Method of regaining reproductive function

Abstract

A method for preventing or restoring age-related loss of reproductive function in a mammal by contacting reproductive cells of the mammal with a composition that contains a flavonoid glycoside, e.g., rutin, and L-arginine. Also provided are methods for preventing or restoring age-related loss of reproductive function, for preventing or restoring age-related decline of fertility, and for reducing the risk of age-related loss of reproductive function by administering the composition to a mammal. Further disclosed is use of the composition for preventing or restoring age-related decline of fertility in a mammal.

Description

METHOD OF REGAINING REPRODUCTIVE FUNCTION

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to US Provisional Application Nos. 63 / 509,450 filed June 21, 2023 and 63 / 549,296 filed February 2, 2024. The entire contents of said applications are incorporated herein by reference.Background

[0002] In Japan, nearly all dairy and beef cattle are bred via artificial insemination using frozen semen. The fertility rate of artificial insemination of cattle has been decreasing in recent years (Endo, J. Reprod. Dev. 2022; 68:85-89). This is leading to prolonged periods without pregnancies, lowered productivity, and is a key issue that directly affects husbandry profits. Investigation of conditions for more efficient artificial insemination or in vitro fertilization (“IVF”) would be beneficial not only for husbandry but also for conserving several rare animals and for human infertility treatment.

[0003] Spermatogenesis reduces with age in humans, resulting in infertility (Yen et al., Aging Cell 2021; 20:e13308; Virant-Klun et al., Antioxidants (Basel) 2022; 11:1617.). Although reactive oxygen species (“ROS”) have been reported to adversely affect ejaculated sperm and fertilization (Rodriguez-Gonzalez et al., Age (Dordr) 2014; 36:9721), it is likely that mature sperm in aging males are more vulnerable to ROS.

[0004] Oocyte and ova maturity also decline with age, leading to infertility (Moghadam et al., JBRA Assist. Reprod. 2022; 26:105-122). Research on infertility in experimental animals has revealed that oxidative stress is a factor in infertility (Zargari et al., J, Xenobiot, 2022; 12:214-222).

[0005] Licorice extracts, which contain flavonoid antioxidants, have recently been shown to improve fertilization rates by including them in sperm pre-cultures for artificial insemination of mice (Tung et al., Biochem. Biophys. Res. Commun. 2015; 467:447-450).

[0006] The need exists to identify methods to improve fertilization rates in mammals such as livestock as well as for preventing, restoring, or reducing the risk of age-related decline in fertility.Summary

[0007] To meet the need set forth above, a method is provided for preventing or restoring age-related loss of reproductive function in a mammal. The method is accomplished by contacting reproductive cells of the mammal with a composition that contains a flavonoid glycoside, e.g., rutin, and L-arginine.

[0008] A second method is disclosed for preventing or restoring age-related loss of reproductive function in a mammal. The method includes administering the composition described above to a mammal in need thereof.

[0009] The same composition is also administered to a mammal for preventing or restoring age-related decline of fertility in a third method.

[0010] Also within the scope of the invention is a fourth method that is for reducing the risk of age-related loss of reproductive function in a mammal by administering the composition mentioned above that contains a flavonoid glycoside and L-arginine to a mammal in need thereof.

[0011] Finally, the invention encompasses use of the composition that contains a flavonoid glycoside and L-arginine for preventing or restoring age-related decline of fertility in a mammal.

[0012] The details of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the detailed description of several embodiments, from the drawings, and also from the appending claims.

[0013] The description below refers to the accompanying drawings, of which:Fig. 1 is a bar graph showing fertility rates expressed as percent 2-cell embryos formed after in-vitro fertilization (“IVF”) of ova with sperm treated as shown. Asterisks indicate a significant effect on fertility rate (n=5), (P < 0.05).Fig. 2 is a bar graph showing fertility rates expressed as percent 2-cell embryos formed after in-vitro fertilization (“IVF”) of ova with sperm treated with sodium ascorbate or eubioquercetin as shown. Asterisks indicate a significant effect on fertility rate (n=5), (*P < 0.05, ** P < 0.01). N.D. = not determined.Fig. 3A is a bar graph of the pregnancy rate of female mice mated with older male mice fed a standard diet or a standard diet supplemented with quercetin or eubioquercetin.Fig. 3B is a bar graph of pregnancy rates for older female mice fed a standard diet or a standard diet supplemented with quercetin or eubioquercetin mated with young male mice.Fig. 4A is a bar graph of the pregnancy rate of female mice mated with older male mice fed a standard diet or a standard diet supplemented with quercetin, rutin, or eubioquercetin.Fig. 4B is a bar graph of the pregnancy rate for older female mice fed a standard diet or a standard diet supplemented with quercetin, rutin, or eubioquercetin, mated with young male mice.DETAILED DESCRIPTION

[0014] As set forth in the SUMMARY section above, a method for preventing or restoring age-related loss of reproductive function in a mammal is provided that features a step of contacting reproductive cells of the mammal with a composition that contains a flavonoid glycoside and L-arginine.

[0015] The contacting step can be carried out in vitro or in vivo. For example, the composition can be added to a conditioned medium used for incubation of sperm during an in vitro fertilization (“IVF”) procedure. Alternatively, the composition can be fed, i.e., orally administered, to a mammal.

[0016] The flavonoid in the composition can be, but is not limited to, a flavonol, for example, 3-hydroxyflavone, azaleatin, fisetin, galangin, gossypetin, kaempferide, kaempferol, isorhamnetin, morin, myricetin, natsudaidain, pachypodol, quercetin, rhamnazin, hesperetin, and rhamnetin. In a specific composition, the flavonol is quercetin.

[0017] The flavonoid glycoside in the composition can be a flavonol glycoside, e.g., astragalin, azalein, hyperoside, isoquercitrin, kaempferitrin, myricitrin, quercitrin, robinin, rutin, spiraeoside, xanthorhamnin, amurensin, icariin, hesperidin, and troxerutinose. In a specific example, the composition includes rutin, i.e., quercetin rutinoside.

[0018] The amount of flavonoid glycoside in the composition is 1% to 60% by weight of the composition, e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60% by weight, or a range falling between any two of these percentage values. In a particular composition, the flavonoid glycoside is present at 50% to 55% by weight of the composition.

[0019] As mentioned above, the composition also includes L-arginine. The L-arginine is present in the composition at 0.5% to 40% by weight of the composition e.g., 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40% weight, or a range falling between any two of these percentage values. In one specific composition, the L-arginine is present at 30% to 35% by weight of the composition.

[0020] A preferred composition includes a flavonoid glycoside at 50% to 55% by weight of the composition and L-arginine at 30% to 35% by weight of the composition.

[0021] The above-described composition includes L-arginine in an amount by weight less than the amount by weight of the flavonoid glycoside. For example, the L-arginine can be present in the composition in an amount 60% by weight of the flavonoid glycoside. In certain embodiments, the L-arginine is present in the composition at 13-80%, 47-73%, or 52-60% by weight of the flavonoid glycoside.

[0022] The composition can include other components such as pharmaceutically acceptable excipients and carriers, e.g., cellulose, carboxy methyl cellulose, maltodextrin, starch, and hydrogenated starch. Preservatives, such as sodium ascorbate, sodium gallate, glycine, tocopherol, and vitamin E, can also be included in the composition. An exemplary composition for oral administration, designated as eubioquercetin, includes, by weight, 55% quercetin rutinoside, 30% L-arginine, 3% sodium ascorbate, and 12% hydrogenated starch.

[0023] In one embodiment, the composition is in an oral formulation that can be, but is not limited to, a liquid, a capsule, a tablet, a pill, and a gel. The composition can be a pharmaceutical drug, a dietary supplement, a natural health product, a food product, or a beverage.

[0024] The second disclosed method is for preventing or restoring age-related loss of reproductive function in a mammal. This method features a step of administering the composition described above to a mammal, e.g., a bovine, a mouse, and a human, in need thereof. In one method, the mammal does not suffer from diabetes, polycystic ovary syndrome, or cancer.

[0025] The mammal is at an age where reproductive function has declined, for example, a decline in spermatogenesis or sperm function in a male or a decline in ovum production or ovarian hormone production in females. See, e.g., Liu K, J. Obstet. Gynaecol. Can. 2011, 33(11):1165-1175. In a particular method, both the male and female mammal in a mating pair is administered with the composition.

[0026] Administering the composition can be carried out by any means known in the art. For example, the composition can be administered orally, intravenously, intranasally, by suppository, or topically. In a particular method, the composition is administered orally. In another method, the composition is orally administered to the mammal daily for a period of at least two months. The period can be 2 months to 12 months, e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months, or a range falling between any two of these time periods.

[0027] The composition to be administered in the second method is the same composition described above for the first method. In a specific example of the second method, eubioquercetin, i.e., 55% quercetin rutinoside, 30% L-arginine, 3% sodium ascorbate, and 12% hydrogenated starch by weight is administered to the mammal.

[0028] To repeat from the SUMMARY section, in a third method, any of the compositions set forth, supra, can be administered to a mammal for preventing or restoring age-related decline of fertility. Like an exemplary second method, the mammal in the third method does not suffer from diabetes, polycystic ovary syndrome, or cancer.

[0029] In one particular example of the third method, the mammal is at an age where reproductive function has declined as defined above. Alternatively, the mammal is at an age before any decline in reproductive function has occurred. In a specific method, both the male and female mammal in a mating pair is administered with the composition.

[0030] To reiterate, administering the composition can be carried out by any means known in the art, e.g., orally, intravenously, and topically. In a preferred method, the composition is orally administered to the mammal daily for a period of at least two months or for any of the time periods mentioned above.

[0031] The composition to be administered in the third method is the same composition described above for the first and second methods, for example, eubioquercetin.

[0032] The invention encompasses a fourth method for reducing the risk of age-related loss of reproductive function in a mammal by administering the composition mentioned above to a mammal. In this method, the mammal, a human for example, does not display any decline in reproductive function. In a particular method, both the male and female mammal in a mating pair is administered with the composition. The composition can be administered by means known in the art such as orally, intravenously, and topically. In a particular method, the composition is orally administered to the mammal daily for a period of at least two months or for any of the time periods mentioned above. The composition to be administered in the fourth method is the same composition described above for the first through third methods, for example, eubioquercetin.

[0033] Finally, the invention encompasses use of any of the compositions described above for preventing or restoring age-related decline of fertility in a mammal.

[0034] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. The publications cited herein are incorporated by reference in their entirety. ExamplesExample 1

[0035] Example 1: Effect of flavonoids on in vitro fertilization rates Male BALB / cByJJcl mice were purchased from CLEA Japan (Tokyo, Japan). Mature sperm from two cauda epididymides per mouse (age at least 8 months) were suspended in 200 μl of albumin-free human tubal fluid medium (“HTF”; Life Global (Registered Trademark) media, IVFonline, CT, covered with paraffin oil. After 5 min., the sperm suspension was transferred to sperm pre-incubation medium. HTF medium containing 1.0 mg / ml polyvinyl alcohol (PVA; Sigma, St. Louis, MO) and 1.0 mM methyl-beta-cyclodextrin (MBCD; Sigma) was used as the pre-incubation medium. See Chen et al., Front. Pharmacol. 2022; 13:865376. In certain samples, rutin, quercetin, ascorbic acid, L-arginine, or eubioquercetin (quercetin-3-O-rutinoside 55%, L-arginine 30%, sodium ascorbate 3%, and hydrogenated starch 12%) was added to the pre-incubation medium at a concentration of 0.02 μg / μl. 25 μl sperm suspension was transferred into 25 μl 2X conditioned medium and incubated for 50 min. at 37°C in a humidified incubator with 5% CO2and 95% air. After the incubation, 2-4 μl of sperm in conditioned medium was used for insemination (final motile sperm concentration = 150 motile sperm / μl.

[0036] Eight-week-old female ICR mice were acquired from SLC Japan, Inc. (Shizuoka, Japan). The female ICR mice were super-ovulated by an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Asuka Inc., Tokyo, Japan), followed 46-48 h later by 5 IU human chorionic gonadotropin (Asuka), and euthanized 14-16 h thereafter. Ovaries with oviducts were transferred to a 30 mm dish filled with paraffin oil (Nacalai Tesque Inc., Kyoto, Japan). Each cumulus-oocyte complex was acquired from the ampulla of a fallopian tube and transferred to a dish containing a 200 μl drop of HTF medium covered with paraffin oil under a stereomicroscope. Five to seven cumulus-oocyte masses were transferred into 200 μl drops of HTF medium with 4% bovine serum albumin (Nacalai) covered with paraffin oil to form an insemination drop. The sperm suspension cultured in each conditioned medium were each transferred to separate insemination drops. At 24 h post insemination, the proportion of 2-cell stage embryos among all oocytes was determined as a measure of the fertilization rate. Data were obtained from at least three independent treatment repetitions. The results were expressed as the mean ± standard deviation (SD) under each condition. Differences between the experimental and control conditions were compared using the Student’s t-test and were considered statistically significant at p < 0.05. The results are shown in Fig. 1.

[0037] Among the treatments tested, eubioquercetin had the greatest effect on the fertilization rate, leading to the formation of as high as 65.8% of 2-cell embryos, compared to 24.4% for the control. See Fig. 1, first and last bars. L-arginine alone did not significantly affect the fertilization rate. On the other hand, rutin, quercetin, and sodium ascorbate each caused a significant increase in fertility, although not as high as eubioquercetin.

[0038] The effect of eubioquercetin and sodium ascorbate concentration on fertility was tested in the same IVF assay described above by adding them to the pre-incubation medium at a concentration between 0.00063 μg / μl to 0.08 μg / μl. The results are shown in Fig. 2. Sodium ascorbate alone had no significant effect on fertility rates compared to control until reaching a concentration of 0.01 μg / μl and failed to show a significant concentration-dependent increase in fertility above 0.02 μg / μl. Eubioquercetin showed a significant increase in fertility at concentrations of 0.02 μg / μl or higher. Of note, 0.02 μg / μl eubioquercetin has a concentration of sodium ascorbate of 0.00063 μg / μl, a concentration at which sodium ascorbate alone has no effect in the IVF assay. See Fig. 2. Eubioquercetin, which includes sodium ascorbate and L-arginine in addition to rutin, showed a significant increase in fertility rate compared to rutin alone. Compare 65.8% to 39.1% in Fig. 1 (see Fig. 1), This result was surprising, as L-arginine alone and sodium ascorbate at the concentration found in eubioquercetin, had no effect on fertility rate. This data points to a synergistic effect on fertility rate of the combination of rutin, sodium ascorbate, and L-arginine.Example 2

[0039] Example 2: Effect of flavonoids on in vivo fertilization rates The effect of oral administration of quercetin and eubioquercetin on fertility in aged mice was assessed. C57BL / 6 male mice (CLEA, Tokyo) and ICR female mice (SLC Japan, Inc.) were maintained under controlled temperature and lighting conditions throughout the experiments and were provided with feed (500N Hi-Durability IRRD M / R, SLC Japan) and water ad libitum. As the feeding average of one mouse per day is approximately 5.0 g, the diet including eubioquercetin was prepared by impregnating 5.0 g of a standard diet with 0.06 ml of aqueous eubioquercetin solution (100 mg / ml). In the case of quercetin, quercetin powder was kneaded into the moistened feed pellets and then provided after drying (1.5 mg quercetin / 5 g of diet). For all diets, the average feed dropped on the floor of the cage per mouse was 1.5 g, leaving an average dietary intake per mouse of 3.5 g (see Starr et al., J. Gerontol. A Biol. Sci. Med. Sci. 2012; 67:1043-1048). Each mouse orally ingested an average of 4.2 mg eubioquercetin (containing 3 μmols of rutin) or 1.1 mg quercetin (3 μmols) per day.

[0040] To test in vivo fertility in aged male mice, C57BL / 6 male mice over 1 year of age were each crossed with two 8-week-old ICR female mice to identify 30 male / female pairs that did not produce a pregnancy for two months. Ten pairs of mice were each fed for two months with a standard diet, a standard diet with added quercetin, or a standard diet with added eubioquercetin and the number of pregnancies recorded. The results are shown in Fig. 3A. No pregnancies were observed in 10 pairs of mice fed with the standard diet or the standard diet supplemented with quercetin. By contrast, 3 pairs, i.e., 30%, fed a eubioquercetin-containing diet produced a pregnancy.

[0041] To test in vivo fertility in aged female mice, two ICR female mice at least 8 months of age were mated to 8-week-old C57BL / 6 male mice to identify 30 male / female pairs for which no offspring were generated for two months. As described above, 10 pairs were each fed a standard diet, a standard diet with added quercetin, or a standard diet with added eubioquercetin and the number of pregnancies recorded. The results are shown in Fig. 3B. No pregnancies were observed in 10 pairs of mice fed with the standard diet or the standard diet supplemented with quercetin. Out of the 10 pairs fed a eubioquercetin-containing diet, 3 pairs, i.e., 30%, produced a pregnancy.

[0042] The results indicate that oral administration of quercetin failed to improve pregnancy rates. Surprisingly, oral administration of eubioquercetin improves pregnancy rates in both aged male and aged female mice.Example 3

[0043] Example 3: Effect of higher levels of flavonoids on in vivo fertilization rates The effect of oral administration at elevated dosages of quercetin, rutin, and eubioquercetin on fertility in aged mice was assessed. C57BL / 6 male mice and ICR female mice were maintained and fed a diet as described above in Example 2. The diet including eubioquercetin was prepared by kneading 6 mg powdered eubioquercetin into 5 g of moistened feed pellets and then drying the pellets. In the case of quercetin and rutin, the feed pellets were prepared in same manner with the blending ratio of 6 mg quercetin / 5 g of pellets and 3 mg rutin / 5 g of pellets. Based on the average dietary intake per mouse of 3.5 g described above, it is estimated that each mouse orally ingested an average of 4.2 mg (12 μmols) quercetin, 2.1 mg (3 μmols) rutin, or 4.2 mg eubioquercetin (containing 3 μmols of rutin) per day.

[0044] In vivo fertility in C57BL / 6 male mice over 1 year of age was assessed essentially as discussed in Example 2. The male mice were each crossed with two 8-week-old ICR female mice to identify 40 male / female pairs that did not produce a pregnancy for two months. Ten pairs of mice were each fed for two months with a standard diet, a standard diet with added quercetin, a standard diet with added rutin, or a standard diet with added eubioquercetin, and the number of pregnancies recorded. The results are shown in Fig. 4A. No pregnancies were observed in 10 pairs of mice fed with the standard diet. Only one pair, i.e., 10%, fed an elevated dose of a quercetin-containing diet (12 μmols) produced a pregnancy. A single pair, again, 10%, fed a rutin -containing diet (3 μmols) produced a pregnancy. By contrast, 3 pairs, i.e., 30%, fed a eubioquercetin -containing diet (containing 3 μmols of rutin) produced a pregnancy.

[0045] In vivo fertility in aged female mice was tested by first mating two ICR female mice at least 8 months of age to 8-week-old C57BL / 6 male mice to identify 40 male / female pairs for which no offspring were generated for two months. As described above, 10 pairs were each fed a standard diet, a standard diet with added quercetin, a standard diet with added rutin, or a standard diet with added eubioquercetin, and the number of pregnancies recorded. The results are shown in Fig. 4B. The pregnancy rate in pairs of mice fed the standard diet, the quercetin-supplemented diet, and the rutin-supplemented diet was 10%. Among mating pairs fed a eubioquercetin-containing diet, the pregnancy rate was 40%.

[0046] The results indicate that oral administration of quercetin produces only a marginal improvement in pregnancy rates, even when the dosage was increased from 3 μmoles to 12 μmoles, i.e., 4-fold. The efficacy of rutin alone is also marginal. By contrast, oral administration of eubioquercetin containing the same amount of rutin greatly improves pregnancy rates by 3- to 4-fold in both aged male and aged female mice.OTHER EMBODIMENTS

[0047] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. Further, from the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

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

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

3. The method of claim 1, wherein the L-arginine is present in an amount by weight less than an amount by weight of the flavonoid glycoside.

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

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

6. The method of claim 5, wherein the mammal does not suffer from diabetes, polycystic ovary syndrome, or cancer.

7. The method of claim 5, wherein the composition is orally administered daily for a period of at least two months, preferably for 2 months to 12 months.

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

9. The method of claim 5, wherein the L-arginine is present in an amount by weight less than an amount by weight of the flavonoid glycoside.

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

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

12. The method of claim 11, wherein the mammal does not suffer from diabetes, polycystic ovary syndrome, or cancer.

13. The method of claim 11, wherein the composition is orally administered daily for at least two months.

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

15. The method of claim 11, wherein the L-arginine is present in an amount by weight less than an amount by weight of the flavonoid glycoside.

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

17. The method of any of the preceding claims, wherein the flavonoid glycoside is present at 1% to 60% by weight of the composition and the L-arginine is present at 0.5% to 40% by weight of the composition.

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

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