KR20100138438A - Composition for anticoagulation comprising fuco-oligosaccharides - Google Patents

Abstract

PURPOSE: An anti-thrombotic composition containing fuco-oligosaccharide is provided to prevent and treat diseases such as hypertension, heart diseases, stroke and myocardial infarction caused by blood clot and platelet aggregation. CONSTITUTION: An anti-thrombotic composition contains fuco-oligosaccharide as an active ingredient. The molecular weight of fuco-oligosaccharide is 1,000-10,000 dalton. The composition is a pharmaceutical composition or food composition. A functional anti-thrombotic food contains the anti-thrombotic composition. The functional foot is beverage, meats, chocolate, snack, pizza, ramen, noodles, gums, ice creams, or alcohol drinks.

Description

Composition for Anticoagulation Comprising Fuco-Oligosaccharides

The present invention relates to an antithrombotic composition comprising a fuco oligosaccharide as an active ingredient.

Cerebrovascular diseases such as arteriosclerosis, cerebral hemorrhage, stroke and cerebral infarction and circulatory diseases such as heart disease are adult diseases that occupy the first and second causes of death. As the adult disease develops into a modern society, it is frequently occurring in middle-aged people due to changes in dietary habits and internal and external environmental stresses. The main pathogen of these diseases is known as thrombus, which is recognized as a pathology mediated by excess platelet aggregation.

A thrombus is an aggregate of blood factors, mainly consisting of aggregates of platelets and fibrin or platelet aggregates, which are typically formed to prevent excessive bleeding of damaged blood vessels. In blood vessel damage, platelets are activated by stimulation of various aggregated derivatives such as collagen, ADP, or arachidonic acid, causing adhesion, secretion, and aggregation, in which the platelet membrane receptor P- Selectin, GPIIb / IIIa, and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors will act. These reactions are associated with hemostasis and play an important role in the development of circulatory diseases, including thrombosis.

Therefore, a substance capable of inhibiting the platelet aggregation reaction, the release reaction and the blood coagulation reaction can be said to be a solution for the prevention and treatment of the diseases involving the thrombosis.

Two methods for removing blood clots have been proposed, and one of them is a method of dissolving, compressing, or removing fibrin, and since the constituents of blood clots are fibrin, they have received considerable response. Currently, thrombolytic agents developed to treat vascular-related diseases include several plasminogen activators that activate plasminogen to make insoluble fibrin a soluble fibrin breakdown product. Another method is to reduce the amount of fibrinogen, a precursor of fibrin, using thrombin-like enzymes.

Anti developed so far platelet agent, theophylline (theopyline), mole sidomin (molsidomine), and the like, verapamil (verapamil), nifedipine (nifedipine), which promotes the formation of cAMP and cGMP to inhibit the mobilization of Ca ^{2 +} Known. Also, nonsteroidal compounds such as aspirin, imidazole and indomethacin are known as drugs having antiplatelet action by inhibiting the production of thromboxane A2. However, although the drugs are chemicals, there are problems that cause various side effects such as hemostasis, infertility, and digestive problems in the human body.

Therefore, a lot of research on the anti-thrombotic substances from natural products with few side effects has been conducted.

Brown algae such as kelp, seaweed and sesame contain high amounts of acidic polysaccharides containing laminin and sulfate groups, which are neutral polysaccharides. Fucoidan is a representative sulfur-containing acidic polysaccharide. Fucoidan is a sulfur-containing hetero acidic polysaccharide containing L-fucose as an esterified sulfuric acid and containing a small amount of D-xylose, D-galactose, and uronic acid. As the macromolecules over, the main component that shows the physiological activity of fucoidan is known to be mainly sulfuric acid group.

Fucoidan differs in structure and physiology from other dietary fiber polysaccharides such as alginic acid, carrageenan, and laminaran. It exhibits anticoagulant and blood purification activity of about 40% (Chizhov et al., Chordajlum Carbohydrates Res, 1999, vol. 319, pp. 108-119). In addition, excellent physiological activities such as antiinflammatory, antitumor, antiviral and immunostimulation have been revealed (McClure et al., AIDA Res. Hum. Retroviruses, 1992, vol. 8). Nagumo and Nishino, In Polysaccharides in Medicinal Applications, 1997, pp.545-574; Patankar et al., J. Biol. Chemi. 1993, vol.268, pp.21770-21776).

However, fucoidan is a macromolecule having a molecular weight of more than 200,000 Daltons, and when used in a pharmaceutical composition, there may be a problem such as capillary closure. Therefore, it is necessary to decompose fucoidan to reduce the molecular weight.

Fucoidan can be used to decompose glycoside bonds in the fucoidan chain using a degrading enzyme to rapidly reduce the molecular weight, or to break oligosaccharides from the end to slowly reduce the molecular weight. Fucoidan is produced by decomposing fucoidan.

The fuco oligosaccharide has a lower molecular weight than that of fucoidan, so when used in a pharmaceutical composition, there is no possibility of capillary obstruction, and thus the fuco oligosaccharide can be used as an injection, and the composition containing the fuco oligosaccharide as an active ingredient effectively inhibits the formation of blood clots in the blood. The present invention was completed by revealing that it can be usefully used for the prevention and treatment of hypertension, heart disease, stroke, myocardial infarction, etc. due to thrombus and platelet aggregation.

It is an object of the present invention to provide an antithrombotic composition which can effectively inhibit the formation of blood clots in the blood using fuco oligosaccharides.

It is another object of the present invention to provide an antithrombotic functional food comprising a fuco oligosaccharide.

The present invention provides an antithrombotic composition comprising a fuco oligosaccharide as an active ingredient.

As used herein, "fuco oligosaccharide" refers to a water-soluble polysaccharide having a sulfuric acid group consisting of 2 to 30 monosaccharides as a degradation product of fucoidan.

The fuco oligosaccharide may be prepared using an enzyme having a resolution capable of decomposing the fucoidan to a small molecular weight.

Enzymes that break down fucoidan are produced mainly from mollusks and bacteria in the sea, and marine microbes that produce enzymes that break down fucoidan are Pseudomonas atlantica ), Pseudomonas carrageenova ), Vibrio sp . ), Pseudo Alteromonas sheet Lea (Pseudoalteromonascitrea), Playa Bo bacteria seae (Flavobacteriaceae), etc., and mollusks in the sense of Guadalupe H. (H. rufescens), H. Koh Lugar Other (H. corrugata), H. Jizan Te and the like (H. gigantea), P. yeso N-Sys (P. yessoensis), P. maximus (P. maximus).

 The present invention can be prepared using fucoidan degrading enzymes derived from the sph.

The sphingomonas genus microorganism may be sphingomonas paucimobilis ( Sphingomonas Paucimobilis ), preferably sphingomonas pasimomobilis ( Sphingomonas according to Korean Patent No. 10-0887682) Paucimobilis ) PF-1 (KCTC 11130BP).

A fuco oligosaccharide having a small molecular weight can be prepared by contacting the fucoidan degrading enzyme and the fucoidan purified from the microorganisms of the sphingomonas to decompose the fucoidan into fuco oligosaccharides.

The fucoidan used to prepare the fuco oligosaccharide may be derived from brown algae such as seaweed, seaweed, and seaweed, preferably fucoidan derived from seaweed, more preferably fucoidan derived from Korean seaweed, for example of Undaria pinnatifida It can be sporophyll.

Fuco oligosaccharide prepared by the manufacturing method effectively inhibits the formation of thrombus, as shown in the following examples, the composition containing the fuco oligosaccharide may be useful for the prevention or treatment of diseases caused by thrombus and platelet aggregation. have.

In one embodiment of the present invention, the fuco oligosaccharide is an antithrombotic composition, characterized in that the molecular weight is 1000 to 10,000 Daltons.

In addition, the fuco oligosaccharides contain elements of C, H, N, S, the content ratio of each element is 1 to 90 parts by weight of carbon, 0.1 to 30 parts by weight of hydrogen, 0.1 to 30 parts by weight of nitrogen, sulfur is It may be contained in a ratio of 0.1 to 30 parts by weight, preferably 1 to 60 parts by weight of carbon, 1 to 30 parts by weight of hydrogen, 1 to 30 parts by weight of nitrogen, 1 to 30 parts by weight of sulfur. More preferably, it may contain 1 to 40 parts by weight of carbon, 1 to 10 parts by weight of hydrogen, 1 to 10 parts by weight of nitrogen, and 1 to 10 parts by weight of sulfur, most preferably 30 To 40 parts by weight, hydrogen 1 to 7 parts by weight, nitrogen 1 to 7 parts by weight and sulfur may be contained in a ratio of 1 to 7 parts by weight.

The antithrombotic composition comprising the fuco oligosaccharide may be a pharmaceutical composition or a food composition.

The pharmaceutical composition according to the present invention may be prepared using pharmaceutically acceptable and physiologically acceptable auxiliaries in addition to fuco oligosaccharides, which may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, Lubricants, flavors and the like can be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. There is no particular limitation on the kind of food to which the composition of the present invention can be added, and it is possible to apply to all kinds of food. For example, there are beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy ice creams, alcoholic beverages, vitamin complexes, health supplements and the like.

The present invention provides a food having an antithrombotic function comprising the food composition.

The anti-thrombotic foods are not particularly limited, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements Or dairy products.

The invention also provides the use of a composition comprising fuco oligosaccharides for the manufacture of an antithrombotic medicament or food. Therefore, the composition containing the fuco oligosaccharide can be used for the use for the manufacture of anti-thrombotic medicine or food.

The present invention also provides a method for preventing or treating blood clots, comprising administering to a mammal a composition comprising a therapeutically effective amount of a fuco oligosaccharide.

As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.

As used herein, the term “therapeutically effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, doctor or other clinician, This includes amounts that induce alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health, sex and diet of the patient. It can be adjusted according to various factors including the time of administration, the route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously. In the treatment method of the present invention, in the case of an adult, the composition of the present invention is preferably administered at a dose of 1 mg / kg to 500 mg / kg once or several times a day.

In the treatment method of the present invention, the composition comprising the fuco oligosaccharide of the present invention is administered in a conventional manner through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. May be administered.

The antithrombotic composition comprising the fuco oligosaccharide according to the present invention maintains the antithrombotic effect of the fucoidan, or is slightly lower than the fucoidan, but has a lower molecular weight than the fucoidan, and thus, when used in a pharmaceutical composition, capillaries There is no possibility of obstruction, so it can be used as an injection and its high absorption rate in the body can effectively inhibit the formation of blood clots in the blood.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the present embodiments are intended to complete the disclosure of the present invention, and those skilled in the art to which the present invention pertains. It is provided to fully inform the scope of the invention, and the invention is defined only by the scope of the claims.

< Example  1> Foucault  Preparation of Oligosaccharides

One) Fucoidan  Containing degrading enzymes Coenzyme  Produce

Sphingomonas sphingomonas to break down fucoidan into fuco oligosaccharides Paucimobilis ) A crude enzyme solution containing fucoidan degrading enzyme was extracted from PF-1 (Accession No. KCTC 11130BP) strain. 80g of PF-1 was mixed with 80ml of 50mM sodium acetate pH 5.6 buffer and sonicated for 2 hours (Duty cycle: 40%, Out put control: 4), followed by centrifugation (12,000rpm, 4 ℃, 30min). , The supernatant was separately taken out and salted out for 1 hour by the addition of 20-80% of ammonium sulfate in small portions, and the precipitates were collected separately by centrifugation (12,000 rpm, 30 minutes, 4 ° C). The precipitate was dialyzed with 50 mM sodium acetate buffer pH 5.6 (MWCO 3,500) and concentrated to an ultrafiltration membrane (MWCO 10,000). Samples obtained from ammonium sulfate precipitation were treated with 10% streptomycin (160 μl / ml protein) and stirred for 1 hour, and centrifuged (12,000 rpm, 30 minutes, 4 ° C.) to separate supernatants to remove nucleic acids. It was. The obtained supernatant was dialyzed with 50 mM sodium acetate buffer pH 5.6 (MWCO 3,000), concentrated to an ultrafiltration membrane (MWCO 10,000), and used as a crude enzyme solution.

2) Foucault  Preparation of Oligosaccharides

Foucault oligosaccharide measurements using HPLC (Dionex) were confirmed using Shodex OHpak SB-805HQ (MW: ˜4 × 10 6, 8.0 × 300 mm, Showa, Japan) and ELSD (Evaporative light scattering detector, Alltech). The enzyme was reacted with the crude enzyme solution and 0.5% fucoidan, followed by centrifugation (12000 rpm, 4 ° C, 30 minutes) to obtain a supernatant. The supernatant was centrifuged by 75% ethanol treatment, and then the supernatant was concentrated to obtain fuco oligosaccharides. 1% fuco oligosaccharide was dissolved in distilled water and analyzed. The elution solvent was distilled water and the flow rate was 0.8 ml / min.

Figure 1 shows the results of the analysis of fuco oligosaccharides by HPLC. A is a 0.5% analysis of fucoidan by HPLC, and B is a 75% ethanol treatment after enzymatic reaction to take a supernatant. Fucoidan was decomposed by the fucoidan degrading coenzyme solution to produce fuco oligosaccharides of different sizes (molecular weight).

< Example  2> Foucault  Oligosaccharide Antithrombotic  effect

In order to analyze the antithrombotic action of Foucault oligosaccharides, APTT (Activated Partial Thromboplastin Time), PT (Prothrombin Time), and TT (Thrombin Time) were measured. APTT is a method of measuring endogenous coagulation, PT is a method of measuring exogenous coagulation, and TT is a method of measuring common parts of two pathways.

One) APTT  ( Activated Partial Thromboplastin Time )

APTT measurement was performed by adding 3 μl of purified fucoidan or fuco oligosaccharide to 100 μl of human plasma (Seoul National University Blood Bank) and incubating for 2 minutes at 37 ° C., then adding 100 μl of APTT reagent (STA-PTT A, USA). Then, it was incubated for 3 minutes at 37 degreeC. Thereafter, 100 µl of 0.025 M CaCl 2 was added to measure the time until the plasma was coagulated (Fibrometer) (BBL Fibrosystem, Fisher Scientific, U.S.A).

As a result of antithrombotic measurement of fucoidan and fuco oligosaccharide, as shown in FIG. 2, in the APTT experiment, the fucoidan was 90 sec at 5 μg / ml and 168.4 sec at 10 μg / ml than the control group (39.9 sec). It was about 2.2 and 4.2 times higher activity, respectively, and the fuco oligosaccharides were 92 s at 100 μg / ml and 156 s at 200 μg / ml, respectively, 2.3 and 3.9 times higher than the control (39.9 sec).

2) PT Measurements of Prothrombin Time )

3 μl of purified fucoidan and fuco oligosaccharides were added to 100 μl human plasma, followed by incubation at 37 ° C. for 2 minutes, followed by addition of 100 μl of STA-PTT A5 reagent (Neoplastine CI plus), followed by incubation at 37 ° C. for 3 minutes. After incubation, 100 µl of 0.025M CaCl₂ was added, and the time until the plasma was coagulated was measured by a fibrometer.

As a result of antithrombotic measurement of fucoidan and fuco oligosaccharide, as shown in FIG. 3, it was found that in the PT experiment measuring exogenous pathway, fucoidan increased anti-thrombotic effect as the concentration was increased. I could see that there is almost no.

3) TT  Measure( Thrombin Time )

Add 3 µl of purified fucoidan and fuco oligosaccharides to 100 µl human plasma, incubate at 37 ° C for 3 minutes, add 200 µl of Thrombin time reagent previously heated to 37 ° C, and measure the time until the plasma coagulates. It was measured with a fibrometer.

TT measurement results of fucoidan and fuco oligosaccharide As shown in FIG. 4, TT results showed that fucoidan was 2.8 and 4.6 times higher at 41.4 sec and 10 sec / ml at 68 μg / ml than the control (14.8 sec), respectively. Foucault oligosaccharides showed high 1.7 and 2.6-fold activity at 25.16 sec at 50 μg / ml and 38.5 sec at 100 μg / ml, respectively.

Experimental results show that fucoidan acts on both the endogenous and exogenous pathways of thrombosis to exert antithrombotic effects.However, the fucooligosaccharides act on the endogenous and common pathways rather than the exogenous pathways. It showed a slightly lower antithrombotic effect than the macromolecule Fucoidan. However, the use of fuco oligosaccharides is safer, maximizes absorption in the body, and still exhibits significant antithrombotic effect, rather than fucoidan, which may have side effects such as decreased efficacy and capillary obstruction, due to its high molecular weight. It has been shown to be highly developed.

1. FIG. 1 shows the results of HPLC analysis of fuco oligosaccharides produced by the treatment of fucoidan degrading coenzyme solution. (A: Fucoidan, B: Fucoidan Degrading Coenzyme Solution)

2. Figure 2 shows the measurement results before endogenous coagulation according to APTT.

3. Figure 3 shows the results of measurement of exogenous coagulant according to PT.

4. Figure 4 shows the measurement results before common coagulant according to TT.

Claims (6)

An antithrombotic composition comprising Foucault oligosaccharides as an active ingredient. The method of claim 1, The fuco oligosaccharide has an antithrombogenic composition, characterized in that the molecular weight of 1,000 to 10,000 Daltons. The method of claim 1, The composition is an antithrombotic composition which is a pharmaceutical composition. The method of claim 1, The composition is an antithrombotic composition which is a food composition. Antithrombotic functional food containing the composition of Claim 4. The method of claim 5, The anti-thrombotic functional food is selected from the group consisting of beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, and dairy products. Thrombotic functional foods.

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