Prevention or treatment agent for pregnancy hypertension syndrome

Abstract

Provided is a formulation that can prevent or treat pregnancy hypertension syndrome. A prevention or treatment agent for pregnancy hypertension syndrome is prepared, said agent containing an effective amount of one or more substances selected from the group consisting of pyrroloquinoline quinone, salts of pyrroloquinoline quinone, apple polyphenols, black rice, and black rice extracts.

Description

Agent for preventing or treating hypertensive disorders in pregnancy

[0001] The present invention relates to an agent for preventing or treating hypertensive disorders in pregnancy.

[0002] Hypertensive disorders in pregnancy (HDP) occur in 5-10% of all pregnancies and are a serious disease that can cause maternal death, perinatal death (death of the fetus or newborn), and other maternal and fetal complications. That is, when hypertensive disorders in pregnancy become severe, there is a possibility of increased blood pressure, proteinuria, convulsive seizures (eclampsia), cerebral hemorrhage, and dysfunction of the liver and kidneys, which poses a danger to the mother and can be fatal in some cases. In addition, hypertensive disorders in pregnancy can cause serious symptoms such as fetal growth restriction, fetal dysfunction, and premature placental abruption not only in the mother but also in the fetus, and it is a serious disease for both the mother and the fetus.

[0003] The treatment method for hypertensive disorders in pregnancy mainly focuses on bed rest and hospitalization. There is no fundamental way to cure this disease, and early termination of pregnancy is considered one of the treatments. Early termination of pregnancy leads to the birth of low-birth-weight infants. In recent years, due to the advancement of neonatal intensive care units (NICUs), it has become possible to give birth to so-called extremely low-birth-weight infants weighing less than 1,000 g. However, in extremely low-birth-weight infants and low-birth-weight infants, the risk of various complications such as respiratory, circulatory, digestive, nervous systems, or future developmental disorders increases. That is, although it is to protect the mother and fetus from the serious disease of hypertensive disorders in pregnancy, early termination of pregnancy is never desirable.

[0004] On the other hand, antihypertensive drugs are generally used as drugs to lower blood pressure. However, in the "Clinical Practice Guidelines for Hypertensive Disorders in Pregnancy 2021", it is stated that "evidence regarding the safety and efficacy of antihypertensive drugs for the mother and fetus is limited". In addition, although methyldopa, labetalol, nifedipine, and hydralazine are recommended for hypertensive disorders in pregnancy in the above clinical practice guidelines, there are provisos regarding side effects and administration methods, and careful administration is required.

[0005] In other words, once gestational hypertension develops, there is no treatment method that is sufficiently safe for both the mother and the fetus and has few side effects. Therefore, there is a strong need for methods to reduce the risk of developing gestational hypertension, or in other words, methods to prevent gestational hypertension.

[0006] Although the mechanisms of gestational hypertension are not fully understood, normal placental formation in early pregnancy is considered to be an extremely important process in reducing the risk of gestational hypertension (Non-Patent Literature 1). In a normal pregnancy, placental trophoblast cells that interact with the endometrium after implantation fuse, leading to normal placental formation. On the other hand, it is thought that a decrease in this fusion of placental trophoblast cells and an increase in apoptosis (programmed cell death) cause placental dysfunction and lead to the development of gestational hypertension.

[0007] Therefore, promoting cell fusion of placental trophoblast cells in the early stages of pregnancy immediately after implantation is crucial for preventing gestational hypertension, and placental trophoblast cell fusion promoters may be safe and effective preventive agents for gestational hypertension for both mother and fetus.

[0008] MANUELA LANGBEIN et. al. , MOLECULAR REPRODUCTION AND DEVELOPMENT 75:175-183 (2008)

[0009] However, research and identification of substances useful in promoting such normal placental formation are still insufficient.

[0010] Therefore, the present inventors have discovered that several components, including pyrroloquinoline quinone or pyrroloquinoline quinone salt (hereinafter also simply referred to as PQQ), apple polyphenols, black rice, and black rice extract, promote the fusion of placental trophoblast cells, which are important for preventing gestational hypertension, and have completed the invention.

[0011] In other words, a preferred embodiment of the present invention provides the following oral composition: [1] A preventive or therapeutic agent for pregnancy-induced hypertension, containing an effective amount of one or more selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract.

[0012] [2] The agent according to [1], wherein the hypertensive syndrome of pregnancy is any of preeclampsia, preeclampsia, superimposed preeclampsia, or pregnancy complicated with hypertension. [3] A placental formation promoting or improving agent containing an effective amount of one or more selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract.

[0013] [4] The agent according to [3] wherein the promotion or improvement of placental formation is due to the promotion or improvement of placental trophoblast cell fusion.

[0014] [5] An agent for improving adaptability to pregnancy, which promotes normal placental formation, containing an effective amount of one or more selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract.

[0015] [6] Any of the drugs [1] to [5] to be taken around the expected date of ovulation, or before the secretion of hCG (human chorionic gonadotropin).

[0016] According to the present invention, PQQ, apple polyphenols, black rice, or black rice extract are useful in promoting normal placental formation, and it is possible to provide a preventive or therapeutic agent for pregnancy-induced hypertension containing these components.

[0017] Figure 1 shows the results of a gene expression test, which serves as an indicator of cell fusion, in Test Example 1. Figure 2 shows the results of a gene expression test, which serves as an indicator of cell fusion, in Test Example 2. Figure 3 is a photograph showing the state of cell fusion in Test Example 3-1. Figure 4 is a graph showing the change in the number of fused cells in Test Example 3-2. Figure 5 is a graph showing the change in the invasiveness of human placental cells in Test Example 4. Figure 6A is a photograph showing the effect of the food material of the example on aged pregnant mice in Test Example 5. Figure 6B is a graph showing the effect of the food material of the example on aged pregnant mice in Test Example 5. Figure 7 shows the results of a gene expression test, which serves as an indicator of cell fusion and placental formation in the placentas of aged pregnant mice in Test Example 6. Figure 8 is a graph showing the change in the marker of invasion in the placentas of aged pregnant mice in Test Example 6.

[0018] [Agent for the prevention or treatment of pregnancy-induced hypertension] The formulation of the present invention contains an effective amount of one or more components selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract.

[0019] (Pyrroloquinoline quinone or its salt) Pyrroloquinoline quinone is given by the following formula 1: It is a well-known compound represented by [formula].

[0020] Examples of pyrroloquinoline quinone salts include alkali metal salts, alkaline earth metal salts, and ammonium salts. Examples of alkali metal salts include sodium salts, potassium salts, and lithium salts. Examples of alkaline earth metal salts include calcium salts and magnesium salts.

[0021] As pyrroloquinoline quinone or its salt, alkali metal salts of pyrroloquinoline quinone are preferred, sodium salts of pyrroloquinoline quinone are more preferred, and disodium pyrroloquinoline quinone is even more preferred.

[0022] Pyrroloquinoline quinone or its salts may be commercially available. Furthermore, pyrroloquinoline quinone or its salts may be used individually or in combination of two or more types.

[0023] From the viewpoint of significantly achieving the effects of the present invention, the content of pyrroloquinoline quinone or its salt can be, for example, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, etc., based on the total amount of the formulation. Alternatively, the content of pyrroloquinoline quinone or its salt can be, for example, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.5% by mass or less, 0.2% by mass or less, etc., based on the total amount of the formulation. Furthermore, the content of pyrroloquinoline quinone or its salt is, for example, based on the total amount of the preparation, 0.001 to 30% by mass, 0.001 to 25% by mass, 0.001 to 20% by mass, 0.001 to 15% by mass, 0.001 to 10% by mass, 0.001 to 5% by mass, 0.001 to 1% by mass, 0.001 to 0.5% by mass, 0.001 to 0.2% by mass, 0.005 to 30% by mass, 0.005 to 25% by mass, 0.005 to 20% by mass, 0.005 to 15% by mass, 0.005 to 10% by mass, 0.005 to 5% by mass, 0.005 to 1% by mass, 0.005 to 0.5 mass%, 0.005 to 0.2 mass%, 0.01 to 30 mass%, 0.01 to 25 mass%, 0.01 to 20 mass%, 0.01 to 15 mass%, 0.01 to 10 mass%, 0.01 to 5 mass%, 0.01 to 1 mass%, 0.01 to 0.5 mass%, 0. 01-0.2% by mass, 0.05-30% by mass, 0.05-25% by mass, 0.05-20% by mass, 0.05-15% by mass, 0.05-10% by mass, 0.05-5% by mass, 0.05-1% by mass, 0.05-0.5% by mass, 0.05-0.2% by mass, etc.

[0024] The daily intake of pyrroloquinoline quinone or its salt may vary depending on the individual's condition (weight, age, sex, etc.) and formulation, but from the viewpoint of significantly achieving the effects of the present invention, examples include 1 mg or more, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 6 mg or more, 7 mg or more, 8 mg or more, 9 mg or more, 10 mg or more, 15 mg or more, 20 mg or more, etc. Alternatively, examples of daily intake of pyrroloquinoline quinone or its salt include 100 mg or less, 80 mg or less, 60 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 25 mg or less, etc. Furthermore, the daily intake of pyrroloquinoline quinone or its salt can be, for example, 5-100 mg, 5-80 mg, 5-60 mg, 5-50 mg, 5-40 mg, 5-30 mg, 5-25 mg, 10-100 mg, 10-80 mg, 10-60 mg, 10-50 mg, 10-40 mg, 10-30 mg, 10-25 mg, 15-100 mg, 15-80 mg, 15-60 mg, 15-50 mg, 15-40 mg, 15-30 mg, 15-25 mg, 20-100 mg, 20-80 mg, 20-60 mg, 20-50 mg, 20-40 mg, 20-30 mg, 20-25 mg, etc.

[0025] (Apple Polyphenols) Apple polyphenols are a polyphenol-rich fraction extracted from the peel or fruit of apples. They can be obtained in large quantities, especially from unripe apples. For example, they can be obtained as a polyphenol fraction column-produced from the clarified juice of unripe apples. The clarified juice can be obtained by pressing. Alternatively, extraction can be performed using a solvent. As extraction solvents for obtaining apple polyphenols, water, methanol, ethanol, propanol, butanol, and other alcohols, as well as polyhydric alcohols such as ethylene glycol, propylene glycol, butylene glycol, and glycerin, can be preferably used.

[0026] As for apple polyphenols, it is preferable to use any of the commercially available products such as "ApplePhenon®" (manufacturer: BBG Japan Co., Ltd.), "ApplePhenon® Applephenon SH" (manufacturer: BBG Japan Co., Ltd.), "ApplePhenon® Apple Procyanidin" (manufacturer: BBG Japan Co., Ltd.), or "ApplePhenon® Applephenon C-100" (manufacturer: BBG Japan Co., Ltd.), but it is not limited to these, and products such as "Apple Polyphenol" (manufacturer: Access One Co., Ltd.) or "Apple Extract Powder" (manufacturer: Vidya Japan Co., Ltd.) can also be used.

[0027] From the viewpoint of significantly demonstrating the effects of the present invention, when using "ApplePhenon®", "ApplePhenon® Applephenon SH", or equivalent products, the apple polyphenol content can be 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, etc., based on the total amount of the formulation. Furthermore, from the viewpoint of significantly demonstrating the effects of the present invention, when using "ApplePhenon® Applephenon SH" from BBG Japan Co., Ltd. or equivalent products, the apple polyphenol content can be 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.5% by mass or less, 0.2% by mass or less, etc., based on the total amount of the formulation. Furthermore, regarding the apple polyphenol content, from the viewpoint of significantly exhibiting the effects of the present invention, for example, when using BBG Japan Co., Ltd.'s "ApplePhenon® Applephenon SH" or an equivalent product, for example, based on the total amount of the formulation, the amounts are 0.001 to 30% by mass, 0.001 to 25% by mass, 0.001 to 20% by mass, 0.001 to 15% by mass, 0.001 to 10% by mass, 0.001 to 5% by mass, 0.001 to 1% by mass, 0.001 to 0.5% by mass, 0.001 to 0.2% by mass, 0.005 to 30% by mass, 0.005 to 25% by mass, 0.005 to 20% by mass, 0.005 to 15% by mass, 0.005 to 10% by mass, 0.005 to 5% by mass, 0.005 to 1% by mass, 0 .. 005-0.5 mass%, 0.005-0.2 mass%, 0.01-30 mass%, 0.01-25 mass%, 0.01-20 mass%, 0.01-15 mass%, 0.01-10 mass%, 0.01-5 mass%, 0.01-1 mass%, 0.01-0.5 mass%, 0.0 Examples include 1 to 0.2 mass%, 0.05 to 30 mass%, 0.05 to 25 mass%, 0.05 to 20 mass%, 0.05 to 15 mass%, 0.05 to 10 mass%, 0.05 to 5 mass%, 0.05 to 1 mass%, 0.05 to 0.5 mass%, 0.05 to 0.2 mass%, etc.

[0028] The daily intake of apple polyphenols may vary depending on the individual's condition (weight, age, sex, etc.) and formulation, but from the viewpoint of significantly achieving the effects of the present invention, examples include 50 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, 500 mg or more, 600 mg or more, 700 mg or more, 900 mg or more, 1000 mg or more, 1200 mg or more, 1500 mg or more, and 2000 mg or more. In addition, examples of daily intake of apple polyphenols include 3000 mg or less, 2500 mg or less, 2200 mg or less, 2000 mg or less, 1800 mg or less, 1600 mg or less, 1500 mg or less, 1200 mg or less, 1000 mg or less, 800 mg or less, and 600 mg or less. Furthermore, the recommended daily intake of apple polyphenols includes, for example, 50-3000 mg, 50-2500 mg, 100-2200 mg, 200-2000 mg, 300-1800 mg, 400-1600 mg, 500-1500 mg, 600-1500 mg, 700-1500 mg, and 800-1500 mg.

[0029] (Black rice or its extract) Black rice is a variety of brown rice that contains anthocyanin pigments in at least one of the seed coat or pericarp of brown rice, and has a black appearance. Known varieties of black rice include Shikuen, Kurodenen, Hunan Black Rice (registered trademark), Kuromurasaki, Hubei Shikuen Rice, etc. Black rice can be used as brown rice (unpolished) or polished rice obtained by milling brown rice. From the viewpoint of significantly achieving the effects of the present invention, it is preferable to use at least one selected from the group consisting of the seed coat, pericarp, and germ of brown rice as the black rice.

[0030] The extraction method for the black rice extract (synonymous with black rice extract) is not limited as long as it achieves the effects of the present invention. For example, the black rice extract refers to an extract obtained by immersing the above-mentioned black rice or its pulverized product in water and / or an organic solvent and filtering off the residue, an extract from which the solvent has been removed, or a fine powder thereof, or an extract or solvent-removed product dissolved, dispersed, or diluted using a suitable solvent. Commercially available products can also be used. As an example of another extraction method, the black rice extract can also be obtained after processing the black rice, such as by steaming it.

[0031] The black rice extract may be the crude extract obtained directly from black rice, a purified extract, or a concentrated extract. Commercially available products can also be used. There are no particular limitations on the method of obtaining the extract; conventional extraction methods, purification methods, concentration methods, synthesis methods, drying and powdering methods, etc., can be employed.

[0032] The extraction solvent for the black rice extract is not limited as long as it achieves the effects of the present invention, but examples include lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropyl alcohol, and butanol; polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol; polar solvents such as water, acetone, ethyl acetate, and methyl acetate; and nonpolar solvents such as hexane and pyridine. Any one or any mixture of two of these may be used. Of these solvents, water, ethanol, 1,3-butylene glycol, propylene glycol, or a mixture thereof is preferred, and a mixture of water and ethanol (hydrated ethanol) is more preferred.

[0033] While not limited to commercially available black rice extracts, "Black Rice Extract P" (manufacturer: Oryza Oil & Fat Chemical Co., Ltd.), "Mixed Ancient Rice Extract" (manufacturer: Satis Pharmaceutical Co., Ltd.), and "Black Rice Extract" (manufacturer: Access One Co., Ltd.) can be used.

[0034] The black rice extract is not limited as long as it achieves the effects of the present invention, but for example, the polyphenol content in the extract is preferably 3% by mass or more, more preferably 5% by mass or more, even more preferably 10% by mass or more, particularly preferably 12% by mass or more, and most preferably 15% by mass or more. The polyphenol content is not limited, but can be measured, for example, by the Folin-Denis method.

[0035] The black rice extract is not limited as long as it achieves the effects of the present invention, but for example, the anthocyanin content in the extract is preferably 1% by mass or more, more preferably 2% by mass or more, even more preferably 3% by mass or more, particularly preferably 4% by mass or more, and most preferably 5% by mass or more. The anthocyanin content can be measured, for example, by a colorimetric method.

[0036] From the viewpoint of significantly demonstrating the effects of the present invention, the content of black rice or its extract can be, for example, 0.001% or more, 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.5% or more, 1% or more, etc., of black rice or its extract with a polyphenol content of about 10% by mass as measured by the Folin-Denis method, based on the total amount of the formulation. Alternatively, the content of black rice or black rice extract can be, for example, 30% or less by mass, 25% or less by mass, 20% or less by mass, 15% or less by mass, 10% or less by mass, 5% or less by mass, 1% or less by mass, 0.5% or less by mass, 0.2% or less by mass, etc., based on the total amount of the formulation. Furthermore, the content of black rice or its extract is, for example, based on the total amount of the preparation, 0.001 to 30% by mass, 0.001 to 25% by mass, 0.001 to 20% by mass, 0.001 to 15% by mass, 0.001 to 10% by mass, 0.001 to 5% by mass, 0.001 to 1% by mass, 0.001 to 0.5% by mass, 0.001 to 0.2% by mass, 0.005 to 30% by mass, 0.005 to 25% by mass, 0.005 to 20% by mass, 0.005 to 15% by mass, 0.005 to 10% by mass, 0.005 to 5% by mass, 0.005 to 1% by mass, and 0.0 05-0.5% by mass, 0.005-0.2% by mass, 0.01-30% by mass, 0.01-25% by mass, 0.01-20% by mass, 0.01-15% by mass, 0.01-10% by mass, 0.01-5% by mass, 0.01-1% by mass, 0.01-0.5% by mass, 0.01 -0.2% by mass, 0.05-30% by mass, 0.05-25% by mass, 0.05-20% by mass, 0.05-15% by mass, 0.05-10% by mass, 0.05-5% by mass, 0.05-1% by mass, 0.05-0.5% by mass, 0.05-0.2% by mass, etc.

[0037] The daily intake of black rice or its extract may vary depending on the individual's condition (weight, age, sex, etc.) and the form of the formulation, but from the viewpoint of significantly achieving the effects of the present invention, examples include 1 mg or more, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 6 mg or more, 7 mg or more, 8 mg or more, 9 mg or more, 10 mg or more, 15 mg or more, 20 mg or more, etc.

[0038] Furthermore, the daily intake of black rice or black rice extract may include, for example, 100 mg or less, 80 mg or less, 60 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 25 mg or less, etc. Furthermore, the recommended daily intake of black rice or black rice extract includes, for example, 5-100 mg, 5-80 mg, 5-60 mg, 5-50 mg, 5-40 mg, 5-30 mg, 5-25 mg, 10-100 mg, 10-80 mg, 10-60 mg, 10-50 mg, 10-40 mg, 10-30 mg, 10-25 mg, 15-100 mg, 15-80 mg, 15-60 mg, 15-50 mg, 15-40 mg, 15-30 mg, 15-25 mg, 20-100 mg, 20-80 mg, 20-60 mg, 20-50 mg, 20-40 mg, 20-30 mg, and 20-25 mg.

[0039] The formulation according to this embodiment may contain pharmacologically active or physiologically active ingredients other than pyrroloquinoline quinone or its salt, apple polyphenols, black rice, or black rice extract, as long as the effects of the present invention are not impaired.

[0040] Specific examples of such pharmacologically active or physiologically active ingredients include, for example, ubiquinone (coenzyme Q10), vitamin B1, vitamin B2, vitamin B6, folic acid, vitamin B12, vitamin C, vitamin D, vitamin A, vitamin E, niacin, calcium pantothenate, taurine, carnosine, anserine, balenine, citrulline, gamma-aminobutyric acid, valine, leucine, isoleucine, glycine, arginine, ornithine, carnitine, glutamic acid, and gluten. Examples include vitamins, creatine, carnitine, luteolin, quercetin, genistin, cyanidin, resveratrol, diosgenin, isoflavone aglycone, isoflavone, lipoic acid, zinc, iron, calcium, selenium, astaxanthin, zeaxanthin, beta-carotene, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, placenta extract, pycnogenol (proanthocyanidin), and equol. From the viewpoint of further enhancing the effects according to the present invention, preferred pharmacologically active or physiologically active ingredients include ubiquinone (coenzyme Q10), vitamin B12, vitamin C, vitamin E, astaxanthin, zinc, carnitine, resveratrol, isoflavone, equol, pycnogenol (proanthocyanidin), and folic acid, and more preferably ubiquinone (coenzyme Q10), astaxanthin, zinc, resveratrol, isoflavone, equol, pycnogenol (proanthocyanidin), and folic acid. The pharmacologically active or physiologically active ingredients may be used individually or in combination of two or more.

[0041] [Use] Pregnancy-induced hypertension refers to the presence of high blood pressure at any point during pregnancy. Pregnancy-induced hypertension is classified into preeclampsia, gestational hypertension, superimposed preeclampsia, or hypertension in pregnancy. Pregnancy-induced hypertension (HDP) can cause organ damage throughout the body, and complications include eclamptic seizures, hypertensive cerebral hemorrhage, heart failure, pulmonary edema, HELLP syndrome, placental abruption, fetal growth restriction, and placental dysfunction. In this specification, the term "pregnancy-induced hypertension" encompasses all of these conditions.

[0042] Here, hypertension includes all of grade I hypertension to grade III hypertension in the normal sense. That is, at any point during pregnancy, it refers to a systolic blood pressure of 140 mmHg or higher or a diastolic blood pressure of 90 mmHg or higher measured in the clinic. In preeclampsia, a systolic blood pressure of 160 mmHg or higher or a diastolic blood pressure of 110 mmHg or higher is classified as severe.

[0043] The placental formation process in the early stage of pregnancy is considered to have a great impact on preeclampsia. As placental trophoblast cells that make up the human placenta, undifferentiated cytotrophoblast cells (mononuclear trophoblast cells), syncytiotrophoblast cells (fused trophoblast cells) that transport nutrients and enzymes to the fetus, and extravillous trophoblast cells that invade the uterus and are responsible for the formation of blood vessels that take in maternal blood into the placenta are known. In a normal pregnancy, cytotrophoblast cells (placental trophoblast cells) that interact with the endometrium after fertilized egg implantation fuse cells, and syncytialization continues to form new types of cells, syncytiotrophoblast cells. This process is essential for placental formation. That is, after fertilized egg formation, the fusion of trophoblast cells in the uterus is an indispensable process for the establishment and maintenance of normal implantation and for placental formation. If the cell fusion of these placental trophoblast cells does not proceed for some reason and the number of cell fusions decreases, apoptosis (cell death) of cells increases, causing placental dysfunction. Such placental dysfunction is considered to be one of the causes of preeclampsia.

[0044] Promoting normal placental formation is important for the prevention of preeclampsia and especially for early treatment. Furthermore, promoting or improving such cell fusion of placental trophoblast cells leads to an improvement in the maternal adaptability to pregnancy.

[0045] In the examples described later, pyrroloquinoline quinone or its salt, apple polyphenol, black rice or its extract has been found as a component that can promote placental trophoblast cell fusion.

[0046] In Test Examples 1 and 2, it has been confirmed that PQQ or black rice extract (black rice extract) contributes to an increase in the expression of Syncytin2 and PAPP-A, which are indicators, or an increase in the expression of Syncytin2 and GCM1 in the induction of syncytialization (cell fusion) of placental trophoblast cells.

[0047] In a test system that simulates in vitro placental trophoblast cell fusion in the following-described examples, cell fusion stimulation is performed. However, in vivo, cell fusion is a phenomenon observed during the placenta formation stage. It is strongly speculated that by promoting cell fusion, placenta formation proceeds smoothly, and in addition to preventing preeclampsia, the maternal adaptability to pregnancy is improved.

[0048] In Test Example 4 described below, it is shown that placental cell invasion is promoted by PQQ or apple polyphenols.

[0049] In Test Example 5, furthermore, it is shown that in aged pregnant mice (preeclampsia model), PQQ, apple polyphenols, or black rice extract has a therapeutic effect on fetal weight.

[0050] Therefore, from the results of the following-described examples, it is strongly suggested that PQQ, apple polyphenols, black rice, or black rice extract is excellent for preventing or treating preeclampsia. It is preferable to start ingesting PQQ, apple polyphenols, black rice, or black rice extract, for example, around the expected ovulation date or from before the secretion of hCG (human chorionic gonadotropin). Alternatively, it is also possible to start ingesting at least 7 days after the first day of menstruation, and it is preferable to continue ingesting after 14 days from the first day of menstruation. It is also preferable to continue drinking during pregnancy.

[0051] (Additives, etc.) The preparation according to the present embodiment can contain various additives in addition to the above components as long as the effects of the present invention are not impaired.

[0052] Specific examples of such additives include, for example, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, odor-correcting agents, saccharides, sugar alcohols / polyhydric alcohols, high-intensity sweeteners, fats and oils, emulsifiers, thickeners, acidulants, fruit juices, and the like.

[0053] Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, gelatin, calcium carbonate, kaolin, crystalline cellulose, silicic acid, and the like.

[0054] Examples of binders include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, cellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, calcium phosphate, and polyvinylpyrrolidone.

[0055] Examples of disintegrants include dried starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose.

[0056] Examples of lubricants include talc, stearate, borax, and polyethylene glycol.

[0057] Examples of flavoring agents include sucrose, orange peel, citric acid, and tartaric acid.

[0058] Examples of sugars include sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, and xylose.

[0059] Examples of sugar alcohols and polyhydric alcohols include sorbitol, xylitol, erythritol, lactitol, palatinose, reduced starch syrup, reduced maltose syrup, glycerin, and propylene glycol.

[0060] Examples of high-intensity sweeteners include aspartame, stevia, acesulfame potassium, and sucralose.

[0061] Examples of oils and fats include safflower oil, grape seed oil, sunflower oil, olive oil, corn oil, sesame oil, soybean oil, rapeseed oil, and perilla oil.

[0062] Examples of emulsifiers include sucrose fatty acid esters, glycerol fatty acid esters, and lecithin.

[0063] Examples of thickening agents include carrageenan, xanthan gum, guar gum, pectin, and locust bean gum.

[0064] Examples of acidulants include citric acid, lactic acid, and malic acid. Examples of fruit juices include lemon juice, orange juice, and berry juices.

[0065] The additives may be used individually or in combination of two or more types.

[0066] As additives, it is preferable to include pyrroloquinoline quinone or its salt, or oils and fats, from the viewpoint of the stability of black rice or black rice extract during formulation.

[0067] (Dosage Form) The dosage form of the formulation according to this embodiment is not particularly limited and includes, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, lozenges, etc.), granules, powders, soft capsules, hard capsules, lozenges, jellies, or liquids (including suspensions, emulsions, syrups, etc.); external preparations such as ointments, suppositories, patches, sprays; and injections. Among these, oral preparations are preferred from the viewpoint of ease of handling and from the viewpoint of further enhancing the effects of the present invention, and tablets, granules, soft capsules, and hard capsules are more preferred.

[0068] The formulation according to this embodiment can be used, for example, as an ingredient in pharmaceuticals, quasi-drugs, cosmetics, and food and beverages (beverages, food). Furthermore, the formulation according to this embodiment can also be used, for example, as a pharmaceutical preparation, a quasi-drug preparation, a food for specified health uses, a nutritional functional food, a food for the elderly, a food for special dietary uses, a food with functional claims, a health supplement (supplement), a food preparation (e.g., confectionery tablets), or a food product. In addition, the formulation according to this embodiment can also be used, for example, as a veterinary drug or a feed additive.

[0069] When the formulation according to this embodiment is used as a food, the food may be a general food blended with pyrroloquinoline quinone or its salt, apple polyphenols, or black rice or black rice extract, and other ingredients as needed. Examples of such foods include solid foods such as cookies, biscuits, snacks, jellies, gummies, chocolates, gums, candies, and cheese; and liquid foods such as nutritional drinks, juices, tea beverages, coffee beverages, and dairy beverages.

[0070] The formulation according to this embodiment can be suitably used in subjects requiring the prevention or treatment of pregnancy-induced hypertension (e.g., humans; non-human animals such as cattle, pigs, horses, sheep, goats, dogs, rabbits, mice, rats, guinea pigs, gerbils, hamsters, and ferrets). From the viewpoint of further enhancing the effects of the present invention, humans are preferred as subjects. Furthermore, from the viewpoint of further enhancing the effects of the present invention, cattle, pigs, and horses are preferred among non-human animals, with pigs being more preferred.

[0071] The formulation according to this embodiment can also be taken by humans before or during pregnancy, and is preferably used for the purpose of a healthy pregnancy, a normal pregnancy course, full-term delivery, or for people with high blood pressure, those who are concerned about their blood pressure, those at risk of high blood pressure, those who want to lower high blood pressure, or those who want to suppress high blood pressure.

[0072] [Manufacturing Method] The method for manufacturing the formulation according to this embodiment is not particularly limited, and known methods can be used. Specifically, one method is to heat and stir each component as necessary to dissolve or disperse them, mix them, then cool and stir them, and then let them stand at room temperature, but this method is not particularly limited.

[0073] [Dosage Method] The formulation according to this embodiment may be stored and / or sold in known containers in doses sufficient for one week or more, 10 days or more, 14 days or more, 20 days or more, 30 days or more, or 35 days or more.

[0074] The formulation according to this embodiment is not limited to, but the administration period may be, for example, at least 3 days, and may be 5 days or more, 7 days or more, 10 days or more, 14 days or more, 20 days or more, 30 days or more, or 35 days or more.

[0075] [Methods for preventing or treating hypertensive disorders of pregnancy, methods for promoting cell fusion, and methods for improving adaptability to pregnancy due to cell fusion promotion] One or more substances selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract can significantly promote the transition from undifferentiated placental trophoblast cells to fused trophoblast cells. This can be verified, for example, by detecting increased expression of Syncytin2, PAPPA (Pregnancy-associated plasma protein), and / or GCM1 (Glial cells missing-1), which are fusion markers for placental trophoblast cells. Accordingly, as one embodiment of the present invention, a method for promoting or improving placental trophoblast cell fusion, a method for improving maternal adaptability to pregnancy due to the promotion of placental trophoblast cell fusion, a method for improving placental cell infiltration ability, and a method for preventing or treating pregnancy-induced hypertension are provided, comprising ingesting a preparation containing one or more selected from the group consisting of pyrroloquinoline quinone or its salt, apple polyphenol, black rice, and black rice extract. Placental trophoblast cells may be human cells, or cells of non-human animals such as cattle, pigs, horses, sheep, goats, dogs, rabbits, mice, rats, guinea pigs, gerbils, hamsters, and ferrets. From the viewpoint of further enhancing the effects of the present invention, human cells are preferred. The types, content, and daily intake of pyrroloquinoline quinone or its salt, black rice or black rice extract, etc., in these embodiments, as well as the types, content, and daily intake of other components, the form of the preparation, etc., are as described above.

[0076] Next, the present invention will be specifically described with reference to examples and test cases, but the present invention is not limited to the following examples and test cases. Unless otherwise specified, the units of each component amount in the table are mass percent.

[0077] [Preparation of placental trophoblast cells (placental trophoblast stem cells)] To simulate the fusion of placental trophoblast cells in vitro, cultured cells maintaining the phenotype of placental trophoblast stem cells were used in [0.3% bovine serum albumin, 1% ITS-X supplement, 0.1 mM 2-mercaptoethanol, 50 ng / mL epidermal growth factor, 0.2% fetal bovine serum, 1.5 μg / mL L-ascorbic acid, 5 μM Y27632, 0.8 mM valproic acid, 2 μM CHIR99021, 0.5 μM A83-01, 1 μM SB431542, Dulbecco's modified Eagle medium / F12 (DMEM / F12) (1:1) medium supplemented with antibiotics and antifungal agents] (all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0078] [Fusion stimulation of placental trophoblast stem cells] In order to induce fusion of placental trophoblast cells in vitro, the cultured cells were given a culture medium containing forskolin (0.5 μM, Cayman Chemical, hereinafter abbreviated as "FSK") as a fusion stimulus. [4% knockout serum replacement (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (Fujifilm Wako Pure Chemical Corporation), 0.3% bovine serum albumin (Fujifilm Wako Pure Chemical Corporation), 1% ITS-X supplement (Fujifilm Wako Pure Chemical Corporation), 2.5 μM Y27632 (Fujifilm Wako Pure Chemical Corporation), 50 ng / mL] To promote cell fusion necessary for placental formation, Dulbecco's modified Eagle medium / F12 (DMEM / F12) (1:1) medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing epidermal growth factor (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), antibiotics, and antifungal agents was added.

[0079] [RNA Extraction and Quantification by RT-PCR] Genes were extracted from cultured trophoblast cells using Isogen II (manufactured by Nippon Gene Co., Ltd.). mRNA was reverse transcribed using ReverTra Ace qPCR RT Kit (manufactured by Toyobo Co., Ltd.), and the resulting cDNA was amplified by qPCR using PowerUP SYBR Green Master Mix (manufactured by Thermo Fisher Scientific). The Ct value of each gene was used as the expression level, and the expression levels of PQQ or cells without black rice extract were used as controls to calculate the expression ratio.

[0080] [Test Example 1. Gene Expression Test in Cell Fusion 1] Human placental trophoblast cells were treated with 10 nM PQQ simultaneously added to the fusion medium, with and without FSK as a fusion stimulus. The fusion status was compared between the group without PQQ and the group with PQQ.

[0081] In each case, RNA extraction and quantification by RT-PCR were performed, and the gene expression of the fusion markers Syncytin2 and PAPPA was evaluated. The results are shown in Figure 1.

[0082] In Figure 1, the following conditions are shown, respectively: no fusion stimulation (Ctrl), PQQ added only (PQQ), cells that have become fused trophoblast cells due to fusion stimulation (FSK), and cells with fusion stimulation plus PPQ added (FSK + PQQ).

[0083] As a result, the addition of PQQ significantly increased the expression of the fusion markers Syncytin2 and PAPPA genes.

[0084] From the above, it can be seen that PQQ significantly promotes cell fusion.

[0085] [Test Example 2. Gene Expression Test in Cell Fusion 2] Human placental trophoblast cells were stimulated for fusion, and 500 μg / mL of black rice extract was added to the fusion medium. The fusion status was compared between the case without black rice extract and the case with black rice extract ("Black Rice Extract P" (manufacturer: Oryza Oil & Fat Chemical Co., Ltd.), hereinafter the same) added.

[0086] In each case, RNA extraction and quantification by RT-PCR were performed, and the gene expression of the fusion markers Syncytin2 and GCM1 was evaluated. The results are shown in Figure 2.

[0087] Figure 2 shows the results for no fusion stimulus (Ctrl), fusion stimulus only (FSK), and fusion stimulus with the addition of black rice extract (FSK + BR).

[0088] As a result, the addition of black rice extract significantly increased the expression of the fusion markers Syncytin2 and GCM1 genes.

[0089] From the above, it can be seen that black rice extract significantly promotes cell fusion.

[0090] [Test Example 3. Cell Fusion Promotion Test] Furthermore, human trophoblast stem cells stimulated by FSK fusion were fixed with methanol and incubated with anti-E cadherin antibody (1:200, #3195, Cell Signaling Technology) and AlexaFluor 488-labeled goat anti-mouse antibody (Thermo Fisher Scientific) to identify the cell surface. The stained images at this time are shown in Figure 3 (Test Example 3-1).

[0091] The nuclei were counterstained with 4',6-diamino-2-phenylindole 2HCl (DAPI). The number of nuclei within syncytiotrophoblast cells (fusion cells) and the total number of nuclei were counted in five randomly selected microscopic regions for each sample and calculated as the fusion index [(number of nuclei in fusion cells / total number of nuclei) × 100] in three independent experiments. The increase in the number of fusion cells measured in this way is shown in Figure 4 (Test Example 3-2). The increase in the number of fusion cells was confirmed by actually using PQQ or black rice extract.

[0092] [Test Example 4. Confirmation Test for Changes in Placental Cell Invasion Ability] A cell invasion assay was performed using human placental trophoblast cells in a Boyden chamber. The membrane at the bottom of the Boyden chamber had an effective area of ​​0.48 cm². ２The membrane is made of track-etched polycarbonate with 8 μm pores. This membrane was coated with Matrigel solution (Corning) and placed in a 24-well plate. Placental cells were seeded in a medium containing 2% FBS in the chamber, and cultured in a medium containing 10% FBS in the wells at 37°C for 1 day. After culturing, the infiltrating cells that passed through the membrane were stained with DAPI, and the number of infiltrating cells was measured.

[0093] These results are shown in Figure 5. It was found that culture media containing PQQ (10 nM) or apple polyphenols (10 μg / mL) ("ApplePhenon®" (manufacturer: BBG Japan Co., Ltd.), hereinafter the same) promoted the infiltration of placental cells.

[0094] [Test Example 5. Efficacy Verification Test on Aged Pregnant Mice] The animals used were C57BL / 6J mice (10 weeks old (Young) and 26 weeks old (Aged), female) and C57BL / 6J mice (10 weeks old, male). They were housed under constant lighting for 12 hours a day, constant temperature (24°C), and constant humidity (55±5%), and were given free access to standard solid samples and water. The estrous cycle of female mice was confirmed by vaginal smear, and mating was performed in the evening during the proestrus phase. The mating day of the mice was designated as gestational day 0 (gestational day 0; GD0), and reagent administration was started from GD1. Blood pressure measurements were performed before pregnancy, and at GD4, GD7, GD10, GD14, and GD18. It was confirmed that blood pressure rose in the Aged group from GD14 onwards, and that symptoms similar to those of gestational hypertension developed.

[0095] Mice were randomly assigned to one of the following four groups: (i) control group (PBS), (ii) PQQ group (PBS adjusted to 10 nM), (iii) black rice extract group (PBS adjusted to 250 μg / mL), and (iv) apple polyphenol group (PBS adjusted to 5 μg / mL). Each reagent was administered intraperitoneally (0.2 mL) daily from GD1 to GD18.

[0096] These results are shown in Figures 6A and 6B. PQQ, black rice extract, and apple polyphenols were found to increase fetal weight in aged pregnant mice, strongly suggesting that they promote normal pregnancy and suppress abnormalities such as gestational hypertension.

[0097] [Test Example 6. Gene Expression Test Using Placental Tissue from Aged Pregnant Mice] RNA was extracted from the placentas of aged pregnant mice obtained in Test Example 5, and quantitative analysis was performed by RT-PCR to evaluate the gene expression of SyncytinA and VEGFA, which are fusion and placental formation markers. The results are shown in Figure 7.

[0098] The cell fusion and placental formation-promoting effects of PQQ, black rice extract, and apple polyphenols were confirmed by the expression of markers.

[0099] Separately, the gene expression of MMP2, a marker of invasion, was evaluated. The results are shown in Figure 8.

[0100] The effects of PQQ, black rice extract, and apple polyphenols in promoting infiltration were confirmed by the expression of markers.

Claims

1. A preventive or therapeutic agent for pregnancy-induced hypertension, containing an effective amount of one or more selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract.

2. The agent according to claim 1, wherein the aforementioned hypertensive syndrome of pregnancy is any of pre-eclampsia, gestational hypertension, superimposed hypertensive syndrome of pregnancy, or pregnancy complicated by hypertension.

3. A placental formation promoting or improving agent containing an effective amount of one or more selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract.

4. The agent according to claim 3, wherein the promotion or improvement of placental formation is due to the promotion or improvement of placental trophoblast cell fusion.

5. An agent that enhances the ability to adapt to pregnancy by promoting normal placental formation, containing an effective amount of one or more selected from the group consisting of pyrroloquinoline quinone, pyrroloquinoline quinone salts, apple polyphenols, black rice, and black rice extract.

6. The agent according to any one of claims 1 to 5, for ingestion around the expected date of ovulation, or before the secretion of hCG (human chorionic gonadotropin).

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