JP2015134725A - Therapeutic agent for infertility - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel therapeutic agent for infertility.SOLUTION: As a therapeutic agent for infertility to be used in ART treatment, provided are: an extract extracted from a fruit of ume (Prunus mume) by using a hydrophilic solvent such as methanol and ethyl acetate; and concentrate of ume vinegar obtained by pickling a fruit of ume in salt, concentrate of ume vinegar obtained by desalinating ume vinegar, and further concentrate obtained by neutralizing the concentrate of ume vinegar. The active ingredient in these is 3,4-dihydroxybenzaldehyde having estradiol production action. Also, the concentrate of ume fruit and dehydroepiandrosterone are combined to be used in ART treatment.

Description

  The present invention relates to an infertility treatment agent.

  In general, infertility is diagnosed when sexual intercourse does not occur for more than two years without contraception. It is said that 10-15% of couples suffer from infertility. Infertility has many factors on both the female and male sides. Factors such as ovulation disorders or endocrine abnormalities and endometriosis are considered as the main factors on the female side. In early stages of infertility treatment, ovulation inducers and endometriosis treatment agents are often used. However, these treatments do not always give birth and there is a need for more effective infertility treatments.

  Under such circumstances, assisted reproductive technology (ART) has begun to spread. ART includes therapies such as in vitro fertilization (IVF), microinsemination (ICSI), and frozen embryo transfer. In ART, it is very important to secure good quality eggs.

  However, as the number of years of infertility treatment increases, the decrease in Ovarian reserve progresses and the responsiveness to hMG (human menopausal gonadotropin) therapy also decreases, making it increasingly difficult to treat.

  DHEA (dehydroepiandrosterone), which is also used as a supplement preparation, is administered to a low responder whose responsiveness to hMG therapy has decreased. DHEA is a sex hormone secreted from the adrenal gland and is a precursor of testosterone and estrogen and has an arteriosclerosis inhibitory action, an anti-obesity effect, and an anti-aging action. There are also reports (Non-Patent Documents 2 and 3) that the ovarian reactivity to gonadotropins is increased to improve the quality of the ovum.

  On the other hand, since it is considered that the decrease in egg quality is largely due to oxidative stress (Non-Patent Document 4), antioxidants are expected to have an effect of improving egg quality (Non-Patent Document 4). Literature 5, 6). Furthermore, there is a report that egg aging is a result of oxidative stress being involved in apoptosis of granulosa cells (Non-patent Documents 7 and 8).

  By the way, plum fruit is a natural material that has been edible for a long time, and ume vinegar obtained by salting plum fruit is also edible. In Patent Document 1 and Non-Patent Document 1, when desalted and concentrated ume vinegar obtained by desalting and concentrating a large amount of by-product ume vinegar during the production of umeboshi is fed to chickens together with feed, egg production rate and egg quality (egg The amount, how unit, egg yolk, amino acid composition of egg protein, etc.) have been improved, and chicken immunity has been shown to improve. In addition, Patent Document 2 shows that plum fruit seeds have an effect on physiological irregularities, and Patent Document 3 shows that ume meat extract has an effect on coldness. Furthermore, it is a well-known fact that plum has an antioxidant effect (Non-patent Document 9).

  However, so far, it has not been known that plum fruit or its extract is effective for infertility treatment, and any of the ingredients contained in plum fruit or its extract is the egg-laying rate. It was also unclear whether it contributed to the improvement of egg quality and egg quality.

JP-A-2005-73651 JP 2002-154980 A JP 2006-348001 A

Tetsuya Itami et al., Effects of desalted concentrated ume vinegar on egg production and egg quality in chickens, Journal of the Japanese Poultry Society, 42, 209-216, 2005 Barad, D., Gleicher, N., Increased oocyte Production after treatment with dehydroepiandrosterone., Fertil. Steril., 84, 756, 2005 Casson, PR. Et al., Dehydroepiandrosterone supplementation augments ovarian stimulation in poor responders: a case series., Hum Replod., 15, 2129-2132, 2000 Utsunomiya, H. et al., Fruit-juice concentrate of Asian plum inhibits growth signals of vascular smooth muscle cells induced by angiotensin II., Life Sciences, 72, 659-667, 2002 Devine, PJ. Et al., Roles of reactive oxygen species and antioxidants in ovarian toxicity., Biol Replod., 86, 1-10, 2011 Juan J. Tarin, et al., Oral antioxidants counteract the negative effects of female aging on oocyte quantity and quality in the mouse., Mol reprod and devel, 61, 385-397, 2002 Tatone C et al., Cellular and molecular aspects of ovarian follicle ageing., Hum Reprod Update., 14, 131-42, 2008 Wiener-Megnazi Z et al., Oxidative stress indices in follicular fluid as measured by the thermochemiluminescence assay correlate with outcome parameters in in vitro fertilization., Fertil Steril., 3, 1171-6, 2004 Nakahata, T. et al., Sphingosine-1-phosphate inhibits H2O2-induced granulasa cell apoptosis via the PI3K / Akt signaking pathway, Fertil. Steril., 98, 1001-1008, 2012

  This invention is made | formed in view of the said background art, Comprising: The subject of this invention is providing a new fertility treatment agent.

  The infertility treatment agent according to the present invention is characterized by comprising an extract of plum fruit, for example, a concentrate of plum vinegar, as an active ingredient, and further in combination with dehydroepiandrosterone.

  Taking the fertility treatment agent according to the present invention improves the pregnancy rate.

FIG. 1 is a diagram showing a method for isolating active ingredients. FIG. 2 is a ¹ H-NMR spectrum of Compound 1. FIG. 3 is a graph showing the estradiol secretion promoting effect of 3,4-DHBA. FIG. 4 is a chart showing an analysis result by a high performance liquid chromatogram of desalted and concentrated plum vinegar (plum BX70S).

  The infertility treatment agent according to the present invention comprises an extract of plum fruit as an active ingredient, and is administered alone or in combination with DHEA.

  The fruit of the plum used as an extraction raw material should just be the fruit of the plum (Prunus mume) of the Rosaceae genus Sakura, and the kind is not ask | required. The part used for extraction can be the plum flesh, the seed of the plum, or the whole plum. A pulverized product is preferably used for extraction.

  Various solvents are used as the extraction solvent. The extraction solvent is, for example, water, an organic solvent, or a mixed solvent thereof. Organic solvents include aliphatic hydrocarbons such as pentane, hexane, and octane, aromatic hydrocarbons such as benzene, toluene, and xylene, halogenated hydrocarbons such as chloroform and dichloroethane, methanol, ethanol, isopropanol, and 1,2. -Alcohols such as propanediol and glycerol; phenols such as phenol; ethers such as diethyl ether and diisopropyl ether; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate, ethyl acetate and methyl propionate. Since the active ingredient according to the present invention is considered to be 3,4-DHBA, a solvent from which a large amount of the ingredient is extracted is preferable, and the extraction solvent is preferably a hydrophilic solvent such as methanol or ethyl acetate.

  The extraction method is not particularly limited, and is generally carried out by leaving the plum fruit as it is, or by taking out the pulp part and / or seed part of the plum and immersing it in a solvent. It may be heated, stirred or pressurized as necessary during the immersion. Further, a method of performing extraction with a medium placed in a supercritical state, for example, a supercritical extraction method may be used.

  The extract according to the invention can also be plum vinegar. Plum vinegar is a leachate obtained by high osmotic pressure generated by salting. Since this leachate contains various components contained in plum fruit, this specification also defines this plum vinegar as an extract of plum fruit.

  Extracts extracted with an extraction solvent and ume vinegar are used as they are, but liquid-liquid extraction and concentration are further performed using various solvents, and 3,4-DHBA (3,4-Dihydroxybenzaldehyde: 3, 4) which is an active ingredient. An extract fraction containing a large amount of (dihydroxybenzaldehyde) may be obtained. Further, when ume vinegar is used, desalting is preferably performed in order to reduce the salt concentration. Desalting is performed using, for example, a reverse osmosis membrane or a dialysis membrane. For example, the desalting method described in Patent Document 1 can be used.

  The obtained extract is used as a fertility treatment. Infertility treatment agents are used as a method of infertility treatment. Since the extract of the present invention contains 3,4-DHBA having an estradiol production promoting action, and the active body in the extract is considered to be 3,4-DHBA, an ovulation inducer and a therapeutic agent for endometriosis Can be used as It is particularly effective for ART treatment and improves the properties of collected eggs. Moreover, the property of an egg is improved by administering in combination with DHEA, and a pregnancy rate is improved.

  The extract can be administered as is, but is usually formulated into various dosage forms by the use of additives. The dosage form may be, for example, a tablet, a granule, a powder, a capsule, a chewable, an extract, a liquid, or an injection. Additives are excipients such as starch and lactose, lubricants, disintegrants, coating agents, purified water, distilled water for injection, pH adjusters, storage One or two or more additives required for the dosage form can be used. In addition, when using ume vinegar, since the citric acid content is high, in order to make it easier to take, some or all of the acid may be neutralized by adding alkali to the obtained ume vinegar or its concentrate. is there. Sweeteners and fragrances may also be added. Examples of the method for neutralizing ume vinegar include the method described in JP2011-177037A.

  The dosage and administration method can be appropriately determined by those skilled in the art. Although it varies depending on the body weight, age, dosage form, and administration method of the recipient, it is 0.001 mg to 1 g per day for the extract using the extraction solvent, and 0.01 mg per day for the extract that is concentrated plum vinegar. ~ 100 g, and when administering 3,4-DHBA, it is 0.1 ng to 0.5 g per day.

  In the present invention, the combination of DHEA and 3,4-DHBA includes not only administering them simultaneously but also administering them at different times. The case of administration at different times includes, for example, when administration time is different in one day, when administration time is different by changing the day or week, and when administration time is different in the treatment cycle. The administration schedule can be appropriately determined by those skilled in the art in consideration of the effects and the like.

  When combined with DHEA, the dose ratio with DHEA can also be determined appropriately by those skilled in the art. The dose ratio varies depending on the body weight, age, dosage form, and administration method of the recipient, but the dose ratio (mass ratio with respect to DHEA) with the extract (in the case of an extract with an extraction solvent or ume vinegar) is 0.001 to 100. The administration ratio with 3,4-DHBA (mass ratio with respect to DHEA) is 0.000001-1.

  Next, the present invention will be specifically described based on the following examples, but the present invention is not limited to the following examples.

Desalted and concentrated ume vinegar was administered to subjects who did not obtain good results with ART treatment, and the influence on the pregnancy effect was examined.
1. Preparation of desalted and concentrated plum vinegar A desalted and concentrated plum vinegar was produced according to the method described in Example 1 of Patent Document 1. In other words, desalted and concentrated plum vinegar (hereinafter referred to as “plum BX70”) was obtained by electrodialysis and heating and concentrating the plum vinegar obtained by salting plum fruits. After this ume BX70 (acidity (as citric acid) 50.5 w / w%) was heated at 90 ° C. for 2 hours, a mixture of potassium hydroxide and sugar alcohol was added to neutralize part of the acid (sample acidity) 26.6 w / w%) (hereinafter referred to as “Plum BX70S”).

2. Improvement of infertility DHEA (dehydroepiandrosterone) at 50 mg / day every day in 16 subjects (33 to 43 years old, mean age 39.4 years old) who were unable to obtain good embryos by ART Administered. After DHEA was taken for 2 months, ovarian stimulation was performed and egg collection, IVF or ICSI was performed. Among the 16 cases described above, 6 cases where consent was obtained among the cases that did not result in pregnancy even after taking DHEA, after receiving DHEA 50 mg / day and the above-mentioned plum BX70S12 ml / day for 2 months, Ovarium stimulation was performed and egg collection, IVF or ICSI was performed. For the ovarian stimulation method, hMG was administered by the long method, the short method, or a GnRH antagonist. The treatment was carried out in the sequence of i) period in which ART was taken without taking anything, ii) period in which DEHA was taken, and iii) period in which DHEA and plum BX70S were used in combination (only 6 patients).

  Cycle of not taking anything and DHEA, DHEA and ume vinegar samples, serum estradiol (E2) value, egg collection number, fertilization rate, pregnancy rate (embryo sac) The clinical pregnancy was confirmed. Statistical analysis of the results of the study used Wilcoxon's test, with P <0.05 considered significant. Statistical evaluation was expressed as mean ± standard error.

  The results were as follows. The target period was 49 periods for IVF and 11 periods for ICSI enforcement, for a total of 60 periods. The average AMH (anti-Muellerian hormone) was 1.49 ng / ml.

  In the period when no medication was taken (non-addition period), 16 cases were subjected to IVF in 20 cycles and ICSI in 5 cycles. Pregnancy was only 1 case per cycle in 25 subjects. The DHEA cycle was performed for 16 cases with 17 IVF cycles and 4 ICSI cycles. Pregnancy was 7 cycles in 7 subjects with 21 cycles. DHEA and ume vinegar sample combined cycle (DHEA / ume BX70S cycle) was performed on 6 cases with 12 cycles of IVF and 2 cycles of ICSI. Pregnancy was established in 4 subjects out of 14 subjects.

  The average number of eggs collected was 7.36 ± 1.28 in the additive-free cycle, 6.52 ± 1.07 in the DHEA cycle, and 4.5 ± 0.73 in the DHEA / ume BX70S cycle, and the treatment cycle passed. Each time the number of eggs collected decreased.

  The average serum E2 value on the day before HCG administration was 2616.4 ± 658.9 pg / ml in the additive-free cycle, 2266.3 ± 467.3 pg / ml in the DHEA cycle, and 1490.7 ± 189.19 in the DHEA / ume BX70S cycle. As with the number of eggs collected, 2 pg / ml, it decreased as the treatment progressed.

  The average fertilization rate was 43.1 ± 9.8% in the additive-free cycle, 54.7 ± 7.29% in the DHEA cycle, and 83.0 ± 7.7% in the DHEA / ume BX70S cycle, which was higher than the cycle without the additive. The cycle, DHEA / Plum BX70S cycle was significantly higher.

  Regarding the pregnancy rate, the additive-free cycle was 4%, the DHEA cycle was 33.3%, the DHEA / ume BX70S cycle was 28.6%, and the DHEA cycle and DHEA / ume BX70S cycle were significantly higher than the additive-free cycle. It was.

  As described above, DHEA and ume vinegar (ume BX70S) were administered to severely infertile patients who did not become pregnant even after administration of DHEA, and the average number of eggs collected was the lowest among the three treatment groups. The highest fertilization rate was obtained, and pregnancy was established in 4 out of 6 cases. From this, it can be said that administration of DHEA and plum BX70S to refractory infertile patients improves egg quality and improves ART treatment results.

1. Isolation and identification of active ingredients using human ovarian granulosa cells (COV434) Next, the improvement effect of infertility was observed in desalted and concentrated plum vinegar, which is an extract of plum fruit. Isolation and identification of active ingredients presumed to be effective were performed. FIG. 1 shows a method for isolating active ingredients. Ovarian granulosa cells are cells that surround egg cells and contribute to follicular development by promoting follicular cavity formation and follicular fluid accumulation. At this time, human ovarian granulosa cells convert androgen that inhibits follicular development into estrogen by aromatase (aromatizing enzyme). As a result, the amount of estrogen secreted from the follicle increases, and a rapid transient release of LH (Luteinizing hormone) is induced from the anterior pituitary gland (LH surge). As a result, mature follicles ovulate. Thus, the androgen / estrogen conversion function of ovarian granulosa cells plays an important role in the normal ovulation of follicles, and when granulosa cell death (apoptosis) occurs during follicular development, the follicles can ovulate It is thought to regress without. Based on these findings, the active ingredient was isolated using the effect on the survival rate of human ovarian granulosa cells (COV434) as an index. The isolated component was subjected to various spectrum analysis (mass spectrum, ¹ H-NMR, ¹³ C-HMR), and the active component was identified as 3,4-Dihydroxybenzaldehyde (3,4-DHBA) (see FIG. 2). .

(Extraction from plum seed)
Plum fruit seeds were used for extraction because the active ingredient content is expected to be higher than plum fruit. 900 ml of methanol was added to 300 g of pulverized plum seeds, and the extraction operation was repeated for 8 hours using a reflux cooling apparatus. Liquid-liquid extraction was performed on the ethanol extract obtained by the extraction operation three times by the method shown in FIG. 1, and each fraction from Fr-1 to 14 was obtained. These fractions were examined for effects on COV434 viability.

(Measurement of COV434 survival rate)
As the cell line, human ovarian granulosa cells (COV434: DS Pharma Biomedical, Osaka, Jpn) were used. Cell culture medium contains 10% (v / v) fetal bovine serum (FBS; HyClone, Logan, UT, USA), 1% (v / v) penicillin / streptomycin (PS: Life Technologies, Carlsbad, CA USA) Dulbecco's modified Eagle's medium (DMEM: Life Technologies) was used. COV434 cells were cultured at 37 ° C. under 5% CO ₂ conditions.

The cultured COV434 cells were seeded at a density of 5 × 10 ⁴ cells / well in 96 well-plates and cultured for 3 days. After the culture, the culture solution was removed, and 100 μL of a culture solution in which each sample was added to the target concentration was added. Further, 100 μL of a culture solution added with DMSO (dimethyl sulfoxide: final concentration 0.1% (v / v)) as a control was added to other wells. After culturing the cells for 18 hours, the culture solution was removed, and 100 μL of a new culture solution was added. Next, MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; Promega, Madison, WI, USA ) Was added and cultured for 1 hour. Since MTS is reduced by living cells to become colored formazan, the absorbance at 490 nm was measured using a microplate reader. Cell viability was determined with the absorbance of the control as 100%. As a result, as shown in FIG.

2. Production effect of estradiol production by 3,4-DHBA COV434 cells were seeded on a 12-well plate at 5 × 10 ⁵ cells / well and cultured for 4 days. Next, the culture solution was removed, 0.5 mL of a culture solution added with 3,4-DHBA was added, and cultured for 18 hours, and the culture solution was collected. After centrifuging the culture solution at 10,000 rpm for 15 minutes, the supernatant was collected, and the concentration of estradiol contained in the culture solution was measured by the Japan Medical Laboratory (wakayama, Jpn). In order to correct the variation in the number of cells between wells, 0.5 mL of a new culture solution was added to the wells after the culture solution was collected, and 125 μL of MTS was further added, followed by incubation for 1 hour, and then the absorbance at 490 nm was measured. The cell viability was calculated from the absorbance obtained here, and the estradiol concentration was corrected with the calculated value. The results are shown relative to the control. The results are shown in FIG.

Next, when the active ingredient in ume vinegar was analyzed by high performance liquid chromatography under the following conditions, about 10 μg of 3,4-DHBA was confirmed in 1 ml of the ume BX70S. An example of the analysis result is shown in FIG. 3,4-DHBA showed absorption peaks at 230.5 nm, 278.5 nm, and 312.5 nm, but 3,4-DHBA could be measured by measuring absorption near 320 nm. In addition, about 4.8 μg of 3,4-DHBA was confirmed in the desalted plum vinegar before concentration.
(Analysis conditions)
Column: Shim-pack VP-ODS 4.6 x 150 mm (manufactured by Shimadzu Corporation)
Detection wavelength (UV): 320 nm
Injection volume: 10 μL
Mobile phase: (A) 0.1% formic acid-containing water, (B) 0.1% formic acid-containing methanol
A: B = 0-3 minutes 90:10
3-15 minutes 90: 10 → 60: 40
15-20 minutes 20:80
20-30 minutes 90:10

  From the above, it was found that the active ingredient contained in the extract of plum fruit is 3,4-DHBA, which is a substance that promotes the production of estradiol. So far, it is not known that 3,4-DHBA is contained in the extract of plum fruit and that it has an effect of promoting the production of estradiol. In particular, plum vinegar, which is an extract of plum fruit, It was concluded that it can be used as a fertility treatment. It has also been proved that combined administration of ume vinegar and DHEA, which is an extract of plum fruit, improves the pregnancy rate in ART therapy.

  The present invention provides a novel therapeutic agent for infertility.

Claims (7)

  An infertility treatment containing an extract of plum fruit as an active ingredient.   The infertility treatment agent according to claim 1, wherein the extract is an extract obtained by salting plum fruit.   The infertility treatment agent according to claim 2, wherein the extract is a concentrate obtained by desalting and extracting an extract obtained by salting plum fruit.   The infertility treatment agent according to claim 2, wherein the extract is a neutralized product of a concentrate obtained by desalting an extract obtained by salting plum fruit.   The fertility treatment agent according to claim 1, wherein the extract is an extract of plum seeds.   An infertility treatment agent comprising 3,4-dihydroxybenzaldehyde as an active ingredient.   The infertility therapeutic agent according to any one of claims 1 to 6, further comprising dehydroepiandrosterone in combination.

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