JP4949579B2 - Antihypertensive agent - Google Patents

Description

【００４２】
実験例２ 高血圧抑制作用
試料は、製造例１、３、４、８、製造例１と４の等量混合物、実施例１、２、及び比較例１を用いた。
雄性高血圧自然発症ラット（ＳＨＲ）を生後１０週齢から１２週齢まで市販の固形飼料と水道水で飼育し、上記試料を１群８匹、体重１ｋｇ当たり、上記の試料を水に分散させて経口投与し、投与前と投与２時間後の血圧を測定した。なお、投与量は、固形分に換算して、製造例１、３、４、８の場合１００ｍｇ、製造例１と４の等量混合物の場合は全量で１００ｍｇ、実施例１、２、及び比較例１で示される散剤１〜３については２５０ｍｇになるように投与した。血圧は、非観血式尾動脈血圧測定装置により、尾動脈で測定した。その最高血圧の平均値を血圧の値とした。対照群には水を投与した。
【００４３】
これらの実験結果を、表２に示した。その結果、黒霊芝の抽出物を含有する製剤は、優れた高血圧抑制作用を示した。更に、黒霊芝の熱水抽出物と霊芝（赤芝）の熱水抽出物を併用した場合、顕著な高血圧抑制作用を示した。また、マンネンタケ抽出物を吸着剤で精製した画分が特に優れた高血圧抑制作用を示した。その他の製造例の抽出物や実施例についても同様に優れた高血圧抑制作用を示した。
【００４４】
【表２】

【００４５】
【発明の効果】
以上のことから、本発明の黒霊芝の抽出物は、霊芝（赤芝）の抽出物と比較してＡＣＥ阻害活性作用に優れていた。さらに、黒霊芝及び霊芝（赤芝）抽出物を併用することにより、相乗効果を示した。また、マンネンタケ抽出物を吸着剤で精製した画分が特に優れたＡＣＥ阻害作用を有することも見出した。これらを含有する食品、医薬部外品又は医薬品は、安全で優れた高血圧抑制作用を示した。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a hypertension inhibitor mainly composed of an extract of black ganoderma, and more particularly, an extract that inhibits the activity of angiotensin converting enzyme (hereinafter referred to as ACE). The present invention relates to an antihypertensive agent containing the same. Furthermore, the present invention relates to a method for producing a fraction obtained by purifying nanentake extract with an adsorbent and a hypertension inhibitor containing the fraction.
[0002]
[Prior art]
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.
[0003]
Examples of such substances having ACE activity inhibition include synthetic compounds such as L-proline derivatives such as captopril, natural products derived from snake venom-derived bradykinin enhancing factors, peptides derived from tryptic digests such as bovine casein and sardines, and the like. It has been.
[0004]
Among basidiomycetes, shiitake mushroom (Lentinus edodes, Japanese Patent Laid-Open No. 58-26820), oyster mushroom (Pleurotus ostereatus, Japanese Patent Laid-Open No. 2-72121), maitake (Grifora frontosa, Japanese Patent Laid-Open No. 8-291788), Enokitake (Flamulinaes, Japanese Kaihei 8-291078), Buna shimeji (Lyophyllum ulmarium, JP-A-8-99895) and the like have been reported.
[0005]
In addition, in relation to garlic mushroom (reishi), a hypertension inhibitor (Japanese Unexamined Patent Publication No. 61-267527) containing an extract of garlic mushroom (reishi), and hypertension compounded with ganoderic acid in an alcoholic extract of mannentake (reishi). Inhibitors (Japanese Patent Laid-Open No. 60-208161) are known. The banyan mushroom used in Japanese Patent Application Laid-Open No. 61-267527 is a mushroom belonging to the department of Sarnosid moss and is described as including red turf, white turf, yellow turf, purple turf and the like. However, in this publication, it is only used as ganoderma, and red turf, white turf, yellow turf, purple turf, etc. were not compared in detail.
[0006]
In general, red mushrooms, white turf, yellow turf, purple turf, and black ganoder turf (black turf) are listed in the above publications, and the fruiting bodies are red, white, yellow, purple, black. Although it has been named simply, it has recently been researched separately from Ganoderma and Shishiba. For example, according to the literature [Japan Brewing Association Journal, 85 (6), 385-392, 1990], black ganoderma does not contain the typical triterpene component (ganoderic acid) contained in red turf or purple turf, and the taste It is reported that it is completely different. From the above, there was room for further research on the antihypertensive effect of black ganoderma.
[0007]
[Problems to be solved by the invention]
From the above, a hypertension inhibitor that is highly safe and excellent in ACE inhibitory action is desired.
[0008]
[Means for Solving the Problems]
Under such circumstances, as a result of intensive studies, the present inventors have found that, among the banyan mushrooms, black reishi extract has a particularly excellent ACE inhibitory action. Furthermore, it has been found that the combined use of black ganoderma and ganoderma (red turf) is more synergistic than the use of black ganoderma alone. In addition, the present inventors have found that a fraction obtained by purifying Mannentake extract with an adsorbent has a particularly excellent ACE inhibitory action, and have completed the present invention.
[0009]
The black ganoderma (Kuroshiba) and the ganoderma (Akashiba) used in the present invention are basidiomycetes used in the herbal medicine “Ganoderma lucidum”, belong to the genus Mannenaceae and the genus Mannentake, and are a kind of Mannentake. According to the literature [Acta Microbiological Sinica, 19 (3), 265-279, 1979], the scientific name is classified into Kuro Ganoderma (Koshiba, scientific name: Ganoderma arum), Ganoderma (Akashiba, scientific name: Ganoderma lucidum). However, the classification method is not limited to this document. As the black ganoderma and ganoderma (red turf) used in the present invention, those widely distributed in China and the Japanese market can be used.
[0010]
The extract of black ganoderma or ganoderma (Akashiba) used in the present invention can be extracted from fruit bodies, mycelia, mycelium cultures, and the like. Preferably, an extract from a fruit body is good. The extraction method is not particularly limited, and for example, it may be a heat extraction or a room temperature extraction. The mycelium or mycelium culture can be produced by a conventional method.
[0011]
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). , Glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Is mentioned. Preferable are polar solvents such as water and lower alcohols, and particularly preferable are water and ethanol. These solvents may be used alone or in combination of two or more.
[0012]
The above extract may be used as it is, or may be used after treatment such as concentration, dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation or the like, if necessary. Further, the extracted solution may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.
[0013]
Furthermore, the fraction excellent in the antihypertensive effect can be obtained by adsorbing the mannentake extract to an adsorbent and eluting it with a solvent.
[0014]
Examples of the adsorbent include adsorbents such as styrene-divinylbenzene synthetic adsorption resin, anion exchange resin, octadecyl group chemically bonded silica gel (ODS), cellulose, dextran, and modified dextran. Among them, a styrene-divinylbenzene synthetic adsorption resin is preferable from the viewpoint of recovery.
[0015]
Examples of the adsorption method include, but are not particularly limited to, a method in which an adsorbent is packed in a column and the extract solution is passed through the column, and a method in which the adsorbent is directly mixed with the extract solution.
[0016]
The elution solvent for the adsorbent can be selected depending on the characteristics of each adsorbent, and examples thereof include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid many Monohydric alcohol (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, etc.), And ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Preferable are polar solvents such as water and lower alcohols, and particularly preferable is water and ethanol. These solvents may be used alone or in combination of two or more. Furthermore, the pH may be adjusted by adding acid or alkali to the elution solvent.
[0017]
The purified product may be used as a solution, or may be used after treatment such as concentration, dilution, filtration, decolorization with activated carbon, deodorization, or the like, if necessary. Furthermore, it may be used as a dried product by performing treatment such as concentration to dryness, spray drying, freeze drying and the like.
[0018]
As a particularly preferred example, an extract of Mannentake (or a solution if the extract is solid) is passed through a column packed with a synthetic adsorption resin of styrene-divinylbenzene and adsorbed. Next, purified water is passed through and washed, and then fractions can be eluted and collected by passing hydrous alcohol (for example, ethanol as alcohol) or alcohol through the column. The obtained fraction can be concentrated under reduced pressure and spray-dried or freeze-dried to obtain the antihypertensive agent of the present invention.
[0019]
In the hypertension inhibitor of the present invention, the above extract or purified product by the adsorbent may be used as it is, and a diluent can be used as long as the effect of the extract is not impaired. Liquid, semi-solid may be used, for example: That is, excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, fragrances, preservatives, solubilizers, solvents, and the like. 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, lauric fat, petrolatum, paraffin, higher alcohol and the like.
[0020]
The antihypertensive agent of the present invention can be used in any of foods, quasi drugs, and pharmaceuticals. Examples of the dosage form include powders, granules, tablets, dragees, capsules, and syrups for oral use. Agents, pills, suspensions, solutions, emulsions and the like. It can be an injection solution for parenteral use. It can also be a suppository.
[0021]
The amount of the extract used in the present invention is 0.0001% by weight or more, preferably 0.001 to 90% by weight, in terms of solid matter, based on the total amount of the antihypertensive agent of the present invention. If it is less than 0.0001% by weight, a sufficient effect is hardly expected. When the blending amount exceeds 90% by weight, the effect is hardly increased and it is uneconomical. In addition, the addition method may be added in advance or during the production, and may be appropriately selected in consideration of workability.
[0022]
Among the above-mentioned extracts used in the present invention, a synergistic antihypertensive effect is exhibited by using a combination of black ganoderma and reishi (Akashiba) extracts. The blend ratio of the extract of black ganoderma and ganoderma (red turf) is 5:95 to 95: 5, preferably 10:90 to 90:10 in terms of the weight ratio of the solid.
[0023]
【Example】
Next, in order to describe the present invention in detail, examples of producing the extract 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 amount shown in the examples means part by weight, and% means% by weight.
[0024]
Production Example 1 Black Ganoderma Hot Water Extract 400 g of purified water was added to 20 g of dried ganoderma fruit body, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated and freeze-dried. 1.4 g of hot water extract of black reishi was obtained.
[0025]
Production Example 2 50% Ethanol Extract of Black Ganoderma 1L of purified water and 1L of ethanol were added to 100g of dried ganoderma fruit bodies, extracted for 7 days at room temperature, filtered, and the filtrate was concentrated and lyophilized. As a result, 1.3 g of a 50% ethanol extract of black ganoderma was obtained.
[0026]
Production Example 3 Black Ganoderma Ethanol Extract To 100 g of dried ganoderma fruit body, 2 L of ethanol was added, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 0.9 g of extract was obtained.
[0027]
Manufacture example 4 Hot water extract of Ganoderma (Akashiba) 400 mL of purified water was added to 20 g of dried ganoderma (Akashiba) fruit body, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated. Then, 2.0 g of hot water extract of Reishi (Akashiba) was obtained by lyophilization.
[0028]
Production Example 5 50% Ethanol Extract of Ganoderma (Akashiba) 1 L of purified water and 1 L of ethanol are added to 100 g of dried ganoderma (Akashiba) fruit body, extracted at room temperature for 7 days, filtered, and the filtrate is concentrated. And lyophilized to obtain 1.8 g of a 50% ethanol extract of Reishi (Akashiba).
[0029]
Production Example 6 Ganoderma (Akashiba) ethanol extract 2 g of ethanol was added to 100 g of dried ganoderma (Akashiba) fruiting body, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 1.0 g of ethanol extract of Reishi (Akashiba) was obtained.
[0030]
Production Example 7 Extract of fungus body culture of black ganoderma cultivated using a jar fermenter. That is, 50 g of malt extract, 20 g of yeast extract, and 20 g of glucose were mixed with 5 L of purified water, placed in a jar, and then sterilized at 121 ° C. for 30 minutes. This was attached to a jar fermenter and inoculated with 150 mL of Kuro Reishi seed mycelium culture cultured in the same medium with shaking at 25 ° C. for 14 days. 300r. p. The mixture was cultured at 25 ° C. for 14 days with aeration of 3 L / min with aeration of m. The obtained culture was filtered to obtain 2.0 kg (wet weight) of mycelia.
2 L of purified water was added to the separated mycelium and extracted at 95-100 ° C. for 2 hours, followed by filtration. The filtrate was concentrated and freeze-dried to obtain 22 g of an extract of the fungus body culture of Kuro Reishi. .
[0031]
Production Example 8 Purified Column of Black Ganoderma Column Dissolved 2.0 g of hot water extract of Black Ganoderma fruit body of Production Example 1 in 50 mL of purified water and packed in 350 mL of styrene-divinylbenzene synthetic adsorption resin. The liquid was passed. Next, 750 mL of purified water was passed through and washed, followed by elution with 750 mL of 25%, 50%, and 100% ethanol. The obtained fraction was concentrated and lyophilized to obtain 0.4 g of 25% ethanol fraction, 0.2 g of 50% ethanol fraction, and 0.2 g of 100% ethanol fraction. These were used in Experimental Example 1.
Similarly, fractions eluted with 750 mL of 25%, 50%, and 100% ethanol were mixed and concentrated, and lyophilized to obtain 0.8 g of purified Kuro Ganoderma column.
[0032]
Example 1 Powder 1
Formulation Formulation 1. Black Reishi hot water extract (Production Example 1) 10 parts2. Reishi (Akashiba) hot water extract (Production Example 4) 10
3. Dried corn starch 30
4). Microcrystalline cellulose 50
[Manufacturing method] Components 1 to 4 are mixed to prepare powder 1.
[0033]
Example 2 Powder 2
In Example 1, the hot water extract of Reishi (Akashiba) (Production Example 4) was replaced with the hot water extract of Black Ganoderma (Production Example 1).
[0034]
Comparative Example 1 Powder 3
In Example 1, powdered powder 3 was obtained by replacing the hot water extract of black ganoderma (Production Example 1) with the hot water extract of Ganoderma (Akashiba) (Production Example 4).
[0035]
Example 3 Tablet formulation Black Reishi lawn ethanol extract (Production Example 3) 4 parts Reishi (Akashiba) ethanol extract (Production Example 6) 1
3. Dried corn starch 25
4). Carboxymethylcellulose calcium 20
5. Microcrystalline cellulose 40
6). Polyvinylpyrrolidone 7
7). Talc 3
[Production Method] Components 1 to 5 are mixed, and then an aqueous solution of Component 6 is added as a binder to form granules. Ingredient 7 is added to the formed granules and tableted. One tablet is 0.52 g.
[0036]
Example 4 Tablet Confection Formulation Black Reishi lawn 50% ethanol extract (Production Example 2) 1 part2. 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 [Production 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.
[0037]
Example 5 Beverage formulation Purified column of black reishi (Production Example 8) 2 parts Reishi (Akashiba) hot water extract (Production Example 4) 2
3. Stevia 0.05
4). Malic acid 5
5. Fragrance 0.1
6). [Method] Make the total amount 100 with water. Dissolve components 3 and 4 in a small amount of water. Components 1, 2, 5 and 6 are then added and mixed.
[0038]
Next, experimental examples will be given to explain the effects of the present invention in detail.
[0039]
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 Tsutsumi et al.'S improved Cushman method (J. Wood Science, 44, 463, 1998).
That is, the extract of each production example is dissolved in a water / dimethyl sulfoxide mixed solution (9: 1) so as to be 400 μg / mL (when the extract of each production example is mixed and used in a total amount of 400 μg / mL). A sample solution is used. 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 (pH 8.3) solution containing 5 mM hippurylhistidylleucine 0 .25 mL was added. This was reacted at 37 ° C. for 30 minutes, and then 2.0 mL of 0.1 M sodium hydroxide 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
[0040]
The results of these experiments are shown in Table 1. As a result, the hot water extract of black ganoderma showed superior ACE inhibitory activity compared with the hot water extract of ganoderma (Akashiba). Furthermore, when the hot water extract of Kuro Reishi and the hot water extract of Reishi (Akashiba) were used in combination, a remarkable ACE inhibitory activity was exhibited. In addition, the fraction obtained by purifying Mannentake extract with an adsorbent showed particularly excellent ACE inhibitory action. The extracts of other production examples also showed excellent ACE inhibitory activity.
[0041]
[Table 1]

[0042]
Experimental Example 2 As the antihypertensive sample, Production Examples 1, 3, 4, 8 and an equal mixture of Production Examples 1 and 4, Examples 1, 2 and Comparative Example 1 were used.
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. In addition, in terms of solid content, the dosage is 100 mg for Production Examples 1, 3, 4, and 8, 100 mg in the case of the equivalent mixture of Production Examples 1 and 4, and Examples 1, 2 and Comparative Powders 1 to 3 shown in Example 1 were administered at 250 mg. 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.
[0043]
The results of these experiments are shown in Table 2. As a result, the preparation containing the black reishi extract showed an excellent antihypertensive action. Further, when the hot water extract of black ganoderma turf and the hot water extract of ganoderma (Akashiba) were used in combination, a remarkable antihypertensive effect was shown. In addition, the fraction obtained by purifying mannentake extract with an adsorbent showed particularly excellent antihypertensive action. The extract of other production examples and the examples also showed excellent antihypertensive action.
[0044]
[Table 2]

[0045]
【Effect of the invention】
From the above, the black ganoderma extract of the present invention was superior in ACE inhibitory activity as compared with the ganoderma (Akashiba) extract. Furthermore, the synergistic effect was shown by using black reishi and reishi (Akashiba) extract together. In addition, the present inventors also found that a fraction obtained by purifying mannentake extract with an adsorbent has a particularly excellent ACE inhibitory action. Foods, quasi-drugs or pharmaceuticals containing these showed safe and excellent antihypertensive action.

Claims (1)

An antihypertensive agent comprising an extract of black reishi and reishi (Akashiba) in a ratio of 1: 1 as a weight ratio of solids.