CN117003901A - Sepia polysaccharide for improving endothelial cell high sugar damage and preparation method and application thereof - Google Patents
Sepia polysaccharide for improving endothelial cell high sugar damage and preparation method and application thereof Download PDFInfo
- Publication number
- CN117003901A CN117003901A CN202310189174.6A CN202310189174A CN117003901A CN 117003901 A CN117003901 A CN 117003901A CN 202310189174 A CN202310189174 A CN 202310189174A CN 117003901 A CN117003901 A CN 117003901A
- Authority
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- China
- Prior art keywords
- sepia
- polysaccharide
- supernatant
- endothelial cell
- mannose
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention discloses sepia polysaccharide for improving endothelial cell high sugar damage, a preparation method and application thereof. The sepia polysaccharide is a polysaccharide skeleton formed by fucose, galactosamine, mannose and N-acetylglucosamine, and has glucuronic acid branched chains at the C-3 position of mannose, and the weight average molecular weight is 10-16 kDa; aminopolysaccharides with 8-15% sulfation degree. Experiments prove that compared with similar compounds, the sepia esculenta polysaccharide can effectively improve the endothelial cell state, keep the normal filtering function of vascular endothelium, has a bleeding side effect risk which is obviously lower than that of similar polysaccharides, has higher use safety, can be used for developing auxiliary therapeutic drugs for later-stage lesions of diabetes, and fills the blank of the products in the current market.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to sepia polysaccharide for improving endothelial cell high-sugar damage, and a preparation method and application thereof.
Background
Diabetes is a systemic progressive disease caused by prolonged higher than normal blood glucose levels, which is frequently and severely complicated. The long-term blood sugar rise can cause damage to large blood vessels and micro blood vessels, endanger various tissues and organs such as heart, brain, kidney, peripheral nerves, eyes, feet and the like, and more than half of diabetic deaths are caused by cardiovascular and cerebrovascular blood vessels. Among them, diabetic nephropathy, diabetic cardiovascular complications, diabetic cerebrovascular diseases are the main complications. At present, clinical treatment mainly controls blood sugar, and is aided by regulating blood fat and platelet functions, and no specific medicine is available. The newly approved new medicine, namely, the doxgliptin Ai Ting tablet for treating the type 2 diabetes in the state of the drug administration is also a Glucokinase (GK) full activator aiming at glucose degradation, is basically a glucose control medicine, and is rarely a therapeutic medicine aiming at organic injury caused by circulatory system and urinary system.
At present, the number of therapeutic compounds for endothelial cell injury, cellular NO injury and the like is large, the types of the compounds are complex, and the compounds mainly comprise traditional Chinese medicine extracts, but the compounds can be clinically developed in a small amount. The therapeutic effect of polysaccharide compounds is also socially confirmed. Among them, heparin analogues, which are a relatively intensive study, have started the four-stage clinic in the united states, but are antithrombotic drugs themselves, have a bleeding risk, and have weak effects on resisting glucose injury and maintaining the semi-permeable membrane characteristics of endothelial tissues.
Disclosure of Invention
In order to overcome the defects, the invention provides the sepia polysaccharide capable of improving the high-sugar damage of endothelial cells, which can maintain the normal filtration effect of endothelial tissues and maintain the normal state of endothelial cells in a high-sugar damage state.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a sepia polysaccharide for improving endothelial cell high sugar injury, which is a polysaccharide skeleton composed of fucose, galactosamine, mannose and N-acetylglucosamine, and has glucuronic acid branched chain at the C-3 position of mannose; the structural schematic formula is as follows:
wherein R is 1 =oh or-SO 3 H or SO 3 H and its salt form; r is R 2 =oh or-SO 3 H or SO 3 H and its salt form; n=10-16.
Further, the weight average molecular weight of the sepia polysaccharide is 10kDa-16kDa, and the sulfation degree is 8% -15%.
The invention also provides a preparation method of the sepia polysaccharide, which comprises the following steps:
(1) Taking sepia ink from the ink sac, adding buffer solution, grinding and suspending uniformly;
(2) Ultrasonic processing the ground sepia, soaking at low temperature, centrifuging, collecting supernatant, adding papain for enzymolysis to obtain enzymolysis sepia;
(3) After the enzymatic hydrolysis of the sepia is denatured by boiling water, centrifuging at low temperature to obtain supernatant, adding deproteinizing agent to remove denatured protein, centrifuging again to obtain supernatant;
(4) Concentrating the supernatant, and separating and precipitating to obtain crude polysaccharide;
(5) Purifying, separating, dialyzing and freeze-drying the crude polysaccharide to obtain the sepia polysaccharide.
Further, in the step (1), the volume ratio of the sepia ink to the buffer solution is 0.5-2: 1 to 2.
Further, in the step (2), the enzymolysis conditions are as follows: the concentration of papain is 1-3 per mill, the enzymolysis temperature is 50-60 ℃, and the incubation time is 1-2 h.
Further, in the step (3), the volume ratio of the supernatant to the deproteinizing agent is 2-4: 0.5 to 1.
Further, the deproteinizing agent is a Sevag reagent.
The invention also provides application of the sepia polysaccharide in preparing medicines for treating vascular inflammation.
Further, the vascular inflammation is vascular endothelial inflammation induced by diabetes mellitus.
Further, the vascular endothelial inflammation is glomerular microvascular endothelial and tubular endothelial inflammation caused by diabetes.
Further, the concentration of the sepia polysaccharide contained in the medicine is 10 mug/ml to 60 mug/ml.
Further, the active ingredient of the medicine is sepia Mo Duotang and pharmaceutically acceptable salt thereof.
Furthermore, the sepia polysaccharide can effectively improve the glucose injury state of endothelial cells and maintain the normal filtering function of endothelial tissues.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention selects sepia ink as raw material, extracts and prepares a new sepia polysaccharide which is a polysaccharide skeleton formed by fucose, galactosamine, mannose and N-acetylglucosamine, contains a glucuronic acid on the mannose C-3 position, and has a weight average molecular weight of 10-16 kDa; aminopolysaccharides with 8-15% sulfation degree.
2. The sepia polysaccharide can effectively improve endothelial cell state, maintain normal filtering function of vascular endothelial, and can be used for treating vascular inflammation caused by vascular endothelial cell injury due to long-term hyperglycemia, and glomerular microvascular endothelial and tubular endothelial inflammation caused by hyperglycemia.
3. The risk of bleeding caused by long-term use of the sepia polysaccharide is obviously lower than that of similar medicines, and the sepia polysaccharide has higher safety.
4. The sepia polysaccharide can be used for developing auxiliary treatment drugs or health care products for later-stage lesions of diabetes, and fills the blank of the products in the current market.
Drawings
FIG. 1 shows elution profile of sepia polysaccharide.
FIG. 2 is an infrared spectrum of sepia polysaccharide.
FIG. 3 is a graph showing the effect of sepia polysaccharide on HUVEC cell status under high sugar damage; wherein A is the undamaged control cell state, B is the glucose damaged cell state, and C is the polysaccharide group added cell state.
FIG. 4 shows improvement of aortic arch endothelial injury in rats under high sugar injury with sepia polysaccharide: blank b model c sulodexide treatment group d: the squid ink polysaccharide treatment group.
Detailed Description
The technical scheme of the invention is further described in detail by combining the following specific examples.
In the following examples, unless otherwise specified, all experimental methods used are conventional and all materials, reagents, etc. are commercially available from biological or chemical reagent companies.
Example 1 preparation of sepia polysaccharide
Fresh sepia ink capsules are stored at-80 ℃ to-50 ℃, thawed at 2 ℃ to 5 ℃, and ground and suspended uniformly by a phosphate buffer solution (PBS, 0.01mol/L, pH 7.4), wherein the dosage volume ratio of the sepia ink to the phosphate buffer solution is 1:2. repeatedly carrying out ultrasonic treatment on the ground cuttlefish ink (ultrasonic treatment is carried out for 2-10 s each time at intervals of 5-10 s and ultrasonic treatment is carried out for 10-100 times), stirring and soaking for 24-72 h at 2-4 ℃, centrifuging for 20min at 10000rpm, and collecting supernatant for 2 times. Adding papain in 1-3 mill into the supernatant, and carrying out enzymolysis for 1-2 h at 45-65 ℃. Carrying out boiling water bath denaturation treatment on the cuttlefish ink after enzymolysis, wherein the boiling water bath time is 10-30 min, cooling, and centrifuging at 4 ℃ and 8000rpm for 40min to centrifugally separate supernatant. Adding Sevag reagent (chloroform: n-butanol=4:1) into the supernatant, stirring for 20-50 min by a stirrer, standing at 4 ℃ for 1-2 h, centrifuging to remove denatured protein, and repeating the operation for 2-3 times. The dosage volume ratio of the supernatant to the Sevag reagent is 4: centrifuging at 6000-15000 rpm for 30-60 min. Concentrating the supernatant by rotary evaporation, adding 4 times volume of absolute ethyl alcohol, standing for 1-2 h at 4 ℃, centrifuging at 6000-10000 rpm for 10-30 min, and separating and precipitating crude polysaccharide. The yield of the crude polysaccharide after freeze drying is 1-3%.
The crude polysaccharide is purified and separated by chromatography to obtain the target compound. Dissolving 1g of dried crude polysaccharide SIP in 5mL of distilled water, heating and dissolving at 60-80 ℃ for 10-30 min, centrifuging at 3000-5000 rpm for 5-10 min, repeating the operation of insoluble substances, combining 2 times of supernatant, removing partial pigment by an activated C18 solid phase extraction column, eluting 3 column volumes by ultrapure water, and concentrating the collected liquid by rotary evaporation. 1g of crude polysaccharide concentrated solution after C18 column chromatography is taken and put on a DEAE-52 cellulose ion exchange column (2.5 cm multiplied by 20 cm), and is eluted with distilled water and NaCl solution (0-2 mol/L) with gradient concentration, the flow rate is 0.5-1 mL/min, and each tube is 2-5 mL. Detecting polysaccharide content in eluent by using a sulfuric acid phenol method, drawing an elution curve (figure 1) by using a tube separation detection method, collecting according to the elution curve by peak, concentrating the collected liquid by rotary evaporation, dialyzing for 48-72 h by using a dialysis bag of 2000-5000 Da, and freeze-drying to obtain the target polysaccharide compound, wherein the yield is 50-80%.
The structure of sepia polysaccharide is shown as follows:
，
wherein R is 1 、R 2 The radical being-SO 3 H or-OH; n=10-16.
The sepia polysaccharide is a polysaccharide skeleton composed of fucose, galactosamine, mannose and N-acetylglucosamine, and has glucuronic acid branched chain at C-3 position of mannose; the molecular weight is as follows: 10-16 kDa; the sulfation degree is 8% -15%.
The infrared spectrogram of the sepia polysaccharide is shown in figure 2: the characteristic absorption peak of the polysaccharide, in particular 3468.35cm, is shown -1 Department and 2927.9cm -1 The absorption bands of (a) correspond to-OH and C-H stretching vibration in the sugar ring respectively; 1613.16cm -1 The absorption peak at this point is the symmetrical vibration of-COOH, which indicates that the sepia polysaccharide contains amide bonds or carboxylic acids; furthermore, at 1211.56cm -1 Department and 807.55cm -1 The absorption peak at this point can be attributed to s=o stretching vibration of the sulfuric acid group; at 1100-1010cm -1 The presence of an absorption band within the range indicates that the polysaccharide is linked by a pyranoside; 890cm -1 The nearby absorption peak is generally denoted as β -glycosidic bond; FT-IR absorption information indicated that the sepia polysaccharide was a beta-type polysaccharide with pyranyl groups and contained sulfate groups and carboxyl characteristic peaks.
EXAMPLE 2 Effect of polysaccharide Compounds on HUVEC cell proliferation under high sugar injury
SRB is a water-soluble protein dye that binds basic amino acids of biological macromolecules, and the amount of the dye bound in cells reflects the total protein and thus the number of cells. The OD at 540nm has a good linear relationship with the number of living cells.
Human vascular endothelial HUVEC cellsPlaced in McCoy's 5A medium containing 25mM glucose, 10% heat-inactivated FBS (fetal bovine serum), 2mM L-glutamine, 100U/ml penicillin and 100g/ml streptomycin at 37℃with 5% CO 2 Is cultured in a cell culture incubator. The liquid is changed once every two days, and after 80% of cells are fused, pancreatin digestion and passage are carried out, so that the cells are kept in a good logarithmic growth phase.
HUVEC cells in the logarithmic growth phase were inoculated into 96-well plates at 5000 wells/well (180. Mu.l/well), and after 24 hours of culture, sepia polysaccharide or glycosaminoglycan (final concentration is shown in Table 1) was added thereto, and 4 wells were set for each concentration. After 72h of drug action, cells were fixed by adding 50% (m/v) ice-cold trichloroacetic acid (TCA) to each well, after SRB staining, 150. Mu.l/well of Tris solution was added and OD at 540nm was measured on a microplate reader.
The inhibition rate of cell growth was calculated as follows:
inhibition = [ (OD 540 control well-OD 540 dosing well)/OD 540 control well ] ×100%
The results are shown in Table 1, and the HUVEC cell viability was significantly reduced after high sugar damage compared to the blank. After the sepia polysaccharide or the glycosaminoglycan with different concentrations is added, the cell viability is recovered to a certain extent; whereas compared with glycosaminoglycan, the sepia polysaccharide has a significantly better effect than the former at 60 mug/ml.
TABLE 1 influence of test Compounds on HUVEC cell proliferation (SRB method)
Note that: * : p is less than or equal to 0.05 compared with a 25mM glucose control group; #: p is less than or equal to 0.05 compared with a concentration sample.
EXAMPLE 3 Effect of Compounds on HUVEC cell status under high sugar injury
Human vascular endothelial HUVEC cells were cultured in McCoy's 5A medium containing 10% heat-inactivated FBS (fetal bovine serum), 2mM L-glutamine, 100U/ml penicillin and 100g/ml streptomycin in a cell incubator at 37℃with 5% CO 2. The liquid is changed once every two days, and after 80% of cells are fused, pancreatin digestion and passage are carried out, so that the cells are kept in a good logarithmic growth phase.
After digestion and passage, the cuttlefish ink polysaccharide with the final concentration of 60 mug/ml is added to the mixture to treat the cuttlefish ink polysaccharide with the final concentration of 60 mug/ml after the digestion and passage, and the cuttlefish ink polysaccharide is treated with 5 percent CO at 37 DEG C 2 Is cultured in a cell culture vessel for 5d. The cell status was compared by photographing.
The observation under an inverted microscope (figure 3) shows that the blank control group cells are uniformly distributed, mostly in a flat polygonal paving stone-like mosaic arrangement, firm in adherence, clear in boundary morphology, rich in cytoplasm, and round or oval in cell nucleus, even in double nuclei, so that the blank control group cells are in division proliferation, and the cell state is good; HUVEC cells in the model group have reduced number, atrophy and deformation of cell morphology, reduced cell body, widened cell gap, blurred boundary, increased cell permeability, cells close to apoptosis and broken cells; compared with the model group, the cell state of the sepia polysaccharide group is obviously recovered, the cell number is increased, and the morphology is normal. Therefore, the sepia polysaccharide has obvious protective effect on HUVEC cells damaged by high sugar.
Example 4 Effect of Compounds on vascular endothelial cell filtration under high sugar injury
HUVEC cells were inoculated into Hanging Cell Culture Inserts at 10000 cells/Well density, a blank control group was added with standard DMEM medium, a model group, a control group 1, a control group 2 and a dosing group were added with 25mM glucose solution, respectively, while the control group 1, the control group 2 and the dosing group were added with 60. Mu.g/ml enoxaparin, sulodite and sepia polysaccharide, respectively, and after 5 days of culture, the medium was changed to 1640 medium without phenol red, and incubated for 3 hours with 400. Mu.g/ml FITC-labeled BSA, the medium in the bottom wells was moved to 96Well Assay Plate, fluorescence intensity was detected, and albumin filtration rate was calculated:
albumin filtration rate (%) = (fluorescence intensity value of test compound group-fluorescence intensity value of NC group)/fluorescence intensity value of NC group ×100
The results are shown in Table 2, and compared with the 25mM glucose control group, the fluorescence density value of the sepia polysaccharide group is remarkably reduced, which shows that the sepia polysaccharide can effectively restore the barrier effect of vascular endothelial cells, reduce the filtration rate of albumin, and has better effect than sulodexide and enoxaparin.
TABLE 2 Effect of test compounds on HUVEC cell filtration under high sugar damage
Note that: * : p is less than or equal to 0.05 compared with a 25mM glucose control group; * **: p is less than or equal to 0.001 compared with a 25mM glucose control group; # #. P is less than or equal to 0.01 compared with a blank control group; # # # #: p is less than or equal to 0.001 compared with a blank control group.
Example 5 therapeutic Effect of Compounds on arterial vascular inflammation caused by diabetes
Experimental animals: male SD rats 75, 200±2g in body weight.
Experimental materials: streptozotocin (sigma); glucose (soribao).
The experimental method comprises the following steps: 40 SD male rats with the weight of about 200g are taken, after being adaptively fed for one week, the rats are weighed on the next day after being fasted and not forbidden for 12 hours, 10 rats are randomly selected as blank groups and fed with normal feed, and the rest rats are taken as diabetes groups and fed with high-fat and high-sugar feed (feed composition: 20% sucrose, 4% cholesterol, 10% lard and 1% sodium cholate) for 30 days. After 30 days, the diabetes group was orally injected with streptozotocin 30mg/kg for 2 days without water control, and the blank group was injected with an equal volume of citric acid-sodium citrate buffer solution, and blood glucose was measured by blood sampling from the tail tip after 7 days.
Grouping drug administration: the diabetic rats with 16.7mmol/L < fasting blood glucose value < 21mmol/L and obvious polydipsia, polyphagia and polyuria are selected and randomly divided into 3 groups (the difference between the groups is not more than 1.1 mmol/L) according to the blood glucose value, and the administration is continued for 45 days.
Table 3 experimental group dosing conditions
| Grouping | Administration of drugs | Administration mode | Dosage of |
| Blank group | Physiological saline | Intravenous injection | -- |
| Model group | Physiological saline | Intravenous injection | -- |
| Sulodexide group | Sulodexide | Intravenous injection | 50 mg/kg/day |
| Sepia polysaccharide | Sepia polysaccharide | Intravenous injection | 50 mg/kg/day |
Type II diabetes is often accompanied by dyslipidemia, and the aorta has blood lipid aggregates and endothelial damage. As shown in FIG. 4, HE staining analysis showed that sepia polysaccharide had an ameliorating effect on aortic arch endothelial injury in rats developed by type II diabetes.
EXAMPLE 6 anticoagulant Properties of Compounds
Sample and control: enoxaparin injection: kesai (0.4 ml/4000 Axalu). Sulodexide injection: italian alpha Wei Shiman, specification: 2ml 600lsu 10 branches/cassette. Physiological saline: shandong Qi is available from pharmaceutical Co Ltd.
Experimental animals: SPF-grade SD rats; 6-8 weeks of age; and (5) male.
The experimental method comprises the following steps: four blood clots were measured by taking blood 1.5h after administration by subcutaneous injection into the abdomen of the rat.
Dosage of administration: enoxaparin, sulodite and sepia polysaccharide are given to 4mg/kg, and the control group is injected with physiological saline with the same volume; each group was made in 6 parallels.
The detection results are shown in tables 4-7, and the APTT of the sepia polysaccharide group is obviously lower than that of the enoxaparin group and the sulodexide group, so that the anticoagulation effect is weaker and the bleeding side effect is lower.
Influence of the compounds of Table 4 on APTT (unit: S)
| Grouping | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 | Mean value of | Standard deviation of |
| Control group | 20.18 | 18.99 | 22.40 | 20.46 | 19.22 | 18.40 | 19.94** | 1.43 |
| Enoxaparin group | 36.33 | 31.47 | 38.22 | 35.44 | 39.52 | 38.33 | 36.55** | 2.89 |
| Sulodexide group | 32.22 | 33.44 | 29.88 | 38.47 | 28.33 | 28.28 | 31.77** | 3.89 |
| Sepia polysaccharide | 26.44 | 25.43 | 22.37 | 22.16 | 22.11 | 23.55 | 23.68 | 1.85 |
* *: compared with the sepia polysaccharide group, P is less than or equal to 0.01;
influence of the compounds of Table 5 on PT (unit: S)
| Grouping | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 | Mean value of | Standard deviation of |
| Control group | 14.22 | 13.17 | 14.23 | 15.26 | 15.22 | 11.25 | 13.89 | 1.51 |
| Enoxaparin group | 13.28 | 16.77 | 11.28 | 11.47 | 14.33 | 15.33 | 13.74 | 2.17 |
| Sulodexide group | 16.22 | 14.23 | 15.23 | 11.42 | 12.53 | 12.88 | 13.75 | 1.80 |
| Sepia polysaccharide | 15.89 | 14.23 | 15.63 | 11.33 | 12.43 | 13.11 | 13.77 | 1.81 |
Influence of the compounds of Table 6 on TT (unit: S)
| Grouping | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 | Mean value of | Standard deviation of |
| Control group | 23.22 | 23.55 | 22.13 | 19.88 | 25.63 | 24.33 | 23.12 | 1.97 |
| Enoxaparin group | 24.56 | 22.58 | 22.36 | 28.33 | 20.43 | 24.22 | 23.75 | 2.69 |
| Sulodexide group | 27.52 | 25.36 | 22.43 | 26.33 | 22.31 | 22.58 | 24.42 | 2.28 |
| Sepia polysaccharide | 25.88 | 36.33 | 25.82 | 25.33 | 20.22 | 21.22 | 25.80 | 5.71 |
The influence of the compounds of Table 7 on the reaction (unit: S)
| Grouping | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 | Mean value of | Standard deviation of |
| Control group | 14.55 | 11.78 | 12.39 | 17.23 | 18.22 | 15.22 | 14.90 | 2.56 |
| Enoxaparin group | 16.83 | 16.52 | 14.25 | 13.24 | 17.25 | 14.33 | 15.40 | 1.66 |
| Sulodexide group | 16.32 | 18.22 | 12.33 | 15.36 | 14.33 | 15.23 | 15.30 | 1.97 |
| Sepia polysaccharide | 14.32 | 15.23 | 15.22 | 15.86 | 15.30 | 15.22 | 15.19 | 0.49 |
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. The sepia polysaccharide for improving endothelial cell high sugar injury is characterized by being a polysaccharide skeleton composed of fucose, galactosamine, mannose and N-acetylglucosamine, and having glucuronic acid branched chain at the C-3 position of mannose; the structural schematic formula is as follows:
wherein R is 1 = -OH or-OSO 3 H or-OSO 3 H and its salt form; r is R 2 = -OH or-OSO 3 H or-OSO 3 H and its salt form; n=10-16.
2. The sepia polysaccharide according to claim 1, wherein the sepia polysaccharide has a weight average molecular weight of 10kDa to 16kDa and a sulfation degree of 8% to 16.5%.
3. The method for preparing sepia polysaccharide as claimed in claim 1 or 2, comprising the steps of:
(1) Taking sepia ink from the ink sac, adding buffer solution, grinding and suspending uniformly;
(2) Ultrasonic processing the ground sepia, soaking at low temperature, centrifuging, collecting supernatant, adding papain for enzymolysis to obtain enzymolysis sepia;
(3) After the enzymatic hydrolysis of the sepia is denatured by boiling water, centrifuging at low temperature to obtain supernatant, adding deproteinizing agent to remove denatured protein, centrifuging again to obtain supernatant;
(4) Concentrating the supernatant, and separating and precipitating to obtain crude polysaccharide;
(5) Purifying, separating, dialyzing and freeze-drying the crude polysaccharide to obtain the sepia polysaccharide.
4. The method according to claim 3, wherein in the step (1), the volume ratio of the sepia ink to the buffer solution is 0.5-2:1-2.
5. The method according to claim 3, wherein in the step (2), the enzymolysis conditions are as follows: the concentration of papain is 1-3 per mill, the enzymolysis temperature is 50-60 ℃, and the incubation time is 1-2 h.
6. The method according to claim 3, wherein in the step (3), the volume ratio of the supernatant to the deproteinizing agent is 2 to 4:0.5 to 1.
7. Use of sepia polysaccharide according to claim 1 or 2 in the manufacture of a medicament for the treatment of vascular inflammation.
8. The use according to claim 7, wherein the vascular inflammation is vascular endothelial inflammation induced by diabetes mellitus.
9. The use according to claim 7, wherein the concentration of sepia polysaccharide contained in the medicament is 10 μg/ml to 60 μg/ml.
10. The use according to claim 7, wherein the active ingredient of the medicament is sepia Mo Duotang and pharmaceutically acceptable salts thereof.
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