WO2021261490A1 - Testosterone secretagogue - Google Patents

Abstract

The present invention addresses the problem of acquiring a promoter that promotes the synthesis and secretion of testosterone without any side effects. The present inventors searched for materials having significantly high ability to promote testosterone secretion among various natural materials and, as a result, found 11 materials that promote testosterone secretion more strongly than Leydig cells. Then, the present inventors identified relevant ingredients in 3 of these materials.

Description

Testosterone secretagogue Related application

This application claims the priority of Japanese Patent Application No. 2020-107193 filed on June 22, 2020 and Japanese Patent Application No. 2021-57563 filed on March 30, 2021. It is something that is folded into.

The present invention relates to an accelerator having a testosterone secretion promoting action. More specifically, the present invention relates to a promoter that promotes the synthesis and secretion of testosterone using a plant extract and components contained therein.

In recent years, the so-called "male menopause" that occurs in middle-aged and older men has become a problem.
Menopausal symptoms range from decreased libido, depression, decreased memory, decreased concentration, fatigue, hot flashes, sleep disorders, and decreased muscle mass, and the cause is thought to be a decrease in male hormones due to aging.

Testosterone is known as a representative of the male hormone (androgen), and in males it is synthesized in the Leydig cells of the testis and secreted into the blood. Testosterone has the strongest androgen action among male hormones, and is said to affect the development of male reproductive organs, the development of skeleton and muscles, and the enhancement of brain and mental vitality. Therefore, a decrease in blood testosterone concentration causes various systemic disorders as described above. It is also known that low testosterone levels increase the risk of metabolic syndrome, cardiovascular disease, diabetes, and respiratory diseases and are related to longevity (Non-Patent Document 1).

The symptom that the testosterone level decreases with aging is called so-called male climacteric (LOH syndrome, age-related hypogonadism). It is reported that the potential number of affected people is 12% in their 50s, 20% in their 60s, 30% in their 70s, and 50% in their 80s.

In this way, despite the fact that it is a very common illness among middle-aged and older men, it has been categorized as "age-related upset" and misdiagnosed as "depression" due to its symptoms. However, due to recent progress in research and its educational activities, male climacteric (LOH syndrome) is a multi-organ dysfunction that significantly lowers QOL, and the importance of testosterone during aging has been recognized (non-patented). Document 2).

Therefore, the present inventors searched for a new material that promotes the synthesis and secretion of testosterone in order to obtain a promoter that promotes the synthesis and secretion of testosterone without side effects.

Japanese Patent No. 4536823 Japanese Patent No. 6309175 Japanese Patent 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, Neurotherapy Vol. 35, No. 3, 182-186, 2018 Bioscience Biotechnol Biochem. 2016,80 (4), 791-797

Currently, as a treatment for male menopause, testosterone hormone replacement therapy by injection is performed, but it is a general treatment because it is a synthetic hormone replacement from outside the body and there is a concern about side effects depending on the amount of hormone replacement. Is hard to say.
In fact, when an aged male mouse was provided with a testosterone subcutaneous injection group and tested, it was reported that the amount of testosterone in the testis, the number and motility of sperm, and the conception ability were significantly reduced in the testosterone subcutaneous injection group as compared with the control group ( Non-Patent Document 3). Furthermore, in Japan, there are no other oral or ointment approved for medical insurance, and even if you have symptoms, you may not be able to receive treatment.

In addition, among natural materials such as plants that are considered to have few side effects, substances that promote the synthesis and secretion of testosterone inhibit the activity of aromatase, which is an enzyme that converts male hormones to female hormones, and reduce testosterone. Known are an extract of sweet tea (Sweet tea) to be suppressed (Patent Document 1) and a compound extract of carob and testosterone as an active ingredient (Patent Document 2). Further, geranylgeraniol (Patent Document 3) derived from annatto (Bixaceae, Achiote) is known as having an action of increasing testosterone, but the number is still small and a problem remains.

Therefore, in view of the above problems, the present inventors have diligently investigated whether there is a substance that promotes the synthesis and secretion of testosterone from natural materials considered to have few side effects, and as a result, newly promote testosterone secretion from Leydig cells. I found a natural material. In addition, one of the mechanisms was found to be due to increased StAR gene expression. Furthermore, regarding the three types of natural materials, the components involved in promoting the secretion of testosterone were identified, and it was found that these components have a combined effect, and the present invention was completed.

　また、本発明にかかるテストステロン分泌促進剤は、
　下記構造式（４）で示されるｍｙｏ－イノシトールと、

下記構造式（３）で示されるロスマリン酸（ＲＡ）

及び／又は、
下記構造式（２）で示されるデルフィニジン－３－ルチノシド（Ｄ３Ｒ）と、

を有効成分とすることを特徴とする。
　また、前記テストステロン分泌促進剤において、男性が摂取するものであることが好適である。
　また、前記促進剤において、ｍｙｏ－イノシトールは、テンヨウケンコウシ及び／又はコメヌカに由来することが好適である。
　また、前記促進剤において、ロスマリン酸（ＲＡ）は、アカジソに由来することが好適である。
　また、前記促進剤において、デルフィニジン－３－ルチノシド（Ｄ３Ｒ）はカシスに由来することが好適である。
　また、本発明にかかるテストステロン分泌促進剤は、オリーブ葉、アイブライト、エキナケア、エゾウコギ、カシス、鶏血藤、コーンシルク、アカジソ、セイヨウニンジン、テンヨウケンコウシ（Ｒｕｂｕｓ　ｓｕａｖｉｓｓｉｍｕｓ）、メリロート、これらの抽出物からなる群より選択される１種または２種以上であることが好適である。
　また、本発明にかかるテストステロン分泌促進剤において、テンヨウケンコウシ（Ｒｕｂｕｓ　ｓｕａｖｉｓｓｉｍｕｓ）の抽出物が水または含水エチルアルコールで抽出された抽出物であることが好適である。
　また、本発明にかかるテストステロン分泌促進剤の製造方法は、テンヨウケンコウシ（Ｒｕｂｕｓ　ｓｕａｖｉｓｓｉｍｕｓ）より、水または含水エチルアルコールで抽出されることを特徴とする。
　また、本発明にかかるテストステロン分泌促進剤は、シアニジン－３－ルチノシド（Ｃ３Ｒ）、デルフィニジン－３－ルチノシド（Ｄ３Ｒ）及びロスマリン酸（ＲＡ）からなる群より選択される１種または２種以上を有効成分とすることが好適である。
　また、本発明にかかるテストステロン分泌促進剤において、デルフィニジン－３－ルチノシド（Ｄ３Ｒ）及び／又はロスマリン酸（ＲＡ）でＳｔＡＲの発現が誘導されることが好適である。
　また、本発明にかかるテストステロン分泌促進剤は、下記構造式（３）で示されるロスマリン酸（ＲＡ）

 
を有効成分とする。
　また、本発明にかかるテストステロン分泌促進剤は、下記構造式（２）で示されるデルフィニジン－３－ルチノシド（Ｄ３Ｒ）

 
を有効成分とする。
That is, the present invention includes the following.
The testosterone secretagogue according to the present invention is characterized by containing myo-inositol represented by the following structural formula (4) as an active ingredient.

Further, the testosterone secretagogue according to the present invention is
Myo-inositol represented by the following structural formula (4) and

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

And / or
Delphinidin-3-lucinoside (D3R) represented by the following structural formula (2) and

Is a feature of the active ingredient.
In addition, it is preferable that the testosterone secretagogue is ingested by men.
Further, in the accelerator, myo-inositol is preferably derived from Tenyokenkoushi and / or Komenuka.
Further, in the accelerator, rosmarinic acid (RA) is preferably derived from perilla.
Further, in the accelerator, delphinidin-3-lutinoside (D3R) is preferably derived from blackcurrant.
The testosterone secretagogue according to the present invention includes olive leaf, eyebright, echinacea, eleuthero, cassis, chicken blood wisteria, corn silk, red ginseng, ginseng, rubus suavissimus, and extracts thereof. It is preferable that one or more of them are selected from the group consisting of two or more.
Further, in the testosterone secretagogue according to the present invention, it is preferable that the extract of Rubus suavissimus is an extract extracted with water or hydrous ethyl alcohol.
Further, the method for producing a testosterone secretagogue according to the present invention is characterized in that it is extracted from Rubus suavissimus with water or hydrous ethyl alcohol.
Further, as the testosterone secretagogue according to the present invention, one or more selected from the group consisting of cyanidin-3-lutinoside (C3R), delphinidin-3-lutinoside (D3R) and rosmarinic acid (RA) is effective. It is preferable to use it as an ingredient.
Further, in the testosterone secretagogue 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).
The testosterone secretagogue according to the present invention is rosmarinic acid (RA) represented by the following structural formula (3).

Is the active ingredient.
The testosterone secretagogue according to the present invention is delphinidin-3-lutinoside (D3R) represented by the following structural formula (2).

Is the active ingredient.

The testosterone secretagogue according to the present invention increases the biosynthesis and secretion of testosterone by using a natural material derived from a plant.
Furthermore, since the testosterone secretagogue according to the present invention is a plant-derived component made of a natural material, there is less concern about side effects as compared with a treatment method in which a hormone preparation is replenished from outside the body to the body, and the testosterone secretagogue can be used safely.

It is a figure which showed the testosterone secretion promoting activity of the tenyokenkoushi extract extracted with water and 40% ethyl alcohol. It is a figure which shows the pathway of testosterone biosynthesis. It is a figure which showed the gene expression of StAR. It is a figure which showed the testosterone secretion promoting activity of the Caco-2 cell permeation component of the extract of Astragalus membranaceus. It is a figure which showed the Caco-2 cytotoxicity rate of the extract of Astragalus membranaceus. It is the result of the testosterone secretagogue activity test of myo-inositol (MI), cyanidin-3-lutinoside (C3R), delphinidin-3-lucinoside (D3R), and rosmarinic acid (RA). ^{It is a figure which compared 1 1} H-NMR (lower part) of the activity peak in the stag beetle extract and reference data (Biological Magnetic Resonance Bank) (upper part). It is the result of the promotion confirmation test of StAR expression for delphinidin-3-lutinoside (D3R). It is the result of the promotion confirmation test of StAR expression for rosmarinic acid (RA). It is the result of the testosterone secretion promotion activity test when each component was used in combination.

As a result of searching for a material having a significantly high testosterone secretion promoting function from various natural materials, the present inventors have found a natural material that promotes testosterone secretion in Leydig cells. Then, it was found that one of the secretory promoting mechanisms is due to an 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 dried and powdered olive leaves of the Oleaceae family.
(2) Eyebright (Eyebright) is an extract powder produced by extracting the above-ground part of Euphrasia rostkoviana HAYNE and other plants of the same genus (Scrophiliaceae) with water.
(3) Echinacea (Latin name of Echinacea purpurea) is an extract powder produced by extracting the dry above-ground part of Echinacea purpurea (Compositee) with water.
(4) Gokasan (Ezokogi) is an extract powder produced by extracting the roots of Eleutherococcus senticosus Maxim. (Alariaceae) with water.
(5) Cassis (blackcurrant) is an extract powder prepared by extracting the fruit of Ribes nigrum (Saxifragaceae) with water and eluting it with hydrous ethyl alcohol in a column.
(6) Chicken blood wisteria (Keiketo) is an extract powder produced by extracting the stem of Keiketo Spatholobus suberectus with hydrous ethyl alcohol.
(7) Corn silk is an extract powder produced by extracting the style and stigma of corn Zea mays L. (Gramineae) with water.
(8) Akajiso is perilla perilla fruitess Britton var. acta Kudo or perilla fruitess Britton var. It is an extract powder produced by extracting the leaves and branch tips of crispa Decaisne with water.
(9) Western ginseng (Ginseng) is an extract powder produced by extracting the roots of the American ginseng Panax quinquefolium Linne (Alariceae) with water.
(10) Rubus suavissimus S. It is an extract powder produced by extracting Lee (Rosaceae) leaves with water or hydrous ethyl alcohol. This ingredient is sometimes called sweet tea. However, the origin is completely different from that of sweet tea used in Patent Document 1.
(11) Yellow sweet clover (Yellow sweet clover) is a yellow sweet clover, Melilotus officinalis Lam. Or Melilotus altissimus Thuillier. It is an extract powder produced by extracting the above-ground part of (Leguminosae) with water.

Among these, cyanidin-3-lucinoside (C3R) and delphinidin-3-lucinoside (D3R) are used for cassis, rosmarinic acid (RA) is used for acadiso, and myo-inositol is used for testosterone for rubus suavissimus. It was identified as a involved substance that promotes secretion. Among them, it was confirmed that delphinidin-3-lucinoside (D3R) and rosmarinic acid (RA) induce the expression of StAR. Hereinafter, these specified components will be described in detail.

Cyanidin-3-lucinoside (C3R) is one of the anthocyanin components and is a derivative of cyanidin represented by the following structural formula (1). Cyanidin-3-glucoside is contained in many red berries including berries, but cyanidin-3-lucinoside (C3R) is a component peculiar to blackcurrant and is not contained in blueberries and bilberries. It is known to have an effect of promoting blood flow in the eye and an antioxidant effect. It accounts for about 35% of the anthocyanins contained in blackcurrant. Cyanidin-3-lucinoside (C3R) has synonyms such as antirrhinin.

Delphinidin-3-lucinoside (D3R) is represented by the following structural formula (2), and is one of the anthocyanins contained in blackcurrant like cyanidin-3-lucinoside (C3R), and is contained in blueberries and bilberries. It is a component peculiar to cassis that has not been used. It is known to have an effect of promoting blood flow in the eye and a preventive effect of glaucoma and axial myopia, and has excellent antioxidant power. It accounts for about 46% of the anthocyanins contained in blackcurrant. Delphinidin-3-lucinoside (D3R) has synonyms such as Tulipanin.

Cyanidin-3-lutinoside (C3R) and delphinidin-3-lucinoside (D3R) are known to be absorbed into the body by ingestion like glucosides. Unlike glucosides, lutinosides are not conjugated or methylated and are confirmed to be rapidly transferred to the blood within 1 to 2 hours after ingestion in the state of anthocyanins and excreted in the urine. It is considered that anthocyanins function as active ingredients in the body as they are (Non-Patent Document 6).

Rosmarinic acid (RA) is a polyphenol represented by the following structural formula (3) and contained in plants of the Labiatae family such as perilla and rosemary. It is known to have antioxidant, antiallergic, and anti-inflammatory effects, and in recent years, its effects on Alzheimer's disease have also been reported. It has been reported that rosmarinic acid is absorbed into the body by oral administration and exhibits functionality (Non-Patent Document 7). Edible rosmarinic acid is contained in lemon balm, sage, spearmint, etc. in addition to perilla and rosemary, and is used.

For rosmarinic acid (RA), (R) -O- (3,4-Dihydroxycinnaminoyl) -3- (3,4-dihydroxyphenyl) lattice acid, 3,4-Dihydroxycinnnamic acid (R) -1-carboxy-
There are synonyms such as 2- (3,4-Dihydroxyphenyl) ester.

Myo-inositol is represented by the following structural formula (4), is one of the nine isomers present in inositol, and is contained in various foods in the natural world. It is absorbed into the body through the intestinal tract by ingestion. It is known to be used as osmolyte to regulate osmotic pressure in the body, and regulates lipid and glucose metabolism. In addition, myo-inositol is known to be useful for the treatment of polycystic ovary syndrome (PCOS).
In addition to the above-mentioned Tenyoukenkoushi, myo-inositol is also contained in rice bran (detailed in the Journal of the Japanese Society of Food and Chemical Engineering Vol.59, No.7, 301-318 (2012)), and has a purity of 97% or higher. The product has been commercialized by Tsukino Rice Fine Chemicals Co., Ltd.

Myo-inositol includes synonyms such as Hexahydroxyculohexane, Cyclohexanehexanol, meso-Inositol, and Myo-Inositol.

As the part of each plant used as the material of the natural material used in the present invention, the above-mentioned part is preferable, but in addition, flowers, spikes, pericarp, fruits, stems, leaves, branches, branches and leaves, trunks, bark, etc. One or more parts selected from rhizomes, bark, roots, seeds, whole plants and the like can be used. As the form of the natural material in the present application, in addition to the one obtained by directly extracting from these natural materials with a solvent, the squeezed liquid and / or the residue obtained after the pressing treatment is extracted by adding a solvent. It includes a wide range of forms, such as those obtained by the above, those obtained by drying and grinding plants into powder, and the like. In addition to the above, it may be industrially synthesized.

The extract of the natural material in the present invention may be produced by a known method, and is extracted at room temperature or by heating using, for example, an alcohol such as water, methanol, ethanol or an extraction solution such as a mixed solvent thereof. It can be produced by the above, and if necessary, it may be extracted under reduced pressure or pressure. The obtained extract can be used as it is, but usually it is concentrated or freeze-dried to dryness.

The accelerator according to the present invention may be ingested alone, or may be ingested in the form of an oral composition by mixing with a pharmaceutically acceptable carrier, excipient, plasticizer, coloring agent, preservative and the like. You may. Examples of the carrier used in the oral composition include sugar alcohols (eg, mannitol), inorganic substances (eg, calcium carbonate), microcrystalline cellulose, cellulose (eg, carboxymethyl cellulose), gelatin, sodium alginate, and the like. Examples thereof include polyvinylpyrrolidone, agar, magnesium stearate, and talc.
The form of the oral composition is not particularly limited, and may be in the form of tablets, pills, capsules, granules, powders, powders, troches, solutions (beverages) and the like.

In addition, the accelerator according to the present invention can be suitably ingested in a state of being blended in general foods, health foods, foods with health claims (foods for specified health use, foods with functional claims, etc.).
Examples of the food include dairy beverages, lactic acid bacteria beverages, soft beverages, carbonated beverages, fruit juice beverages, vegetable beverages, alcoholic beverages, powdered beverages, coffee beverages, tea beverages, green tea beverages, wheat tea beverages and other beverages; pudding, jelly. , Bavaroa, yogurt, ice cream, gum, gummy, chocolate, candy, caramel, biscuits, cookies, okaki, rice cakes and other confectionery; consomme soup, potage soup and other soups; miso, soy sauce, dressing, ketchup, sauce, Various seasonings such as sauces and sprinkles; jams such as strawberry jam, blueberry jam, marmalade, apple jam; fruit drinks such as red wine; fruit for processing such as syrup-pickled cherries, apricots, apples, strawberries, peaches; udon, Noodles such as cold barley, somen, buckwheat, Chinese soba, spaghetti, macaroni, beefun, harusame and wantan; and various processed foods.

In addition, in the case of Tenyoukenkoushi extract, the intake is preferably about 50 to 1500 mg per day for a 60 kg human. When taken as other ingredients, myo-inositol (МI) is about 100 to 10000 mg a day, delphinidin-3-lucinoside (D3R) is about 1 to 200 mg a day, and rosmarinic acid (RA) is about 1 to 200 mg a day. About 3 to 500 mg is preferable.

The testosterone secretagogue according to the present invention can exert the above-mentioned effect not only on humans but also on animals other than humans. Therefore, the testosterone secretagogue according to the present invention can also be added to feeds for livestock and pets.
In recent years, male infertility has been increasing, and some of the causes include testicular dysfunction and erectile dysfunction (ED). It is known that increasing the testosterone concentration in the supraclavicular body improves the spermatogenic process in the supraclavicular body. On the other hand, since erectile dysfunction (ED) is a symptom of testosterone lowering, it is considered that the promotion of testosterone secretion may be useful not only for male menopause but also for improvement of male infertility.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.
[Example 1] Testosterone secretagogue activity test The inventors conducted a testosterone secretagogue experiment on 100 natural materials. For the selection of ingredients, among the natural ingredients available in Japan and overseas, the ingredients that are judged to be "food" in Japan in the current food category were selected.

The sample preparation method was as follows.
Each material was dissolved in a 50% aqueous solution of dimethyl sulfoxide (DMSO) at a concentration of 100 mg / mL.

The method for measuring the activity was as follows.
I-10 cells (JCRB cell bank, JCRB9097) were seeded on a 48-well plate (manufactured by Thermo Scientific) at 2 × 10 ⁴ cells / well, the medium was removed after 24 hours, and the medium to which the sample was added was added. The medium was F-10 (manufactured by SIGMA-Aldrich), 10% bovine serum (manufactured by Gibco), penicillin 100unit / mL, streptomycin sulfate 100 μg / mL, and gentamicin sulfate 50 μg / mL (all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ) Is added. The medium was collected 24 hours after the sample was added, and the amount of testosterone was quantified with a microplate reader (manufactured by Biotech) using a testosterone ELISA kit (manufactured by Cayman Chemical).
As a control, a 50% DMSO aqueous solution was used.
The results are shown in Table 1.

As shown in Table 1, 11 materials showed a significant increase in testosterone more than twice as much as that of the control (50% DMSO aqueous solution only).

[Example 2] Method for extracting components in Tenyokenkoushi The inventors of the present invention have the amount of the extract extracted with water and ethyl alcohol hydrous when extracting Tenyokenkoushi, which had particularly high testosterone synthesis / secretion activity in Example 1. Was examined.

Extraction was performed as follows.
1. Crush the dried leaves of Tenyoukenkoushi (manufactured by Matsuura Pharmaceutical Co., Ltd.) with a mill to make a powder.
2. Place 5 g of the obtained powdered leaves in two beakers, add 100 ml of water to one and 100 ml of 40% ethyl alcohol to the other, and stir.
3. After stirring, cover with plastic wrap and let stand in the refrigerator for 5 days.
4. Extract and powdered leaves are No. Separate with 2 filter papers (manufactured by Advantech Toyo), and transfer the obtained extract to a 200 mL eggplant-shaped flask.
5. Freeze-drying was carried out for 24 hours in a freeze-dryer (manufactured by Tokyo Rika), and after sufficiently drying, the total weight of the extract including the flask was measured. Let the total weight at this time be (i).
The amount of the obtained extract was calculated by subtracting the flask weight (ii) from the total weight (i) obtained in 6.5.
The results are shown in Table 2.

Table 2 showed that the amount of extract obtained was slightly higher when extracted with 40% ethyl alcohol than when extracted with water.
Moreover, when the amount of testosterone was quantified 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 the testosterone secretagogue activity can be obtained higher by extracting with a solvent containing ethyl alcohol than with water. 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 testosterone secretion promotion Furthermore, in order to analyze the activity mechanism of testosterone secretion promotion, the inventors investigated changes in the expression level of testosterone biosynthesis-related genes.
By the way, as shown in FIG. 2, the testosterone biosynthesis-related gene is a factor involved in the step that corresponds to the rate-determining step of steroid hormone synthesis, and promotes the transfer of cholesterol from the outer membrane of the mitochondria to the inner membrane of the steroid (Staro). CYP11A1 (P450scc), which continuously hydroxylates the C22 and C20 positions using cholesterol as a substrate, catalyzes another step of the oxygenase reaction, and cleaves the covalent bond between C20 and C22 to produce pregnenolone. CYP17A1 (P450c17), δ-5-3-, which catalyzes two reactions, a 17α-hydroxylation reaction and a cleavage reaction between C17 and C20, and produces androstenedione from pregnenolone to Dehydropedian steroid (DHEA) and progesterone with one enzyme. 3β-hydroxy-δ5-steroid that catalyzes the oxidative conversion of β-hydroxysteroid precursors to δ-4-ketosteroids and the interconversion of 3-β-hydrokey and 3-keto-5-α-androstenedione. Examples include dehydrogenase (HSD3β), 17β-hydroxy steroid dehidrogenase (HSD17β), which catalyzes the reduction of 17-ketosteroids and the dehydrogenation of 17β-hydroxysteroids and produces androstendiol from DHEA and testosterone from androstenedione.

Among them, StAR, which is in the rate-determining step, is greatly involved in steroid hormone synthesis and is considered to be the most important step in the steroid hormone synthesis system. There is a disease called lipoid hyperplasia (Prader's disease) in which almost all steroid hormones in the adrenal glands and gonads are not synthesized due to an abnormality in the StAR gene. This disease presents with adrenal insufficiency from birth, and in XY solids, the external genitalia become feminized due to impaired androgen production in the testes.
It has also been reported that when the expression of StAR decreases in animals, the amount of steroid hormones including testosterone decreases (Non-Patent Document 4).

Furthermore, when 24-month-old aged mice are used as a male menopausal animal model and oral administration of the Chinese herbal medicine Saikokaryukotsuboi-to increases the expression of StAR in male menopausal model mice, the serum testosterone level is improved, and male menopause. There is a report that the decrease in sexual activity, which is one of the symptoms, was improved. This study suggests that activating the expression of StAR can improve serum testosterone levels and may improve the symptoms caused by male menopause (Non-Patent Document 5).

The analysis experiment was performed as follows.
I-10 cells (JCRB cell bank, JCRB9097) were seeded in a 6-well plate (manufactured by Nippon Genetics) at 1 × 10 ⁵ cell / mL at 2 mL / well, and F-10 medium (10% FBS, penicillin 100 unit / mL, The cells were cultured in an incubator (manufactured by Sanyo Electric Co., Ltd.) for 3 days (under 37 ° C. and 10% CO ₂ conditions) in a streptomycin sulfate 100 μg / mL and gentamicin sulfate 50 μg / mL.
A sample (100 mg / mL, 50% DSMO aqueous solution) diluted 100-fold with F-10 medium was added to the above cells, and the cells were cultured in an incubator (manufactured by Sanyo Electric Co., Ltd.) for 3 days (37 ° C., 10% CO ₂ conditions). ..
The cells were washed with PBS solution, then exfoliated with Accutase (manufactured by Nacalai Tesque) and collected.
The collected cells were centrifuged at 300 g for 3 minutes using a desktop micro-cooled centrifuge (manufactured by Kubota Shoji), and then the supernatant was removed.
Total RNA was extracted using realaPrep® RNA Cell Miniprep System (Promega) or Maxwell® RCS simple RNA Cells Kit (Promega).
Total RNA (0.5-1.0 μg) is reverse transcribed using RiverTra Ace® qPCR RT Master Mix (Toyobo), and then listed as GeneAce SYBR® qPCR Mix α No ROX (Nippon Gene). Analysis was performed by Thermal Cycler Dice Real Time System (manufactured by Takara Bio Inc.) using the primers shown in 3.
In this experiment, the control was a 50% DSMO aqueous solution.
The results are shown in FIG.

From FIG. 3, StAR showed significantly higher expression in 11 species as compared with the control. This suggests that StAR is involved in promoting testosterone synthetic secretion.

[Example 4] Confirmation of intestinal epithelial permeability In the examples of the present invention, a sample is directly added to I-10 cells, which is a Leydig cell model, but in a living body, Leydig cells are not absorbed from the intestinal tract after oral ingestion. The ingredients cannot reach.
Therefore, in order to confirm whether the accelerator according to the present invention permeates the intestinal epithelium, the inventors investigated whether the extract of Tenyokenkoushi shows activity using Caco-2 cells, which is an intestinal epithelial cell model.
Caco-2 cells (RIKEN Cell Bank, RCB0988) were seeded on an insert (BioCoat Collagen I inserts, manufactured by Corning) used for the permeability test, and transepithelial resistance (manufactured by Merck Millipore) was used. TEER) is measured every few days and cultured until it reaches about ^{600 Ω · cm 2.} Add 0.2 mL of medium containing a sample (concentrations of 5, 10 and 20 mg / mL, respectively, of an extract of Tenyokenkoushi extracted with 40% by mass ethyl alcohol) into the insert, allow it to stand overnight, and then the basement membrane. The side medium (0.6 mL) was collected, added to I-10 cells, and the testosterone secretagogue activity was measured by the method of Example 1. The results are shown in FIG. 40% by mass ethyl alcohol was used as a control.

Furthermore, the cytotoxicity of these concentrations of Tenyokenkoushi extract to Caco-2 cells was investigated using Cytotoxicity LDH Assay kit-WST (manufactured by Dojin Chemical Co., Ltd.). The results are shown in FIG.

As shown in FIG. 4, it was shown that the medium on the basement membrane side promoted the secretion of testosterone in I-10 cells in a concentration-dependent manner.
Further, as shown in FIG. 5, no significant cytotoxicity could be confirmed.
From these results, it was shown that the Tenyoukenkoushi extract contains a component that permeates the intestinal tract and promotes the secretion of testosterone in Leydig cells.

[Example 5] Test for specifying the components involved The inventors conducted the following tests to identify the components involved in 11 kinds of natural materials that increase testosterone. The results are shown in FIG. 6, and a comparison between the peak diagram obtained for Tenyokenkoushi and the reference data is shown in FIG.

The test was conducted by the following method.
For the extract of sweet tea, 5 g of sweet tea was extracted with a 40% ethanol aqueous solution at 4 ° C. for 4 days, ethanol was removed from the obtained extract, and then the solvent was sequentially partitioned with hexane, ethyl acetate, and 1-butanol. An aqueous layer (750 mg) was obtained. The entire aqueous layer was passed through DIAION HP-20 column chromatography (2.4 × 20 cm), washed with water, and a 50% aqueous methanol solution and methanol were sequentially flowed. After concentrating the non-adsorbed fraction (water fraction, 444 mg), it is added to Cosmosil 75C18-OPN column chromatography (1.5 x 3.0 cm), and water and a 60% methanol aqueous solution are flowed to flow the water-eluted fraction (444 mg). ) Was obtained. HPLC using this water-eluting fraction as a column and Inertsustain C18 (20 × 250 mm, GL Science), and the mobile phase using a gradient (20% aqueous methanol solution to 65% aqueous methanol solution, 0.1% trifluoroacetic acid addition, 60 minutes). Fractionated by. The obtained fraction was followed by HPLC using Cosmosil PBr (10 × 250 mm, Nacalai Tesque), and the mobile phase was gradient (1% aqueous methanol solution to 15% aqueous methanol solution, 0.1% trifluoroacetic acid addition, 60 minutes). Fractionated by. Finally, the obtained fraction was fractionated by Shodex Asahipak NH2P-50 4E (4.6 × 250 mm, SHOWA DENKO KK), and the mobile phase was fractionated by HPLC using an 85% acetonitrile aqueous solution, and the obtained peak was obtained. ¹ H-NMR and ESI-MS were measured and compared with the reference data (Biological Magnetic Resonance Bank) to identify myo-inositol.
In addition, since a plurality of components contained in these materials are known for cassis and perilla, the components involved were searched for by a chemical biology method using a plurality of compounds. As a result, cyanidin-3-lucinoside (C3R) and delphinidin-3-lucinoside (D3R) were identified in blackcurrant, and rosmarinic acid (RA) was identified in red perilla.
The testosterone secretagogue activity test was performed by the method of [Example 1].
GGOH (geranylgeraniol) was used as a positive control. GGOH (geranylgeraniol) is a plant that grows naturally in Central and South America, but it is a natural isoprenoid compound obtained from the seeds of Bixaceae Achiote (Annatto: Bixa orella), which is currently cultivated all over the world such as India and Africa. Is. It is disclosed as a testosterone enhancer (Patent Document 3), and its testosterone enhancing action has been reported (Non-Patent Document 8).

As can be seen from FIG. 6, significant promotion of testosterone secretion was observed in cassis-derived cyanidin-3-lutinoside (C3R), delphinidin-3-lutinoside (D3R), and rosmarinic acid (RA) derived from acadiso as compared with the control. Was done. In addition, it was confirmed that myo-inositol (MI) derived from Rubus suavissimus also promoted the secretion of testosterone. These components are considered to be the components involved in the promotion of testosterone secretion.

[Example 6] Test for confirming promotion of StAR expression For the components involved in Example 5, the inventors further confirmed whether the expression of StAR was promoted with delphinidin-3-lutinoside (D3R) and rosmarin. For acid (RA), a stAR expression promotion confirmation test was performed by the method of [Example 3]. The results are shown in FIGS. 8 and 9.

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

[Example 7] Combined effect of the components involved The inventors further determined by the method of [Example 1] whether the effect of promoting the secretion of testosterone is increased when the components involved found in Example 5 are used in combination rather than alone. A testosterone secretagogue activity test was conducted and examined. The results are shown in FIG.

As can be seen from FIG. 10, myo-inositol (МI), rosmarinic acid (RA), and delphinidin-3-lucinoside (D3R) alone are more than myo-inositol (МI), rosmarinic acid (RA), and myo-inositol (МI), respectively. ) And delphinidin-3-lutinoside (D3R) were confirmed to increase the promotion of testosterone secretion. This suggests that more testosterone is secreted by using the ingredients together.

Prescription example 1: Vegetable juice [ingredients] [blending amount]
(1) Tenyoukenkoushi Hydrous Ethanol Extract 0.5
(2) Vegetable juice 84.5
(3) Apple 5 times concentrated juice 5.0
(4) Lemon triple concentrated juice 2.0
(5) Sodium ascorbate 0.05
(6) Residual of purified water

[Manufacturing method]
(1) to (6) are mixed to obtain vegetable juice.

Prescription example 2: cookie [ingredient] [blending amount]
(1) Tenyoukenkoushi Hydrous Ethanol Extract 10.0
(2) Shortening 40.0
(3) Milk 5.0
(4) Aspartame 7.5
(5) Egg 7.5
(6) Baking powder 0.001
(7) Residual of cake flour

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

Prescription example 3: Gummy [ingredient] [blending amount]
(1) Tenyoukenkoushi Hydrous Ethanol Extract 2.5
(2) Apple 5 times concentrated juice 45.0
(3) Honey 41.5
(4) Lemon juice 5.0
(5) Gelatin 6.0
(6) Appropriate amount of cinnamon

[Manufacturing method]
Heat and mix (1) to (4), add (5) and (6), and heat and mix until more uniform. The mixture was poured into a mold and cooled at 4 ° C. for 1 hour. I removed it from the mold and got a gummy candy.

Prescription example 4: Tablet-type supplement [ingredients] [blending amount]
(1) Tenyoukenkoushi Hydrous 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

[Manufacturing method]
After uniformly mixing (1) to (6), tableting was performed using a single-shot tableting machine to obtain a tablet having a diameter of 5 mm and a mass of 15 mg.

Prescription Example 5: Granule-type supplement [ingredients] [blending amount]
(1) Tenyoukenkoushi Hydrous Ethanol Extract 15.0
(2) Ascorbic acid 25.0
(3) d-α-tocopherol acetate 1.5
(4) Powder reduced maltose starch syrup 54.0
(5) Aspartame 0.6
(6) Hydroxypropyl cellulose 1.5
(7) Ribofluorobin butyrate 0.2
(8) Sucralose 0.2
(9) Sucrose fatty acid ester 2.0

[Manufacturing method]
A mixture of (1) to (6) and a solution prepared by dissolving (7) and (8) in 25 mL of ethanol are mixed, kneaded, and then granulated using an extruder. (9) is added to and mixed with the obtained granules to obtain granules.

Prescription example 6: Tablet-type supplement [ingredients] [blending amount]
(1) Siberian ginseng 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

[Manufacturing method]
After uniformly mixing (1) to (6), tableting was performed using a single-shot tableting machine to obtain a tablet having a diameter of 5 mm and a mass of 15 mg.

Prescription Example 7: Granule-type supplement [ingredients] [blending amount]
(1) Western ginseng dry powder 15.0
(2) Ascorbic acid 25.0
(3) d-α-tocopherol acetate 1.5
(4) Powder reduced maltose starch syrup 54.0
(5) Aspartame 0.6
(6) Hydroxypropyl cellulose 1.5
(7) Ribofluorobin butyrate 0.2
(8) Sucralose 0.2
(9) Sucrose fatty acid ester 2.0

[Manufacturing method]
A mixture of (1) to (6) and a solution prepared by dissolving (7) and (8) in 25 mL of ethanol are mixed, kneaded, and then granulated using an extruder. (9) is added to and mixed with the obtained granules to obtain granules.

Prescription Example 8: Gummies [Ingredients] [Amount]
(1) Perilla water extract 2.5
(2) Apple 5 times concentrated juice 45.0
(3) Honey 41.5
(4) Lemon juice 5.0
(5) Gelatin 6.0
(6) Appropriate amount of cinnamon

[Manufacturing method]
Heat and mix (1) to (4), add (5) and (6), and heat and mix until more uniform. The mixture was poured into a mold and cooled at 4 ° C. for 1 hour. I removed it from the mold and got a gummy candy.

Prescription example 9: Tablet-type supplement [ingredients] [blending amount]
(1) myo-inositol 67.0
(2) Perilla extract powder (containing rosmarinic acid) 0.6
(3) Blackcurrant 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) Appropriate amount of fragrance

[Manufacturing method]
After uniformly mixing (1) to (9), tableting was performed using a single-shot tableting machine to obtain a tablet having a diameter of 11 mm and a mass of 400 mg.

Claims (13)

A testosterone secretagogue characterized by containing myo-inositol represented by the following structural formula (4) as an active ingredient.

Myo-inositol represented by the following structural formula (4) and

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

And / or
Delphinidin-3-lucinoside (D3R) represented by the following structural formula (2) and

A testosterone secretagogue characterized by containing. The testosterone secretagogue according to claim 1 or 2, wherein the testosterone secretagogue is ingested by a man. In the accelerator according to claim 1, myo-inositol is a testosterone secretagogue characterized by being derived from Tenyokenkoushi and / or Komenuka. In the accelerator according to claim 2, rosmarinic acid (RA) is a testosterone secretagogue characterized by being derived from perilla. Among the accelerators according to claim 2, delphinidin-3-lucinoside (D3R) is a testosterone secretagogue characterized by being derived from blackcurrant. One or two selected from the group consisting of olive leaves, eyebright, echinacea, eleuthero, cassis, chicken blood wisteria, corn silk, red ginseng, ginseng, rubus suavissimus, melilot, and extracts thereof. A testosterone secretagogue characterized by consisting of more than one species. The testosterone secretagogue according to claim 7, wherein the extract of Rubus suavissimus is an extract extracted with water or hydrous ethyl alcohol. A method for producing a testosterone secretagogue, which is extracted from Rubus suavissimus with water or hydrous ethyl alcohol. A testosterone secretagogue containing one or more selected from the group consisting of cyanidin-3-lutinoside (C3R), delphinidin-3-lutinoside (D3R) and rosmarinic acid (RA) as an active ingredient. The testosterone secretagogue according to claim 10, wherein the expression of StAR is induced by delphinidin-3-lucinoside (D3R) and / or rosmarinic acid (RA). Rosmarinic acid (RA) represented by the following structural formula (3)

A testosterone secretagogue containing the active ingredient. Delphinidin-3-lucinoside (D3R) represented by the following structural formula (2)

A testosterone secretagogue characterized by containing.

Images