JP2008255041A - Hypertension inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe hypertension inhibitor having excellent hypertension-inhibitory effect. <P>SOLUTION: The hypertension inhibitor, which contains an enzymolysis product of bracket fungus, is obtained by treating an extract of bracket fungus selected from Ganoderma lucidum, Ganoderma atrum and Ganoderma neo-japonicus and the like with a proteolytic enzyme (e.g. End-type protease). Foods, quasi-drugs or pharmaceuticals each containing the above hypertension inhibitor are also provided, respectively. The enzymolysis product of bracket fungus, as compared to bracket fungus extract, exhibits more excellent angiotensin converting enzyme-inhibitory effect and hypertension-inhibitory effect. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a hypertension inhibitor mainly composed of an enzyme-treated product of ganoderma and a food, quasi-drug, and pharmaceutical product containing the same. It has an inhibitory effect on activity (referred to as ACE) and relates to an antihypertensive agent containing the same.

  One of the important factors of high blood pressure is the renin-angiotensin system that plays a role in increasing blood pressure. ACE plays a central role in the renin-angiotensin system. ACE is an enzyme that converts inactive angiotensin I into active angiotensin II, which has a high blood pressure increasing action. Therefore, it is possible to suppress an increase in blood pressure by inhibiting the ACE activity. Recently, attempts have been made to regulate the renin-angiotensin system to inhibit hypertension by inhibiting the activity of ACE.

  As such substances having ACE activity inhibition, L-proline derivatives such as captopril are known as synthetic compounds, and snake venom-derived bradykinin enhancing factors are derived from natural products. Of these, captopril has already been put to practical use as an oral antihypertensive agent, but may cause side effects such as allergic symptoms, headache, dizziness, and lightheadedness.

Among basidiomycetes, Lentinus edodes (Patent Document 1), Pleurotus osteoreas (Patent Document 2), Maitake (Grifora frontosa) (Patent Document 3), Enokitake (Flamulina velutipes), Patent 4 The inhibitory effect of ACE has been reported in (Lyophyllum ulmarium) (Patent Document 5).
JP 58-26820 A JP-A-2-72121 JP-A-8-291078 JP-A-8-291078 JP-A-8-99895

In addition, in relation to Ganoderma, antihypertensive agent containing Ganoderma extract (Patent Document 6), antihypertensive agent containing ganoderic acid in Ganoderma alcoholic extract (Patent Document 7), An antihypertensive agent containing an extract (Patent Document 8) and the like are known.
JP-A 61-267527 JP-A-60-208161 JP2003-104904A

However, these examinations are about the extract itself, and more effective antihypertensive effect has been desired.

In view of the above, an object of the present invention is to provide an antihypertensive agent having high safety and excellent ACE inhibitory action.

  The present inventors found an ACE inhibitory effect superior to that of the extract itself by performing enzyme treatment on Ganoderma lucidum in studying a more effective antihypertensive agent, and completed the present invention. .

Therefore, the present invention provides a hypertension inhibitor containing an enzyme-treated product of ganoderma.

The present invention also provides foods, quasi-drugs, and pharmaceuticals for use in the prevention or treatment of hypertension including the hypertension inhibitor.

Ganoderma used in the present invention is a basidiomycete used in the herbal medicine “Ganoderma lucidum”, belongs to the family Ganodermaceae and the genus Ganoderma, and is a kind of ganoderma. Its scientific name is classified into red ganoderma (Akashiba, scientific name: Ganoderma lucidum), black ganoderma (Kuroshiba, scientific name: Ganoderma atrum), maggot (scientific name: Ganoderma Japanicum), etc. (Non-patent Document 1) is classified. The method is not limited to this document alone. In addition, the ganoderma used in the present invention is not limited to red ganoderma, black ganoderma, maggot, etc., and those widely distributed in the Chinese and Japanese markets can be used.
Acta Microbiological Sinica, 19 (3), 265-279, 1979

  Ganoderma used in the present invention is selected from fruit bodies, mycelia, mycelium cultures, and the like, and preferably fruit bodies are used. The enzyme-treated product of ganoderma of the present invention is an enzyme-treated ganoderma and / or ganoderma extract, and preferably the ganoderma extract is treated with enzyme. The extraction method is not particularly limited, but extraction by heating is preferable.

  Examples of the solvent to be extracted include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol). ), Ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Can be mentioned. A polar solvent is preferable, and water is more preferable. These solvents may be used alone or in combination of two or more. Furthermore, the pH may be adjusted by adding an acid or alkali to the extraction solvent.

  A commercially available thing can be used as an enzyme to be used. Examples include protease, peptidase, tannase, chitinase, glucanase, cellulase, and lipase. Proteolytic enzymes such as protease and peptidase are preferable, and end-type protease is more preferable. These enzymes may be used alone or in combination of two or more. In the treatment method, for example, water or a buffer solution is added to ganoderma and / or ganoderma extract and treated at the optimum pH and temperature of each enzyme.

  The enzyme-treated product of the present invention may be used as a solution, or may be used after treatment with concentration, dilution, filtration, activated carbon or the like, if necessary. Furthermore, you may perform processes, such as spray-drying and freeze-drying, and you may use as a dried material.

  As the antihypertensive agent of the present invention, the enzyme-treated product may be used as it is, and a diluent may be used as long as the effect is not impaired. The diluent may be solid, semi-solid, or liquid, For example: That is, excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, fragrances, preservatives, solubilizers, solvents and the like can be mentioned. Specifically, lactose, sucrose, sorbit, mannitol, starch, precipitated calcium carbonate, heavy magnesium oxide, talc, calcium stearate, magnesium stearate, cellulose or derivatives thereof, amylopectin, polyvinyl alcohol, gelatin, surface activity Agents, water, physiological saline, ethanol, glycerin, propylene glycol, cacao butter, laurin butter, petrolatum, paraffin, higher alcohol and the like.

The antihypertensive agent of the present invention can be used for any of foods, quasi drugs, and pharmaceuticals. As examples of food, ordinary food forms such as tablet confectionery, capsules, chocolate, gum, candy, and beverages can be adopted. Examples of quasi-drug or pharmaceutical dosage forms include powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, liquids, emulsions and the like for oral use. For parenteral use, it can be an injection solution. It can also be a suppository.

About the addition method of the ganoderma enzyme processed material used for this invention, it may add in advance or may be added in the middle of manufacture, and should just select it suitably considering workability | operativity.

The antihypertensive agent of the present invention has an ACE inhibitory activity containing an enzyme-treated product of ganoderma as an active ingredient, and can be used for prevention or treatment of hypertension. The present invention is described in detail below.

Next, in order to describe the present invention in detail, examples of production of the enzyme-treated product used in the present invention, formulation examples and experimental examples of the present invention will be given as examples, but the present invention is not limited thereto. The part of the blending amount shown in the formulation examples indicates part by weight, and% indicates% by weight.

Comparative Production Example 1 Preparation of hot water extract of red ganoderma fruit body 2 L of purified water was added to 100 g of dried red ganoderma fruit body, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated. And freeze-dried to obtain 5.0 g of a hot water extract of red ganoderma fruiting bodies. This was used in Production Examples 1 and 6.

Comparative Production Example 2 Preparation of hot water extract of black reishi fruit bodies 2 L of purified water was added to 100 g of dried black reishi fruit bodies, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated. And lyophilized to obtain 4.8 g of a hot water extract of Kuroshiki Shibashi fruiting body. This was used in Production Examples 2 and 7.

Comparative Production Example 3 Preparation of hot water extract of pearl oyster fruit body 2 L of purified water was added to 100 g of dried potato fruit fruit body, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated and lyophilized. As a result, 5.4 g of a hot water extract of the fruit body was obtained. This was used in Production Examples 3 and 8.

Comparative Production Example 4 Preparation of hot water extract of red ganoderma mycelium Add 2 L of purified water to 100 g of dried red ganoderma mycelium, extract at 95-100 ° C. for 2 hours, filter, and concentrate the filtrate. And freeze-dried to obtain 6.0 g of a hot water extract of red ganoderma mycelium. This was used in Production Example 4.

Comparative Production Example 5 Preparation of ethanol extract of red ganoderma fruit body 2 L of ethanol was added to 100 g of dried red ganoderma fruit body, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 1.0 g of an ethanol extract of red ganoderma fruiting bodies was obtained. This was used in Production Example 5.

Production Example 1 Preparation of End-type Protease Product of Red Ganoderma Fruit Body Hot Water Extract 3.0 g of Red Ganoderma fruit body hot water extract (Comparative Production Example 1) is mixed with 100 mL of purified water, and 1M NaOH or 1M. After adjusting to pH 7.0 with HCl, 0.1 g of protease N “Amano” G (Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, treated at 95-100 ° C. for 1 hour, and the enzyme was lost. After being activated, the mixture was filtered, and the filtrate was concentrated and lyophilized to obtain 2.9 g of an end-type protease-treated product.

Production Example 2 Preparation of End-type protease treatment product of Black Reishi fruit body hot water extract 3.0 g of Black Reishi fruit body hot water extract (Comparative Production Example 2) was mixed with 100 mL of purified water, and 1M NaOH or 1M. After adjusting to pH 7.0 with HCl, 0.1 g of protease N “Amano” G (Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, and treated at 95-100 ° C. for 1 hour to lose the enzyme. After being activated, the mixture was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.8 g of an end-type protease-treated product.

Production Example 3 Preparation of Endo-Protease Treated Product of Magojakhi Fruit Body Hot Water Extract 3.0 g of Magojakite fruit body hot water extract (Comparative Production Example 3) was mixed with 100 mL of purified water, and pH was adjusted with 1M NaOH or 1M HCl. After preparing to 7.0, 0.1 g of protease N “Amano” G (Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, treated at 95-100 ° C. for 1 hour to inactivate the enzyme. The filtrate was concentrated and freeze-dried to obtain 2.8 g of an end-type protease-treated product.

Production Example 4 Preparation of End-type Protease Product of Red Ganoderma Mycelium Hot Water Extract 3.0 g of Red Ganoderma mycelium hot water extract (Comparative Production Example 4) is mixed with 100 mL of purified water, and 1M NaOH or 1M After adjusting to pH 7.0 with HCl, 0.1 g of protease N “Amano” G (Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, and treated at 95-100 ° C. for 1 hour to lose the enzyme. After being activated, the mixture was filtered, and the filtrate was concentrated and lyophilized to obtain 2.9 g of an end-type protease-treated product.

Production Example 5 Preparation of Endoprotease Treated Product of Red Ganoderma Fruit Body Ethanol Extract 1.0 g of red ganoderma fruit body ethanol extract (Comparative Production Example 5) is mixed with 100 mL of purified water, and added to 1M NaOH or 1M HCl. After adjusting to pH 7.0, 0.1 g of protease N “Amano” G (Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, treated at 95-100 ° C. for 1 hour to inactivate the enzyme. After filtration, the filtrate was concentrated and freeze-dried to obtain 0.9 g of an end-type protease-treated product.

Production Example 6 Preparation of Peptidase Processed Product of Red Ganoderma Body Hot Water Extract 3.0 g of Red Ganoderma fruit body hot water extract (Comparative Production Example 1) was mixed with 100 mL of purified water and dissolved in 1M NaOH or 1M HCl. After adjusting to pH 7.0, 0.1 g of equinezyme G (manufactured by Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, treated at 95-100 ° C. for 1 hour to deactivate the enzyme, and then filtered. The filtrate was concentrated and lyophilized to obtain 2.9 g of a peptidase-treated product.

Production Example 7 Preparation of Peptidase Processed Product of Black Reishi Fruit Body Hot Water Extract 3.0 g of Black Reishi fruit body hot water extract (Comparative Production Example 2) is mixed with 100 mL of purified water, and added to 1M NaOH or 1M HCl. After adjusting to pH 7.0, 0.1 g of horse zyme G (manufactured by Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, treated at 95-100 ° C. for 1 hour to deactivate the enzyme, and then filtered. The filtrate was concentrated and lyophilized to obtain 2.8 g of a peptidase-treated product.

Production Example 8 Preparation of Peptidase Processed Product of Pepperase Hot Water Extract 3.0 g of Pepper fruit body hot water extract (Comparative Production Example 3) was mixed with 100 mL of purified water, and adjusted to pH 7.0 with 1M NaOH or 1M HCl. After preparation, 0.1 g of equinezyme G (manufactured by Amano Enzyme) was added, reacted at 50 ° C. for 3 hours, treated at 95-100 ° C. for 1 hour to inactivate the enzyme, filtered, and the filtrate was concentrated. And lyophilized to obtain 2.9 g of a peptidase-treated product.

  In addition, protease N “Amano” G in Production Example 1 is converted to tannase (manufactured by Kikkoman), chitinase (manufactured by Sigma), tunica FN (manufactured by Amano Enzyme), cellulase A “Amano” 3 (manufactured by Amano Enzyme) or lipase. It replaced with AY "Amano" 3G (made by Amano Enzyme Co., Ltd.), and adjusted temperature and pH to the optimal conditions, and was set as manufacture examples 9-13, respectively.

Production Example 14 Preparation of End-type protease treated product of red ganoderma fruit body 400 ml of purified water was added to 20 g of dried red ganoderma fruit body, adjusted to pH 7.0 with 1 M NaOH or 1 M HCl, and then protease N “Amano” G (Amano Enzyme) 0.1 g was added, reacted at 50 ° C. for 3 hours, treated at 95-100 ° C. for 2 hours to inactivate the enzyme and extracted from the active ingredient, then filtered. The filtrate was concentrated and freeze-dried to obtain 2.0 g of an end-type protease-treated product of red ganoderma fruiting bodies.

  In addition, protease N “Amano” G in Production Example 14 was converted to tannase (manufactured by Kikkoman), chitinase (manufactured by Sigma), tunica FN (manufactured by Amano Enzyme), cellulase A “Amano” 3 (manufactured by Amano Enzyme) or lipase. AY “Amano” 3G (manufactured by Amano Enzyme Co., Ltd.) was used, and those prepared under optimum conditions of temperature and pH were referred to as Production Examples 15 to 20, respectively.

Formulation Example 1 Powder 1
Formulation Formulation 1. Red Ganoderma Hot Water Extract Treated with End Type Protease (Production Example 1) 20
2. Dried corn starch 30
3. Microcrystalline cellulose 50
[Manufacturing method] Components 1 to 3 are mixed to make powder 1.

Formulation Example 2 Tablet formulation Formulation amount 1. Processed End Protease of Red Ganoderma Hot Water Extract (Production Example 1) 5
2. Dried corn starch 25
3. Carboxymethylcellulose calcium 20
4). Microcrystalline cellulose 40
5. Polyvinylpyrrolidone 7
6). Talc 3
[Production Method] Components 1 to 4 are mixed, and then an aqueous solution of Component 5 is added as a binder to form granules. Ingredient 6 is added to the formed granules and compressed. One tablet is 0.52 g.

Formulation Example 3 Tablet Confectionery Formulation amount 1. Red Ganoderma hydrothermal extract treated with End-type protease (Production Example 1) 2
2. Dried corn starch 50
3. Erythritol 40
4). Citric acid 5
5. Sucrose fatty acid ester 3
6). Perfume appropriate amount 7. Water Appropriate amount [Manufacturing method] Components 1 to 4 and 7 are mixed and granulated. Ingredients 5 and 6 are added to the formed granules and compressed. One tablet is 1.0 g.

Formulation Example 4 Beverage Formulation Processed End Protease of Red Ganoderma Hot Water Extract (Production Example 1) 5
2. Stevia 0.05
3. Malic acid 5
4). Fragrance 0.1
5. [Manufacturing method] totaling 100 with water Components 2 and 3 are dissolved in a small amount of water. Components 1, 4 and 5 are then added and mixed.

Comparative Example 1 Powder 2
In Formulation Example 1, powdered product 2 is obtained by replacing End Gaseous Hydrothermal Extract End-type protease-treated product (Production Example 1) with Red Ganoderma Hot Water Extract (Comparative Production Example 1).

  Next, experimental examples will be given to explain the effects of the present invention in detail.

Experimental Example 1 ACE inhibitory activity The antihypertensive effect was measured using ACE inhibitory activity as an index. The stronger the ACE inhibitory activity, the stronger the blood pressure lowering effect. The method for measuring the ACE inhibitory activity was in accordance with the modified Cushman method (Non-patent Document 2) of Tsutsumi et al.
That is, the extract of each production example is dissolved in water so as to be 500 μg / mL (in the case of mixing and using the extract of each production example, the total amount is 500 μg / mL) to obtain a sample solution. After adding 0.25 mL of a 3.0 mU / mL ACE solution to 0.5 mL of the sample solution and maintaining at 37 ° C. for 3 minutes, a 100 mM HEPES buffer solution (pH 8.3) solution containing 5 mM hippurylhistidylleucine 0 .25 mL was added. At this time, the sample concentration is 250 μg / mL. This was reacted at 37 ° C. for 30 minutes, and then 2.0 mL of 0.1M NaOH was added to stop the reaction. Next, 0.1 mL of a 0.2% orthophthalaldehyde methanol solution was added, and the mixture was left to stand at 0 ° C. for 15 minutes. To this, 0.4 mL of 1.5 M phosphoric acid solution was added to prepare a test solution, and fluorescence intensity (excitation wavelength: 360 nm, fluorescence wavelength: 480 nm) was measured. ACE inhibitory activity (%) is the fluorescence intensity of the test solution (C), the value when water is added instead of the sample (A), the enzyme blank of (C) (water added instead of enzyme) ) Is (D), and the value of the enzyme blank of (A) is (B).
ACE inhibition rate (%) = {1− (C−D) / (A−B)} × 100
J. et al. Wood Science, 44, 463, 1998

The results of these experiments are shown in Table 1. As a result, the enzyme-treated product of Ganoderma hot water extract showed an excellent ACE inhibitory activity as compared with Ganoderma hot water extract. The extracts of other production examples also showed excellent ACE inhibitory activity.

Experimental Example 2 Antihypertensive Action Production Examples 1, 6, and 10, Prescription Example 1, and Comparative Example 1 were used as samples.
Male spontaneously hypertensive rats (SHR) were bred with commercially available solid feed and tap water from the age of 10 to 12 weeks of age, 8 samples per group, 1 kg of body weight, and the samples were dispersed in water. Orally, blood pressure was measured before administration and 2 hours after administration. The dosage was 100 mg in the case of Production Examples 1, 6, and 10, and the powders 1 and 2 shown in Formulation Example 1 and Comparative Example 1 were 250 mg in terms of solid content. The blood pressure was measured at the tail artery using a noninvasive tail artery blood pressure measuring device. The average value of the maximum blood pressure was taken as the blood pressure value. Water was administered to the control group.

The results of these experiments are shown in Table 2. As a result, the preparation containing an enzyme-treated product of Ganoderma showed an excellent antihypertensive action. The extract of other production examples and the examples also showed excellent antihypertensive action.

From the above, the enzyme-treated product of Ganoderma of the present invention was superior in ACE inhibitory activity as compared with Ganoderma extract. Therefore, it is expected to be used as an active ingredient for foods, quasi-drugs or pharmaceuticals having a safe and excellent antihypertensive action.

Claims (7)

An antihypertensive agent containing an enzyme-treated product of ganoderma. The antihypertensive agent according to claim 1, wherein the enzyme-treated product of ganoderma is an enzyme-treated product obtained by enzyme-treating an extract of ganoderma lucidum. The antihypertensive agent according to claim 1 or 2, wherein the ganoderma is a fruit body of ganoderma. The antihypertensive agent according to any one of claims 1 to 3, wherein the ganoderma is one or more selected from the group consisting of red ganoderma, black ganoderma and maggot. The hypertension inhibitor according to any one of claims 1 to 4, wherein the enzyme is a proteolytic enzyme. The antihypertensive agent according to claim 5, wherein the proteolytic enzyme is an End-type protease. A food, quasi-drug or pharmaceutical comprising the antihypertensive agent according to any one of claims 1 to 6.