WO2023276861A1 - Composition for oral ingestion for improving sperm finding - Google Patents
Composition for oral ingestion for improving sperm finding Download PDFInfo
- Publication number
- WO2023276861A1 WO2023276861A1 PCT/JP2022/025218 JP2022025218W WO2023276861A1 WO 2023276861 A1 WO2023276861 A1 WO 2023276861A1 JP 2022025218 W JP2022025218 W JP 2022025218W WO 2023276861 A1 WO2023276861 A1 WO 2023276861A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sperm
- ergothioneine
- creatine
- composition
- day
- Prior art date
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
- A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to an orally ingestible composition for improving sperm findings, containing ergothioneine as an active ingredient.
- Ergothioneine is a type of amino acid that is used in cosmetics as an antioxidant, contained in mushrooms such as Tamogitake.
- Patent Document 1 reports that the ergothioneine content in Pleurotus cornucopia is 15.8 ⁇ 0.06 mg per 1 g of dry weight.
- Non-Patent Document 1 indicates that when ergothioneine is ingested by humans and animals as food, it takes a long time to metabolize, so it tends to accumulate in many tissues and body fluids of humans and animals. It has been confirmed in vitro that ergothioneine has an antioxidant effect and a cytoprotective effect, and it has been suggested that it is involved in the repair of tissue damage and the like in the bodies of humans and animals.
- Non-Patent Document 1 reports that the amount of ergothioneine contained in animal semen is 6 times or more the blood concentration.
- Non-Patent Document 2 reports that contacting freeze-thawed sheep sperm with ergothioneine increases sperm motility, advancement rate, and motility velocity (VAP).
- VAP motility velocity
- Non-Patent Document 3 it has been reported that contacting mouse sperm with creatine, which is related to energy metabolism, increases sperm motility.
- the purpose of the present invention is to provide an orally ingestible composition for improving sperm findings.
- the present inventors found that oral ingestion of ergothioneine improved sperm findings in mammals, leading to the completion of the present invention. That is, the present invention provides the following.
- a composition for oral ingestion for improving sperm findings in mammals comprising ergothioneine as an active ingredient.
- the composition according to (3) or (4), wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
- the composition according to (7) or (8), wherein ergothioneine and creatine are blended at a weight ratio of 1:3000 to 1:100 per day.
- a method of improving sperm findings in a mammal comprising orally administering ergothioneine to a subject in need of improving sperm findings.
- the method of (14), wherein the subject is human.
- the human is a male infertility patient.
- the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
- the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
- composition for oral ingestion is a food.
- composition for oral ingestion is a pharmaceutical.
- a sperm culture medium containing ergothioneine (31) A sperm culture medium containing ergothioneine. (32) The sperm culture medium according to (31), further comprising creatine.
- This specification includes the disclosure of Japanese Patent Application No. 2021-106397, which is the basis of priority of this application.
- composition for oral ingestion that improves sperm findings can be provided.
- VSL linear velocity
- VCL curve velocity
- % indicates % by weight.
- molar concentration (M) indicates the molar amount contained per cubic decimeter (mol/dm 3 ).
- composition for oral ingestion for improving sperm findings in mammals of the present invention contains ergothioneine as an active ingredient, and is used to improve sperm findings in mammals. characterized by being used.
- the term "mammal” refers to animals belonging to the phylum Chordata subphylum Mammalia, including humans and non-humans.
- mammals including humans and chimpanzees, pets such as dogs and cats
- Animals include livestock animals such as cows, pigs, horses, sheep and goats, rodents such as mice and rats, mammals kept in zoos, and the like.
- the subject to whom the composition for oral ingestion of the present invention is administered is preferably human.
- sperm findings refers to the state of mammalian, preferably human semen. Velocity (average velocity (VAP), linear velocity (VSL), and curvilinear velocity (VCL)), head amplitude (ALH), etc.
- VAP average velocity
- VSL linear velocity
- VCL curvilinear velocity
- AH head amplitude
- each index in sperm findings is described in "Human Semen Examination and Procedure” World Health Organization (WHO) Laboratory Manual 5th Edition (WHO laboratory manual for the Examination and processing of human semen Fifth edition (2010)) It refers to the one measured by the method according to the method. Such indicators are generally measured using a sperm motility analysis system (CASA: Computer-Aided Sperm Analysis) system.
- CASA Computer-Aided Sperm Analysis
- the subject When the subject is a human, there is no particular limitation on the male subject to ingestion, and it can be a male who wishes to conceive a female partner.
- the subject is preferably a male infertility patient.
- infertility is defined as the inability to conceive after a year of normal cohabitation and unprotected sex in a young, apparently healthy man and woman after marriage. Infertility accounts for about 15% of healthy people, of which about one-third are known to be caused by men, and one-fifth are known to be caused by both men and women. It is Therefore, infertility is caused by men in about 50% of cases, and is said to be on the rise in recent years.
- the fertility of healthy men declines on average from around the age of 40, and it is said that the fertility of a 45-year-old man declines by about 25% compared to that of a 30-year-old man.
- the causes of infertility in young men under the age of 40 include spermatogenesis dysfunction, which is a problem in the process of sperm formation and maturation; Disorders caused by accessory genitalia, in which sperm are affected by inflammation, and sexual dysfunction such as inability to have sexual intercourse or ejaculate in the vagina are known.
- the composition according to the present invention is preferably used for symptoms in sperm findings in male infertility caused by idiopathic spermatogenic dysfunction, such as a decrease in sperm concentration in semen (oligozoospermia: sperm 2 ⁇ 10 7 / mL or less), decreased sperm motility (asthenozoospermia: sperm motility rate of 40% or less), decreased sperm motility, abnormal morphology (teratozoospermia: normal sperm ratio of 40% below), or a combination thereof, particularly for improving sperm findings in idiopathic oligozoospermia and/or idiopathic asthenozoospermia.
- Symptoms of such sperm findings include low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
- “ergothioneine” refers to the compound represented by Formula I below and derivatives thereof. All stereoisomers having the structural formula of Formula I below are included. Derivatives here include salts (calcium salts, sodium salts, potassium salts, magnesium salts, hydrochlorides, citrates, etc.), hydrates, esters, and the like. It is not particularly limited.
- Ergothioneine is a thiol/thioketone produced by some fungi and bacteria, and is particularly found in mushrooms such as oyster mushroom, oyster mushroom, oyster mushroom, crispa crisp, enoki mushroom, and shiitake mushroom, and is known to be especially abundant in oyster mushroom.
- mushrooms such as oyster mushroom, oyster mushroom, oyster mushroom, crispa crisp, enoki mushroom, and shiitake mushroom, and is known to be especially abundant in oyster mushroom.
- the transporter OCTN1 Due to the slow metabolism of ergothioneine in animals, ergothioneine tends to accumulate in body tissues and body fluids.
- the composition of the present invention is preferably formulated so that ergothioneine is ingested in an amount of 10 to 5000 ⁇ g/kg body weight/day.
- it is preferably 20 ⁇ g/kg body weight/day or more, 30 ⁇ g/kg body weight/day or more, 40 ⁇ g/kg body weight/day or more, or 50 ⁇ g/kg body weight/day or more.
- it is preferably 3000 ⁇ g/kg body weight/day or less, 2000 ⁇ g/kg body weight/day or less, 1000 ⁇ g/kg body weight/day or less, 500 ⁇ g/kg body weight/day or less, or 300 ⁇ g/kg body weight/day or less.
- composition is preferably blended so that the concentration of ergothioneine in the seminal plasma of a subject who has ingested the composition is 20-2000 ⁇ M, particularly 50-1500 ⁇ M.
- Compositions of the present invention formulated in such dosages can be taken in a single dose or in multiple doses per day.
- Ergothioneine may be purified from raw materials such as ergothioneine-containing mushrooms and incorporated into the composition of the present invention.
- a method for purifying ergothioneine for example, the method shown in Patent Document 1 can be used.
- ergothioneine may be included in the compositions of the present invention as unrefined comminutes, dry powders, concentrated extracts, etc. of sources such as ergothioneine-containing mushrooms.
- the compositions of the invention may comprise ergothioneine as a dry powder or concentrated extract of mushrooms, in particular as a dry powder or concentrated extract of Pleurotus cornucopia.
- the composition of the present invention preferably further contains creatine as an active ingredient in addition to ergothioneine.
- the inventors have found that contacting sperm with ergothioneine and creatine simultaneously improved sperm findings more significantly.
- the composition of the present invention can exhibit a higher sperm finding improvement effect by blending creatine in addition to ergothioneine.
- Creatine also called 1-methylguanidinoacetic acid or methylglycocyamine, is a type of organic acid that is mainly present in muscle. Creatine is biosynthesized from arginine and glycine in the kidney and liver, and is converted into energy-storing creatine phosphate in muscle tissue by reacting with ATP through the action of creatine kinase. Creatine phosphate acts as a storage material for high-energy phosphate bonds in organs such as muscles that consume a large amount of energy instantaneously, and when energy is insufficient, ATP is generated by supplying phosphate groups, and energy is generated. supply.
- creatine is phosphorylated again by creatine kinase and either recycled as creatine phosphate or undergoes irreversible nonenzymatic dehydration to creatinine. Creatinine is ultimately excreted in the urine by the kidneys. As described above, since creatine is involved in energy metabolism in explosive exercise, it is known that oral intake of creatine enhances the explosive power during exercise in subjects.
- the form of creatine contained in the composition of the present invention includes any physiologically available form of creatine, and may be, for example, creatine free acid, salt, or derivative thereof.
- the form of creatine is not particularly limited, but examples thereof include salts such as free acid, calcium salt, sodium salt, potassium salt, magnesium salt, hydrochloride and citrate, ester, lactone, hydrate and the like. Among them, creatine calcium salt, creatine-hydrate and tricreatine malate are preferred.
- the creatine used in the present invention one synthesized by a known method or a commercially available product can be used.
- the composition for oral ingestion according to the present invention preferably contains creatine in an amount of 50 to 300 mg/kg body weight/day. In particular, it is preferably 50 mg/kg body weight/day or more, or 100 mg/kg body weight/day or more. Also, it is preferably 250 mg/kg body weight/day or less, or 200 mg/kg body weight/day or less.
- Ergothioneine and creatine in the present invention are preferably blended so that the daily intake is 1:3000 to 1:100, particularly 1:2500 to 1:1000, by weight.
- the composition of the present invention is a food (for example, a food for special uses, a food for specified health use, a food with nutrient function claims, a functional food whose efficacy has been approved by a predetermined organization, a supplement, etc.), or a pharmaceutical (quasi-drug). including), or can be used as a general food or food additive.
- composition of the present invention is not particularly limited as long as it can be easily ingested by the target mammalian animal.
- the composition of the present invention can optionally contain a carrier.
- carriers include excipients, binders, disintegrants, fillers, emulsifiers, flow additive modifiers, lubricants and the like.
- Excipients include sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides (more particularly but not limited to glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, maltitol, dextrin, maltodextrin, starch and cellulose, and their processed products), metal salts (e.g.
- Examples include glycine, low, medium and high molecular weight polyethylene glycols (PEG), Pluronics, kaolin, silicic acid, or combinations thereof.
- binders include starch paste using corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac and/or polyvinylpyrrolidone. It is mentioned as.
- Disintegrants include starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride. Or those salts are mentioned as an example.
- fillers include the above sugars and/or calcium phosphate (eg, tricalcium phosphate or calcium hydrogen phosphate).
- emulsifiers examples include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.
- flow additives and lubricants examples include silicates, talc, stearates or polyethylene glycol.
- Such a carrier is used to maintain the shape of the product and the effect of ergothioneine, and may be used appropriately as necessary.
- flavoring agents solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption promoters, bulking agents, wetting agents, humectants, adsorbents, Disintegration retardants, anti-caking agents, coating agents, coloring agents, preservatives, antioxidants, flavoring agents, flavoring agents, sweetening agents, buffering agents and the like may also be included.
- the composition of the present invention may be ingested as it is, or may be ingested after being mixed with other compositions or added to drinking water.
- the composition of the present invention preferably contains 1/5 to 3 times, particularly 1/3 to 1 time of the daily intake of the active ingredient per dose. That is, the intake frequency is preferably once every three days to five times a day, particularly once a day to three times a day.
- the frequency with which the subject ingests the composition of the present invention is not particularly limited, and for example, the frequency may be once a day.
- the intake period is also not particularly limited, but can be, for example, 1 to 30 days, 3 to 21 days, or 5 to 14 days.
- the method for producing a composition for oral ingestion for improving sperm findings in mammals of the present invention (hereinafter also referred to as the "production method of the present invention") is characterized by comprising the step of blending ergothioneine. More specifically, the production method of the present invention includes ⁇ 1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>.
- the composition produced by the production method of the present invention can improve the sperm findings of subjects who have ingested it.
- the production method of the present invention may include a step of further blending creatine.
- the manufactured composition can exhibit a higher effect of improving sperm findings.
- the order in which ergothioneine and creatine are added is not particularly limited, and they can be added in the order that facilitates formulation.
- the method for improving sperm findings in mammals of the present invention comprises the step of orally administering ergothioneine to a subject in need of improving sperm findings. .
- mammals are the subjects to which ergothioneine is orally administered.
- mammals include humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, pigs, horses, sheep and goats, rodents such as mice and rats, and animals raised in zoos. mammals, etc.
- the subject of the improvement method of the present invention is preferably human.
- the subject When the subject is a human, it is preferably a male infertility patient.
- the male infertility is preferably idiopathic oligozoospermia and/or asthenozoospermia.
- the symptom of male infertility is a decrease in sperm concentration in semen, a decrease in sperm motility, and/or a decrease in sperm advance rate.
- ergothioneine is preferably orally administered to the subject in an amount of 10-5000 ⁇ g/kg body weight/day.
- it is preferably 20 ⁇ g/kg body weight/day or more, 30 ⁇ g/kg body weight/day or more, 40 ⁇ g/kg body weight/day or more, or 50 ⁇ g/kg body weight/day or more.
- it is preferably 3000 ⁇ g/kg body weight/day or less, 2000 ⁇ g/kg body weight/day or less, 1000 ⁇ g/kg body weight/day or less, 500 ⁇ g/kg body weight/day or less, or 300 ⁇ g/kg body weight/day or less.
- ergothioneine it is preferable to orally administer ergothioneine so that the concentration of ergothioneine in the subject's seminal plasma is 20 to 2000 ⁇ M, particularly 50 to 1500 ⁇ M.
- concentration of ergothioneine in the subject's seminal plasma is 20 to 2000 ⁇ M, particularly 50 to 1500 ⁇ M.
- Such amounts of ergothioneine can be administered in single or multiple doses per day.
- creatine is further orally administered to the subject.
- Creatine may be administered in the same composition as ergothioneine or may be administered in a separate composition.
- creatine may be administered simultaneously with ergothioneine or may be administered separately.
- Creatine is preferably administered orally in an amount of 50-300 mg/kg body weight/day. In particular, it is preferably 50 mg/kg body weight/day or more, or 100 mg/kg body weight/day or more. Also, it is preferably 250 mg/kg body weight/day or less, or 200 mg/kg body weight/day or less.
- the daily dosage of ergothioneine and creatine is preferably 1:3000 to 1:100, especially 1:2500 to 1:1000 by weight.
- the detailed conditions such as the method of formulation of the active ingredient and the presence or absence of administration of other ingredients, unless there is a particular contradiction, ⁇ 1.
- the present invention provides the use of ergothioneine in the manufacture of an orally ingestible composition for improving sperm findings in mammals. Said uses are hereinafter also referred to as "uses of the invention". Specifically, the use of the present invention is ⁇ 1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>. Unless otherwise contradicted, detailed conditions such as subjects for ingestion, uses, effects, and preparation methods of the composition for oral ingestion produced by the use of the present invention are described in ⁇ 1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>.
- the present invention provides a sperm medium containing ergothioneine.
- the sperm culture medium of the present invention is used for mammalian sperm, preferably human male sperm.
- the sperm culture medium of the present invention can maintain or improve the motility of sperm, and is used, for example, but not limited to, for washing, preserving, and culturing sperm, in vitro fertilization, or artificial insemination.
- the sperm culture medium of the present invention can be produced by adding ergothioneine to a known sperm culture medium such as HTF medium, TYH medium, or the medium described in JP-A-2014-128219.
- the concentration of ergothioneine contained in the sperm culture medium is not particularly limited, it is preferably 0.01 to 3 mM, particularly 0.1 to 1 mM.
- the sperm culture medium of the present invention may further contain creatine.
- creatine By further containing creatine, it is possible to obtain an additive or synergistic effect of maintaining and improving sperm motility.
- the concentration of creatine is not particularly limited, but is preferably 0.01 to 1 mM, particularly 0.05 to 0.5 mM.
- Example 1 Tamogitake intake test> Thirteen male subjects aged 28 to 51 years were orally administered 1.98 g/day of Pleurotus cornucopia powder (equivalent to 10 mg/day of ergothioneine) for 2 weeks. Semen was collected from each subject before and after the administration of powdered Pleurotus cornucopia. Semen was collected by masturbation in the clinic. The collected semen was allowed to stand at 37° C. for 30 minutes to fully liquefy, and the liquid weight (semen volume) was measured. A sperm motility analysis system CASA/IVOS (registered trademark) II (manufactured by Hamilton Thorne) was used to measure sperm concentration, motility, advance rate and average velocity (VAP).
- CASA/IVOS registered trademark
- VAP average velocity
- Table 1 shows the semen volume, sperm concentration, sperm motility, advancement rate, and VAP before and after administration of Tamogitake powder.
- Sperm concentration, motility, advancement rate, and VAP increased after administration. In particular, it was confirmed that sperm motility and advance rate were significantly increased.
- Example 2 Ergothioneine addition test to porcine semen (in vitro)> Fresh sperm were collected from 5 boars, and a sperm preservation solution containing 0, 0.1 or 1.0 mM ergothioneine (30 mM glucose, 120 mM lactose, 6.9 g/L sodium citrate, 1 g/L NaHCO 3 , 2.35 g/L EDTA-2Na, 2.9 g/L citric acid, 5.65 g/L Tris, 5 mM lactic acid, 1 mM glycine) to prepare a sperm suspension. After allowing each sperm suspension to stand at 37° C. under 5% CO 2 conditions for 6 hours, the linear velocity of sperm (VSL ( ⁇ m/S)) and curve velocity (VCL ( ⁇ m/S)) were measured.
- VSL linear velocity of sperm
- VCL curve velocity
- VSL and VCL of sperm in each sperm suspension are shown in Figures 1 and 2, respectively. It was confirmed that both VSL and VCL were significantly improved by adding 0.1 mM or more of ergothioneine (p ⁇ 0.05). It has been reported that the sperm preservation solution used in this study has the effect of maintaining sperm motility (see JP-A-2014-128219). It was confirmed that the addition of ergothioneine further improved sperm motility.
- Example 3 Coexistence test of ergothioneine and creatine (in vitro)> Fresh spermatozoa were collected from 5 boars and inoculated in HTF (Human tubal fluids) medium supplemented with 0.1% albumin containing ergothioneine and creatine shown in FIG. The medium was cultured at 37° C. and 5% CO 2 for 4 hours, and sperm motility was measured in the same manner as in Example 1.
- HTF Human tubal fluids
- the HTF medium is known as a culture medium that induces sperm motility during fertilization, and it was confirmed that coexistence of ergothioneine and creatine has the effect of further increasing sperm motility.
- Example 4 Tamogitake mushroom + creatine intake test>
- a composition containing 275 mg of oyster mushroom powder and creatine including 275 mg of oyster mushroom powder, 5 g of creatine powder, flavor, sweetener, and salts
- Semen was collected from each subject before and after administration of the composition. The abstinence period before semen collection was within 3 days.
- sperm motility analysis system CASA / IVOS (registered trademark) II manufactured by Hamilton Thorne
- the undiluted semen findings sperm concentration, forward motility, total motile sperm count
- sperm motility VAP, VCL, VSL and ALH
- the semen after examination was centrifuged at 400 rpm for 10 minutes to separate the seminal plasma.
- the seminal plasma obtained was stored frozen at about -20°C for up to _60 days. Separated sperm were seeded in HTF medium (Kitasato Corporation), 37 °C, 5% CO2 .
- sperm motility was measured by the same method as in Example 1.
- sperm motility VAP, VCL, VSL and ALH
- CASA/IVOS registered trademark
- the frozen seminal plasma was thawed and the contents of 8-hydroxy-2'-deoxyguanosine (8-OHdG), spermine, creatine, testosterone and zinc were measured.
- 8-OHdG concentration in seminal plasma was measured by the following method.
- the 8-OHdG-BSA complex was diluted with PBS, dispensed into 96-well microplates in 50 ⁇ L aliquots, and shaken at room temperature for 1 hour. After washing four times with 0.01% Tween20-PBS (TPBS), 250 ⁇ L of 1% BSA/PBS was added and incubated at room temperature for 1 hour. After washing four times with TPBS, 50 ⁇ L of seminal plasma and 8-OHdG standard solution of known concentration were added.
- TPBS 0.01% Tween20-PBS
- each HRP-labeled anti-8-OHdG antibody solution 50 ⁇ L was dispensed. After shaking for 10 minutes, it was incubated at room temperature for 50 minutes. After washing four times with TPBS, 100 ⁇ L of tetramethylbenzidine (TMB) was dispensed and allowed to stand for 10 minutes to develop color, and 50 ⁇ L of 1 M phosphoric acid was dispensed to stop. Absorbance at 450 nm/570 nm was measured in a microwell plate. A calibration curve was prepared from the absorbance data of the standard solution, and the 8-OHdG concentration of each seminal plasma was calculated.
- TMB tetramethylbenzidine
- spermine is a component of semen and is a substance involved in cell division and protein synthesis.
- spermine is 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxa, as described in J. Chromatogr A. 2008 Sep 26;1205(1-2):94-102.
- the chromatographic conditions were as follows.
- Liquid chromatograph equipment ACQUITYTM Ultra Performance Liquid Chromatography and Micromass LCT PremierTM XE Mass Spectrometer (High sensitivity orthogonal time-of-flight instrument; Waters, Milford, USA)
- the creatine concentration in the seminal plasma was measured by diluting the seminal plasma sample 40 times and using the enzymatic method. Specifically, creatine contained in the diluted sample was converted to hydrogen peroxide using a series of enzymatic reactions involving creatine kinase and sarcosine oxidase. The resulting hydrogen peroxide was quantified by condensing 4-aminoantipyrine with phenols in the presence of peroxidase to form a dye and measuring the absorbance of the resulting dye.
- the testosterone concentration in seminal plasma was measured by the following method.
- the anti-testosterone antibody was diluted with PBS, dispensed into 96-well microplates in 50 ⁇ L aliquots, and shaken at room temperature for 1 hour. After washing four times with 0.01% Tween20-PBS (TPBS), 250 ⁇ L of 1% BSA/PBS was added and incubated at room temperature for 1 hour. After washing four times with TPBS, 50 ⁇ L each of HRP-labeled testosterone, seminal plasma, or testosterone standard solution of known concentration was added. After shaking for 10 minutes, it was incubated at room temperature for 50 minutes.
- TMB tetramethylbenzidine
- the zinc concentration in seminal plasma was measured by the following method. After thawing, the seminal plasma sample was diluted 20-fold, and iron and copper in the seminal plasma were removed by coprecipitation with trichloroacetic acid and potassium fluoride. It is then reacted with a reagent containing 2-(5-bromo-2-pyridylazo)-5-[Nn-propyl-N-(3-sulfopropylamino]-phenol (5-Br-PAPS) and the absorbance is Measured and quantified.
- Table 2 shows the sperm concentration, forward motility, and total motility index immediately after collection for the semen before and after administration of the composition.
- Table 3 shows the motility rate, VAP, VCL, VSL and ALH. Numerical values represent the mean ⁇ standard deviation. It was confirmed that sperm concentration, forward motility, and total motility index all increased significantly after administration of the composition. It was also confirmed that the motility rate, VAP, VCL, VSL, and ALH were also significantly increased. In particular, VCL, VSL and ALH are known to be indicators of sperm fertility, and it was suggested that their elevations improved sperm fertility.
- Table 4 shows the VAP, VCL, VSL, and ALH of sperm cultured for 3 hours after collection for semen before and after administration of the composition. Numerical values represent the mean ⁇ standard deviation. When artificial insemination is performed, the sperm is brought into contact with the egg after undergoing a 3-hour incubation process. Also in natural pregnancy, it is said that it takes about 3 hours for the sperm after ejaculation to reach the egg. As shown in Table 4, VSL and ALH were significantly increased after administration of the composition. This suggested that the sperm after administration of the composition could maintain a significantly high fertility even after 3 hours of culture.
- Table 5 shows the measurement results of the contents of 8-OHdG, spermine, creatine, testosterone and zinc in the seminal plasma before and after administration of the composition. Numerical values represent the mean ⁇ standard deviation. The creatine concentration in the seminal plasma increased significantly after ingestion, indicating that the ingested creatine was transferred to semen. rice field. 8-OHdG, spermine, testosterone and zinc are all components that are suggested to have a positive or negative effect on fertility, but it has been confirmed that there is no significant effect by composition administration. rice field.
Abstract
The present invention improves the sperm finding of mammalian animals, and in particular, male infertility patients. This composition for oral ingestion, which contains ergothioneine as an active component, is administered orally to a subject requiring an improvement in sperm finding.
Description
本発明は、エルゴチオネインを有効成分として含む、精子所見を改善するための経口摂取用組成物に関する。
The present invention relates to an orally ingestible composition for improving sperm findings, containing ergothioneine as an active ingredient.
男性不妊症の９０％を占める乏精子症や精子無力症の多くが、原因が特定されない特発性である。特発性の乏精子症や精子無力症に対する治療法は、非特異的な薬物療法が主体である。一般的に使用される薬剤は、精子のエネルギー代謝、ＤＮＡ合成や抗酸化作用などに着目した薬剤が多く、ビタミン剤やカリクレイン製剤、各種酵素剤や微量元素製剤、漢方薬などを用いた非分泌療法と、クロミフェンやゴナトロピン製剤などを用いた内分泌療法が行われているが、特発性の症例に対しての大規模な無作為化比較対象試験（ＲＣＴ）は報告も少なく、現在、特発性男性不妊症に対しての治療的効果の根拠はほとんど知られていない。
Many cases of oligozoospermia and asthenozoospermia, which account for 90% of male infertility, are idiopathic with no specific cause. Treatment for idiopathic oligozoospermia and asthenozoospermia is mainly nonspecific pharmacotherapy. Many of the commonly used drugs focus on sperm energy metabolism, DNA synthesis, and antioxidant effects. and endocrine therapy using clomiphene and gonatropin preparations, etc., but there are few reports of large-scale randomized controlled trials (RCTs) for idiopathic cases, and currently, idiopathic male infertility Little is known about the evidence for its therapeutic effect on disease.
エルゴチオネインは、タモギタケ等のキノコ類に含まれる、抗酸化物質として化粧品等に使用されるアミノ酸の一種である。特許文献１には、タモギタケにおけるエルゴチオネイン含有量は、乾燥重量１ｇあたり１５．８±０．０６ｍｇと報告されている。非特許文献１には、エルゴチオネインは、食物としてヒト及び動物に摂取された場合、代謝に時間がかかるため、ヒト及び動物の多くの組織及び体液に蓄積しやすいことが示されている。エルゴチオネインには、抗酸化作用及び細胞保護作用があることがｉｎ ｖｉｔｒｏで確認されており、ヒト及び動物の体内において、組織損傷の修復等に関与することが示唆されている。ヒトにおいて、エルゴチオネインは、その抗酸化作用と細胞保護作用により、神経変性疾患、眼疾患、循環器疾患、腎臓疾患、糖尿病等への治療効果を有する安全性の高い食物由来成分として期待されている。非特許文献１には、動物の精液に含まれるエルゴチオネイン量が、血中濃度の６倍以上となることが報告されている。また、非特許文献２には、凍結融解したヒツジの精子をエルゴチオネインと接触させることで、精子の運動率、前進率、運動速度（ＶＡＰ）が上昇することが報告されている。しかしながら、エルゴチオネインの経口摂取による精液及び精子に対する臨床的な効果については、これまでに報告されていない。
Ergothioneine is a type of amino acid that is used in cosmetics as an antioxidant, contained in mushrooms such as Tamogitake. Patent Document 1 reports that the ergothioneine content in Pleurotus cornucopia is 15.8±0.06 mg per 1 g of dry weight. Non-Patent Document 1 indicates that when ergothioneine is ingested by humans and animals as food, it takes a long time to metabolize, so it tends to accumulate in many tissues and body fluids of humans and animals. It has been confirmed in vitro that ergothioneine has an antioxidant effect and a cytoprotective effect, and it has been suggested that it is involved in the repair of tissue damage and the like in the bodies of humans and animals. In humans, ergothioneine is expected to be a highly safe food-derived ingredient with therapeutic effects on neurodegenerative diseases, eye diseases, cardiovascular diseases, kidney diseases, diabetes, etc., due to its antioxidant and cytoprotective effects. . Non-Patent Document 1 reports that the amount of ergothioneine contained in animal semen is 6 times or more the blood concentration. In addition, Non-Patent Document 2 reports that contacting freeze-thawed sheep sperm with ergothioneine increases sperm motility, advancement rate, and motility velocity (VAP). However, clinical effects of oral intake of ergothioneine on semen and sperm have not been reported so far.
一方、マウスの精子をエネルギー代謝に関連するクレアチンと接触させることで、精子の運動率が上昇することが報告されている（非特許文献３）。
On the other hand, it has been reported that contacting mouse sperm with creatine, which is related to energy metabolism, increases sperm motility (Non-Patent Document 3).
本発明は、精子所見を改善するための経口摂取用組成物を提供することを目的とする。
The purpose of the present invention is to provide an orally ingestible composition for improving sperm findings.
本発明者らは、鋭意検討を重ねた結果、哺乳類動物において、エルゴチオネインの経口摂取によってその精子所見が改善することを見出し、本発明を完成させるに至った。すなわち、本発明は、以下を提供するものである。
As a result of extensive studies, the present inventors found that oral ingestion of ergothioneine improved sperm findings in mammals, leading to the completion of the present invention. That is, the present invention provides the following.
（１）エルゴチオネインを有効成分として含む、哺乳類動物の精子所見を改善するための経口摂取用組成物。
（２）前記哺乳類動物がヒトである、（１）に記載の組成物。
（３）前記ヒトが、男性不妊症患者である、（２）に記載の組成物。
（４）前記男性不妊症が、特発性の乏精子症及び／又は精子無力症である、（３）に記載の組成物。
（５）前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下の症状を有する、（３）又は（４）に記載の組成物。
（６）エルゴチオネインが、１０～５０００μｇ／ｋｇ体重／日の量で摂取されるように配合される、請求項（１）～（５）のいずれかに記載の組成物。
（７）クレアチンを有効成分としてさらに含む、（１）～（６）のいずれかに記載の組成物。
（８）クレアチンが、５０～３００ｍｇ／ｋｇ体重／日の量で摂取されるように配合される、（７）に記載の組成物。
（９）エルゴチオネインとクレアチンが、一日あたりの摂取量が重量比で１：３０００～１：１００となるように配合される、（７）又は（８）に記載の組成物。
（１０）食品である、（１）～（９）のいずれかに記載の組成物。
（１１）医薬品である、（１）～（９）のいずれかに記載の組成物。
（１２）エルゴチオネインを配合する工程を含む、哺乳類動物の精子所見を改善するための経口摂取用組成物の製造方法。
（１３）クレアチンをさらに配合する工程を含む、（１２）に記載の方法。 (1) A composition for oral ingestion for improving sperm findings in mammals, comprising ergothioneine as an active ingredient.
(2) The composition according to (1), wherein the mammal is human.
(3) The composition according to (2), wherein the human is a male infertility patient.
(4) The composition according to (3), wherein the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
(5) The composition according to (3) or (4), wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
(6) The composition according to any one of (1) to (5), wherein ergothioneine is formulated so as to be ingested in an amount of 10 to 5000 μg/kg body weight/day.
(7) The composition according to any one of (1) to (6), further comprising creatine as an active ingredient.
(8) The composition according to (7), wherein creatine is formulated to be taken in an amount of 50-300 mg/kg body weight/day.
(9) The composition according to (7) or (8), wherein ergothioneine and creatine are blended at a weight ratio of 1:3000 to 1:100 per day.
(10) The composition according to any one of (1) to (9), which is a food.
(11) The composition according to any one of (1) to (9), which is a pharmaceutical.
(12) A method for producing a composition for oral ingestion for improving sperm findings in mammals, comprising the step of incorporating ergothioneine.
(13) The method according to (12), which further comprises the step of blending creatine.
（２）前記哺乳類動物がヒトである、（１）に記載の組成物。
（３）前記ヒトが、男性不妊症患者である、（２）に記載の組成物。
（４）前記男性不妊症が、特発性の乏精子症及び／又は精子無力症である、（３）に記載の組成物。
（５）前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下の症状を有する、（３）又は（４）に記載の組成物。
（６）エルゴチオネインが、１０～５０００μｇ／ｋｇ体重／日の量で摂取されるように配合される、請求項（１）～（５）のいずれかに記載の組成物。
（７）クレアチンを有効成分としてさらに含む、（１）～（６）のいずれかに記載の組成物。
（８）クレアチンが、５０～３００ｍｇ／ｋｇ体重／日の量で摂取されるように配合される、（７）に記載の組成物。
（９）エルゴチオネインとクレアチンが、一日あたりの摂取量が重量比で１：３０００～１：１００となるように配合される、（７）又は（８）に記載の組成物。
（１０）食品である、（１）～（９）のいずれかに記載の組成物。
（１１）医薬品である、（１）～（９）のいずれかに記載の組成物。
（１２）エルゴチオネインを配合する工程を含む、哺乳類動物の精子所見を改善するための経口摂取用組成物の製造方法。
（１３）クレアチンをさらに配合する工程を含む、（１２）に記載の方法。 (1) A composition for oral ingestion for improving sperm findings in mammals, comprising ergothioneine as an active ingredient.
(2) The composition according to (1), wherein the mammal is human.
(3) The composition according to (2), wherein the human is a male infertility patient.
(4) The composition according to (3), wherein the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
(5) The composition according to (3) or (4), wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
(6) The composition according to any one of (1) to (5), wherein ergothioneine is formulated so as to be ingested in an amount of 10 to 5000 μg/kg body weight/day.
(7) The composition according to any one of (1) to (6), further comprising creatine as an active ingredient.
(8) The composition according to (7), wherein creatine is formulated to be taken in an amount of 50-300 mg/kg body weight/day.
(9) The composition according to (7) or (8), wherein ergothioneine and creatine are blended at a weight ratio of 1:3000 to 1:100 per day.
(10) The composition according to any one of (1) to (9), which is a food.
(11) The composition according to any one of (1) to (9), which is a pharmaceutical.
(12) A method for producing a composition for oral ingestion for improving sperm findings in mammals, comprising the step of incorporating ergothioneine.
(13) The method according to (12), which further comprises the step of blending creatine.
（１４）哺乳類動物の精子所見を改善する方法であって、精子所見の改善が必要な対象にエルゴチオネインを経口投与する工程を含む、方法。
（１５）前記対象がヒトである、（１４）に記載の方法。
（１６）前記ヒトが男性不妊症患者である、（１５）に記載の方法。
（１７）前記男性不妊症が、特発性の乏精子症及び／又は精子無力症である、（１６）に記載の方法。
（１８）前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下の症状を有する、（１６）又は（１７）に記載の方法。
（１９）エルゴチオネインが、１０～５０００μｇ／ｋｇ体重／日の量で経口投与される、（１６）～（１８）のいずれかに記載の方法。
（２０）前記対象にクレアチンをさらに経口投与する、（１４）～（１９）のいずれかに記載の方法。
（２１）クレアチンが、５０～３００ｍｇ／ｋｇ体重／日の量で経口投与される、（２０）に記載の方法。
（２２）エルゴチオネインとクレアチンが、一日あたりの投与量が重量比で１：３０００～１：１００となるように経口投与される、（２０）又は（２１）に記載の方法。 (14) A method of improving sperm findings in a mammal, comprising orally administering ergothioneine to a subject in need of improving sperm findings.
(15) The method of (14), wherein the subject is human.
(16) The method of (15), wherein the human is a male infertility patient.
(17) The method according to (16), wherein the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
(18) The method according to (16) or (17), wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
(19) The method according to any one of (16) to (18), wherein ergothioneine is orally administered in an amount of 10 to 5000 μg/kg body weight/day.
(20) The method according to any one of (14) to (19), wherein creatine is further orally administered to the subject.
(21) The method according to (20), wherein creatine is orally administered in an amount of 50-300 mg/kg body weight/day.
(22) The method according to (20) or (21), wherein ergothioneine and creatine are orally administered at a weight ratio of 1:3000 to 1:100 per day.
（１５）前記対象がヒトである、（１４）に記載の方法。
（１６）前記ヒトが男性不妊症患者である、（１５）に記載の方法。
（１７）前記男性不妊症が、特発性の乏精子症及び／又は精子無力症である、（１６）に記載の方法。
（１８）前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下の症状を有する、（１６）又は（１７）に記載の方法。
（１９）エルゴチオネインが、１０～５０００μｇ／ｋｇ体重／日の量で経口投与される、（１６）～（１８）のいずれかに記載の方法。
（２０）前記対象にクレアチンをさらに経口投与する、（１４）～（１９）のいずれかに記載の方法。
（２１）クレアチンが、５０～３００ｍｇ／ｋｇ体重／日の量で経口投与される、（２０）に記載の方法。
（２２）エルゴチオネインとクレアチンが、一日あたりの投与量が重量比で１：３０００～１：１００となるように経口投与される、（２０）又は（２１）に記載の方法。 (14) A method of improving sperm findings in a mammal, comprising orally administering ergothioneine to a subject in need of improving sperm findings.
(15) The method of (14), wherein the subject is human.
(16) The method of (15), wherein the human is a male infertility patient.
(17) The method according to (16), wherein the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
(18) The method according to (16) or (17), wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
(19) The method according to any one of (16) to (18), wherein ergothioneine is orally administered in an amount of 10 to 5000 μg/kg body weight/day.
(20) The method according to any one of (14) to (19), wherein creatine is further orally administered to the subject.
(21) The method according to (20), wherein creatine is orally administered in an amount of 50-300 mg/kg body weight/day.
(22) The method according to (20) or (21), wherein ergothioneine and creatine are orally administered at a weight ratio of 1:3000 to 1:100 per day.
（２３）哺乳類動物の精子所見を改善するための経口摂取用組成物の製造におけるエルゴチオネインの使用。
（２４）前記哺乳類動物がヒトである、（２３）に記載の使用。
（２５）前記ヒトが男性不妊症患者である、（２４）に記載の使用。
（２６）前記男性不妊症が、特発性の乏精子症及び／又は精子無力症である、（２５）に記載の使用。
（２７）前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下の症状を有する、（２５）又は（２６）に記載の使用。
（２８）前記経口摂取用組成物にエルゴチオネインが１日用量として１０～５０００μｇ／ｋｇ体重／日の量で経口摂取されるように配合される、（２３）～（２７）のいずれかに記載の使用。
（２９）前記経口摂取用組成物が食品である、（２３）～（２８）のいずれかに記載の使用。
（３０）前記経口摂取用組成物が医薬品である、（２３）～（２８）のいずれかに記載の使用。 (23) Use of ergothioneine in the manufacture of an orally ingestible composition for improving sperm findings in mammals.
(24) The use according to (23), wherein said mammal is a human.
(25) The use according to (24), wherein the human is a male infertility patient.
(26) The use according to (25), wherein the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
(27) The use according to (25) or (26), wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
(28) The composition according to any one of (23) to (27), wherein ergothioneine is added to the composition for oral ingestion at a daily dose of 10 to 5000 μg/kg body weight/day for oral ingestion. use.
(29) Use according to any one of (23) to (28), wherein the composition for oral ingestion is a food.
(30) The use according to any one of (23) to (28), wherein the composition for oral ingestion is a pharmaceutical.
（２４）前記哺乳類動物がヒトである、（２３）に記載の使用。
（２５）前記ヒトが男性不妊症患者である、（２４）に記載の使用。
（２６）前記男性不妊症が、特発性の乏精子症及び／又は精子無力症である、（２５）に記載の使用。
（２７）前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下の症状を有する、（２５）又は（２６）に記載の使用。
（２８）前記経口摂取用組成物にエルゴチオネインが１日用量として１０～５０００μｇ／ｋｇ体重／日の量で経口摂取されるように配合される、（２３）～（２７）のいずれかに記載の使用。
（２９）前記経口摂取用組成物が食品である、（２３）～（２８）のいずれかに記載の使用。
（３０）前記経口摂取用組成物が医薬品である、（２３）～（２８）のいずれかに記載の使用。 (23) Use of ergothioneine in the manufacture of an orally ingestible composition for improving sperm findings in mammals.
(24) The use according to (23), wherein said mammal is a human.
(25) The use according to (24), wherein the human is a male infertility patient.
(26) The use according to (25), wherein the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
(27) The use according to (25) or (26), wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
(28) The composition according to any one of (23) to (27), wherein ergothioneine is added to the composition for oral ingestion at a daily dose of 10 to 5000 μg/kg body weight/day for oral ingestion. use.
(29) Use according to any one of (23) to (28), wherein the composition for oral ingestion is a food.
(30) The use according to any one of (23) to (28), wherein the composition for oral ingestion is a pharmaceutical.
（３１）エルゴチオネインを含む、精子用培地。
（３２）クレアチンをさらに含む、（３１）に記載の精子用培地。
本明細書は本願の優先権の基礎となる日本国特許出願番号2021-106397号の開示内容を包含する。 (31) A sperm culture medium containing ergothioneine.
(32) The sperm culture medium according to (31), further comprising creatine.
This specification includes the disclosure of Japanese Patent Application No. 2021-106397, which is the basis of priority of this application.
（３２）クレアチンをさらに含む、（３１）に記載の精子用培地。
本明細書は本願の優先権の基礎となる日本国特許出願番号2021-106397号の開示内容を包含する。 (31) A sperm culture medium containing ergothioneine.
(32) The sperm culture medium according to (31), further comprising creatine.
This specification includes the disclosure of Japanese Patent Application No. 2021-106397, which is the basis of priority of this application.
本発明によれば、精子所見を改善する経口摂取用組成物を提供することができる。
According to the present invention, a composition for oral ingestion that improves sperm findings can be provided.
本明細書において、特に記載にない限り、「％」は、重量％を示す。また、特に記載のない限り、モル濃度（Ｍ）は、１立方デシメートルあたりに含まれるモル量（ｍｏｌ／ｄｍ３）を示す。
In this specification, unless otherwise specified, "%" indicates % by weight. In addition, unless otherwise specified, the molar concentration (M) indicates the molar amount contained per cubic decimeter (mol/dm 3 ).
＜１．経口摂取用組成物＞
本発明の哺乳類動物の精子所見を改善するための経口摂取用組成物（以下、「本発明の組成物」とも称する）は、エルゴチオネインを有効成分として含み、哺乳類動物の精子所見を改善するために使用されることを特徴とする。 <1. Composition for Oral Ingestion>
The composition for oral ingestion for improving sperm findings in mammals of the present invention (hereinafter also referred to as "the composition of the present invention") contains ergothioneine as an active ingredient, and is used to improve sperm findings in mammals. characterized by being used.
本発明の哺乳類動物の精子所見を改善するための経口摂取用組成物（以下、「本発明の組成物」とも称する）は、エルゴチオネインを有効成分として含み、哺乳類動物の精子所見を改善するために使用されることを特徴とする。 <1. Composition for Oral Ingestion>
The composition for oral ingestion for improving sperm findings in mammals of the present invention (hereinafter also referred to as "the composition of the present invention") contains ergothioneine as an active ingredient, and is used to improve sperm findings in mammals. characterized by being used.
本発明において、「哺乳類動物」とは、ヒト及びヒト以外を包含する、脊索動物門脊椎動物亜門哺乳網に属する動物を指し、例えば、ヒト、チンパンジーを含む霊長類、イヌ、ネコなどのペット動物、ウシ、ブタ、ウマ、ヒツジ、ヤギなどの家畜動物、マウス、ラットなどの齧歯類、動物園で飼育される哺乳類動物などが含まれる。本発明の経口摂取用組成物を投与する対象は、好ましくは、ヒトである。
In the present invention, the term "mammal" refers to animals belonging to the phylum Chordata subphylum Mammalia, including humans and non-humans. For example, primates including humans and chimpanzees, pets such as dogs and cats Animals include livestock animals such as cows, pigs, horses, sheep and goats, rodents such as mice and rats, mammals kept in zoos, and the like. The subject to whom the composition for oral ingestion of the present invention is administered is preferably human.
本発明において「精子所見」とは、哺乳類動物、好ましくはヒトの精液の状態のことを指し、具体的には、精液の量（射精量）、精子濃度、精子運動率、精子前進率、精子速度（平均速度（ＶＡＰ）、直線速度（ＶＳＬ）、及び曲線速度（ＶＣＬ））、頭部振幅（ＡＬＨ）等の指標によって評価される状態を指す。本発明において、精子所見における各指標は、「ヒト精液検査と手技」世界保健機関（ＷＨＯ）ラボマニュアル第５版（WHO laboratory manual for the Examination and processing of human semen Fifth edition (2010)）に記載の方法に準ずる方法で測定されるものを指す。このような指標は、一般に精子運動解析システム（ＣＡＳＡ：Computer-Aided Sperm Analysis)システムを使用して計測される。ＣＡＳＡで測定可能な指標のうち、特にＶＳＬ、ＶＣＬ及びＡＬＨが、精子の受精能の指標として有用であることが報告されている（Hirano Y., et al., J. Assist. Reprod. Genet., Vol. 18, No. 4, pp. 213-218 (2001))。
In the present invention, "sperm findings" refers to the state of mammalian, preferably human semen. Velocity (average velocity (VAP), linear velocity (VSL), and curvilinear velocity (VCL)), head amplitude (ALH), etc. In the present invention, each index in sperm findings is described in "Human Semen Examination and Procedure" World Health Organization (WHO) Laboratory Manual 5th Edition (WHO laboratory manual for the Examination and processing of human semen Fifth edition (2010)) It refers to the one measured by the method according to the method. Such indicators are generally measured using a sperm motility analysis system (CASA: Computer-Aided Sperm Analysis) system. Among the indicators measurable by CASA, VSL, VCL and ALH have been reported to be particularly useful as indicators of sperm fertility (Hirano Y., et al., J. Assist. Reprod. Genet. , Vol. 18, No. 4, pp. 213-218 (2001)).
前記対象がヒトである場合、特に、摂取の対象となる男性は限定されず、女性パートナーの妊娠を希望する男性等とすることができる。特に、対象は、男性不妊症患者とすることが好ましい。ＷＨＯによると、不妊は、外形上健康な若い男女が結婚した後、通常の同居と避妊をしていない性生活を１年間持続したにもかかわらず、妊娠しない場合と定義されている。不妊は、健常者の約１５％を占めており、このうち約３分の１で男性に不妊の原因があり、５分の１は、男性と女性の両方に不妊の原因があることが知られている。したがって、不妊は男性による原因が約５０％を占めており、近年増加傾向にあるといわれている。
When the subject is a human, there is no particular limitation on the male subject to ingestion, and it can be a male who wishes to conceive a female partner. In particular, the subject is preferably a male infertility patient. According to the WHO, infertility is defined as the inability to conceive after a year of normal cohabitation and unprotected sex in a young, apparently healthy man and woman after marriage. Infertility accounts for about 15% of healthy people, of which about one-third are known to be caused by men, and one-fifth are known to be caused by both men and women. It is Therefore, infertility is caused by men in about 50% of cases, and is said to be on the rise in recent years.
健康な男性の生殖能力は平均値で４０歳頃から低下し、３０歳の男性と比べた場合、４５歳の男性の生殖能力は２５％ほど低下するといわれている。結婚年齢の高齢化に伴い、女性だけでなく男性の生殖能力の低下が問題となっている。さらに、４０歳未満の若年の男性不妊症の原因は、精子の形成や成熟の過程に問題がある造精機能障害、精子の輸送経路が障害されている精路通過障害、精嚢や前立腺の炎症によって精子が影響を受ける副性器に起因する障害、性交渉や膣内射精ができないなどの性機能障害が知られている。病因別では造精機能障害が約９０％、精路通過障害が約５％、性機能障害が約３％、その他の原因が約２％とされており、原因がはっきりしない特発性の造精機能障害が男性不妊症の大多数を占めている。
The fertility of healthy men declines on average from around the age of 40, and it is said that the fertility of a 45-year-old man declines by about 25% compared to that of a 30-year-old man. Along with the aging of the marriage age, not only women but also men's fertility decline is becoming a problem. Furthermore, the causes of infertility in young men under the age of 40 include spermatogenesis dysfunction, which is a problem in the process of sperm formation and maturation; Disorders caused by accessory genitalia, in which sperm are affected by inflammation, and sexual dysfunction such as inability to have sexual intercourse or ejaculate in the vagina are known. By etiology, about 90% are spermatogenic dysfunction, about 5% are seminiferous passage disorders, about 3% are sexual dysfunction, and about 2% are due to other causes. Idiopathic spermatogenesis with unclear causes Dysfunction accounts for the majority of male infertility.
本発明に係る組成物は、上記の原因のうち、好ましくは、特発性造精機能障害に起因する男性不妊症における精子所見における症状、例えば、精液中の精子濃度の低下（乏精子症：精子数が２×１０７／ｍＬ以下)、精子運動率の低下（精子無力症：精子運動率が４０％以下）、精子運動率の低下、異常形態（奇形精子症：正常精子の比率が４０％以下）、又はこれらの組み合わせ、特には、特発性乏精子症及び／又は特発性精子無力症における精子所見の改善のために用いることができる。このような精子所見の症状としては、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下などが挙げられる。
Among the above causes, the composition according to the present invention is preferably used for symptoms in sperm findings in male infertility caused by idiopathic spermatogenic dysfunction, such as a decrease in sperm concentration in semen (oligozoospermia: sperm 2 × 10 7 / mL or less), decreased sperm motility (asthenozoospermia: sperm motility rate of 40% or less), decreased sperm motility, abnormal morphology (teratozoospermia: normal sperm ratio of 40% below), or a combination thereof, particularly for improving sperm findings in idiopathic oligozoospermia and/or idiopathic asthenozoospermia. Symptoms of such sperm findings include low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
本発明において、「エルゴチオネイン」は、下記式Ｉで表される化合物及びその誘導体を指す。下記式Ｉの構造式を有する全ての立体異性体が包含される。ここでいう誘導体としては、塩（カルシウム塩、ナトリウム塩、カリウム塩、マグネシウム塩、塩酸塩、クエン酸塩等）、水和物、エステル等が挙げられるが、下記式Ｉの骨格を有する限り、特に限定されない。
In the present invention, "ergothioneine" refers to the compound represented by Formula I below and derivatives thereof. All stereoisomers having the structural formula of Formula I below are included. Derivatives here include salts (calcium salts, sodium salts, potassium salts, magnesium salts, hydrochlorides, citrates, etc.), hydrates, esters, and the like. It is not particularly limited.
エルゴチオネインは、一部の真菌及び細菌によって生成されるチオール／チオケトンであり、特にタモギタケ、ヒラタケ、エリンギ、ハナビラタケ、エノキタケ、シイタケ等のキノコ類に含まれ、特にタモギタケに多く含まれることが知られる。哺乳類動物に摂取されると、トランスポーターＯＣＴＮ１の働きにより、動物の体内に取り込まれる。動物体内でのエルゴチオネインの代謝スピードが遅いことから、エルゴチオネインは体内組織や体液に蓄積されやすい。
Ergothioneine is a thiol/thioketone produced by some fungi and bacteria, and is particularly found in mushrooms such as oyster mushroom, oyster mushroom, oyster mushroom, crispa crisp, enoki mushroom, and shiitake mushroom, and is known to be especially abundant in oyster mushroom. When ingested by a mammalian animal, it is taken into the body of the animal by the action of the transporter OCTN1. Due to the slow metabolism of ergothioneine in animals, ergothioneine tends to accumulate in body tissues and body fluids.
動物の精液において、血中濃度の６倍以上のエルゴチオネインが検出されたことが報告されているが、エルゴチオネインの経口摂取が、哺乳類動物、特にヒトの精液の状態に与える影響は明らかにされていなかった。本発明者らは、エルゴチオネインの経口摂取により、哺乳類動物、より具体的にはヒトの精液所見が改善されることを見出した。
It has been reported that ergothioneine was detected in animal semen at a concentration six times higher than that in blood, but the effect of oral intake of ergothioneine on the condition of mammalian semen, especially in humans, has not been elucidated. rice field. The present inventors have found that oral intake of ergothioneine improves semen findings in mammals, more specifically in humans.
本発明の組成物は、エルゴチオネインが１０～５０００μｇ／ｋｇ体重／日の量で摂取されるように配合されることが好ましい。特に２０μｇ／ｋｇ体重／日以上、３０μｇ／ｋｇ体重／日以上、４０μｇ／ｋｇ体重／日以上又は５０μｇ／ｋｇ体重／日以上とすることが好ましい。また、３０００μｇ／ｋｇ体重／日以下、２０００μｇ／ｋｇ体重／日以下、１０００μｇ／ｋｇ体重／日以下、５００μｇ／ｋｇ体重／日以下、又は３００μｇ／ｋｇ体重／日以下とすることが好ましい。より具体的には、組成物を摂取した対象の精漿中のエルゴチオネイン濃度が、２０～２０００μＭ、特に５０～１５００μＭとなるように配合されることが好ましい。このような用量が配合された本発明の組成物は、１日に単回又は複数回に分けて摂取することができる。
The composition of the present invention is preferably formulated so that ergothioneine is ingested in an amount of 10 to 5000 μg/kg body weight/day. In particular, it is preferably 20 µg/kg body weight/day or more, 30 µg/kg body weight/day or more, 40 µg/kg body weight/day or more, or 50 µg/kg body weight/day or more. Also, it is preferably 3000 µg/kg body weight/day or less, 2000 µg/kg body weight/day or less, 1000 µg/kg body weight/day or less, 500 µg/kg body weight/day or less, or 300 µg/kg body weight/day or less. More specifically, the composition is preferably blended so that the concentration of ergothioneine in the seminal plasma of a subject who has ingested the composition is 20-2000 μM, particularly 50-1500 μM. Compositions of the present invention formulated in such dosages can be taken in a single dose or in multiple doses per day.
エルゴチオネインは、エルゴチオネインを含有するキノコ類等の原料から精製されて、本発明の組成物に配合されてもよい。エルゴチオネインの精製方法としては、例えば、特許文献１に示される方法を用いることができる。あるいは、エルゴチオネインは、エルゴチオネインを含有するキノコ類等の原料の未精製の細砕物、乾燥粉末、濃縮エキス等として、本発明の組成物に含まれていてもよい。本発明の組成物は、エルゴチオネインを、キノコ類の乾燥粉末又は濃縮エキスとして、特にタモギタケの乾燥粉末又は濃縮エキスとして含んでいてもよい。
Ergothioneine may be purified from raw materials such as ergothioneine-containing mushrooms and incorporated into the composition of the present invention. As a method for purifying ergothioneine, for example, the method shown in Patent Document 1 can be used. Alternatively, ergothioneine may be included in the compositions of the present invention as unrefined comminutes, dry powders, concentrated extracts, etc. of sources such as ergothioneine-containing mushrooms. The compositions of the invention may comprise ergothioneine as a dry powder or concentrated extract of mushrooms, in particular as a dry powder or concentrated extract of Pleurotus cornucopia.
本発明の組成物は、エルゴチオネインに加えて、クレアチンを有効成分としてさらに含むことが好ましい。本発明者らは、精子にエルゴチオネインとクレアチンと同時に接触させることで、精子所見がより顕著に改善することを見出した。本発明の組成物は、エルゴチオネインに加えてクレアチンが配合されることで、より高い精子所見改善効果を示すことができる。
The composition of the present invention preferably further contains creatine as an active ingredient in addition to ergothioneine. The inventors have found that contacting sperm with ergothioneine and creatine simultaneously improved sperm findings more significantly. The composition of the present invention can exhibit a higher sperm finding improvement effect by blending creatine in addition to ergothioneine.
クレアチンは、１－メチルグアニジノ酢酸又はメチルグリコシアミンとも称され、主に筋肉中に存在する有機酸の１種である。クレアチンは、腎臓及び肝臓において、アルギニンとグリシンから生合成され、さらに筋肉組織内において、クレアチンキナーゼの作用によりＡＴＰと反応して、エネルギー貯蔵型のクレアチンリン酸に変換される。クレアチンリン酸は、筋肉のように瞬時に多量にエネルギーを消費する器官において、高エネルギーリン酸結合の貯蔵物質として働き、エネルギーの不足時にはリン酸基を供給することによりＡＴＰを生成し、エネルギーを供給する。これにより生じたクレアチンはクレアチンキナーゼにより再びリン酸化され、クレアチンリン酸として再利用されるか、不可逆的な非酵素的脱水を経てクレアチニンになる。クレアチニンは最終的には腎臓にて尿中に排世される。このように、クレアチンは瞬発的な運動におけるエネルギー代謝に関与することから、クレアチンの経口摂取が対象の運動時の瞬発力を高めることが知られる。
Creatine, also called 1-methylguanidinoacetic acid or methylglycocyamine, is a type of organic acid that is mainly present in muscle. Creatine is biosynthesized from arginine and glycine in the kidney and liver, and is converted into energy-storing creatine phosphate in muscle tissue by reacting with ATP through the action of creatine kinase. Creatine phosphate acts as a storage material for high-energy phosphate bonds in organs such as muscles that consume a large amount of energy instantaneously, and when energy is insufficient, ATP is generated by supplying phosphate groups, and energy is generated. supply. The resulting creatine is phosphorylated again by creatine kinase and either recycled as creatine phosphate or undergoes irreversible nonenzymatic dehydration to creatinine. Creatinine is ultimately excreted in the urine by the kidneys. As described above, since creatine is involved in energy metabolism in explosive exercise, it is known that oral intake of creatine enhances the explosive power during exercise in subjects.
本発明の組成物に含まれるクレアチンの形態は、クレアチンのあらゆる生理的に利用可能な形態を含み、例えば、クレアチンの遊離酸、塩、又はこれらの誘導体であってもよい。クレアチンの形態は特に制限されないが、例えば、遊離酸、カルシウム塩、ナトリウム塩、カリウム塩、マグネシウム塩、塩酸塩、クエン酸塩等の塩、エステル、ラクトン、又は水和物等が挙げられる。その中でもクレアチンカルシウム塩、クレアチン－水和物及びリンゴ酸トリクレアチンが好ましい。本発明において用いられるクレアチンとしては、公知の方法により合成したものや、市販品を用いることができる。
The form of creatine contained in the composition of the present invention includes any physiologically available form of creatine, and may be, for example, creatine free acid, salt, or derivative thereof. The form of creatine is not particularly limited, but examples thereof include salts such as free acid, calcium salt, sodium salt, potassium salt, magnesium salt, hydrochloride and citrate, ester, lactone, hydrate and the like. Among them, creatine calcium salt, creatine-hydrate and tricreatine malate are preferred. As the creatine used in the present invention, one synthesized by a known method or a commercially available product can be used.
本発明に係る経口摂取用組成物においては、クレアチンが、５０～３００ｍｇ／ｋｇ体重／日の量で摂取されるように配合されることが好ましい。特に、５０ｍｇ／ｋｇ体重／日以上、又は１００ｍｇ／ｋｇ体重／日以上とすることが好ましい。また、２５０ｍｇ／ｋｇ体重／日以下、又は２００ｍｇ／ｋｇ体重／日以下とすることが好ましい。
The composition for oral ingestion according to the present invention preferably contains creatine in an amount of 50 to 300 mg/kg body weight/day. In particular, it is preferably 50 mg/kg body weight/day or more, or 100 mg/kg body weight/day or more. Also, it is preferably 250 mg/kg body weight/day or less, or 200 mg/kg body weight/day or less.
本発明におけるエルゴチオネインとクレアチンは、１日あたりの摂取量が重量比で１：３０００～１：１００、特に１：２５００～１：１０００となるように配合されることが好ましい。
Ergothioneine and creatine in the present invention are preferably blended so that the daily intake is 1:3000 to 1:100, particularly 1:2500 to 1:1000, by weight.
本発明の組成物は、食品（例えば、特別用途食品、特定保健用食品、栄養機能食品、所定機関より効能の表示が認められた機能性食品、サプリメントなど）、又は医薬品（医薬部外品を含む）、あるいは、一般的な食品又は食品添加剤等として用いることができる。
The composition of the present invention is a food (for example, a food for special uses, a food for specified health use, a food with nutrient function claims, a functional food whose efficacy has been approved by a predetermined organization, a supplement, etc.), or a pharmaceutical (quasi-drug). including), or can be used as a general food or food additive.
本発明の組成物の形状は、対象の哺乳類動物が無理なく摂取できるものであれば特に限定されないが、例えば、液体（飲料）、粉末、顆粒、錠剤又はカプセル剤等とすることができる。また、本発明の組成物は、必要に応じて担体を含むことができる。担体としては、賦形剤、結合剤、崩壊剤、充填剤、乳化剤、流動添加調節剤、滑沢剤等が挙げられる。
The form of the composition of the present invention is not particularly limited as long as it can be easily ingested by the target mammalian animal. In addition, the composition of the present invention can optionally contain a carrier. Examples of carriers include excipients, binders, disintegrants, fillers, emulsifiers, flow additive modifiers, lubricants and the like.
賦形剤としては、単糖、二糖類、シクロデキストリン及び多糖類のような糖（より具体的には、限定はしないが、グルコース、スクロース、ラクトース、ラフィノース、マンニトール、ソルビトール、イノシトール、マルチトール、デキストリン、マルトデキストリン、デンプン及びセルロース、及びこれらの加工品を含む）、金属塩（例えば、塩化ナトリウム、塩化カリウム、リン酸ナトリウム若しくはリン酸カルシウム、硫酸カルシウム、硫酸マグネシウム、炭酸カルシウム）、クエン酸、酒石酸、グリシン、低、中、高分子量のポリエチレングリコール（ＰＥＧ）、プルロニック（登録商標）、カオリン、ケイ酸、あるいはそれらの組み合わせが例として挙げられる。
Excipients include sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides (more particularly but not limited to glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, maltitol, dextrin, maltodextrin, starch and cellulose, and their processed products), metal salts (e.g. sodium chloride, potassium chloride, sodium or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, Examples include glycine, low, medium and high molecular weight polyethylene glycols (PEG), Pluronics, kaolin, silicic acid, or combinations thereof.
結合剤としては、トウモロコシ、コムギ、コメ、若しくはジャガイモのデンプンを用いたデンプン糊、単シロップ、グルコース液、ゼラチン、トラガカント、メチルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロースナトリウム、セラック及び／又はポリビニルピロリドン等が例として挙げられる。
Examples of binders include starch paste using corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac and/or polyvinylpyrrolidone. It is mentioned as.
崩壊剤としては、前記デンプンや、乳糖、カルボキシメチルデンプン、架橋ポリビニルピロリドン、アガー、ラミナラン末、炭酸水素ナトリウム、炭酸カルシウム、アルギン酸若しくはアルギン酸ナトリウム、ポリオキシエチレンソルビタン脂肪酸エステル、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド又はそれらの塩が例として挙げられる。
Disintegrants include starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride. Or those salts are mentioned as an example.
充填剤としては、前記糖及び／又はリン酸カルシウム（例えば、リン酸三カルシウム、若しくはリン酸水素カルシウム）が例として挙げられる。
Examples of fillers include the above sugars and/or calcium phosphate (eg, tricalcium phosphate or calcium hydrogen phosphate).
乳化剤としては、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、ショ糖脂肪酸エステル、プロピレングリコール脂肪酸エステルが例として挙げられる。
Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.
流動添加調節剤及び滑沢剤としては、ケイ酸塩、タルク、ステアリン酸塩又はポリエチレングリコールが例として挙げられる。
Examples of flow additives and lubricants include silicates, talc, stearates or polyethylene glycol.
このような担体は、製品の形状やエルゴチオネインの効果を維持するために用いられるものであり、必要に応じて適宜使用すればよい。上記の添加剤の他、必要であれば矯味矯臭剤、可溶化剤、懸濁剤、希釈剤、界面活性剤、安定剤、吸収促進剤、増量剤、付湿剤、保湿剤、吸着剤、崩壊抑制剤、固結防止剤、コーティング剤、着色剤、保存剤、抗酸化剤、香料、風味剤、甘味剤、緩衝剤等を含むこともできる。
Such a carrier is used to maintain the shape of the product and the effect of ergothioneine, and may be used appropriately as necessary. In addition to the above additives, flavoring agents, solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption promoters, bulking agents, wetting agents, humectants, adsorbents, Disintegration retardants, anti-caking agents, coating agents, coloring agents, preservatives, antioxidants, flavoring agents, flavoring agents, sweetening agents, buffering agents and the like may also be included.
本発明の組成物は、そのまま摂取してもよいが、他の組成物と混合するか、飲料水に添加して摂取してもよい。本発明の組成物は、摂取１回分において、１日の摂取量の１／５～３倍、特に１／３～１倍の有効成分が含まれることが好ましい。すなわち、摂取頻度は、３日に１回～１日５回、特に１日１回～１日３回とすることが好ましい。
The composition of the present invention may be ingested as it is, or may be ingested after being mixed with other compositions or added to drinking water. The composition of the present invention preferably contains 1/5 to 3 times, particularly 1/3 to 1 time of the daily intake of the active ingredient per dose. That is, the intake frequency is preferably once every three days to five times a day, particularly once a day to three times a day.
本発明の組成物を対象に摂取させる頻度は、特に限定されず、例えば、頻度としては１日１回摂取させてもよい。摂取期間についても特に限定されないが、例えば、１日～３０日間、３～２１日間又は５～１４日間とすることができる。
The frequency with which the subject ingests the composition of the present invention is not particularly limited, and for example, the frequency may be once a day. The intake period is also not particularly limited, but can be, for example, 1 to 30 days, 3 to 21 days, or 5 to 14 days.
＜２．哺乳類動物の精子所見を改善するための経口摂取用組成物の製造方法＞
本発明の哺乳類動物の精子所見を改善するための経口摂取用組成物の製造方法（以下、「本発明の製造方法」とも称する）は、エルゴチオネインを配合する工程を含む、ことを特徴とする。より具体的には、本発明の製造方法は、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の本発明の組成物を製造する方法である。本発明の製造方法で製造された組成物は、摂取した対象の精子所見を改善することが可能である。 <2. Method for Producing Composition for Oral Ingestion for Improving Sperm Findings in Mammals>
The method for producing a composition for oral ingestion for improving sperm findings in mammals of the present invention (hereinafter also referred to as the "production method of the present invention") is characterized by comprising the step of blending ergothioneine. More specifically, the production method of the present invention includes <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>. The composition produced by the production method of the present invention can improve the sperm findings of subjects who have ingested it.
本発明の哺乳類動物の精子所見を改善するための経口摂取用組成物の製造方法（以下、「本発明の製造方法」とも称する）は、エルゴチオネインを配合する工程を含む、ことを特徴とする。より具体的には、本発明の製造方法は、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の本発明の組成物を製造する方法である。本発明の製造方法で製造された組成物は、摂取した対象の精子所見を改善することが可能である。 <2. Method for Producing Composition for Oral Ingestion for Improving Sperm Findings in Mammals>
The method for producing a composition for oral ingestion for improving sperm findings in mammals of the present invention (hereinafter also referred to as the "production method of the present invention") is characterized by comprising the step of blending ergothioneine. More specifically, the production method of the present invention includes <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>. The composition produced by the production method of the present invention can improve the sperm findings of subjects who have ingested it.
本発明の製造方法は、クレアチンをさらに配合する工程を含んでもよい。クレアチンをさらに配合することで、製造された組成物は、より高い精子所見改善効果を示すことが可能となる。エルゴチオネイン及びクレアチンの配合順は特に限定されず、より製剤化が容易となる順で配合することができる。
The production method of the present invention may include a step of further blending creatine. By further blending creatine, the manufactured composition can exhibit a higher effect of improving sperm findings. The order in which ergothioneine and creatine are added is not particularly limited, and they can be added in the order that facilitates formulation.
本発明の製造方法における有効成分の配合量、製剤手法、他の成分の添加の有無等の詳細な条件は、特に矛盾のない限り、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の条件と同様である。
Detailed conditions such as the amount of active ingredients in the manufacturing method of the present invention, the formulation method, and the presence or absence of the addition of other ingredients, unless there is a particular contradiction <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>.
＜３．哺乳類動物の精子所見を改善する方法＞
本発明の哺乳類動物の精子所見を改善する方法（以下、「本発明の改善方法」とも称する）は、精子所見の改善が必要な対象にエルゴチオネインを経口投与する工程を含む、ことを特徴とする。 <3. Methods for Improving Sperm Findings in Mammals>
The method for improving sperm findings in mammals of the present invention (hereinafter also referred to as "improvement method of the present invention") comprises the step of orally administering ergothioneine to a subject in need of improving sperm findings. .
本発明の哺乳類動物の精子所見を改善する方法（以下、「本発明の改善方法」とも称する）は、精子所見の改善が必要な対象にエルゴチオネインを経口投与する工程を含む、ことを特徴とする。 <3. Methods for Improving Sperm Findings in Mammals>
The method for improving sperm findings in mammals of the present invention (hereinafter also referred to as "improvement method of the present invention") comprises the step of orally administering ergothioneine to a subject in need of improving sperm findings. .
本発明の改善方法において、エルゴチオネインを経口投与する対象は哺乳類動物である。哺乳類動物としては、例えば、ヒト、チンパンジーを含む霊長類、イヌ、ネコなどのペット動物、ウシ、ブタ、ウマ、ヒツジ、ヤギなどの家畜動物、マウス、ラットなどの齧歯類、動物園で飼育される哺乳類動物などが挙げられる。本発明の改善方法の対象は、好ましくは、ヒトである。
In the improvement method of the present invention, mammals are the subjects to which ergothioneine is orally administered. Examples of mammals include humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, pigs, horses, sheep and goats, rodents such as mice and rats, and animals raised in zoos. mammals, etc. The subject of the improvement method of the present invention is preferably human.
前記対象がヒトである場合、男性不妊症患者であることが好ましい。また、前記男性不妊症は、前記男性不妊症が、特発性の乏精子症及び／又は精子無力症であることが好ましい。さらに、前記男性不妊症の症状が、前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下であることが好ましい。
When the subject is a human, it is preferably a male infertility patient. Moreover, the male infertility is preferably idiopathic oligozoospermia and/or asthenozoospermia. Furthermore, it is preferable that the symptom of male infertility is a decrease in sperm concentration in semen, a decrease in sperm motility, and/or a decrease in sperm advance rate.
本発明の改善方法において、エルゴチオネインは、対象に１０～５０００μｇ／ｋｇ体重／日の量で経口投与されることが好ましい。特に２０μｇ／ｋｇ体重／日以上、３０μｇ／ｋｇ体重／日以上、４０μｇ／ｋｇ体重／日以上又は５０μｇ／ｋｇ体重／日以上とすることが好ましい。また、３０００μｇ／ｋｇ体重／日以下、２０００μｇ／ｋｇ体重／日以下、１０００μｇ／ｋｇ体重／日以下、５００μｇ／ｋｇ体重／日以下、又は３００μｇ／ｋｇ体重／日以下とすることが好ましい。より具体的には、対象の精漿中のエルゴチオネイン濃度が、２０～２０００μＭ、特に５０～１５００μＭとなるように経口投与されることが好ましい。このような量のエルゴチオネインは、１日に単回又は複数回に分けて投与することができる。
In the improving method of the present invention, ergothioneine is preferably orally administered to the subject in an amount of 10-5000 μg/kg body weight/day. In particular, it is preferably 20 µg/kg body weight/day or more, 30 µg/kg body weight/day or more, 40 µg/kg body weight/day or more, or 50 µg/kg body weight/day or more. Also, it is preferably 3000 µg/kg body weight/day or less, 2000 µg/kg body weight/day or less, 1000 µg/kg body weight/day or less, 500 µg/kg body weight/day or less, or 300 µg/kg body weight/day or less. More specifically, it is preferable to orally administer ergothioneine so that the concentration of ergothioneine in the subject's seminal plasma is 20 to 2000 μM, particularly 50 to 1500 μM. Such amounts of ergothioneine can be administered in single or multiple doses per day.
本発明の改善方法において、対象にクレアチンがさらに経口投与されることが好ましい。クレアチンは、エルゴチオネインと同一の組成物として投与されてもよく、また、別の組成物として投与されてもよい。別の組成物として投与される場合、クレアチンは、エルゴチオネインと同時に投与されてもよく、また、別に投与されてもよい。
In the improvement method of the present invention, it is preferable that creatine is further orally administered to the subject. Creatine may be administered in the same composition as ergothioneine or may be administered in a separate composition. When administered as separate compositions, creatine may be administered simultaneously with ergothioneine or may be administered separately.
クレアチンは、５０～３００ｍｇ／ｋｇ体重／日の量で経口投与されることが好ましい。特に、５０ｍｇ／ｋｇ体重／日以上、又は１００ｍｇ／ｋｇ体重／日以上とすることが好ましい。また、２５０ｍｇ／ｋｇ体重／日以下、又は２００ｍｇ／ｋｇ体重／日以下とすることが好ましい。
Creatine is preferably administered orally in an amount of 50-300 mg/kg body weight/day. In particular, it is preferably 50 mg/kg body weight/day or more, or 100 mg/kg body weight/day or more. Also, it is preferably 250 mg/kg body weight/day or less, or 200 mg/kg body weight/day or less.
エルゴチオネインとクレアチンの１日あたりの投与量は、重量比で１：３０００～１：１００、特に、１：２５００～１：１０００とすることが好ましい。
The daily dosage of ergothioneine and creatine is preferably 1:3000 to 1:100, especially 1:2500 to 1:1000 by weight.
本発明の改善方法における、有効成分の製剤手法、他の成分の投与の有無等の詳細な条件は、特に矛盾のない限り、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の条件と同様である。
In the improvement method of the present invention, the detailed conditions such as the method of formulation of the active ingredient and the presence or absence of administration of other ingredients, unless there is a particular contradiction, <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>.
＜４．エルゴチオネインの使用＞
本発明は、哺乳類動物の精子所見を改善するための経口摂取用組成物の製造におけるエルゴチオネインの使用を提供する。以下、前記使用を「本発明の使用」とも称する。本発明の使用は、具体的には、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の本発明の組成物を製造するためのエルゴチオネインの使用である。特に矛盾のない限り、本発明の使用で製造される経口摂取用組成物の摂取対象、用途、効果、製剤手法等の詳細な条件は、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の条件と同様である。 <4. Use of Ergothioneine>
The present invention provides the use of ergothioneine in the manufacture of an orally ingestible composition for improving sperm findings in mammals. Said uses are hereinafter also referred to as "uses of the invention". Specifically, the use of the present invention is <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>. Unless otherwise contradicted, detailed conditions such as subjects for ingestion, uses, effects, and preparation methods of the composition for oral ingestion produced by the use of the present invention are described in <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>.
本発明は、哺乳類動物の精子所見を改善するための経口摂取用組成物の製造におけるエルゴチオネインの使用を提供する。以下、前記使用を「本発明の使用」とも称する。本発明の使用は、具体的には、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の本発明の組成物を製造するためのエルゴチオネインの使用である。特に矛盾のない限り、本発明の使用で製造される経口摂取用組成物の摂取対象、用途、効果、製剤手法等の詳細な条件は、＜１．哺乳類動物の精子所見を改善するための経口摂取用組成物＞の項に記載の条件と同様である。 <4. Use of Ergothioneine>
The present invention provides the use of ergothioneine in the manufacture of an orally ingestible composition for improving sperm findings in mammals. Said uses are hereinafter also referred to as "uses of the invention". Specifically, the use of the present invention is <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>. Unless otherwise contradicted, detailed conditions such as subjects for ingestion, uses, effects, and preparation methods of the composition for oral ingestion produced by the use of the present invention are described in <1. Composition for Oral Ingestion for Improving Sperm Findings in Mammals>.
＜５．精子用培地＞
本発明は、エルゴチオネインを含む、精子用培地を提供する。本発明の精子用培地は、哺乳類動物の精子、好ましくはヒト男性の精子に使用される。また、本発明の精子用培地は、精子の運動性を維持又は向上することができ、特に限定されないが、例えば、精子の洗浄、保存、培養又は体外受精若しくは人工授精に使用される。本発明の精子用培地は、既知の精子用培地、例えばＨＴＦ培地、ＴＹＨ培地又は特開２０１４－１２８２１９号公報に記載の培地等を用いて、これにエルゴチオネインを添加して製造することができる。特に、精子の運動性を維持させる効果を有することが報告されていることから、特開２０１４－１２８２１９号公報に記載の培地を使用することが好ましい。精子用培地に含まれるエルゴチオネインの濃度は、特に限定されないが、０．０１～３ｍＭ、特に０．１～１ｍＭとすることが好ましい。 <5. Sperm culture medium>
The present invention provides a sperm medium containing ergothioneine. The sperm culture medium of the present invention is used for mammalian sperm, preferably human male sperm. In addition, the sperm culture medium of the present invention can maintain or improve the motility of sperm, and is used, for example, but not limited to, for washing, preserving, and culturing sperm, in vitro fertilization, or artificial insemination. The sperm culture medium of the present invention can be produced by adding ergothioneine to a known sperm culture medium such as HTF medium, TYH medium, or the medium described in JP-A-2014-128219. In particular, it is preferable to use the medium described in JP-A-2014-128219 because it is reported to have an effect of maintaining sperm motility. Although the concentration of ergothioneine contained in the sperm culture medium is not particularly limited, it is preferably 0.01 to 3 mM, particularly 0.1 to 1 mM.
本発明は、エルゴチオネインを含む、精子用培地を提供する。本発明の精子用培地は、哺乳類動物の精子、好ましくはヒト男性の精子に使用される。また、本発明の精子用培地は、精子の運動性を維持又は向上することができ、特に限定されないが、例えば、精子の洗浄、保存、培養又は体外受精若しくは人工授精に使用される。本発明の精子用培地は、既知の精子用培地、例えばＨＴＦ培地、ＴＹＨ培地又は特開２０１４－１２８２１９号公報に記載の培地等を用いて、これにエルゴチオネインを添加して製造することができる。特に、精子の運動性を維持させる効果を有することが報告されていることから、特開２０１４－１２８２１９号公報に記載の培地を使用することが好ましい。精子用培地に含まれるエルゴチオネインの濃度は、特に限定されないが、０．０１～３ｍＭ、特に０．１～１ｍＭとすることが好ましい。 <5. Sperm culture medium>
The present invention provides a sperm medium containing ergothioneine. The sperm culture medium of the present invention is used for mammalian sperm, preferably human male sperm. In addition, the sperm culture medium of the present invention can maintain or improve the motility of sperm, and is used, for example, but not limited to, for washing, preserving, and culturing sperm, in vitro fertilization, or artificial insemination. The sperm culture medium of the present invention can be produced by adding ergothioneine to a known sperm culture medium such as HTF medium, TYH medium, or the medium described in JP-A-2014-128219. In particular, it is preferable to use the medium described in JP-A-2014-128219 because it is reported to have an effect of maintaining sperm motility. Although the concentration of ergothioneine contained in the sperm culture medium is not particularly limited, it is preferably 0.01 to 3 mM, particularly 0.1 to 1 mM.
本発明の精子用培地は、さらにクレアチンを含んでいてもよい。クレアチンをさらに含むことで相加的又は相乗的な精子の運動性の維持、向上効果を得ることが可能となる。精子用培地にクレアチンが含まれる場合、クレアチンの濃度は、特に限定されないが、０．０１～１ｍＭ、特に０．０５～０．５ｍＭとすることが好ましい。
The sperm culture medium of the present invention may further contain creatine. By further containing creatine, it is possible to obtain an additive or synergistic effect of maintaining and improving sperm motility. When the sperm culture medium contains creatine, the concentration of creatine is not particularly limited, but is preferably 0.01 to 1 mM, particularly 0.05 to 0.5 mM.
＜実施例１：タモギタケ摂取試験＞
２８～５１歳の男性被験者１３人に、タモギタケ粉末１．９８ｇ／日（エルゴチオネイン１０ｍｇ／日相当量）、２週間経口投与した。タモギタケ粉末投与前後の各被験者の精液を採取した。精液はクリニック内でマスターベーションにより採取した。採取された精液は、３７℃で３０分間静置して十分に液化を行い、液体重量（精液量）を測定した。精子運動解析システムＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩ（Ｈａｍｉｌｔｏｎ Ｔｈｏｒｎｅ社製）を用いて精子の濃度、運動率、前進率及び平均速度（ＶＡＰ）を測定した。 <Example 1: Tamogitake intake test>
Thirteen male subjects aged 28 to 51 years were orally administered 1.98 g/day of Pleurotus cornucopia powder (equivalent to 10 mg/day of ergothioneine) for 2 weeks. Semen was collected from each subject before and after the administration of powdered Pleurotus cornucopia. Semen was collected by masturbation in the clinic. The collected semen was allowed to stand at 37° C. for 30 minutes to fully liquefy, and the liquid weight (semen volume) was measured. A sperm motility analysis system CASA/IVOS (registered trademark) II (manufactured by Hamilton Thorne) was used to measure sperm concentration, motility, advance rate and average velocity (VAP).
２８～５１歳の男性被験者１３人に、タモギタケ粉末１．９８ｇ／日（エルゴチオネイン１０ｍｇ／日相当量）、２週間経口投与した。タモギタケ粉末投与前後の各被験者の精液を採取した。精液はクリニック内でマスターベーションにより採取した。採取された精液は、３７℃で３０分間静置して十分に液化を行い、液体重量（精液量）を測定した。精子運動解析システムＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩ（Ｈａｍｉｌｔｏｎ Ｔｈｏｒｎｅ社製）を用いて精子の濃度、運動率、前進率及び平均速度（ＶＡＰ）を測定した。 <Example 1: Tamogitake intake test>
Thirteen male subjects aged 28 to 51 years were orally administered 1.98 g/day of Pleurotus cornucopia powder (equivalent to 10 mg/day of ergothioneine) for 2 weeks. Semen was collected from each subject before and after the administration of powdered Pleurotus cornucopia. Semen was collected by masturbation in the clinic. The collected semen was allowed to stand at 37° C. for 30 minutes to fully liquefy, and the liquid weight (semen volume) was measured. A sperm motility analysis system CASA/IVOS (registered trademark) II (manufactured by Hamilton Thorne) was used to measure sperm concentration, motility, advance rate and average velocity (VAP).
タモギタケ粉末投与前後の、精液量、精子濃度、精子の運動率、前進率及びＶＡＰを表１に示す。精子濃度、運動率、前進率、ＶＡＰが投与後に上昇した。特に、精子の運動率と前進率は有意に上昇することが確認された。
Table 1 shows the semen volume, sperm concentration, sperm motility, advancement rate, and VAP before and after administration of Tamogitake powder. Sperm concentration, motility, advancement rate, and VAP increased after administration. In particular, it was confirmed that sperm motility and advance rate were significantly increased.
＜実施例２：ブタ精液へのエルゴチオネイン添加試験（ｉｎ ｖｉｔｒｏ）＞
５個体の雄ブタから新鮮な精子を採取し、エルゴチオネインを０、０．１又は１．０ｍＭ含む精子保存液（３０ｍＭグルコース、１２０ｍＭラクトース、６．９ｇ／Ｌクエン酸ナトリウム、１ｇ／Ｌ ＮａＨＣＯ３、２．３５ｇ／Ｌ ＥＤＴＡ－２Ｎａ、２．９ｇ／Ｌクエン酸、５．６５ｇ／Ｌ Ｔｒｉｓ、５ｍＭ乳酸、１ｍＭグリシン）に懸濁し、精子懸濁液を調製した。各精子懸濁液を３７℃、５％ＣＯ２条件下で６時間静置した後、精子運動解析システムＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩ（Ｈａｍｉｌｔｏｎ Ｔｈｏｒｎｅ社製）を用いて精子の直線速度（ＶＳＬ（μｍ／Ｓ））及び曲線速度（ＶＣＬ（μｍ／Ｓ））を測定した。 <Example 2: Ergothioneine addition test to porcine semen (in vitro)>
Fresh sperm were collected from 5 boars, and a sperm preservation solution containing 0, 0.1 or 1.0 mM ergothioneine (30 mM glucose, 120 mM lactose, 6.9 g/L sodium citrate, 1 g/L NaHCO 3 , 2.35 g/L EDTA-2Na, 2.9 g/L citric acid, 5.65 g/L Tris, 5 mM lactic acid, 1 mM glycine) to prepare a sperm suspension. After allowing each sperm suspension to stand at 37° C. under 5% CO 2 conditions for 6 hours, the linear velocity of sperm (VSL (μm/S)) and curve velocity (VCL (μm/S)) were measured.
５個体の雄ブタから新鮮な精子を採取し、エルゴチオネインを０、０．１又は１．０ｍＭ含む精子保存液（３０ｍＭグルコース、１２０ｍＭラクトース、６．９ｇ／Ｌクエン酸ナトリウム、１ｇ／Ｌ ＮａＨＣＯ３、２．３５ｇ／Ｌ ＥＤＴＡ－２Ｎａ、２．９ｇ／Ｌクエン酸、５．６５ｇ／Ｌ Ｔｒｉｓ、５ｍＭ乳酸、１ｍＭグリシン）に懸濁し、精子懸濁液を調製した。各精子懸濁液を３７℃、５％ＣＯ２条件下で６時間静置した後、精子運動解析システムＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩ（Ｈａｍｉｌｔｏｎ Ｔｈｏｒｎｅ社製）を用いて精子の直線速度（ＶＳＬ（μｍ／Ｓ））及び曲線速度（ＶＣＬ（μｍ／Ｓ））を測定した。 <Example 2: Ergothioneine addition test to porcine semen (in vitro)>
Fresh sperm were collected from 5 boars, and a sperm preservation solution containing 0, 0.1 or 1.0 mM ergothioneine (30 mM glucose, 120 mM lactose, 6.9 g/L sodium citrate, 1 g/L NaHCO 3 , 2.35 g/L EDTA-2Na, 2.9 g/L citric acid, 5.65 g/L Tris, 5 mM lactic acid, 1 mM glycine) to prepare a sperm suspension. After allowing each sperm suspension to stand at 37° C. under 5% CO 2 conditions for 6 hours, the linear velocity of sperm (VSL (μm/S)) and curve velocity (VCL (μm/S)) were measured.
各精子懸濁液中の精子のＶＳＬ及びＶＣＬを図１及び２にそれぞれ示す。ＶＳＬ、ＶＣＬともに、エルゴチオネインを０．１ｍＭ以上加えることで有意に向上することが確認された（ｐ＜０．０５）。本検討に使用した精子保存液は、精子の運動性を維持させる効果を有することが報告されている（特開２０１４－１２８２１９号公報参照）。これにエルゴチオネインを加えることで、さらに精子の運動性が向上することが確認された。
The VSL and VCL of sperm in each sperm suspension are shown in Figures 1 and 2, respectively. It was confirmed that both VSL and VCL were significantly improved by adding 0.1 mM or more of ergothioneine (p<0.05). It has been reported that the sperm preservation solution used in this study has the effect of maintaining sperm motility (see JP-A-2014-128219). It was confirmed that the addition of ergothioneine further improved sperm motility.
＜実施例３：エルゴチオネインとクレアチンの共存試験（ｉｎ ｖｉｔｒｏ）＞
５個体の雄ブタから新鮮な精子を採取し、図３に示すエルゴチオネイン及びクレアチンを含む、０．１％のアルブミンを添加したＨＴＦ（Ｈｕｍａｎ ｔｕｂａｌ ｆｌｕｉｄｓ）培地に播種した。培地を３７℃、５％ＣＯ２条件下で４時間培養し、精子の運動率を実施例１と同様の方法で測定した。 <Example 3: Coexistence test of ergothioneine and creatine (in vitro)>
Fresh spermatozoa were collected from 5 boars and inoculated in HTF (Human tubal fluids) medium supplemented with 0.1% albumin containing ergothioneine and creatine shown in FIG. The medium was cultured at 37° C. and 5% CO 2 for 4 hours, and sperm motility was measured in the same manner as in Example 1.
５個体の雄ブタから新鮮な精子を採取し、図３に示すエルゴチオネイン及びクレアチンを含む、０．１％のアルブミンを添加したＨＴＦ（Ｈｕｍａｎ ｔｕｂａｌ ｆｌｕｉｄｓ）培地に播種した。培地を３７℃、５％ＣＯ２条件下で４時間培養し、精子の運動率を実施例１と同様の方法で測定した。 <Example 3: Coexistence test of ergothioneine and creatine (in vitro)>
Fresh spermatozoa were collected from 5 boars and inoculated in HTF (Human tubal fluids) medium supplemented with 0.1% albumin containing ergothioneine and creatine shown in FIG. The medium was cultured at 37° C. and 5% CO 2 for 4 hours, and sperm motility was measured in the same manner as in Example 1.
図３に各試料の運動率を示す。エルゴチオネイン単独及びクレアチン単独を投与した試料では運動率の有意な改善は認められなかった。一方で、１ｍＭのエルゴチオネインを添加した試料では、クレアチン濃度依存的に運動精子の割合が向上し、１ｍＭのエルゴチオネインと５００μＭのクレアチンとを添加した試料で最も高い運動率が認められた。二元配置分散分析の結果、無添加区とクレアチン単独区で有意差が認められると共に（ｐ＝０．０３７４）、エルゴチオネインとクレアチンの相互効果も認められた（ｐ＝０．０４７８）。ＨＴＦ培地は、精子の受精時の動きを誘導する培養液として知られるが、エルゴチオネイン及びクレアチンを共存させることで、さらに精子の運動率を高める効果があることが確認された。
Figure 3 shows the motility of each sample. No significant improvement in locomotion rate was observed in samples receiving ergothioneine alone and creatine alone. On the other hand, in the sample added with 1 mM ergothioneine, the percentage of motile spermatozoa improved in a creatine concentration-dependent manner, and the highest motility rate was observed in the sample added with 1 mM ergothioneine and 500 μM creatine. As a result of two-way layout analysis of variance, a significant difference was observed between the non-added group and the creatine-only group (p=0.0374), and a mutual effect between ergothioneine and creatine was also observed (p=0.0478). The HTF medium is known as a culture medium that induces sperm motility during fertilization, and it was confirmed that coexistence of ergothioneine and creatine has the effect of further increasing sperm motility.
＜実施例４：タモギタケ＋クレアチン摂取試験＞
男性被験者９人に、タモギタケ粉末及びクレアチン配合組成物（タモギタケ粉末２７５ｍｇ、クレアチン粉末５ｇ、香料、甘味料、塩類を含む）７ｇを、１日２回、２週間経口投与した。組成物投与前後の各被験者の精液を採取した。精液採取前の禁欲期間は３日間以内とした。精液採取後、直ちに、精子運動解析システムＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩ（Ｈａｍｉｌｔｏｎ Ｔｈｏｒｎｅ社製）を用いて、原液の精液所見（精子濃度、前進運動率、総運動精子数）及び精子運動性（ＶＡＰ、ＶＣＬ、ＶＳＬ及びＡＬＨ）を測定した。検査後の精液を４００ｒｐｍで１０分間遠心分離し、精漿を分離した。得られた精漿は約－２０℃で最長＿60日間冷凍保存した。分離した精子をＨＴＦ培地（北里コーポレーション）に播種し、３７℃、５％ＣＯ２。Ｏ２条件下で３時間培養し、精子の運動率を実施例１と同様の方法で測定した。培養後の精子について、ＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩを用いて、精子運動性（ＶＡＰ、ＶＣＬ、ＶＳＬ及びＡＬＨ）を測定した。 <Example 4: Tamogitake mushroom + creatine intake test>
Nine male test subjects were orally administered 7 g of a composition containing 275 mg of oyster mushroom powder and creatine (including 275 mg of oyster mushroom powder, 5 g of creatine powder, flavor, sweetener, and salts) twice a day for 2 weeks. Semen was collected from each subject before and after administration of the composition. The abstinence period before semen collection was within 3 days. Immediately after semen collection, using the sperm motility analysis system CASA / IVOS (registered trademark) II (manufactured by Hamilton Thorne), the undiluted semen findings (sperm concentration, forward motility, total motile sperm count) and sperm motility ( VAP, VCL, VSL and ALH) were measured. The semen after examination was centrifuged at 400 rpm for 10 minutes to separate the seminal plasma. The seminal plasma obtained was stored frozen at about -20°C for up to _60 days. Separated sperm were seeded in HTF medium (Kitasato Corporation), 37 °C, 5% CO2 . After culturing for 3 hours under O2 conditions, sperm motility was measured by the same method as in Example 1. For the cultured sperm, sperm motility (VAP, VCL, VSL and ALH) was measured using CASA/IVOS (registered trademark) II.
男性被験者９人に、タモギタケ粉末及びクレアチン配合組成物（タモギタケ粉末２７５ｍｇ、クレアチン粉末５ｇ、香料、甘味料、塩類を含む）７ｇを、１日２回、２週間経口投与した。組成物投与前後の各被験者の精液を採取した。精液採取前の禁欲期間は３日間以内とした。精液採取後、直ちに、精子運動解析システムＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩ（Ｈａｍｉｌｔｏｎ Ｔｈｏｒｎｅ社製）を用いて、原液の精液所見（精子濃度、前進運動率、総運動精子数）及び精子運動性（ＶＡＰ、ＶＣＬ、ＶＳＬ及びＡＬＨ）を測定した。検査後の精液を４００ｒｐｍで１０分間遠心分離し、精漿を分離した。得られた精漿は約－２０℃で最長＿60日間冷凍保存した。分離した精子をＨＴＦ培地（北里コーポレーション）に播種し、３７℃、５％ＣＯ２。Ｏ２条件下で３時間培養し、精子の運動率を実施例１と同様の方法で測定した。培養後の精子について、ＣＡＳＡ／ＩＶＯＳ（登録商標）ＩＩを用いて、精子運動性（ＶＡＰ、ＶＣＬ、ＶＳＬ及びＡＬＨ）を測定した。 <Example 4: Tamogitake mushroom + creatine intake test>
Nine male test subjects were orally administered 7 g of a composition containing 275 mg of oyster mushroom powder and creatine (including 275 mg of oyster mushroom powder, 5 g of creatine powder, flavor, sweetener, and salts) twice a day for 2 weeks. Semen was collected from each subject before and after administration of the composition. The abstinence period before semen collection was within 3 days. Immediately after semen collection, using the sperm motility analysis system CASA / IVOS (registered trademark) II (manufactured by Hamilton Thorne), the undiluted semen findings (sperm concentration, forward motility, total motile sperm count) and sperm motility ( VAP, VCL, VSL and ALH) were measured. The semen after examination was centrifuged at 400 rpm for 10 minutes to separate the seminal plasma. The seminal plasma obtained was stored frozen at about -20°C for up to _60 days. Separated sperm were seeded in HTF medium (Kitasato Corporation), 37 °C, 5% CO2 . After culturing for 3 hours under O2 conditions, sperm motility was measured by the same method as in Example 1. For the cultured sperm, sperm motility (VAP, VCL, VSL and ALH) was measured using CASA/IVOS (registered trademark) II.
冷凍保存した精漿を解凍し、８－ヒドロキシ－２’－デオキシグアノシン（８－ＯＨｄＧ）、スペルミン、クレアチン、テストステロン及び亜鉛の含有量を測定した。精漿中の８－ＯＨｄＧ濃度は、以下の方法で測定した。８－ＯＨｄＧ－ＢＳＡ複合体をＰＢＳで希釈して、９６ウェルマイクロプレートに５０μＬずつ分注し、室温で１時間振とうした。０．０１％Ｔｗｅｅｎ２０－ＰＢＳ（ＴＰＢＳ）で４回洗浄した後、１％ＢＳＡ／ＰＢＳ ２５０μＬを添加して室温で１時間インキュベートした。ＴＰＢＳで４回洗浄した後、精漿、濃度既知の８－ＯＨｄＧ標準液をそれぞれ５０μＬ添加した。ＨＲＰ標識抗８－ＯＨｄＧ抗体溶液を各５０μＬ分注した。１０分間振とう後、室温で５０分間インキュベートした。ＴＰＢＳで４回洗浄した後、テトラメチルベンジジン（ＴＭＢ）１００μＬを分注して１０分間静置して発色させて、１Ｍリン酸５０μＬを分注して停止させた。マイクロウェルプレートで４５０ｎｍ／５７０ｎｍの吸光度を測定した。標準液の吸光度データより検量線を作成し、各精漿の８－ＯＨｄＧ濃度を算出した。
The frozen seminal plasma was thawed and the contents of 8-hydroxy-2'-deoxyguanosine (8-OHdG), spermine, creatine, testosterone and zinc were measured. 8-OHdG concentration in seminal plasma was measured by the following method. The 8-OHdG-BSA complex was diluted with PBS, dispensed into 96-well microplates in 50 μL aliquots, and shaken at room temperature for 1 hour. After washing four times with 0.01% Tween20-PBS (TPBS), 250 μL of 1% BSA/PBS was added and incubated at room temperature for 1 hour. After washing four times with TPBS, 50 μL of seminal plasma and 8-OHdG standard solution of known concentration were added. 50 μL of each HRP-labeled anti-8-OHdG antibody solution was dispensed. After shaking for 10 minutes, it was incubated at room temperature for 50 minutes. After washing four times with TPBS, 100 μL of tetramethylbenzidine (TMB) was dispensed and allowed to stand for 10 minutes to develop color, and 50 μL of 1 M phosphoric acid was dispensed to stop. Absorbance at 450 nm/570 nm was measured in a microwell plate. A calibration curve was prepared from the absorbance data of the standard solution, and the 8-OHdG concentration of each seminal plasma was calculated.
スペルミンは、精液の成分であり、細胞分裂やタンパク質合成に係わる物質である。スペルミンは、J. Chromatogr A. 2008 Sep 26;1205(1-2):94-102に記載の、４－（Ｎ，Ｎ－ジメチルアミノスルホニル）－７－フルオロ－２，１，３－ベンゾキサジアゾール（ＤＢＤ－Ｆ）を用いる誘導体化によるＵＰＬＣによって測定した。すなわち、精液にメタノールを加え、４℃、１５０００×ｇで２０分間遠心分離して得られる除タンパク後の上清を、ＤＢＤ－Ｆと反応させて誘導体化した後、ＵＰＬＣで分析すした。クロマトグラフ条件は、以下の通りとした。
液体クロマトグラフ装置：ACQUITYTM Ultra Performance Liquid Chromatography and Micromass LCT PremierTM XE Mass Spectrometer (High sensitivity orthogonal time-of-flight instrument; Waters, Milford, USA)
カラム：ACQUITY UPLC BEH C18 column (1.7μm,100×2.1mm i.d.; Waters)
流速：０．４ｍＬ／分
移動相：Ａ；０．１％ギ酸水溶液、Ｂ；０．１％ギ酸アセトニトリル溶液（グラジエント条件：Ｂ％＝２０（０分）－６０（８分）－９０（１０分）－９８（１１分）－９８（１２分）－２０（１３分）－２０（２０分）
検出：５５０ｎｍ（励起波長：４５０ｎｍ） Spermine is a component of semen and is a substance involved in cell division and protein synthesis. Spermine is 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxa, as described in J. Chromatogr A. 2008 Sep 26;1205(1-2):94-102. Measured by UPLC by derivatization with diazole (DBD-F). That is, methanol was added to the seminal fluid, and the protein-removed supernatant obtained by centrifugation at 4° C. and 15,000×g for 20 minutes was reacted with DBD-F for derivatization, and then analyzed by UPLC. The chromatographic conditions were as follows.
Liquid chromatograph equipment: ACQUITYTM Ultra Performance Liquid Chromatography and Micromass LCT PremierTM XE Mass Spectrometer (High sensitivity orthogonal time-of-flight instrument; Waters, Milford, USA)
Column: ACQUITY UPLC BEH C18 column (1.7 μm, 100×2.1 mm id; Waters)
Flow rate: 0.4 mL/min Mobile phase: A; 0.1% formic acid aqueous solution, B; 0.1% formic acid acetonitrile solution (gradient conditions: B% = 20 (0 min) -60 (8 min) -90 (10 minutes)-98 (11 minutes)-98 (12 minutes)-20 (13 minutes)-20 (20 minutes)
Detection: 550 nm (excitation wavelength: 450 nm)
液体クロマトグラフ装置：ACQUITYTM Ultra Performance Liquid Chromatography and Micromass LCT PremierTM XE Mass Spectrometer (High sensitivity orthogonal time-of-flight instrument; Waters, Milford, USA)
カラム：ACQUITY UPLC BEH C18 column (1.7μm,100×2.1mm i.d.; Waters)
流速：０．４ｍＬ／分
移動相：Ａ；０．１％ギ酸水溶液、Ｂ；０．１％ギ酸アセトニトリル溶液（グラジエント条件：Ｂ％＝２０（０分）－６０（８分）－９０（１０分）－９８（１１分）－９８（１２分）－２０（１３分）－２０（２０分）
検出：５５０ｎｍ（励起波長：４５０ｎｍ） Spermine is a component of semen and is a substance involved in cell division and protein synthesis. Spermine is 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxa, as described in J. Chromatogr A. 2008 Sep 26;1205(1-2):94-102. Measured by UPLC by derivatization with diazole (DBD-F). That is, methanol was added to the seminal fluid, and the protein-removed supernatant obtained by centrifugation at 4° C. and 15,000×g for 20 minutes was reacted with DBD-F for derivatization, and then analyzed by UPLC. The chromatographic conditions were as follows.
Liquid chromatograph equipment: ACQUITYTM Ultra Performance Liquid Chromatography and Micromass LCT PremierTM XE Mass Spectrometer (High sensitivity orthogonal time-of-flight instrument; Waters, Milford, USA)
Column: ACQUITY UPLC BEH C18 column (1.7 μm, 100×2.1 mm id; Waters)
Flow rate: 0.4 mL/min Mobile phase: A; 0.1% formic acid aqueous solution, B; 0.1% formic acid acetonitrile solution (gradient conditions: B% = 20 (0 min) -60 (8 min) -90 (10 minutes)-98 (11 minutes)-98 (12 minutes)-20 (13 minutes)-20 (20 minutes)
Detection: 550 nm (excitation wavelength: 450 nm)
精漿中のクレアチン濃度は、精漿検体を40倍で希釈し、酵素法を用いて測定した。具体的には希釈した検体中に含まれるクレアチンを、クレアチンキナーゼおよびサルコシンオキシダーゼが関与する一連の酵素反応を使用して過酸化水素に変換した。生じた過酸化水素を、ペルオキシダーゼの存在下で４－アミノアンチピリンとフェノール類を縮合させて色素を生成させ、生じた色素の吸光度を測定することで定量した。
The creatine concentration in the seminal plasma was measured by diluting the seminal plasma sample 40 times and using the enzymatic method. Specifically, creatine contained in the diluted sample was converted to hydrogen peroxide using a series of enzymatic reactions involving creatine kinase and sarcosine oxidase. The resulting hydrogen peroxide was quantified by condensing 4-aminoantipyrine with phenols in the presence of peroxidase to form a dye and measuring the absorbance of the resulting dye.
精漿中のテストステロン濃度は、以下の方法で測定した。抗テストステロン抗体をＰＢＳで希釈して、９６ウェルマイクロプレートに５０μＬずつ分注し、室温で１時間振とうした。０．０１％Ｔｗｅｅｎ２０－ＰＢＳ（ＴＰＢＳ）で４回洗浄した後、１％ＢＳＡ／ＰＢＳ ２５０μＬを添加して室温で１時間インキュベートした。ＴＰＢＳで４回洗浄した後、ＨＲＰ標識テストステロン、精漿、または濃度既知のテストステロン標準液をそれぞれ５０μＬ添加した。１０分間振とう後、室温で５０分間インキュベートした。ＴＰＢＳで４回洗浄した後、テトラメチルベンジジン（ＴＭＢ）１００μＬを分注して１５分間静置して発色させて、１Ｍリン酸５０μＬを分注して停止させた。マイクロウェルプレートで４５０ｎｍ／５７０ｎｍの吸光度を測定した。標準液の吸光度データより検量線を作成し、各精漿のテストステロン濃度を算出した。
The testosterone concentration in seminal plasma was measured by the following method. The anti-testosterone antibody was diluted with PBS, dispensed into 96-well microplates in 50 μL aliquots, and shaken at room temperature for 1 hour. After washing four times with 0.01% Tween20-PBS (TPBS), 250 μL of 1% BSA/PBS was added and incubated at room temperature for 1 hour. After washing four times with TPBS, 50 μL each of HRP-labeled testosterone, seminal plasma, or testosterone standard solution of known concentration was added. After shaking for 10 minutes, it was incubated at room temperature for 50 minutes. After washing four times with TPBS, 100 μL of tetramethylbenzidine (TMB) was dispensed and allowed to stand for 15 minutes to develop color, and 50 μL of 1 M phosphoric acid was dispensed to stop. Absorbance at 450 nm/570 nm was measured in a microwell plate. A calibration curve was created from the absorbance data of the standard solution, and the testosterone concentration of each seminal plasma was calculated.
精漿中の亜鉛濃度の測定は、以下の方法で行った。精漿検体を融解後２０倍に希釈し、精漿中の鉄や銅をトリクロロ酢酸とフッ化カリウムを用いて共沈させることにより除去した。その後、２－（５－ブロモ－２－ピリジルアゾ）－５－［Ｎ－ｎ－プロピル－Ｎ－（３－スルホプロピルアミノ］－フェノール（５－Ｂｒ－ＰＡＰＳ）を含む試薬と反応させ、吸光度を測定し定量した。
The zinc concentration in seminal plasma was measured by the following method. After thawing, the seminal plasma sample was diluted 20-fold, and iron and copper in the seminal plasma were removed by coprecipitation with trichloroacetic acid and potassium fluoride. It is then reacted with a reagent containing 2-(5-bromo-2-pyridylazo)-5-[Nn-propyl-N-(3-sulfopropylamino]-phenol (5-Br-PAPS) and the absorbance is Measured and quantified.
組成物投与前後の精液について、採取直後の精子濃度、前進運動率、総運動性指数を表２に示す。また、運動率、ＶＡＰ、ＶＣＬ、ＶＳＬ、ＡＬＨを表３に示す。数値は、平均値±標準偏差を示す。精子濃度、前進運動率、及び総運動性指数は、いずれも組成物投与後に有意に上昇することが確認された。また、運動率、ＶＡＰ、ＶＣＬ、ＶＳＬ、ＡＬＨについても有意に上昇することが確認された。特にＶＣＬ、ＶＳＬ及びＡＬＨは、精子の受精能の指標であることが知られ、これらの上昇により、精子の受精能が向上したことが示唆された。
Table 2 shows the sperm concentration, forward motility, and total motility index immediately after collection for the semen before and after administration of the composition. Table 3 shows the motility rate, VAP, VCL, VSL and ALH. Numerical values represent the mean±standard deviation. It was confirmed that sperm concentration, forward motility, and total motility index all increased significantly after administration of the composition. It was also confirmed that the motility rate, VAP, VCL, VSL, and ALH were also significantly increased. In particular, VCL, VSL and ALH are known to be indicators of sperm fertility, and it was suggested that their elevations improved sperm fertility.
組成物投与前後の精液について、採取後、３時間培養した精子のＶＡＰ、ＶＣＬ、ＶＳＬ、ＡＬＨを表４に示す。数値は、平均値±標準偏差を示す。人工授精を行う場合、精子を３時間の培養工程を経た後に卵子に接触させる。また、自然妊娠においても、射精後の精子が卵子に到達するまでの時間は約３時間といわれている。表４に示す通り、ＶＳＬ及びＡＬＨについて、組成物投与後に有意に上昇していた。これにより、組成物投与後の精子は、３時間培養後も有意に高い受精能を維持できることが示唆された。
Table 4 shows the VAP, VCL, VSL, and ALH of sperm cultured for 3 hours after collection for semen before and after administration of the composition. Numerical values represent the mean±standard deviation. When artificial insemination is performed, the sperm is brought into contact with the egg after undergoing a 3-hour incubation process. Also in natural pregnancy, it is said that it takes about 3 hours for the sperm after ejaculation to reach the egg. As shown in Table 4, VSL and ALH were significantly increased after administration of the composition. This suggested that the sperm after administration of the composition could maintain a significantly high fertility even after 3 hours of culture.
組成物投与前後の精液について、精漿中の８－ＯＨｄＧ、スペルミン、クレアチン、テストステロン及び亜鉛の含有量の測定結果を表５に示す。数値は、平均値±標準偏差を示す。精漿中のクレアチン濃度は、摂取後に有意に上昇しており、摂取したクレアチンが精液に移行することが示された、一方、他の成分については、組成物投与前後で有意差は見られなかった。８－ＯＨｄＧ、スペルミン、テストステロン及び亜鉛は、いずれも妊孕性へのプラス又はマイナスの影響を与えることが示唆される成分であるが、組成物投与によっては有意な影響を受けないことが確認された。
Table 5 shows the measurement results of the contents of 8-OHdG, spermine, creatine, testosterone and zinc in the seminal plasma before and after administration of the composition. Numerical values represent the mean±standard deviation. The creatine concentration in the seminal plasma increased significantly after ingestion, indicating that the ingested creatine was transferred to semen. rice field. 8-OHdG, spermine, testosterone and zinc are all components that are suggested to have a positive or negative effect on fertility, but it has been confirmed that there is no significant effect by composition administration. rice field.
Claims (13)
- エルゴチオネインを有効成分として含む、哺乳類動物の精子所見を改善するための経口摂取用組成物。 A composition for oral ingestion for improving sperm findings in mammals, containing ergothioneine as an active ingredient.
- 前記哺乳類動物がヒトである、請求項１に記載の組成物。 The composition according to claim 1, wherein said mammal is human.
- 前記ヒトが、男性不妊症患者である、請求項２に記載の組成物。 The composition according to claim 2, wherein the human is a male infertility patient.
- 前記男性不妊症が、特発性の乏精子症及び／又は精子無力症である、請求項３に記載の組成物。 The composition according to claim 3, wherein the male infertility is idiopathic oligozoospermia and/or asthenozoospermia.
- 前記男性不妊症が、精液中の精子濃度の低下、精子運動率の低下、及び／又は精子前進率の低下の症状を有する、請求項３又は４に記載の組成物。 The composition according to claim 3 or 4, wherein the male infertility has symptoms of low sperm concentration in semen, low sperm motility, and/or low sperm advance rate.
- エルゴチオネインが、１０～５０００μｇ／ｋｇ体重／日の量で摂取されるように配合される、請求項１に記載の組成物。 The composition according to claim 1, wherein ergothioneine is formulated to be ingested in an amount of 10-5000 μg/kg body weight/day.
- クレアチンを有効成分としてさらに含む、請求項１に記載の組成物。 The composition according to claim 1, further comprising creatine as an active ingredient.
- クレアチンが、５０～３００ｍｇ／ｋｇ体重／日の量で摂取されるように配合される、請求項７に記載の組成物。 The composition according to claim 7, wherein creatine is formulated to be ingested in an amount of 50-300 mg/kg body weight/day.
- エルゴチオネインとクレアチンが、一日あたりの摂取量が重量比で１：３０００～１：１００となるように配合される、請求項７又は８に記載の組成物。 The composition according to claim 7 or 8, wherein ergothioneine and creatine are blended at a daily intake of 1:3000 to 1:100 by weight.
- 食品である、請求項１に記載の組成物。 The composition according to claim 1, which is a food.
- 医薬品である、請求項１に記載の組成物。 The composition according to claim 1, which is a pharmaceutical.
- エルゴチオネインを配合する工程を含む、哺乳類動物の精子所見を改善するための経口摂取用組成物の製造方法。 A method for producing a composition for oral ingestion for improving sperm findings in mammals, including the step of incorporating ergothioneine.
- クレアチンをさらに配合する工程を含む、請求項１２に記載の方法。 The method according to claim 12, comprising the step of further blending creatine.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060154993A1 (en) * | 2004-12-17 | 2006-07-13 | Littarru Gian P | Method for treating and preventing male infertility |
| US7226947B1 (en) * | 1999-05-08 | 2007-06-05 | Alzchem Trostberg Gmbh | Use of creatine as a fat substitute |
| US20100227307A1 (en) * | 2007-04-12 | 2010-09-09 | Hausman Marvin S | Ergothioneine and/or its derivatives as a cell preservative |
| US20170239201A1 (en) * | 2014-08-18 | 2017-08-24 | Max-Planck-Gesellschat Zur Forderung Der Wissenschaften E.V. | Glycolic acid enhances sperm mobility |
| WO2021132655A1 (en) * | 2019-12-27 | 2021-07-01 | 国立研究開発法人国立成育医療研究センター | Inhibitor of fertilized egg fragmentation |
-
2022
- 2022-06-24 JP JP2023531886A patent/JPWO2023276861A1/ja active Pending
- 2022-06-24 WO PCT/JP2022/025218 patent/WO2023276861A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7226947B1 (en) * | 1999-05-08 | 2007-06-05 | Alzchem Trostberg Gmbh | Use of creatine as a fat substitute |
| US20060154993A1 (en) * | 2004-12-17 | 2006-07-13 | Littarru Gian P | Method for treating and preventing male infertility |
| US20100227307A1 (en) * | 2007-04-12 | 2010-09-09 | Hausman Marvin S | Ergothioneine and/or its derivatives as a cell preservative |
| US20170239201A1 (en) * | 2014-08-18 | 2017-08-24 | Max-Planck-Gesellschat Zur Forderung Der Wissenschaften E.V. | Glycolic acid enhances sperm mobility |
| WO2021132655A1 (en) * | 2019-12-27 | 2021-07-01 | 国立研究開発法人国立成育医療研究センター | Inhibitor of fertilized egg fragmentation |
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