TW201619390A - Method for preparing seaweed-oligosaccharide with anticoagulant activity - Google Patents

Abstract

The present invention relates to a method for preparing seaweed-oligosaccharide with anticoagulant activity. Specifically, according to this invention the method uses enzyme to hydrolyze seaweed polysaccharide samples which were made from Sargassum, agar, or Monostroma nitidum to obtain seaweed-oligosaccharide having anticoagulant activity, wherein the enzyme is generated by subjecting the marine bacterium strain of Pseudomonas vesicularis and/or Aeromonas salmonicida to the induction of marine polysaccharides of Sargassum, agar, and Monostroma nitidum.

Description

Method for preparing seaweed oligosaccharide with anticoagulant activity

The present invention relates to a method of preparing an algae oligosaccharide having anticoagulant activity. The present invention also relates to an alginate oligosaccharide having anticoagulant activity produced by the method, and a composition containing the same.

Certain seaweed polysaccharides and oligosaccharides have been studied for many years due to their complex structural composition and the importance of their physiological activities. Many polysaccharides and oligosaccharides with sulfate groups have unique physiological activities, such as heparin. With anticoagulant effect. For example, a seaweed polysaccharide gum having a sulfate group and acting as an anticoagulant is carrageenan and fucoidan.

Fucoidan is a substance contained in brown algae and certain marine invertebrates (such as sea urchins and sea cucumbers). The main structure contains a large amount of L-fucose and sulfate groups. However, the structural composition of fucoidan of each brown algae is different. In addition to fucose and sulfate groups, other monosaccharides are contained, and thus there are different changes in physiological activities. It is known that fucoidan produced from Undaria pinnatifida sporophyll is hydrolyzed by an enzyme to obtain a lower molecular weight fucoidan, and the anticoagulant test results have a delayed blood coagulation effect. Studies in this field have shown that after hydrolysis of seaweed polysaccharides by enzymes, it is generally necessary to chemically increase the content of sulfate groups to increase the anticoagulant activity. However, by chemical methods (such as the addition of chlorosulfonic acid-dimethylformamidine) Amine reagents) Chemical reagents must be used to increase the sulfate content. These reagents are highly toxic and require further removal and purification, resulting in higher preparation costs.

In addition to providing habitats, foraging, reproduction, and refuge in the sea, algae Health and medical care also have an indelible contribution. Some special ingredients in seaweed are important sources of essential elements, amino acids and vitamins in the human body. In ancient China and Japan, there were evidences of seaweed as food and medicine. The ancient medical code includes "Compendium of Materia Medica", "Ben Cao Jing Ji", "Hai Yao Materia Medica" and "Compendium of Materia Medica", etc. A record of the disease. Seaweed is also a traditional medicine in Indonesia and other Southeast Asian countries. It is used for fever, cough, and treatment of asthma, hemorrhoids, runny nose, stomach upset and urinary diseases. Japanese people like to eat seaweed to strengthen their anti-cancer and anti-tumor ability, and can effectively improve the symptoms of diabetes and relieve stress.

Therefore, there is a need to provide a method for preparing an algae oligosaccharide having anticoagulant activity. Instead of using natural seaweed as a source, it is possible to produce a seaweed oligosaccharide having anticoagulant activity without chemically increasing the sulfate group content.

The present invention is unexpectedly found to culture marine strains, Pseudomonas vesicularis and/or acaricidal cells, in a bacterial culture containing seaweed polysaccharides from Sargassum, broccoli or Qinghaicai . Aeromonas salmonicida , which induces the production of suitable saccharolytic enzymes by these marine strains, and hydrolyzes samples of seaweed polysaccharides prepared from Sargassum, broccoli or Qinghai using an enzyme solution containing the saccharolytic enzymes. The reaction can achieve an improved hydrolysis efficiency, and the obtained algae oligosaccharide has excellent anticoagulant activity without adding an acid group via an additional chemical method.

Accordingly, in one aspect, the present invention provides a method of preparing an algae oligosaccharide having anticoagulant activity, comprising: (a) cultivating a marine strain in a bacterial culture solution containing seaweed polysaccharide, wherein the marine strain is induced to produce sugar Decomposing the enzyme, and then separating the obtained marine strain culture containing the saccharolytic enzyme, and removing the bacterial body to obtain a crude enzyme liquid containing the saccharolytic enzyme, wherein the seaweed polysaccharide is selected from the group consisting of Sargassum, broccoli , seaweed prepared by a group consisting of Qinghai cuisine and any combination thereof, and the marine strain is selected from the group consisting of Pseudomonas vesicularis , Aeromonas salmonicida , and combinations thereof. a group consisting of; (b) providing a seaweed polysaccharide sample prepared from seaweed selected from the group consisting of Sargassum, broccoli, Qinghai, and any combination thereof; and (c) in step (b) In the seaweed polysaccharide sample, the crude enzyme solution obtained in the step (a) is added to carry out a hydrolysis reaction to produce an alginate oligosaccharide having anticoagulant activity.

In some embodiments, the seaweed polysaccharide used in the invention to induce marine strains and produce algal oligosaccharides is from the same seaweed.

In a specific embodiment, the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from Sargassum, and the seaweed polysaccharide sample of the step (b) is prepared from Sargassum.

In a specific embodiment, the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from the broccoli, and the seaweed polysaccharide sample of the step (b) is prepared from the broccoli.

In a specific embodiment, the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from Qinghai Cai, and the seaweed polysaccharide sample of the step (b) is prepared from Qinghai Cai.

In some embodiments, the seaweed contained in the bacterial culture solution of the step (a) The polysaccharide or the seaweed polysaccharide sample of the step (b) is a seaweed polysaccharide extract obtained by subjecting the seaweed to a hot extraction treatment.

In some embodiments, the method of the present invention may further comprise the step (d), It is the separation of the algae oligosaccharide obtained in the step (c) according to the molecular weight.

In a specific embodiment, the algae oligosaccharide obtained in step (c) can be separated into components. A fraction below 3 kDa or a fraction having a molecular weight between 5 kDa and 3 kDa.

In some embodiments, the algae oligosaccharide obtained in step (c) or (d) can be dried. Dry processing.

In another aspect, the present invention also provides a seaweed oligosaccharide having anticoagulant activity, the sea Algal oligosaccharides are prepared by the methods described herein.

In yet another aspect, the present invention provides a composition for anticoagulant activity, which comprises The seaweed oligosaccharide is used as an active ingredient.

In some embodiments, the anticoagulant active composition of the invention is for oral administration versus.

The details of one or more specific embodiments of the invention are set forth in the description below. this invention Other features and advantages will be apparent from the following detailed description of the specific embodiments and the appended claims.

All technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art to which the invention pertains, unless otherwise indicated.

The term "a" as used herein, unless otherwise specified, refers to the quantity of at least one (one or more).

The present invention relates to a method of preparing an algae oligosaccharide having anticoagulant activity. Specifically, the method of the present invention is to obtain a seaweed oligosaccharide having anticoagulant activity by hydrolyzing a sample of seaweed polysaccharide prepared from sargasso, broccoli or qinghai cabbage, wherein the enzyme is Pseudomonas vesicularis ( Pseudomonas vesicularis ) and/or marine strains of Aeromonas salmonicida are produced by marine polysaccharides from Sargassum, broccoli or Qinghai.

Specifically, the method of the present invention comprises: (a) cultivating a marine strain in a bacterial culture solution containing seaweed polysaccharide, causing the marine strain to be induced to produce a saccharolytic enzyme, and then obtaining the obtained marine strain containing the saccharolytic enzyme The culture is separated, and the cells are removed to obtain a crude enzyme solution containing the saccharolytic enzyme, wherein the seaweed polysaccharide is made of seaweed selected from the group consisting of Sargassum, broccoli, Qinghai and any combination thereof. And the marine strain is selected from the group consisting of Pseudomonas vesicularis , Aeromonas salmonicida , and combinations thereof; (b) providing a sample of seaweed polysaccharide, which is Prepared from seaweed selected from the group consisting of Sargassum, broccoli, Qinghai, and any combination thereof; and (c) in the seaweed polysaccharide sample of step (b), the crude enzyme obtained in the step (a) is added The solution is subjected to a hydrolysis reaction to produce an alginate oligosaccharide having anticoagulant activity.

The Pseudomonas vesicularis and Aeromonas aeruginosa described herein are common aquatic animal senses. Dyeing bacteria can be isolated from the estuary, seawater and other environments in a conventional way. They can also be separated from other environments such as onshore freshwater, wastewater, soil, etc., and the strains with better ability to decompose acacia can be further screened by using acacia medium.

As used herein, "Sargasso" refers to the algae of Sargassum , belonging to brown algae. Common species include: Sargassum siliquosum , Sargassum polycystum , Sargassum hemiphyllum )Wait. As used herein, "Cauliflower" refers to the algae of the genus Gelidium , belonging to red algae. Common species include: Gelidium amansii , Gelidium pacificum , Gelidium pusillum Wait. As used herein, "Qinghai Cai" refers to the algae of the genus Monostroma , belonging to the green algae. Common species include: Monostroma nitidum and Monostroma oxyspermum .

The term "bacterial culture solution" as used herein refers to a culture solution generally used for cultivating marine bacteria. For example, marine liquid medium (Marine Broth, MB).

According to the method of the present invention, step (a) is added to the bacterial culture solution of seaweed polysaccharide or The seaweed polysaccharide sample of the step (b) may be a seaweed polysaccharide extract obtained by subjecting the seaweed to a hot extraction treatment.

According to the method of the present invention, seaweed can be obtained by hot extraction to obtain seaweed polysaccharide. In the heat Before the extraction treatment, the seaweed is preferably a dry sample. For example, the seaweed is washed, air-dried or sun-dried to dryness, or dried in an oven (for example, 60 ° C), preferably further cut into an appropriate size or ground to a fine The powder, for example, is less than 40 mesh in size for ease of handling. The dried seaweed sample is soluble in water and heated to 100-150 ° C, preferably 100-121 ° C, for about 10 minutes or more, for example, 20-40 minutes, to dissolve the polysaccharide component.

According to the method of the present invention, in step (a), marine bacteria are cultured to contain A bacterial culture solution of seaweed polysaccharides from Sargassum, broccoli and/or Qinghai is induced to produce a saccharolytic enzyme. The specific culture conditions can be cultured at 23-27 ° C for 1-3 days. The bacterial culture solution obtained in the step (a) is then separated, and the bacterial cells are removed to obtain a crude enzyme solution containing a saccharolytic enzyme which is induced by marine bacteria. The separation of the bacterial culture described herein can be carried out by a general separation method such as filtration or centrifugation to remove the bacterial cells, and a crude enzyme solution containing a sugar-decomposing enzyme induced by marine bacteria can be obtained.

According to the method of the present invention, the seaweed polysaccharide sample used in the step (b) is selected from the group consisting of horses The seaweed of the group consisting of spirulina, broccoli, qinghaicai, and any combination thereof is prepared, for example, as described above, the seaweed polysaccharide extract is prepared by hot extraction of seaweed.

In some embodiments, the invention is used in the bacterial culture of step (a) The seaweed polysaccharide added by the inducing marine strain to produce the saccharolytic enzyme, and the seaweed polysaccharide sample used in the step (b) are from the same seaweed species.

In a specific embodiment, the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from Sargassum, and the seaweed polysaccharide sample of the step (b) is prepared from Sargassum.

In a specific embodiment, the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from the broccoli, and the seaweed polysaccharide sample of the step (b) is prepared from the broccoli.

In a specific embodiment, the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from Qinghai Cai, and the seaweed polysaccharide sample of the step (b) is prepared from Qinghai Cai.

According to the method of the present invention, the step (c) is a step of adding the crude enzyme solution obtained in the step (a) to the seaweed polysaccharide sample of the step (b) to carry out a hydrolysis reaction to produce an algal oligosaccharide having anticoagulant activity.

Specifically, in the hydrolysis reaction, the amount of the crude enzyme solution from the step (a) is about 5-15% (v/v) based on the total volume of the reaction, the reaction time may be 1-3 days, and the reaction temperature may be 23-27 ° C.

In some embodiments, the method of the present invention may further comprise the step (d) of isolating the algae oligosaccharide obtained in step (c) by molecular weight. The manner in which the saccharide molecules are separated is well known in the art, for example, ultrafiltration.

In a particular embodiment, the algal oligosaccharide obtained in step (c) can be separated into a fraction having a molecular weight of 3 kDa or a fraction having a molecular weight of between 5 kDa and 3 kDa.

The algae oligosaccharide obtained by the method of the present invention exhibits the desired anticoagulant activity without chemically increasing the sulfate group. In a particular embodiment, the algae oligosaccharide obtained by the method of the invention has anticoagulant activity superior to existing anticoagulants (eg, heparin), eg, the coagulation delay time is longer or not observed during the analysis period. Condensation phenomenon. See Example 2-6.

In some embodiments, the method of the present invention may further comprise the step (d) of isolating the algae oligosaccharide obtained in step (c) by molecular weight.

In a particular embodiment, the algal oligosaccharide obtained in step (c) can be separated into a fraction having a molecular weight of 3 kDa or a fraction having a molecular weight of between 5 kDa and 3 kDa.

In some embodiments, the algal oligosaccharide obtained in step (c) or (d) may be dried, for example, for storage.

In another aspect, the invention provides an alginate oligosaccharide having anticoagulant activity produced by the methods described herein.

In still another aspect, the present invention provides a composition for anticoagulant activity comprising the algae oligosaccharide described herein as an active ingredient.

In some embodiments, the anticoagulant active composition of the invention is for oral administration versus.

The composition of the present invention can be adjusted with any one or more carriers according to conventional conventional methods. Made with. For example, the algal oligosaccharide described herein is mixed as an active ingredient with a suitable carrier, which is then formulated into the desired dosage form.

As used herein, the term "carrier" means to carry the active ingredient to form the desired distribution. Inactive ingredients in the form of a form include any standard pharmaceutically or physiologically acceptable carrier which is compatible with the active ingredient of the formulation and is not deleterious to the individual to be administered. The carrier can act as a diluent, vehicle, excipient or base for the active ingredient. Examples of some suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, gum arabic, calcium phosphate, alginate, gum tragacanth, gelatin, calcium citrate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, sterilized water, syrup and methylcellulose. The composition may additionally comprise a lubricant, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, for example, methyl and propyl hydroxybenzoates; sweeteners; .

The compositions of the present invention can be made using conventional techniques in accordance with the teachings provided in this specification. Various forms of want. In a particular embodiment, the composition according to the invention may be in the form of tablets, pills, powders, lozenges, sachets, tablets, elixirs, suspensions, emulsions, solvents, syrups, soft and hard gelatin capsules, suppositories, sterilization In the form of injections and packaging powders. In a particular embodiment, the compositions of the invention are in solution or powder form.

In particular, the seaweed used in the present invention is obtained from natural ingredients and does not chemically increase sulfuric acid. The base step, which is not toxic, can be added directly to food or nutraceuticals. The composition of the present invention has the potential to develop a health product for preventing thrombosis by using the anticoagulant activity of algae oligosaccharide as an active ingredient.

The invention is further illustrated by the following examples, which are merely provided The invention is intended to be illustrative, not limiting. It will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Example 1: Powder preparation and heat treatment of Sargassum, Acacia, and Qinghai

In this experiment, Sargassum siliquosum , Gelidium amansii , and Monostroma nitidum were selected as the source of seaweed of Sargassum, Acacia, and Qinghai, and the seaweed was heat treated. The polysaccharide is extracted to obtain a seaweed polysaccharide liquid from Sargassum, broccoli, and Qinghai. In short, the Sargasso, the broccoli, and the Qinghai cuisine are washed and dried in running water, and then ground to obtain the powder of Sargasso, Lihua, and Qinghai. 5 g of Sargasso, safflower and Qinghai vegetable powder were respectively added to 100 mL of water, and hot-extracted at 121 ° C for 10 to 30 minutes in a sterilizer to obtain seaweed polysaccharide liquids from Sargassum, broccoli and Qinghai.

Example 2: Inducing a saccharolytic enzyme from a marine strain by polysaccharides from Sargassum, broccoli, and Qinghai

In this experiment, a marine strain is separately cultured in a bacterial culture solution containing polysaccharides from Sargassum, Acacia, and Qinghai, and is induced to produce a saccharolytic enzyme to obtain a crude enzyme solution. Briefly, the seaweed polysaccharide liquids obtained from Example 1 from Sargassum, broccoli, and Qinghai were separately added to marine broth (MB) at a concentration of 0.3% or 0.15% (v/v). Pseudomonas vesicularis (MA103) and Aeromonas aeruginosa (MAEF108) were inoculated therein and cultured at 25 ° C for 24 hours to induce the production of saccharolytic enzymes by the marine strains. Then, the culture is separated, the cells are removed, and the supernatant containing the saccharolytic enzyme is obtained to obtain a crude enzyme solution, which is a crude enzyme solution obtained by inducing the marine strains with polysaccharides from Sargassum. Crude enzyme solution S), these marine strains are from stone flowers The crude enzyme solution (hereinafter referred to as the crude enzyme solution G) obtained by the polysaccharide of the vegetable, the crude enzyme solution (hereinafter referred to as the crude enzyme solution M) obtained by the polysaccharide derived from the seaweed vegetable.

Example 3: Hydrolysis of seaweed polysaccharides from Sargassum, broccoli, and Qinghai vegetables by crude enzyme solution and separation treatment

In this experiment, a polysaccharide sample from Sargassum, Acacia, and Qinghai was hydrolyzed in a corresponding crude enzyme solution to produce oligosaccharides, which were then separated to obtain hydrolyzed regions of algae oligosaccharides of different molecular weight ranges. Briefly, in the seaweed polysaccharide liquid from Sargassum, broccoli, and Qinghai, which were obtained in Example 1, the crude enzyme solution S, the crude enzyme solution G, and the crude enzyme solution M (the crude enzyme solution) obtained in Example 2 were respectively added. The volume percentage of the seaweed polysaccharide liquid is about 10%. For example, about 100 ml of the crude enzyme solution is added to about 1,000 ml of the seaweed polysaccharide liquid, and the mixture is reacted at room temperature for 1-2 days to hydrolyze the polysaccharide to produce Sargassum, stone. Cauliflower, Qinghai cuisine, seaweed oligosaccharide hydrolysate. Then, using 5kDa and 3kDa cassette filtration membrane, the respective algal oligosaccharide hydrolysates were separated by an ultrafiltration system to obtain an ultrafiltration separation liquid, respectively obtaining a hydrolyzed fraction of algae oligosaccharide having a molecular weight of 3-5 kDa and a low molecular weight. The 3 kDa algal oligosaccharide hydrolyzed fraction. Finally, the obtained algal oligosaccharide hydrolysis fraction was passed through a freeze-drying system to obtain a dried powder of algal oligosaccharide, which was stored in a dry box for subsequent experiments. Table 1 lists the various dried algal oligosaccharide powders and their preparation.

Example 4: Testing anticoagulant activity with rabbit plasma

4.1 Activated partial thromboplastin time (APTT) analysis

The dried algae oligosaccharide powder of Example 3 was used as a sample of the test group, and APTT analysis was performed. First, the dried powders of S. cerevisiae, broccoli, and Qinghai oligosaccharides of Example 3 were respectively dissolved in secondary water (ddH ₂ O) to prepare Sargasso, Lihua, and the concentration of 5 mg/mL or 20 mg/mL. Qinghai vegetable oligosaccharide solution. Take 10 μL of S. cerevisiae, broccoli, and Qinghai oligosaccharide solution, add 90 μL of rabbit plasma, and react at 37 ° C for 1 minute. Then, add 37 ° C preheated APTT reagent, react at 37 ° C for 5 minutes, and add 37. The precalcined calcium chloride solution (CaCl ₂ , 0.025 M) was then measured and the clotting time was determined. The blank group replaced the sample with sterile water, and the positive control group replaced the test sample with 10 μg/mL heparin (Heparin). Table 2 shows the results.

The results show that the algae oligosaccharide prepared according to the present invention exhibits an anti-coagulation effect in the APTT analysis. In particular, except for Group 7, the APTT analysis results of most of the experimental groups were comparable to or better than those of commercially available heparin. For example, the collocation time of the 5th group of broccoli oligosaccharide solution 3-5kDa at 5mg/mL 378 seconds, comparable to commercially available heparin, and the second group of Sargassum oligosaccharide solution <3kDa at a concentration of 5mg/mL, the clotting time of 475 seconds, compared with the commercial heparin condensation time extended by 95 seconds; in particular, other The test group did not observe coagulation within 10 minutes after the reaction, showing excellent anticoagulant activity. It should be noted that the clotting time of the APTT analysis measured by the 3-5 kDa of the 3-5 kDa of the geranium oligosaccharide solution in the seventh group was compared. Short, may be the experimental error caused by the high concentration of the test sample, because the same test sample still proves its excellent anti-condensation effect in other tests (such as PT analysis).

4.2 Prothrombin Time analysis

The dried algae oligosaccharide powder of Example 3 was used as a sample of the test group, and APTT analysis was performed. First, the dried powders of S. cerevisiae, broccoli, and Qinghai oligosaccharides of Example 3 were respectively dissolved in secondary water (ddH ₂ O) to prepare Sargasso, Lihua, and the concentration of 5 mg/mL or 20 mg/mL. Qinghai vegetable oligosaccharide solution. Take 10 μL of Sargasso, broccoli, and Qinghai oligosaccharide solution, add 90 μL of rabbit plasma, and react at 37 ° C for 1 minute. Then, add PT reagent preheated at 37 ° C, react at 37 ° C for 5 minutes, and add 37. The precalcined calcium chloride solution (CaCl ₂ , 0.025 M) was then measured and the clotting time was determined. The blank group replaced the sample with sterile water, and the positive control group replaced the test sample with 10 μg/mL heparin (Heparin). Table 2 shows the results.

The results show that the algae oligosaccharide prepared according to the present invention exhibits anticoagulation in the PT test. The effect is better than the commercially available heparin. In detail, the 3-5kDa of the 9th group of Qinghai Cai oligosaccharide solution at a concentration of 5mg/mL has a clotting time of 72 seconds, which is 52 seconds longer than the coagulation time of commercially available heparin; the sixth group of broccoli oligosaccharide liquid <3kDa at a concentration of 5mg/mL, the clotting time is 90 seconds, the coagulation time of the commercially available heparin is extended by 70 seconds; the 10th group of Qinghaicao oligosaccharide solution <3kDa at a concentration of 5mg/mL, the clotting time is 108 seconds. The coagulation time of the commercially available heparin was extended by 88 seconds; the collocation time of the 3-5 kDa of the 11th group of Qinghaicao oligosaccharide solution at a concentration of 20 mg/mL was 114 seconds, which was 94 seconds longer than that of the commercially available heparin; Group 8 of Cauliflower oligosaccharide solution <3kDa at a concentration of 20mg/mL, clotting time of 141 seconds, compared with the coagulation time of commercially available heparin extended by 121 seconds; Group 12 of Qinghai Cai oligosaccharide solution <3kDa at 20mg / At the concentration of mL, the clotting time was 189 seconds, which was 169 seconds longer than that of the commercially available heparin; in particular, the other test groups showed no coagulation within 10 minutes after the reaction, showing excellent anticoagulant activity.

Example 5: Testing anticoagulant activity with human plasma

The blood of three volunteers and 3.2% sodium citrate anticoagulant were mixed slowly and uniformly in a volume ratio of 9:1, and centrifuged for the first time at 1,000 × g, 10 minutes, at room temperature. Centrifuge into two layers (serum and hematocrit), collect the upper platelet-rich serum (Platelet-rich-plasma, PRP); then centrifuge at 2,000 × g, 10 minutes, room temperature for a second time, collect the upper layer without Platelet serum (Platelet-poorplasma, PPP). Perform APTT analysis and PT analysis.

5.1 APTT analysis

The APTT analysis method is the same as that described in Example 4.1, and the results are shown in Table 4.

Table 4: Results of APTT analysis with human plasma.

5.2 PT analysis

The PT analysis method is the same as that described in Example 4.2, and the results are shown in Table 5.

The results show that the algae oligosaccharide prepared according to the present invention exhibits anticoagulant activity in both human APTT and PT tests.

Example 6: Testing platelet aggregation with human plasma (Platelet aggregation)

The blood of three volunteers and 3.2% sodium citrate anticoagulant were mixed slowly and uniformly in a volume ratio of 9:1, and centrifuged for the first time at 1,000 × g, 10 minutes, at room temperature. Centrifuge into two layers (serum and hematocrit), collect the upper platelet-rich serum (Platelet-rich-plasma, PRP); then centrifuge at 2,000 × g, 10 minutes, room temperature for a second time, collect the upper layer without Platelet-poor plasma (PPP). Since the number of platelets in normal people is about 200-400×10 ³ /mL, and the number of platelets in each subject is different, all blood samples need to determine the number of platelets in PRP, and PRP is its own PPP. The platelet count was diluted and adjusted, and the platelet concentration was adjusted to 300 × 10 ³ /mL, and stored at 4 ° C for subsequent testing. A platelet coagulant (Adenosine diphosphate reagent ADP reagent, 200 μM) was used as a control group, and the absorbance after the reaction was 100%, and the absorbance of the test group was analyzed and the percentage of platelet aggregation reduced was calculated. The results are shown in Table 6.

The results showed that the algae oligosaccharide prepared according to the present invention exhibited anticoagulant activity for analysis of anti-platelet coagulation activity in human plasma, and each group showed at least 17% or even 70% inhibition of platelet aggregation compared with the control group.

Claims (10)

A method for preparing an algae oligosaccharide having anticoagulant activity, comprising: (a) cultivating a marine strain in a bacterial culture solution containing seaweed polysaccharide, causing the marine strain to be induced to produce a saccharolytic enzyme, and then obtaining the obtained The marine strain culture of the saccharolytic enzyme is separated, and the bacterial cell is removed to obtain a crude enzyme liquid containing the saccharolytic enzyme, wherein the seaweed polysaccharide is composed of selected from the group consisting of Sargassum, broccoli, Qinghai and any combination thereof. a group of seaweeds, and the marine strain is selected from the group consisting of Pseudomonas vesicularis , Aeromonas salmonicida , and combinations thereof; (b) Providing a seaweed polysaccharide sample prepared from seaweed selected from the group consisting of Sargassum, broccoli, Qinghai and any combination thereof; and (c) in the seaweed polysaccharide sample of step (b), adding step (a) The obtained crude enzyme solution is subjected to a hydrolysis reaction to produce an alginate oligosaccharide having anticoagulant activity. The method of claim 1, wherein the seaweed polysaccharide contained in the bacterial culture solution of the step (a) and the seaweed polysaccharide sample of the step (b) are prepared from the same seaweed. The method of claim 1, wherein (i) the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from Sargassum, and the seaweed polysaccharide sample of the step (b) is prepared from Sargassum; (ii) the seaweed polysaccharide contained in the bacterial culture solution of the step (a) is prepared from the broccoli, and the seaweed polysaccharide sample of the step (b) is prepared from the broccoli; or (iii) the step (a) The seaweed polysaccharide contained in the bacterial culture solution is prepared from Qinghai Cai, and the seaweed polysaccharide sample in the step (b) is prepared from Qinghai Cai. The method of claim 1, wherein the seaweed polysaccharide contained in the bacterial culture solution of the step (a) or the seaweed polysaccharide sample of the step (b) is a seaweed polysaccharide extract obtained by subjecting the seaweed to a hot extraction treatment. The method of claim 1, further comprising the step (d) of isolating the algae oligosaccharide obtained in the step (c) by molecular weight. The method of claim 5, wherein the algal oligosaccharide is separated into a region having a molecular weight of 3 kDa or less or a region having a molecular weight of between 5 kDa and 3 kDa. The method of any one of clauses 1 to 6, wherein the algal oligosaccharide obtained in step (c) or (d) is subjected to a drying treatment. A seaweed oligosaccharide having anticoagulant activity, which is produced by the method of any one of claims 1 to 7. A composition having anticoagulant activity comprising the algal oligosaccharide of claim 8 as an active ingredient. The composition of claim 8 which is for oral administration.