JP2021130652A - Hypotensive agent, anti saccharization agent, antioxidant, anti aging agent, antiallergic agent, immunostimulator, and periodontal disease bacteria inhibitor - Google Patents

Abstract

To provide a hypotensive agent, an anti saccharization agent, an antioxidant, an anti aging agent, an antiallergic agent, an immunostimulator, a periodontal disease bacteria inhibitor, and a food product composition, a medicine composition, and a cosmetic composition including an extract of mountain herb.SOLUTION: There are provided a hypotensive agent, an anti saccharization agent, an antioxidant, an anti aging agent, an antiallergic agent, an immunostimulator, a periodontal disease bacteria inhibitor, and a food product composition, a medicine composition, and a cosmetic composition including an extract of mountain herb, and having at least any of hypotension action, saccharized product production suppression action, antioxidant action, anti aging action, antiallergic action, immunopotentiative action, and periodontal disease bacteria inhibition action.SELECTED DRAWING: None

Description

The present invention includes antihypertensive agents, anti-glycation agents, antioxidants, anti-aging agents, anti-allergic agents, immunostimulators, periodontal disease suppressants, and food compositions and pharmaceutical compositions containing extracts of mountain herbs. , Cosmetic composition.

Hypertension is one of the lifestyle-related diseases. Angiotensin converting enzyme inhibitors and the like are used for the prevention and treatment of hypertension. It is possible to suppress the increase in blood pressure by inhibiting the activity of angiotensin converting enzyme. Various naturally-derived components and chemical compounds have been reported as such antihypertensive agents. As naturally derived components, for example, tea extract, mushroom extract, corn extract and the like are known (see Non-Patent Document 1, Non-Patent Document 2 and Non-Patent Document 3). Among such ingredients derived from natural products, those with high safety can be expected to be blended not only in pharmaceutical products but also in foods and drinks, and therefore, development of a new antihypertensive agent derived from natural products is required.

Advanced glycation end products (AGEs) are produced in vivo by a Maillard reaction between a reducing sugar such as glucose and a protein. Not only do AGEs cause diabetic complications, but the accumulation of AGEs in the skin contributes to the deterioration of the entire skin, and in diabetic patients, AGEs caused by hyperglycemia cause cataracts, arteriosclerosis, and renal function. Causes complications such as dysfunction (Non-Patent Document 4). Therefore, it is considered that a substance that inhibits the glycation reaction and suppresses the production of AGEs and a substance that decomposes the produced AGEs can suppress various age-related diseases and diabetic complications.
In recent years, it has been desired to develop an anti-glycation agent that prevents the accumulation of AGEs in vivo, which can induce diabetic complications. As anti-glycation materials derived from natural products, for example, Kumaizasa extract, Saracia extract and the like are known (see Non-Patent Document 5 and Non-Patent Document 6).

In addition, active oxygen is drawing attention as a cause of aging phenomena such as skin stains and wrinkles, and lifestyle-related diseases such as arteriosclerosis and cancer (Non-Patent Document 7). It has been clarified that active oxygen generated in a living body reacts with unsaturated fatty acids to generate lipid peroxide, which has an adverse effect on the human body. In particular, the skin is directly exposed to stimuli such as ultraviolet rays, so active oxygen is likely to be generated. The increase in the active oxygen concentration has a remarkable effect on inflammation, edema, necrosis, etc. as well as aging phenomena such as spots and wrinkles (see Non-Patent Document 8). In addition, many cosmetics, pharmaceuticals, foods and drinks contain oils and fats, which react with active oxygen during storage and use to generate lipid peroxide, which causes deterioration of quality, nutrition, and human body. The development of toxicity has become a major problem, and exploratory research on drugs with antioxidant activity has been widely conducted. In particular, those derived from natural products and having high safety can be applied to foods and drinks, and therefore, development of new antioxidants derived from natural products is desired.

One of the causes of skin aging such as age spots and wrinkles is considered to be the decrease and degeneration of collagen, which is the main component of the dermis. It is very important to prevent the decrease and denaturation of collagen that causes these aging phenomena. Since the decrease in collagen is caused by collagenase, suppressing the action of collagenase is one of the effective methods. In particular, those derived from natural products and having high safety can be applied to foods and drinks, so the development of new anti-aging agents derived from natural products is desired. As a collagenase inhibitory material derived from a natural product, for example, unlan extract, staphylea pinnata extract, lemongrass extract and the like are known (see Non-Patent Document 9 and Non-Patent Document 10).

There are four types of allergies, from type I to type IV. The type I to type III reactions are humoral immune reactions caused by IgE antibodies, and the type I reaction is particularly referred to as an immediate allergic reaction. The type IV reaction is a cell-mediated immune reaction caused by sensitized lymphocytes and is called a delayed allergic reaction. In an allergic reaction, IgE antibody existing on mast cells reacts with an allergen to release granules containing a chemical substance such as hexosaminidase (histamine) secreted in mast cells from the cell surface. This reaction is called a degranulation reaction. It is known that the released chemical substances cause inflammation and itching by the degranulation reaction. Inflammatory diseases caused by such allergies include contact dermatitis (rash), psoriasis, pemphigus vulgaris, and various other skin diseases accompanied by rough skin. From these facts, attempts have been made to prevent or treat allergic diseases and inflammatory diseases with substances that inhibit or suppress the degranulation reaction. The onset of allergic diseases is caused by genetic constitution and environmental factors. It is known that people with allergies produce a large amount of IgE antibody in their bodies, and it is necessary to control their symptoms by themselves in order to spend their days more comfortably. In other words, it is important to continue the prevention or treatment of allergic diseases for a long period of time. In addition, there is a need to develop new anti-allergic materials that are derived from natural products and are highly safe and can be continuously ingested. As anti-allergic materials derived from natural products, for example, tea extract, cabbage extract, cresson extract and the like are known (see Non-Patent Document 11 and Non-Patent Document 12).

Immune function is divided into innate immunity and acquired immunity. The former innate immunity is the transmission of antigen information and the removal of bacteria and viruses by taking in bacteria and viruses that have invaded the body by immune cells such as neutrophils and macrophages by phagocytosis. The latter acquired immunity memorizes the antigen information transmitted by innate immunity and rapidly produces an antibody at the time of the second infection to remove the antigen. It is known that such immune function is reduced by aging, stress, lack of sleep, disorder of eating habits, and the like. In recent years, there has been increasing interest in healthy life expectancy (a period in which there are no restrictions on daily life) advocated by WHO. One of the goals set by the Ministry of Health, Labor and Welfare is to extend healthy life expectancy. Maintaining or enhancing immunity is extremely important in extending healthy life expectancy. There is a need to develop materials that are derived from natural products, are highly safe, and have an immunostimulatory effect that can be ingested in daily life. As a material derived from a natural substance that increases macrophages that exert innate immunity as an immunostimulatory action, for example, Kikyo extract, Medinilla speciosa extract and the like are known (see Non-Patent Document 14 and Non-Patent Document 15). ..

Periodontal disease is an inflammatory disease caused by a bacterial infection. It is known that bacteria that specifically cause tooth decay and periodontal disease are present in the accumulated plaque. As periodontal disease progresses, it causes serious symptoms such as tartar accumulation, swelling due to inflammation of the gums, bleeding, gum retraction, and tooth melting. Currently, it is said that about 80% of the Japanese people have periodontal disease. Removal of plaque and bacteria that cause periodontal disease is effective for prevention or treatment of periodontal disease, and daily oral maintenance is indispensable. Prevention or treatment of periodontal disease is very important for people to eat, stay healthy and comfortable for a long time. Material development is required.

By the way, the mountain herb is a plant of the Labiatae family, and its scientific name is Sideritis cardica, and its English name is Mountain Herb, Mursalski, and the like. It is also called "mountain tea" because it grows in the harsh mountain highlands of over 1000 meters. It has been drunk as a healthy tea since ancient times in Greece, the Mediterranean coast, Turkey and Bulgaria.
So far, mountain herbs have been reported to have antibacterial activity against gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Pasteurella multocida (see Non-Patent Document 13). However, the antibacterial action against periodontal disease bacteria is not known.

Nippon Nogeikagaku Kaishi Vol. 61, No. 7, pp. 803-808, 1987 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. 53, No. 9,459-465 Moleculars 2019, 24, 1886; doi: 10.3390 / molecules 24101886 Kanazawa Med Univ 40: 95-103, 2015 THE SCIENCE AND ENGINEERING REVIEW OF DOSHISHA UNIVERSITY, VOL. 52, NO. 3 October 2011 YAKUGAKU ZASSHI 123 (10) 871-880 (2003) YAKUGAKU ZASSHI 122 (3) 203-218 (2002) Jpn. J. Immumol. , 29 (6) 349-338, 2006 Cosmetology Research Report, 2017, 25: 9-15. Bulletin of the University of Nagano, 2009, 64 49-59 Food Hygiene and Safety Science (Shokuhin Eiseigaku Zashi), 1989, 30.4: 295-299_1. Journal of Food Hygiene, 1998, 39 (2) 72-77_1. Molecules 2012, 17, 2683-2703 International Immunopharmacology, 4 1477-1487, 2004 AIP Conference Proceedings 2260, 040019, 2020

As mentioned above, until now, mountain herbs have no known blood pressure lowering effect, anti-glycation effect, antioxidant effect, anti-aging effect, anti-allergic effect, immunostimulatory effect, and periodontal disease suppressing effect. This is a new finding based on the diligent research of the inventors.

An object of the present invention is to solve the above-mentioned conventional problems and to achieve the following object. That is, the present invention provides a natural antihypertensive agent, an antiglycation agent, an antioxidant, an anti-aging agent, an antiallergic agent, an immunostimulator, and a periodontal disease inhibitor, which are highly safe and whose raw materials are easily available. The purpose is to do.

Further, in the present invention, at least one of the antihypertensive agent, the anti-glycation agent, the antioxidant, the anti-aging agent, the anti-allergic agent, the immunostimulatory agent, and the periodontal disease inhibitor is used as an active ingredient. An object of the present invention is to provide a blended food composition, pharmaceutical composition, and cosmetic composition.

In order to solve the above problems, antihypertensive agents, anti-glycation agents, antioxidants, anti-aging agents, anti-allergic agents, immunostimulators, which are highly safe natural products and can be widely used in pharmaceuticals, foods and drinks, and cosmetics. As a result of diligent studies by the present inventors on the agent and the periodontal disease inhibitor, the present invention has been reached.

The present invention is based on the above-mentioned findings of the present inventors, and the means for solving the above-mentioned problems are as follows. That is,
<1> An antihypertensive agent characterized by containing an extract of mountain herbs.
<2> An anti-glycation agent characterized by containing an extract of mountain herbs.
<3> An antioxidant characterized by containing an extract of mountain herbs.
<4> An anti-aging agent characterized by containing an extract of mountain herbs.
<5> An antiallergic agent characterized by containing an extract of mountain herbs.
<6> An immunostimulant characterized by containing an extract of mountain herbs.
<7> A periodontal disease bacterium inhibitor characterized by containing an extract of mountain herbs.
<8> A food composition, a pharmaceutical composition, or a cosmetic composition, which comprises the extract of the mountain herb according to any one of <1> to <7> as an active ingredient.

The antihypertensive agent, anti-glycation agent, antioxidant agent, anti-aging agent, anti-allergic agent, immunostimulatory agent, and periodontal disease bacteria inhibitor of the present invention are naturally derived extracts and are excellent in safety. It is suitable for use in a pharmaceutical composition or a cosmetic composition by blending or adding it.

It is a figure which shows the result of HPLC of the production example 1 in an Example. It is a figure which shows the result of HPLC of the production example 2 in an Example. It is a figure which shows the result of HPLC of the production example 3 in an Example.

Hereinafter, embodiments of the present invention will be described in detail. The present invention can be implemented in many different embodiments and is not limited to the specific examples of embodiments and examples shown below.

The antihypertensive agent, anti-sugar agent, antioxidant, collagenase inhibitor, anti-allergic agent, immunostimulator, periodontal disease bacterium inhibitor, and the pharmaceuticals, foods and drinks, and cosmetics containing the present invention are mountain herbs. It contains the extract as an active ingredient and, if necessary, other ingredients.

The extract of mountain herbs can be easily obtained by a method generally used for extracting plants. The extract of the mountain herb includes an extract of the mountain herb, a dried product obtained by drying a diluted solution of the extract, or a crude product or a purified product thereof.

The raw material for extracting the mountain herb is not particularly limited and may be appropriately selected depending on the intended purpose. For example, whole grass, above-ground part, stem, leaf and flower of the mountain herb can be used.

The mountain herb, which is the extraction raw material, can be obtained by drying and then crushing the herb as it is or using a coarse crusher and subjecting it to solvent extraction. Drying may be performed in the sun, or may be performed using a commonly used dryer.

The extraction method of the mountain herb extract is not particularly limited and can be carried out according to a method well known to those skilled in the art. As the extraction solvent, water or warm water, an alcohol solvent, and other organic solvents such as acetone can be used. Examples of the alcohol solvent include methanol, ethanol, propanol, isopropanol, butanol, isobutanol and the like, and mixtures thereof. Examples of other organic solvents other than acetone include esters such as ethyl acetate; polyhydric alcohols such as ethylene glycol, propylene glycol and 1,3-butylene glycol; and ethers such as diethyl ether. These solvents may be used alone or in combination. The extraction solvent may be used in the form of a hydrophilic hydrophilic solvent in combination with water and a hydrophilic organic solvent. When the solvents are used in combination, the mixing ratio of the solvents can be set arbitrarily. Among these, ethanol is preferably used, and it is more preferable to use hydrous ethanol having an ethanol content of 10% (w / w) or more and 95% (w / w) or less.

The amount of the extraction solvent is not limited as long as the composition exhibiting the above effects can be obtained, but is 2 parts by weight or more and 100 parts by weight or less with respect to 1 part by weight of the dried above-ground part of the mountain herb. Is preferable. The extraction temperature is preferably 4 ° C. or higher and 95 ° C. or lower. The extraction time is preferably 30 minutes or more and 1 week or less. The extraction method can be any method such as stirring extraction, immersion extraction, countercurrent extraction, ultrasonic extraction, and supercritical extraction.

The mountain herb extract can be any of the filtrate itself obtained by filtering the obtained extract, the concentrate obtained by concentrating the filtrate, the dried product obtained by drying the concentrate, and these crude or purified products. It may be a mixture thereof, or it may be a mixture thereof. The concentration method can be any method such as vacuum concentration, evaporation type concentration, and membrane concentration. The drying method can be any method such as vacuum drying, freeze drying, and spray drying. Excipients such as dextrin may be added if necessary. Purification can be carried out according to means known to those skilled in the art. For example, synthetic adsorption resins, activated carbons, ion exchange resins, gel filters such as Sephadex and biogels, column chromatography, recrystallization and the like may be used alone or in combination.

In addition, the mountain herb extract in the present composition is the sum of glycosides of three types of 8-hydroxyflavones (4'-O-methylisoscutellarein, isoscutellarein, 4'-O-methhypolatein) in the mountain herb extract. The amount is preferably 0.5% by weight or more, more preferably 1% by weight or more and 50% by weight or less. When it is contained in this range, the effect of the present composition can be preferably exhibited.

The extract of the mountain herb of the present invention has at least one of an excellent blood pressure lowering effect, an anti-glycation effect, an antioxidant effect, a collagenase inhibitory effect, an anti-allergic effect, an immunostimulatory effect, and a periodontal disease bacteria suppressing effect. In particular, it is suitable for blending in the food composition, pharmaceutical composition, and cosmetic composition of the present invention described below.

The food composition is not particularly limited and may be appropriately selected depending on the intended purpose. For example, beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks; candy, candy, gum, etc. Confectionery such as chocolate; various forms of health foods such as tablets, capsules and drinks, foods with functional claims and nutritional supplements can be mentioned.

The pharmaceutical composition is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tablets, capsules, drinks and troches.

The cosmetic composition is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include cosmetics, cosmetics, creams, milky lotions, hair styling products, hair washing products, and finishing cosmetics. Be done.

The antihypertensive agent, anti-sugar agent, antioxidant agent, collagenase inhibitor, anti-allergic agent, immunostimulator, periodontal disease bacterium inhibitor, and foods, pharmaceuticals, and cosmetics containing the present invention are used for humans. Although it is preferably applied to animals, it can also be applied to animals other than humans as long as the respective effects can be obtained.

As described above, according to the present invention, it is possible to provide a blood pressure-lowering agent, an anti-glycation agent, an antioxidant, an anti-aging agent, an anti-allergic agent, an immunostimulatory agent, and a periodontal disease inhibitor using a new extract as an active ingredient. .. In particular, the mountain herb extract of the present invention is a naturally derived extract, which is excellent in safety and is suitable for use in a food composition, a pharmaceutical composition, or a cosmetic composition.

Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

(Manufacturing Example 1)
-Manufacturing 80% (w / w) ethanol extract of mountain herbs-
250 g of the above-ground pulverized product of mountain herb was put into 2500 mL of 80% (w / w) ethanol, heated under reflux for 2 hours with stirring, and then an extract was obtained. The extract was filtered, activated carbon was added to the filtrate, and the mixture was further stirred for 1 hour and then filtered. Excipients were added to the filtrate, concentrated under reduced pressure, and further dried under reduced pressure to obtain an extract (powder).

Hydrochloric acid / methanol was added to the 80% (w / w) ethanol extract of the mountain herb obtained in Production Example 1, and the mixture was refluxed for 2 hours with stirring to hydrolyze. Then, the mixture was concentrated under reduced pressure, and ethyl acetate was added to the concentrated solution for partitioning. The obtained ethyl acetate layer was concentrated and isolated and purified by reverse phase liquid chromatography (ODS) to obtain Compound 1, Compound 2, and Compound 3.

Regarding the chemical structure of each compound, the structure was determined according to a conventional method. The results of NMR are shown below.

^{― 1} H-NMR and ¹³ C-NMR of Compound 1 _{(DMSO-d 6} ) ―
< ¹³ C-NMR>
55.7 (OCH ₃ ), 98.6 (6-C), 103.0 (3-C), 103.3 (10-C), 112.1 (5'-C), 113.2 (2) '-C), 118.8 (6'-C), 151.1 (4'-C), 123.3 (1'-C), 125.0 (8-C), 145.5 (9-C) C) 146.7 (3'-C), 153.0 (5-C), 153.4 (7-C), 163.3 (2-C), 182.0 (4-C)
< ^{1 1} H-NMR>
3.86 (3H, s, 4'-OCH ₃ ), 6.26 (1H, s, 6-H), 6.70 (1H, s, 3-H), 7.08 (1H, d, J) = 8.6Hz, 5'-H), 7.53 (1H, d, J = 2.5Hz, 2'-H), 7.61 (1H, dd, J = 8.6Hz, J = 2.2Hz , 6'-H), 8.71 (1H, s, 8-OH), 9.41 (1H, s, 3'-OH), 10.48 (1H, s, 7-OH), 12.34 (1H, s, 5-OH)

^{― 1} H-NMR and ¹³ C-NMR of Compound 2 _{(DMSO-d 6} ) ―
< ¹³ C-NMR>
98.6 (6-C), 102.3 (3-C), 103.2 (10-C), 115.8 (3'-C), 115.8 (5'-C), 121.4 (1'-C), 125.0 (8-C), 128.5 (2'-C), 128.5 (6'-C), 145.4 (9-C), 153.0 (5) -C), 153.3 (7-C), 161.1 (4'-C), 163.5 (2-C), 182.0 (4-C)
< ^{1 1} H-NMR>
6.26 (1H, s, 6-H), 6.72 (1H, s, 3-H), 6.92 (1H, d, 3'-H), 6.92 (1H, d, 5' -H), 8.00 (1H, d, 2'-H), 8.00 (1H, d, 6'-H), 8.7 (1H, s, 8-OH), 10.3 (1H) , S, 4'-OH), 10.4 (1H, s, 7-OH), 12.4 (1H, s, 5-OH)

^{― 1} H-NMR and ¹³ C-NMR of Compound 3 _{(DMSO-d 6} ) ―
< ¹³ C-NMR>
55.5 (4'-OCH ₃ ), 98.7 (6-C), 103.0 (3-C), 103.3 (10-C), 114.5 (3'-C), 114. 5 (5'-C), 123.1 (1'-C), 125.0 (8-C), 128.4 (2'-C), 128.4 (6'-C), 145.5 (9-C), 153.1 (5-C), 153.5 (7-C), 162.3 (4'-C), 163.1 (2-C), 182.1 (4-C) )
< ^{1 1} H-NMR>
3.10 (3H, s, 4'-OCH3), 6.26 (1H, s, 6-H), 6.80 (1H, s, 3-H), 7.11 (1H, d, J = 8.9Hz, 3'-H), 7.11 (1H, d, J = 8.9Hz, 5'-H), 8.11 (1H, d, J = 8.9Hz, 2'-H), 8.11 (1H, d, J = 8.9Hz, 6'-H), 12.3 (1H, s, 5-OH))

From the comparison with the chemical shift value, spectral shape, coupling constant, coupling state, and literature value in the nuclear magnetic resonance spectra ( ¹ H-NMR and ^{13 C-NMR) of each compound, compound 1 is 4'-O-.} The structure was determined to be methylhyporatein, compound 2 is isoscaturarein, and compound 3 is 4'-O-methylithoscaturarein.

(Manufacturing Example 2)
-Manufacturing of 50% (w / w) ethanol extract of mountain herbs-
4 kg of the above-ground crushed mountain herb was put into 80 L of 50% (w / w) ethanol, heated under reflux for 1 hour with stirring, and then an extract was obtained. The extract was filtered, activated carbon was added to the filtrate, and the mixture was further stirred for 1 hour and then filtered. Excipients were added to the filtrate, concentrated under reduced pressure, and then spray-dried to obtain an extract (powder).

(Manufacturing Example 3)
-Manufacturing of 50% (w / w) ethanol extract of mountain herbs-
9 kg of the above-ground crushed mountain herb was put into 200 L of 50% (w / w) ethanol, heated under reflux for 1 hour with stirring, and then an extract was obtained. The extract was filtered, activated carbon was added to the filtrate, and the mixture was further stirred for 1 hour and then filtered. Excipients were added to the filtrate, concentrated under reduced pressure, and then spray-dried to obtain an extract (powder).

-Measurement of 8-hydroxyflavone glycoside content by HPLC analysis-
In order to measure the content of glycosides of the three types of 8-hydroxyflavones shown in Production Example 1, the extracts of Production Example 1, Production Example 2 and Production Example 3 were subjected to HPLC analysis.

About 0.05 g of the extracts of Production Example 1, Production Example 2 and Production Example 3 were precisely weighed, and 20 mL of 11% hydrochloric acid and 10 mL of methanol were added. After dissolution, methanol was added to make exactly 50 mL. Take 10 mL of this solution in a hydrolysis tube and hydrolyze at 100 ° C. for 1 hour. After cooling to room temperature, filter through a 0.45 μm membrane filter to prepare a sample solution. Separately, a standard product of 4'-O-methylisoscutellarein, 4'-O-methylhyporatein and isoscutellarein (manufactured by Tokiwa Phytochemical Research Institute) is used and dissolved in methanol to prepare a standard solution. Accurately take 10 μL each of the sample solution and the standard solution, and perform the test by the liquid chromatograph method by the method shown in the following condition 1. The peak areas of 4'-O-methylisoscutellarein, 4'-O-methylhyporatein and isoscutellarein of each solution were measured.

流速：１．０ｍＬ／ｍｉｎ
カラム温度：３０℃ <HPLC analysis conditions>
Detector: Ultraviolet-visible absorptiometer Measurement wavelength: 280 nm
Column: CAPCELL PACK C18 UG120 4.6 x 250 nm
Mobile phase: Liquid A: TFA / water mixture (0.1: 100)
Liquid B: TFA / acetonitrile mixed liquid (0.1: 100)

Flow velocity: 1.0 mL / min
Column temperature: 30 ° C

<Calculation of 8-hydroxyflavone glycoside content>
The molecular weight ratio was used as a conversion coefficient, and the contents of 4'-O-methylisoscutellarein, 4'-O-methylhyporatein, and isoscutellarein were converted into glycoside contents, respectively.

(result of analysis)

(Example 1)
-Blood pressure lowering effect-
Using the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 and Compound 1, Compound 2 and Compound 3 isolated and purified from Production Example 1 as samples, the blood pressure lowering effect was exhibited by the following test method. evaluated.

50 μL of the sample solution, 50 μL of HEPES buffer (pH = 8.3), 100 μL of 1 mU / mL ACE solution, or 100 μL of purified water was added to a 96-well plate, and the mixture was left at 37 ° C. for 10 minutes. Next, 25 μL of angiotensin converting enzyme substrate solution was added, and the reaction was carried out at 37 ° C. for 40 minutes. Then, 50 μL of 1N NaOH was added, and the mixture was stirred to stop the reaction. To this, 10 μL of 0.2% O-phthalaldehyde was added, and the mixture was shielded from light and left at room temperature for 15 minutes. To this, 15 μL of 3.6 M phosphoric acid was added, and after stirring, the absorbance at an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm was measured. Then, from the obtained results, the angiotensin I converting enzyme inhibition rate was calculated by the following mathematical formula 1. However, in the following formula, A represents the absorbance of the sample solution, B represents the absorbance of the sample solution blank, C represents the absorbance of the control solution, and D represents the absorbance of the control blank.
(Number 1)
Angiotensin I converting enzyme inhibition rate (%) = {1- (AB) / (CD)} × 100

From the results in Table 3, it was confirmed that the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 have a blood pressure lowering effect. From the results in Table 4, it was confirmed that Compound 1, Compound 2 and Compound 3 isolated and purified from mountain herbs have a blood pressure lowering effect.

(Example 2)
-Anti-glycation effect-
Using the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 and Compound 1, Compound 2 and Compound 3 isolated and purified from Production Example 1 as samples, the anti-glycation effect was exhibited by the following test method. evaluated.

To 100 μL of 0.1 mol / mL phosphate buffer (pH 7.4), 20 μL of distilled water and 40 μL of 40 mg / mL human serum albumin were added. To this, 20 μL of a 2 mol / L glucose aqueous solution or 20 μL of distilled water was added, 20 μL of a sample solution or 20 μL of distilled water was further added, and the mixture was left at 60 ° C. for 40 hours. Then, the absorbance was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 440 nm.
Then, from each of the measured absorbances, the AGEs production suppression rate (%) was calculated by the following mathematical formula 2. However, in the following formula, A represents the absorbance of the sample solution, B represents the absorbance of the sample solution blank, C represents the absorbance of the control solution, and D represents the absorbance of the control blank.
(Number 2)
AGEs production suppression rate (%) = {1- (AB) / (CD)} × 100

From the results in Table 5, it was confirmed that the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 have an anti-glycation effect. From the results in Table 6, it was confirmed that Compound 1, Compound 2 and Compound 3 isolated and purified from mountain herbs have an anti-glycation effect.

(Example 3)
-antioxidant effect-
Using the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 and Compound 1, Compound 2 and Compound 3 isolated and purified from Production Example 1 as samples, the antioxidant action was exhibited by the following test method. evaluated.

The sample was extracted with 25 mL of MWA (methanol: water: acetic acid = 90: 9.5: 0.5) and used as a sample solution. 35 μL of 75 mM potassium phosphate buffer (pH 7.0) or Trolox solution of each concentration and sample solution was added, and 115 μL of Fluorescein working solution was added. Then, the mixture was left at 37 ° C. for 10 minutes and measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 530 nm. Then, 50 μL of 31.7 mM 2,2'-Asobis (2-amidinopropane) Dihydrochlide (AAPH) solution was added, and the measurement was performed once every 2 minutes at an excitation wavelength of 485 nm and a fluorescence wavelength of 530 nm, for a total of 45 measurements. Then, the ORAC value was calculated from the measured fluorescence intensity. The calculated ORAC values are shown in Table 7.

From the results in Table 7, it was confirmed that the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 have an antioxidant effect. From the results in Table 8, it was confirmed that Compound 1, Compound 2 and Compound 3 isolated and purified from mountain herbs have an antioxidant effect.

(Example 4)
-Collagenase inhibitory effect-
Using the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 and Compound 1, Compound 2 and Compound 3 isolated and purified from Production Example 1 as samples, the collagenase inhibitory action was exhibited by the following test method. evaluated.

In a 15 mL tube, 400 μL of 1.25 mg / mL Pz-peptide solution, 50 μL of sample solution or 0.1 mol / L Tris-HCl buffer ( _{containing pH 7.1, 20 mmol / L CaCl 2} ) 50 μL, 0.1 mg / 50 μL of mL of collagenase solution or 50 μL of 0.1 mol / L Tris-HCl buffer ( _{containing pH 7.1 and 20 mmol / L of CaCl 2} ) was added, and the reaction was carried out at 37 ° C. for 30 minutes. Then, 1 mL of 25 mM citric acid water was added to stop the reaction.

To this, 5 mL of ethyl acetate was added, and the mixture was shaken vigorously and centrifuged (1600 rpm) for 10 minutes. Then, the ethyl acetate layer was recovered, dehydrated with sodium sulfate, and then the absorbance at a wavelength of 320 nm was measured. Then, from the measured absorbance, the collagenase inhibition rate was calculated by the following mathematical formula 4. However, in the following formula, A represents the absorbance of the sample solution, B represents the absorbance of the sample solution blank, C represents the absorbance of the control solution, and D represents the absorbance of the control blank.

(Number 3)
Collagenase inhibition rate (%) = {1- (AB) / (CD)} × 100

From the results in Table 9, it was confirmed that the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 had a collagenase inhibitory effect. In addition, from the results in Table 10, it was confirmed that Compound 1, Compound 2 and Compound 3 isolated and purified from mountain herbs have a collagenase inhibitory effect.

(Example 5)
-Anti-allergic effect-
Using the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 and Compound 1, Compound 2 and Compound 3 isolated and purified from Production Example 1 as samples, the antiallergic effect was exhibited by the following test method. evaluated.
A rat basophil cell line (RBL-2H3) of 2.5 × 10 ⁵ cells / mL (Dalveco MEM containing 10% fetal bovine serum (FBS)) was seeded on a 24-well plate and cultured overnight. After completion of the culture, PBS washing was performed twice, 500 μL of 50 ng / mL Anti DNP-IgE or 500 μL of medium was added, and the mixture was allowed to stand in a _{5% CO 2 incubator at 37 ° C. for 2 hours.} After removing Anti DNP-IgE and washing with MT buffer, 490 μL of MT buffer or 490 μL of sample solution prepared with MT buffer was added, and the mixture was allowed to stand in a _{5% CO 2 incubator at 37 ° C. for 10 minutes.} Next, 10 μL of 2.5 μg / mL DNP-HSA was added, mixed well, and reacted in a _{5% CO 2 incubator at 37 ° C. for 30 minutes.} The reaction was then stopped on ice and the cell supernatant was transferred to a new 24-well plate. 500 μL of 0.1% Triton was added to each hole from which the supernatant was removed, and the cells were crushed by ultrasonic waves. Transfer 50 μL of the cell supernatant and the pulverized extracellular fluid to a 96-well plate, add 100 μL of 1.13 mg / mL p-Nitrophenyl-2-acetamido-2-deoxy-β-D-glucopylanoside (substrate solution), and add 37 ° C. Reacted with. Then, 100 μL of 2M glycine buffer was added, and the absorbance at a wavelength of 405 nm was measured. In addition, the degranulation suppression rate was calculated from the following mathematical formula 5. However, in the following formula, CL is the absorbance of the cell crushed solution, CLc is the absorbance when the substrate solution is added after adding 2M glycine buffer in the cell crushed solution, S is the absorbance of the cell supernatant, and Sc is the cell supernatant. The absorbance when the substrate solution is added after adding the 2M glycine buffer solution is shown in. Further, A represents the degranulation rate of the sample solution, and B represents the degranulation rate of the control.

(Number 4)
Degranulation rate (%) = [(S-Sc) / {(S-Sc) + (CL-CLc)}] x 100
Degranulation inhibition rate (%) = (BA) / B × 100

From the results in Table 11, it was confirmed that the extracts of the mountain herbs of Production Example 1, Production Example 2 and Production Example 3 have an anti-allergic effect. From the results in Table 12, it was confirmed that Compound 1, Compound 2 and Compound 3 isolated and purified from mountain herbs have an anti-allergic effect.

(Example 6)
-Immune activating effect-
Using the extracts of the mountain herbs of Production Example 2 and Production Example 3 as samples, the immunostimulatory effect was evaluated by the following test method.
100 μL of mouse macrophage cell line (RAW264.7) of 1.0 × 10 ⁶ cells / mL (αMEM containing 10% fetal bovine serum (FBS)) was inoculated on a 96-well plate, cultured overnight, and then 100 μL of sample solution was added. It was added and cultured overnight. After completion of the culture, 100 μL of each cell supernatant was transferred to a new 96-well plate. Next, 100 μL of Chriss Reagent was added, and the mixture was allowed to stand at room temperature for 15 minutes, and then the absorbance at a wavelength of 520 nm was measured.

From the results in Table 13, it was confirmed that the extracts of the mountain herbs of Production Example 2 and Production Example 3 had an immunostimulatory effect.

(Example 7)
-Suppressive effect on periodontal disease bacteria-
Using the extract of the mountain herb of Production Example 2 as a sample, the inhibitory effect on periodontal disease bacteria was evaluated by the following test method.

A medium obtained by mixing Production Example 2 and a modified GAM liquid medium was used as a test medium. Periodontal bacteria Porphyromonas gingivalis is ingested variant GAM agar medium, 37 ° C., after 5 days of culture under anaerobic conditions, using saline, test bacteria what was adjusted to the number of bacteria is ¹⁰ 5 / mL It was made into a liquid. The test bacterial solution was ingested in the test medium and cultured at 37 ° C. under anaerobic conditions for 1 week. After culturing, the presence or absence of growth of the test bacterium was visually observed to determine the minimum inhibitory concentration (MIC). The growth of the test bacteria was well observed in the control.

From the results in Table 14, it was confirmed that the extract of the mountain herb of Production Example 2 had an inhibitory effect on periodontal disease bacteria.

As described above, the effects of the present invention could be confirmed by each of the above-mentioned production examples and examples.

The present invention is a food composition, a pharmaceutical composition, a cosmetic composition containing at least one of an antihypertensive agent, an anti-glycation agent, an antioxidant, an anti-aging agent, an anti-allergic agent, an immunostimulatory agent, and a periodontal disease inhibitor. I will provide a.

Claims (8)

An antihypertensive agent characterized by containing an extract of mountain herbs. An anti-glycation agent characterized by containing an extract of mountain herbs. An antioxidant characterized by containing an extract of mountain herbs. An anti-aging agent characterized by containing an extract of mountain herbs. An antiallergic agent characterized by containing an extract of mountain herbs. An immunostimulant characterized by containing an extract of mountain herbs. A periodontal disease bacterium inhibitor characterized by containing an extract of mountain herbs. A food composition, a pharmaceutical composition, or a cosmetic composition, which comprises the extract of the mountain herb according to any one of claims 1 to 7 as an active ingredient.

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