JP7645483B2 - Food or feed containing ergothioneine - Google Patents

Description

The present invention relates to a food or feed containing ergothioneine for improving the fertility of female mammals, and a method for improving the fertility of female mammals by ingesting ergothioneine.

In recent years, the trend in Japan towards later marriage has led to an increase in the number of women using assisted reproductive technology. This is because the average age of women hoping to become pregnant is rising, while women's fertility declines with age, leading to an increase in cases where medical intervention is required to become pregnant. Treatments to increase women's fertility include the use of ovulation-inducing drugs and hormones, but these treatments are known to cause a number of side effects.

In order to improve productivity in non-human mammals, particularly in animals used for meat, such as cattle, pigs, and sheep, it is desirable to increase the average number of offspring born in multiparous animals and to increase the birth frequency in singleparous animals. It is also desirable to increase the number of pregnancies per unit time in both multiparous and singleparous animals.

Ergothioneine is a type of amino acid contained in mushrooms such as Pleurotus pilosus and used as an antioxidant in cosmetics. Patent Document 1 reports that the ergothioneine content in Pleurotus pilosus is 15.8±0.06 mg per 1 g dry weight. Non-Patent Document 1 shows that ergothioneine, when ingested by humans and animals as food, takes a long time to metabolize, so it is likely to accumulate in many tissues and body fluids of humans and animals. It has been confirmed in vitro that ergothioneine has antioxidant and cytoprotective effects, and it has been suggested that it is involved in the repair of tissue damage in the human and animal bodies. In humans, ergothioneine is expected to be a highly safe food-derived component that has therapeutic effects on neurodegenerative diseases, eye diseases, cardiovascular diseases, kidney diseases, diabetes, etc. due to its antioxidant and cytoprotective effects. However, the effect of ergothioneine in improving fertility in humans or mammals other than humans has not been reported.

Patent Publication 2012-105618

B. Halliwell et al., FEBS letters, vol.592, pp. 3357-3366 (2018)

The present invention aims to provide a highly safe food or feed that can improve female fertility or reduce the number of days for estrus to return in humans or non-human mammals.

As a result of intensive research, the present inventors have found that fertility of mammals is increased and/or the number of days for estrus to return is reduced by ingesting ergothioneine, and have completed the present invention.
(1) A food or feed containing ergothioneine for improving fertility or reducing the number of days for estrus to return in female mammals.
(2) The food or feed of (1), in which the intake of ergothioneine by a female mammal is 0.18 mg/kg body weight/day or more.
(3) A method for improving fertility in a female mammal, comprising administering ergothioneine to a female mammal other than a human.
(4) The method according to (3), wherein the intake amount of ergothioneine of the female non-human mammal is 0.18 mg/kg body weight/day or more.
(5) A method for reducing the number of days to return to estrus in a female mammal other than a human, comprising administering ergothioneine to the female mammal.
(6) The method according to (5), wherein the intake amount of ergothioneine of the female non-human mammal is 0.18 mg/kg body weight/day or more.

The present invention makes it possible to provide a highly safe food or feed that can improve female fertility or reduce the number of days for estrus to return in humans or non-human mammals.

FIG. 1 is a schematic diagram showing the procedure for producing mice by repeated superovulation stimulation in Example 1. 1 is a line graph showing the estrous cycle patterns of a 3-month-old control mouse (A) and a mouse that had been subjected to six rounds of superovulation stimulation (B). 1 is a bar graph showing the length of one estrous cycle in control mice and mice after superovulation stimulation. In the figure, "*" indicates that the p-value in the Student's t-test was less than 0.05. 1 is a bar graph showing the number of ovulated eggs in 3-month-old mice and mice that underwent repeated superovulation stimulation. In the figure, "**" indicates that the p-value in the Student's t-test was less than 0.01. FIG. 1 is a line graph showing the estrous cycle patterns of mice without ergothioneine administration (A) and mice with ergothioneine administration (B). 1 is a bar graph showing the length of one estrous cycle in mice without and with ergothioneine administration, where "*" indicates that the p-value in the Student's t-test was less than 0.05. 1 is a bar graph showing the number of ovulated eggs in control mice and mice repeatedly superovulated with and without ergothioneine administration. In the figure, "*" and "**" indicate that the p-values in the Student's t-test were less than 0.05 and 0.01, respectively. FIG. 1 is a schematic diagram showing the procedure for administering ergothioneine in drinking water to mice in Example 4. 1 is a bar graph showing the number of ovulated eggs in control mice and mice administered ergothioneine in each test group. In the figure, "*" indicates that the p value in the Student's t-test was less than 0.05. 1 is a bar graph showing the number of eggs ovulated in control mice and mice administered ergothioneine at various doses. In the figure, "*" indicates that the p-value was less than 0.05 in the Student's t-test. 1 is a bar graph showing the average number of pups per litter in control mice and mice treated with ergothioneine. In the figure, "*" indicates a p-value of less than 0.05 in the Student's t-test.

<1. Food or Feed>
The food or feed of the present invention contains ergothioneine and is characterized in that it improves fertility or reduces the number of days for estrus to return in female mammals. In the present invention, "food" refers to a substance or composition in a form suitable for ingestion by humans. Also, in the present invention, "feed" refers to a substance or composition in a form suitable for ingestion by animals other than humans.

In the present invention, "ergothioneine" refers to a compound represented by the following formula I and its derivatives. All stereoisomers having the structural formula of the following formula I are included. The derivatives referred to here include metal salts, hydrates, etc., but are not particularly limited as long as they have the skeleton of the following formula I.

Ergothioneine is contained in mushrooms such as Tamogitake, Oyster Mushroom, King Oyster Mushroom, Sparassis crispa, Enokitake, and Shiitake mushrooms, and is known to be contained in Tamogitake in particular. Ergothioneine may be refined from raw materials such as mushrooms containing ergothioneine, and added to the food or feed of the present invention. As a method for purifying ergothioneine, for example, the method shown in Patent Document 1 can be used. Alternatively, ergothioneine may be contained in the food or feed of the present invention as unrefined finely ground raw materials such as mushrooms containing ergothioneine, dried powder, concentrated extract, etc. In the present invention, ergothioneine is preferably contained in the food or feed as a dried powder or concentrated extract of mushrooms, particularly as a dried powder or concentrated extract of Tamogitake.

In the present invention, "mammals" refers to animals belonging to the class Mammalia, subphylum Vertebrates, phylum Chordata, including humans and non-humans, and includes, for example, primates including humans and 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 mammals kept in zoos. The subjects to be fed the food or feed of the present invention are preferably humans or livestock animals such as cows, pigs and sheep.

In the present invention, "fertility" refers to the ease with which female mammals become pregnant. In the present invention, improvement in fertility is determined by indicators such as stabilization or shortening of the estrus cycle and, in multiparous animals, an increase in the number of offspring per birth. In human adult females, one of the causes of infertility is a disruption or prolongation of the menstrual cycle. One of the objectives of the present invention is to stabilize this, thereby improving fertility.

In the present invention, "the number of days until estrus recurs" refers to the number of days from when a female mammalian animal weans a previously born child until the animal goes into estrus again. By decreasing the number of days until estrus recurs, it is possible to increase the number of births per unit period (e.g., one year), and as a result, to increase the number of offspring born per unit period.

In a study using mice in the examples of this specification, repeated superovulation stimulation lengthened the estrous cycle of the mice, but in mice that were administered ergothioneine at the same time as the superovulation stimulation, the estrous cycle was shortened to the same length as that of normal mice. Furthermore, the number of ovulations in the oviducts of mice administered ergothioneine was greater than that of normal mice. Furthermore, in a study using pigs, a decrease in the number of days for estrus to return and an increase in the number of offspring were observed in pigs that ingested ergothioneine. Thus, it has been revealed that ergothioneine has the effect of improving the fertility of female mammals and the effect of reducing the number of days for estrus to return. The present invention comprises incorporating ergothioneine, which has the above-mentioned effect, into food or feed, thereby improving the fertility and/or reducing the number of days for estrus to return in female mammals that ingest the food or feed.

The form of the food or feed of the present invention is not particularly limited as long as it can be ingested without difficulty by the target mammalian animal, and may be, for example, a liquid (beverage), powder, granules, tablet, or capsule. In addition, the food or feed of the present invention may contain a carrier as necessary. Examples of the carrier include excipients, binders, disintegrants, fillers, emulsifiers, flow additive regulators, lubricants, etc.

Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium, and high molecular weight polyethylene glycols (PEGs), pluronics, kaolin, silicic acid, or combinations thereof.

Examples of binders include starch paste made from corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, and/or polyvinylpyrrolidone.

Examples of disintegrants include the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, or salts thereof.

Examples of fillers include the above-mentioned sugars and/or calcium phosphates (e.g., tricalcium phosphate or calcium hydrogen phosphate).

Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

Such carriers are used to maintain the shape of the product and the effects of ergothioneine, and may be used as needed. In addition to the above additives, flavorings, solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption promoters, bulking agents, wetting agents, humectants, adsorbents, disintegration inhibitors, coating agents, colorants, preservatives, antioxidants, fragrances, flavorings, sweeteners, buffers, etc. may also be included as needed.

The food or feed of the present invention may be ingested as is, or may be mixed with other foods or feeds or added to drinking water. In particular, when ingested by mammals other than humans, the food or feed may be mixed with other feeds or dissolved in drinking water to ensure that the required amount is ingested.

In the food or feed of the present invention, the content of ergothioneine is preferably adjusted so that the intake amount in a mammalian animal ingesting the food or feed is 0.18 mg/kg body weight/day or more, particularly 0.36 mg/kg body weight/day or more. There is no particular upper limit to the intake amount, but the amount that can be ingested as food or feed can be, for example, 10.0 mg/kg body weight/day or less, particularly 5.0 mg/kg body weight/day or less, and further 3.2 mg/kg body weight/day or less. Note that 3.2 mg/kg body weight corresponds to the amount of ergothioneine that an adult human female (assumed to weigh 50 kg) can ingest when eating 100 g (raw weight) of Pleurotus cornucopiae.

The food or feed of the present invention preferably contains 1/5 to 3 times, and particularly 1/3 to 1 times, the daily intake amount of ergothioneine per serving. In other words, it is preferable that the intake frequency is once every 3 days to five times a day, and particularly once to three times a day.

The timing and period for which the food or feed of the present invention is ingested by a mammalian animal is not particularly limited, and the mammalian animal may, for example, be ingested daily. Alternatively, the mammalian animal may, for example, be ingested only during the period from a few days before ovulation until mating.

2. Method for improving fertility in female mammals
The present invention includes a method for improving fertility in a female mammalian animal, comprising having a female mammalian animal ingest ergothioneine. In the present invention, the means for having a mammalian animal ingest ergothioneine is preferably having the mammalian animal ingest the feed of the present invention.

In the present invention, "non-human mammals" refers to all animals other than humans that belong to the phylum Chordata, subphylum Vertebrates, class Mammalia, and includes, for example, 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 mammals kept in zoos. The subjects to which the method of the present invention is applied are preferably livestock animals such as cows, pigs, and sheep.

In the present invention, the amount of ergothioneine ingested by a mammalian animal is preferably 0.18 mg/kg body weight/day or more, particularly 0.36 mg/kg body weight/day or more. There is no particular upper limit to the intake amount, but it can be, for example, 10.0 mg/kg body weight/day or less, particularly 5.0 mg/kg body weight/day or less, and further 3.2 mg/kg body weight/day or less.

In the method of the present invention for improving the fertility of female mammals, the definitions of "ergothioneine" and "fertility," the preferred form of feed containing ergothioneine, the method of ingestion, and the frequency of ingestion are all as described in the section <1. Food or Feed>.

<3. Method for reducing the number of days for estrus recurrence in female mammals>
The present invention includes a method for reducing the number of days until estrus recurs in a female mammalian animal, comprising administering ergothioneine to a non-human female mammalian animal. In the present invention, the means for administering ergothioneine to a mammalian animal is preferably the administration of the feed of the present invention.

In the method of the present invention for reducing the number of days for which estrus recurs in female mammals, the target mammal, the amount of ergothioneine to be ingested by the mammal, the definitions of "ergothioneine" and "number of days for which estrus recurs," the preferred form of the feed containing ergothioneine, the method of ingestion, and the frequency of ingestion are all as described in the sections <1. Food or feed> and <2. Method for improving fertility in female mammals>.

4. Methods for improving fertility in human females
The present invention also includes a method for improving fertility in a human female. The method for improving fertility in a human female of the present invention is characterized in that the human female ingests ergothioneine. In the present invention, the means for ingesting ergothioneine in a human female is preferably by ingesting the food product.

In the method of the present invention for improving fertility in human females, the definitions of "ergothioneine" and "fertility," the preferred form, intake amount, intake method, and intake frequency of foods containing ergothioneine, etc. are all as described in the sections <1. Food or feed> and <2. Method for improving fertility in female mammals>.

Example 1: Generation of mice by repeated superovulation stimulation
Three-week-old immature C57BL6 mice were administered with equine chorionic gonadotropin (eCG) (follicle-stimulating hormone (FSH) preparation) and human chorionic gonadotropin (hCG) (luteinizing hormone (LH) preparation) to stimulate superovulation, and mice were produced that were repeatedly subjected to superovulation stimulation. Specifically, 4 I.U of eCG was administered by subcutaneous injection, and 2 days later, 5 I.U of hCG was administered. This process was repeated six times every 10 days (FIG. 1). After each administration, the mice were subjected to vaginal cavity examination, and their estrous cycle was observed. In addition, vaginal cavity examination was also performed on 3-month-old and 6-month-old mice that had not been administered hormone drugs as control mice. The number of control mice and superovulation-stimulated mice tested was three each, and the average value ± standard error of each measurement was calculated.

Figure 2 shows the estrous cycle patterns of 3-month-old control mice (Figure 2A) and mice that had been subjected to six superovulation stimulations (Figure 2B). Figure 3 shows the length of one estrous cycle for each control mouse and each mouse after superovulation stimulation. "*" in the figure indicates that the p value in Student's t-test was less than 0.05. The estrous cycle in the control mice was 4.7 ± 0.9 days, whereas in the mice that had been subjected to six repeated superovulation stimulations, microestrus was observed continuously and the estrous cycle was prolonged to 8.1 ± 1.1 days.

Mice that had been subjected to six repeated superovulation stimulations were further subjected to one additional superovulation stimulation (administration of eCG and hCG). 16 hours after administration of hCG, the oviducts were collected and the number of eggs ovulated was measured. The number of eggs ovulated was also measured in the same manner for three-month-old mature female control mice. Three control mice and three superovulation-stimulated mice were tested, and the mean ± standard error of each measurement was calculated.

Figure 4 shows the number of eggs ovulated in 3-month-old mice and mice that had been repeatedly superovulated. In the figure, "**" indicates that the p-value was less than 0.01 in the Student's t-test. In the 3-month-old mice, 16.6 ± 2.3 eggs were ovulated, but in the mice that had been repeatedly superovulated, only 5.3 ± 0.3 eggs were ovulated, a number that was significantly reduced. Therefore, it was revealed that repeated superovulation stimulation reduces the fertility of female mice.

Example 2: Examination of the effect of ergothioneine on the lengthening of the estrous cycle
Three-week-old immature C57BL6 mice were continuously administered 1.8 mg/kg body weight/day of ergothioneine in drinking water, and superovulation stimulation was performed six times every 10 days. The mice were subjected to vaginal cavity examination, and their estrous cycles were observed. As control mice, mice treated under the same conditions except for omitting administration of ergothioneine were also observed for their estrous cycles. Three mice were tested each with and without ergothioneine administration, and the mean ± standard error of each measured value was calculated.

Figure 5 shows the estrous cycle patterns of mice without ergothioneine administration (Figure 5A) and mice with ergothioneine administration (Figure 5B). Figure 6 shows the length of one estrous cycle of mice with and without ergothioneine administration. "*" in the figure indicates that the p value in Student's t-test was less than 0.05. In mice that underwent six repeated superovulation stimulations, the microestrus period continued and the estrous cycle was prolonged to 11 ± 0.7 days. On the other hand, when superovulation stimulation was repeated six times while ergothioneine was administered in drinking water, no prolongation of the estrous cycle was observed (4.9 ± 0.2 days).

Example 3: Examination of the effect of ergothioneine on decreased hormone sensitivity
Mice were continuously administered 1.8 mg/kg body weight/day of ergothioneine in drinking water, and were subjected to six superovulation stimulations every 10 days, and were further subjected to one superovulation stimulation (eCG and hCG administration). Then, 16 hours after hCG administration, the oviducts were collected and the number of ovulated eggs was measured. For comparison, the number of ovulated eggs was measured for mice treated under the same conditions except that ergothioneine administration was omitted. In addition, the number of ovulated eggs was similarly measured for 3-month-old mature female mice as control mice. The number of control mice and mice without and with ergothioneine administration was five each, and the mean value ± standard error of each measurement was calculated.

Figure 7 shows the number of ovulated eggs in control mice and mice that underwent repeated superovulation stimulation without and with ergothioneine administration. In the figure, "*" and "**" indicate that the p-values in the Student's t-test were less than 0.05 and less than 0.01, respectively. In mice that underwent six repeated superovulation stimulations, the number of ovulations was significantly reduced compared to control mice (9±1.7 ovulations). On the other hand, in mice that underwent repeated superovulation stimulation and were administered ergothioneine, the number of ovulations increased dramatically. The number of ovulations in control mice, mice without ergothioneine administration, and mice with ergothioneine administration was 23±1.2, 9±1.7, and 30±2.6, respectively. Therefore, it was revealed that long-term administration of ergothioneine in drinking water has the effect of preventing the decline in fertility caused by repeated superovulation stimulation.

Example 4: Examination of the timing of administration of ergothioneine
To examine the effect of ergothioneine on young mice, 1.8 mg/kg body weight/day of ergothioneine was administered in drinking water at the same time as eCG administration (Test Group 1) and 2 days before (Test Group 2). Figure 8 shows an outline of ergothioneine administration in drinking water in Test Group 1 and Test Group 2. 48 hours (2 days) after eCG administration, hCG was administered, 16 hours later the oviducts were collected and the number of ovulated eggs was measured. The number of ovulated eggs was also measured for control mice of the same age that were not administered ergothioneine. The number of control mice and mice in Test Group 1 and Test Group 2 was 5 mice each, and the average value ± standard error of each measured value was calculated.

Figure 9 shows the number of eggs ovulated in the control mice and in the mice in test groups 1 and 2. The "*" in the figure indicates that the p-value in the Student's t-test was less than 0.05. It was revealed that the number of ovulations was significantly increased by administering ergothioneine in drinking water starting two days before eCG administration.

Example 5: Examination of optimal concentration of ergothioneine
To investigate the optimal concentration of ergothioneine, 0.09, 0.18, 0.36, 0.9, and 1.8 mg/kg body weight/day of ergothioneine was administered in drinking water two days before eCG administration, and hCG was administered 48 hours later. The oviducts were collected from each mouse 16 hours after hCG administration, and the number of ovulated eggs was measured. In addition, the number of ovulated eggs was measured for control mice of the same age that were not administered ergothioneine. The number of control mice and mice administered ergothioneine at each dose was three each, and the average value ± standard error of each measurement value was calculated.

Figure 10 shows the number of eggs ovulated in control mice and mice administered ergothioneine at various doses. In the figure, "*" indicates that the p value in the Student's t-test was less than 0.05. The number of ovulations increased depending on the amount of ergothioneine administered, with the highest number of ovulations observed at a dose of 1.8 mg/kg body weight/day.

Example 6: Study of the effect of ergothioneine on mouse litter size
To clarify the effect of ergothioneine on the number of offspring, 3-month-old female mice were bred with males for 3 months under conditions where 1.8 mg/kg body weight/day of ergothioneine was administered in drinking water. The number of offspring per birth was measured during that period. As control mice of the same age were treated in the same way except that ergothioneine was not administered, and the number of offspring per birth was measured. Four control mice and four mice administered ergothioneine were tested, and the mean value ± standard error of each measurement was calculated.

Figure 11 shows the average number of offspring per birth for control mice and mice administered ergothioneine. In the figure, "*" indicates that the p-value in Student's t-test was less than 0.05. The number of offspring born was significantly increased by administering ergothioneine to drinking water. In other words, it was revealed that ergothioneine has the effect of improving fertility by increasing the number of ovulations in females.

Example 7: Study of the effect of ergothioneine on pig fertility and days to estrus return
Thirteen parous sows (excluding primiparous ones) were fed Tamogitake powder so that their ergothioneine intake was 0.36 mg/kg body weight/day from weaning to mating (Test group 3). The number of days to estrus recurrence (number of days from weaning to estrus) and the number of offspring at the first birth after Tamogitake intake were counted for each individual. In addition, the number of days to estrus recurrence and the number of offspring were counted for each individual in the control group that did not receive Tamogitake.

The number of pigs, number of days to estrus recurrence, and number of offspring in the test group 3 and the control group are shown in Table 1. The average number of days to estrus recurrence and number of offspring were calculated for each group ± standard deviation. Note that there were two pigs in the control group whose number of days to estrus recurrence exceeded 50 days, but these were not included in the calculation of the average number of days to estrus recurrence.

As shown in Table 1, in test group 3 where Tamogitake mushroom was administered, the number of offspring born increased by about one on average compared to the control group. In addition, a decrease in the number of days until estrus recurred was also observed. From the above results, it was found that ergothioneine also has a fertility-enhancing effect in pigs. It was also found that ergothioneine has an effect of decreasing the number of days until estrus recurred.

Claims (7)

A food or feed for stabilizing or shortening the estrus cycle of a female mammal , comprising ergothioneine. A feed containing ergothioneine for increasing the number of offspring per birth in female mammalian prolific animals. 3. The food according to claim 1 or the feed according to claim 1 or 2 , wherein the intake of ergothioneine is 0.18 mg/kg body weight/day or more. A method for stabilizing or shortening the estrous cycle of a female non-human mammal, comprising administering ergothioneine to the female non-human mammal . 5. The method of claim 4 , wherein the non-human mammalian female has an intake of ergothioneine of 0.18 mg/kg body weight/day or more. A method for increasing the number of offspring per birth in a female mammalian animal that is capable of multiparity, comprising administering ergothioneine to the female mammalian animal that is capable of multiparity . 7. The method of claim 6, wherein the intake of ergothioneine by the female of the mammalian multiparous animal is at least 0.18 mg/kg body weight/day .

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