JP6806965B2 - Sperm function improving agents and pharmaceutical compositions containing them, feeds, food compositions and methods for improving sperm function in livestock or poultry. - Google Patents

Description

The present invention relates to a sperm function improving agent, a pharmaceutical composition containing the same, a feed, a food composition, and a method for improving sperm function of livestock or poultry. More specifically, the present invention relates to a sperm function improving agent containing an extract of Angelica keiskei as an active ingredient.

Currently, in Japan, about 15% of those who wish to become pregnant are diagnosed with infertility. According to the WHO definition, infertility is "a condition in which you cannot get pregnant within two years without contraception." Treatment options for infertility include fertility drugs, in vitro fertilization, and artificial insemination.

The in vitro fertilization includes embryo transfer (In Vitro Fertilization Embryo Transfer: IVF-ET) in which the collected eggs and sperm are fertilized in a chalet and the fertilized egg is returned to the uterus, and the sperm is directly injected into the egg under a microscope for fertilization. There is a method called Intracytoplasmic Sperm Injection (ICSI). In the treatment of infertility by such in vitro fertilization, the process of collecting an egg and the process of returning the fertilized egg to the uterus are indispensable, and it is said that the physical and mental burden on the woman is large.

Although the technique of in vitro fertilization has been developed in recent years, the success rate of the in vitro fertilization is generally about 20%, which is not a sufficiently high probability. Against the background of late birth in recent years, the quality of eggs is declining with age in women at the stage of wishing to become pregnant. There is also a view that there is no improvement in the success rate of in vitro fertilization due to this.

There are many factors of infertility on the male side and the female side. The main factors on the male side are (1) spermatogenic dysfunction such as azoospermia (spermatogenic dysfunction), (2) impaired sperm passage such as congenital abnormalities of the epididymis, and (3) anti-sperm. If you have sperm dysfunction such as sperm antibody immunodeficiency. According to a WHO survey, male factors account for about 24% of all infertility factors, and both male and female factors account for about 24% of the total. That is, among the factors of infertility, males are involved in about 48%.

Based on these facts, if a sperm function improving agent that can promote normal sperm formation function in the testis and / or improve the function of mature sperm and avoid the risk of side effects is developed for women. It is expected to lead to a useful treatment alternative to fertility treatment, which has a heavy physical and mental burden.

On the other hand, infertility has become a problem not only in humans but also in industrial animals such as pigs, cattle and chickens. Infertility (reproductive disorder) in pigs and cows causes production efficiency problems such as a decrease in meat production due to a decrease in the number of pups and a decrease in milk production due to inability to milk.

In industrial animals, spermatogenic dysfunction such as (1) azoospermia is one of the main causes of infertility (reproductive disorder). As infertility caused by this spermatogenic dysfunction, so-called "summer infertility" is known. In a narrow sense, summer infertility is a phenomenon in which the spermatogenic function of male individuals temporarily declines in the hot and humid summer, resulting in poor semen properties and a decrease in conception rate. In particular, dairy cow hostaine. It is known that in animals that are sensitive to heat, such as species, ovulation disorders occur due to heat stress even in female individuals. In the breeding of cattle, artificial insemination is generally performed, and frozen semen is used for the artificial insemination, and the ratio reaches 99%. Therefore, it can be said that most of the factors of summer infertility are female factors, especially in the breeding of cattle.

On the other hand, in pig breeding, since the sperm motility after thawing of frozen semen is significantly reduced, artificial insemination using frozen semen is generally performed, but artificial insemination using raw semen is generally performed. is there. However, it is known that in the semen of pigs produced in summer, the survival rate of sperms is low, and even if the survival rate of sperms is high, the proportion of sperms with low fertility and motility is high. In addition, it is known that the proportion of malformed sperm in semen is high. In addition, it is known that female pigs also have ovulation disorders due to heat stress, as in the case of female dairy cow hostine species, and that heat stress increases embryo mortality in the mother's body. Is also known. For this reason, it is said that the cause of summer infertility in pigs is both male and female.

In this way, although the factors of summer infertility have been elucidated, as a measure to prevent summer infertility, a large fan or mist shower is installed in the livestock barn or poultry house to control the temperature inside the livestock barn or poultry house. It is only a matter of lowering, and a fundamental solution focusing on biological function has not been devised. Therefore, for domestic animals and poultry, especially pigs, sperm function improving agents have been developed that can promote normal sperm forming function and / or improved sperm function of mature sperm in male individuals and avoid the risk of side effects. If so, a useful solution to summer infertility is expected.

It is extremely important to promote the improvement of normal sperm formation function and / or the function of mature sperm in such industrial animals in order to increase the production efficiency of the livestock industry.

Here, the process of sperm formation in mammals will be described. Sperm is produced in the testis and epididymis, which are the male reproductive tract. The surface of the testis is covered with a white membrane, while the inside of the testis is partitioned by a plurality of testis lobules. There is a seminiferous tubule in each testis lobules, and sperm are formed in this seminiferous tubule. Spermatogonia, which exist as cells in the fine tubes, undergo meiosis by the action of Sertoli cells, which are supporting cells, and mature into sperms via primary spermatogonia, secondary spermatogonia, and sperm cells. Both ends of the plurality of seminiferous tubules provided in the testis are connected to the testis network, and the testis network forms a plurality of testis export tubes. The plurality of testis export tubes form one epididymis tube, and each seminiferous tubule in the testis is connected to the epididymis. Here, the sperm discharged from the seminiferous tubule does not have fertility and motility. It is known that such sperms are transferred to the epididymis through the testis export tube and acquire fertility and motility as they pass through the epididymis tube in the epididymis. Then, the sperm matured in the epididymis are ejected to the outside of the body together with the prostatic fluid and the sperm sac fluid.

However, as shown in Non-Patent Document 1 and Non-Patent Document 2, it is known that the spermatogenic cells or sperm genes are damaged by reactive oxygen species due to an increase in oxidative stress. As a result, normal sperm formation is inhibited, and it is known that male infertility develops. It is also known that reactive oxygen species cause a decrease in sperm fertilization function. Here, "oxidative stress increases" means a state in which the oxidative action of reactive oxygen species in the living body and the antioxidant action of antioxidant enzymes are out of balance and the oxidative reaction in the living body is enhanced. ..

Next, "Ashitaba" and "Old-growth forest tomorrow" related to the present invention will be described.

Ashitaba (Ashitaba) is a perennial plant of the genus Umbelliferae native to Hachijojima in Japan, and is widely cultivated in the Izu area. The leaves and stems of Angelica keiskei are traditionally used as tempura for food. Ashitaba is rich in minerals and vitamins as a green-yellow vegetable, and is said to have effects such as constipation prevention, diuretic and tonic effects. Therefore, it has been attracting attention as a health food in recent years (see, for example, Patent Document 1). ). Furthermore, it is known that Angelica keiskei contains a plurality of compounds belonging to chalcones and coumarins, and these compounds have anticancer activity, antiulcer activity, antithrombotic activity, antibacterial activity and anti-AIDS activity. It has been reported to have such effects.

The virgin forest Ashitaba is a species found as a mutant that does not die even at temperatures below freezing, while the wild species Ashitaba withers at low temperatures, and is registered as the Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 14641. There is. The virgin forest Ashitaba is excellent in taste and nutritional value, and is expected to have the same or better effects as the wild species in the above-mentioned various actions. Furthermore, since the virgin forest Ashitaba has excellent cold resistance and overwintering resistance, it is not limited to warm areas such as the Izu area like wild species, but can be cultivated on a large scale in a wide area including cold areas. It is expected to be possible.

Japanese Unexamined Patent Publication No. 2005-237292

Ashok Agarwal, "Effect of Oxidative Stress on Male Reproduction" World J Mens Health 2014 April 32 (1): p1-17 Watanabe Oyster Research Institute Co., Ltd. Mitsugu Watanabe, "Effects of Watanabe Active Oyster on Sperm Motility in Healthy Adult Men" November 2010

As mentioned above, promoting the improvement of normal sperm formation function and / or the function of mature sperm itself in the testis is for the treatment of infertility in humans and industrial animals and for the improvement of production efficiency in the livestock industry. It has become a very important issue. Therefore, an object of the present invention is to provide a novel sperm function improving agent having excellent safety.

As a result of diligent studies to solve the above problems, the present inventors have newly found that the extract of Angelica keiskei has an action of improving the function of normal sperm formation in the testis and an action of improving the function of mature sperm. The invention of the present application has been completed. That is, the present invention provides a sperm function improving agent containing an extract of Angelica keiskei as an active ingredient.
In this sperm function improving agent, the Angelica keiskei can be the Angelica keiskei No. 14641 of the Ministry of Agriculture, Forestry and Fisheries.
In addition, in this sperm function improving agent, the Angelica keiskei extract can be xanthangelol or 4-hydroxydericin.
Furthermore, the present invention contains a pharmaceutical composition for infertility treatment or prevention containing this sperm function improving agent, and Ashitaba or an extract thereof for livestock or poultry used for improving sperm function. Feed and food compositions are also provided.
Furthermore, the present invention also provides a method for improving sperm function of livestock or poultry, which comprises a procedure for feeding Ashitaba or an extract thereof to livestock or poultry.

In the present invention, the "Ashitaba extract" is a liquid component obtained by pressing or crushing the whole plant of Ashitaba or one or more parts selected from leaves, stems, rhizomes, inflorescences, fruits, seeds and the like. Shall be included. In addition, the "Ashitaba extract" also contains an elution component extracted from one or more sites selected from the whole plant of Ashitaba or leaves, stems, rhizomes, inflorescences, fruits, seeds, etc. using an appropriate solvent. Is done. Further, the "Ashitaba extract" can be obtained by drying the whole plant of Ashitaba, or one or more parts selected from leaves, stems, rhizomes, inflorescences, fruits, seeds, etc. by freeze-drying, spray-drying, or the like. Contains solid components.

INDUSTRIAL APPLICABILITY According to the present invention, a novel sperm function improving agent and the like having excellent safety are provided. Since the active ingredient of this sperm function improving agent is derived from Angelica keiskei extract, it is highly possible that it has few side effects and is excellent in safety.

6 is a drawing-substituting graph showing the relationship between heat stress and oxidative stress in the testis (Test Example 1). It is a drawing substitute graph which shows the evaluation result (test example 2) of the spermatogenesis ability improving action of the Angelica keiskei extract. It is a drawing substitute graph which shows the evaluation result (test example 2) of the spermatogenesis ability improving action of the Angelica keiskei extract. It is a drawing substitute graph which shows the result (Test Example 3) which evaluated the protein expression level of Catalase in the testis treated with Ashitaba extract. It is a drawing substitute graph which shows the result (Test Example 3) which evaluated the protein expression level of HO-1 in the testis treated with the Angelica keiskei extract. 3 is a drawing-substituting graph showing the results of evaluating the time dependence of the protein expression level of catalase in the testis treated with the Angelica keiskei extract (Test Example 4). It is a drawing substitute graph which shows the result (Test Example 4) which evaluated the time dependence of the protein expression level of HO-1 in the testis treated with the Angelica keiskei extract. It is a drawing substitute graph which shows the evaluation result (test example 5) of the sperm function improving action of the Angelica keiskei extract. It is a drawing substitute graph which shows the evaluation result (test example 5) of the sperm function improving action of the Angelica keiskei extract. It is a drawing substitute graph which shows the evaluation result (test example 6) of the spermatogenesis ability improving action of a chalcone compound. It is a drawing substitute graph which shows the evaluation result (test example 6) of the spermatogenesis ability improving action of a chalcone compound. It is a drawing substitute photograph of a stained tissue section showing the result of the morphological change of the seminiferous tubule due to heat stress and the protective effect by oral administration of Angelica keiskei powder. It is a drawing substitute photograph of a stained tissue section showing the result of the morphological change of the seminiferous tubule due to heat stress and the protective effect by oral administration of a chalcone compound.

The present inventors have found for the first time that the Angelica keiskei extract has an action of improving spermatogenic function and mature sperm function in the testis by examination using mice, as will be described in detail in Examples. The action of this Angelica keiskei extract was considered to be expressed through the characteristic of increasing the protein expression level of the antioxidant enzyme of the orally administered Angelica keiskei extract.

As described above, it has been known that gene damage to spermatogenic cells or sperms in an environment with high oxidative stress is a cause of infertility caused by males not only in humans but also in industrial animals. In this regard, the present invention reduces the amount of reactive oxygen species generated in vivo by increasing the protein expression level of antioxidant enzymes by orally administered Ashitaba extract, thereby reducing spermatogenic function in the testis and / or It is thought to improve the function of mature sperm itself.

Therefore, the present invention can be suitably used for improving spermatogenic function and / or sperm function by applying it to infertile patients due to spermatogenic dysfunction and / or sperm dysfunction. It can also be applied to industrial animals to improve the decrease in the number of pups and eggs laid due to the decrease in spermatogenic function and / or sperm function, and to improve the production efficiency. Furthermore, it is applied to healthy subjects and healthy animals to prevent infertility.

In the present invention, Angelica keiskei can be used raw or dried. As Ashitaba, in particular, the primeval forest Ashitaba of Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 14641 can be used. The site of Angelica keiskei to be used is not particularly limited as long as the object of the present invention is not impaired, and can be a whole plant or one or more sites selected from leaves, stems, rhizomes, inflorescences, fruits, seeds, etc. Can be used in combination. The sites to be used are preferably leaves, stems and rhizomes.

Ashitaba extract can be obtained as a solid component by squeezing or crushing Ashitaba, or as a liquid component by squeezing or crushing Ashitaba, and further by extracting Ashitaba with an appropriate solvent. It can also be obtained as an elution component.

Solvent extraction can be performed by a conventionally known method. Solvent extraction can be performed, for example, by immersing the whole plant of Angelica keiskei or one or more parts in a solvent at low temperature or heating for a predetermined time or refluxing by heating. The obtained solvent extract may be subjected to filtration, concentration, purification / separation using an ion exchange resin, liquid chromatography, or the like, freeze-drying, or the like, if necessary.

As the solvent, usually one kind or two or more kinds of solvents used for plant extraction can be selected and used. Examples of the solvent include water, alcohols, glycols, ketones, esters, ethers, carbon halides and the like. Examples of alcohols include ethanol, methanol and propanol. Examples of glycols include ethylene glycol, diethylene glycol, butylene glycol and propylene glycol. Examples of the ketones include acetone, methyl ethyl ketone and the like. Examples of the esters include ethyl acetate, propyl acetate, ethyl formate and the like.

The Angelica keiskei extract can be used in any form such as liquid (juice), paste or gel. The Angelica keiskei extract may be dried to a solid state, or may be dried by freeze-drying, spray-drying, or the like to form a powder.

Ashitaba has been edible for a long time, which indicates the high safety of Ashitaba extract. Therefore, the sperm function improving agent according to the present invention containing Ashitaba extract as an active ingredient, and the pharmaceutical composition, feed, food composition and cosmetic composition containing the same have the following advantages.

That is, since the sperm function improving agent or the like according to the present invention is a component derived from a natural product, it is highly possible that the pharmaceutical composition can be continuously applied for a long period of time and that there are few side effects. Further, for example, by continuously applying it as a dietary supplement (functional food) for a long period of time, it may be possible to prevent a decrease in spermatogenic function and / or sperm function. In addition, when applied to the feed of industrial animals, it may be possible to prevent the occurrence of spermatogenic function and / or sperm function decline caused by stress due to poor management of the breeding environment.

The pharmaceutical composition according to the present invention can be used orally as, for example, sugar-coated tablets, capsules, elixirs, microcapsules, etc. as necessary, or with water or other pharmaceutically acceptable liquids. Can be used parenterally in the form of injectables such as sterile solutions or suspensions. The pharmaceutical composition is a unit dose of the above sperm function improving agent, along with physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., which are generally accepted and required for formulation implementation. Manufactured by mixing in form. Additives that can be mixed with tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragant, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Binders, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavors such as peppermint, reddish oil or cherry are used.

The dose of the pharmaceutical composition is determined in consideration of the type of dosage form, the administration method, the age and weight of the administration target (including animals), symptoms and the like.

In addition, the food composition according to the present invention contains ascorbic acid, fructose, sucrose, maltose, sorbitol, stebioside, rubusoside, corn syrup, lactose, citric acid, tartrate acid, malic acid, succinic acid, and lactic acid. , L-ascorbic acid, dl-α-tocopherol, sodium erythorbicate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, Arabic gum, carrageenan, casein , Gelatin, pectin, agar, vitamin Bs, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives, etc., which are usually used as food raw materials, can be appropriately blended. .. It may also be mixed with rice, wheat, corn, potatoes, sweet potatoes, soybeans, kelp, wakame seaweed, gelidiaceae, etc., which are generally used as food materials.

Examples of the food composition include health foods, functional foods, foods for specified health uses, dietary supplements, enteral nutrition foods and the like. In addition, these food compositions can also be fed to animals.

The feed according to the present invention is mixed with silage or its extract and raw grass or hay, green-cut feed crop (green-cut corn, etc.), straws, raw grass or hay, green-cut feed crop (green-cut corn, etc.), straws, etc. Can be obtained by doing.

In general, livestock feed is roughly classified into three types: roughage, concentrated feed, and special feed. Of these, roughage refers to feed that has a relatively high crude fiber content and a small amount of digestible nutrients for its large volume. Raw grass, hay, green-cut forage crops (green-cut corn, etc.), root vegetables, straws, etc. are used as the roughage. In addition, concentrated feed refers to feed having a relatively high nutrient content and a low water content and crude fiber content. Concentrated feed includes cereals such as corn, mylo, barley, embaku, rice, millet, barnyard millet, millet, and kolyan, grain by-products (bran) such as rice bran and bran, peanut cake, cotton cake, and sunflower cake. Oil cakes such as rapeseed meal, sesame meal, and flaxseed meal are used.

The feed according to the present invention can be produced by adding whole grass of Angelica keiskei or leaves, stems, rhizomes, inflorescences, fruits and seeds to these feeds in a raw state or as a dried product. In this case, Angelica keiskei may be added by cutting or pulverizing. The feed according to the present invention can also be produced by adding Ashitaba extract to the above-mentioned feed.

The food composition and feed according to the present invention described above are indicated to be used for improving spermatogenic function and / or sperm function, if necessary.

<Test Example 1>
1. 1. Examination of the relationship between heat stress and oxidative stress in the testis As mentioned above, reactive oxygen species affect spermatogenic cells or sperm genes in an environment with high oxidative stress, which can cause male infertility. Are known. There are various factors that increase oxidative stress, and for example, ingestion of ultraviolet rays, radiation, tobacco, drugs, metals, oxidized foods, etc. is considered. One of the causes of the summer infertility in industrial animals, especially pigs, is deterioration of sperm properties due to an increase in external temperature. From these points, we investigated the possibility that heat stress induces an increase in oxidative stress in the testis.

As animals, ICR mice (male, 8 weeks old) were used. For heat stress, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 42 ° C. for 20 minutes (hereinafter referred to as “heat stress group”). As a control, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 33 ° C. for 20 minutes (hereinafter referred to as “control group”).
Then, for the mice in the heat stress group, the testes were removed 24 hours after the heat stress was applied. On the other hand, for the mice in the control group, the testes were removed at the same time as the mice in the heat stress group.

For each group, after testis removal, the redox state of the testis was evaluated using the reduced / oxidized glutathione ratio (GSH / GSSG). This Test Example 1 was carried out using a GSSG / GSH Quantification Kit manufactured by Dojin Chemical Laboratory Co., Ltd.
[Evaluation of reduced / oxidized glutathione ratio]
[1] Pretreatment of measurement sample (1) Testes (samples) removed from mice in each group are homogenized with 1 ml of 5% SSA (5-Sulfosalicylic acid dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution per 100 mg.
(2) After centrifuging at 8000 xg for 10 minutes, transfer the supernatant to a new tube, dilute it to 1/10 with pure water, and use this as the measurement sample.
[2] Measurement of concentration of reduced / oxidized glutathione (1) Measurement of concentration of reduced / oxidized glutathione was performed according to the protocol attached to the GSSG / GSH Quantification Kit.

The results are shown in FIG. In FIG. 1, (A) shows the ratio of reduced / oxidized glutathione in the testis of mice in the control group, and (B) shows the ratio of reduced / oxidized glutathione in the testis of mice in the heat stress group. As shown in FIG. 1, the ratio of reduced glutathione to oxidized glutathione in the testis was significantly reduced in the mice (B) in the heat stress group as compared with the mice (A) in the control group.

From the results of Test Example 1, it was found that due to heat stress, most of them are present in the reduced form in the living body, and glutathione, which is an antioxidant, tends to be converted to the oxidized form. That is, there was a tendency that heat stress was converted into oxidative stress in the living body of mice.

<Test Example 2>
2. 2. Examination of the improving effect of the testis extract on spermatogenic function in the testis Test Example 1 showed that heat stress tended to be converted into oxidative stress in the living body of mice. Therefore, using an animal model, heat stress was used. The effect of improving spermatogenic function in the testis of Ashitaba extract in the environment was investigated.

As animals, ICR mice (male, 8 weeks old) were used. This mouse was purchased from Oriental Yeast Co., Ltd. For heat stress, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 42 ° C. for 20 minutes (hereinafter referred to as “heat stress zone”). As a control, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 33 ° C. for 20 minutes (hereinafter referred to as “control group”).
Then, mice not administered with Ashitaba extract were used as a control group (hereinafter referred to as "control group control group"). In addition, in order to observe the effect of heat stress on spermatogenesis function, mice in the heat stress group to which Ashitaba extract was not administered were used (hereinafter referred to as "heat stress group control group"). Further, in the heat stress group, mice to which the Ashitaba extract was administered were used (hereinafter, referred to as “heat stress group Ashitaba administration group”).

For the Ashitaba administration group in this heat stress group, the heat stress load was applied 24 hours after the first oral administration of the Ashitaba extract. As an Ashitaba extract, Nippon Biological Co., Ltd. "Ashitaba powder" manufactured by the Institute of Science was used. In 100 mg of this Angelica keiskei powder, 20 mg of the chalcones and 28 mg of the coumarins are contained. Then, this Angelica keiskei powder was mixed with the powdered feed normally ingested by mice and orally administered daily for 28 days by free intake. This powdered feed for mice was purchased from Oriental Yeast Co., Ltd. The dose of this Angelica keiskei powder was 75 mg / kg / day body weight. The environmental conditions (lighting, ventilation, feeding method, etc.) other than the temperature in the constant temperature bath were the same for all groups.

(1) Individuals in the heat-stressed Ashitaba-administered group were dissected on the 28th day, and the epididymis tail was first removed, and then the epididymis head, body and testis were removed.
(2) Individuals in the heat stress group control group and the control group control group were also dissected on the 28th day, and the epididymis tail was first removed, and then the epididymis head, body and testis were removed.

Then, in each group of individuals, the sperm concentration of epididymal sperm stored in the epididymal tail was detected by the following procedure.
[Method of detecting sperm concentration]
(1) Make three cuts with scissors in the removed epididymis tail.
(2) Buffer for sperm (2.2 mM HEPES pH7.35, 1.2 mM MgCl ₂ , 100 mM NaCl, 4.7 mM KCl, 1 mM Pyruvic acid, 4.8 mM) in which the tail of the epididymis was placed on a heating plate at 37 ° C. It was soaked in Lactic acid hemi calcium salt, 5.5 mM D-Glucose, 20 mM Sodium bicarbonate), and sperm that had swam out after 15 minutes were collected.
(3) Semen was diluted 10-fold with pure water, and the sperm count was measured using a hemocytometer under a microscope.
(4) The sperm count was measured 3 times per sample, and the average value was used as the result.

Furthermore, the acrosome reaction rate of epididymal sperm was measured by the following procedure, and based on this, the fertilization ability of epididymal sperm in each group of mice was detected.
[Acrosome reaction rate detection method]
(1) In order to remove impurities in the semen collected from the tail of the epididymis in (1) of the method for detecting sperm concentration, centrifuge at 100 xg for 1 minute and then transfer the supernatant to a new tube.
(2) Centrifuge at 400 xg for 8 minutes to adjust the concentration, and adjust to 2.0 × 10 ⁶ sperm / 300 μl using a hemocytometer.
(3) Add 2-Hydroxypropyl-β-cyclodextrin (final concentration 3 mM) and leave it in a constant temperature bath at 37 ° C for 40 minutes to acquire fertility.
(4) After adding Progesterone (final concentration 20 μM), let stand again in a constant temperature bath at 37 ° C. for 20 minutes.
(5) Add 300 μl of 8% Paraformaldehyde to the semen and leave it at room temperature for 15 minutes to fix the semen tissue.
(6) Then, centrifuge at 200 xg for 3 minutes to remove the supernatant, and add 800 μl of ammonium acetate (100 mM).
(7) Make an enclosure on the slide glass with a bop pen (manufactured by Matsunami Glass Industry Co., Ltd.), put 200 μl of the fixed semen in it, and dry it on a heating plate.
(8) Add 100 μl of Coomassie brilliant blue (CBB) staining solution (Coomassie brilliant blue 0.22%, MeOH 50%, pure water 40%, Acetic acid 10%) to stain.
(9) Rinse the staining solution with Phosphate-buffered saline (PBS) 5 minutes after CBB staining. The PBC cleaning is performed 5 times.
(10) A mounting solution (manufactured by Genemed Biotechnologies) is rubbed on a cover glass and placed on a slide glass, 100 or more sperms are observed under a microscope, and the fertilization ability acquisition sperm ratio, that is, the acrosome reaction rate is measured.

The result of sperm concentration is shown in FIG. 2, and the result of the acrosome reaction is shown in FIG. As described above, since Test Example 2 quantifies the sperm concentration and the acrosome reaction rate 28 days after the heat stress load, if a decrease in the sperm concentration and the acrosome reaction rate is observed, the spermatogenesis function It is considered that heat stress contributes to the inhibition of. Then, in FIG. 2, (A) shows the sperm concentration of the mouse of the control group control group, and (B) shows the sperm concentration of the mouse of the heat stress group control group. In addition, (C) shows the sperm concentration of the mice in the heat stress group Ashitaba administration group. As shown in FIG. 2, the mouse (B) in the heat stress group control group has a lower sperm concentration than the mouse (A) in the control group control group, and the sperm formation function is lowered by applying heat stress. Was shown. On the other hand, the mice (C) in the heat stress group Ashitaba administration group showed a significant increase in sperm concentration as compared with the mice (B) in the heat stress group control group, and were in a state of being loaded with heat stress. However, the spermatogenic function was improved. Then, it became clear that this increase in sperm concentration was dependent on the administration of Angelica keiskei extract.

In FIG. 3, (A) shows the acrosome reaction rate of the mice in the control group control group, and (B) shows the acrosome reaction rate of the mice in the heat stress group control group. In addition, (C) shows the acrosome reaction rate of the mice in the heat stress group Ashitaba administration group. As shown in FIG. 3, the mouse (B) in the heat stress group control group has a lower acrosome reaction rate than the mouse (A) in the control group control group, and the acrosome reaction of sperm by applying heat stress. It has been shown that the rate, or fertility, is reduced. On the other hand, the mice (C) in the heat stress group Ashitaba administration group showed a significant improvement in the acrosome reaction rate and the sperm fertility was improved as compared with the mice (B) in the heat stress group control group. .. In addition, the acrosome reaction rate recovered to the same level as the mice in the control group.

From the results of Test Example 2, it was clarified that the Angelica keiskei extract showed an effect of remarkably suppressing the decrease in spermatogenic function due to heat stress loading, and had an effect of improving spermatogenic function.

<Test Example 3>
3. 3. Examination of the function of increasing the expression level of antioxidant enzymes in Ashitaba extract As described above, in an environment where oxidative stress is increasing in vivo, active oxygen species may damage spermatogenic cells or sperm genes. Are known. High oxidative stress is a state in which the oxidative action of active oxygen species in the living body and the antioxidant action of antioxidant enzymes are out of balance and the oxidative reaction in the living body is enhanced. Therefore, we investigated the possibility that the spermatogenic function-improving effect of the Angelica keiskei extract observed in Test Example 2 was expressed due to an increase in the expression level of antioxidant enzymes that suppress the action of reactive oxygen species. ..

In Test Example 2, after the epididymis tail was removed, the head and body of the epididymis and the testis were removed, and these were stored at −80 ° C. Then, the protein expression level of the antioxidant enzyme in the testis was evaluated using Western blot analysis. Catalase and heme oxygenase (hereinafter referred to as "HO-1") were evaluated as antioxidant enzymes. This Western blot analysis was performed by the following procedure.
[Catalase and heme oxygenase Western blot analysis]
[1] Protein preparation (1) Thaw the testes stored at -80 ^o C on ice.
(2) Tissue lysis buffer (50 mM HEPES pH7.5, 50 mM NaCl, 1 mM EDTA, 5% Glycerol, 50 mM NaF, 10 mM Sodium pyrophosphate, 0.02% TritonX-100, 1 mM Na ₃ VO ₄ , 1 mM
PMSF, 10 μg / ml Antipain, 10 μg / ml Leupeptin, 10 μg / ml Aprotinin, 200 μM Dithiothreitol, 25 mM β-Glycerophosphate) are added at a ratio of 500 μl of Buffer to 100 mg of testicular weight to homogenate.
(3) After that, centrifuge at 14000 rpm for 30 minutes.
(4) Collect the supernatant and centrifuge at 14000 rpm for 30 minutes to collect the supernatant to prepare the protein.
[2] Electrophoresis and antigen-antibody reaction (1) The protein concentration in the collected cell extract is measured by the so-called BCA method using the BCA TM Protein Assay Kit (manufactured by Takara Bio Co., Ltd.).
(2) After that, 30 μg of protein is subjected to 10% Acrylamide gel SDS-PAGE and electrophoresis is performed.
(3) After electrophoresis, transfer to a PVDF membrane (manufactured by Merck Group).
(4) Then, blocking is performed with 5% BSA / TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20) to react the primary antibody.
(5) After washing the membrane with TBS-T for 15 minutes, an antigen-antibody reaction is carried out using a secondary antibody that recognizes each primary antibody.
Antibodies for Catalase and HO-1 were prepared using rabbits by synthesizing the following peptides, respectively. Peptide synthesis and antibody production were ordered from Sigma-Aldrich Japan GK.
Catalase: ADNRDPASDQMKHWKEQRAC
HO-1: QPNSMPQDLSEALKEATKEC
(6) Wash the membrane again with TBS-T for 15 minutes, use Chemi-Lumi One (manufactured by Nacalai Tesque, Inc.) to detect the band, and use LAS 4000 (Fuji Film) to emit chemiluminescence by Horse radish peroxidase (HRP). To detect.
(7) 5% BSA / TBS-T was used to dilute the antibody. In addition, α-tubulin was used as an internal standard protein.
In Test Example 3, the experiment was performed three times or more, and the result was shown by the mean value ± standard error (SE). The comparison of each data was performed by T-test, and it was judged that there was a significant difference when p <0.05.

The results are shown in FIGS. 4 and 5. FIG. 4 shows the results of the protein expression level of Catalase, and FIG. 5 shows the result of the protein expression level of HO-1. In FIG. 5, (A) shows the protein expression level of catalase in the testis of mice in the control group control group, and (B) shows the protein expression level of catalase in the testis of mice in the heat stress group control group. In addition, (C) shows the protein expression level of catalase in the testis of mice in the heat-stress group Ashitaba administration group.

As shown in FIG. 4, the protein expression level of Catalase, which is an antioxidant enzyme, was significantly increased in the mice (C) in the heat stress group Ashitaba administration group as compared with the mice (A) in the control group control group. In addition, the protein expression level of catalase showed a clear increasing tendency as compared with the mouse (B) in the heat stress group control group.

In FIG. 5, (A) shows the protein expression level of HO-1 in the testis of mice in the control group control group, and (B) shows the protein expression level of HO-1 in the testis of mice in the heat stress group control group. .. In addition, (C) shows the protein expression level of HO-1 in the testis of mice in the heat-stress group Ashitaba administration group.

As shown in FIG. 5, the protein expression level of the antioxidant enzyme HO-1 was significantly increased in the mice (C) in the heat stress group Ashitaba administration group as compared with the mice (A) in the control group control group. In addition, the protein expression level of HO-1 showed a clear increasing tendency as compared with the mice (B) in the heat stress group control group.

From the results of Test Example 3, it was clarified that the Angelica keiskei extract has an effect of increasing the protein expression level of the antioxidant enzyme even under the load environment of heat stress. Then, it was clarified that the Angelica keiskei extract has an effect of improving spermatogenic function even in an environment under the load of heat stress.

<Test Example 4>
4. Examination of time dependence of antioxidant enzyme expression level increasing function by Angelica keiskei Extract In Test Example 3, the antioxidant enzyme expression level increasing function of Angelica keiskei extract was observed. The possibility that the action of this Angelica keiskei extract was expressed over time was investigated by Western blot analysis.

In this Test Example 4, ICR mice (male, 8 weeks old) were used as animals, and the procedure was as follows. The ashitaba extract used in this test example 4, the dose of the ashitaba extract and the administration method of the ashitaba extract are the same as those of the test example 2. Furthermore, the procedure for Western blot analysis and the antioxidant enzyme to be evaluated are the same as in Test Example 3.
(1) As a target group, mice not administered with Ashitaba extract (after 0 days) were used. Furthermore, mice are dissected 1 day after the start of administration of Angelica keiskei extract, 3 days after the administration of Angelica keiskei extract, and 7 days after the administration of Angelica keiskei extract, and the testes of each individual are removed.
(2) After orchiectomy, store at -80 ° C.
(3) The protein expression level of antioxidant enzyme in the excised testis is evaluated by Western blot analysis.

The results are shown in FIGS. 6 and 7. FIG. 6 shows the results of the protein expression level of Catalase, and FIG. 7 shows the result of the protein expression level of HO-1. As shown in FIGS. 6 and 7, it was revealed that the administration of Ashitaba extract increased the protein expression levels of Catalase and HO-1 according to the administration period.

From the results of Test Example 4, it was clarified that the function of increasing the expression level of the antioxidant enzyme of the Angelica keiskei extract had a strong effect over time, and that the Angelica keiskei extract also had the effect of improving the spermatogenic function. became.

<Test Example 5>
5. Examination of sperm function improving effect of Angelica keiskei extract Using an animal model, we investigated the possibility that Angelica keiskei extract exerts a sperm function improving effect in a heat stress environment, especially in a short period after the load of heat stress.

As animals, ICR mice (male, 8 weeks old) were used. As one of the heat stresses, the rear 1/2 including the testis of the mouse was exposed to a constant temperature bath at 39 ° C. for 20 minutes (hereinafter referred to as “first heat stress zone”). As another heat stress, the rear 1/2 including the testis of the mouse was exposed to a constant temperature bath at 40 ° C. for 20 minutes (hereinafter referred to as “second heat stress zone”). As a control, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 33 ° C. for 20 minutes (hereinafter referred to as “control group”).

Then, mice not administered with Ashitaba extract were used as a control group (hereinafter referred to as "control group control group"). In addition, in order to observe the effect of heat stress on sperm function, mice in the heat stress group to which Ashitaba extract was not administered were used (hereinafter referred to as "heat stress group control group"). The heat stress zone control group was provided for each of the first heat stress zone and the second heat stress zone (hereinafter, each is a "first heat stress zone control group" and a "second heat stress zone control group". ). Further, in the heat stress group, mice to which the Ashitaba extract was administered were used (hereinafter, referred to as “heat stress group Ashitaba administration group”). This heat stress group Ashitaba administration group was also established for each of the first heat stress group and the second heat stress group (hereinafter, each is "the first heat stress group Ashitaba administration group" and "the second heat stress group Ashitaba". Administration group ").

Regarding the first heat stress group Ashitaba administration group and the second heat stress group Ashitaba administration group, the heat stress load was applied 7 days after the first oral administration of the Ashitaba extract. As an Ashitaba extract, Nippon Biological Co., Ltd. Using "Ashitaba powder" manufactured by the Institute of Science, it was mixed with the powdered feed normally ingested by mice and orally administered by free intake every day for 7 days. The dose of the Angelica keiskei extract used in Test Example 5 and the dose of this Angelica keiskei extract are the same as those in Test Example 2. The environmental conditions (lighting, ventilation, feeding method, etc.) other than the temperature in the constant temperature bath were the same for all groups.

(1) Individuals in the first heat stress group Ashitaba administration group and the second heat stress group Ashitaba administration group are dissected 24 hours after the heat stress load, and the epididymis tail of each individual is removed.
(2) Individuals in the first heat stress group control group and the second heat stress group control group are also dissected 24 hours after the heat stress load, and the epididymal tail of each individual is removed. In addition, the individuals in the control group of the control group are dissected at the same time as the individuals in each group loaded with heat stress, and the epididymal tail of each individual is removed.

Then, the epididymal tail of each group of individuals was dissected, and the sperm concentration and acrosome reaction rate of epididymal sperm stored in the epididymis were measured by the same method as in Test Example 2.

The result of the testis concentration is shown in FIG. 8, and the result of the acrosome reaction is shown in FIG. In FIG. 8, (A) shows the sperm concentration of the mouse of the control group control group, and (B) shows the sperm concentration of the mouse of the first heat stress group control group. In addition, (C) shows the sperm concentration of the mice in the first heat stress group Ashitaba administration group. Further, (D) shows the sperm concentration of the mouse in the second heat stress group control group, and (E) shows the sperm concentration of the mouse in the second heat stress group Ashitaba administration group.

As shown in FIG. 8, in the groups (B) to (E) loaded with heat stress, the sperm concentration tended to decrease as compared with the control group (A). Mice (C) in the first heat stress group Ashitaba administration group showed an increasing tendency of sperm concentration as compared with mice (B) in the first heat stress group control group, which depended on the administration of Ashitaba extract. it is conceivable that.

Further, as shown in FIG. 8, the mice (E) in the second heat stress group Ashitaba administration group showed an increasing tendency of sperm concentration as compared with the mice (D) in the second heat stress group control group, and this was extracted from Ashitaba. It is considered that it depended on the administration of the substance.

In FIG. 9, (A) shows the acrosome reaction rate of the mice in the control group control group, and (B) shows the acrosome reaction rate of the mice in the first heat stress group control group. In addition, (C) shows the acrosome reaction rate of the mice in the first heat stress group Ashitaba administration group. Further, (D) shows the acrosome reaction rate of the mice in the second heat stress group control group, and (E) shows the acrosome reaction rate of the mice in the second heat stress group Ashitaba administration group.

As shown in FIG. 9, in the groups (B) to (E) loaded with heat stress, the acrosome reaction rate tended to be lower than that in the control group (A). Furthermore, the mice (C) in the first heat stress group Ashitaba administration group had a higher acrosome reaction rate than the mice (B) in the first heat stress group control group, and even when they were loaded with heat stress, An increasing tendency of the acrosome reaction rate was observed, which is considered to be dependent on the administration of Ashitaba extract. This tendency was also observed in the group loaded with the second heat stress.

This Test Example 5 quantifies the sperm concentration and the acrosome reaction rate 24 hours after the heat stress load, and quantifies the sperm concentration and the acrosome reaction rate 28 days after the heat stress load as in Test Example 2. Not what I did. From this point, it was clarified that heat stress not only affects the stage of sperm formation in the testis, but also affects the mature sperm itself stored in the epididymis. In this regard, the measurement of the acrosome reaction rate shown in FIG. 9 was performed based on the sperms that had swam into the sperm buffer as described above. Therefore, due to the decreasing tendency of the acrosome reaction rate in the mice (C) and (E) of the Ashitaba-administered group shown in FIG. 9, heat stress also affects the mature sperm itself stored in the epididymis. Became clear.

Furthermore, despite the different temperature conditions, the ashitaba-administered groups (C) and (D) showed an increasing tendency of sperm concentration and acrosome reaction with respect to the control groups (B) and (D). From the above, it was clarified that the Angelica keiskei extract has an effect of suppressing the decrease in sperm function due to the load of heat stress regardless of the strength of heat stress, and has an effect of improving sperm function.

<Test Example 6>
6. Examination of the improving effect of chalcones xanthangelol and 4-hydroxydelicin on the spermatogenic function in the testis In Test Example 2 and the like, the improving effect of the ashitaba extract on the spermatogenic function was observed.
On the other hand, as described above, it is known that Angelica keiskei contains a plurality of compounds belonging to chalcones and coumarins.
For this reason, the present inventors particularly among compounds belonging to chalcones contained in Angelica keiskei (hereinafter referred to as "chalcone compounds"), xanthangelol (hereinafter abbreviated as "XA") and 4-hydroxy. Regarding delicin (hereinafter abbreviated as "4HD"), the effect of improving spermatogenic function was examined.

As animals, ICR mice (male, 8 weeks old) were used. This mouse was purchased from Oriental Yeast Co., Ltd. For heat stress, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 42 ° C. for 20 minutes (hereinafter referred to as “heat stress zone”). As a control, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 33 ° C. for 20 minutes (hereinafter referred to as “control group”).
Then, mice not administered with the chalcone compound were used as a control group (hereinafter, referred to as "control group control group"). In addition, in order to observe the effect of heat stress on spermatogenesis function, mice in the heat stress group to which the chalcone compound was not administered were used (hereinafter, referred to as "heat stress group control group"). Further, in the heat stress group, mice to which XA was administered were used (hereinafter, referred to as "heat stress group XA administration group"). Moreover, in the heat stress group, the mouse to which 4HD was administered was used (hereinafter, referred to as "heat stress group 4HD administration group").

Regarding the heat stress group XA administration group and the heat stress group 4HD administration group, the heat stress load was applied 7 days after the first oral administration of the chalcone compound.
As a chalcone compound, Nippon Biological Co., Ltd. After extraction with 80% methanol (methanol) from "Ashitaba chalcone powder" manufactured by the Institute of Science, HPLC (High Performance Liquid Chromatography) was repeated twice to purify the product to a purity of 95% or higher.
Here, the conditions of HPLC are as follows.
<Conditions for purification of 4HD and XA by HPLC>
Column: Intersil OSD-4 (10.0 x 250 mm)
Mobile phase: 80% Methanol
Flow velocity: 4.0 ml / min
Solvent: Methanol
Detection: 280 nm
Injection amount: 0.75 ml

Then, purified XA and 4HD were mixed with corn oil normally ingested by mice and orally administered daily using a sonde for 7 days. The dose of this chalcone compound was 5 mg / kg / day body weight. The environmental conditions (lighting, ventilation, feeding method, etc.) other than the temperature in the constant temperature bath were the same for all groups.

(1) Individuals in the heat stress group XA administration group, the heat stress group 4HD administration group, and the heat stress group control group were dissected 24 hours after the heat stress load, and the epididymal tail of each individual was excised.
(2) The individuals in the control group of the control group were dissected at the same time as the individuals in each group loaded with heat stress, and the epididymal tail of each individual was removed.

Then, the epididymal tail of each group of individuals was dissected, and the sperm concentration and acrosome reaction rate of epididymal sperm stored in the epididymis were measured by the same method as in Test Example 2.
In Test Example 6, 4 mice were used in the control group and the heat stress group 4HD administration group, and 5 mice were used in the heat stress group XA administration group and the heat stress group control group, and the experiment was performed 3 times or more. The results are shown as mean ± standard error (SE). For comparison of each data, T-test was performed, and it was judged that there was a significant difference when p <0.05 with respect to the result of the heat stress group control group.

The results of testis concentration are shown in FIG. 10, and the results of the acrosome reaction rate are shown in FIG. In FIG. 10, (A) shows the sperm concentration of the mouse of the control group control group, and (B) shows the sperm concentration of the mouse of the heat stress group control group. In addition, (C) shows the sperm concentration of the mice in the heat stress group XA administration group. Furthermore, (D) shows the sperm concentration of the mice in the heat stress group 4HD administration group.

As shown in FIG. 10, in the groups (B) to (D) loaded with heat stress, the sperm concentration tended to decrease as compared with the control group (A). On the other hand, the mice (D) in the heat stress group 4HD administration group showed an increasing tendency of sperm concentration as compared with the mice (B) in the heat stress group control group, which is considered to be dependent on the administration of 4HD. ..

In FIG. 11, (A) shows the acrosome reaction rate of the mice in the control group control group, and (B) shows the acrosome reaction rate of the mice in the heat stress group control group. Further, (C) shows the acrosome reaction rate of the mice in the heat stress group XA administration group, and (D) shows the acrosome reaction rate of the mice in the heat stress group 4HD administration group.

As shown in FIG. 11, the acrosome reaction rate tended to decrease in the heat stress group control group (B) as compared with the control group control group (A).
On the other hand, the mice (C) in the heat stress group XA administration group showed the same acrosome reaction rate as the mice (A) in the control group control group, and even when the heat stress was applied, the XA It was confirmed that the acrosome reaction rate increased depending on the administration.
Furthermore, the acrosome reaction rate of the heat stress group 4HD administration group (D) was significantly higher than that of the control group control group (A) not loaded with heat stress.
That is, it was confirmed that the acrosome reaction rate was increased by the administration of 4HD even under the load of heat stress.

This Test Example 6 quantifies the sperm concentration and the acrosome reaction rate 24 hours after the heat stress load, and quantifies the sperm concentration and the acrosome reaction rate 28 days after the heat stress load as in Test Example 2. Not what I did.
From this point, it was clarified that heat stress not only affects the stage of sperm formation in the testis, but also affects the mature sperm itself stored in the epididymis. In this regard, the measurement of the acrosome reaction rate shown in FIG. 11 was performed based on the sperms that had swam into the sperm buffer as described above. Therefore, from the tendency of the acrosome reaction rate of the mice (B) in the heat stress group control group shown in FIG. 11 to decrease, it is clear that the heat stress also affects the mature sperms themselves stored in the epididymis. It became.

Then, since the heat stress group 4HD administration group (D) showed an increasing tendency of sperm concentration and an increasing tendency of the acrosome reaction with respect to the heat stress group control group (B), 4HD was loaded with heat stress. It was clarified that it has an effect of suppressing a decrease in sperm function in a short period of time and has an effect of improving sperm function. In addition, since the heat stress group XA administration group (C) showed an increasing tendency of the acrosome reaction with respect to the heat stress group control group (B), XA also shows an effect of improving sperm function at least for the acrosome reaction. It became clear.

<Test Example 7>
7. Examination of the function of Ashitaba powder against heat stress From Test Examples 2 to 5, it was confirmed that Ashitaba powder has an improving effect on spermatogenic function in the testis.
Therefore, the present inventors investigated the function of Angelica keiskei powder against heat stress in the seminiferous tubule where sperm is produced.
Here, since multinucleated giant cells are generally observed in stressed seminiferous tubules, they are regarded as indicators of disorders in the testis. Therefore, the present inventors examined the function of Angelica keiskei powder against heat stress based on the appearance of multinucleated giant cells.

As animals, ICR mice (male, 8 weeks old) were used. This mouse was purchased from Oriental Yeast Co., Ltd. For heat stress, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 42 ° C. for 20 minutes (hereinafter referred to as “heat stress zone”). As a control, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 33 ° C. for 20 minutes (hereinafter referred to as “control group”).
Then, mice not administered with Ashitaba powder were used as a control group (hereinafter referred to as "control group control group"). In addition, in order to observe the effect of heat stress on spermatogenesis function, mice in the heat stress group to which Ashitaba powder was not administered were used (hereinafter, referred to as "heat stress group control group"). Further, in the heat stress group, mice to which Ashitaba powder was administered were used (hereinafter, referred to as "heat stress group Ashitaba administration group").

Regarding the group administered with Ashitaba in the heat stress group, the load of heat stress was applied 7 days after the first oral administration of Ashitaba powder. As for Ashitaba powder, Nippon Biological Co., Ltd. The one manufactured by the Institute of Science was mixed with the powdered feed normally ingested by mice, and was orally administered by free ingestion every day for 7 days. In this Test Example 7, the dose of Angelica keiskei powder was 75 mg / kg / day body weight. The environmental conditions (lighting, ventilation, feeding method, etc.) other than the temperature in the constant temperature bath were the same for all groups.

(1) Heat stress group Ashitaba-administered individuals are dissected 48 hours after the heat stress load, and the testes of each individual are removed.
(2) The individuals in the heat stress zone control group are also dissected 48 hours after the heat stress load, and the testes of each individual are removed.
(3) The individuals in the control group of the control group are dissected at the same time as the individuals in each group loaded with heat stress, and the testes of each individual are removed.

For each group, the excised testis was immersed in a 10% neutral buffered formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) and fixed. Then, a tissue section was prepared, and the section was subjected to Hematoxylin Eosin (HE) staining (hereinafter referred to as "HE staining").
The tissue section was prepared by a paraffin embedding method. The HE staining method is not particularly limited, and a known method can be adopted.

The result of HE staining is shown in FIG. In FIG. 12, (A) is a stained tissue section of a mouse in the control group control group, (B) is a stained tissue section of a mouse in the heat stress group control group, and (C) is a mouse in the heat stress group Ashitaba administration group. The stained tissue section of is shown.
As shown in FIG. 12, in the heat stress zone control group (B), many multinucleated giant cells were present in the fine tubes, and cavities due to lack of cells were observed (arrow line portion). On the other hand, in the heat stress group Ashitaba administration group (C), the appearance of polynuclear giant cells and cavities was suppressed.

From the results of this Test Example 7, by oral administration of Ashitaba powder, the function of protecting spermatocytes, which are present inside the seminiferous tubules and further inside the seminiferous tubules and are considered to be vulnerable to stress such as heat, from heat stress. It was confirmed that normal spermatogenesis is performed even under heat stress.

<Test Example 8>
8. Examination of the functions of XA and 4HD to heat stress Test Example 6 confirmed the improving effect of chalcones on the spermatogenic function in the testis of XA and 4HD in the epididymis where sperm maturation is performed. Furthermore, Test Example 7 confirmed that Ashitaba powder exists inside the seminiferous tubule and further inside the seminiferous tubule, and has a function of protecting spermatocytes, which are considered to be vulnerable to stress such as heat, from heat stress. ..
From the results of these test examples, the present inventors examined the functions of XA and 4HD with respect to heat stress.

As animals, ICR mice (male, 8 weeks old) were used. This mouse was purchased from Oriental Yeast Co., Ltd. For heat stress, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 42 ° C. for 20 minutes (hereinafter referred to as “heat stress zone”). As a control, the posterior 1/2 including the testis of the mouse was exposed to a constant temperature bath at 33 ° C. for 20 minutes (hereinafter referred to as “control group”).
Then, mice not administered with the chalcone compound were used as a control group (hereinafter, referred to as "control group control group"). In addition, in order to observe the effect of heat stress on spermatogenesis function, mice in the heat stress group to which the chalcone compound was not administered were used (hereinafter, referred to as "heat stress group control group"). Further, in the heat stress group, mice to which XA was administered were used (hereinafter, referred to as "heat stress group XA administration group"). Moreover, in the heat stress group, the mouse to which 4HD was administered was used (hereinafter, referred to as "heat stress group 4HD administration group").

Regarding the heat stress group XA administration group and the heat stress group 4HD administration group, the heat stress load was applied 7 days after the first oral administration of the chalcone compound.
As a chalcone compound, Nippon Biological Co., Ltd. After extraction with 80% methanol (methanol) from "Ashitaba chalcone powder" manufactured by the Institute of Science, HPLC (High Performance Liquid Chromatography) was repeated twice to purify the product to a purity of 95% or higher. The HPLC conditions are the same as those of Test Example 6.

Then, purified XA and 4HD were mixed with corn oil normally ingested by mice and orally administered daily using a sonde for 7 days. The dose of this chalcone compound was 5 mg / kg / day body weight. The environmental conditions (lighting, ventilation, feeding method, etc.) other than the temperature in the constant temperature bath were the same for all groups.

(1) Individuals in the heat stress group XA administration group, the heat stress group 4HD administration group, and the heat stress group control group were dissected 48 hours after the heat stress load, and the testes of each individual were excised.
(2) The individuals in the control group of the control group were dissected at the same time as the individuals in each group loaded with heat stress, and the testes of each individual were excised.

For each group, the excised testis was immersed in Buan fixative (manufactured by Wako Pure Chemical Industries, Ltd.) and fixed. Then, a tissue section was prepared and the section was subjected to HE staining.
The tissue section was prepared by a paraffin embedding method. The method for HE staining is not particularly limited, and a known method can be adopted.

The result of HE staining is shown in FIG. In FIG. 13, (A) is a stained tissue section of a mouse in the control group control group, (B) is a stained tissue section of a mouse in the heat stress group control group, and (C) is a mouse in the heat stress group 4HD administration group. (D) shows the stained tissue section of the mouse in the heat stress group XA administration group.
As shown in FIG. 13, in the heat stress zone control group (B), many multinucleated giant cells were present in the fine tubes, and cavities due to lack of cells were observed (arrow line portion). On the other hand, in the heat stress group 4HD administration group (C) and the heat stress group XA administration group (D), the appearance of polynuclear giant cells and cavities was suppressed.

From the results of this Test Example 8, by oral administration of the calcon compound, the function of protecting spermatocytes, which are present inside the seminiferous tubule and further inside the seminiferous tubule and are considered to be vulnerable to stress such as heat, from heat stress. It was confirmed that normal spermatogenesis is performed even under heat stress.

The sperm function improving agent according to the present invention can be suitably used as a pharmaceutical composition, a feed and a food composition for treating infertility in humans and animals, or for improving the production efficiency of the livestock industry.

Claims (7)

An oral preparation for improving sperm function containing Ashitaba or an extract thereof as an active ingredient. The oral preparation for improving sperm function according to claim 1, wherein the extract of Angelica keiskei is xanthangelol or 4-hydroxydelicin. An oral preparation for improving sperm function containing xanthangelol or 4-hydroxydelicin as an active ingredient. An oral pharmaceutical composition for treating or preventing infertility, which comprises the oral preparation for improving sperm function according to any one of claims 1 to 3. Livestock or poultry feed containing Angelica keiskei or its extract and used to improve sperm function. A food composition containing Ashitaba or an extract thereof and used for improving sperm function. A method for improving sperm function in livestock or poultry, which comprises a procedure for feeding Ashitaba or an extract thereof to livestock or poultry.