KR101464129B1 - Nanocapsules comprising mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid - Google Patents

Abstract

The present invention relates to nanocapsules for covering a mixture of a red ginseng extract, conjugated linoleic acid, and gamma linolinic acid with a covering agent of a mixed coating material of chitosan-arginine polymers and fucoidan, and to a method for manufacturing the same. The nanocapsules can be usefully applied as a composition for preventing or treating vascular diseases since the nanocapsules can enhance an effect of improving blood circulation by delivering the unsaturated fatty acids such as the red ginseng extract, the conjugated linoleic acid, and the gamma linolenic acid in a stable condition in the stomach environment.

Description

Nanocapsules containing a mixture of red ginseng extract, conjugated linoleic acid, and gamma linolenic acid (conjugated linoleic acid and gamma linolenic acid)

The present invention relates to a nanocapsule containing a mixture of red ginseng extract, conjugated linoleic acid, and gamma linolenic acid. More specifically, the present invention relates to a nanocapsule in which a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is coated with a mixed coating of chitosan-arginine polymer and fucoidan .

In general, diseases are expressed in various forms as the factors such as society, economy, culture, and environment change. This can be confirmed by the recent diversification of lifestyle including dietary life due to rapid economic development and improvement of living standard, and it is confirmed that past infectious diseases are decreased and chronic degenerative diseases of developed countries are increased.

In particular, it is known that these changes are contributing to a dramatic increase in mortality from vascular diseases such as hypertension, arteriosclerosis, ischemic heart disease, cerebral infarction, cerebral hemorrhage and stroke. These vascular diseases are caused by increased blood cholesterol, changes in blood components due to changes in lipid composition, and excessive mental tension.

Hematologic disorders, the cause of cardiovascular disease, have been reported to be caused when the hemostasis and dissolution of blood is not normally regulated. In steady state, activation and inhibition of hemostatic mechanism of blood are balanced to maintain homeostasis of blood flow. When blood vessels are damaged by trauma or disease, blood vessels are contracted to stop the bleeding, and collagen, von Willebrand factor, etc. on the inner wall of the blood vessels are exposed to the blood vessels by the first hemostatic process such as platelet adhesion and aggregation Platelet plugs are formed (Sarratt et al., 2004, Sarratt et al., 2005). At the same time, blood coagulation factors of the plasma are activated and a hemostasis plug is formed and a second hemostasis is achieved. These primary and secondary hemostatic processes are interrelated. That is, the activation of the platelet, which is the first hemostatic process, accelerates the second coagulation, which is the second hemostatic process, and the thrombin generated in the second hemostatic process induces platelet activation. First, the fibrin produced by the second hemostatic procedure must be completely dissolved through the fibrinolysis process before normal blood flow occurs. That is, blood flow in the human body is a complicated process in which blood vessels, platelets, blood coagulation factors, coagulation inhibitors, and dissolution processes must be balanced. However, when excessive hemostatic action occurs due to excessive activation of platelet aggregation, abnormal red blood cell deforming ability, and coagulation factors in plasma, thrombosis is generated and resulting thrombosis causes blood flow abnormality, leading to thrombosis and cardiovascular diseases Ross, 1993, Duhamel & Xu, 2007).

Drugs such as warfarin, coumarin or an antiplatelet agent such as aspirin, which are anticoagulants, are generally used as therapeutic agents for vascular diseases related to hemodynamic disorders. However, once the hemorrhagic disorder has developed, it is not easy to cure, and long-term administration of these drugs results in resistance to drugs, bleeding or gastrointestinal disturbances (Warner et al., 2011) (Kim et al., 2001). In addition, there are some problems in terms of side effects such as not only dissolving blood clots but also reacting with other proteins in the body (Kim et al., 2001). Therefore, there is an active research on therapeutic agents having anti-platelet aggregation or anti-coagulant activity related to blood circulation improvement without side effects even with continuous ingestion.

(Kung & Yang, 2006, Cumashi, 2006), which is involved in the coagulation mechanism of blood, such as red ginseng (Yu et al., 2006), conjugated linoleic acid (CLA) and gamma linolenic acid et al., 2007, Riaz et al., 2009).

It is known that red ginseng is produced by steaming and drying white ginseng. It is known that the toxicity of ginseng is alleviated and its pharmacological activity is improved in this process. Red ginseng contains a large amount of saponin. Most saponin present in the plant is oleanane, and red ginseng saponin is triterpenoid saponin of the dammarane family And ginsenosides Rh ₂ , Rg ₂ , Rg ₃ , Rg ₁ , and Rh ₁ as the specific saponin components of red ginseng (Kim et al., 2010). The general characteristic of saponin is that it is soluble in water, alcohol, it is consistently bubbling, physiologically detoxifying, and is not toxic to overdose. In particular, ginsenoside has been known to exert various effects on the body control function by affecting the central nervous system, the endocrine system, the immune system, and the metabolic system. In addition, red ginseng contains various components such as acid polysaccharide, polyphenol component, vitamins and minerals. Genesenoside is known to have pharmacological effects such as immune enhancement, anti-stress action, anticancer action, inhibition of aging, and improvement of blood circulation through inhibition of platelet aggregation (Chang-Xiao & Pei-Gen, 1992, Kitts & Hu, 2000). In particular, platelets can inhibit the production of intracellular Ca ²⁺ ions, Ca ²⁺ ions and prevent them from binding to the platelet membrane is known to be effective for improving blood circulation by inhibiting platelet activation and aggregation that (Matsuda et al., 1986 , Park et al., 2005, Jin et al., 2007). Panax ginseng , Panax quinquefolia , Panax notoginseng , Panax vietnamensis , Panax ginseng , Panax ginseng , and Panax ginseng is a perennial plant belonging to the genus Panax. Panax elegatior , Panax wangianus or Panax bipinratifidus , Panax angustifolium , and the like.

Conjugated linoleic acid is naturally present in dairy products or beef and is an isomer having a donor double bond derived from linoleic acid, an essential omega-6 fatty acid, cis 9, cis 11-conjugated linoleic acid, trans 9, cis 11-conjugated linoleic acid, trans 9, trans 11-conjugated linoleic acid, cis 10, cis 12-conjugated linoleic acid, trans 10, cis 12-conjugated linoleic acid and trans 10, trans 12-conjugated linoleic acid. Conjugated linoleic acid is known to exert excellent effects on symptoms such as immune function improvement, anticancer effect, growth promotion and anti-obesity effect, and it is known to inhibit the occurrence of arteriosclerosis by inhibiting platelet aggregation and delaying blood coagulation time (Truitt et al. 1999, MacDonald, 2000, Kung & Yang, 2006). Gamma linolenic acid is an intermediate substance metabolized from linoleic acid, an omega-6 fatty acid. It is contained in vegetable oils such as vegetable oil, sunflower seed oil and evening primrose oil. It lowers cholesterol in blood and prevents osteoporosis, skin aging, (Goodnight et al., 1982). In addition, it has been reported that anti-coagulant effects such as bleeding time and prolongation of blood clotting time have been reported (McDonald et al., 1989, Riaz et al., 2009).

On the other hand, ginsenosides in red ginseng have been reported to be unstable in acidic conditions, so they are degraded in gastrointestinal tract after ingestion and have low water uptake due to low bioavailability (Akao et al , 1998, Xu et al., 2003, Leung & Wong, 2010). Unsaturated fatty acids such as conjugated linoleic acid and gamma linolenic acid are structurally very unstable due to double bonds and are vulnerable to oxidation. Oxidation of fat increases the acid value and the peroxide value, and not only the quality is lowered due to the increase of viscosity and specific gravity, but also the essential fatty acid is destroyed and various physiological activities are lost. Therefore, in order to use red ginseng extract, conjugated linoleic acid and gamma linolenic acid, which are unsaturated fatty acids, as a blood circulation improving agent, it is necessary to search for a method of protecting the red ginseng extract from the acidic environment and preventing oxidation of conjugated linoleic acid and gamma linolenic acid.

Recently, several studies have reported on improving the stability of unstable physiologically active ingredients by nano-encapsulation using natural polysaccharides, and improving the absorption rate by improving the solubility of poorly soluble components. Examples of the encapsulation method include spray drying, spray cooling, extrusion, fluid phase deposition coating, complex formation, liposome entrapment, coacervation, centrifugal suspension separation, ionic gelation, etc. . Particularly, the ionic gelation is produced by bonding an electrolyte having opposite charge, such as a natural polysaccharide having a positive electric charge or a negative electric charge, so that the manufacturing process is relatively simple and the advantage that a harmful substance such as an organic solvent is not used It is widely used in food and medicine fields.

Chitosan, which is mainly used as a coating material for nanocapsules in the food sector, is the only component showing positive charge among natural polysaccharides. The chitosan is a polysaccharide produced by deacetylation of chitin which is a main constituent of crustaceans such as lobster, N-acetylglucosamine and glucosamine are composed of repeating structures based on β-1,4-glycosidic bonds. These chitosan are biodegradable, biocompatible and non-toxic, and are widely used as food ingredients and drug delivery agents (Sinha et al., 2004, Yoksan et al., 2010). Since a typical chitosan is not dissolved in a neutral solution but dissolves only in an acidic condition, it is limited for use as a drug delivery vehicle. A low molecular weight water-soluble chitosan having a free-amino group is easily dissolved and easily dissolved in a neutral aqueous solution And can be used as a drug delivery system, a peptide delivery system, and a gene delivery system. In addition, chitosan has been reported to be effective as a hemostatic agent and to promote wound healing by promoting platelet aggregation (Chou et al., 2003, Okamoto et al., 2003). It is assumed that the chitosan promoting activity of chitosan is due to the charge of chitosan (He et al., 2013), because chitosan is positively charged while platelets and erythrocytes are negatively charged, . In addition, platelet aggregation and the activation process is influenced by Ca ^2+, platelets are not stimulated physiologically very low cytoplasmic free Ca ²⁺ (cytosol free Ca ²⁺⁾ maintaining the concentration, however, agonists, such as collagen (agonist ) Stimulates the platelets to increase the cytoplasmic free Ca ²⁺ concentration, and thus acts like fibrinogen binding. Although the mechanism is not clear yet, it is known that chitosan increases platelet Ca ²⁺ concentration and promotes platelet aggregation (Chou et al., 2003). Therefore, although chitosan has a very excellent function to be used as a nanocapsule covering component, it is known that chitosan is not suitable for use as a carrier for drugs for improving blood circulation and food.

On the other hand, research has been reported that chitosan-arginine polymer produced when chitosan is polymerized with arginine has an effect of delaying blood coagulation time (Liu et al., 2004, Xiao et al., 2011). Arginine is a precursor of nitric oxide (NO), which acts as an anti-arteriosclerotic and vasodilating agent in the human body. It is an amino acid that interferes with platelet activation (Stief et al., 2001) . However, there has been no case of using a chitosan-arginine polymer as a covering component of nanocapsules as a delivery system for improving blood circulation.

Polyglutamic acid (polyglutamic acid, γ-PGA) and fucoidan are both soluble, non-toxic, biodegradable, water-soluble natural compounds that are widely used in the food, medicine and cosmetics industries. . The anti-blood coagulant activity of polyglutamic acid was reported in U.S. Patent No. 8618057. As a result of observing blood coagulation phenomena by mixing polyglutamic acid and mouse blood of various molecular weights, Although there was no difference in the coagulation action, it was confirmed that most polyglutamic acid delayed the coagulation of blood. Fucoidan is a polymeric polysaccharide mainly contained in brown algae. It is mainly composed of fucose and sulfate. It also contains monosaccharides, uronic acids, acetyl groups, and proteins. Fucoidan is known to inhibit the activity of thrombin and delay the coagulation of blood. Anti-coagulant activity is different depending on sulfate content, molecular weight, chemical composition, etc. Especially, Activity was reported to increase (Cumashi et al., 2007). On the other hand, no studies have been reported on the application of polyglutamic acid and fucoidan as coating materials for nanocapsules.

Accordingly, the present inventors have found that the nanocapsules prepared by coating a mixture of red ginseng extract, conjugated linoleic acid, and gamma linolenic acid with a mixed coating of chitosan-arginine polymer and fucoidan were excellent in blood circulation improvement effect of the nanocapsules, Completed.

Korean Patent No. 1222845 discloses a composition for treating vascular diseases containing a persimmon vinegar reaction mixture of red ginseng extract containing arginine derivatives. However, this composition differs from the present invention corresponding to the nanocapsule technology. Korean Patent No. 1138258 discloses nanoparticles containing red ginseng extract and conjugated linoleic acid as a method for solubilizing an oily / insoluble active material through formation of an oligomer complex. However, in the above-mentioned nanocapsules, chitosan-arginine Polymers, fucoidan and polyglutamic acid, or that the nanocapsules can be used as a composition for the treatment of vascular diseases. Korean Patent No. 466719 discloses a method for manufacturing spherical microcapsules containing red ginseng or ginseng root regimens. However, Korean Patent No. 466719 discloses only that the stability of red ginseng is maintained and the bitter taste is suppressed . Korean Patent Laid-Open Publication No. 2011-0122499 discloses that red ginseng has an effect of inhibiting platelet aggregation. However, Korean Patent Publication No. 2011-0122499 does not disclose the preparation of a composition containing red ginseng into nanocapsules . Korean Patent Laid-Open Publication No. 2006-0060644 discloses a red ginseng extract in which the content of ginsenosides Rg ₃ and Rh ₁ in the red ginseng extract is increased. However, there is no disclosure about the preparation thereof as a nanocapsule not. U.S. Patent No. 8,017,147 discloses that nanocapsules containing ginseng, conjugated linoleic acid, and unsaturated fatty acids such as gamma linolenic acid and chitosan can be used as a composition for treating vascular diseases. However, the therapeutic effect of specific vascular diseases Is not disclosed as an example, nor does it show that the nanocapsule contains a chitosan-arginine polymer, fucoidan or polyglutamic acid. European Patent No. 1358876 discloses a technique for capsules using chitosan and a polymer. However, this is an invention relating to a composition for improving acne, and the technical characteristics are completely different from those of the present invention.

Korean Registered Patent No. 1222845 (Composition for prevention or treatment of vascular diseases containing red ginseng reaction mixture of red ginseng extract, registered on Jan. 31, 2013) Korean Patent No. 1138258 (a method for solubilizing an oily / insoluble active material through formation of an oligomer complex, registered on Apr. 13, 2012) Korean Registered Patent No. 466719 (Method for manufacturing spherical microcapsules containing red ginseng or ginseng root, registered on 07.01.2005) Korean Patent Publication No. 2011-0122499 (Method for manufacturing health functional food using red ginseng powder, disclosed on November 11, 2011) Korean Patent Publication No. 2006-0060644 (Red ginseng manufacturing method according to the simplified method of Korean ginseng, published on Jun. 5, 2006) U.S. Patent No. 8017147 (Nutritional supplement for the prevention of cardiovascular disease, Alzheimer's disease, diabetes, and regulation and reduction of blood sugar and insulin resistance, registered on September 13, 2011) U.S. Patent No. 8618057, entitled Anticoagulant and composition for preventing thrombus containing poly-gamma-glutamic acid, European Registered Patent No. 1358876 (Microcapsules containing anti-acne agents registered at Cognis IP Management GmbH, 2007.07.07)

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It is an object of the present invention to provide a nanocapsule containing a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid, in detail, a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid as a coating of a mixed coating of chitosan- arginine polymer and fucoidan Coated nano-capsules.

The present invention relates to a nanocapsule in which a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is coated with a coating of a mixed coating of chitosan-arginine polymer and fucoidan.

The nanocapsules may be prepared by mixing 0.1 to 100 parts by weight of conjugated linoleic acid and 10 to 500 parts by weight of gamma linolenic acid based on 100 parts by weight of red ginseng extract.

The coated body of the nanocapsule may be a mixture of 10 to 50 parts by weight of the chitosan-arginine polymer and 6 to 10 parts by weight of fucoidan based on 100 parts by weight of the red ginseng extract.

0.01 to 1 part by weight of polyglutamic acid may be added to the coating of the nanocapsules on the basis of 100 parts by weight of the red ginseng extract.

The chitosan-arginine polymer was prepared by mixing chitosan and arginine in a TEMED / HCl buffer solution (N, N, N'-tetramethylethylenediamine / HCl buffer) ) May be a reaction product obtained by reacting 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide with N-hydroxy-succinimide.

The chitosan-arginine polymer is preferably prepared by mixing 10 to 100 parts by weight of arginine on the basis of 100 parts by weight of chitosan and mixing the mixture in a TEMED / HCl buffer solution (N, N ', N'-tetramethylethylenediamine / HCl buffer) , 10 to 100 parts by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 100 to 200 parts by weight of N-hydroxy succinimide were added to 100 parts by weight of the chitosan, It may be a dry matter.

The nanocapsules may have a particle size of 250 to 500 nm.

The nanocapsules may be used as a pharmaceutical composition for the prevention or treatment of vascular diseases. Similarly, the nanocapsule can be used as a health functional food for preventing or improving vascular diseases.

(1) The present invention relates to a method for producing a chitosan-containing composition, which comprises mixing 10 to 100 parts by weight of arginine per 100 parts by weight of chitosan and dissolving the mixture in a TEMED / HCl buffer solution (N, N'-tetramethylethylenediamine / HCl buffer) , 10 to 100 parts by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 100 to 200 parts by weight of N-hydroxy-succinimide are added to 100 parts by weight of the chitosan, Obtaining a chitosan-arginine polymer, and then redissolving the chitosan-arginine polymer in water to prepare a chitosan-arginine polymer solution;

(Step 2) Mixing a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid in the chitosan-arginine polymer solution prepared in the above step 1 to prepare a mixed solution; And

(Step 3) Mixing a fucoidan aqueous solution into the mixed solution of the two steps;

A nanocapsule in which a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is coated with a coating of a mixed coating of chitosan-arginine polymer and fucoidan can be produced.

The concentration of the chitosan-arginine polymer solution in the above step 1 may be 1 to 5 mg / ml.

The concentration of the red ginseng extract in the mixture of the red ginseng extract, the conjugated linoleic acid and the gamma linolenic acid is 1 to 15 mg / ml, the concentration of conjugated linoleic acid is 0.01 to 10 mg / ml, the concentration of gamma linolenic acid is 1 to 50 mg / ml Lt; / RTI >

The concentration of the fucoidan aqueous solution in the above three steps may be 0.6 to 1 mg / ml.

In the above step 3, polyglutamic acid may be added to the fucoidan aqueous solution at a concentration of 0.001 to 0.1 mg / ml.

Hereinafter, the present invention will be described in detail.

The red ginseng extract contained in the nanocapsules may be obtained by concentrating a red ginseng extract having a solid content of 50 to 70% by weight in a falling film evaporator (hereinafter referred to as FFE). Preferably, the red ginseng extract is obtained by concentrating the red ginseng extract, which is extracted with water, an aqueous alcohol solution, and an alcohol, as an extraction solvent, to a solid content of 60% by weight and then concentrating it with FFE, And may be selected from among ethanol, methanol, propanol, isopropanol and butanol.

The coated body of the mixed coating of chitosan-arginine polymer and fucoidan may be a mixture of chitosan-arginine polymer and fucoidan.

The nanocapsule may be mixed with 0.01 to 1000 parts by weight, more preferably 0.1 to 500 parts, and most preferably 0.1 to 100 parts by weight of conjugated linoleic acid based on 100 parts by weight of the red ginseng extract. May be mixed with 1 to 5000 parts by weight, more preferably 10 to 2500 parts by weight, and most preferably 10 to 500 parts by weight. When the concentration is outside this range, a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid The synergistic effect is not apparent.

The coated body of the nanocapsule preferably contains 1 to 500 parts by weight, more preferably 10 to 250 parts by weight, most preferably 10 to 50 parts by weight of the chitosan-arginine polymer based on 100 parts by weight of the red ginseng extract Can be mixed. In addition, fucoidan may be mixed in an amount of preferably 0.6 to 100 parts by weight, more preferably 6 to 50 parts by weight, and most preferably 6 to 10 parts by weight based on 100 parts by weight of the red ginseng extract. In the coated body of the nanocapsule, 0.001 to 10 parts by weight, more preferably 0.01 to 5 parts by weight, and most preferably 0.01 to 1 part by weight of polyglutamic acid may be added based on 100 parts by weight of the red ginseng extract If the concentration of the chitosan-arginine polymer exceeds the above range, the nanocapsules may not be well-formed or the nanocapsules may not be prepared in a stable state. Thus, coagulation phenomena of chitosan-arginine polymer, fucoidan and polyglutamic acid may occur.

The chitosan-arginine polymer used in the production of the nanocapsules is prepared by dissolving arginine in an amount of preferably 1 to 200 parts by weight, more preferably 10 to 100 parts by weight based on 100 parts by weight of chitosan, Preferably 1 to 200 parts by weight, more preferably 10 to 100 parts by weight, of N-hydroxysuccinimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) ( N- hydroxy-succinimide) in an amount of 1 to 400 parts by weight, more preferably 100 to 200 parts by weight. When chitosan, arginine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or N-hydroxy-succinimide is used in a condition out of the above range, the preparation of chitosan- It is not possible to produce a chitosan-arginine polymer having a good inhibitory effect.

In the preparation of the chitosan-arginine polymer, TEMED / HCl buffer (N, N, N ', N'-tetramethylethylenediamine / HCl buffer) of 1 to 200 mM (pH 5.5 to 7.0) Is used in an amount of 1000 to 100000 parts by weight based on 100 parts by weight of chitosan.

In the method for producing a nanocapsule, the concentration of the chitosan-arginine polymer in one step may be preferably 0.5 to 10 mg / ml, more preferably 1 to 5 mg / ml, and most preferably 1 to 2 Mg / ml. In addition, the concentration of the red ginseng extract in the two steps may be preferably 0.1 to 20 mg / ml, more preferably 1 to 15 mg / ml, and most preferably 1 to 10 mg / ml. The concentration of the conjugated linoleic acid may be 0.01 to 10 mg / ml, more preferably 0.01 to 1 mg / ml, and most preferably 0.06 to 0.12 mg / ml. The concentration of gamma linolenic acid may be preferably 1 to 50 mg / ml, more preferably 5 to 50 mg / ml, and most preferably 10 to 50 mg / ml. The concentration of the fucoidan aqueous solution may be preferably 0.6 to 2 mg / ml, more preferably 0.6 to 1 mg / ml. The fucoidan aqueous solution may be supplemented with polyglutamic acid at a concentration of 0.001 to 1 mg / ml, preferably 0.001 to 0.1 mg / ml. When the concentration is less than the minimum concentration of the red ginseng extract, the conjugated linoleic acid, the gamma linolenic acid, the chitosan-arginine derivative, the fucoidan, or the polyglutamic acid, or when the concentration exceeds the maximum concentration, the nanocapsulation may not be performed well, Or even when nanocapsules are produced, aggregation may occur during storage.

The red ginseng extract, fucoidan, polyglutamic acid and chitosan-arginine polymer are used in the preparation of nanocapsules in aqueous solution. Preferably, the conjugated linoleic acid and gamma linolenic acid are dissolved in ethanol or an aqueous ethanol solution and mixed with the red ginseng extract, and the aqueous ethanol solution is preferably an aqueous 30 to 99% (v / v) ethanol solution.

In the method for producing a nanocapsule, a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is mixed with the chitosan-arginine polymer solution at 100 to 10000 rpm, preferably 300 to 5000 rpm, more preferably 500 to 2000 rpm, They can be prepared as a mixed solution.

The nanocapsules exhibit a collection efficiency of 30 to 50% (w / w) of Rg1 and 15 to 40% (w / w) of Rb1 for the extract of red ginseng, which is classified as' red ginseng extract, conjugated linoleic acid and gamma linolenic acid Quot; mixture " of < / RTI >

The nanocapsule may have a particle size of preferably 250 to 500 nm, the particle dispersion degree is 0.1 to 0.4, more preferably 0.2 to 0.4, the scattering intensity is preferably 100,000 to 500,000, more preferably 200,000 to 500,000, 300,000. ≪ / RTI > At this time, the nanocapsule may have a particle size of 250 to 500 nm, but when it is less than 250 nm, it is difficult to produce nanocapsules. When the particle diameter exceeds 500 nm, the nanocapsule can be produced up to 1000 nm. However, since the particle dispersion degree exceeds 0.4 and the particle formation is uneven, the nanocapsulation of the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is not performed well, It may be difficult to maintain a stable state when the nanocapsule is stored, so that each component may merely aggregate. Therefore, it is most preferable that the size of the nanocapsules does not exceed 500 nm.

In the method for preparing the nanocapsules, the solution in which the fucoidan aqueous solution or the solution containing polyglutamic acid is added to the mixed solution in the step 3 may be mixed and the mixing speed may be preferably 0.1 to 10 ml / min , More preferably 0.3 to 5 ml / min, and most preferably 0.5 to 2 ml / min.

The mixing volume of the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid: chitosan-arginine polymer solution: fucoidan solution in the two steps and three steps in the method of producing the nanocapsules is 1: 0.01 to 5: 0.1 to 5, 1: 0.5 to 2: 0.5 to 3, more preferably 1: 1 to 2: 1 to 2.5, and most preferably 1: 1 to 1.5: 1 to 2.

The nanocapsule may be used as a pharmaceutical composition for preventing or treating vascular diseases, or as a health functional food for preventing or improving vascular diseases. The health functional food may be various foods, beverages, food additives, and the like.

The composition containing the nanocapsules may be contained in an amount of 0.001 to 100% by weight based on the total amount of the pharmaceutical composition or the health functional food product.

The vascular disease may be a disease selected from the group consisting of arteriosclerosis, hypertension, vascular restenosis, peripheral vascular occlusion, cerebral infarction, cerebral hemorrhage and stroke.

The pharmaceutical composition containing the nanocapsule may be administered orally in the form of a solid preparation, and may be in the form of tablets, pills, powders, granules, capsules, liquid preparations such as suspensions, Syrups, and the like. The nanocapsules can be used directly in the form of a dispersion prepared, and in particular, can be prepared as a soft capsule, or a hard capsule. Or the liquid phase of the dispersion may be dried to prepare a powder or a solid preparation containing the nanocapsules. Alternatively, the nanocapsules may be separated from the dispersion liquid to remove the nanocapsules separately or may be directly used or pulverized.

The pharmaceutical compositions containing the nanocapsules may comprise conventional pharmaceutical excipients, carriers and diluents. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

The dosage of the pharmaceutical composition containing the nanocapsule may vary depending on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease of the patient, It may be administered once or several times a day at a predetermined time interval. For example, the daily dose based on the active ingredient content may be 0.001 to 500 mg / kg, preferably 0.1 to 200 mg / kg, based on the dry weight of the red ginseng extract.

The present invention also provides a health functional food comprising the nanocapsule and a pharmaceutically acceptable food-aid additive. The nanocapsule may be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention may be prepared in the form of a liquid preparation such as a suspension, a solution, an emulsion, a syrup, a capsule or the like, and in particular, a soft capsule or a hard capsule. The health functional foods of the present invention include, for example, various foods, beverages, and vitamin complexes.

The present invention relates to a nanocapsule characterized in that a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is coated with a coating of a mixed coating of chitosan-arginine polymer and fucoidan and a method for producing the same. The nanocapsule can be effectively used as a composition for preventing or treating vascular diseases because it can enhance the blood circulation improving effect by transferring the unsaturated fatty acid such as red ginseng extract, conjugated linoleic acid and gamma linolenic acid in a stable state in the gastric environment. Meanwhile, the present invention is prepared by supplementing a nanocapsule preparation containing red ginseng extract of Korean Patent Application No. 2013-0062926 (red ginseng nanocapsule coated with a coating of red ginseng extract with chitosan, fucoidan and polyglutamic acid) Compared with Patent Application No. 2013-0062926, nanoparticles of uniform size can be stably prepared by using chitosan-arginine polymer, and addition of unsaturated fatty acids in addition to red ginseng extract can enhance blood circulation improving effect.

Fig. 1 shows the results of measurement of the structure of the chitosan-arginine polymer by FTIR spectroscopy.
Fig. 2 shows the effect of inhibiting platelet aggregation of conjugated linoleic acid (CLA) and gamma linolenic acid (GLA).
FIG. 3A shows the acidity of the nanocapsule dispersions (Fu600, Fu800 and Fu1000) according to the concentration of fucoidan measured by the TBARS method, and FIG. 3B shows the degree of acidity of the nanocapsule dispersion of the present invention according to the concentration of polyglutamic acid measured by the TBARS method (P1, P10 and P100).

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Preparation of red ginseng extract >

Example 1-1. Preparation of Rg1-fortified red ginseng extract by FFE concentration method

For the production of red ginseng extract, red ginseng (4 year old, 2010) was used. To 10 kg of the red ginseng shell, 100 L of distilled water 10 times was added and repeatedly extracted 4 times at 85 캜 for 8 hours. The extracts were frozen at 20 ° C for 12 hours and then centrifuged at 7,600 rpm for 30 minutes. Thereafter, the supernatant was taken and concentrated at a steam temperature of 90 ° C with a falling film evaporator (hereinafter referred to as FFE) to have a solid content of 65% by weight. The FFE concentration method is a method of evaporating the concentrated liquid from the upper part of the heating tube to the thin film state and circulating type and non-recirculating type. Since the heat transfer rate is high and the retention time is short, the heat sensitive solution, fruit juice, milk, It is suitable for the evaporative concentration of pharmaceutical compositions.

Examples 1-2. General red ginseng extract manufacturing

Red ginseng extract was prepared in the same manner as in Example 1-1 except that the red ginseng extract was centrifuged, and the supernatant was taken. The supernatant was taken and concentrated without concentrating the FFE. The concentrate was placed in a normal concentrator and concentrated under reduced pressure at a temperature of 60 ° C and a vacuum of 600-700 mmHg To give a solid content of 65% by weight.

Examples 1-3. Production of ginseng extract using FFE concentration method

Ginseng extract was prepared using the ginseng instead of red ginseng according to the method of Example 1-1.

≪ Example 2: Analysis of components of red ginseng extract >

Example 2-1. Identify general characteristics

3 g of the red ginseng extract of Examples 1-1 and 1-2 was dried in a dry oven at an internal temperature of 105 ° C and then calculated as the amount of the residue of evaporation. The average value was calculated.

The organic acidity of the red ginseng extracts of Examples 1-1 and 1-2 was calculated by adding 100 ml of distilled water boiled and boiled for 1 g of the sample, 0.5 ml of the phenolphthalein indicator, and then 0.1 N NaOH consumption according to the method of food revolution. Each analytical value was calculated by the average value obtained by performing three repeated experiments.

The pH of the red ginseng extract of Examples 1-1 and 1-2 was measured by taking a sample of 10 ml at room temperature using a pH meter (Orion 3 Star pH Benchtop, Singapore) repeatedly three times and calculating the average value Respectively.

The water-insoluble components of the red ginseng extracts of Examples 1-1 and 1-2 were measured by the AOAC method (AOAC 2000). To this end, 1 g of each red ginseng extract was dissolved in distilled water (15 ml), filtered through a glass filter, washed with distilled water until no chlorine was removed, and dried at 105 ° C for 3 hours to obtain a constant weight. Respectively.

To analyze the crude saponin of the red ginseng extract of Examples 1-1 and 1-2, 100 ml of methanol was added to 10 g of the sample, stirred for 1 hour, mixed with the filtrate using methanol, and concentrated under reduced pressure. Then, 50 ml of distilled water and 50 to 70 ml of water-saturated butanol were added to the concentrated residue, and this procedure was repeated three times. Then, the butanol layer was concentrated under reduced pressure, ether (50 ml) was added, and the mixture was degreased by heating in a reflux condenser (35 ° C) for 30 minutes. After removing the ether, the residue was dried at 105 ° C for 1 hour and then cooled in a desiccator and weighed. Each analytical value was calculated as the mean value obtained by repeating 3 times.

The results are shown in Table 1. Referring to the results shown in Table 1, the Rg1-enriched red ginseng extract of Example 1-1, which was concentrated by a falling film evaporator (FFE) method, The results of the insoluble sediment measurements showed no significant difference. In particular, insoluble sediments are known to have an impact on quality when they occur in excess. Therefore, it is considered that the FFE method used in this experiment does not directly affect the general quality characteristics of red ginseng extract.

On the other hand, when the crude saponin content of the red ginseng extracts of Examples 1-1 and 1-2 was analyzed, it was found that the red ginseng extract of Example 1-1, which was concentrated by the FFE method, The crude saponin content was increased by 5 mg / g compared to the extract. In the case of ginsenoside Rg1, which was decomposed in the extraction and general decompression, the decomposition was suppressed by the ultra high pressure decomposition method using the FFE extraction method, which resulted in the increase of the crude saponin content.

Analysis target Example 1-1
FFE red ginseng extract Examples 1-2
Common red ginseng extract Solid content (% by weight) 65.30 ± 0.10 65.40 + - 0.10 Organic acid acidity (% by weight) 0.12 ± 0.05 0.10 ± 0.05 pH 4.90 + - 0.10 4.80 + - 0.10 Water insoluble component (% by weight) 0.80 ± 0.00 0.80 + - 0.10 Crude saponin content (mg / g)  85 ± 7 80 ± 5

Example  2-2. HPLC  analysis

For the ginsenoside analysis of the red ginseng extracts or ginseng extracts of Examples 1-1 to 1-3, a Thermo Sycronis C18 column (4.6 × 250, 5 μm) was used with Agilent HPLC, the injection amount was 20 μl, The flow rate was 1.6 ml / min, the column temperature was 35 ° C, and the ultraviolet absorption wavelength was 203 nm. Solvent A (water) and solvent B (acetonitrile) were degassed using an ultrasonic washing machine and analyzed using a gradient system (Table 2). Meanwhile, standard products of Ginsenosides Rg1 and Rb1 were purchased from Embo Lab (Daejeon, Korea). Acetonitrile (Avantor Central Valley, PA, USA) and water (Water, JT Baker, USA) were used for HPLC.

Instrument Column Thermo Sycronis C ₁₈ column (4.6 x 250, 5 [mu] m) Column Temp. 35 ℃ Pump Agilent 1260 Quat pump Detector Agilent 1260 VWD Mobile phase A: Water B: Acetonitrile Gradient Time: 0/5/13/85/90/95/98/100
A (%): 80/80/75/55/10/55/80/80 Flow rate 1.6 ml / min Injection volume 20 쨉 l

The ginsenoside content of each red ginseng extract was quantitatively analyzed based on the ginsenoside standard, and the results are shown in Table 3 below.

Gin Senocide Example 1-1
FFE red ginseng extract Examples 1-2
Common red ginseng extract Examples 1-3
FFE Ginseng Extract Rg1 (mg / g) 1.89 0.47 0.37 Rb1 (mg / g) 6.19 3.29 2.00

As shown in Table 3, it can be seen that the content of Rg1 and Rb1 in the red ginseng extract of Example 1-1 prepared by the FFE method was significantly increased compared to the general red ginseng extract of Example 1-2. It is known that ginsenoside Rg1 is decomposed in extraction and general decompression process. In the case of using ultra high pressure decompression method using FFE process, Rg1 decomposition is performed in both hot water and ethanol extracts with minimization of concentration time. And the content was increased by inhibition. In order to compare the effect with the ginseng extract, the ginseng extract using the FFE method of Example 1-3 was analyzed, but it was also confirmed that the ginsenoside content of Example 1-1 was significantly higher than that of Example 1-1. On the other hand, when red ginseng was extracted with a 70% (v / v) ethanol aqueous solution and then subjected to reduced pressure concentration using the FFE method, a 70% (v / v) ethanol aqueous solution extract (Rg1: 1.05 (Rg1: 6.25 mg / g, Rb1: 10.82 mg / g) compared to that of Rb1 (㎎ / g, Rb1: 8.25 mg / g). At this time, the aqueous extract of 70% (v / v) ethanol was extracted with a 70% (v / v) ethanol aqueous solution at 40 ° C instead of extracting at 85 ° C using water in the method of Example 1-1 Ethanol was distilled off by a conventional method.

Meanwhile, all the following nanocapsules were prepared using the red ginseng extract of Example 1-1.

≪ Example 3: Preparation of chitosan-arginine polymer &

Chitosan-arginine polymers were prepared by modifying the method reported by Liu et al. (2004). Water-soluble chitosan (MW 1,000 ~ 3,000) was purchased from Kittolife Co. (Seoul, Korea), and arginine was purchased from Sigma aldrich.

1 g of chitosan and 0.3 g of arginine were dissolved in TEMED / HCl buffer (N, N, N ', N'-tetramethylethylenediamine / HCl buffer, 10 mM, pH 6.0) to make 100 ml. To a solution of chitosan and arginine dissolved were added 0.6 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 1 g of N-hydroxy- 1.4 g of N-hydroxy-succinimide (NHS) was added and stirred at room temperature for 24 hours to obtain a reaction product. The resulting reaction product was dialyzed for 5 days (Spectra / Por ^® 1, MWCO 6,000 ~ 8,000), and the obtained dialyzate was lyophilized to obtain a chitosan-arginine polymer shown in the following Scheme 1.

[Reaction Scheme 1]

The structure of the chitosan-arginine polymer was confirmed by FTIR (fourier transform infrared spectroscopy, Nicolet FT-IR spectrometer, MAGNA-IR 760 ESP, Nicolet Instrument Corp., Madison, Wis., USA) Respectively.

1, the FTIR spectrum analysis results show that, as in the results of Liu et al. (2004), a new FTIR spectrum of chitosan and arginine in the range of 1,500 to 1,600 cm ^-1 (specifically, 1520 cm ^-1 ) It can be confirmed that a peak is formed. It has been reported that the new peak is due to the amide bond formed by combining the amine group of chitosan and the carboxyl group of arginine. Also, it was confirmed through arginine quantitative analysis that arginine of 241.1 ± 10.86 μmol / mg was contained in the chitosan-arginine polymer of the present invention. This is similar to the study of Zhu et al. (2002) which reported that arginine contained about 200 μmol / mg of chitosan-arginine polymer. The arginine quantitative analysis was carried out using the Sakaguchi reaction (arginine detection reaction) of Wang et al. (2008). Specifically, 5 ml of chitosan-arginine solution, 1 ml of 8-hydroxyquinoline solution and 1 ml of 2 mol / l sodium hydroxide solution were mixed and left on ice for 10 minutes. Then 0.5 ml of cold sodium hypobromite was added, and the mixture was vigorously shaken for 30 seconds. Then, 1 ml of a 40% (w / v) urea solution was added thereto, and the absorbance was measured by measuring the absorbance at a wavelength of 500 nm.

<Example 4 Confirmation of Platelet Aggregation Inhibitory Effect of Chitosan, Arginine, Chitosan-Arginine Polymer, Fucoidan and Polyglutamic Acid>

The platelet aggregation effect of each coating material was measured in order to confirm the blood circulation improving activity of the materials used as coating materials for nanocapsules. Each coating material was dissolved in water and used.

The polyglutamic acid (MW 50 kDa) and fucoidan (extracted from Laminaria Japonica ) were purchased from Bioleaders Corp. (Daejeon, Korea) and Haewon biotech (Seoul, Korea).

The platelet aggregation inhibition effect was measured by the degree of change in the light transmission of the dispersion solution caused when platelets aggregated using an aggregometer (Chronolog Co.). First, blood was collected from the central artery of the rabbit using an injection needle. Male rabbits weighing 2 to 3 kg were bred in an animal laboratory and blood was collected once every two weeks without exceeding the recommended maximum volume of blood collected, which was 10% of the circulating blood volume. The anticoagulants were ACD anticoagulant citrate dextrose) was used. The blood was centrifuged to separate the platelet rich plasma (PRP) and blood cells, and the PRP was again centrifuged and separated into platelets and platelet poor plasma (PPP). The precipitated platelets were washed with HEPES buffer and then dispersed in HEPES buffer and adjusted to a concentration of calcium chloride (CaCl ₂ ) of 200 mM to prepare a platelet dispersion for platelet aggregation inhibition experiment. Thereafter, the platelet dispersion was adjusted to a platelet concentration of 3 x ^{10 8} cells / ml using a hemocyte counter, placed in a cuvette containing a magnetic bar, heated to 37 ° C, and a sample to be measured was added while rotating the magnetic bar. The collagen (collagen, 5 μg / ml) which causes aggregation of platelets was added to the treated platelet dispersion to measure the turbidity change according to the degree of platelet aggregation as compared with the buffer used as a control. Respectively. At this time, 290 占 퐇 of platelet dispersion, 10 占 퐇 of sample, and 1.5 占 퐇 of collagen were added. On the other hand, through previous experiments, the water-soluble chitosan solution was not tested by itself as it was confirmed that the platelet aggregation was further increased as the concentration increased from 1 to 8 mg / ml. In addition, the simple mixing of chitosan and arginine showed similar effects. Arginine inhibited platelet aggregation at low concentrations and inhibited platelet aggregation by 5 ~ 10% even at high concentrations.

Condition Platelet Aggregation Inhibitory Effect (%) 1 mg / ml chitosan-arginine polymer 12.0 Chitosan-arginine polymer 1.5 mg / ml 25.6 Chitosan-arginine polymer 2 mg / ml 31.9 Chitosan-arginine polymer 4 mg / ml 39.4 Chitosan-arginine polymer 10 mg / ml 40.5 Fucoidan 600 占 퐂 / ml <5.0 Fucoidan 2 mg / ml <5.0 Fucoidan 4 mg / ml <5.0 Fucoidan 10 mg / ml 5.0 1 μg / ml of polyglutamic acid <5.0 2 mg / ml of polyglutamic acid <5.0 4 mg / ml of polyglutamic acid <5.0 10 mg / ml of polyglutamic acid 5.0

As shown in Table 4, the platelet aggregation inhibitory effect of the chitosan-arginine polymer was increased as the chitosan-arginine polymer concentration was increased, unlike the aqueous chitosan solution alone. As a result, it was shown that chitosan platelet aggregation induction was abolished when arginine was polymerized with chitosan, and the effect of inhibiting platelet aggregation was increased. Thus, it can be confirmed that the chitosan-arginine polymer can be used as an effective coating material for nano-carriers for improving blood circulation. Fucoidan and polyglutamic acid showed very low platelet aggregation inhibitory effect within 5%.

On the other hand, when the platelet aggregation inhibitory effect of fucoidan was assayed by using the platelet dispersion in the above platelet aggregation experiment, platelet aggregation by fucoidan appeared before the addition of collagen causing platelet aggregation during the experiment. This phenomenon is presumed to be caused by aggregation of platelets due to binding of Ca ²⁺ ions liberated in the process of adding calcium chloride into the platelet dispersion and negatively charged fucoidan. To solve this problem, fucoidan was measured using PRP (platelet rich plasma) instead of a washed platelet suspension.

Example 5. Confirmation of Platelet Aggregation Inhibitory Effect and Mixing Condition of Red Ginseng Extract, Conjugated Linoleic Acid and Gamma Linolenic Acid, respectively.

In order to confirm the blood circulation improving activity of the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid, the platelet aggregation effect of 0 to 10 mg / ml of conjugated linoleic acid and 0 to 80 mg / ml of gamma linolenic acid was first measured using the method described in Example 4 And the results are shown in Fig. A 50% (v / v) aqueous ethanol solution was used as a solvent for the conjugated linoleic acid and gamma linolenic acid.

2, it can be seen that both the conjugated linoleic acid and gamma linolenic acid increase the effect of inhibiting platelet aggregation in a concentration-dependent manner.

Next, the inhibitory effect of platelet aggregation on the concentration of red ginseng extract (solvent: water) was ascertained by the same method, which is shown in Table 5 below.

Composition Platelet Aggregation Inhibitory Effect (%) Red ginseng extract 1 mg / ml 15.3 Red ginseng extract 10 mg / ml 25.2 Red ginseng extract 12 mg / ml 26.5 Red ginseng extract 15 mg / ml 30.5

Referring to Table 5, it is confirmed that red ginseng extract has an inhibitory effect on platelet aggregation at a concentration of 1 to 15 mg / ml.

On the other hand, since nano-encapsulation of nano-capsules was observed when nano-capsules were mixed with red ginseng extract at concentrations of conjugated linoleic acid exceeding 10 mg / ml and gamma linolenic acid exceeding 50 mg / ml through previous experiments, The concentrations for these concentrations were chosen to be lower.

Referring to the results of FIG. 2 and Table 5, the concentrations of the red ginseng extract, conjugated linoleic acid and gamma linolenic acid for application to the nanocapsules were determined as follows.

The concentration of red ginseng extract was fixed at 10mg / ㎖, which is a concentration of 25.2% inhibiting platelet aggregation. Gamma linolenic acid was administered at a dose of 0.12 mg / ml: 0 mg / ml, 0.06 mg / ml: 20 mg / ml, in order to inhibit platelet aggregation by conjugated linoleic acid and gamma linolenic acid, (20%: 0%, 10%: 10%, 0%: 20%).

Thereafter, a mixture of 'red ginseng extract and conjugated linoleic acid', 'red ginseng extract and gamma linolenic acid', 'red ginseng extract, conjugated linoleic acid and gamma linolenic acid' was prepared by mixing 10 mg / ml of red ginseng extract and the conjugated linoleic acid: gamma linolenic acid , And the inhibitory effect on platelet aggregation was measured using the same. At this time, the red ginseng extract in aqueous solution and the conjugated linolenic acid and gamma linolenic acid in the state of aqueous solution of 50% (v / v) ethanol were mixed, and the solvent state of the mixture shown in Table 6 below was 10% (v / v) In subsequent experiments, the solvents of these mixtures remained the same.

Composition Platelet Aggregation Inhibitory Effect (%) Red ginseng extract 10 mg / ml + conjugated linoleic acid 0.12 mg / ml 49.3 Red ginseng extract 10 mg / ml + gamma linolenic acid 80 mg / ml 52.1 Red ginseng extract 10 mg / ml + conjugated linoleic acid 0.06 mg / ml
+ Gamma linolenic acid 20 mg / ml 80.2

As shown in Table 6, in the case of the mixture of 'red ginseng extract and conjugated linoleic acid', 'red ginseng extract and gamma linolenic acid', the inhibitory effect of 25% platelet aggregation of red ginseng extract and the inhibitory effect of platelet aggregation of 20% (45%), but the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid showed a synergistic effect, resulting in a total platelet aggregation effect of 80.2%. On the other hand, when ginseng extract was used instead of ginseng extract, the effect of inhibiting platelet aggregation was confirmed. The use of ginseng extract was significantly lower than that of using red ginseng extract. When mixed with conjugated linoleic acid and gamma linolenic acid, There was no synergistic effect when using extracts. The inhibitory effect of platelet aggregation on the platelet aggregation inhibitory effect is an intrinsic effect due to the mixing of red ginseng extract with conjugated linoleic acid and gamma linolenic acid. Especially, the effect of using ginsenosides in combination with conjugated linoleic acid and gamma linolenic acid in red ginseng extract can do.

From the above results, it was confirmed that a mixture of 'red ginseng extract, conjugated linoleic acid and gamma linolenic acid' (hereinafter, referred to as &quot; ginseng linolenic acid &quot;) mixed with 10 mg / ml of red ginseng extract, 0.06 mg / ml of conjugated linoleic acid and 20 mg / Core "in the tables and drawings).

&Lt; Example 6: Preparation of nanocapsules >

The nanocapsules were prepared by ionic gelation. (F) or polyglutamic acid (P) alone or in combination as chitosan-arginine polymer as a basic coating material, the nanocapsules of 'red ginseng extract-conjugated linoleic acid-gamma linolenic acid / chitosan-arginine / fucoidan' - conjugated linoleic acid-gamma linolenic acid / chitosan-arginine / fucoidan / polyglutamic acid '.

First, red ginseng-conjugated linoleic acid-gamma linolenic acid / chitosan-arginine / fucoidan (Core / CS-arg / Fu) nanocapsules were prepared. To this end, the chitosan-arginine polymer prepared in Example 3 was dissolved in distilled water to prepare a 2 mg / ml chitosan-arginine polymer solution. 1.5 ml of the above 2 mg / ml chitosan-arginine polymer solution was stirred at 1,000 rpm (WiseStir MS-MP8, Wisd Laboratory Instruments, Wertheim, Germany), and the amount of 'red ginseng extract, conjugated linoleic acid and gamma linolenic acid' (Core / CS-arg) mixture of red ginseng extract-conjugated linoleic acid-gamma linolenic acid / chitosan-arginine was prepared by slowly mixing 1 ml of the mixture (10 mg / ml of red ginseng + 0.06 mg / ml of conjugated linoleic acid + 20 mg / . 2 ml of a fucoidan aqueous solution (600 to 1000 μg / ml) was dropped into the mixed solution at a rate of 1.0 min / ml using a peristaltic pump (Master flex 77200-60, Cole Paramer Inc., Vernon Hills, IL, USA) After that, the mixture was stirred at 1,000 rpm for 10 minutes to prepare red ginseng extract-conjugated linoleic acid-gamma linolenic acid / chitosan-arginine / fucoidan (Fu600, Fu700, Fu800, Fu900 and Fu1000) nanocapsules. On the other hand, when the concentration of the fucoidan aqueous solution was not more than 500 / / ml, the nanocapsules could not be prepared and the coagulation phenomenon of the coating materials could not be produced. Meanwhile, it was confirmed that the preparation conditions of the nanocapsules vary depending on the concentration of each solution rather than the volume difference.

The same procedure was followed except that a mixed aqueous solution of fucoidan (600 μg / ml) and polyglutamic acid (1 to 1000 μg / ml) was used instead of the fucoidan aqueous solution to prepare red ginseng extract-conjugated linoleic acid-gamma linolenic acid / chitosan- Glutamic acid (P1, P10, P100, P500 and P1000) nanocapsules were prepared.

The conditions of the nanocapsules prepared under the above conditions are shown in Table 7 below.

Nano Capsule
designation Red ginseng
extract
density
(Mg / ml) Conjugate
Linoleic acid
density
(占 퐂 / ml) gamma
Linolenic acid
density
(Mg / ml) The chitosan-
Arginine
Polymer concentration
(Mg / ml) Fucoidan
density
(占 퐂 / ml) Poly
Glutamic acid
density
(占 퐂 / ml) Fu600 10 60 20 2 600 0 Fu700 10 60 20 2 700 0 Fu800 10 60 20 2 800 0 Fu900 10 60 20 2 900 0 Fu1000 10 60 20 2 1,000 0 P1 10 60 20 2 600 One P10 10 60 20 2 600 10 P100 10 60 20 2 600 100 P500 10 60 20 2 600 500 P1000 10 60 20 2 600 1,000

&Lt; Example 7: Physical properties of nanocapsules >

1 ml of the nanocapsules in the dispersion state prepared in Example 6 was placed in a disposable cuvette and the size and dispersion of the nanocapsules in the dispersion were measured using a Marvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK) The polydispersity index (PDI) and the derived count rate were measured. The measurement conditions were performed in multiple narrow modes where multiple peaks could be displayed simultaneously, and the size of the nanocapsules was compared and analyzed at 25 ± 1 ° C through an intensity distribution. All the samples were repeatedly measured at least three times, and the results are shown in Table 8.

Conditions (Nano Capsule Name) Size (nm) Dispersion degree Scattering intensity (kcps) Fu600 460 ± 4.4 0.38 ± 0.01 202,477 ± 73,033 Fu700  410 ± 7.0 0.29 ± 0.01 244,932 ± 33,669 Fu800  416 ± 2.6 0.33 + 0.03 215,023 ± 13,519 Fu900  433 ± 33.6 0.35 + 0.04 214,334 ± 19,685 Fu1000  425 ± 34.2 0.36 + 0.04 220,729 ± 32,318 P1  412 ± 7.0 0.35 + 0.02 229, 410 ± 2,293 P10  414 ± 5.7 0.33 + 0.04 217,416 ± 6,065 P100  444 ± 13.7 0.35 + 0.02 231,943 ± 17,982 P500  613 ± 30.8 0.42 ± 0.01 236,197 ± 15,711 P1000 1,718 ± 248.8 0.90 + 0.07 256,350 ± 59,217

As shown in Table 8, in the nanocapsules having a concentration of fucoidan of 600 to 1,000 μg / ml (Fu600, Fu700, Fu800, Fu900 and Fu1000), there was no significant change in the particle characteristics of the nanocapsules with increasing fucoidan concentration , A nanocapsule size of 410 to 460 nm, and a dispersion degree of 0.4 or less. It is known that the scattering intensity increases as the size or number of nanocapsules increase, as the scattering intensity is the amount of scattering intensity from the particles Bergeron et al., 2010). Therefore, when the concentration of fucoidan is 700 μg / ㎖, the nanocapsule has the smallest size of 410 nm, the dispersion degree is 0.29, and the scattering strength is 244,932 kcps. Respectively.

On the other hand, when the concentration of polyglutamic acid was increased to 1 to 1,000 占 퐂 / ml (P1, P10, P100, P500 and P1000) after fixing the fucoidan concentration to 600 占 퐂 / Was significantly increased at the concentration of 1,000 ㎍ / ㎖, but the scattering intensity was not significantly different. This is because as the concentration of polyglutamic acid as a coating material increases, the amount of polyglutamic acid forming ionic gelation reaction with the chitosan-arginine polymer in the production of nanocapsules is also increased to increase the particle size, and as the particle size increases, It is considered to be a general phenomenon that the dispersion degree is increased. However, it can be inferred that the number of nanocapsules produced is relatively reduced because the intensity of scattered light does not change in spite of the formation of large particles. Therefore, it was judged that nanocapsules having a uniform size were effectively produced when the concentration of polyglutamic acid was in the range of 1 to 100 占 퐂 / ml, which is a condition that the capsule size is 500 nm or less and the dispersion degree is 0.4 or less.

The nanocapsules of the present invention prepared under the above conditions are found to have a constant size and dispersity in comparison with the nanocapsules containing red ginseng extract of Korean Patent Application No. 2013-0062926 which is a prior art of the present inventor. Therefore, the nanocapsules are not agglomerated, and the nanocapsules are kept in a stable state. It is expected that unlike in the above-mentioned Patent Application No. 2013-0062926 where chitosan is used as a coating of nanocapsules, the chitosan-arginine polymer is used in the present invention to eliminate the instability of chitosan. Particularly, since the size of the nanocapsules is relatively small, the nanocapsules can be stably retained even in a long-term storage state (data not shown).

&Lt; Example 8: Capture efficiency of nanocapsules >

To measure the collection efficiency of red ginseng extract, conjugated linoleic acid and gamma linolenic acid in the nanocapsules, the nanocapsules in the dispersion state of Example 6 were centrifuged (4 ° C, 15,000 × g, Optima TL Ultracentrifuge, Beckman, CA, USA). After that, the ginsenosides Rg ₁ and Rb ₁ , which are the index components of the red ginseng component contained in the supernatant, were checked by HPLC for the collection efficiency. The HPLC analysis conditions were the same as those in Table 2. The entrapment efficiency (EE) was calculated according to the following equation, and the results are shown in Table 9 (Kuo, 2005, Jang & Lee, 2008). At this time, the names of the nanocapsules were the same as those in Table 7, and the following nanocapsules were used as they are in Table 7 (NPs: nanocapsules).

Condition Ginsenoside Rg ₁ Collection efficiency (%) Ginsenoside Rb ₁ Collection efficiency (%) Fu600 39.3 18.2 Fu800 42.8 37.3 Fu1000 46.7 48.2 P1 36.0 32.3 P10 35.4 33.2 P100 42.3 47.3

As shown in Table 9, the collection efficiency for ginsenosides Rg ₁ and Rb ₁ tended to increase slightly as the concentration of fucoidan and polyglutamic acid used in the preparation of nanocapsules increased, Higher collecting efficiency was increased to about 50%.

<Example 9> Release characteristics of nanocapsules under gastric-like conditions>

In order to confirm that the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid captured in the nanocapsules was released well in the body, the nanocapsule condition of the dispersed state immediately after preparation was adjusted by simulated gastric fluid (SGF) Among the substances contained in the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid, ginsenosides Rg ₁ and Rb ₁ were determined as standard substances and their release amounts were confirmed. First, to adjust the environment of the nanocapsule of Example 6 by gastric-like conditions, the nanocapsules in the dispersion state were adjusted to pH 2.0 using 1M HCl and allowed to stand for 2 hours. Then, the nanocapsules were centrifuged for 30 minutes The amounts of ginsenosides Rg ₁ and Rb ₁ contained in the supernatant were analyzed according to HPLC conditions in Table 2 and calculated according to the following formula These are shown in Table 10 (NPs: nanocapsules).

Condition Ginsenoside Rg ₁ Emission rate (%) Ginsenoside Rb ₁ Emission rate (%) Fu600 90.2 60.3 Fu800 82.8 72.1 Fu1000 84.2 73.9 P1 93.2 63.6 P10 82.0 60.5 P100 83.8 65.3

Referring to Table 10, it can be seen that at least 60% of the nanocapsules (Fu600, Fu800, Fu1000, P1, P10 and P100) are released under gastric conditions, and the active material is released well from the nanocapsules.

&Lt; Example 10: Oxidation reaction confirmation >

In order to measure the antioxidant activity of the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid in the nanocapsules, the inhibitory effect of the non-encapsulated red ginseng extract, the conjugated linoleic acid and gamma linolenic acid mixture on the oxidation inhibition of the nanocapsules prepared in Example 6 The thiobarbituric acid reactive substances (TBARS) method, which was modified by Kikuzaki (1993), was used for measurement.

To this end, a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid (10 mg / ml of red ginseng extract, 0.06 mg / ml of conjugated linoleic acid, 20 mg / ml of gamma linolenic acid) 0.6 ml of 20% TCA (trichloroacetic acid) solution and 0.6 ml of 0.67% TBA (thiobarbituric acid) solution were added to each 0.3 ml of the dispersion (Fu600, Fu700, Fu800, Fu900, Fu1000, P1, P10, P100, P500 and P1000) The mixture was heated at 100 ° C. for 10 minutes, cooled to room temperature, and centrifuged at 1,500 × g for 20 minutes to obtain a supernatant. The absorbance of the supernatant was measured at 532 nm (Kikuzaki & Nakatani, 1993) Respectively.

3, it was found that the degree of oxidation by TBARS was accelerated in the case of non-nano-encapsulated red ginseng extract, conjugated linoleic acid and gamma linolenic acid mixture (core) than in the control (control, 10% [v / v] ethanol aqueous solution) . This is because the unsaturated fatty acids conjugated linoleic acid and gamma linolenic acid are vulnerable to oxidation due to the double bonds present in the molecular structure. On the other hand, it is confirmed that the nanocapsules (Fu600, Fu800, Fu1000, P1, P10 and P100) have oxidation inhibition as compared to the non-encapsulated mixture. Oxidation of non - encapsulated red ginseng extract, conjugated linoleic acid and gamma linolenic acid mixture (Core) is accelerated as the oxidation time elapses, but the rate of oxidation is decreased in the case of nanocapsulized. This suggests that the rate and extent of oxidation of the red ginseng extract, conjugated linoleic acid and gamma - linolenic acid mixture are protected from the oxidative environment by nano - encapsulation. Although not shown in FIG. 3, a mixed solution (Core / Cs-Arg) in which only a chitosan-arginine polymer is mixed with a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is similar to the red ginseng extract, conjugated linoleic acid and gamma linolenic acid mixture It was confirmed that oxidation occurred. It is expected that this phenomenon is caused by the lack of nanocapsulation.

Example 11 Confirmation of Platelet Aggregation Inhibitory Effect of Nanocapsules < RTI ID = 0.0 >

The nanocapsules in the dispersion state of Example 6 immediately after preparation and the nanocapsules in the dispersion state were each diluted 4 times in water (including Core, Core / CS-Arg), and the diluted material and the diluted material were dispersed for 2 hours The platelet aggregation inhibitory effect was measured (using the method of Example 4) against the simulated gastric fluid (SGF) at pH 2.0. The results are shown in Table 11.

Condition Immediately after the preparation,
Platelet Aggregation Inhibitory Effect (%) Immediately after the preparation,
Under gastric analogy
Platelet Aggregation Inhibitory Effect (%) Core 44.8 <5.0 Core / CS-Arg 39.2 25.3 Fu600 19.5 81.2 Fu800 17.2 82.2 Fu1000 15.8 83.4 P1 33.2 78.3 P10 35.2 92.9 P100 38.2 93.2

In Table 11, the nanocapsules (Fu600, Fu800, Fu1000, P1, P10 and P100) of the present invention had a poor platelet aggregation inhibition effect in the dispersion immediately after preparation, but the platelet aggregation inhibition Activity was expressed by almost 80% or more. It was judged that the red ginseng extract, the conjugated linoleic acid and the gamma linolenic acid collected in the capsules in the nanocapsule state were released to the outside of the capsule as shown in Example 9 under the gastric analogous condition to express the activity.

A mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid (non-nano-encapsulated 'red ginseng extract, conjugated linoleic acid and gamma linolenic acid mixture' (red ginseng extract 10 mg / ml + conjugated linoleic acid 0.06 mg / ml + gamma linolenic acid 20 mg / (Core / CS-Arg) with chitosan-arginine polymer significantly reduces platelet aggregation inhibitory activity under gastric-like conditions, the nanocapsules of the present invention are effective for inhibiting the platelet aggregation inhibitory activity of gonad extract, conjugated linoleic acid and gamma linolenic acid It can be seen that the activity is well protected.

Thus, it can be seen that the nanocapsulation using chitosan-arginine polymer, fucoidan and polyglutamic acid is unstable in acidic condition, and thus it is a delivery method of increasing the bioavailability of the red ginseng extract and the easily degradable unsaturated fatty acid which are low in bioavailability .

Claims (14)

Wherein the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is coated with a coating of a mixed coating of chitosan-arginine polymer and fucoidan. The method according to claim 1,
Wherein the nanocapsule comprises 0.1 to 100 parts by weight of conjugated linoleic acid and 10 to 500 parts by weight of gamma linolenic acid based on 100 parts by weight of red ginseng extract. The method according to claim 1,
Wherein the coating of the nanocapsules comprises 10 to 50 parts by weight of the chitosan-arginine polymer and 6 to 10 parts by weight of fucoidan based on 100 parts by weight of red ginseng extract. The method according to claim 1,
Wherein the coated body of the nanocapsule is supplemented with 0.01 to 1 part by weight of polyglutamic acid based on 100 parts by weight of red ginseng extract. The method according to claim 1,
The chitosan-arginine polymer was prepared by mixing chitosan and arginine in a TEMED / HCl buffer solution (N, N, N'-tetramethylethylenediamine / HCl buffer) ) Carbodiimide and N-hydroxy-succinimide, and reacting the reaction product. 6. The method of claim 5,
The chitosan-arginine polymer is prepared by mixing 10 to 100 parts by weight of arginine with 100 parts by weight of chitosan and dissolving the mixture in a TEMED / HCl buffer solution (N, N ', N'-tetramethylethylenediamine / HCl buffer) And 10 to 100 parts by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 100 to 200 parts by weight of N-hydroxy-succinimide are added to the reaction mixture, Nano-capsules. The method according to claim 1,
Wherein the nanocapsule has a particle size of 250 to 500 nm. A pharmaceutical composition for preventing or treating vascular diseases selected from thrombosis, arteriosclerosis, hypertension, vascular restenosis, peripheral vascular occlusion, cerebral infarction, cerebral hemorrhage and stroke, which comprises the nanocapsule of claim 1. A health functional food for preventing or ameliorating a vascular disease selected from thrombosis, arteriosclerosis, hypertension, vascular restenosis, peripheral vascular occlusion, cerebral infarction, cerebral hemorrhage and stroke, characterized by containing the nanocapsule of claim 1. (1 step) To 100 parts by weight of chitosan, 10 parts by weight of arginine was mixed with 100 parts by weight of chitosan, and the mixture was dissolved in TEMED / HCl buffer solution (N, N, N'-tetramethylethylenediamine / HCl buffer) 10 to 100 parts by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 100 to 200 parts by weight of N-hydroxy-succinimide were added to the reaction mixture and the resulting reaction product was dried to obtain a chitosan- Preparing a chitosan-arginine polymer solution by redissolving the chitosan-arginine polymer in water;
(Step 2) Mixing a mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid in the chitosan-arginine polymer solution prepared in the above step 1 to prepare a mixed solution; And
(Step 3) Mixing a fucoidan aqueous solution into the mixed solution of the two steps;
Wherein the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid is coated with a coating of a mixed coating of chitosan-arginine polymer and fucoidan. 11. The method of claim 10,
Wherein the concentration of the chitosan-arginine polymer solution in the one step is 1 to 5 mg / ml. 11. The method of claim 10,
The concentration of the mixture of red ginseng extract, conjugated linoleic acid and gamma linolenic acid mixed in the chitosan-arginine polymer solution in the above two steps is 1 to 10 mg / ml of red ginseng extract, 0.06 to 0.12 mg / ml of conjugated linoleic acid, 10 to 50 mg of gamma linolenic acid / Ml, wherein the mixture is prepared by mixing red ginseng extract in an aqueous solution state, conjugated linoleic acid and gamma linolenic acid in a 30 to 99% (v / v) aqueous ethanol solution state. 11. The method of claim 10,
Wherein the concentration of the fucoidan aqueous solution in the above three steps is 0.6 to 1 mg / ml. 11. The method of claim 10,
Wherein the polyglutamic acid is added to the fucoidan aqueous solution at a concentration of 0.001 to 0.1 mg / ml in the step 3 above.

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