Methods to restore reproductive function

Abstract

A method for preventing or restoring age-related fertility loss in mammals by contacting mammalian germ cells with a composition containing a flavonoid glycoside, such as rutin and L-arginine. Also provided are a method for preventing or restoring age-related fertility loss, preventing or restoring age-related fertility decline, and reducing the risk of age-related fertility loss by administering the composition to a mammal. Furthermore, the use of the composition for preventing or restoring age-related fertility decline in mammals is disclosed.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 509,450, filed on June 21, 2023, and U.S. Provisional Application No. 63 / 549,296, filed on February 2, 2024. The entire contents of the said applications are incorporated herein by reference.

Background Art

[0002] In Japan, almost all dairy cows and beef cattle are bred by artificial insemination using frozen semen. In recent years, the fertilization rate of artificial insemination in cattle has been decreasing (Endo, J. Reprod. Dev. 2022; 68: 85 - 89). This leads to a lengthening of the non - pregnancy period and a decrease in productivity, which is an important problem directly affecting livestock farming profits. Investigating the conditions for more efficient artificial insemination or in vitro fertilization ("IVF") would be beneficial not only for livestock farming but also for the protection of some rare animals and the treatment of human infertility.

[0003] Spermatogenesis declines with age in humans, leading to infertility (Yen et al., Aging Cell 2021; 20: e13308; Virant - Klun et al., Antioxidants (Basel) 2022; 11: 1617). Reactive oxygen species ("ROS") have been reported to have an adverse effect on ejaculated sperm and fertilization (Rodriguez - Gonzalez et al., Age (Dordr) 2014; 36: 9721), and mature sperm in males are likely to become more vulnerable to ROS with aging.

[0004] The maturity of oocytes and eggs also declines with age, leading to infertility (Moghadam et al., JBRA Assist. Reprod. 2022; 26: 105 - 122). Studies on infertility in experimental animals have revealed that oxidative stress is a factor in infertility (Zargari et al., J, Xenobiot, 2022; 12: 214 - 222).

[0005] A recent study showed that including licorice extract containing flavonoid antioxidants in pre-sperm cultures for artificial insemination in mice improved fertilization rates (Tung et al., Biochem. Biophys. Res. Commun. 2015;467:447-450). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Endo,J.Reprod.Dev.2022;68:85-89

[0007] [Non-Patent Document 2] Yen et al.,Aging Cell 2021;20:e13308

[0008] [Non-Patent Document 3] Virant-Klun et al., Antioxidants (Basel)2022;11:1617

[0009] [Non-Patent Document 4] Rodriguez-Gonzalez et al.,Age(Dordr)2014;36:9721

[0010] [Non-Patent Document 5] Moghadam et al.,JBRA Assist.Reprod.2022;26:105-122

[0011] [Non-Patent Document 6] Zargari et al.,J,Xenobiot,2022;12:214-222

[0012] [Non-Patent Document 7] Tung et al.,Biochem.Biophys.Res.Commun.2015;467:447-450

[0013] There is a need to identify methods to improve fertilization rates in mammals such as livestock, as well as methods to prevent, restore, or reduce the risk of age-related decline in fertility. [Overview of the Initiative]

[0014] To satisfy the above needs, a method is provided for preventing or restoring age-related loss of reproductive function in mammals. This method is achieved by contacting mammalian germ cells with a composition containing a flavonoid glycoside, for example, rutin and L-arginine.

[0015] A second method for preventing or restoring age-related loss of reproductive function in mammals is disclosed. This method comprises administering the composition to a mammal in need.

[0016] In the third method, the same composition is administered to mammals to prevent or restore age-related decline in fertility.

[0017] A fourth method for reducing the risk of age-related loss of reproductive function in mammals by administering the above composition containing a flavonoid glycoside and L-arginine to mammals in need is also within the scope of the present invention.

[0018] Finally, the present invention encompasses the use of a composition containing a flavonoid glycoside and L-arginine to prevent or restore age-related decline in fertility in mammals.

[0019] Details of the present invention are described below. Other features, purposes, and advantages of the present invention will become apparent from the detailed description of some embodiments, the drawings, and the appended claims.

[0020] The following description relates to the accompanying drawings.

Brief Description of the Drawings

[0021] [Figure 1] Figure 1 is a bar graph showing the fertilization rate expressed as the percentage of 2-cell stage embryos formed after in vitro fertilization (“IVF”) of oocytes with sperm treated as shown. Asterisks indicate significant effects on the fertilization rate (n = 5) (P < 0.05).

[0022] [Figure 2] Figure 2 is a bar graph showing the fertilization rate expressed as the percentage of 2-cell stage embryos formed after in vitro fertilization (“IVF”) of oocytes with sperm treated with sodium ascorbate or eubioquercetin as shown. Asterisks indicate significant effects on the fertilization rate (n = 5) (*P < 0.05, **P < 0.01). N.D. = not determined.

[0023] [Figure 3A] Figure 3A is a bar graph of the pregnancy rate of female mice mated with aged male mice fed a standard diet or a standard diet supplemented with quercetin or eubioquercetin.

[0024] [Figure 3B] Figure 3B is a bar graph of the pregnancy rate of aged female mice mated with young male mice and fed a standard diet or a standard diet supplemented with quercetin or eubioquercetin.

[0025] [Figure 4A] Figure 4A is a bar graph of the pregnancy rate of female mice mated with aged male mice fed a standard diet or a standard diet supplemented with quercetin, rutin, or eubioquercetin.

[0026] [Figure 4B]Figure 4B is a bar graph showing the pregnancy rates of older female mice mated with young male mice and fed either a standard diet or a standard diet supplemented with quercetin, rutin, or ubiocercetin. [Modes for carrying out the invention]

[0027] As described in the summary section of the invention above, a method is provided for preventing or restoring age-related loss of reproductive function in mammals, the method comprising the step of contacting mammalian germ cells with a composition containing a flavonoid glycoside and L-arginine.

[0028] The contact process can be carried out in vitro or intracellularly. For example, the composition can be added to the conditioning medium used for sperm incubation during in vitro fertilization ("IVF") procedures. Alternatively, the composition can be ingested by mammals, i.e., administered orally.

[0029] The flavonoids in the composition may be, but are not limited to, flavonols such as 3-hydroxyflavone, azareatin, fisetin, galangin, goshipetin, kaemperia, kaemperool, isorhamnetin, morin, myricetin, natsudaidain, pachypodol, quercetin, rhamnadin, hesperetin, and ramnetin. In a particular composition, the flavonol is quercetin.

[0030] The flavonoid glycosides in the composition may be flavonol glycosides, such as astragalin, azalein, hyperoside, isoquercitrin, kaempperitrin, myricitrin, quercitrin, robinin, rutin, spireoside, xantramunin, amurensin, icariin, hesperidin, and troxerutinose. In certain examples, the composition contains rutin, i.e., quercetin rutinoside.

[0031] The amount of flavonoid glycosides in a composition is between 1% by weight and 60% by weight of the composition, for example, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%, or within any two of these percentage values. In a particular composition, flavonoid glycosides are present in an amount of 50% to 55% by weight of the composition.

[0032] As described above, the composition also contains L-arginine. L-arginine is present in the composition in amounts ranging from 0.5% to 40% by weight, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40%, or between any two of these percentage values. In one particular composition, L-arginine is present in amounts ranging from 30% to 35% by weight.

[0033] A preferred composition comprises 50% to 55% by weight of flavonoid glycosides and 30% to 35% by weight of L-arginine.

[0034] The above composition contains L-arginine in a weight less than the weight of the flavonoid glycosides. For example, L-arginine may be present in the composition in an amount of 60% by weight of the flavonoid glycosides. In certain embodiments, L-arginine may be present in the composition in an amount of 13-80% by weight, 47-73% by weight, or 52-60% by weight of the flavonoid glycosides.

[0035] The composition may contain pharmaceutically acceptable excipients and carriers, such as cellulose, carboxymethylcellulose, maltodextrin, starch, and hydrogenated starch. Preservatives such as sodium ascorbate, sodium gallate, glycine, tocopherol, and vitamin E may also be included in the composition. An exemplary composition for oral administration named ubiocercetin comprises 55% by weight of quercetin rutinoside, 30% by weight of L-arginine, 3% by weight of sodium ascorbate, and 12% by weight of hydrogenated starch.

[0036] In one embodiment, the composition is an oral formulation that may, but is not limited to, a liquid, capsule, tablet, pill, or gel. The composition may be a pharmaceutical, dietary supplement, natural health product, food, or beverage.

[0037] A second disclosed method is for preventing or restoring age-related loss of reproductive function in mammals. This method is characterized by the step of administering the composition to mammals in need, such as cattle, mice, and humans. In one method, the mammals are not suffering from diabetes, polycystic ovary syndrome, or cancer.

[0038] Mammals are at an age where reproductive function is declining (e.g., decreased spermatogenesis or sperm function in males, or decreased oocyte production or ovarian hormone production in females). See, for example, Liu K, J. Obstet. Gynaecol. Can. 2011, 33(11):1165-1175. In certain methods, the composition is administered to both male and female mammals of a mating pair.

[0039] The composition may be administered by any means known in the art. For example, the composition may be administered orally, intravenously, intranasally, by suppositories, or topically. In a particular method, the composition is administered orally. In another method, the composition is administered orally to mammals daily for a period of at least two months. This period may range from two to twelve months, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, and twelve months, or any two of these periods.

[0040] The composition administered by the second method is the same composition as described above for the first method. In a specific example of the second method, ubiocercetin, i.e., 55% by weight of quercetin rutinoside, 30% by weight of L-arginine, 3% by weight of sodium ascorbate, and 12% by weight of hydrogenated starch, is administered to a mammal.

[0041] As repeated from the summary section of the invention, the third method involves administering any of the above compositions to a mammal to prevent or restore age-related decline in fertility. Similar to the exemplary second method, the mammal in the third method is free from diabetes, polycystic ovary syndrome, or cancer.

[0042] In one specific example of the third method, the mammal is of an age at which reproductive function declines as defined above, or an age before reproductive function declines. In the specific method, the composition is administered to both the male and female mammals of the mating pair.

[0043] To reiterate, the composition may be administered by any means known in the art, for example, orally, intravenously, and topically. In a preferred method, the composition is administered orally to mammals daily for at least two months or for whichever period is described above.

[0044] The composition administered by the third method is the same composition as described above for the first and second methods, for example, ubiocercetin.

[0045] The present invention encompasses a fourth method for reducing the risk of age-related loss of reproductive function in mammals by administering the above-mentioned composition to the mammals. In this method, the mammals, for example, humans, do not exhibit a decline in reproductive function. In a particular method, the composition is administered to both male and female mammals of a mating pair. The composition can be administered by any means known in the art, for example, orally, intravenously, and topically. In a particular method, the composition is administered orally to the mammals daily for at least two months or for any of the above periods. The composition administered in the fourth method is the same composition as described above in the first to third methods, for example, ubiocercetin.

[0046] Finally, the present invention encompasses the use of any of the above compositions to prevent or restore age-related decline in fertility in mammals.

[0047] Without further detail, those skilled in the art will be able to make the most of the present invention based on the above description. Therefore, the following specific examples should be construed as merely illustrative and not in any way limiting the remainder of this disclosure. Publications referenced herein are incorporated in their entirety by reference.

[0048] Examples [Examples]

[0049] Example 1: Effect of flavonoids on in vitro fertilization rates Male BALB / cByJJcl mice were purchased from CREA Japan Co., Ltd. (Tokyo, Japan). Mature sperm from two epididymal tails per mouse (at least 8 months old) were suspended in 200 μl of albumin-free human fallopian tube fluid medium ("HTF", Life Global® medium, IVFonline, CT) coated with paraffin oil. After 5 minutes, the sperm suspension was transferred to sperm pre-incubation medium. HTF medium containing 1.0 mg / ml polyvinyl alcohol (PVA, Sigma, St. Louis, Missouri) and 1.0 mM methyl-β-cyclodextrin (MBCD, Sigma) was used as the pre-incubation medium. Chen et al. See al., Front.Pharmacol.2022;13:865376. For specific samples, rutin, quercetin, ascorbic acid, L-arginine, or ubiocercetin (quercetin-3-O-rutinoside 55%, L-arginine 30%, sodium ascorbate 3%, and hydrogenated starch 12%) was added to pre-incubation medium at a concentration of 0.02 μg / μl. 25 μl of sperm suspension was transferred to 25 μl of 2x condition medium and incubated at 37°C for 50 minutes in a humidified incubator containing 5% CO2 and 95% air. After incubation, sperm in 2–4 μl of condition medium were used for fertilization (final motile sperm concentration = 150 motile sperm / μl).

[0050] Eight-week-old female ICR mice were obtained from SLC Japan, Inc. (Shizuoka, Japan). Superovulation was induced in the female ICR mice by intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (Asuka Inc., Tokyo, Japan), followed by intraperitoneal injection of 5 IU of human chorionic gonadotropin (Asuka) 46-48 hours later. The mice were then euthanized 14-16 hours later. The ovaries, along with the fallopian tubes, were transferred to 30 mm dishes filled with paraffin oil (Nacalai Tesque Co., Ltd., Kyoto, Japan). Cumulus oophorus oocyte complexes were obtained from the ampulla of the fallopian tube and transferred under a stereomicroscope to dishes containing 200 μl of HTF medium droplets covered with paraffin oil. Five to seven cumulus oocyte clusters were transferred to 200 μl droplets of HTF medium containing 4% bovine serum albumin (Nacalai) coated with paraffin oil to form fertilization droplets. Sperm suspensions cultured in each conditional medium were transferred to separate fertilization droplets. Twenty-four hours after fertilization, the fertilization rate was determined by the percentage of two-cell stage embryos among all oocytes. Data were obtained from at least three independent treatment replicates. Results are expressed as mean ± standard deviation (SD) for each condition. Differences between experimental and control conditions were compared using Student's t-test, and p < 0.05 was considered statistically significant. The results are shown in Figure 1.

[0051] Of the treatments tested, ubiocercetin had the greatest effect on fertilization rates, resulting in a significantly higher 2-cell stage embryo formation rate of 65.8% compared to 24.4% in the control group. (See the first and last bars in Figure 1.) L-arginine alone did not have a significant effect on fertilization rates. Rutin, quercetin, and sodium ascorbate, on the other hand, each caused a significant increase in fertilization capacity, although not as high as ubiocercetin.

[0052] The effects of ubiocercetin and sodium ascorbate concentrations on fertilization ability were tested using the same IVF assay as above, by adding them to pre-incubation medium at concentrations ranging from 0.00063 μg / μl to 0.08 μg / μl. The results are shown in Figure 2. Sodium ascorbate alone did not have a significant effect on fertilization rate compared to the control until it reached a concentration of 0.01 μg / μl, and could not show a significant concentration-dependent increase in fertilization ability above 0.02 μg / μl. Ubiocercetin showed a significant increase in fertilization ability at concentrations of 0.02 μg / μl and above. Notably, 0.02 μg / μl of ubiocercetin is equivalent to a sodium ascorbate concentration of 0.00063 μg / μl, and at this concentration, sodium ascorbate alone had no effect in the IVF assay. See Figure 2. Ubiocercetin, which contains rutin, sodium ascorbate, and L-arginine, showed a significant increase in fertilization rates compared to rutin alone. Compare 65.8% and 39.1% in Figure 1 (see Figure 1). This result was surprising because L-arginine alone, and the concentrations of sodium ascorbate found in ubiocercetin, had no effect on fertilization rates. This data demonstrates a synergistic effect on fertilization rates of the combination of rutin, sodium ascorbate, and L-arginine. [Examples]

[0053] Example 2: Effect of flavonoids on in vitro fertilization rate The effects of oral administration of quercetin and ubiocercetin on fertilization ability in aged mice were evaluated. C57BL / 6 male mice (Tokyo Crea) and ICR female mice (Nippon SLC Co., Ltd.) were maintained under controlled temperature and lighting conditions throughout the experiment and were given free access to feed (500N Hi-Durability IRRD M / R, Nippon SLC) and water. Since the average daily food intake per mouse was approximately 5.0 g, a feed containing ubiocercetin was prepared by impregnating 5.0 g of standard feed with 0.06 ml of ubiocercetin aqueous solution (100 mg / ml). In the case of quercetin, quercetin powder was kneaded into moistened feed pellets, and then supplied after drying (quercetin 1.5 mg / 5 g feed). For all feeds, the average amount of feed that fell to the cage floor per mouse was 1.5 g, and the average amount of food consumed per mouse was 3.5 g (see Starr et al., J.Gerontol.A Biol.Sci.Med.Sci.2012;67:1043-1048). Each mouse received an average of 4.2 mg of ubiocercetin (containing 3 μmol of rutin) or 1.1 mg of quercetin (3 μmol) orally per day.

[0054] To test the ability of aged male mice to conceive in vivo, 30 pairs of male / female mice were identified that did not result in pregnancy after 2 months. Each pair of C57BL / 6 male mice over 1 year old was mated with two 8-week-old ICR female mice. Ten pairs of mice were fed either a standard diet, a standard diet supplemented with quercetin, or a standard diet supplemented with ubiocercetin for 2 months, and the number of pregnancies was recorded. The results are shown in Figure 3A. No pregnancies were observed in the 10 pairs of mice fed the standard diet or the standard diet supplemented with quercetin. In contrast, three pairs, or 30%, that were fed the ubiocercetin-containing diet became pregnant.

[0055] To test the ability of fertilization in aged female mice, two ICR female mice at least 8 months old were mated with 8-week-old C57BL / 6 male mice, and 30 pairs of males / females that did not produce offspring after 2 months were identified. As described above, each 10 pairs were fed standard diet, standard diet supplemented with quercetin, or standard diet supplemented with ubiocercetin, and the number of pregnancies was recorded. The results are shown in Figure 3B. No pregnancies were observed in the 10 pairs of mice fed standard diet or standard diet supplemented with quercetin. Of the 10 pairs fed ubiocercetin-containing diet, 3 pairs, or 30%, became pregnant.

[0056] The results indicate that oral administration of quercetin did not improve pregnancy rates. Surprisingly, oral administration of ubiocercetin improved pregnancy rates in both aged male and aged female mice. [Examples]

[0057] Example 3: Effects of high levels of flavonoids on in vitro fertilization rates The effects of high-dose oral administration of quercetin, rutin, and ubiocercetin on fertilization ability in aged mice were evaluated. C57BL / 6 male mice and ICR female mice were maintained and fed as described in Example 2. Feed containing ubiocercetin was prepared by mixing 6 mg of powdered ubiocercetin with 5 g of moist feed pellets, and then drying the pellets. For quercetin and rutin, feed pellets were similarly prepared with a ratio of 6 mg of quercetin / 5 g of pellets and 3 mg of rutin / 5 g of pellets. Based on the average daily intake of 3.5 g per mouse, it is estimated that each mouse orally ingested an average of 4.2 mg (12 μmol) of quercetin, 2.1 mg (3 μmol) of rutin, or 4.2 mg of ubiocercetin (containing 3 μmol of rutin) per day.

[0058] In essentially the same manner as described in Example 2, the in vivo fertilization ability of C57BL / 6 male mice over one year of age was evaluated. Forty pairs of male / female mice were identified that did not result in pregnancy after two months of mating with two eight-week-old ICR female mice. Each of the ten pairs of mice was fed either a standard diet, a standard diet supplemented with quercetin, a standard diet supplemented with rutin, or a standard diet supplemented with ubiocercetin for two months, and the number of pregnancies was recorded. The results are shown in Figure 4A. No pregnancies were observed in the ten pairs of mice fed the standard diet. Pregnancy occurred in only one pair (10%) given a high-dose quercetin-containing diet (12 μmol). Pregnancy also occurred in one pair (10%) given a rutin-containing diet (3 μmol). In contrast, pregnancy occurred in three pairs (30%) given a ubiocercetin-containing diet (containing 3 μmol of rutin).

[0059] First, the in vivo fertilization ability in aged female mice was tested by mating two ICR female mice, at least 8 months old, with 8-week-old C57BL / 6 male mice and identifying 40 male / female pairs that did not produce offspring after 2 months. As described above, each 10 pairs were fed standard diet, standard diet supplemented with quercetin, standard diet supplemented with rutin, or standard diet supplemented with ubiocercetin, and the number of pregnancies was recorded. The results are shown in Figure 4B. The pregnancy rate was 10% in mouse pairs fed standard diet, quercetin-supplemented diet, and rutin-supplemented diet. In mating pairs fed ubiocercetin-containing diet, the pregnancy rate was 40%.

[0060] The results show that oral administration of quercetin, even when the dose was increased fourfold from 3 μmol to 12 μmol, resulted in only a minimal improvement in pregnancy rates. The effectiveness of rutin alone was also minimal. In contrast, oral administration of ubiocercetin containing the same amount of rutin significantly improved pregnancy rates by 3 to 4 times in both aged male and aged female mice.

[0061] Other Embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise specified, each disclosed feature is merely an example of a general set of equivalent or similar features. Furthermore, from the above description, those skilled in the art can easily identify the essential features of the present invention and can make various changes and modifications to the invention to suit various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also within the scope of the claims.

Claims

1. A method for reducing, preventing, or restoring the risk of age-related loss of reproductive function in a mammal, comprising contacting the germ cells of the mammal with a composition containing a flavonoid glycoside and L-arginine.

2. The method according to claim 1, wherein the flavonoid glycoside is quercetin rutinoside.

3. The method according to claim 1, wherein the L-arginine is present in a weight less than the weight of the flavonoid glycoside.

4. The method according to claim 3, wherein the L-arginine is present in an amount equivalent to 60% w / w of the flavonoid glycoside.

5. A method for reducing, preventing, or restoring the risk of age-related loss of reproductive function in mammals, comprising administering a composition containing a flavonoid glycoside and L-arginine to a mammal in need thereof.

6. The method according to claim 5, wherein the mammal is not suffering from diabetes, polycystic ovary syndrome, or cancer.

7. The method according to claim 5, wherein the composition is administered orally on a daily basis for a period of at least two months, preferably two to twelve months.

8. The method according to claim 5, wherein the flavonoid glycoside is quercetin rutinoside.

9. The method according to claim 5, wherein the L-arginine is present in a weight less than the weight of the flavonoid glycoside.

10. The method according to claim 9, wherein the L-arginine is present in an amount equivalent to 60% w / w of the flavonoid glycoside.

11. A method for preventing or restoring age-related decline in fertility in mammals, comprising administering a composition containing a flavonoid glycoside and L-arginine to a mammal in need thereof.

12. The method according to claim 11, wherein the mammal is not suffering from diabetes, polycystic ovary syndrome, or cancer.

13. The method according to claim 11, wherein the composition is administered orally on a daily basis for at least two months.

14. The method according to claim 11, wherein the flavonoid glycoside is quercetin rutinoside.

15. The method according to claim 11, wherein the L-arginine is present in a weight less than the weight of the flavonoid glycoside.

16. The method according to claim 15, wherein the L-arginine is present in an amount equivalent to 60% w / w of the flavonoid glycoside.

17. The method according to any one of claims 1 to 16, wherein the flavonoid glycoside is present in the composition in an amount of 1% to 60% by weight, and the L-arginine is present in the composition in an amount of 0.5% to 40% by weight.

18. The method according to claim 17, wherein the flavonoid glycoside is present in 50% to 55% by weight of the composition, and the L-arginine is present in 30% to 35% by weight of the composition.

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