lowering and blood-glucose-lowering activity experiment result of the polygonatum kingianum polysaccharide shows that the polygonatum kingianum polysaccharide can obviously reduce lipid accumulation of human liver cancer HepG2 cells and obviously improve glucose consumption of insulin-resistant cells, the polygonatum kingianum polysaccharide has good in-vitro lipid-lowering and blood-glucose-lowering activity, and a new way is provided for treating and preventing diabetes mellitus.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for extracting, separating, purifying and identifying the primary structure of Polygonatum cyrtonema polysaccharide, and an application thereof in treating diabetes. Background Art

[0002] Type 2 diabetes (T2DM) is a chronic metabolic disease caused by insulin resistance and insufficient insulin secretion. In recent years, the number of patients with diabetes has been increasing at an alarming rate, becoming a challenge to human health. At present, the main drugs for the treatment of T2DM in clinical practice are biguanides, sulfonylureas, insulin, etc. None of them can fundamentally treat diabetes, and may even cause a series of complications such as hypertension, diabetic peripheral neuropathy, and diabetic nephropathy, which will damage the health of patients. Therefore, finding a drug with low side effects that has both high blood sugar-lowering activity and can reasonably improve the overall health level of patients has become a top priority in the clinical treatment of diabetes.

[0003] Polygonatum yunnanensis ( Polygonatum kingianum Coll. et Hemsl.) is a perennial herbaceous plant of the genus Polygonatum in the Liliaceae family, and its rhizome is used for medicinal purposes. It has the effects of nourishing qi and yin, moistening the lungs and tonifying the spleen, and nourishing the kidneys and filling essence. As a Chinese medicinal material with both medicinal and edible properties, Yunnan Polygonatum is mild in nature, has no toxic side effects, and contains a variety of chemical components, mainly including polysaccharides, steroidal saponins, anthraquinones, alkaloids, cardiac glycosides, lignans, vitamins and amino acids. Among them, polysaccharides are the most abundant component of Polygonatum, and their functionality is also the most significant. Modern pharmacological studies have shown that Polygonatum polysaccharides have hypoglycemic and lipid-lowering activities, but most of the current studies on the use of Polygonatum polysaccharides to treat diabetes have been conducted using crude Polygonatum polysaccharides for experiments. The crude polysaccharides contain a variety of polysaccharide components and a large amount of impurities, and the specific active components are still unclear. These impurity components will interfere with the polysaccharide structure characterization and pharmacodynamic experiments. The present invention extracts, separates and purifies uniform polysaccharides from the roots of fresh Yunnan Polygonatum and identifies its primary structure, which can be used to develop drugs for the treatment of diabetes with high safety and low side effects. Summary of the invention

[0004] The present invention provides a polygonatum sichuanensis polysaccharide and application thereof in treating diabetes. The polygonatum sichuanensis polysaccharide is extracted, separated and purified from fresh polygonatum sichuanensis. The polysaccharide is a homogeneous polysaccharide with a molecular weight of 2365 Da. The polysaccharide is composed of arabinose, fructose, galactose, glucose and ribose in a mass ratio of 39.19:24.28:2.85:1.37:3.38.

[0005] The purpose of the present invention is achieved through the following technical solutions: 1. Wash fresh Polygonatum sibiricum, cut into tubers, add Polygonatum sibiricum tubers and water into a flask, reflux extract at 80℃ for 4h, filter, extract the residue again according to the above method, combine the extracts and concentrate to 1 / 3 of the original volume; 2. Add ethanol to the concentrated extract to make the ethanol volume concentration in the system 20%, let it stand overnight, centrifuge, take the supernatant, add ethanol to the supernatant to make the ethanol volume concentration in the system 80%, precipitate by alcohol, centrifuge to take the precipitate, dissolve the precipitate with distilled water, remove protein by Sevag method, concentrate, dialyze, and freeze-dry to obtain Yunnan Polygonatum cyrtonema crude polysaccharide; 3. The crude polysaccharide of Yunnan polygonatum is prepared into an aqueous solution, loaded on a DEAE-52 cellulose chromatography column, first eluted with distilled water, and then eluted with 0.1, 0.2, and 0.4 mol / L NaCl solutions in a continuous gradient, and the eluate is collected. The elution is tracked and monitored by a phenol-sulfuric acid method and a spectrophotometer, and an elution curve is drawn according to the absorbance value, and the liquid corresponding to the elution peak is collected, and concentrated and freeze-dried to obtain the Yunnan polygonatum polysaccharide FP4 of the present invention; 4. The molecular weight of the homogeneous polysaccharide FP4 was determined to be 2365 Da using the HPGPC analytical instrument; the monosaccharide composition was determined by comparing it with the monosaccharide standard using a gas chromatography-mass spectrometer, which was composed of arabinose, fructose, galactose, glucose, and ribose in a mass ratio of 39.19:24.28:2.85:1.37:3.38; the chemical structure was inferred by nuclear magnetic resonance analysis, and the nuclear magnetic resonance spectrum structure analysis was as follows: In the study of Polygonatum cyrtonema polysaccharide FP4 1 In H-NMR, polysaccharide signals mainly appear in δ In the range of 3.1~5.2ppm, δ The range of 4.4~5.2ppm is the anomeric hydrogen of polysaccharide. α Configuration sugar 1 H chemical shift is greater than 5.0ppm, β Configuration of sugar 1 H is less than 5.0ppm, based on which the type of glycosidic bond in the polysaccharide can be inferred; 1 The H-NMR spectrum shows more peaks at chemical shifts less than 5 ppm, indicating that its glycosidic bonds are mainly β Type. In polysaccharide 13 In the C NMR spectrum, the chemical shift of 90 ~110 ppm is the anomeric carbon signal area. Polysaccharide FP4 has 5 signal peaks in the anomeric carbon model area, which are δ97.94, 103.02, 103.53, 103.67, 103.86 ppm, indicating that FP4 should have 5 sugar residues, which is consistent with the monosaccharide composition results. The chemical shifts of the 5 sugar residue signal peaks are basically greater than 103ppm, indicating that the anomeric carbon configuration of Yunnan Polygonatum polysaccharide is mainly β configuration. In the HSQC spectrum, δ97.94 / 3.64ppm, 103.02 / 4.65ppm, 103.53 / 4.66ppm, 103.67 / 4.67ppm, 103.86 / 4.68ppm can be attributed to the residue signals of glucose, arabinose, fructose, ribose, and galactose.

[0006] 5. The results of the in vitro lipid-lowering and blood sugar-lowering activity experiment of Polygonatum sibiricum polysaccharide FP4 showed that it can significantly reduce the lipid accumulation of human liver cancer HepG2 cells and significantly increase the glucose consumption of insulin-resistant (IR-HepG2) cells. Polygonatum sibiricum polysaccharide FP4 has good in vitro blood sugar-lowering and blood sugar-lowering activity.

[0007] Advantages and technical effects of the present invention: 1. The preparation method of the polygonatum cyrtonema polysaccharide of the present invention is simple, low-cost and easy to industrialize; 2. The raw materials for preparing Yunnan Polygonatum cyrtonema polysaccharide in the present invention are medicinal and edible medicinal materials with high safety; 3. The polygonatum yunnanensis polysaccharide of the present invention is a uniform polysaccharide with high purity and significant blood sugar and lipid lowering activity, and can be used to treat diseases related to diabetes and dyslipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the DEAE-52 anion exchange chromatography elution curve of Polygonatum cyrtonema polysaccharide FP4; Figure 2 This is the HPGPC diagram of Polygonatum cyrtonema polysaccharide FP4; Figure 3 This is the monosaccharide composition diagram of Yunnan Polygonatum polysaccharide FP4, in which a: arabinose; b: fructose; c: galactose; d: glucose; e: ribose; Figure 4 Polygonatum cyrtonema polysaccharide FP4 1 H NMR spectrum; Figure 5 Polygonatum cyrtonema polysaccharide FP4 13 C NMR spectrum; Figure 6 This is the HSQC spectrum of Polygonatum cyrtonema polysaccharide FP4; Figure 7 The results of the effect of Polygonatum yunnanensis polysaccharide FP4 on lipid accumulation in HepG2 cells, where Figure A: Oil Red O staining; Figure B: Statistical chart of the relative area percentage of red lipid droplets; Figure 8This is the result of the effect of Polygonatum cyrtonema polysaccharide FP4 on the glucose consumption of HepG2 cells. DETAILED DESCRIPTION

[0009] The present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the protection scope of the present invention is not limited to the above contents. The reagents used in the examples, unless otherwise specified, are conventional commercially available reagents or reagents prepared by conventional methods. The methods used in the examples, unless otherwise specified, are conventional experimental methods. Example 1: Preparation of Polygonatum cyrtonema polysaccharide 1. Wash the fresh Polygonatum sibiricum, cut it into tubers of 0.5-1 cm, take 1 kg of Polygonatum sibiricum tubers and water into a flask at a solid-liquid ratio of 1:5, reflux extract at 80°C for 4 hours, filter, add water to the residue and extract once, collect and combine the extracts, and concentrate to 1 / 3 of the original volume at 50°C; 2. Add ethanol to the concentrated extract to make the ethanol volume concentration in the system 20%, let it stand overnight, centrifuge at 8000rpm, take the supernatant, add ethanol to the supernatant to make the ethanol volume concentration in the system 80%, precipitate with alcohol, centrifuge at 8000rpm, take the precipitate, dissolve the precipitate in water, add Sevag reagent (n-butanol: chloroform = 1:4), mix well, let it stand for stratification, remove the organic phase, and then add Sevag reagent, repeat 2 times, concentrate the liquid after protein removal at 50℃, transfer the concentrate to a dialysis bag (molecular weight cutoff is 1000Da), replace distilled water every 6 hours, dialyze for 2 days, and freeze-dry the liquid after dialysis to obtain 21.5g of Yunnan Polygonatum cyrtonema crude polysaccharide; 3. Take 200 mg of crude polysaccharide from Polygonatum yunnanensis, prepare it into a 10 mg / mL aqueous solution, load it on a DEAE-52 cellulose chromatography column, and the loading volume is 1 / 20-1 / 10 column volume. Elute it with distilled water first, and then elute it with 0.1, 0.2, and 0.4 mol / L NaCl solution in a continuous gradient. Use the phenol-sulfuric acid method and a spectrophotometer to track and monitor the elution. Draw an elution curve based on the absorbance value. See Figure 1 , collect the liquid corresponding to the elution peak, combine, concentrate, and freeze-dry to obtain 126 mg of Polygonatum cyrtonema polysaccharide FP4; The phenol-sulfuric acid method is to take 0.5 mL of the eluent, add 1 mL of the freshly prepared 6% phenol solution, then add 2.5 mL of concentrated sulfuric acid, shake well, let it stand for 10 minutes, heat it in a water bath at 100°C for 15 minutes, take it out and cool it to room temperature, and measure its absorbance at a detection wavelength of 490 nm.

[0010] Example 2: Structural Identification of Polygonatum cyrtonema Polysaccharide FP4 1. Molecular weight determination 15 mg of Polygonatum cyrtonema polysaccharide FP4 was weighed and dissolved in 1 mL of 0.1 mol / L NaNO3 aqueous solution. The molecular weight was analyzed by high performance gel permeation chromatography with a differential refractometer. Figure 2 ).

[0011] 2. Determination of Polygonatum cyrtonema polysaccharide composition Take 10 mg of Polygonatum yunnanensis polysaccharide, add 4 mL of trifluoroacetic acid, heat to 110 ° C and hydrolyze for 6 h, then remove the trifluoroacetic acid by rotary evaporation, then add 3 mL of pyridine and heat to 80 ° C to dissolve, then add 100 mL of N-methyl-N-(trimethylsilyl) trifluoroacetamide, react at 80 ° C for 1 h, cool, and analyze by gas chromatography-mass spectrometry. The results are shown in Figure 3 , indicating that Polygonatum cyrtonema polysaccharide FP4 is composed of five monosaccharides, namely arabinose (a), fructose (b), galactose (c), glucose (d), and ribose (e), with a mass ratio of 39.19:24.28:2.85:1.37:3.38.

[0012] use 1 H NMR, 13 The structure of Polygonatum cyrtonema polysaccharide FP4 was analyzed by C NMR and HSQC spectra. Figure 4 , Figure 5 , Figure 6 , in polysaccharides 1 In H-NMR, polysaccharide signals mainly appear in δ In the range of 3.1~5.2 ppm, δ The range of 4.4~5.2ppm is the anomeric hydrogen of polysaccharide. α Configuration sugar 1 H chemical shift is greater than 5.0ppm, β Configuration of sugar 1 H is less than 5.0ppm, from which it can be inferred that the types of glycosidic bonds in polysaccharides are mainly β Type. In polysaccharide 13 In the C NMR spectrum, the chemical shift of 90 ~110 ppm is the anomeric carbon signal area. Yunnan Polygonatum cyrtonema polysaccharide FP4 has 5 signal peaks in the anomeric carbon model area, which are δ 97.94, 103.02, 103.53, 103.67, 103.86 ppm, indicating that FP4 should have 5 sugar residues, which is consistent with the monosaccharide composition results. The chemical shifts of the 5 sugar residue signal peaks are basically greater than 103 ppm, indicating that the anomeric carbon configuration of FP4 is mainly βIn the HSQC spectrum, δ97.94 / 3.64ppm, 103.02 / 4.65ppm, 103.53 / 4.66ppm, 103.67 / 4.67ppm, and 103.86 / 4.68ppm can be assigned to the residue signals of glucose, arabinose, fructose, ribose, and galactose.

[0013] Example 3: In vitro lipid-lowering and blood sugar-lowering activity experiment of Polygonatum cyrtonema polysaccharide FP4 1. Effect of Polygonatum cyrtonema polysaccharide FP4 on lipid accumulation in human hepatocellular carcinoma HepG2 cells Human hepatocellular carcinoma HepG2 cells were cultured at a density of 1×10 5 / mL were inoculated in 12-well plates, and cultured at 37℃ and 5% CO2 for 24h. Then, blank group (Control), model group (Model), positive drug group (Fenofibrate), and polygonatum cyrtonema polysaccharide group (FP4) were set up respectively. Except for the blank group, 1mmol / L free fatty acid (FFA) was added to other groups and cultured for 24h. DMEM medium was added to the blank group, 10μmol / L fenofibrate was added to the positive drug group, and 1mg / mL FP4 was added to the polygonatum cyrtonema polysaccharide group to treat HepG2 cells for 24h. The effect of polygonatum cyrtonema polysaccharide FP4 on lipid accumulation of HepG2 cells was observed by Oil Red O staining; the cell morphology and distribution of lipid droplets in each group were observed under a microscope, the results were recorded and photographed, and the relative area percentage of lipid droplets was analyzed by Image J.

[0014] Preparation of FFA: Dissolve oleic acid and palmitic acid in 0.1mmol / L NaOH solution, heat in a 70℃ water bath for 20min, prepare FFA mother solution, dilute with 10% BSA solution to 10mmol / L working solution and store in a 4℃ refrigerator for later use; The results are as follows Figure 7 As shown in the figure, compared with the blank group, a large number of red lipid droplets appeared in the model group; compared with the model group, the number and size of red lipid droplets in the Polygonatum cyrtonema polysaccharide FP4 group were significantly reduced ( P <0.001). The above results show that Polygonatum yunnanensis polysaccharide can reduce lipid accumulation in HepG2 cells and has good lipid-lowering activity in vitro.

[0015] 2. Effect of Polygonatum cyrtonema polysaccharide FP4 on glucose consumption in HepG2 cells HepG2 cells were cultured at 2×10 5 The cells were inoculated into a 96-well plate at a density of 100 / mL and cultured at 37°C and 5% CO2 for 12 h. Then, a blank group (Control), a model group (Model), a positive drug group (Met), and a Polygonatum cyrtonema polysaccharide group (FP4) were set up. -3mmol / L insulin and 10mmol / L dexamethasone. In addition, 0.1mmol / L metformin hydrochloride was added to the positive drug group, and 0.25mg / mL, 0.5mg / mL and 1mg / mL of Polygonatum cyrtonema polysaccharide FP4 were added to the Polygonatum cyrtonema polysaccharide group; after treating HepG2 cells for 48 hours, the glucose concentration of HepG2 cell supernatant was detected using a glucose kit.

[0016] The results are as follows Figure 8 As shown, compared with the blank group, the glucose consumption in the model group was reduced ( P <0. 05); compared with the model group, Polygonatum cyrtonema polysaccharide increased the glucose consumption of HepG2 cells ( P <0.001). The above results show that Polygonatum sibiricum polysaccharide can enhance the uptake and consumption of glucose by cells and has good hypoglycemic activity in vitro.

Claims

1. A polysaccharide derived from Polygonatum yunnanensis, with a molecular weight of 2365 Da, and is a homogeneous polysaccharide; the polysaccharide is composed of arabinose, fructose, galactose, glucose, and ribose in a mass ratio of 39.19:24.28:2.85:1.37:3.

38.

2. Use of the polysaccharide according to claim 1 in the preparation of drugs for preventing and / or treating diabetes and dyslipidemia.

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