JP7463058B2 - Compositions with ACE inhibitory activity - Google Patents

Description

The present invention relates to a composition that contains a plant extract and has angiotensin converting enzyme (ACE) inhibitory activity.

Licorice, derived from a plant belonging to the genus Glycyrrhiza in the family Fabaceae, has been used as a herbal medicine since ancient times and is known to have various effects such as antispasmodic, analgesic, antitussive, and expectorant.

Also known are compositions containing a licorice hydrophobic extract for preventing or improving hypertension and the like (Patent Documents 1 and 2), and a preparation for lowering blood pressure and the like that contains a licorice extract that does not contain glycyrrhizinic acid (Patent Document 3).

Furthermore, it is known that licorice extract has the effect of suppressing the unpleasant odor inherent to sardine peptides by adding licorice extract to a composition containing sardine peptides (sardine peptides) that have a blood pressure lowering effect (Patent Document 4).

However, it has not been known until now that licorice extract has ACE inhibitory effects.

International Publication No. 2002/047699 International Publication No. 2008/143182 JP 2002-114695 A JP 2009-284900 A

The present invention aims to provide a composition having ACE inhibitory activity. It also aims to provide a food or medicine containing the composition as an active ingredient for preventing or alleviating ACE-related diseases.

As a result of extensive investigations, the present inventors have found that an extract from a plant belonging to the genus Glycyrrhiza of the family Leguminosae has ACE inhibitory activity, leading to the completion of the present invention.
That is, the present invention includes the following aspects.
[1] A composition having angiotensin-converting enzyme (ACE) inhibitory activity, characterized by containing an extract of a plant belonging to the genus Glycyrrhiza of the family Leguminosae.
[2] The composition described in [1], wherein the extract of a plant belonging to the genus Glycyrrhiza of the family Fabaceae is an extract extracted with water, an organic solvent, or a mixture thereof.
[3] The composition according to [1] or [2], wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, acetone, 1-butanol, 2-butanol, 2-methyl-2-propanol, propylene glycol, glycerin, methyl acetate, ethyl acetate, hexane, diethyl ether, 2-butanone, cyclohexane, dichloromethane, edible fats and oils, propane, butane, 1,1,1,2-tetrafluoroethane, and 1,1,2-trichloroethene.
[4] The composition according to any one of [1] to [3], wherein the extract of a plant belonging to the genus Glycyrrhiza of the family Fabaceae is an extract from the roots and/or stolons.
[5] The composition according to any one of [1] to [4], wherein the concentration of the extract of a plant belonging to the genus Glycyrrhiza of the family Fabaceae is 0.10 mg/mL or more.
[6] The composition according to any one of [1] to [5], wherein the plant belonging to the genus Glycyrrhiza of the family Fabaceae is the species Glycyrrhiza uralensis.
[7] A food or medicine for preventing or alleviating a disease associated with angiotensin-converting enzyme (ACE), comprising the composition according to any one of [1] to [6] as an active ingredient.

The composition of the present invention has ACE inhibitory activity and can therefore prevent or alleviate ACE-related diseases. Furthermore, the composition of the present invention contains a naturally derived extract and is therefore highly safe. Therefore, the composition of the present invention is useful as a food or medicine for preventing or alleviating ACE-related diseases.

The composition of the present invention contains an extract from a plant belonging to the genus Glycyrrhiza of the family Fabaceae. Examples of plants belonging to the genus Glycyrrhiza of the family Fabaceae include Glycyrrhiza acanthocarpa, G. aspera, G. astragalina, G. bucharica, G. echinata (Russian Glycyrrhiza species), G. eglandulosa, G. foetida, G. foetidissima, G. glabra (Spanish Glycyrrhiza species), G. gontscharovii, G. iconica, G. inflate (Xinjiang Glycyrrhiza species), G. korshinskyi, G. lepidota (American Glycyrrhiza species), G. pallidiflora, G. squamulosa, G. triphylla, G. uralensis (Ural Glycyrrhiza species), and G. yunnanensis. Variants of these plants belonging to the genus Glycyrrhiza of the family Fabaceae can also be used. Glycyrrhiza species and Ural Glycyrrhiza species are preferred, and Ural Glycyrrhiza species is more preferred. In the present invention, for example, Miyako kanzo (also called "Miyako No. 1"), which belongs to the genus Glycyrrhiza of the family Fabaceae, is used.

The characteristics of Miyako kanzo are "very tall, very many roots, very thick roots, brown root skin, strong yellow coloring in root cross section, medium number of stems, no anthocyanin coloring in stems, none or coarse stem hairs, long leaf, very wide leaf, medium leaf waviness, medium number of leaflets, very long leaf blade, very wide leaf blade, somewhat many inflorescences, medium inflorescence, quite many florets, N81A flower color, very heavy dry root weight, somewhat high glycyrrhizic acid content (RHS color chart used)." Miyako kanzo is distinguishable from the Kitano Agricultural Experiment Station strain by its considerably taller plant height and considerably longer leaf blade. It is also distinguishable from the Hokkaido University strain by its considerably taller plant height and considerably longer leaf blade. Furthermore, compared to "Medical Series," it is distinguishable by its significantly taller plant height, significantly longer leaf blade length, and later wilting.

The extract contained in the composition of the present invention is, for example, an extract from the roots, stolons, leaves, stems or a mixture of these parts of a plant belonging to the genus Glycyrrhiza of the Leguminosae family. In the composition of the present invention, the extract may be from the roots and/or stolons, or from the leaves and/or stems. Preferably, it is an extract from the roots and/or stolons of a plant belonging to the genus Glycyrrhiza of the Leguminosae family, and more preferably, it is an extract from the roots and/or stolons of Licorice japonica.

The extract of the present invention is obtained by extracting the plant using a solvent normally used for plant extraction according to a standard method. For example, the extract can be obtained by immersing the plant in the extraction solvent and leaving it to stand or stirring it.

The plants used for extraction can be in their raw form (fresh), dried form, or chopped or crushed form.

Solvents used for extraction include those conventionally used for plant extraction, such as water, an organic solvent, or a mixture thereof.
Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, acetone, 1-butanol, 2-butanol, 2-methyl-2-propanol, propylene glycol, glycerin, methyl acetate, ethyl acetate, hexane, diethyl ether, 2-butanone, cyclohexane, dichloromethane, edible fats and oils, propane, butane, 1,1,1,2-tetrafluoroethane, and 1,1,2-trichloroethene.
Preferred examples include water, ethanol, or a mixture thereof. When a mixture of water and ethanol is used, the ethanol concentration in the mixture is, for example, 0.1 to 99.5 v/v%, preferably 5 to 80 v/v%, and more preferably 10 to 70 v/v%.

The extraction method may be any method that is commonly used, and for example, extraction can be performed using any device at room temperature or under heating. Specifically, the plant is placed in a treatment tank filled with a solvent, stirred from time to time to dissolve, and then centrifuged and filtered as necessary to remove the extraction residue, thereby obtaining an extract. Alternatively, a method using a supercritical fluid consisting of water, nitrous oxide, carbon dioxide, etc. may be used as an extraction method.

The amount of the solvent used for extraction is not particularly limited, but is, for example, 1 to 100 mL, preferably 5 to 50 mL, per gram of plant (calculated as dry weight).
The extraction conditions are not particularly limited as long as they are sufficient for extraction, but generally, the lower the extraction temperature, the longer the extraction time, and the higher the extraction solvent, the shorter the extraction time. The extraction temperature is appropriately set in the range of 0 to 100°C, and is preferably in the range of room temperature (about 25°C) to the boiling point of the extraction solvent. The extraction time is set in the range of 10 minutes to 24 hours, and is preferably in the range of 30 minutes to 5 hours. When water is used as the extraction solvent, the extraction conditions are, for example, about 20 to 95°C and about 30 minutes to 3 hours. When ethanol or a mixed solvent of water and ethanol is used as the extraction solvent, the extraction conditions are, for example, about 20 to 80°C and about 30 minutes to 3 hours.
The extraction may be performed once or multiple times to increase the yield.

The obtained extract can be used as it is as the composition of the present invention. If necessary, the extract may be subjected to purification for the purpose of decolorization or deodorization. Examples of purification methods include activated carbon treatment, adsorption treatment, and ion exchange resin treatment.

The extract may be further concentrated or concentrated to dryness, diluted with a suitable solvent, or dried to a powder after adding an excipient as necessary. Methods for concentration or concentration to dryness include, for example, vacuum concentration (e.g., a method using an evaporator) and membrane concentration. Methods for powdering include, for example, spray drying, freeze drying, and fluidized bed drying. Excipients used to powderize the extract include those that have been conventionally used, such as modified starch and dextrin.

The "extract" in this invention includes various solvent extracts obtained by the above-mentioned extraction methods, their purified fractions, their dilutions, their concentrates, and their dried products (powders, etc.).

The amount of the extract from the plant belonging to the genus Licorice of the family Leguminosae contained in the composition of the present invention is not particularly limited as long as it is an amount that can exert a desired effect.For example, when the composition of the present invention is a liquid composition, the lower limit of the concentration in the liquid composition (dry weight of the extract/volume of the liquid composition) is, for example, 0.01 mg/mL or more, preferably 0.10 mg/mL or more, and the upper limit is, for example, 100 mg/mL or less, preferably 10 mg/mL or less.
When the composition of the present invention is a solid composition, the lower limit of the blending ratio in the solid composition (dry weight of extract/dry weight of composition) is, for example, 0.01 wt % or more, preferably 0.1 wt % or more, and the upper limit is, for example, 99 wt % or less, preferably 50 wt % or less.

The extract of the present invention has an ACE activity inhibitory effect, and therefore can prevent or alleviate ACE-related diseases. ACE produces angiotensin II, which has a vasoconstricting effect in the renin-angiotensin system, which is a hypertensive system, while it is also an enzyme that breaks down bradykinin, which has a hypotensive effect. Therefore, inhibiting ACE activity produces a hypotensive effect through two mechanisms: suppressing the production of angiotensin II and suppressing the inactivation of bradykinin. Therefore, the composition of the present invention exhibits a blood pressure improving effect, for example, a blood pressure lowering effect, through its ACE activity inhibitory effect.

In this context, "improving blood pressure" includes improving vasodilation in mammals such as humans, maintaining normal vasodilation, lowering blood pressure, maintaining stable blood pressure, and protecting endothelial cells of blood vessels from degeneration. Vasodilatation also refers to the dilation of blood vessels, particularly the dilation of arterioles, which results in increased blood flow to a portion of a blood vessel.

In addition, the extract of the present invention is highly safe because it is derived from a natural plant belonging to the genus Glycyrrhiza of the family Leguminosae. Therefore, the composition of the present invention is useful as a food and medicine, particularly as a food and medicine for preventing or alleviating ACE-related diseases.

The pharmaceutical product of the present invention is prepared by using the above extract or the above composition according to a conventional method. For example, the above extract or the above composition can be added to or mixed with a pharma- ceutically acceptable carrier or additive commonly used in the field of formulation technology, and then made into a dosage form for oral administration, such as tablets, capsules, granules, powders, oral liquids, syrups, etc.
Examples of the carrier or additive include excipients, disintegrants, binders, lubricants, colorants, pH adjusters, surfactants, stabilizers, acidulants, flavors, etc. These additives are used in amounts conventionally used in the technical field of formulations.

Examples of excipients include starches such as corn starch, potato starch, wheat corn starch, rice starch, partially pregelatinized starch, pregelatinized starch, and porous starch; sugars or sugar alcohols such as lactose hydrate, sucrose, fructose, glucose, mannitol, sorbitol, erythritol, xylitol, trehalose, maltitol, powdered reduced maltose syrup, and lactitol; anhydrous calcium hydrogen phosphate, crystalline cellulose, powdered cellulose, precipitated calcium carbonate, and calcium carbonate.
Examples of disintegrants that can be used include carmellose, carmellose calcium, sodium carboxymethyl starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl starch, croscarmellose sodium, crospovidone, low-substituted hydroxypropyl cellulose (L-HPC), and hydroxypropyl starch, and preferably, croscarmellose sodium and L-HPC.
Examples of binders that can be used include hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, copolyvidone, powdered acacia, methylcellulose, low-substituted hydroxypropyl cellulose, hypromellose, sodium carmellose, dextrin, partially pregelatinized starch, pullulan, gum acacia, agar, gelatin, tragacanth, and sodium alginate, and preferred are hydroxypropyl cellulose and hydroxypropyl methylcellulose.
Examples of the fluidizing agent that can be used include light anhydrous silicic acid, hydrous silicon dioxide, calcium silicate, magnesium silicate, magnesium aluminometasilicate, and talc, with light anhydrous silicic acid and magnesium aluminometasilicate being preferred.
Examples of lubricants include stearic acid, magnesium stearate, calcium stearate, talc, and sucrose fatty acid esters.
Examples of coloring agents include yellow ferric oxide, ferric oxide, Food Blue No. 1, Food Blue No. 2, Food Yellow No. 4, Food Yellow No. 5, Food Green No. 3, Food Red No. 2, Food Red No. 3, Food Red No. 102, Food Red No. 104, Food Red No. 105, Food Red No. 106, food lake color, riboflavin, and riboflavin sodium phosphate.
Examples of pH adjusters include citric acid, phosphoric acid, carbonic acid, tartaric acid, fumaric acid, acetic acid, amino acids, and salts thereof.
Surfactants include sodium lauryl sulfate, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and the like.
Examples of the stabilizer include tocopherol, tetrasodium edetate, nicotinamide, and cyclodextrins.
Examples of acidulants include ascorbic acid, citric acid, tartaric acid, and malic acid.
Examples of flavorings include L-menthol, peppermint oil, lemon oil, vanillin, and the like.
The above-mentioned carriers or additives may be used in a suitable mixture of two or more kinds.

The pharmaceutical product of the present invention is preferably in a unit dosage form containing 0.05 to 5 g, preferably 0.1 to 1.5 g, of the extract calculated as a dry weight. Here, "unit dosage form" means a physically separate unit, each unit containing a predetermined amount of active ingredient calculated to obtain a desired effect together with a carrier or additive.

The dosage of the pharmaceutical of the present invention varies depending on the age, sex, weight, and condition of the subject to be administered, as well as the administration route, administration schedule, dosage form, and formulation composition, but is, for example, 1 to 1000 mg, preferably 5 to 500 mg. The dosage may be administered once a day, or in two or more divided doses.

The food of the present invention is produced by a conventional method using the extract or composition. For example, it is produced by a known means such as adding the extract or composition to a food, its food ingredients, or its intermediate food materials. For example, it is produced by extracting a plant belonging to the genus Glycyrrhiza of the family Leguminosae with water or ethanol, concentrating the extract, adding the concentrated extract to a food material, and then preparing the food by a conventional method.

Examples of the above foods include snacks (e.g., snacks such as potato chips, baked goods such as biscuits and cookies, chocolate, gum, candy, etc.), desserts (e.g., pudding, jelly, yogurt, ice cream, etc.), staple foods such as noodles (e.g., soba, udon, ramen, pasta, etc.), cereal foods (e.g., corn flakes, oatmeal, etc.), seasonings (e.g., soup, curry, stew, etc.), processed agricultural products (e.g., jam, etc.), dairy oil foods (e.g., spreads, cheese, etc.), health foods (e.g., protein, fiber, etc.), calorie-adjusted foods, non-alcoholic beverages (e.g., roasted soybean tea beverages, grain tea, coffee beverages, black tea beverages, green tea beverages, barley tea beverages, matcha beverages, vegetable juice beverages, orange juice, grapefruit juice, soft drinks, etc.), and alcoholic beverages (e.g., beer, wine, sake, sparkling wine, plum wine, whiskey, brandy, shochu, vodka, rum, gin, liqueurs, cocktails, etc.).

The food of the present invention can be, for example, a general food, including nutritional supplements, health supplements, and nutritionally adjusted foods, as well as a functional health food, including a food for specified health uses, a food with nutritional functions, and a food with functional claims. For example, the food of the present invention can be a functional food that displays, based on scientific evidence, that it is expected to have a specific health purpose (for example, "It has been reported to have the function of lowering blood pressure, and is suitable for people with high blood pressure"). The food of the present invention may also be a supplement in a form for oral intake, such as a tablet, capsule, powder, granule, liquid, or syrup.

The amount of the extract-containing composition contained in the food of the present invention may be any amount that can exert the desired effect, and may be appropriately determined depending on the general intake amount of the food, the type of food, the composition, etc. For example, the food of the present invention may contain the extract-containing composition in the range of 0.1 to 99.0% by dry weight.

The intake amount of the food of the present invention may vary depending on the age, sex, weight, and condition of the subject taking the food, as well as the type and composition of the food, and is, for example, 1 to 1000 mg, preferably 5 to 500 mg. The intake amount may be taken once a day, or in two or three or more divided doses.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[Examples 1 to 5] Preparation of aqueous extract from liquorice roots and stolons Approximately 30 L of water was added to 3 kg of liquorice roots and stolons, and the mixture was extracted at 90°C for approximately 2 hours, followed by mesh filtration to obtain 23.2 L of extract. This extract was then concentrated under reduced pressure at approximately 60°C to obtain 2544 g of concentrate. 2511 g of this concentrate was spray-dried to obtain 608 g of dry extract.
Approximately 1.0 g of the above dried extract was extracted with 20 ml of 50% ethanol solution and then appropriately diluted with 0.1 mol/l HEPES buffer (pH 8.3) to obtain aqueous extract-containing compositions from the roots and stolons of Licorice glabra having sample concentrations of 0.1 mg/mL (Example 1), 0.125 mg/mL (Example 2), 0.15 mg/mL (Example 3), 0.2 mg/mL (Example 4) and 0.225 mg/mL (Example 5).

[Examples 6-10] Preparation of an ethanol extract from liquorice roots and stolon Approximately 30 L of 30% ethanol was added to 3 kg of liquorice roots and stolon, and the mixture was extracted at 60°C for approximately 2 hours, followed by mesh filtration to obtain 25.2 L of extract. This extract was further concentrated under reduced pressure at approximately 60°C to obtain 2849 g of a concentrate. 2802 g of this concentrate was spray-dried to obtain 636 g of a dry extract.
Approximately 1.0 g of the above dried extract was extracted with 20 ml of 50% ethanol solution and then appropriately diluted with 0.1 mol/l HEPES buffer (pH 8.3) to obtain compositions containing ethanol extracts from the roots and stolons of Licorice glabra having sample concentrations of 0.1 mg/mL (Example 6), 0.125 mg/mL (Example 7), 0.15 mg/mL (Example 8), 0.175 mg/mL (Example 9) and 0.2 mg/mL (Example 10).

[Test Example]
1. Test Overview The angiotensin-converting enzyme (ACE) activity test was carried out based on the method described in Nakno et al. Biosci Biotechnol. Biochem. , 70, 1118-1126 (2006), in which the dipeptide decomposed by ACE from the substrate (His-His-Leu) was fluorescentized with orthophthalaldehyde (hereinafter abbreviated as "OPA"). The fluorescence intensity of the reaction product was then measured. The ACE activity inhibition was evaluated based on the relative ACE activity when the activity of the untreated group to which no test solution was added was taken as 100%. The IC ₅₀ value was calculated from a graph of the sample concentration (mg/ml) and the inhibition rate (%).

2. Samples The compositions obtained in the above examples were used as test solutions for samples 1) and 2) below. In addition, Sarden Peptide (product name: Sarden Peptide N100, manufacturer: Senmi Extract Co., Ltd.) was used as a positive control (sample 3).
1) Aqueous extract from the root and stolon of Licorice Root (Examples 1 to 5)
2) Ethanol extract from the root and stolon of Licorice Root (Examples 6 to 10)
3) Sardine peptide (positive control)

3. Test method 25 μl of 0.1 mol/l HEPES buffer (pH 8.3) (untreated group) or each test solution was added to a 96-well microplate, 25 μl of 20 mU/ml ACE solution was added, and the mixture was incubated at 37°C for 5 minutes. 25 μl of 8 mmol/l His-His-Leu solution was added, and the mixture was reacted at 37°C for 30 minutes. Then, 25 μl of 0.1 mol/l sodium hydroxide solution was added to stop the reaction, 25 μl of 1% OPA solution was added, and the mixture was left at room temperature for 20 minutes. Furthermore, 25 μl of 0.1 mol/l hydrochloric acid was added, and the mixture was left at room temperature for 10 minutes, and the fluorescence intensity was measured with a microplate reader. Note that the blank was tested in the same manner using PBS instead of 20 mU/ml ACE solution.

<Microplate reader operating conditions>
Model: SpectraMax M2e
Measurement conditions: fluorescence, endpoint mode, bottom read Excitation wavelength: 355 nm
Fluorescence wavelength: 460 nm

4. Test results The inhibition rate in each test solution is shown in Table 1. As a result, a concentration-dependent enhancement of ACE activity inhibition was observed. The _IC50 value calculated from the graph showing the relationship between the sample concentration and the inhibition rate is shown in Table 2.

The composition of the present invention has ACE inhibitory activity and can therefore prevent or alleviate ACE-related diseases. Furthermore, the composition of the present invention contains extracts of natural origin and is therefore highly safe. Therefore, the composition of the present invention can be used as a food or medicine for preventing or alleviating ACE-related diseases.

Claims (2)

A food or medicine for improving or lowering blood pressure, which contains as an active ingredient a composition having angiotensin-converting enzyme (ACE) inhibitory activity, characterized in that it contains an extract of a plant belonging to the genus Glycyrrhiza of the family Fabaceae, wherein the extract of a plant belonging to the genus Glycyrrhiza of the family Fabaceae is a water extract of the root and/or stolon of a plant belonging to the species Glycyrrhiza uralensis of the genus Glycyrrhiza of the family Fabaceae . 2. The food or medicine according to claim 1, wherein the concentration of the extract of a plant belonging to the genus Glycyrrhiza of the family Fabaceae is 0.10 mg/mL or more.