CN101328227A - A kind of reef film polysaccharide and its preparation method and application - Google Patents

Abstract

The invention relates to Monostroma nitidum polysaccharide, which is characterized in that: sugar chains of the Monostroma nitidum polysaccharide contain rhamnose groups, xylose groups, glucose groups, galactose groups, mannose groups and glucuronic acid groups, and the rhamnose group polysaccharide is a linear long chain molecule which is connected by alpha-1, 2-glycosidic bonds and alpha-1, 3-glycosidic bonds. Under the condition of preparation, algae frond is immersed into water for homogenate, and then heated and extracted, and a clear solution is separated, concentrated and desalinated; the desalinated solution is concentrated and deproteinized; the deproteinized solution is concentrated, alcohol deposited, dried, dissolved and then separated through an ion exchange chromatographic column and a gel chromatographic column in turn; the collected solution is concentrated and desalinated; and the desalinated solution is concentrated, alcohol deposited, cleaned, dried and crushed. The Monostroma nitidum polysaccharide can be used for preparing a novel anticoagulant or antithrombotic reagent.

Description

A kind of reef film polysaccharide and its preparation method and application

technical field

The invention relates to a reef membrane polysaccharide, a preparation method and application thereof.

Background technique

Studies have found that heparin, which has been widely used as an anticoagulant drug for more than 70 years, has many side effects, such as inducing thrombocytopenia and bleeding. In addition, the concentration of heparin in the main raw material for preparing heparin is low, and the occurrence of "mad cow disease" related to cattle makes it urgent to find alternatives to anticoagulant drugs. In recent years, in addition to continuing to research and develop low-molecular-weight heparins, such as enoxaparin, nadroparin calcium, dalteparin sodium and tinzaparin sodium, people have also modified the structure of some natural polymers according to the structural characteristics of heparin to achieve similar purpose. Experiments have proved that these heparins have certain anticoagulant activity, but they also have certain toxicity. Thrombotic diseases seriously endanger human health and are one of the main fatal diseases in modern society. At present, the annual global market value of antithrombotic drugs is about 12 billion U.S. dollars, and is increasing at an annual rate of 15%; the global market value of anticoagulant drugs is about 4 billion U.S. dollars, and is increasing at a rate of 13%. Coagulation and antithrombotic drugs are far from meeting people's growing needs. Seaweed is a natural and rich source of anticoagulant polysaccharides. At present, the research and development of seaweed polysaccharides mainly focus on red algae polysaccharides and fucoidan, while the research and development of green algae polysaccharides is rare, and the literature on reef polysaccharides has not been reported yet.

Contents of the invention

The purpose of the present invention is to provide a kind of reef membrane polysaccharide and its preparation method and application, which can make up for the above-mentioned deficiencies of the prior art.

A reef membrane polysaccharide is characterized in that the sugar chain of the reef membrane polysaccharide contains rhamnose, xylose, glucose, galactosyl, mannosyl and glucuronic acid groups, and is based on α-1,2 - Linear long-chain molecules linked by glycosidic bonds and α-1,3-glycosidic bonds; rhamnosyl accounts for 60-90% of the total molar percentage of monosaccharides, and L-molecules linked by α-1,2-glycosidic bonds The molar ratio of lysyl to α-1,3-glycosidic linked L-rhamnosyl is 1-8:1; the sulfate group is located at the C-3 position of α-1,2-L-rhamnosyl And/or the C-4 position, and/or the C-2 position and/or the C-4 position at the α-1,3-L-rhamnosyl, the weight percentage of the sulfate group in the reef membrane polysaccharide is 10 ～40%; molecular weight is 50000～800000 Daltons.

The preparation method of the above-mentioned reef film polysaccharide is characterized in that seaweed algae are soaked in water to homogenate, heated and extracted, the clear liquid is separated, the obtained clear liquid is concentrated, desalted, the desalted solution is concentrated, deproteinized, and the deproteinized The protein solution is concentrated, alcohol precipitated, dried, dissolved, and then separated by ion exchange column and gel chromatography column in sequence, the collected solution is concentrated, desalted, and then the desalted solution is concentrated, alcohol precipitated, washed, and dried , crushed.

Application of the above-mentioned reef membrane polysaccharide in the preparation of anticoagulant drugs or antithrombotic drugs.

The raw material natural seaweed used for preparing the product of the present invention has no toxic and side effects on the human body and can be used for a long time. The product has good anticoagulant activity and antithrombotic activity, and can be made into tablets, powders, granules, capsules, etc. by adding various additives as anticoagulant or antithrombotic drugs for the prevention and treatment of thromboembolic diseases. It has a good market application prospect.

Detailed ways

Below by embodiment the present invention will be further described.

Weigh the dried seaweed algae, add water 1 to 80 times the weight of the seaweed to soak and homogenate, stir and extract polysaccharides in a water bath at 40 to 100°C for 0.1 to 6 hours, centrifuge to discard impurities, take the clear liquid, and reduce the obtained clear liquid to Concentrate under pressure, then carry out dialysis or ultrafiltration to remove salt, concentrate the polysaccharide solution after removing the salt under reduced pressure, then perform enzymatic and/or Sevage method to remove the protein in the polysaccharide, and then concentrate the polysaccharide solution after removing the protein under reduced pressure, Adding 4 times the volume of ethanol with a concentration of 95% (weight percentage, the same below) to precipitate the polysaccharide, washing the resulting precipitate with absolute ethanol for 1 to 3 times, drying in an oven at 40 to 80°C for 0.01 to 8 hours, and then Add a small amount of water to dissolve the obtained polysaccharide, separate it through the ion-exchange chromatography column Q-Sepharose Fast Flow, and sequentially elute with distilled water and 0.01-3.0mol/L sodium chloride aqueous solution, collect the eluent of the elution peak, and dissolve the obtained polysaccharide solution Concentrate under reduced pressure, then perform dialysis or ultrafiltration to remove salt, concentrate the polysaccharide solution after removing salt under reduced pressure, then pass through the gel chromatography column Sephacryal S-400HR for further separation, and elute with 0.01-0.5mol/L sodium acetate aqueous solution , collecting the eluate of the elution peak, concentrating the resulting polysaccharide solution under reduced pressure, then performing dialysis or ultrafiltration to remove salt, concentrating the polysaccharide solution after removing the salt under reduced pressure, adding 4 times the volume of 95% ethanol to precipitate the polysaccharide, The obtained precipitate is washed with absolute ethanol for 1 to 3 times, dried in an oven at 40 to 80° C. for 0.01 to 8 hours, and pulverized to obtain the reef film polysaccharide of the present invention.

After the reef film polysaccharide of the present invention is fully mixed with starch and/or powdered sugar, a soft material is made with 75% ethanol aqueous solution, then passed through a 12-mesh stainless steel sieve for granulation, dried at 50-60°C, and passed through a 14-mesh sieve. Stainless steel sieve whole grain. Then add magnesium stearate and/or talcum powder with 1% weight of reef film polysaccharide, and after fully mixing uniformly, perform tablet compression according to the general requirements for pressing tablet cores, so that the tablet cores have suitable hardness. The compressed plain tablets are alternately coated with 15-18 layers of powder coating layers and 10-15 layers of sugar-coated layers with syrup and talcum powder, and finally polished to obtain tablets with anticoagulant or antithrombotic effects.

The seaweed algae described in this embodiment can be wide reef membrane, reef membrane, pocket reef membrane, Arctic reef membrane, broken reef membrane, capsule reef membrane, thick reef membrane, pointed species reef membrane, wrinkled reef membrane or brown reef The ethanol can also be replaced by methanol, acetone or isopropanol; the ion-exchange column DEAE Sepharose Fast Flow can also be replaced by Q-Sepharose Fast Flow, DEAECellulose, DEAE Sephadex, Q-Sepharose XL, Q Sepharose, Q Sepharose 4 Fast Flow, QAESephadex or Dowex; the elution with distilled water and 0.01-3.0mol/L sodium chloride aqueous solution can also be eluted with distilled water and 0.01-3.0mol/L potassium chloride aqueous solution; The above-mentioned gel chromatography column Sephacryal S-400HR can also be replaced by Toyopearl HW-65, Sephacryal S-300HR, Sephacryal S-200HR, Sephacryal S-100HR, Sephacryal S-500 HR, Sephacryal S-1000SF, Superdex 200, Superose 6 , Superose 12, Sepharose 6 Fast Flow, Sepharose 4 Fast Flow, Sepharose 2B, Sepharose 4B, Sepharose 6B, Sepharose CL-2B, Sepharose CL-4B or Sepharose CL-6B; wash with 0.01～0.5mol/L sodium acetate aqueous solution It can also be eluted with distilled aqueous solution, 0.01～0.5mol/L sodium chloride aqueous solution, 0.01～0.5mol/L potassium chloride aqueous solution or 0.01～0.5mol/L ammonium bicarbonate aqueous solution; Switch to powders, granules, or capsules.

The reef film polysaccharide of the present invention is off-white or white amorphous powder, and the monosaccharide composition of the reef film polysaccharide that obtains is measured 6 times with gas chromatography, measures uronic acid composition with thin-layer chromatography and capillary electrophoresis, uses barium sulfate- The sulfuric acid group content was determined by gelatin turbidimetry, the molecular weight was determined by high performance gel permeation chromatography, the sugar chain structure was determined by periodic acid oxidation, Smith (Smith) degradation, methylation analysis, infrared spectroscopy, nuclear magnetic resonance spectroscopy and mass spectrometry, and Determined by analysis, the obtained reef membrane polysaccharide contains rhamnosyl, xylosyl, glucosyl, galactosyl, mannosyl and glucuronic acid groups in the sugar chain, which is based on α-1,2-glycosidic bond and Linear long-chain molecules linked by α-1,3-glucosidic bonds; rhamnose groups account for 60-90% of the total molar percentage of monosaccharides, and L-rhamnosyl groups linked by α-1,2-glycosidic bonds and The molar ratio of α-1,3-glucoside-linked L-rhamnosyl is 1-8:1; the sulfate group is located at the C-3 position and/or C of α-1,2-L-rhamnosyl -4 position, and/or at C-2 position and/or C-4 position of α-1,3-L-rhamnosyl, the weight percentage of sulfate group in reef membrane polysaccharide is 10-40%; The molecular weight is 50,000 to 800,000 Daltons.

The anticoagulant effect of the reef membrane polysaccharide of the present invention is evaluated by the influence on the activated partial thromboplastin time and thrombin time of healthy human venous blood, and the antithrombotic effect is evaluated by the experimental thrombus formation in rats in vivo and in vitro. The effect is evaluated, and the experimental results prove that the product of the present invention has obvious anticoagulant activity and antithrombotic effect. The specific experimental methods and results are as follows:

1. The influence on the activated partial thromboplastin time of venous blood of healthy people: take the venous blood of healthy people and place it in citrate containing 1/10 (the volume ratio of blood and anticoagulant is 9:1) and the concentration by weight is 3.3%. In the plastic tube of sodium citrate anticoagulant solution, gently invert and mix well, centrifuge at 3000 rpm for 15 minutes, collect the upper layer of plasma, and set aside. Take 9 samples of plasma to be tested, 90 μL each, add 10 μL of reef membrane polysaccharide aqueous solution with a concentration of 10, 25, 50, 100, 200, 300, 400, 500, 600 μg/mL, incubate at 37 °C for 1 min, add the activated part Take 100 μL of thromboplastin time reagent, incubate at 37°C for 2 minutes, add 100 μL of CaCl ₂ aqueous solution with a concentration of 0.025mol/L preheated at 37°C, and record the clotting time, which is the activated partial thromboplastin time value. The control group was added with 10 μL of normal saline, and the activated partial thromboplastin time was measured according to the above method. The experimental results show that the reef membrane polysaccharide can significantly prolong the activation time of activated partial thromboplastin, and there is a significant difference compared with the control group (P<0.001), indicating that the reef membrane polysaccharide has a significant anticoagulant effect and can inhibit endogenous coagulation pathway or shared coagulation pathway. The effect of different concentrations of reef membrane polysaccharides on activated partial thromboplastin time was that the greater the concentration, the longer the coagulation time, indicating that the anticoagulant effect of reef membrane polysaccharides had a linear relationship with the concentration.

2. Effect on venous blood thrombin time of healthy people: The separation of plasma is as above. Take 9 samples of plasma to be tested, 90 μL each, add 10 μL of reef membrane polysaccharide aqueous solution with a concentration of 10, 25, 50, 100, 200, 300, 400, 500, 600 μg/mL, incubate at 37 °C for 1 minute, add 37 Preheat 200 μL of thrombin time reagent at ℃, record the clotting time, which is the thrombin time value. To the control group, 10 μL of normal saline was added, and the thrombin time value was determined according to the above method. The experimental results show that the reef membrane polysaccharide can significantly prolong the plasma thrombin time, and there is a significant difference compared with the control group (P<0.001), indicating that the reef membrane polysaccharide has a significant anticoagulant effect, can inhibit thrombin activity or inhibit fibrin Converted to fibrin. The effect of different concentrations of reef membrane polysaccharides on thrombin time is that the greater the concentration, the longer the coagulation time, indicating that the anticoagulant effect of reef membrane polysaccharides has a linear relationship with the concentration.

3. Effect on experimental thrombosis in rats: Take healthy male Wistar rats weighing 200-250 grams, and randomly divide them into normal saline group, 24.0mg/Kg reef membrane polysaccharide group, and 12.0mg/Kg reef membrane polysaccharide group , 6.0mg/Kg reef membrane polysaccharide group and 6.0mg/Kg heparin group were administered twice a day in the morning and evening, 0.4mL each time, and the normal saline group was given 0.4mL normal saline. On the 11th day, 20 minutes after intragastric administration of the daily total amount, the rats were anesthetized with pentobarbital sodium intraperitoneal injection (40.0mg/Kg), and the right common carotid artery was stripped to a length of 15mm, and the stimulating electrodes of the in vivo thrombosis instrument were used to anesthetize the rats. Gently stir up the proximal end of the artery, and stir up the distal end of the artery with a temperature measuring probe. Thirty minutes after the drug, the rats were stimulated with a 1.6mA direct current for 7 minutes. When blood flow is blocked by thrombus formation in an artery, the surface temperature of the blood vessel drops suddenly. The time required from the start of stimulation to the sudden drop in temperature is called Ocelusion time (OT for short), and the length of OT is used as the efficacy index. The experimental data of each group is expressed as the mean plus or minus the standard deviation, and the statistical processing is carried out, and the significance test of the difference between the groups is tested by the t test. Experimental results show that the reef membrane polysaccharide has obvious anti-arterial thrombosis effect, and the effect will be enhanced with the increase of dose, and the reef membrane polysaccharide has obvious anti-arterial thrombosis effect than heparin (P<0.001). 4. Effects on experimental thrombus formation in rats in vitro: Drug grouping: control group (normal saline group), 12.0mg/Kg reef membrane polysaccharide group, 6.0mg/Kg reef membrane polysaccharide group, 3.0mg/Kg reef membrane polysaccharide group and 3.0mg/Kg heparin group. Take healthy male Wistar rats, anesthetize the rats intravenously with pentobarbital sodium (40.0 mg/Kg), separate the common carotid artery, take 2 mL of blood from the common carotid artery with a dry glass syringe, and divide it equally between two siliconized polyethylene In the tube ring, the blood was rotated at 17 rpm at 37°C for 30 minutes and then stopped. According to the dosing sequence designed by the Latin square, 0.1 mL of medicine was added to both ends of each tube ring, and the blood was rotated for 60 minutes. The thrombus was poured into the vinyl tube ring, weighed immediately, measured the length of the thrombus, dried in a constant temperature drying oven at 60°C, and weighed its dry weight. Follow the above steps to draw blood for the remaining four groups, and then take 5 rats to repeat In the above experiment, the thrombolytic rate of each drug group was calculated according to the following formula. The experimental data of each group is expressed as the mean plus or minus the standard deviation, and the statistical processing is carried out, and the significance test of the difference between the groups is tested by the t test.

The experimental results showed that the wet weight, dry weight and length of the 12.0mg/Kg reef membrane polysaccharide group, 6.0mg/Kg reef membrane polysaccharide group and 3.0mg/Kg reef membrane polysaccharide group were significantly reduced compared with the normal saline group (P<0.001) , indicating that the reef membrane polysaccharide has in vitro thrombolytic effect, and the 12.0mg/Kg reef membrane polysaccharide group and the 6.0mg/Kg reef membrane polysaccharide group have obvious thrombolytic effect than the 3.0mg/Kg heparin group (P<0.001), and the heparin group Compared with the normal saline group, there was no significant difference in thrombus wet weight, dry weight and length.

Claims (4)

1. A reef membrane polysaccharide is characterized in that the sugar chain of the reef membrane polysaccharide contains rhamnose, xylose, glucose, galactosyl, mannose and glucuronic acid, and is based on α-1 , linear long-chain molecules linked by 2-glycosidic bonds and α-1,3-glycosidic bonds; rhamnosyl accounts for 60-90% of the total molar percentage of monosaccharides, and L -The molar ratio of the rhamnose group to the L-rhamnosyl group connected by α-1,3-glucosidic bonds is 1 to 8:1; the sulfate group is located at the C- 3-position and/or C-4 position, and/or C-2 position and/or C-4 position in α-1,3-L-rhamnosyl, the weight percent content of sulfate group in reef membrane polysaccharide 10-40%; molecular weight 50,000-800,000 Daltons. 2. the preparation method of the described reef membrane polysaccharide of claim 1 is characterized in that seaweed algal bodies are soaked in water homogenate, heat extraction, separate clear liquid, gained clear liquid is concentrated, desalination, the solution after desalination is concentrated , deproteinize, then concentrate the deproteinized solution, ethanol precipitation, dry, dissolve, then separate through ion exchange chromatography column and gel chromatography column in turn, concentrate the collected solution, desalt, and then concentrate the desalted solution , alcohol precipitation, washing, drying, crushing. 3. The application of the reef membrane polysaccharide according to claim 1 in the preparation of anticoagulant medicine or antithrombotic medicine. 4. the preparation method of reef film polysaccharide as claimed in claim 2 is characterized in that described seaweed algal body is wide reef film, reef film, pocket reef film, arctic reef film, broken reef film, capsule reef film, thick Reef film, pointed reef film, wrinkled reef film or brown reef film.