KR101883371B1 - The composition of Cordyceps militaris comprising codycepin for inhibting dissolution codycepin, and the functional foods composition - Google Patents

Abstract

The present invention provides a method and a composition for suppressing the decomposition of codistin in vivo by adding Naringin, which is a natural substance with a weak toxicity to codistin contained in cordycepin or millitary cordyceps, as an ADA inhibitor, Functional food composition and a pharmaceutical composition in which the duration of potency is increased by inhibiting the decomposition of cordycepin or millitaryl Cordyceps codicifine.

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting the degradation of cordycepin contained in a molluscan cordyceps and a functional food composition containing the same,

The present invention provides a method for inhibiting degradation of cordycepin contained in cordycepin or millitary cordyceps in vivo.

In addition, the present invention provides a composition comprising a component that inhibits degradation of cordycepin or cordycepin contained in cordyceps cordyceps in vivo.

Also, the present invention provides a functional beverage composition, a functional food composition, and a pharmaceutical composition, wherein the duration of efficacy of the millitary caterpillar fungus is increased by inhibiting the decomposition of cordycepin contained in cordycepin or millitary cordyceps.

Cordyceps is a mushroom strain of Cordyceps mushroom which grows in the body of insects in winter and absorbs nutrients during winter and grows as a mushroom in the body of dead insects in summer. Taxonomically speaking, there are Ascomycota, Pyrenomycetes, It belongs to Clavicipitaceae and Codyceps. There are about 300 species of 100 genera, including Korea, China and Japan. Among them, it is a type of entomopathogenic fungi that forms an amygdaloid type of stigma with the larva or pupa of Lepidoptera as a host, and the peritubular angle is semi-protruding type, Shaped asphalt spores are present.

Cordycepin, a physiologically active substance, is known as an effective ingredient for the efficacy against Cordyceps.

Codisepin represented by the following formula (1) is a natural compound of 3-deoxyadenosine type and has a molecular weight of 251.2. After its first effect by Cunningham et al. In 1950, many studies on pharmacological activity have been conducted to inhibit the synthesis of mRNA Antibacterial, antifungal, antiviral effect, platelet aggregation inhibitory effect and anti-cancer effect through immunity enhancement are well known.

[Chemical Formula 1]

In particular, the fruiting body of Millitarian Cordyceps sinensis has a relatively high content of cordycepin, which protects the lungs and strengthens the kidney. It is widely recognized as a treatment for tuberculosis and jaundice and as a tonic agent, It is used as an expensive herbal medicine used for body control, care, and the like. Especially recently, antiviral and immune function-enhancing components are known, and they are also effective against pulmonary tuberculosis, chronic cough, asthma, anemia, male sexual dysfunction and hypertension. In addition, ergosterol peroxide, which is an anticancer component, has been identified in the mulberry tree Cordyceps in Korea, and it is known that the use of ergosterol peroxide is also effective in relieving stress.

Adenosine deaminase (ADA, EC 3.5.4.4) is commonly found in plants, germs, mammals, and especially in the human body, such as the brain, heart, small intestine, and large intestine, where ADA is adenosine It is responsible for the enzymatic action of hydrolyzing codistepine and metabolizing it with inosine.

[Reaction Scheme 1]

ADA is present in not only red blood cells but also lymphocytes and white blood cells, and plays an important role in showing immune function. Therefore, deficiency of ADA weakens immune function and makes it susceptible to disease invasion.

On the other hand, 3'-deoxyadenosine is a derivative of adenosine (2'-deoxyadenosine), which can be easily metabolized by ADA because of its similar structure. Therefore, if it is possible to increase the half-life of codeseepine by inhibiting ADA, You will have enough time to demonstrate the action in this body.

For this reason, many researchers have developed ADA inhibitors and have reported that deoxycoformycin (pentostatin) and erythro-9- (2-hydroxy-3-nonyl) adenine (EHNA) And thus it is used only for diseases such as leukemia. Therefore, development of a natural substance having a weak toxicity is under development.

Codisepin, which is a major active ingredient of Cordyceps militaris, is degraded into inosine in a few seconds to several minutes by adenosine deaminase (ADA) in vivo of human or animal, thereby failing to exert various pharmacological functions properly.

DISCLOSURE OF THE INVENTION The present invention has been made to overcome these disadvantages, and it is intended to improve the disadvantage that codisepin is not decomposed into inosine by ADA in vivo in human or animal, thereby failing to exert various pharmacological functions properly.

The present invention improves the disadvantage that cordisepin, which is a main active ingredient of the miliitis Cordyceps, is decomposed into inosine within a few seconds to several minutes by the adenosine deaminase (ADA) in vivo of a human or animal, A method for inhibiting the decomposition of codistin in vivo by adding a natural substance ADA inhibitor having a weak toxicity to cordisepin (hereinafter referred to as " millitaris cordyceps cordisecin ") contained in cordycepin or millitaris cordyceps .

The present invention also provides a composition comprising a natural substance ADA inhibitor which inhibits the decomposition of cordycepin and cordiercine and which is weakly toxic. Further, the present invention relates to a composition for inhibiting degradation of Cordyceps sinensis, And a natural product ADA inhibitor.

In addition, the present invention relates to a functional beverage composition, a functional food composition and a functional food composition, which are industrially effective in inhibiting the degradation of cordisepin or myrrhitis ciliaris cordis cephe by adding a natural substance with a weak toxicity to an ADA inhibitor, To provide a pharmaceutical composition.

The present inventors have searched and experimented with a natural toxin-resistant ADA inhibitor that inhibits the degradation of cordisepin or millitarid Cordyceps sinensis. As a result, naringin, kaempferol, quercetin, myricetin and naringenin were selected as candidates . As a result, it was confirmed that naringin among these candidate substances is the most effective to inhibit the degradation of cordisepin or Cordyceps sinensis of Millitaredus cordifolia, and the present inventors have completed the present invention by clarifying the concentration thereof will be.

Naringin represented by the following formula 2 is naringenin-7-rhamnoglucoside, and its molecular formula is C ₂₇ H ₃₂ O ₁₄ : its molecular weight is 580.54, and it is a glucoside of a pale, colorless crystal flavonon. Naringin is a natural product belonging to citrus fruits and can be extracted from orange, lemon, lime, grafe fruit, citrus aurantium, citrus grandis, citrus junos, citrus unshiu marcor and citrus konokuni tanaka.

Naringin has been known to have antioxidant, antiinflammatory, anti-cancer, anti-allergic and anti-diabetic effects, and it has recently been shown to inhibit angiogenesis.

(2)

The present inventors have found that Naringin is a substance having a high molecular weight and can hydrogen bond with the active site of ADA and the surface of ADA protein and acts as a strong ADA inhibitor to inhibit the decomposition action of codeseepin or millitaryl Cordyceps cordisepine by ADA It is possible to exhibit a long-term effect at a high concentration without toxicity when it is used in combination with cordycepin or millitaris cordyceps cordisepine.

The present invention provides a method for inhibiting the degradation of codistepin in vivo by adding nordinine to cordisepin or millitallic cordyceps cordisepin.

Further, the present invention provides a composition comprising naringin, which inhibits the decomposition of cordycepin and cordycepin, and provides a composition comprising millitary cordyceps cordisepine and naringin inhibiting its degradation.

According to the present invention, the ratio of the molar concentration (M) of b) the molar concentration of ninulinin, that is, 1: 1 or less when a: b is 1: 2 or more The decolorization of codistin is suppressed by 10 times or more, so that the concentration of the residual codistin is increased by 10 times or more. Thus, it has a critical meaning, preferably 1: 2 to 16, more preferably 1 : 4 to 16, and most preferably 1: 8 to 16.

In addition, the present invention provides a functional beverage composition, a functional food composition, and a pharmaceutical composition in which naringin is added to inhibit the degradation of cordycepin or millitary cordyceps cordocentepin to increase the duration of efficacy of the millitary cordyceps.

Naringin can be extracted from citrus fruits such as citrus, citron, citrus, orange, lemon, grapefruit or citron, or can be prepared by a known synthetic method. Naringin is especially contained in grapefruit, sugar beet, citron and the like.

A pharmaceutical composition can be prepared by adding naringin as a decomposition inhibitor of cordisepin to cordycepin or millitaredis cordis cordisepin and mixing with a pharmaceutically acceptable carrier. The pharmaceutical composition may further contain commonly used excipients, disintegrants, sweeteners, lubricants, flavors and the like, and may be formulated into tablets, capsules, powders, granules, suspensions, emulsions, syrups, Or parenterally administered preparations such as a single dose or multiple dose pharmaceutical formulation.

The composition for inhibiting the degradation of cordycepin by adding cordycepin or millitary cordyceps cordisepine enanthrin as an active ingredient may be administered parenterally or orally, as desired. Naringin may be administered in one to several doses so as to be administered in an amount of 0.5 mg to 100 mg, preferably 2 to 20 mg per kg of body weight per day. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate, severity of disease, and the like.

The composition for inhibiting the degradation of cordycepin, in which codisepin or millitary cordyceps codisepin enanthrin is added as an active ingredient, may be added to foods or beverages for the same purpose. Examples of foods that can be supplemented with naringin for the development of health functional foods include foodstuffs, meat, beverages, chocolates, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, alcoholic beverages, And health supplement foods.

The health functional food using the composition according to the present invention may be provided in various forms such as health drinks, functional supplements, tea, confectionery or medicines.

The compositions according to the invention may comprise ingredients which are conventionally added in the manufacture of food for use as a food composition. For example, proteins, fats, nutrients, flavoring agents and flavoring agents. Natural flavors (tautatin, stevia extract) and synthetic flavors (saccharin, aspartame, etc.) can be used as the flavor.

Formulations such as pills, granules, beverages, tablets, capsules and the like may be prepared using the composition according to the invention, in which case additives may be added to make each formulation.

In order to use the present invention as a pharmaceutical composition, it may be prepared by a method well known in the pharmaceutical field, and may be prepared by mixing with powder, granules, tablets, capsules or the like, or by mixing with a pharmaceutically acceptable carrier, Injections, and the like. They may also be administered orally or parenterally.

May be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterilized injection solutions according to a conventional method, Carriers, excipients and diluents that may be included include lactose, textol, sucrose, sorbitol, mannitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl Pyrrolidone, water, methylhydroxy, hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are 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 talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The effective dose of the composition according to the present invention can be appropriately selected depending on the degree of absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, severity of the disease to be treated,

The composition according to the invention can be used in feed for livestock and in each case can be prepared using additives or additives which are acceptable in livestock feed.

In order to use the composition according to the present invention as an active ingredient of a cosmetic composition, the ingredients contained in the cosmetic composition include ingredients commonly used in cosmetic compositions in addition to the red ginseng extract. Such as, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetics according to the present invention can be prepared in any formulations conventionally produced in the art. For example, as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing oils, powdered foundations, emulsion foundations, waxed foundations and sprays, no.

More specifically, it can be manufactured in the form of a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol chiba ester, polyethylene glycol or fatty acid esters of sorbitan can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulosic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane Propane, such as butane or dimethyl ether.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

The cosmetic composition containing the composition according to the present invention as an active ingredient may include a composition selected from the group consisting of water-soluble vitamins, fat-soluble vitamins, sphingolipids, high-molecular polysaccharides, and high molecular peptides.

As water-soluble vitamins, any of water-soluble vitamins that can be incorporated into cosmetics can be used, but preferably vitamin B 1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamini B12, pantothenic acid, nicotinic acid, nicotinamide, folic acid, vitamin C, And salts thereof (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate etc.) can also be used as water-soluble vitamins . The water-soluble vitamin can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzymatic method, or a chemical synthesis method.

Examples of the fat-soluble vitamin include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (DL-alpha tocopherol and D-alpha tocopherol), and derivatives thereof such as palmitic acid Alpha-tocopherol, vitamin E, DL = pantothenyl alcohol, pantothenyl ethyl ether, etc.) used in the present invention may also be used in the present invention. It is included in vitamins. The fat-soluble vitamin can be obtained by a microorganism conversion method, purification from a culture of a microorganism, an enzyme, or a chemical synthesis method.

The cosmetic of the present invention may contain, in addition to the above-mentioned essential ingredients, other ingredients usually added to cosmetics, if necessary. Examples of the compounding ingredients that may be added include organic solvents such as a preservative component, a moisturizer, an emollient, a surfactant, an organic and inorganic pigment, an organic powder, an ultraviolet absorbent, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, A blood circulation promoter, a cold agent, a restriction agent, and purified water.

The components to be added in addition to the above are not limited to the above, and all of the above components can be blended within the range not to impair the objects and effects of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

A method and a composition for inhibiting the degradation of cordysepine in vivo by the addition of nordinine to cordisepin or millitareis Cordyceps sinensis in accordance with the present invention is effective for inhibiting the degradation of cordisepin or millitary cordyceps codistin, Or the efficacy duration of milliliter Cordyceps sinensis codisepin is greatly increased.

1 is a graph showing a calibration curve of the standard product cordisepin and millitary cordyceps cordisepine.
FIG. 2 is a graph showing the decomposition inhibition results of naringin concentration at 50 μM of cordycepin. FIG.

Hereinafter, the present invention will be described in more detail.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings, and the inventor may properly define the concept of the term to describe its invention in the best possible way And should be construed in accordance with the principles and meanings and concepts consistent with the technical idea of the present invention.

Therefore, the examples described in the present specification and drawings are only the most preferred examples of the present invention and do not represent all the technical ideas of the present invention. Accordingly, various equivalents and substitutes It should be understood that variations can be made.

Hereinafter, the present invention will be described in more detail.

Example

5 to 10 ml of blood was collected from the heart of the rat using a Vacutainer (BD), and 6 ml of blood and 6 ml of stop solution (200 μM of dipyridamole and 10 μM of theophylline mixture) were reacted at room temperature for 30 minutes. Then, the mixture of blood and stop solution was mixed with 50 μl of each of aliquots (25, 50, 100, 200, 400, and 800 μM) and reacted in a water bath at 37 ° C for 5 minutes. Naringin was dissolved in 2% ethanol at a concentration of 1 mg / ml, diluted again with 2% ethanol, and then used in the experiment. Then, centrifugation was carried out at a speed of 3,000 g for 10 minutes using a refrigerated centrifuge maintained at 4 캜 to collect only the supernatant as a plasma component. 300 μl of plasma and 300 μl of cordycepin (sigma) or Cordycepin powder dilution were mixed and centrifuged in a microcentrifuge, followed by a 2 minute reaction at 37 ° C in a waterbath, and the reaction was stopped on ice. After that, 1200 μl of acetonitrile (ACN) and 600 μl of the reacted sample were mixed and reacted at -70 ° C. for 30 minutes to precipitate the protein. After the protein precipitate was dissolved again in ice, it was centrifuged at 3,000 g for 30 minutes. Then, the supernatant was filtered with 0.2 μm filter and measured by UPLC of Waters.

1-1: Experimental animals

Male rats weighing 250 ~ 300 g (SD rat, Koatech, Korea) were maintained at room temperature of 25 ± 2 ° C, humidity of 50 ± 10%, day and night for 12 hours Feed and water were kept in an environment where they could be freely consumed.

1-2: Reagents and Materials

The reagents and materials used in the above examples are as follows.

end. Stop solution

Dipyridamole (Sigma) was dissolved in Ethyl Alcohol (Duksan, GR grade) to a concentration of 40 mM. Theophyline (sigma) was dissolved in 0.9% NaCl aqueous solution to a concentration of 2 mM. After that, Dipyridamole and Therophyline were diluted in 0.9% NaCl aqueous solution to 400 μM and 20 μM, respectively. The mixture of the two solutions was used as a stop solution.

I. Naringin solution

Naringin (Sigma) was dissolved in 0.9% NaCl aqueous solution to a concentration of 1 mM and dissolved in 70 ℃ water bath for 30 min. Since the crystals are formed when the temperature of the solution is lowered, it is melted again in a water bath at 70 ° C before use, and then the temperature is lowered to 37 ° C.

All. Cordycepin solution

Standard product Cordycepin (codisepin purchased from Sigma Co., Ltd. is labeled with cordisepine as a standard product to distinguish it from cordisepin milletalis Cordyceps) is dissolved in 1M NH ₄ OH (Sigma) to make 10 mg / ml, 0.9% NaCl aqueous solution.

la. Millitarian Cordyceps sinensis solution

The millitary Cordyceps sinensis solution used in the experiment was diluted with distilled water (DW) to a concentration of 10 mg / ml, then boiled for 20 minutes in a water bath at 80 ° C, vortexed for 30 seconds, (Millipore). Cordyepin content was analyzed by UPLC equipment from Waters. Columns used for the analysis were UPLC BEH column 18 column. The mobile phase used in the assay was 0.5 mM acetic acid (Junsei) and Acetonitrile (Duksan). In the UPLC analysis, the samples were prepared in the same manner and the average value was calculated as cordycepin amount contained in the extract of Cordyceps militaris.

Experimental Example 1: Calibration curve measurement of cordisepin and millitaris Cordyceps cordisepin

In the blood, codistepine is metabolized to inosine by decomposition within a few seconds to several minutes. Therefore, the concentration of cordisepin was measured by UPLC of Waters Co., Ltd. and the concentration of cordisepin of Millitarian Cordyceps sinensis was measured. As shown in Table 1 and FIG. 1, the results of the experiment showed a correlation coefficient of 0.999 in a concentration range of 25 to 200 μg / ml.

cordycepin  density UPLC  Measures Standard Codisepin 25 μg / ml 1546052.967306 Millitarian Cordyceps sinensis 25 μg / ml 1544564.310463 Standard product Codisepin 50 μg / ml 3098455.889420 Millitarian Cordyceps sinensis 50 μg / ml 3091452.485755 Standard product Codisceptin 100 μg / ml 6095927.482388 Millitarian Cordyceps sinensis 100 μg / ml 6100922.530967 Standard product Codisepin 200 μg / ml 10597802.879015 Milliliter Cordyceps sinensis 200 μg / ml 10605547.250057

Experimental Example 2: Measurement of naringin concentration to inhibit the decomposition of cordycepin

In order to measure the concentration of naringin, which can effectively inhibit the degradation of codistepin, the concentration of naringin was treated at concentrations of 25 μM to 800 μM at a concentration of 50 μM of Cordyceps militaris, .

As shown in Table 2 and FIG. 2, when the concentration of naringin is less than 50 μM, which corresponds to one time the concentration of Cordyceps militaris, it can not inhibit the degradation of codistepin, so that it is less than 0.18 μg / ml Very low concentrations were measured.

Since the concentration of naringin is rapidly suppressed from 100 μM, which corresponds to twice the concentration of Cordyceps sinensis, to the very high concentration of residual codistin of 1.81 μg / ml or more Respectively.

That is, when the concentration of naringin was 100 μM, the concentration of naringin increased sharply to 1.81 μg / ml, which is 10 times (1000%) or more, compared with 0.18, which is the residual concentration of cheddin at 50 μM.

In addition, it was confirmed that the decomposition of the codistin is further suppressed from 200 μM or more, which corresponds to four times the concentration of the corditis codicin cordocetin, and that the concentration of the residual codistin is measured more rapidly.

Further, it was confirmed that the decomposition of the codistin is further suppressed from 400 μM or more, which corresponds to 8 times the concentration of the corditis codicin cordocintepil, and the concentration of the residual codistin measured is further increased sharply.

On the other hand, even in the case of the standard product cordisepin, the same tendency as in the case of millitary cordyceps cordisepin was observed in inhibiting the degradation by the concentration of naringin, but the degree of inhibition of decomposition was somewhat lowered, and the concentration of the residual cordycepin was somewhat lowered .

Neglination treatment concentration  Residual Codecine Average Concentration  0 μM 0.16 μg / ml 25 μM 0.17 μg / ml 50 μM 0.18 μg / ml 100 [mu] M 1.81 μg / ml 200 μM 4.31 μg / ml 400 μM 8.11 μg / ml 800 μM 8.62 μg / ml

Claims (6)

a) cordycepin or millitaris cordis cordisepine; And
b) Naringin;
Wherein the coddepine decomposition inhibiting composition further comprises:
characterized in that the ratio of a) cordycepin or millitaredis cordis cordissefin: b) naringin is 1: 2-16. delete The composition according to claim 1, wherein the ratio of a) cordycepin or milliliter cordyceps cordiercine: b) naringin is 1: 4 to 16. The composition according to claim 3, wherein the ratio of a) cordycepin or milliliter cordyceps cordiercine: b) naringin is 1: 8-16.
A functional food composition comprising the composition for inhibiting the decomposition of ceedy sebacic acid according to any one of claims 1, 3 and 4. delete

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