Sepia esculenta ink polysaccharide for improving high-glucose injury of endothelial cells, preparation method and application thereof

Abstract

The present invention discloses a Sepia esculenta ink polysaccharide for improving high-glucose damage of endothelial cells, and a preparation method and application thereof. The Sepia esculenta ink polysaccharide has a polysaccharide backbone composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and has a glucuronic acid branch chain at the C-3 position of mannose. It is an aminopolysaccharide with a weight-average molecular weight of 10-16 kDa and a sulfation degree of 8%-15%. Through experimental verification, compared with similar compounds, the Sepia esculenta ink polysaccharide can effectively improve the state of endothelial cells and maintain the normal filtration function of vascular endothelium. At the same time, the risk of bleeding side effects of the Sepia esculenta ink polysaccharide is significantly lower than that of similar polysaccharides, and it has higher safety in use. Furthermore, the Sepia esculenta ink polysaccharide can be used to develop adjuvant therapeutic drugs for late-stage diabetic lesions, filling the gap in such products on the current market.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a Sepia esculenta ink polysaccharide for improving high-glucose-induced endothelial cell injury, and a preparation method and application thereof. Background Art

[0002] Diabetes is a systemic progressive disease caused by prolonged blood glucose levels above the normal range, with many and severe complications. Prolonged hyperglycemia can cause damage to large and small blood vessels, and endanger various tissues and organs such as the heart, brain, kidneys, peripheral nerves, eyes, and feet. More than half of the deaths due to diabetes are caused by cardiovascular and cerebrovascular diseases. Among them, diabetic nephropathy, diabetic cardiovascular complications, and diabetic cerebrovascular diseases are the main complications. At present, clinical treatment mainly focuses on controlling blood glucose, supplemented by regulating blood lipids and platelet function, and there is no specific and effective drug. The recently approved class 1 new drug for the treatment of type 2 diabetes, dorzagliatin tablets, is also a glucose kinase (GK) full activator targeting glucose degradation, basically a blood glucose control drug, and there are few treatment drugs for organic damage caused to the circulatory system and urinary system.

[0003] At present, there are a large number and complex types of compounds for the treatment of endothelial cell injury and cell NO injury, mainly traditional Chinese medicine extracts, but few can truly enter clinical development. The therapeutic effects of polysaccharide compounds have also been recognized by society. Among them, heparin analogs with relatively high attention and thorough research have started phase IV clinical trials in the United States. However, it is an antithrombotic drug itself, with a risk of bleeding, and has a weak effect on resisting glucose injury and maintaining the semipermeable membrane property of endothelial tissue. Summary of the Invention

[0004] In view of the above deficiencies, the present invention provides a Sepia esculenta ink polysaccharide that can improve high-glucose-induced endothelial cell injury, which can maintain the normal filtration function of endothelial tissue and the normal state of endothelial cells in a high-glucose injury state.

[0005] To achieve the above invention object, the present invention is implemented by the following technical solutions:

[0006] The present invention provides a Sepia esculenta ink polysaccharide for improving high-glucose-induced endothelial cell injury, which is a polysaccharide backbone composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and has a glucuronic acid branch chain at the C-3 position of mannose; its structural schematic formula is as follows:

[0007] ,

[0008] wherein, R1 = OH or -SO3H or SO3H and its salt forms; R2 = OH or -SO3H or SO3H and its salt forms; n = 10 - 16.

[0009] Furthermore, the weight-average molecular weight of the sepia officinalis ink polysaccharide is 10 kDa - 16 kDa, and its degree of sulfation is 8% - 15%.

[0010] The present invention also provides a preparation method of the sepia officinalis ink polysaccharide, comprising the following steps:

[0011] (1) Take sepia officinalis ink from the ink sac, add buffer solution and grind to make a uniform suspension;

[0012] (2) Ultrasonically treat the ground sepia officinalis ink, soak it at low temperature and then centrifuge, take the supernatant and add papain for enzymatic hydrolysis to obtain enzymatically hydrolyzed sepia officinalis ink;

[0013] (3) After subjecting the enzymatically hydrolyzed sepia officinalis ink to boiling water bath denaturation, centrifuge at low temperature to take the supernatant, add a protein remover to remove the denatured protein, and centrifuge again to take the supernatant;

[0014] (4) Concentrate the supernatant and separate the precipitate to obtain crude polysaccharide;

[0015] (5) Purify, dialyze and freeze-dry the crude polysaccharide to obtain sepia officinalis ink polysaccharide.

[0016] Furthermore, in the step (1), the volume ratio of sepia officinalis ink to buffer solution is 0.5 - 2:1 - 2.

[0017] Furthermore, in the step (2), the enzymatic hydrolysis conditions are: the concentration of papain is 1‰ - 3‰, the enzymatic hydrolysis temperature is 50°C - 60°C, and the incubation time is 1 h - 2 h.

[0018] Furthermore, in the step (3), the volume ratio of the supernatant to the protein remover is 2 - 4:0.5 - 1.

[0019] Furthermore, the protein remover is Sevag reagent.

[0020] The present invention also provides the application of the sepia officinalis ink polysaccharide in the preparation of a drug for treating vascular inflammation.

[0021] Furthermore, the vascular inflammation is vascular endothelial inflammation induced by diabetes primers.

[0022] Furthermore, the vascular endothelial inflammation is glomerular microvascular endothelial and renal tubular endothelial inflammation caused by diabetes.

[0023] Furthermore, the concentration of the sepia officinalis ink polysaccharide contained in the drug is 10 mg / ml - 60 mg / ml.

[0024] Furthermore, the active ingredient of the drug is sepia officinalis ink polysaccharide and its pharmaceutically acceptable salts.

[0025] Furthermore, the Sepia esculenta ink polysaccharide can effectively improve the state of glucose damage in endothelial cells and maintain the normal filtration function of endothelial tissues.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] 1. The present invention selects the ink of Sepia esculenta as a raw material, extracts and prepares a new Sepia esculenta ink polysaccharide, which is a polysaccharide backbone composed of fucose, galactosamine, mannose, and N-acetylglucosamine, contains a glucuronic acid at the C-3 position of mannose, and has a weight average molecular weight of 10-16 kDa; it is an amino polysaccharide with a sulfation degree of 8%-15%.

[0028] 2. The Sepia esculenta ink polysaccharide can effectively improve the state of endothelial cells and maintain the normal filtration function of vascular endothelium, and can be used to treat vascular inflammation caused by vascular endothelial cell damage caused by long-term hyperglycemia, as well as glomerular microvascular endothelial and renal tubular endothelial inflammation caused by high glucose.

[0029] 3. The bleeding risk of the Sepia esculenta ink polysaccharide is significantly lower than that of similar drugs after long-term use, and it is safer.

[0030] 4. The Sepia esculenta ink polysaccharide can be used to develop adjuvant therapeutic drugs or health products for late-stage diabetic complications, filling the gap in such products on the current market. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the elution curve of Sepia esculenta polysaccharide.

[0032] Figure 2 is the infrared spectrogram of Sepia esculenta ink polysaccharide.

[0033] Figure 3 is the effect of Sepia esculenta ink polysaccharide on the state of HUVEC cells under high glucose damage; among them, A is the state of undamaged control cells, B is the state of glucose-damaged cells, and C is the state of cells in the polysaccharide-added group.

[0034] Figure 4 is the improvement of endothelial injury of rat aortic arch under high glucose damage by Sepia esculenta ink polysaccharide: a: blank group, b: model group, c: sulodexide treatment group, d: Sepia esculenta ink polysaccharide treatment group. EMBODIMENTS

[0035] The technical solutions of the present invention will be further described in detail in conjunction with the following specific examples.

[0036] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies.

[0037] Example 1: Preparation of Sepia esculenta ink polysaccharide

[0038] Fresh cuttlefish ink sacs are stored at -80°C to -50°C and thawed at 2°C - 5°C. Sepia esculenta ink and phosphate buffer solution (PBS, 0.01 mol / L, pH 7.4) are ground and suspended evenly. The volume ratio of Sepia esculenta ink to phosphate buffer is 1:2. The ground cuttlefish ink is repeatedly ultrasonically treated (ultrasonic for 2 - 10 s each time, interval of 5 - 10 s, ultrasonic for 10 - 100 times), and stirred and soaked at 2 - 4°C for 24 - 72 h, then centrifuged at 24°C, 10000 rpm for 20 min, and the supernatant is collected twice. 1‰ - 3‰ papain is added to the supernatant, and enzymolysis is carried out at 45 - 65°C for 1 - 2 h. The enzymolyzed cuttlefish ink juice is subjected to boiling water bath denaturation treatment for 10 - 30 min. After cooling, it is centrifuged at 4°C, 8000 rpm for 40 min to separate the supernatant. Sevag reagent (chloroform: n-butanol = 4:1) is added to the supernatant and stirred with a stirrer for 20 - 50 min, left to stand at 4°C for 1 - 2 h, and the supernatant is centrifuged to remove denatured proteins, and the operation is repeated 2 - 3 times. The volume ratio of the supernatant to Sevag reagent is 4:1, and centrifuged at 6000 - 15000 rpm for 30 - 60 min. After the supernatant is concentrated by rotary evaporation, 4 times the volume of absolute ethanol is added, left to stand at 4°C for 1 - 2 h, and centrifuged at 6000 - 10000 rpm for 10 - 30 min to separate the precipitated crude polysaccharide. The yield of the crude polysaccharide after freeze-drying is 1% - 3%.

[0039] The crude polysaccharide is purified and separated by chromatography to obtain the target compound. 1 g of dry crude polysaccharide SIP is dissolved in 5 mL of distilled water, heated and dissolved at 60 - 80°C for 10 - 30 min, centrifuged at 3000 - 5000 rpm for 5 - 10 min, and the insoluble matter is repeated the above operation. The two supernatants are combined, and part of the pigment is removed by an activated C18 solid-phase extraction column, eluted with ultrapure water for 3 column volumes, and the collected liquid is concentrated by rotary evaporation. 1 g of the concentrated crude polysaccharide solution after C18 column chromatography is loaded onto a DEAE-52 cellulose ion exchange column (2.5 cm × 20 cm), and gradient elution is carried out with distilled water and gradient concentration NaCl solution (0 - 2 mol / L) at a flow rate of 0.5 - 1 mL / min, 2 - 5 mL per tube. The polysaccharide content in the eluate is detected by the phenol-sulfuric acid method, and the elution curve is plotted by detecting every other tube ( Figure 1 ), and collected according to the peak according to the elution curve, the collected liquid is concentrated by rotary evaporation, dialyzed through a 2000 - 5000 Da dialysis bag for 48 - 72 h, and freeze-dried to obtain the target polysaccharide compound, and the yield is 50% - 80%.

[0040] The structure of Sepia esculenta ink polysaccharide is as follows:

[0041] ,

[0042] wherein the R1 and R2 groups are -SO3H or -OH; n = 10 - 16.

[0043] Sepia esculenta ink polysaccharide is a polysaccharide backbone composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and has a glucuronic acid side chain at the C-3 position of mannose; its molecular weight is: 10 - 16 kDa; the degree of sulfation is 8% - 15%.

[0044] The infrared spectrum of Sepia esculenta ink polysaccharide is as Figure 2 shown: The figure shows the characteristic absorption peaks of the polysaccharide. Specifically, the absorption bands at 3468.35 cm -1 and 2927.9 cm -1 correspond to the -OH and C–H stretching vibrations in the sugar ring, respectively; the absorption peak at 1613.16 cm -1 is the symmetric vibration of -COOH, indicating that Sepia esculenta ink polysaccharide contains amide bonds or carboxylic acids; in addition, the absorption peaks at 1211.56 cm -1 and 807.55 cm -1 can be attributed to the S=O stretching vibration of the sulfate group; the absorption band present in the range of 1100 - 1010 cm -1 indicates that the polysaccharide is linked by pyranose ring glycosides; the absorption peak near 890 cm -1 generally represents a β-glycosidic bond; the FT-IR absorption information indicates that Sepia esculenta polysaccharide is a β-type polysaccharide with pyranose groups and contains characteristic peaks of sulfate groups and carboxyl groups.

[0045] Example 2. Effect of polysaccharide compounds on the proliferation of HUVEC cells under high glucose injury

[0046] SRB is a water-soluble protein dye that can bind to the basic amino acids of biological macromolecules. The amount of its binding to cells can reflect the total protein amount and thus reflect the number of cells. The OD value at 540 nm has a good linear relationship with the number of live cells.

[0047] Human vascular endothelial HUVEC cells were placed in McCoy's 5A medium containing 25 mM glucose, 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin, and cultured in a cell incubator at 37 °C and 5% CO2. The medium was changed every two days. After the cells reached 80% confluence, they were digested with trypsin and passaged to maintain the cells in a good logarithmic growth phase.

[0048] HUVEC cells in the logarithmic growth phase were seeded in 96-well plates at 5000 cells / well (180 μl / well). After culturing for 24 h, sepia ink polysaccharide or glycosaminoglycan was added (the final concentrations are shown in Table 1), and 4 replicate wells were set for each concentration. After the drugs acted for 72 h, 50% (m / v) ice-cold trichloroacetic acid (TCA) was added to each well to fix the cells. After SRB staining, 150 μl / well of Tris solution was added, and the OD value at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0049] The inhibition rate of cell growth was calculated according to the following formula:

[0050] Inhibition rate = [(OD540 control well - OD540 drug-treated well) / OD540 control well] × 100%

[0051] The test results are shown in Table 1. Compared with the blank group, the viability of HUVEC cells was significantly decreased after high glucose injury. After adding different concentrations of sepia ink polysaccharide or glycosaminoglycan, the cell viability was restored to a certain extent; compared with glycosaminoglycan, sepia ink polysaccharide had a significantly better effect at 60 mg / ml.

[0052] Table 1 Effects of test compounds on the proliferation of HUVEC cells (SRB method)

[0053]

[0054] Note: *: Compared with the 25 mM glucose control group, P ≤ 0.05; #: Compared with samples at the same concentration, P ≤ 0.05.

[0055] Example 3: Effects of compounds on the state of HUVEC cells under high glucose injury

[0056] Human vascular endothelial HUVEC cells were placed in McCoy's 5A medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin, and cultured in a cell incubator at 37 °C and 5% CO2. The medium was changed every two days. After the cells reached 80% confluence, they were digested with trypsin and passaged to maintain the cells in a good logarithmic growth phase.

[0057] After digestion and passage, cells were treated without injury, with 25 mM glucose injury, or with 25 mM glucose injury plus sepia ink polysaccharide at a final concentration of 60 mg / ml, and cultured in a cell incubator at 37 °C and 5% CO2 for 5 days. Cell status was compared by taking pictures.

[0058] Under an inverted microscope ( Figure 3Observation showed that the cells in the blank control group were evenly distributed, mostly arranged in a flat polygonal pavement-like mosaic pattern, adhered firmly to the wall, had clear boundary morphology, abundant cytoplasm, and round or oval nuclei, with occasional binucleation, indicating that they were undergoing mitotic proliferation and had good cell status; the number of HUVEC cells in the model group decreased, the cell morphology atrophied and deformed, the cell body became smaller, the cell gap widened, and the boundary was blurred, suggesting increased cell permeability and that the cells were approaching apoptosis, and some cells had fragmented; the cell status in the Sepia esculenta ink polysaccharide group was significantly restored compared with the model group, the number of cells increased, and the morphology tended to be normal. This indicates that Sepia esculenta ink polysaccharide has a significant protective effect on HUVEC cells damaged by high glucose.

[0059] Example 4. Effect of the compound on the filtration function of vascular endothelial cells damaged by high glucose

[0060] HUVEC cells were seeded at a density of 10,000 cells / well into Hanging Cell Culture Inserts. The blank control group was added with standard DMEM medium, and the model group, control group 1, control group 2, and drug administration group were respectively added with 25 mM glucose solution. At the same time, control group 1, control group 2, and the drug administration group were respectively added with 60 μg / ml enoxaparin, sulodexide, and Sepia esculenta ink polysaccharide. After culturing for 5 days, the medium was changed to 1640 culture solution without phenol red, and 400 μg / ml FITC-labeled BSA was added and incubated for 3 hours. The culture solution in the bottom well was transferred to a 96 Well Assay Plate, the fluorescence intensity was detected, and the albumin filtration rate was calculated as follows:

[0061] Albumin filtration rate (%) = (fluorescence intensity value of the test compound group - fluorescence intensity value of the NC group) / fluorescence intensity value of the NC group × 100

[0062] The results are shown in Table 2. Compared with the 25 mM glucose control group, the fluorescence density value in the Sepia esculenta ink polysaccharide group was significantly reduced, indicating that Sepia esculenta ink polysaccharide can effectively restore the barrier function of vascular endothelial cells, reduce the albumin filtration rate, and has a better effect than sulodexide and enoxaparin.

[0063] Table 2. Effect of the test compound on the filtration function of HUVEC cells damaged by high glucose

[0064]

[0065] Note: *: Compared with the 25 mM glucose control group, P ≤ 0.05; ***: Compared with the 25 mM glucose control group, P ≤ 0.001; ##: Compared with the blank control group, P ≤ 0.01; : Compared with the blank control group, P ≤ 0.001.

[0066] Example 5. Therapeutic effect of the compound on arterial vascular inflammation caused by diabetes

[0067] Experimental animals: 75 male SD rats, weighing 200 ± 2 g.

[0068] Experimental materials: Streptozotocin (Sigma); Glucose (Solarbio).

[0069] Experimental method: Select 40 male SD rats weighing about 200 g. After one week of adaptive feeding, fast the rats for 12 h without water restriction. Weigh them the next morning. Randomly select 10 rats as the blank group and feed them with normal feed. The remaining rats are used as the diabetes group and fed with a high-fat and high-sugar diet (diet composition: 20% sucrose, 4% cholesterol, 10% lard, 1% sodium cholate) for 30 days. After 30 days, fast the rats for 12 h without water restriction. Inject streptozotocin at a dose of 30 mg / kg intraperitoneally into the diabetes group for 2 consecutive days. The blank group is injected with an equal volume of citrate-sodium citrate buffer. After 7 days, collect blood from the tip of the tail to measure the blood glucose level.

[0070] Grouping and administration: Select rats in the diabetes group with a fasting blood glucose level of 16.7 mmol / L < fasting blood glucose value < 21 mmol / L and obvious symptoms of polyuria, polydipsia, and polyphagia. Randomly divide them into 3 groups (the difference between groups is no more than 1.1 mmol / L) and administer drugs continuously for 45 days.

[0071] Table 3 Experimental grouping and administration

[0072]

[0073] Type 2 diabetes is often accompanied by dyslipidemia, and there will be lipid accumulation and endothelial damage in the aorta. As Figure 4 shown, by HE staining analysis, it was observed that Sepia esculenta ink polysaccharide has an improving effect on the endothelial damage of the aortic arch in rats with type 2 diabetes.

[0074] Example 6, Anticoagulant characteristics of the compound

[0075] Samples and reference substances: Enoxaparin injection: Clexane (0.4 ml / 4000 AxalU). Sulodexide injection: Alfresa Pharma Italy, specification: 2 ml: 600 LSU * 10 vials / box. Normal saline: Shandong Qidu Pharmaceutical Co., Ltd.

[0076] Experimental animals: SPF-grade SD rats; 6 - 8 weeks old; male.

[0077] Experimental method: After subcutaneous injection of drugs in the abdomen of rats, collect blood at 1.5 h to detect the four coagulation items.

[0078] Dose of administration: Enoxaparin, Sulodexide, and Sepia esculenta ink polysaccharide are administered at a dose of 4 mg / kg. The control group is injected with an equal volume of normal saline; 6 parallels are set for each group.

[0079] The test results are shown in Table 4-7. The APTT of the Sepia esculenta ink polysaccharide group was significantly lower than that of the enoxaparin group and the sulodexide group, indicating that its anticoagulant effect was weaker and the bleeding side effect was lower.

[0080] Table 4 Effects of Compounds on APTT (Unit: s)

[0081]

[0082] **: Compared with the Sepia esculenta ink polysaccharide group, P≤0.01;

[0083] Table 5 Effects of Compounds on PT (Unit: s)

[0084]

[0085] Table 6 Effects of Compounds on TT (Unit: s)

[0086]

[0087] Table 7 Effects of Compounds on (Unit: s)

[0088]

[0089] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. Use of sepia officinalis polysaccharide for improving high glucose-induced injury of endothelial cells in the preparation of a drug for treating vascular inflammation, characterized in that, The Sepia esculenta ink polysaccharide is a polysaccharide backbone composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and has a glucuronic acid side chain at the C-3 position of mannose; its structural schematic formula is as follows: ， Among them, R1 = -OH or -OSO3H or -OSO3H and its salt forms; R2 = -OH or -OSO3H or -OSO3H and its salt forms; n = 10 - 16. The weight-average molecular weight of the Sepia esculenta ink polysaccharide is 10 kDa - 16 kDa, and its degree of sulfation is 8% - 16.5%.

2. The application according to claim 1, characterized in that The preparation method of the Sepia esculenta ink polysaccharide comprises the following steps: (1) Take Sepia esculenta ink from the ink sac, add a buffer solution and grind to make a homogeneous suspension; (2) Ultrasonically treat the ground Sepia esculenta ink, soak it at low temperature and then centrifuge. Take the supernatant and add papain for enzymatic hydrolysis to obtain enzymatically hydrolyzed Sepia esculenta ink; (3) After denaturing the enzymatically hydrolyzed Sepia esculenta ink in a boiling water bath, centrifuge at low temperature to take the supernatant, add a protein remover to remove the denatured protein, and centrifuge again to take the supernatant; (4) Concentrate the supernatant and separate the precipitate to obtain crude polysaccharide; (5) Purify, separate, dialyze, and freeze-dry the crude polysaccharide to obtain Sepia esculenta ink polysaccharide.

3. The application according to claim 2, wherein In the step (1), the volume ratio of Sepia esculenta ink to the buffer solution is 0.5 - 2:1 - 2.

4. The application according to claim 2, wherein In the step (2), the enzymatic hydrolysis conditions are as follows: the concentration of papain is 1‰ - 3‰, the enzymatic hydrolysis temperature is 50°C - 60°C, and the incubation time is 1 h - 2 h.

5. The application according to claim 2, characterized in that, In the step (3), the volume ratio of the supernatant to the protein remover is 2 - 4:0.5 - 1.

6. The application according to claim 1, characterized in that, The vascular inflammation is vascular endothelial inflammation caused by diabetes.

7. The application according to claim 1, characterized in that The concentration of Sepia esculenta ink polysaccharide contained in the drug is 10 mg / ml - 60 mg / ml.

8. The application according to claim 1, characterized in that, The active ingredient of the drug is Sepia esculenta ink polysaccharide and its pharmaceutically acceptable salts.

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