KR20230015368A - testosterone secretagogue - Google Patents

Abstract

The object of the present invention is to obtain an accelerator that promotes the synthesis and secretion of testosterone without side effects. As a result of searching for a material with significantly high testosterone secretion promoting ability among various natural materials, a material that promotes testosterone secretion from Leydig cells. 11 species were found, and the involved components were specified for 3 of them.

Description

testosterone secretagogue

This application claims priority from Japanese Patent Application No. 2020-107193 filed on June 22, 2020 and Japanese Patent Application No. 2021-57563 filed on March 30, 2021, and is incorporated herein.

The present invention relates to an stimulating agent having a testosterone secretion stimulating action. More specifically, it relates to an accelerator that promotes the synthesis and secretion of testosterone using a plant extract or a component contained therein.

In recent years, so-called "male menopause" that occurs in men after middle age has become a problem.

Male menopausal symptoms range from a variety of aspects, such as decreased libido, depression, poor memory, poor concentration, fatigue, hot flashes, sleep disorders, and decreased muscle mass, and the cause can be cited as a decrease in male hormones due to aging.

Testosterone is known as a representative male hormone (androgen), and is synthesized in Leydig cells of the testes and secreted into the blood in men. Testosterone has the strongest male hormone action among male hormones, and is known to affect the development of male genital organs, the development of bones and muscles, and the enhancement of brain and mental vitality. For this reason, a decrease in testosterone concentration in blood causes various conditions throughout the body as described above. In addition, it is known that a low testosterone level increases the risk of metabolic syndrome, cardiovascular disease, diabetes, and respiratory disease, and is related to lifespan (Non-Patent Document 1).

Symptoms of a decrease in testosterone levels with aging are called andropause (LOH syndrome, senescent hypogonadism). It is reported that the potential number of affected patients is 12% in their 50s, 20% in their 60s, 30% in their 70s, and 50% in their 80s.

In this way, although it is actually a very common disease in middle-aged and older men, it has been dismissed as "aging-related physical condition" or misdiagnosed as "depression" based on its symptoms. However, andropause (LOH syndrome) is a multi-organ dysfunction that significantly lowers QOL, and the importance of testosterone in aging has been recognized from recent advances in research and development activities thereof (Non-Patent Document 2).

Therefore, the inventors of the present invention searched for new materials that promote testosterone synthesis/secretion in order to obtain an accelerator that promotes testosterone synthesis/secretion without side effects.

Japanese Patent Publication No. 4536823 Japanese Patent Publication No. 6309175 Japanese Patent Publication No. 5382512

J Clin Endocrinol Metab 86: 724-731, 2001 Journal of the Japanese Society of Internal Medicine, Volume 102: 914-921, 2013 Chinese Medical Journal (Engl). 129(7): 846-853, 2016 Endocrinology 137(10), 4522-4523, 1996 The Aging Male. (18)2: 106-110, 2015 J. Agric. Food Chem. 2001, 49, 3, 1546-1551 Kenjiro Ono, Masahito Yamada, Neurology, Vol. 35, No. 3, 182-186, 2018 Biosci Biotechnol Biochem. 2016, 80(4), 791-797

Currently, testosterone hormone supplementation therapy by injection is performed as a treatment for male menopause, but it is difficult to say that it is a general treatment in that it is a synthetic hormone supplementation from outside the body and there is a risk of side effects depending on the amount of hormone supplementation.

In fact, when a testosterone subcutaneous injection group was formed in aged male rats, it has been reported that the testosterone amount in the testis, the number and motility of sperm, and fertility are significantly lowered in the testosterone subcutaneous injection group compared to the control group (Non-Patent Documents). 3). In addition, in Japan, there are no other oral medications or topical medications approved for medical treatment by insurance, so there are cases where you cannot receive treatment even if you have symptoms.

In addition, among natural materials such as plants that are thought to have few side effects, substances that promote the synthesis and secretion of testosterone inhibit the activity of aromatase, an enzyme that converts male hormones into female hormones, to suppress the decrease in testosterone. (Patent Document 1) or a composite extract of cherub and Cudrania mulberry as an active ingredient (Patent Document 2) is known. In addition, geranylgeraniol (Patent Document 3) derived from annatto (Big Apple, Big Sar) and the like are known as having a testosterone increasing effect, but the number is still small, leaving a problem.

Therefore, in view of the above problems, the inventors of the present invention intensively examined whether there was a substance that promotes the synthesis/secretion of testosterone from natural substances that are thought to have few side effects, and as a result, they found a new natural substance that promotes testosterone secretion from Leydig cells. Found. In addition, one of the mechanisms was found to be due to the elevation of StAR gene expression. In addition, with respect to the three natural materials, components involved in promoting secretion of testosterone were identified, and it was found that these components had a combined effect, and the present invention was completed.

That is, the present invention includes the following.

The testosterone secretion promoter according to the present invention is characterized in that it contains myo-inositol represented by the following structural formula (4) as an active ingredient.

In addition, the testosterone secretion stimulator according to the present invention

myo-inositol represented by the following structural formula (4);

Rosmarinic acid (RA) represented by the following structural formula (3)

and/or

Delphinidin-3-lutinoside (D3R) represented by the following structural formula (2)

It is characterized by using as an active ingredient.

In addition, it is preferable for males to take the testosterone secretion promoter.

In addition, in the above accelerator, myo-inositol is preferably derived from Rubus suavissimus and/or rice bran.

In addition, in the above accelerator, rosmarinic acid (RA) is preferably derived from perilla leaf.

Further, in the above accelerator, delphinidin-3-lutinoside (D3R) is preferably derived from cassis.

In addition, the testosterone secretion stimulant according to the present invention is a group consisting of olive leaf, eyebright, echinacea, cassia galpi, cassis, gypsy, corn beard, perilla leaf, western ginseng, rubus suavissimus, merlot, and extracts thereof It is preferable that it is 1 type(s) or 2 or more types selected from.

In addition, in the testosterone secretion stimulator according to the present invention, it is preferable that the extract of Rubus suavissimus is an extract extracted with water or hydrous ethyl alcohol.

In addition, the preparation method of the testosterone secretion enhancer according to the present invention is characterized in that it is extracted from Rubus suavissimus with water or hydrous ethyl alcohol.

In addition, the testosterone secretion promoter according to the present invention contains one or two or more selected from the group consisting of cyanidin-3-lutinoside (C3R), delphinidin-3-lutinoside (D3R) and rosmarinic acid (RA). It is preferable to use it as an active ingredient.

In addition, in the testosterone secretion stimulator according to the present invention, it is preferable that the expression of StAR is induced by delphinidin-3-lutinoside (D3R) and/or rosmarinic acid (RA).

In addition, the testosterone secretion promoter according to the present invention is rosmarinic acid (RA) represented by the following structural formula (3)

as an active ingredient.

In addition, the testosterone secretion stimulator according to the present invention is delphinidin-3-lutinoside (D3R) represented by the following structural formula (2)

as an active ingredient.

The testosterone secretion stimulator according to the present invention increases the biosynthesis and secretion of testosterone by using plant-derived natural materials.

In addition, since the testosterone secretion stimulator according to the present invention is a plant-derived component made of natural materials, it can be safely used with less side effects compared to treatment methods in which hormone preparations are supplemented from the outside of the body to the body.

Fig. 1 is a graph showing the testosterone secretion stimulating activity of extracts extracted with water and 40% ethyl alcohol.
Figure 2 is a diagram showing the pathway of testosterone biosynthesis.
Figure 3 is a diagram showing the gene expression of StAR.
Figure 4 is a diagram showing the testosterone secretion promoting activity of the Caco-2 cell-penetrating component of the chamomile extract.
5 is a diagram showing the cell damage rate of Caco-2 of the extract of cheomcha.
6 shows the test results of myo-inositol (MI), cyanidin-3-lutinoside (C3R), delphinidin-3-lutinoside (D3R), and rosmarinic acid (RA) for promoting testosterone secretion.
7 is a diagram comparing ¹ H-NMR (bottom) and reference data (Biological Magnetic Resonance Bank) (top) of the activity peak in the extract of the extract.
8 shows the results of a test to confirm the promotion of StAR expression for delphinidin-3-lutinoside (D3R).
9 shows the results of a test confirming the promotion of StAR expression for rosmarinic acid (RA).
Fig. 10 shows the results of a testosterone secretion promoting activity test when each component was used in combination.

As a result of searching for a material with significantly high testosterone secretion promoting function among various natural materials, the present inventors discovered a natural material that promotes testosterone secretion in Leydig cells. And, as one of the secretion promotion mechanisms, it was found that it is due to the increase in StAR gene expression. Details of the natural materials used in the Examples of the present invention are as follows. However, the form of the natural material according to the present invention is not limited to these.

(1) Olive leaves are powdered by drying the leaves of Olea europea of the Oleaceae family.

(2) Eyebright (miller millet) is an extract powder prepared by water extraction of the aerial parts of Euphrasia rostkoviana HAYNE and other plants of the same genus (Scophulariaceae).

(3) Echinacea (Latin name for Jaju Rudbeckia) is a liquid powder prepared by extracting the dried aerial parts of Echinacea purpurea (Compositae) with water.

(4) Ogasam (Prickly pear ginseng) is an extract powder prepared by extracting the root of Prickly pear Eleutherococcus senticosus Maxim. (Araliaceae) with water.

(5) Cassis (black currant) is a concentrated powder prepared by extracting fruits of black currant Ribes nigrum (Saxifragaceae) with water and eluting with hydrated ethyl alcohol in a column.

(6) Gyehyeoldeung is a liquid powder prepared by extracting the stem of Gyehyeoldeung Spatholobus suberectus with hydrous ethyl alcohol.

(7) Corn silk is a liquid powder prepared by extracting pollen and pollen of corn Zea mays L. (Gramineae) with water.

(8) Perilla frutescens Britton var. It is an extract powder prepared by extracting the leaves and branches of acuta Kudo or Perilla frutescens Britton var.crispa Decaisne with water.

(9) Western ginseng is an extract powder prepared by extracting the root of American ginseng Panax quinquefolium Linne (Araliaceae) with water.

(10) Cheomcha is a concentrated powder prepared by extracting the leaves of Rubus suavissimus S. Lee (Rosaceae) with water or hydrous ethyl alcohol. In addition, this component may be called cheomcha. However, the origin is completely different from that used in Patent Literature 1.

(11) Melilotus officinalis Lam. or extract powder of the aerial part of Melilotus altissimus Thuill. (Leguminosae) with water.

Among them, cyanidin-3-lutinoside (C3R) and delphinidin-3-lutinoside (D3R) in cassis, rosmarinic acid (RA) in perilla leaf, and myo-inositol in rubus suavisissimus increase testosterone. It was specified as a substance involved in promoting secretion. Among them, it was confirmed that delphinidin-3-rutinoside (D3R) and rosmarinic acid (RA) induce the expression of StAR. Hereinafter, these specified components are explained in detail.

Cyanidin-3-lutinoside (C3R) is one of the anthocyanin components and is a cyanidin derivative represented by the following structural formula (1). Cyanidin-3-glucoside is included in many red liquid fruits including berries, but cyanidin-3-lutinoside (C3R) is a unique component of Cassis and is not included in blueberries or bilberries. It is mainly known for its effect of promoting blood flow to the eyes and its antioxidant action. It accounts for about 35% of the anthocyanins contained in cassis. Cyanidin-3-lutinoside (C3R) has synonyms such as Keracyyanin.

Delphinidin-3-rutinoside (D3R) is represented by the following structural formula (2) and, like cyanidin-3-rutinoside (C3R), is one of the anthocyanins included in cassis, and is not found in blueberries or bilberries. It is a unique ingredient of Cassis that is not included. The effect of promoting blood flow to the eye and the effect of preventing glaucoma and axial myopia are known, and it is excellent in antioxidant power. Among the anthocyanins contained in cassis, it occupies about 46%. Delphinidin-3-lutinoside (D3R) has synonyms such as Tulipanin.

It was found that cyanidin-3-rutinoside (C3R) and delphinidin-3-rutinoside (D3R) were absorbed into the body through oral ingestion in the same way as glucosides. Unlike glucosides, rutinosides are not conjugated or methylated, and it is confirmed that anthocyanin is rapidly transferred into the blood until 1 to 2 hours after ingestion and excreted in the urine in the form of anthocyanin intact. It is considered to function as an active ingredient (Non-Patent Document 6).

Rosmarinic acid (RA) is represented by the following structural formula (3) and is a polyphenol contained in plants of the Perilla family, such as perilla and rosemary. Antioxidant action, anti-allergic action, and anti-inflammatory action are known, and recently, an effect on Alzheimer's disease has also been reported. It has been reported that rosmarinic acid is absorbed into the body by oral administration and exerts its functionality (Non-Patent Document 7). Rosmarinic acid as food is used in lemon balm, sage, spearmint, etc. in addition to perilla leaf and rosemary.

Rosmarinic acid (RA) contains (R)-O-(3,4-Dihydroxycinnamoyl)-3-(3,4-dihydroxyphenyl)latic acid, 3,4-Dihydroxycinnnamic acid(R)-1-carboxy-2-(3, There are synonyms such as 4-Dihydroxyphenyl)ethyl ester.

Myo-inositol is represented by the following structural formula (4), is one of nine isomers present in inositol, and is included in various foods in nature. Oral ingestion is absorbed into the body from the intestinal tract. It is known to be used as an osmotic substance that adjusts osmotic pressure in the body, and regulates lipid or sugar metabolism. Also, myo-inositol is known to be useful in the treatment of polycystic ovarian syndrome (PCOS).

In addition to the above green tea, myo-inositol is also contained in rice bran (Journal of the Japanese Society of Food and Chemical Engineers Vol.59, No.7, 301 to 318 (2012) in detail), etc., and has a purity of 97% or more is Tsuno Rice Pine It is being commercialized by Chemicals Co., Ltd.

Myo-inositol has synonyms such as Hexahydroxycuclohexane, Cyclohexanehexanol, meso-Inositol, and Myo-Inosit.

The site of use of each plant, which is the material of the natural material used in the present invention, is preferably the above-mentioned site, but in addition, flowers, flower trees, pericarp, fruits, plant stems, leaves, branches, branch leaves, tree trunks, and bark , one or two or more parts selected from rhizomes, root coats, roots, seeds, or outposts may be used. In the form of the natural material herein, besides those obtained by direct extraction from these natural materials using a solvent, those obtained by extracting by adding a solvent to the squeezed liquid and/or residue obtained after pressing, and drying plants It includes a wide range of forms, such as those made by grinding and crushing into powder. In addition to the above, it may be industrially synthesized.

The natural material extract in the present invention may be prepared by a known method, for example, by extracting at room temperature or warming using an extraction solution such as alcohol such as water, methanol, or ethanol or a mixed solvent thereof It can be manufactured, and you may extract under reduced pressure or pressure as needed. The obtained extracted extract can be used as it is, but usually, concentrated or freeze-dried ones are used.

The accelerator according to the present invention may be ingested alone or may be ingested in the form of an oral composition mixed with a pharmaceutically acceptable carrier, excipient, plasticizer, colorant, preservative, or the like. Carriers used in the oral composition include, for example, sugar alcohols (eg, mannitol), inorganic substances (eg, calcium carbonate), microcrystalline cellulose, cellulose (eg, carboxymethylcellulose), gelatin, sodium alginate , polyvinylpyrrolidone, agar, magnesium stearate, talc, and the like.

The form of the oral composition is not particularly limited, and may be in the form of tablets, pills, capsules, granules, powders, powders, troches, or solutions (drinks).

In addition, the promoter according to the present invention can be preferably ingested in a state in which it is blended with general food, health food, and health functional food (food for specific health use, food with functional claims, etc.).

Beverages such as milk drinks, lactic acid bacteria drinks, soft drinks, carbonated drinks, fruit drinks, vegetable drinks, alcoholic drinks, powder drinks, coffee drinks, black tea drinks, green tea drinks, and barley tea drinks; confectionery such as pudding, jelly, bavarois, yogurt, ice cream, gum, gummies, chocolate, candy, caramel, biscuits, cookies, okaki (baked glutinous rice crackers), and rice crackers (baked non-glutinous rice crackers); soups such as consommé soup and potage soup; various seasonings such as miso, soy sauce, dressing, ketchup, seasoning, sauce, and furikake; jams such as strawberry jam, blueberry jam, marmalade, and apple jam; fruit wines such as red wine; processed fruits such as syrup-pickled cherries, apricots, apples, strawberries, and peaches; noodles such as udon, cold noodles, somen, soba, Chinese soba, spaghetti, macaroni, mifun (rice noodles), harusame (mung bean vermicelli) and wontons; Other various processed foods etc. are mentioned.

In addition, the amount of intake is preferably about 50 to 1500 mg per day for a 60 kg human in the case of cheomcha essence. When ingested as other components, myo-inositol (MI) is about 100 to 10000 mg per day, delphinidin-3-lutinoside (D3R) is about 1 to 200 mg per day, and rosmarinic acid (RA) is about 3 to 500 mg per day. degree is desirable.

The testosterone secretion stimulator according to the present invention is not limited to humans, and can exhibit the above effects to animals other than humans. Therefore, the testosterone secretion stimulator according to the present invention may be incorporated into feed for livestock or pets.

In addition, although male infertility is increasing in recent years, some of the causes include testicular dysfunction and erectile dysfunction (ED). It is known that increasing the concentration of testosterone in the testicular corpus improves the sperm maturation process in the testicular corpus. On the other hand, since there is erectile dysfunction (ED) as a symptom of low testosterone, it can be considered that it is useful not only for male menopause but also for improving male infertility by promoting secretion of testosterone.

Example

Examples are described below, but the present invention is not limited thereto.

[Example 1] Testosterone secretion promoting activity test

The inventors conducted testosterone secretion stimulation experiments with respect to 100 natural materials. For the selection of materials, among natural materials available at home and abroad, in the current food and drug classification, materials that are judged to be “formula” in Korea were selected.

The manufacturing method of the sample was performed as follows.

Each material was dissolved in a 50% dimethylsulfoxide (DMSO) aqueous solution at a concentration of 100 mg/mL.

The activity measurement method was performed as follows.

I-10 cells (JCRB cell bank, JCRB9097) were seeded in a 48-well plate (manufactured by Thermo Scientific) at 2×10 ⁴ cells/well, and after 24 hours, the medium was removed and the medium containing the sample was added. In addition, the medium is F-10 (manufactured by SIGMA-Aldrich), 10% bovine serum (manufactured by Gibco), penicillin 100 units/mL, streptomycin sulfate 100 μg/mL, gentamycin sulfate 50 μg/mL (all manufactured by Fujifilm Wako Junyaku). production) was added. 24 hours after the addition of the sample, the medium was recovered, and the amount of testosterone was quantified with a microplate reader (manufactured by Biotech) using a testosterone ELISA kit (manufactured by Cayman Chemical).

In addition, as a control, 50% DMSO aqueous solution was used.

The results are shown in Table 1.

As shown in Table 1, 11 materials recognized a 2-fold or more significant increase in testosterone compared to the control (only 50% DMSO aqueous solution).

[Example 2] Method for extracting components from chewed tea

The inventors examined whether the amounts of extract extracted with water and ethyl alcohol-containing water were different when extracting the tea, which had a particularly high testosterone synthesis/secretion activity in Example 1.

Extraction was performed as follows.

1. Grind dried leaves (manufactured by Matsuura Yakugyo) with a mill to make powder.

2. Put the obtained powder leaves into two beakers, 5 g each, and add 100 ml of water to one side and 100 ml of 40% ethyl alcohol to the other side, and stir.

3. After stirring, cover and leave in the refrigerator for 5 days.

4. The extract and powder leaves were prepared in No. It isolate|separates with 2 filter paper (made by Advantech Toyo), and transfers the obtained extract to a 200 mL eggplant flask.

5. Lyophilization was performed for 24 hours in a freeze dryer (manufactured by Tokyo Rika), and after drying sufficiently, the total weight of the concentrate including the flask was measured. Let the total weight at this time be (i).

6. The weight of the flask (ii) was subtracted from the total weight (i) obtained in 5 to calculate the amount of the concentrate obtained.

The results are shown in Table 2.

From Table 2, it was shown that the amount of extract obtained by extracting with 40% ethyl alcohol was slightly larger than that of extracting with water.

In addition, as a result of quantifying the amount of testosterone by the method of Example 1, as shown in Fig. 1, the extract extracted with 40% ethyl alcohol showed higher testosterone secretion promoting activity.

Therefore, it was shown that extracting with a solvent containing ethyl alcohol, rather than water, with respect to sweet tea can obtain a higher testosterone secretion promoting activity. The concentration of ethyl alcohol is 10 to 90% by mass, preferably 20 to 80% by mass, more preferably 30 to 70% by mass, and most preferably 40 to 60% by mass.

[Example 3] Mechanism of action of stimulating testosterone secretion

In addition, the inventors investigated changes in the expression level of genes related to testosterone biosynthesis in order to analyze the activation mechanism of testosterone secretion promotion.

By the way, as shown in FIG. 2, the testosterone biosynthesis-related gene is a factor related to the step corresponding to the rate-limiting step of steroid hormone synthesis, and Steroidogenic acute regulatory protein (StAR), which promotes the transition of cholesterol from the mitochondrial outer membrane to the inner membrane, cholesterol CYP11A1 (P450scc) that continuously hydroxylates position C22 and position C20 using as substrate, further catalyzes another step of oxygenase reaction, and cleaves the covalent bond between C20-C22 to produce pregnenolone , CYP17A1 (P450c17), δ-5, which catalyzes two reactions of 17α-hydroxylation and C17-C20 cleavage with a single enzyme, and generates Dehydroepiandrosterone (DHEA) from pregnenolone and androstenedione from progesterone. 3β-hydroxy catalyzes the oxidative conversion of 3-β-hydroxysteroid precursors to δ-4-ketosteroids and the interconversion of 3-β-hydroxy- and 3-keto-5-α-androstansteroids -δ5-steroid dehydrogenase (HSD3β), 17β-hydroxy steroid dehydrogenase (HSD17β) that catalyzes the reduction of 17-ketosteroid and dehydrogenation of 17β-hydroxysteroid, and produces androstendiol from DHEA and testosterone from androstenedione can be heard

Among them, the relation of StAR to the synthesis of steroid hormones in the rate-limiting step is great, and it is considered to be the most important step in the synthesis system of steroid hormones. There is a disease called lipoid hyperplasia (Prader's disease) in which almost all of the steroid hormones in the adrenal glands and gonads are not synthesized because of an abnormality in the StAR gene. This disease shows symptoms of adrenal insufficiency from birth, and also in the solid of 46, XY, the external genital organs are feminized due to the production of male hormones in the testes. In addition, it has been reported that when the expression of StAR is reduced in animals, the amount of steroid hormones, including testosterone, is reduced (Non-Patent Document 4).

In addition, when 24-month-old aged rats were used as male menopausal animal models and oral administration of herbal medicine Shihoga Yonggol Moryo-tang increased the expression of StAR in male menopausal model rats, improving serum testosterone levels and improving male menopausal symptoms. There are reports of improvement in the symptom, a decrease in sexual activity. This study suggests the possibility of improving serum testosterone levels and improving various symptoms caused by male menopause by activating the expression of StAR (Non-Patent Document 5).

Analysis experiments were conducted as follows.

I-10 cells (JCRB cell bank, JCRB9097) were seeded in 2mL/well at 1×10 ⁵ cells/mL in a 6-well plate (manufactured by Nippon Genetics), and F-10 medium (10% FBS, penicillin 100unit/mL, streptomycin Sulfate 100 μg/mL, gentamycin sulfate 50 μg/mL) were cultured for 3 days (under 37°C, 10% CO ₂ conditions) in an incubator (manufactured by Sanyo Denki).

A solution of the sample (100 mg/mL, 50% aqueous solution of DSMO) diluted 100-fold in F-10 medium was added to the cells, and cultured in an incubator (manufactured by Sanyo Denki) for 3 days (under 37°C, 10% CO ₂ conditions).

These cells were washed with a PBS solution, then detached with Accutase (manufactured by Nacalai Tesque) and recovered.

The collected cells were centrifuged at 300 g for 3 minutes in a tabletop microcooling centrifuge (manufactured by Kubota Corporation), and then the supernatant was removed.

Total RNA was extracted using ReliaPrep (registered trademark) RNA Cell Miniprep Systems (manufactured by Promega) or Maxwell (registered trademark) RCS Simply RNA Cells Kit (manufactured by Promega).

Total RNA (0.5 to 1.0 μg) was reverse transcribed using ReverTra Ace (registered trademark) qPCR RT Master Mix (Toyobo), GeneAce SYBR (registered trademark) qPCR Mix α No ROX (Nippon Gene) and Table 3 Analysis was performed with a Thermal Cycler Dice Real Time System (manufactured by Takara Bio) using the indicated primers.

The control in this experiment was a 50% DSMO aqueous solution.

Results are shown in FIG. 3 .

From Figure 3, StAR showed significantly higher expression compared to the control in 11 species. In this respect, it was suggested that StAR is involved in promoting secretion of testosterone synthesis.

[Example 4] Confirmation of intestinal epithelial permeability

In the embodiment of the present invention, the sample is directly added to the Leydig cell model, I-10 cells, but in vivo, components cannot reach the Leydig cells unless they are absorbed from the intestinal tract after oral ingestion.

Therefore, the inventors investigated whether the extract of the extract shows activity using Caco-2 cells, which are an intestinal epithelial cell model, in order to confirm whether the promoter according to the present invention permeates the intestinal epithelium.

Caco-2 cells (Riken Cell Bank, RCB0988) were seeded on inserts (BioCoat Collagen I inserts, manufactured by Corning) used for permeability testing, and transepithelialization was performed using Millicell ERS-2 (manufactured by Merck Millipore). Resistance (TEER) is measured at intervals of several days, and cultured until it reaches about 600 Ω·cm 2 . Into the insert, 0.2 mL of a medium containing a sample (an extract obtained by extracting the tea with 40% by mass of ethyl alcohol at a concentration of 5, 10, or 20 mg/mL, respectively) was added, and after standing overnight, the medium (0.6 mL) on the basement membrane side was recovered. and added to I-10 cells, and testosterone secretion promoting activity was measured by the method of Example 1. Results are shown in FIG. 4 . 40% by mass ethyl alcohol was used for control.

In addition, the cytotoxicity of the extracts of these teas at these concentrations to Caco-2 cells was investigated using Cytotoxicity LDH Assay kit-WST (manufactured by Dojin Chemical Co., Ltd.). Results are shown in FIG. 5 .

As shown in Fig. 4, it is disclosed that the basement membrane-side medium promotes the secretion of testosterone from I-10 cells in a concentration-dependent manner.

In addition, as shown in Fig. 5, significant cytotoxicity could not be confirmed.

In this regard, it is disclosed that the extract of chamomile contains a component that permeates the intestinal tract and promotes the secretion of testosterone in Leydig cells.

[Example 5] Involved component specific test

The inventors conducted the following tests to identify components involved in 11 natural materials that increase testosterone. The results are shown in Fig. 6, and Fig. 7 shows a comparison between peak figures and reference data obtained for each wheel.

The test was conducted by the following method.

For the extract of tea, 5 g of tea was extracted with a 40% aqueous ethanol solution at 4° C. for 4 days, and ethanol was removed from the obtained extract, followed by sequential solvent partitioning with hexane, ethyl acetate, and 1-butanol to obtain an aqueous layer (750 mg). The entire aqueous layer was passed through DIAION HP-20 column chromatography (2.4 x 20 cm), washed with water, and then 50% methanol aqueous solution and methanol were sequentially flowed through. The unadsorbed fraction (harvested fraction, 444 mg) was concentrated and added to Cosmosil 75C18-OPN column chromatography (1.5 x 3.0 cm), and water and a 60% methanol aqueous solution were flowed through to obtain a water-soluble fraction (444 mg). This aqueous fraction was fractionated by HPLC using Inertsustain C18 (20 × 250 mm, GL Science) as a column, and a gradient (20% methanol aqueous solution to 65% methanol aqueous solution, 0.1% trifluoroacetic acid addition, 60 minutes) for the mobile phase. . The obtained fractions were then fractionated by HPLC using Cosmosil PBr (10 × 250 mm, Nacalai Tesque), and the mobile phase using a gradient (1% methanol aqueous solution, 15% methanol aqueous solution, 0.1% trifluoroacetic acid addition, 60 minutes). Finally, the obtained fractions were fractionated by HPLC using Shodex Asahipak NH2P-50 4E (4.6 × 250 mm, SHOWA DENKO KK) and an 85% acetonitrile aqueous solution as the mobile phase, and ¹ H-NMR and ESI-MS of the obtained peaks were measured. and identified as myo-inositol by comparison with reference data (Biological Magnetic Resonance Bank).

In addition, since a plurality of components contained in these materials are known for cassis and perilla leaf, involved components were searched for using a chemical biological method using a plurality of compounds. As a result, cyanidin-3-lutinoside (C3R) and delphinidin-3-lutinoside (D3R) were identified in cassis, and rosmarinic acid (RA) in perilla leaf.

The testosterone secretion stimulating activity test was conducted by the method of [Example 1].

GGOH (geranylgeraniol) was used as a positive control. GGOH (Geranylgeraniol) is a natural isoprenoid compound obtained from the seeds of Bixa and Bixa (Annato: Bixa orella), which is a plant native to Central and South America, but is currently cultivated around the world, including India and Africa. It is disclosed as a testosterone enhancing agent (Patent Document 3) and reported for its testosterone enhancing action (Non-Patent Document 8).

As can be seen in Figure 6, cyanidin-3-lutinoside (C3R) and delphinidin-3-lutinoside (D3R) derived from cassis and rosmarinic acid (RA) derived from perilla locust compared to the control, and the testosterone Significant secretion stimulation was observed. In addition, it was confirmed that myo-inositol (MI) derived from Rubus suavisismus also stimulated the secretion of testosterone. These components are considered to be involved in the promotion of testosterone secretion.

[Example 6] Test to confirm promotion of StAR expression

Regarding the involved components identified in Example 5, the inventors further investigated delphinidin-3-lutinoside (D3R) and rosmarinic acid (RA) in the method of [Example 3] to confirm whether the expression of StAR was promoted. A test to confirm the promotion of StAR expression was performed. Results are shown in FIGS. 8 and 9 .

As can be seen from FIGS. 8 and 9 , the expression of StAR was increased for both delphinidin-3-rutinoside (D3R) and rosmarinic acid (RA) compared to other testosterone biosynthesis-related genes. From this point of view, it is considered that expression of StAR is induced in both delphinidin-3-rutinoside (D3R) and rosmarinic acid (RA).

[Example 7] Combination effect of involved components

The inventors further conducted a testosterone secretion stimulating activity test by the method of [Example 1] to examine whether or not the testosterone secretion stimulating effect of the involved components identified in Example 5 was increased when used in combination rather than alone. Results are shown in FIG. 10 .

As can be seen in FIG. 10, myo-inositol (MI), rosmarinic acid (RA), myo-inositol ( MI) and delphinidin-3-lutinoside (D3R), it was confirmed that the secretion of testosterone was increased. From this point, it was suggested that more testosterone is secreted by using the components together.

Formulation Example 1: Vegetable Juice

〔ingredient〕 [Formulation amount]

(1) Superhydrous ethanol extract 0.5

(2) vegetable juice 84.5

(3) 5 times concentrated apple juice 5.0

(4) Lemon 3 times concentrated juice 2.0

(5) Sodium ascorbate 0.05

(6) purified water leftover

〔quite〕

(1) to (6) are mixed to obtain vegetable juice.

Formulation Example 2: Cookies

〔ingredient〕 [Formulation amount]

(1) Superhydrous ethanol extract 10.0

(2) shortening 40.0

(3) Milk 5.0

(4) Aspartame 7.5

(5) eggs 7.5

(6) baking powder 0.001

(7) Soft flour leftover

〔quite〕

After mixing (2) to (4) using a stirrer, (5) was added little by little and mixed until uniform. To the mixture, previously mixed (6), (7) and (1) were added and kneaded to obtain a cookie dough. After standing in the refrigerator for 30 minutes, mold and bake.

Prescription Example 3: Gummi

〔ingredient〕 [Formulation amount]

(1) Superhydrous ethanol extract 2.5

(2) 5 times concentrated apple juice 45.0

(3) Honey 41.5

(4) Lemon juice 5.0

(5) Gelatin 6.0

(6) Cinnamon Appropriate amount

〔quite〕

Heat and mix (1) to (4), add (5) and (6), and heat and mix until uniform again. The mixture was poured into a mold and cooled at 4°C for 1 hour. It was taken out of the mold and gummy was obtained.

Formulation Example 4: Tablet Supplement

〔ingredient〕 [Formulation amount]

(1) Superhydrous ethanol extract 10.0

(2) microcrystalline cellulose 75.0

(3) Sodium ascorbate 10.0

(4) Glycerin fatty acid ester 3.0

(5) Talc 1.8

(6) Sodium stearate 0.2

〔quite〕

After uniformly mixing (1) to (6), the tablet was tableted using a single-screw tablet press to obtain a tablet having a diameter of 5 mm and a mass of 15 mg.

Formulation Example 5: Granular Supplement

〔ingredient〕 [Formulation amount]

(1) Superhydrous ethanol extract 15.0

(2) ascorbic acid 25.0

(3) d-α-tocopherol acetate 1.5

(4) powdered reduced maltose starch syrup 54.0

(5) Aspartame 0.6

(6) Hydroxypropyl cellulose 1.5

(7) riboflavin butyrate 0.2

(8) Sucralose 0.2

(9) Sucrose fatty acid ester 2.0

〔quite〕

A mixture of (1) to (6) and a solution obtained by dissolving (7) and (8) in 25 mL of ethanol are mixed, kneaded, and then extruded and granulated using a granulator. (9) is added and mixed to the obtained granules to obtain granules.

Formulation Example 6: Tablet Supplement

[Ingredient]

(1) Casiogalbi dry powder 10.0

(2) microcrystalline cellulose 75.0

(3) Sodium ascorbate 10.0

(4) Glycerin fatty acid ester 3.0

(5) Talc 1.8

(6) Sodium stearate 0.2

〔quite〕

After uniformly mixing (1) to (6), the tablet was tableted using a single-screw tablet press to obtain a tablet having a diameter of 5 mm and a mass of 15 mg.

Formulation Example 7: Granular Supplement

〔ingredient〕 [Formulation amount]

(1) Western ginseng dry powder 15.0

(2) ascorbic acid 25.0

(3) d-α-tocopherol acetate 1.5

(4) powdered reduced maltose starch syrup 54.0

(5) Aspartame 0.6

(6) Hydroxypropyl cellulose 1.5

(7) riboflavin butyrate 0.2

(8) Sucralose 0.2

(9) Sucrose fatty acid ester 2.0

〔quite〕

A mixture of (1) to (6) and a solution obtained by dissolving (7) and (8) in 25 mL of ethanol are mixed, kneaded, and then extruded and granulated using a granulator. (9) is added and mixed to the obtained granules to obtain granules.

Formulation Example 8: Gummi

[Ingredient]

(1) Perilla leaf extract 2.5

(2) 5 times concentrated apple juice 45.0

(3) honey 41.5

(4) Lemon juice 5.0

(5) Gelatin 6.0

(6) Cinnamon Appropriate amount

〔quite〕

Heat and mix (1) to (4), add (5) and (6), and heat and mix until uniform again. The mixture was poured into a mold and cooled at 4°C for 1 hour. It was taken out of the mold and gummy was obtained.

Formulation Example 9: Tablet Supplement

〔ingredient〕 [Formulation amount]

(1) myo-inositol 67.0

(2) Perilla leaf essence powder (containing rosmarinic acid) 0.6

(3) Cassis extract powder (containing Cassis anthocyanin) 1.2

(4) microcrystalline cellulose 26.2

(5) Glycerin fatty acid ester 2.5

(6) Talc 1.5

(7) Sodium stearate 0.6

(8) acidulant 0.4

(9) Spices

〔quite〕

After uniformly mixing (1) to (9), tableting was performed using a single-screw tableting machine to obtain tablets having a diameter of 11 mm and a mass of 400 mg.

Claims (8)

A testosterone secretion promoter comprising delphinidin-3-lutinoside (D3R) represented by the following structural formula (2) as an active ingredient.

myo-inositol represented by the following structural formula (4),

Delphinidin-3-lutinoside (D3R) represented by the following structural formula (2)

A testosterone secretagogue comprising as an active ingredient. According to claim 1 or 2,
A testosterone secretagogue, intended for consumption by men. According to claim 2,
Rosmarinic acid (RA) further represented by the following structural formula (3)

Including, testosterone secretagogue. According to claim 2,
Myo-inositol is a testosterone secretion stimulant derived from cilantro and/or rice bran. According to claim 1 or 2,
Delphinidin-3-lutinoside (D3R) is a testosterone secretagogue derived from cassis. According to claim 4,
Rosmarinic acid (RA) is a testosterone secretion stimulant derived from perilla leaves. According to claim 4,
A testosterone secretion stimulator in which the expression of StAR is induced by delphinidin-3-rutinoside (D3R) and/or rosmarinic acid (RA).

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