CN117003901A - Sepia polysaccharide for improving endothelial cell high sugar damage and preparation method and application thereof - Google Patents

Abstract

The invention discloses a golden cuttlefish polysaccharide for improving endothelial cell high-glucose damage and its preparation method and application. The golden sepia polysaccharide is a polysaccharide skeleton composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and has a glucuronic acid branch chain at the C-3 position of mannose. It is a weight average molecular weight It is an amino polysaccharide with a sulfation degree of 10 to 16 kDa and a sulfation degree of 8% to 15%. It has been experimentally verified that compared with similar compounds, the golden sepia polysaccharide can effectively improve the state of endothelial cells and maintain the normal filtration function of vascular endothelium. At the same time, its risk of bleeding side effects is significantly lower than that of similar polysaccharides, making it safer to use. , furthermore, the golden cuttlefish ink polysaccharide can be used to develop auxiliary treatment drugs for late-stage diabetic lesions, filling the gap of such products in the current market.

Description

A kind of golden cuttlefish polysaccharide that improves endothelial cell high-glucose damage and its preparation method and application

Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a golden cuttlefish polysaccharide for improving endothelial cell high-glucose damage and its preparation method and application.

Background technique

Diabetes is a systemic progressive disease caused by blood sugar levels being higher than normal for a long time. It has many and serious complications. Long-term elevated blood sugar will cause damage to large blood vessels and microvessels, and endanger various tissues and organs such as the heart, brain, kidneys, peripheral nerves, eyes, and feet. More than half of the deaths from diabetes are caused by cardiovascular and cerebrovascular diseases. Among them, diabetic nephropathy, diabetic cardiovascular complications, and diabetic cerebrovascular disease are the main complications. At present, clinical treatment is mainly based on blood sugar control, supplemented by regulating blood lipids and platelet function. There are no targeted specific drugs. The latest Class 1 new drug approved by the State Food and Drug Administration for the treatment of type 2 diabetes, Dogglietine Tablets, is also a full activator of glucokinase (GK) that targets glucose degradation. It is basically a sugar control drug, but rarely targets the circulatory system, Drugs for the treatment of organic damage caused by the urinary system.

At present, there are a large number of therapeutic compounds targeting endothelial cell damage and cellular NO damage, and the types are complex. Traditional Chinese medicine extracts are the main ones, but few can actually be developed clinically. The therapeutic effect of polysaccharide compounds has also been recognized by society. Among them, heparin analogues, which have attracted more attention and have been studied thoroughly, have started Phase IV clinical trials in the United States. However, they are anti-thrombotic drugs and have bleeding risks. They are weak in resisting glucose damage and maintaining the semipermeable membrane properties of endothelial tissue. .

Contents of the invention

In view of the above shortcomings, the present invention provides a golden sepia polysaccharide that can improve endothelial cell damage caused by high glucose. It can maintain the normal filtration of endothelial tissue and maintain the normal state of endothelial cells in the state of high glucose injury.

In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions to achieve it:

The invention provides a golden cuttlefish polysaccharide for improving endothelial cell high-sugar damage. It is a polysaccharide skeleton composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and has a polysaccharide skeleton at the C-3 position of mannose. It has a glucuronic acid branch chain; its structural formula is as follows:

Among them, R ₁ =OH or -SO ₃ H or SO ₃ H and its salt form; 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 golden sepia polysaccharide is 10kDa-16kDa, and its sulfation degree is 8%-15%.

The invention also provides a preparation method of the golden sepia ink polysaccharide, which includes the following steps:

(1) Take golden cuttlefish ink from the ink sac, add buffer solution, grind and suspend evenly;

(2) Ultrasonically treat the ground golden cuttlefish ink, soak it at low temperature and then centrifuge, add the supernatant to papain for enzymatic hydrolysis, and obtain enzymatically hydrolyzed golden cuttlefish ink;

(3) After denaturing the enzymatically hydrolyzed golden cuttlefish ink in a boiling water bath, centrifuge at low temperature to obtain the supernatant, add a protein remover to remove denatured proteins, and centrifuge again to obtain the supernatant;

(4) Concentrate the supernatant, separate and precipitate to obtain crude polysaccharide;

(5) The crude polysaccharide is purified, separated, dialyzed, and freeze-dried to obtain golden sepia polysaccharide.

Further, in the step (1), the volume ratio of golden sepia ink and buffer solution is 0.5-2:1-2.

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

Further, in the step (3), the volume ratio of the supernatant liquid and the protein removing agent is 2-4:0.5-1.

Further, the protein removing agent is Sevag reagent.

The invention also provides the use of the golden sepia polysaccharide in preparing medicine for treating vascular inflammation.

Further, the vascular inflammation is vascular endothelial inflammation induced by diabetes.

Further, the vascular endothelial inflammation is glomerular microvascular endothelial and renal tubular endothelial inflammation caused by diabetes.

Further, the concentration of golden sepia polysaccharide contained in the medicine is 10 μg/ml-60 μg/ml.

Furthermore, the active ingredient of the drug is golden sepia polysaccharide and its pharmaceutically acceptable salts.

Furthermore, the golden cuttlefish ink polysaccharide can effectively improve the glucose damage state of endothelial cells and maintain the normal filtration function of endothelial tissue.

Compared with the existing technology, the present invention has the following advantages and beneficial effects:

1. The present invention selects golden cuttlefish ink as raw material, extracts and prepares a new golden cuttlefish ink polysaccharide, which is a polysaccharide skeleton composed of fucose, galactosamine, mannose, and N-acetylglucosamine. It is an amino polysaccharide containing a glucuronic acid at the C-3 position of mannose, with a weight average molecular weight of 10 to 16 kDa and a sulfation degree of 8% to 15%.

2. The golden squid ink polysaccharide can effectively improve the state of endothelial cells, maintain the normal filtration function of vascular endothelium, and can be used to treat vascular inflammation caused by damage to vascular endothelial cells caused by long-term high blood sugar, as well as renal insufficiency caused by high sugar. Inflammation of glomerular microvascular endothelium and renal tubular endothelium.

3. The risk of bleeding caused by long-term use of the golden sepia ink polysaccharide is significantly lower than that of similar drugs, and it is safer.

4. The golden squid ink polysaccharide can be used to develop auxiliary treatment drugs or health products for late-stage diabetic lesions, filling the gap of such products in the current market.

Description of the drawings

Figure 1 shows the elution curve of golden squid polysaccharide.

Figure 2 shows the infrared spectrum of golden cuttlefish ink polysaccharide.

Figure 3 shows the effect of golden sepia ink polysaccharide on the state of HUVEC cells under high glucose injury; A is the undamaged control cell state, B is the glucose-damaged cell state, and C is the cell state in the polysaccharide group.

Figure 4 shows the improvement of golden cuttlefish ink polysaccharide on aortic arch endothelial damage in rats under high glucose injury: a: blank group b: model group c: sulodexide treatment group d: golden cuttlefish ink polysaccharide treatment group.

Detailed ways

The technical solution of the present invention will be further described in detail with reference to the following specific examples.

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

Example 1. Preparation of golden cuttlefish ink polysaccharide

Fresh cuttlefish ink sacs are stored at -80°C to -50°C and thawed at 2°C to 5°C. Grind and suspend the golden cuttlefish ink and phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) evenly. The volume ratio of the golden sepia ink and phosphate buffer solution is 1:2. Repeat the ultrasonic treatment of the ground sepia ink (ultrasonic for 2 to 10 seconds each time, 5 to 10 seconds apart, ultrasonic for 10 to 100 times), stir and soak at 2 to 4°C for 24 to 72 hours, 24°C, 10,000 rpm, centrifuge for 20 minutes, and collect Supernatant 2 times. Add 1‰ to 3‰ of papain to the supernatant, and perform enzymatic hydrolysis at 45 to 65°C for 1 to 2 hours. The enzymatically hydrolyzed cuttlefish ink is denatured in a boiling water bath. The boiling water bath time is 10 to 30 minutes. After cooling, the supernatant is separated by centrifugation at 4°C and 8000 rpm for 40 minutes. Add Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant and stir for 20 to 50 minutes with a stirrer, let stand at 4°C for 1 to 2 hours, centrifuge the supernatant to remove denatured proteins, and repeat the operation 2 to 3 times. The dosage volume ratio of the supernatant and Sevag reagent is 4:1, and centrifugation is performed at 6000-15000 rpm for 30-60 minutes. 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 to 2 hours, and centrifuged at 6000 to 10000 rpm for 10 to 30 minutes to separate and precipitate crude polysaccharides. The yield of crude polysaccharide after freeze-drying is 1% to 3%.

The crude polysaccharide is purified and separated by chromatography to obtain the target compound. Dissolve 1g of dry crude polysaccharide SIP in 5mL of distilled water, heat and dissolve at 60-80°C for 10-30 minutes, centrifuge at 3000-5000rpm for 5-10 minutes, repeat the above operation for insoluble matter, combine the two supernatants, and add the activated C18 solid Use a phase extraction cartridge to remove part of the pigment, use ultrapure water to elute for 3 column volumes, and rotary evaporate to concentrate the collected solution. Take 1g of the crude polysaccharide concentrate after C18 column chromatography and put it on a DEAE-52 cellulose ion exchange column (2.5cm×20cm), and use distilled water and gradient concentration NaCl solution (0~2mol/L) to gradient elute, with a flow rate of 0.5~ 1mL/min, 2~5mL per tube. Use the phenol sulfate method to detect the polysaccharide content in the eluate, draw an elution curve through septum detection (Figure 1), and collect according to peaks according to the elution curve. The collected liquid is concentrated by rotary evaporation, and dialyzed in a 2000-5000Da dialysis bag for 48-72 hours. , freeze-drying to obtain the target polysaccharide compound, with a yield of 50% to 80%.

The structure of golden sepia ink polysaccharide is as follows:

,

Among them, R ₁ and R ₂ groups are -SO ₃ H or -OH; n=10-16.

Golden sepia polysaccharide is a polysaccharide skeleton composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and has a glucuronic acid branch chain at the C-3 position of mannose; its molecular weight is: 10~ 16kDa; sulfation degree is 8% to 15%.

The infrared spectrum of golden squid ink polysaccharide is shown in Figure 2: The figure shows the characteristic absorption peaks of the polysaccharide. Specifically, the absorption bands at 3468.35cm ^-1 and 2927.9cm ^-1 respectively correspond to -OH in the sugar ring. and C–H stretching vibration; the absorption peak at 1613.16cm ^-1 is the symmetric vibration of -COOH, which indicates that the golden sepia polysaccharide contains amide bonds or carboxylic acids; in addition, the absorption peak at 1211.56cm ^-1 and 807.55cm ^-1 The absorption peak of can be attributed to the S=O stretching vibration of the sulfate group; the absorption band existing in the range of 1100-1010cm ^-1 indicates that the polysaccharide is connected by cyclopyranoside; the absorption peak near 890cm ^-1 is generally expressed as β glycoside bond; FT-IR absorption information shows that golden cuttlefish polysaccharide is a β-polysaccharide with a pyranyl group and contains characteristic peaks of sulfate groups and carboxyl groups.

Example 2. Effect of polysaccharide compounds on HUVEC cell proliferation under high glucose injury

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

Human vascular endothelial HUVEC cells were placed 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°C. , cultured in a cell culture incubator with 5% _CO2 . The medium was changed every two days. After the cells were 80% confluent, they were digested with trypsin and passaged to keep the cells in a good logarithmic growth phase.

HUVEC cells in the logarithmic growth phase were seeded into a 96-well plate at 5000 cells/well (180 μl/well). After culturing for 24 hours, golden sepia polysaccharide or glycosaminoglycan (final concentration is shown in Table 1) was added. Set up 4 duplicate wells for each concentration. After 72 hours of drug action, 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 on a microplate reader.

The inhibition rate of cell growth is calculated according to the following formula:

Inhibition rate = [(OD540 control hole - OD540 administration hole)/OD540 control hole] × 100%

The test results are shown in Table 1. Compared with the blank group, the viability of HUVEC cells was significantly reduced after high glucose injury. After adding different concentrations of golden cuttlefish polysaccharides or glycosaminoglycans, cell viability was restored to a certain extent; compared with glycosaminoglycans, the effect of golden cuttlefish polysaccharides at 60 μg/ml was significantly better than the former.

Table 1 Effect of test compounds on HUVEC cell proliferation (SRB method)

Note: *: compared with 25mM glucose control group, P≤0.05; #: compared with samples of the same concentration, P≤0.05.

Example 3. Effect of compounds on HUVEC cell status under high glucose injury

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

After digestion and passage, the cells were treated with no damage, 25mM glucose damage, and 25mM glucose damage with a final concentration of 60 μg/ml golden sepia polysaccharide, and were cultured in a cell culture incubator at 37°C and 5% _CO2 for 5 days. Take photos to compare cell status.

Observed under an inverted microscope (Figure 3), the cells in the blank control group were evenly distributed, mostly arranged in a flat polygonal paving stone-like mosaic, with strong adhesion to the wall, clear borders, abundant cytoplasm, round or oval nuclei, and occasionally Double nuclei, indicating that they are dividing and proliferating, and the cell status is good; the number of HUVEC cells in the model group decreased, the cell shape shrank and deformed, the cell body became smaller, the intercellular gaps widened, and the boundaries were blurred, indicating that the cell permeability increased and the cells were close to apoptosis. The cells in the golden sepia ink polysaccharide group recovered significantly compared with the model group, with an increase in the number of cells and a normal morphology. This shows that golden cuttlefish ink polysaccharide has a significant protective effect on HUVEC cells damaged by high glucose.

Example 4. Effect of compounds on filtration of vascular endothelial cells under high glucose injury

HUVEC cells were inoculated into Hanging Cell Culture Inserts at a density of 10,000 cells/well. Standard DMEM culture medium was added to the blank control group. 25mM glucose solution was added to the model group, control group 1, control group 2 and administration group respectively. At the same time, control group 1 and control group were added. Group 2 and the administration group were added with 60 μg/ml enoxaparin, sulodexide and golden sepia polysaccharide respectively. After 5 days of culture, the culture medium was changed to 1640 culture medium without phenol red, and 400 μg/ml FITC label was added. Incubate with BSA for 3 hours, move the culture medium in the bottom well to the 96Well Assay Plate, detect the fluorescence intensity, and calculate the albumin filtration rate:

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

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

Table 2 Effects of test compounds on HUVEC cell filtration under high glucose injury

Note: *: compared with 25mM glucose control group, P≤0.05; ***: compared with 25mM glucose control group, P≤0.001; ##: compared with blank control group, P≤0.01; ###: compared with blank control Group comparison, P≤0.001.

Example 5. Therapeutic effect of compounds on arterial and vascular inflammation caused by diabetes

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

Experimental materials: streptozotocin (Sigma Company); glucose (Solebao).

Experimental method: Take 40 SD male rats with a body weight of about 200g. After one week of adaptive feeding, they are fasted and water-free for 12 hours. Then, they are weighed the next morning. 10 rats are randomly selected as a blank group and fed with normal feed. The remaining rats are As the diabetic group, they were fed high-fat and high-sugar feed (feed composition: 20% sucrose, 4% cholesterol, 10% lard, 1% sodium cholate) for 30 days. After 30 days, the diabetic group was fasted and water-free for 12 hours. The diabetic group was intraperitoneally injected with streptozotocin 30 mg/kg for 2 consecutive days. The blank group was injected with an equal volume of citric acid-sodium citrate buffer. After 7 days, blood was collected from the tail tip to measure blood glucose. value.

Group administration: Select rats from the diabetic group with 16.7mmol/L <fasting blood glucose <21mmol/L and with obvious symptoms of polydipsia, polyphagia, and polyuria, and randomly divide them into 3 groups according to their blood glucose values (the difference between the groups is not greater than 1.1mmol/L), administered continuously for 45 days.

Table 3 Dosing status of experimental groups

Group Give medication Dosing method dose Blank group saline intravenous injection -- model group saline intravenous injection -- Sulodexide group Sulodexide intravenous injection 50mg/kg/day Golden sepia ink polysaccharide Golden sepia ink polysaccharide intravenous injection 50mg/kg/day

Type 2 diabetes is often accompanied by dyslipidemia, with lipid accumulation and endothelial damage in the aorta. As shown in Figure 4, HE staining analysis showed that golden sepia polysaccharide has an ameliorative effect on aortic arch endothelial damage in rats with type 2 diabetes.

Example 6. Anticoagulant characteristics of compounds

Sample and reference substance: enoxaparin injection: Kesai (0.4ml/4000AxalU). Sulodexide injection: Italian Alfa Weissman, specification: 2ml: 600LSU*10 pieces/box. Normal saline: Shandong Qidu Pharmaceutical Co., Ltd.

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

Experimental method: After subcutaneous injection into the abdomen of rats, blood was collected at 1.5 hours and four items of coagulation were detected.

Dosage: Enoxaparin, sulodexide, and sepia polysaccharide were administered at 4 mg/kg, and the control group was injected with the same volume of normal saline; 6 parallel injections were made in each group.

The test results are shown in Table 4-7. The APTT of the golden sepia polysaccharide group was significantly lower than that of the enoxaparin group and sulodexate group, indicating that its anticoagulant effect was weak and its bleeding side effects were low.

Table 4 Effect of compounds on APTT (unit: S)

Group number 1 number 2 number 3 No 4 Number 5 number 6 mean standard deviation 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 Golden sepia ink polysaccharide 26.44 25.43 22.37 22.16 22.11 23.55 23.68 1.85

**: Compared with the golden sepia ink polysaccharide group, P≤0.01;

Table 5 Effect of compounds on PT (unit: S)

Group number 1 number 2 number 3 No 4 Number 5 number 6 mean standard deviation 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 Golden sepia ink polysaccharide 15.89 14.23 15.63 11.33 12.43 13.11 13.77 1.81

Table 6 Effect of compounds on TT (unit: S)

Group number 1 number 2 number 3 No 4 Number 5 number 6 mean standard deviation 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 Golden sepia ink polysaccharide 25.88 36.33 25.82 25.33 20.22 21.22 25.80 5.71

Table 7 Effect of compounds on (unit: S)

Group number 1 number 2 number 3 No 4 Number 5 number 6 mean standard deviation 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 Golden sepia ink polysaccharide 14.32 15.23 15.22 15.86 15.30 15.22 15.19 0.49

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still make modifications to the foregoing embodiments. Modifications are made to the recorded technical solutions, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims (10)

1. A golden cuttlefish polysaccharide that improves endothelial cell high-glucose damage, characterized in that the golden cuttlefish polysaccharide is a polysaccharide skeleton composed of fucose, galactosamine, mannose, and N-acetylglucosamine, and There is a glucuronic acid branch chain at the C-3 position of mannose; its structural formula is as follows: Among them, R ₁ =-OH or -OSO ₃ H or -OSO ₃ H and its salt form; R ₂ =-OH or -OSO ₃ H or -OSO ₃ H and its salt form; n=10-16. 2. The golden cuttlefish ink polysaccharide according to claim 1, characterized in that the weight average molecular weight of the golden cuttlefish ink polysaccharide is 10kDa-16kDa, and its sulfation degree is 8%-16.5%. 3. The preparation method of golden cuttlefish ink polysaccharide according to claim 1 or 2, characterized in that it includes the following steps: (1) Take golden cuttlefish ink from the ink sac, add buffer solution, grind and suspend evenly; (2) Ultrasonically treat the ground golden cuttlefish ink, soak it at low temperature and then centrifuge, add the supernatant to papain for enzymatic hydrolysis, and obtain enzymatically hydrolyzed golden cuttlefish ink; (3) After denaturing the enzymatically hydrolyzed golden cuttlefish ink in a boiling water bath, centrifuge at low temperature to obtain the supernatant, add a protein remover to remove denatured proteins, and centrifuge again to obtain the supernatant; (4) Concentrate the supernatant, separate and precipitate to obtain crude polysaccharide; (5) The crude polysaccharide is purified, separated, dialyzed, and freeze-dried to obtain golden sepia polysaccharide. 4. The preparation method according to claim 3, characterized in that in the step (1), the volume ratio of golden sepia ink and buffer solution is 0.5~2:1~2. 5. The preparation method according to claim 3, characterized in that 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 incubation The time is 1h~2h. 6. The preparation method according to claim 3, characterized in that in the step (3), the volume ratio of the supernatant and the protein removing agent is 2-4:0.5-1. 7. Application of the golden sepia polysaccharide according to claim 1 or 2 in the preparation of a medicine for treating vascular inflammation. 8. The application according to claim 7, wherein the vascular inflammation is vascular endothelial inflammation caused by diabetes. 9. The application according to claim 7, characterized in that the concentration of golden sepia polysaccharide contained in the medicine is 10 μg/ml-60 μg/ml. 10. The application according to claim 7, characterized in that the active ingredient of the drug is golden sepia polysaccharide and its pharmaceutically acceptable salts.