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WO2023276861A1 - Composition pour ingestion orale pour améliorer la recherche de sperme - Google Patents

Composition pour ingestion orale pour améliorer la recherche de sperme Download PDF

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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
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Prior art keywords
sperm
ergothioneine
creatine
composition
day
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PCT/JP2022/025218
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English (en)
Japanese (ja)
Inventor
昌之 島田
崇 梅原
陽介 瀧本
浩之 塚本
陽子 秋葉
一隆 寺井
Original Assignee
国立大学法人広島大学
株式会社ダンテ
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Application filed by 国立大学法人広島大学, 株式会社ダンテ filed Critical 国立大学法人広島大学
Priority to JP2023531886A priority Critical patent/JPWO2023276861A1/ja
Priority to CN202280057864.XA priority patent/CN118201499A/zh
Publication of WO2023276861A1 publication Critical patent/WO2023276861A1/fr

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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • 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 or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [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
    • 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
    • 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
    • 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.

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Abstract

La présente invention améliore la recherche de sperme chez des mammifères, et en particulier, des patients masculins souffrant d'infertilité.　Cette composition pour ingestion orale, qui contient de l'ergothionéine en tant que composant actif, est administrée par voie orale à un sujet nécessitant une amélioration de la recherche de sperme.
PCT/JP2022/025218 2021-06-28 2022-06-24 Composition pour ingestion orale pour améliorer la recherche de sperme WO2023276861A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
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 (fr) * 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 Inhibiteur de la fragmentation d'œufs fertilisés

Patent Citations (5)

* Cited by examiner, † Cited by third party
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 (fr) * 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 Inhibiteur de la fragmentation d'œufs fertilisés

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