Composition containing choline ester for oral ingestion

Abstract

To provide compositions with hypotensive and / or anti-stress effects that allows humans to easily orally ingest while containing choline ester such as acetylcholine as an active ingredient.SOLUTION: Objects of the present invention are to provide a novel composition for oral ingestion that allows humans to ingest choline ester such as acetylcholine easily orally and has hypotensive and vasodilatory effects, and to provide a food that is a useful source for that purpose.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a composition having antihypertensive and / or anti-stress effects, which contains as an active ingredient a choline ester, which is a compound in which choline and an organic acid are ester-bonded, and a method for producing the same. [Background technology]

[0002] Among choline esters, acetylcholine is known to be an essential neurotransmitter for mammalian vital activities. In 1929, a hypotensive substance distinct from histamine was isolated from horse spleen, and its active substance was chemically identified as acetylcholine (Non-Patent Document 1). Furthermore, it was discovered that the hypotensive substance in ergot is acetylcholine, and it has been confirmed that fungi produce acetylcholine (Non-Patent Document 2). Acetylcholine is found in edible plants, edible fungi, royal jelly, milk, etc., and is also present in Bacillus subtilis and yeast. Among edible plants, eggplant and bamboo shoots have been reported to have high acetylcholine contents (Non-Patent Documents 3-7).

[0003] In addition to acetylcholine, several choline esters have also been discovered. Propionylcholine, which has a propionyl group with a carbon chain one carbon longer than the acetyl group, was discovered in bovine spleen in 1953 (Non-Patent Documents 8 and 9). Subsequently, propionylcholine production has been confirmed in bull semen, the hemolymph and smooth muscle of European crayfish, American horseshoe crab, European cockle, giant clam, purple mussel, Anodonta americana, and giant apple snail, in electrogenic tissue cultures of electric rays, as well as in sedge, mung bean, plantain, poplar, and white birch (Non-Patent Documents 10 to 13).

[0004] Butyrylcholine was discovered in brain extracts in 1954 (Non-Patent Document 14), and has been shown to exist in arthropods and mollusks together with acetylcholine and propionylcholine (Non-Patent Document 11). Furthermore, in addition to propionylcholine and butyrylcholine, several other choline esters have been identified from mollusks. For example, the structures of urocanoylcholine from a species of Muricidae, ββ-dimethylacroylcholine (senecioylcholine) from a species of Ganoderma lucidum, acroylcholine from a species of Bivalvia, and imidazolepropionylcholine from a species of Ganoderma lucidum have been determined (Non-Patent Documents 15 to 18).

[0005] The present inventors have studied the active ingredients in fermented osmanthus (a lactic acid fermentation product of buckwheat plants) that have antihypertensive and vasodilatory effects, and have provided an extract composition containing quaternary alkylammonium compounds based on multiple choline esters, including at least acetylcholine and propionylcholine. They have also demonstrated that purified acetylcholine, propionylcholine, and butyrylcholine exhibit antihypertensive effects when administered orally in a single dose to spontaneously hypertensive rats (SHR) (Patent Documents 1 and 2). Meanwhile, as described in an interview form for a drug containing acetylcholine chloride as an active ingredient (Non-Patent Document 19), "acetylcholine is broken down in the digestive tract when administered orally and is hardly absorbed, so an injection was used," the application of choline esters to humans has so far been via injection, and no studies have been conducted on their oral administration. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2015 / 147251 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-189745 [Patent Document 3] Japanese Patent Application Publication No. 06-065068 [Non-patent literature]

[0007] [Non-Patent Document 1] Dale HH, Dudley HW. The presence of histamine and acetylcholine in the spleen of the ox and the horse. J Physiol 68:97-123, 1929. [Non-patent document 2] Ewins AJ. Acetylcholine, a new active principle of ergot. Biochem J 8:44-49, 1914. [Non-patent document 3] Yoshie Momoki, Acetylcholine in Plants, Chemical Regulation of Plants, 30(1), 49-61 (1995) [Non-patent document 4] Koichiro Kawashima, The roots of acetylcholine and non-neuronal acetylcholine, Basic Aging Research, 34(4), 12-24 (2010) [Non-Patent Document 5] Masato Shinoda et al., Blood flow increasing factors in royal jelly, Pharmaceutical Journal, 98(2), 139-145 (1978) [Non-patent document 6] Whittaker VP. Acetylcholine in Milk. Nature 181:856-857, 1958. [Non-Patent Document 7] Horiuchi Y, Kimura R, Kato N, Fujii T, Seki M, Endo T, Kato T, Kawashima, K. Evolutional study on acetylcholine expression. Life Sci 72:1745-1756, 2003. [Non-patent document 8] Banister J, Whittaker VP, Wijesundera S. The occurrence of homologues of acetylcholine in ox spleen. J Physiol 121(1):55-71, 1953. [Non-Patent Document 9] Gardiner JE, Whittaker VP. The identification of propionylcholine as a constituent of ox spleen. Biochem J 58(1):24-29, 1954. [Non-Patent Document 10] Bishop MR, Sastry BV, Stavinoha WB. Identification of acetylcholine and propionylcholine in bull spermatozoa by integrated pyrolysis, gas chromatography and mass spectrometry. Biochim Biophys Acta 500(2):440-444, 1977. [Non-Patent Document 11] Wolfgang W, Jutta N, Dettmar W. Distribution of cholinesters and cholinesterases in haemolymphs and smooth muscles of molluscs. Comp Biochem Phys C 61(1):121-131, 1978. [Non-Patent Document 12] O'Regan S. The synthesis, storage, and release of propionylcholine by the electric organ of Torpedo marmorata. J Neurochem 39(3):764-772, 1982. [Non-Patent Document 13] Miural GA, Shin TM. Identification of proprionylcholine in higher plants. Physiol Plant 62:341-343, 1984. [Non-Patent Document 14] Holtz P, Schumann HJ. Butyrylcholine in brain extracts. Naturwissenschaften 41:306, 1954. [Non-Patent Document 15] Erspamer V, Benati O. Identification of murexine as beta-[imidazolyl-(4)]acrylcholine. Science 117:161-162, 1953. [Non-Patent Document 16] Keil MJ, Michaelson IA, Whittaker VP. Physiologically active choline esters in certain marine gastropods and other invertebrates. J Physiol 139:434, 1957. [Non-Patent Document 17] Whittaker VP. Acrylylcholine: a new naturally occurring pharmacologically active choline ester from Buccinum undatum. Biochem Pharmacol 1(4):342-346, 1959. [Non-Patent Document 18] Roseghini M. Occurrence of dihydromurexine (imidazole propionylcholine) in the hypobranchial gland of Thais (purpura) haemastoma. Experientia 27(9):1008-1009, 1971. [Non-Patent Document 19] Interview form for Obisote® Injection 0.1g, January 2013 [Non-Patent Document 20] Latest Basic Pharmacology (Supervised by Keijiro Takagi and Tsutomu Kameyama, edited by Sachiko Oishi and Susumu Okabe, published by Hirokawa Shoten, 96 pages, lines 6-10) [Non-Patent Document 21] Abstracts of the 2016 Annual Meeting of the Society of Agricultural Chemistry of Japan, 4E076, published March 5, 2016. [Non-Patent Document 22] Kleiber, M. The fire of life. An introduction to animal energetics. New York: Wiley, 1961. [Non-Patent Document 23] Kim JM, Lee SW, Kim KM, Chang UJ, Song JC, Suh HJ. Anti-stress effect and functionality of yeast hydrolysate SCP-20. Europe Food Res Technol 217(2):168?172, 2003. [Non-Patent Document 24] Armando I, Carranza A, Nishimura Y, Hoe KL, Barontini M, Terron JA, Falcon-Neri A, Ito T, Juorio AV, Saavedra JM. Peripheral administration of an angiotensin II AT(1) receptor antagonist decreases the hypothalamic-pituitary- adrenal response to isolation Stress. Endocrinology 142(9):3880-3889, 2001. [Non-Patent Document 25] Nakamura K, Okitsu S, Ishida R, Tian S, Igari N, Amano Y. Identification of natural lactoylcholine in lactic acid bacteria-fermented food.Food Chem 201:185-189, 2016. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have noticed that, if choline esters could be used by oral ingestion in humans, it would be possible to lower blood pressure and the like safely and easily without putting a strain on the body. However, a technique for appropriately administering acetylcholine orally to humans and a suitable supply source for said technique have not yet been found, and they have considered it important to clarify these points. Therefore, the object of the present invention is to provide a novel orally ingestible composition that contains a choline ester such as acetylcholine as an active ingredient, can be easily taken orally by humans, and has antihypertensive and vasodilatory effects, and to provide a food product that can be a useful source for this purpose. [Means for solving the problem]

[0009] In the course of intensive research to solve the above-mentioned problems, the present inventors have surprisingly discovered that, despite the conventional belief that acetylcholine cannot be orally administered to humans, there is an appropriate dose at which choline esters can exert antihypertensive and vasodilatory effects when orally administered, and that such doses also exert anti-stress effects. They have also discovered edible plants that are suitable as raw materials for efficiently supplying choline esters for these purposes, and as a result of further research, have completed the present invention.

[0010] The present invention therefore relates to the following: [1] A composition having a blood pressure lowering effect and / or an anti-stress effect, which contains a choline ester as an active ingredient, the composition having a choline ester content of 5 μg to 50 mg, and is for oral intake. [2] The composition according to [1] above, which is a food composition. [3] The composition according to [1] above, which is a pharmaceutical composition for lowering blood pressure and / or for anti-stress purposes.

[0011] [4] The composition according to any one of [1] to [3] above, wherein the choline ester is derived from an edible plant. [5] The composition according to any one of [1] to [4] above, which comprises freeze-dried powder and / or hot-air-dried powder of an edible plant. [6] The composition according to [5] above, which consists of a freeze-dried powder and / or a hot air-dried powder that can pass through a 20-mesh sieve. [7] The composition according to any one of [1] to [4] above, which is an extract obtained by extracting an edible plant with ethanol or aqueous ethanol. [8] The composition according to any one of [4] to [7] above, wherein the edible plant is the fruit of Solanum melongena (family Solanaceae) and / or the young shoots of Poaceae (family Poaceae, subfamily Bambusoideae, family Bambuseae).

[0012] [9] The composition according to any one of [1] to [3] above, wherein the choline ester comprises one or more choline esters selected from the group consisting of acetylcholine, butyrylcholine, and propionylcholine.

[10] The composition described in [9] above, wherein the choline ester does not include lactoylcholine.

[11] The composition according to any one of [1] to

[10] above, wherein the concentration of the choline ester is 5 μg / g to 250 mg / g and the daily intake amount is adjusted to 5 μg to 50 mg.

[12] The composition according to any one of the above [1] to

[11] , which is processed by freezing.

[13] The composition according to any one of [1] to [3] above, which is a part or all of a frozen fruit of Solanum melongena (Solanaceae).

[0013]

[14] A method for producing an oral composition having antihypertensive and / or anti-stress effects, which contains a choline ester as an active ingredient, comprising: freeze-drying an edible plant into a powder and / or hot-air-dried powder; and distributing the freeze-dried powder and / or hot-air-dried powder so that the choline ester content is 5 μg to 50 mg.

[15] The method according to

[14] above, wherein the edible plant is the fruit of Solanum melongena in the Solanaceae family and / or the young shoots of Poaceae, Bambusoideae, Bambuseae in the Poaceae family.

[16] The method according to

[14] or

[15] , further comprising heating the edible plant.

[17] The method according to any one of

[14] to

[16] above, further comprising suspending the freeze-dried powder and / or hot-air-dried powder of the edible plant in water and adding an acid to the resulting suspension.

[18] The method according to

[17] , further comprising adjusting the pH of the suspension to 5.5 to 4.5 after adding the acid.

[19] A composition for oral administration having antihypertensive and / or anti-stress effects, which contains a choline ester as an active ingredient and is produced by the method according to any one of

[14] to

[18] above.

[0014]

[20] A method for producing an extract for oral administration having antihypertensive and / or anti-stress effects, which contains a choline ester as an active ingredient, the method comprising extracting an edible plant, or a freeze-dried powder and / or a hot-air-dried powder of an edible plant, with ethanol or aqueous ethanol.

[21] The method according to

[20] , which comprises extracting the edible plant or freeze-dried powder and / or hot air-dried powder of the edible plant with ethanol.

[22] The method according to

[20] , comprising extracting an edible plant, or a freeze-dried powder and / or a hot-air-dried powder of an edible plant, with aqueous ethanol, wherein the ethanol concentration of the aqueous ethanol is 25 to 60% (w / w) or 95% (w / w) or more.

[23] The method according to any one of

[20] to

[22] above, wherein L-ascorbic acid is added to the ethanol or aqueous ethanol used for extraction.

[24] The method according to any one of

[20] to

[23] above, which comprises adjusting the choline ester content of the extract to 5 μg to 50 mg.

[25] An extract for oral administration having a blood pressure lowering effect and / or an anti-stress effect, which contains a choline ester as an active ingredient and is produced by the method according to any one of

[20] to

[24] above. [Effects of the Invention]

[0015] The present invention provides a composition that has a blood pressure lowering effect even when the active ingredient, a choline ester, is taken orally without being made into an injection, and that can be easily taken over a long period of time. In particular, a composition made from a freeze-dried powder of an edible plant can be processed at extremely low cost because it does not require extraction or purification processes. Furthermore, in order to increase the choline ester and sterilize it, heating can be performed under certain conditions before freeze-drying, and an acid can be added before or after freeze-drying to increase the stability of the choline ester. Because choline esters have a long history of use in food, the composition of the present invention is highly safe and can be used as both a food composition and a pharmaceutical composition for lowering blood pressure. Furthermore, the composition of the present invention can be used in foods, beverages, and pharmaceuticals in various forms, such as cut pieces of edible plants (fresh produce) containing a predetermined amount of choline ester, heated or frozen edible plants, dried powders of edible plants (freeze-dried powders, hot-air dried powders), suspensions of such dried powders, extracts obtained by extracting choline esters from fresh produce, and concentrated extracts obtained by concentrating such extracts.

[0016] Choline esters act on cholinergic receptors (muscarinic acetylcholine receptors and nicotinic acetylcholine receptors), and nicotinic acetylcholine receptor stimulation requires a higher concentration of acetylcholine than muscarinic acetylcholine receptor stimulation (Non-Patent Document 20). Furthermore, while blood pressure is lowered by the action of muscarinic acetylcholine receptors, this effect is thought to be counteracted by the action of nicotinic acetylcholine receptors, as sympathetic nervous activity becomes dominant. The present invention is based on the finding that oral administration of extremely small amounts of choline esters exhibits different effects than oral administration of large amounts. Specifically, the amount of choline ester in the composition of the present invention is extremely small compared to Obisot® Injection 0.1 g / dose, yet it is present in an amount that sufficiently acts on muscarinic acetylcholine receptors to exert a blood pressure lowering effect, yet does not act on nicotinic acetylcholine receptors, thereby not counteracting the blood pressure lowering effect. The composition of the present invention contains a choline ester content that is appropriate for balancing the effects of the two receptors, and therefore exhibits a blood pressure lowering effect when taken orally, contrary to the conventional technical common sense that acetylcholine cannot be administered orally.

[0017] In the present invention, edible plants with high choline ester content, such as eggplant and bamboo shoots, are preferred for oral administration because only small amounts of plant matter are required. For example, if a plant with a low choline ester content, such as lettuce, is selected, a daily intake of more than 7.5 kg of fresh weight would be required, which is not practical for daily intake. Even when freeze-dried, lettuce has a yield of only about 3.60%, and a daily intake of approximately 270 g is desirable. On the other hand, the recommended daily intake of eggplant, determined from animal testing using SHRs, was only 0.41 g of fresh weight, suggesting that a consistent daily intake can provide a blood pressure-lowering effect. Furthermore, when eggplant is freeze-dried, either raw or after heating, the recommended daily intake can be further reduced. Therefore, even when processed, it can be used as an extremely rational and economical oral food. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a graph showing the yield of each fresh agricultural product after freeze-drying. [Figure 2] Figure 2 is a graph showing the choline ester and choline content of each fresh produce. The vertical axis is a logarithmic scale. AcCh: acetylcholine, BuCh: butyrylcholine, PrCh: propionylcholine, Ch: choline. [Figure 3] Figure 3 shows the time course of the choline ester and choline content in fresh eggplant fruit stored at room temperature for 1 to 5 days (p<0.05, p<0.01 vs. the first day). AcCh: acetylcholine, BuCh: butyrylcholine, PrCh: propionylcholine, Ch: choline. [Figure 4] Figure 4 is a graph showing the choline ester content in fresh eggplant fruit stored at room temperature and after 6 days of refrigeration (p<0.05, p<0.01). The vertical axis is a logarithmic scale. AcCh: acetylcholine, BuCh: butyrylcholine, PrCh: propionylcholine. [Figure 5] 5 is a graph showing the time course of the amount of acetylcholine as a function of pH when a standard sample is used. AcCh: acetylcholine. [Figure 6] 6 is a graph showing the change in acetylcholine amount over time as a function of pH when eggplant extract was used. AcCh: acetylcholine. [Figure 7] 7 is a graph showing the change in the amount of acetylcholine in eggplant due to heating. AcCh: acetylcholine. [Figure 8] FIG. 8 is a graph showing the results (systolic blood pressure) of a single oral administration test of freeze-dried eggplant powder containing the equivalent of 1.00×10 −9 mol / kg of acetylcholine to SHR (p*<0.05 vs. administration of pure water). [Figure 9] FIG. 9 is a graph showing the results (diastolic blood pressure) of a single oral administration test to SHR of freeze-dried eggplant powder containing the equivalent of 1.00×10 −9 mol / kg of acetylcholine. [Figure 10]FIG. 10 is a graph showing the results (heart rate) of a test in which a single oral administration of freeze-dried eggplant powder containing the equivalent of 1.00×10 −9 mol / kg of acetylcholine to SHR was performed. [Figure 11] FIG. 11 is a graph showing the results (systolic blood pressure) of a single oral administration test of freeze-dried eggplant powder to WKY rats (p*<0.05, p**<0.01 vs. administration of pure water). [Figure 12] FIG. 12 is a graph showing the results of a vascular isometric tension test on freeze-dried eggplant (p*<0.05, p**<0.01 vs. freeze-drying with a 20-mesh filter; p#<0.05, p##<0.01 vs. hot-air drying with a 20-mesh filter). [Figure 13] FIG. 13 is a graph showing the results (systolic blood pressure) of a test in which freeze-dried eggplant powder was repeatedly administered orally to SHR (p*<0.05, p**<0.01 vs. administration of pure water). [Figure 14] FIG. 14 is a graph showing the results (diastolic blood pressure) of a test in which freeze-dried eggplant powder was repeatedly administered orally to SHR (p*<0.05 vs. administration of pure water). [Figure 15] FIG. 15 is a graph showing the results of repeated oral administration of freeze-dried eggplant powder to SHR (changes in urinary adrenaline levels) (p*<0.05, p**<0.01 vs. administration of pure water). [Figure 16] FIG. 16 is a graph showing the results of repeated oral administration of freeze-dried eggplant powder to SHR (changes in urinary noradrenaline levels) (p*<0.05, p**<0.01 vs. administration of pure water). DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to a composition having a blood pressure lowering effect, which contains a choline ester as an active ingredient, and to said composition which further has an anti-stress effect. The composition of the present invention is for oral ingestion and may contain 5 μg to 50 mg of choline ester. The choline ester content of the composition of the present invention is 5 to 500 μg, preferably 5 to 250 μg, more preferably 10 to 50 μg, and particularly preferably 25 μg, which is preferable when the composition is used for a subject with hypertension, for example. The choline ester content may be 5 μg to 50 mg, preferably 500 μg to 50 mg, and more preferably 500 μg to 5 mg. Such a choline ester content is preferable, for example, when the composition is used for a healthy (non-hypertensive) subject with high blood pressure.

[0020] The composition of the present invention may be a food composition or a pharmaceutical composition. When the composition of the present invention is a pharmaceutical composition, it may be a pharmaceutical composition for lowering blood pressure and / or for anti-stress purposes. The choline ester, which is the active ingredient of the composition of the present invention, can be derived from animals, plants, or microorganisms, but it is preferable to use one derived from an organism that has been eaten by humans, and it is particularly preferable to use one derived from an edible plant.

[0021] The edible plants are not particularly limited as long as they contain choline esters. Examples of edible plants include cucumbers, tomatoes, paprika, bell peppers, eggplants, asparagus, yam, cabbage, lettuce, carrots, apples, shishito peppers, Japanese pears, grapes, radish sprouts, broccoli, alfalfa, pea sprouts, buckwheat, and bamboo shoots. From the viewpoint of acetylcholine content, Solanum melongena (Solanaceae) and Poaceae (Poaceae, Bambusoideae, Bambuseae) are preferred, and the fruit of Solanum melongena and / or young shoots of Poaceae (Poaceae, Bambusoideae, Bambuseae) are particularly preferred.

[0022] Among the varieties of eggplant (Solanum melongena) in the Solanaceae family, Senshu Mizunasu (water eggplant), Batten Nasu (eggplant), Koryo Salad Nasu (also known as Bijin), Higo Murasaki (Japanese sardine), Onaga Nasu (Japanese sardine), Chikuyo, etc. are preferred, and since they can be eaten raw, Senshu Mizunasu (water eggplant), Batten Nasu (eggplant), Koryo Salad Nasu (eggplant), and Higo Murasaki are preferred. Higo Murasaki is particularly preferred.

[0023] Choline esters that can be contained in the composition of the present invention include acetylcholine, butyrylcholine, propionylcholine, lactoylcholine, etc., and the composition may contain one or more of these. In particular, when the choline ester is derived from a plant, the composition of the present invention contains one or more selected from the group consisting of acetylcholine, butyrylcholine, and propionylcholine, but does not contain lactoylcholine.

[0024] The composition of the present invention has a daily intake of choline ester adjusted to fall within a predetermined range. For example, the choline ester content in one package is adjusted to 5 to 125 μg, preferably 10 to 75 μg, and particularly preferably 15 to 50 μg, and can be orally taken once to several times (preferably about three times) per day. Alternatively, for example, multiple packages may be used so that the total amount of choline ester falls within the above range. In this case, the choline ester concentration will be 5 to 2500 μg / g. Furthermore, for example, when the daily intake of choline ester is adjusted to be higher than the above-mentioned range, i.e., for example, to 5 μg to 50 mg, preferably 500 μg to 12.5 mg, and more preferably 500 μg to 1.25 mg, the choline ester content per package can be adjusted to be higher in accordance with the above-mentioned method. In this case, the choline ester concentration may be 500 to 250 mg / g.

[0025] The composition of the present invention is adjusted to a predetermined choline ester content. For example, the composition of the present invention can be prepared by adjusting the choline ester content of fresh agricultural products, such as cut pieces, frozen products, freeze-dried products, or extracts. The composition of the present invention is preferably a composition consisting of freeze-dried powder and / or extract of an edible plant. The composition of the present invention may be cut into daily portions to provide the choline ester content required for daily intake, or may be individually packaged in vacuum packs or the like to prevent deterioration of quality and browning of the flesh. The composition of the present invention is preferably frozen. The freezing process can suppress the activity of cholinesterase contaminating the composition and preserve the choline ester for a long period of time. The composition of the present invention is preferably a part or whole of a frozen fruit of Solanum melongena.

[0026] In one aspect, the present invention relates to a method for producing a composition for oral administration having antihypertensive and / or anti-stress effects, which contains a choline ester as an active ingredient. The method includes freeze-drying an edible plant into a freeze-dried powder and / or a hot air-dried powder or an extract, and distributing the freeze-dried powder and / or the hot air-dried powder or the extract so as to obtain a predetermined amount of choline ester. Here, the predetermined amount may be 5 μg to 250 mg, preferably 5 μg to 50 mg, and more preferably 10 μg to 50 mg. In one embodiment, the predetermined amount may be, for example, 5 to 500 μg, 5 to 250 μg, 10 to 50 μg, or 25 μg.

[0027] The method of the present invention can further include heating the edible plant. Heating can be carried out in a microwave oven or by boiling in hot water. For example, when heating in a 550 W microwave oven, heating is carried out for 1 to 15 minutes, preferably 2 to 10 minutes, and more preferably 4 to 6 minutes per 100 g of edible plant. When boiling in hot water, heating is preferably carried out in water at 90 to 100°C. Heating the edible plant in this manner not only sterilizes the plant but also increases the choline esters in the plant. In one aspect, the method of the present invention is characterized in that, in addition to the usual freeze-drying or hot air drying that prevents spoilage by microorganisms, the edible plants are further heat-treated (to have a sterilizing effect and increase choline esters).

[0028] The method of the present invention can further comprise suspending the freeze-dried powder and / or hot-air-dried powder of the edible plant in water and adding an acid to the resulting suspension. The pH of the suspension to which the acid has been added is adjusted to, for example, 5.5 to 4.5, preferably 5.4 to 4.6. Adjusting the pH in this manner stabilizes the choline ester, resulting in a composition (suspension) with excellent long-term storage stability.

[0029] The present invention also relates to a method for producing an extract for oral administration having antihypertensive and / or anti-stress effects, which contains a choline ester as an active ingredient. The method of the present invention comprises extracting an edible plant, or a freeze-dried and / or hot-air-dried powder of an edible plant, with ethanol or hydroethanol. More specifically, the method of the present invention can include an extract enriched in choline esters, which can be obtained by adding ethanol or hydroethanol to freeze-dried powder and / or hot-air-dried powder of an edible plant, or by adding ethanol to a fresh edible plant, grinding the plant, and removing any residue.

[0030] When extraction is performed with aqueous ethanol, the ethanol concentration of the aqueous ethanol is not particularly limited, but can be appropriately selected from the viewpoint of the extraction rate and concentration rate of choline ester, etc. The ethanol concentration of the aqueous ethanol may be, for example, 10% (w / w) or more, preferably 10 to 99% (w / w), more preferably 25 to 60% (w / w) or 95% (w / w) or more, and particularly preferably 30 to 60% (w / w) or 99% (w / w) or more. L-ascorbic acid may be added to the ethanol or aqueous ethanol used for extraction, for example, at a concentration of 1 to 5 wt %, preferably 3 wt %. The method of the present invention may include adjusting the choline ester content of the extract to 5 μg to 50 mg.

[0031] The present invention also relates to an orally ingested composition having antihypertensive and / or anti-stress effects, which contains as an active ingredient a choline ester produced by the above-mentioned method. The dry powder of the composition of the present invention is preferably a powder that has been passed through a sieve of an appropriate mesh, for example, the composition of the present invention is preferably a freeze-dried powder and / or a hot-air dried powder that can pass through a 20-mesh sieve. Here, the hot air dried powder can be prepared, for example, by drying an edible plant by exposing it to hot air at about 90°C for about 1 to 2 hours, and then pulverizing it to form a powder.

[0032] The composition of the present invention can be used as an active ingredient in various functional health foods or pharmaceutical compositions. In the case of food, it can be used as a food composition by combining with an appropriate food additive.In addition to such food compositions, it can also be provided in a form that can be ingested daily, such as by adding it to green tea, black tea, oolong tea, multigrain tea, etc. to make a beverage, or by adding it to biscuits, bread, candy, etc. to make a food.It can also be used as a so-called supplement in an appropriate dosage form according to the preparation of pharmaceuticals described below.

[0033] When used as a pharmaceutical, it can be combined with appropriate pharmaceutical additives and used in various dosage forms according to conventional compounding techniques, such as solid preparations such as powders, granules, capsules, pills, and tablets, and liquid preparations for oral administration such as solutions, suspensions, and emulsions.

[0034] When the composition of the present invention is used as a food product, it can be used not only as a general food or drink product but also as a functional health food product that exerts a specific function to promote health. Specific forms in this case include supplements consisting of capsules, tablets, powders, granules, etc. containing the composition of the present invention as an active ingredient; bakery foods such as bread, cakes, cookies, etc.; seasonings such as sauces, soups, dressings, mayonnaise, etc.; dairy products such as milk, yogurt, cream, etc.; confectioneries such as chocolate, candy, etc.; and various beverages such as green tea, black tea, oolong tea, barley tea, multigrain tea, fruit juice, vegetable drinks, dairy drinks, soft drinks, and carbonated drinks.

[0035] When the composition of the present invention is used as an active ingredient in a pharmaceutical composition, the dosage will vary depending on the ratio of each ingredient, and also on various factors such as the patient's age, weight, sex, symptoms, and administration method. However, for adults, when administered orally daily, the choline ester content can be selected generally within the range of 5 μg to 50 mg, and in one embodiment, within the range of 5 μg to 500 μg. The dosage can also be increased or decreased as appropriate depending on the degree of symptom improvement. The number of doses can be administered once a day or in divided doses several times a day.

[0036] When the composition of the present invention is used as a food, the intake amount can be selected in accordance with the case of oral administration of the above-mentioned medicines. However, unlike medicines, in the case of foods and drinks, there are no particular restrictions on the dosage and frequency of administration, so as long as no particularly serious symptoms occur, the intake amount may be selected without being limited to the above range, taking into consideration the purpose of maintaining health, as well as taste and preference. [Example]

[0037] Hereinafter, embodiments of the present invention will be described with reference to examples and test examples, but the present invention is not limited to these examples. The meanings of the abbreviations used in the examples are as follows: EN: (2-aminoethyl)trimethylammonium pivaloylamide, AcCh: acetylcholine, BuCh: butyrylcholine, Ch: choline, LaCh: lactoylcholine, PrCh: propionylcholine. Hereinafter, EN, AcCh, BuCh, Ch, LaCh, and PrCh will be collectively referred to as choline compounds. EtOH: ethanol.

[0038] [Experimental Materials and Methods] 1. Analysis Sample The various agricultural products listed in Table 1 were obtained as analytical samples. [Table 1]

[0039] 2.Extraction method (1) Sample preparation Immediately after receiving the fresh agricultural products (analysis samples), the surface was washed with tap water. After wiping off the moisture, the edible portions were sliced ​​into 1-3 cm widths using a knife, as needed. The edible portions were freeze-dried using a freeze dryer (FDU-2000, Tokyo Rikakikai Co., Ltd.). The freeze-dried material was pulverized into powder using a mill mixer (MASTER, Tokyo Unicom Co., Ltd.).

[0040] (2) Preparation of reagents Sodium dihydrogen phosphate (59.99 mg) and disodium hydrogen phosphate (70.98 mg) were weighed and dissolved in pure water (100 mL) to prepare a 10 mM phosphate buffer solution. EN (0.80 mg) was dissolved in 10 mM phosphate buffer (1 mL) to prepare a solution of 800.00 μg / mL, and then diluted 100-fold to prepare a solution of 8.00 μg / mL, which was used as the EN internal standard.

[0041] (3) Shaking extraction The lyophilized material (10 mg) was weighed into a 2 mL tube and EN internal standard (10 μL) was added. 10 mM phosphate buffer (190 μL) was added, and the mixture was stirred for 3 minutes using a vortex mixer (FLX-S, FRONT LAB, AS ONE Corporation). The mixture was then centrifuged (1000 × g, room temperature, 3 minutes) using a centrifuge (CFM-200, Iwaki Corporation) to obtain the supernatant. 200 μL of 10 mM phosphate buffer was added to the residue, and the stirring, centrifugation, and supernatant collection procedures were repeated twice. The collected supernatants were combined (approximately 600 μL) to prepare the extracted sample.

[0042] (4) Solid phase extraction The solid-phase extraction cartridge used was a weakly acidic cation exchange cartridge, Inert Sep CBA 100 mg / 1 mL (GL Sciences). The solid-phase extraction cartridge was activated with methanol (1 mL) and purified water (1 mL) and equilibrated with 10 mM phosphate buffer (8 mL). The extracted sample (approximately 600 μL) prepared in (3) above was then added. The cartridge was stabilized with 10 mM phosphate buffer (600 μL), washed with purified water (2.5 mL), and eluted with hydrochloric acid (500 μL).

[0043] (5) Quantitative sample preparation The eluate (500 μL) obtained by solid-phase extraction with hydrochloric acid was filled to exactly 1 mL using a 1 mL volumetric flask with LC / MS / MS analysis solvent, and then divided into three 300 μL aliquots. The choline compound mixture solution was added to each aliquot, and the LC / MS / MS analysis solvent was added to dilute the eluate two-fold (Table 2), to prepare quantitative samples.

[0044] [Table 2]

[0045] The choline compound mixture solution was prepared as shown in Table 3, and the concentration of each choline compound stock solution was determined based on the analysis results of a sample without the choline compound mixture solution (Table 2-A). The choline compound stock solution was diluted with the LC / MS / MS analysis solvent to prepare each concentration. If a choline compound was not detected, an equal volume of the LC / MS / MS analysis solvent without that compound was added.

[0046] [Table 3]

[0047] 3.LC / MS / MS analysis (1)LC / MS / MS analysis conditions The column used was a YMC-Triart PFP (4.6 mm × 250 mm, 5 μm, YMC Corporation). The analytical solvent was 0.01% formic acid and 33% methanol in water. The flow rates were 0.5 mL / min (LC) and 0.3 mL / min (MS), injection volume was 50 μL, separation temperature was 40 °C, analysis time was 30 min, ionization mode was ESI+MRM, capillary voltage was 3500 V, cone voltage was 10 V, collision voltage was 10 V, N2 gas flow (desolvation) was 600 L / hr, N2 gas flow (cone) was 50 L / hr, N2 source temperature was 120 °C, and N2 desolvation temperature was 350 °C. LC / MS / MS analysis was performed using an ACQUITY UPLC [UPLC, Waters Corp.]-Quattro micro API [MS, Waters Corp.]. The MRM mode designation m / z for each choline compound is shown in Table 4.

[0048] [Table 4]

[0049] (2) Standard addition method A calibration curve was created from the chromatographic peak area values ​​obtained by LC / MS / MS analysis, and choline compounds were quantified by the standard addition method. The concentration of each choline compound was corrected by the recovery rate calculated from the calibration curve of the EN internal standard, and the exact concentration of the choline compound in the quantified sample was calculated. The obtained concentrations were converted to the content in the lyophilized product (mg / g D.W.), and the amount of each choline compound per 100g fresh weight (μg / 100g F.W.) was calculated from the yield before and after lyophilization.

[0050] [Experiment 1] Fresh weight and dry weight The fresh and dry weights of various fresh agricultural products were determined, and the yields before and after freeze-drying were calculated. The results are shown in Figure 1. As shown in Figure 1, lettuce (Shinano Hope) (fresh weight: 79.19g, dry weight: 2.85g) had the lowest yield at 3.60%, while mountain yam (Nagaimo) (fresh weight: 79.76g, dry weight: 21.23g) had the highest yield at 26.62%. For most agricultural products, the yield was around 5 to 10%.

[0051] [Experiment 2] Determination of choline compounds in various fresh agricultural products Choline compounds were extracted from 20 types of fresh agricultural products using the method described above, and five choline compounds, AcCh, Ch, BuCh, PrCh, and LaCh, were analyzed by LC / MS / MS (n = 3). The results are shown in Figure 2. AcCh and Ch were detected in all tested produce. On the other hand, LaCh was not detected in any tested produce. BuCh was detected in all tested produce except for tomatoes, lettuce, alfalfa sprouts, pea sprouts, buckwheat sprouts, apples, Japanese pears, grapes, and bamboo shoots. PrCh was detected in all tested produce except for tomatoes and pea sprouts. The quantitative results of this experiment showed that bamboo shoots and eggplants contained over 1,000 times more AcCh than the other 18 fresh produce, with bamboo shoots having the highest AcCh content.

[0052] [Experiment 3] Quantitative analysis of choline compounds in six eggplant varieties In Experiment 2, we investigated differences in choline compound content among eggplant varieties, which are rich in AcCh, can be eaten raw, and can be grown year-round. Choline compounds were extracted from six eggplant varieties using the method described above, and five choline compounds (AcCh, Ch, BuCh, PrCh, and LaCh) were analyzed by LC / MS / MS in triplicates. The results are shown in Table 5. LaCh was not detected in any of the cultivars. Among the six cultivars, Higomurasaki had the highest AcCh content.

[0053] [Table 5]

[0054] Previously, AcCh in eggplant was quantified using high-performance liquid chromatography-electrochemical detection (HPLC-ECD) and found to be 6.09 × 10 3 It has been reported that the content of AcCh is μg / 100 g F.W. (Non-Patent Document 7), and the quantitative results of the LC / MS / MS analysis in this experiment showed an equivalent AcCh content.

[0055] [Experiment 4] Changes in choline compound content in eggplant over time The storage period for eggplant is generally 2-3 days at room temperature and about a week at 7-10°C, but storing it at temperatures below 6°C is not recommended as it can cause chilling damage such as pitting, where the skin becomes crater-like, browning, and black depressions. The optimum temperature for storing eggplant fruit is between 10°C and 20°C. In this experiment, we investigated the changes in choline compound levels over time when eggplant cultivar Higomurasaki was stored at room temperature. The plants were wrapped in newspaper and stored in a dark place at room temperature. Approximately 10g of each sample was taken once per day from the same plant. One of the halves was divided into two and sampled on the sixth day. Another sample was also prepared by wrapping another half in newspaper and storing it in the refrigerator for six days. Choline compounds were extracted using the method described above, and five choline compounds (AcCh, Ch, BuCh, PrCh, and LaCh) were analyzed by LC / MS / MS (n=3). The results are shown in Table 6, Figures 3 and 4.

[0056] [Table 6]

[0057] LaCh was not detected in either case. The content of each choline compound tended to decrease gradually with the length of storage time (Fig. 3). In this experiment, no chilling damage such as pitting occurred in the refrigerated specimens, but the amount of choline esters remaining was higher in specimens stored at room temperature than in specimens stored in the refrigerator (Fig. 4).

[0058] The results of this experiment showed that when eggplants were stored at room temperature for 5 days, the choline ester content tended to decrease gradually over time. However, since more than 80% of the AcCh content remained in the eggplants stored at room temperature for 5 days compared to the first day, there was no sudden change in the AcCh content in the eggplants during storage at room temperature, and it is considered to be highly stable.

[0059] [Experiment 5] Stability of AcCh under acidic conditions We investigated methods to further stabilize choline esters in eggplant. Generally, ester compounds are easily hydrolyzed under basic conditions but are relatively stable under acidic conditions. Furthermore, over 99% of the choline esters in eggplant are AcCh. Therefore, we first investigated the change in concentration of AcCh preparations over time under neutral and acidic conditions. A neutral solution (pH 7.0) was prepared using phosphate buffer, and acidic solutions (pH 4.1, pH 3.6, pH 3.1, and pH 2.6) were prepared by adding acetic acid. Solutions with an AcCh concentration of 1.9 mg / mL were stored at room temperature (23–25°C) for 0, 3, 6, and 12 days, after which the AcCh content was quantified. The results are shown in Table 7 and Figure 5.

[0060] [Table 7]

[0061] Under neutral conditions, AcCh decomposed over time, decreasing to approximately half after 12 days. On the other hand, at pH 4.1 (acetic acid concentration 0.0015%), AcCh remained stable, and the AcCh content tended to increase after 12 days. At pH 4.1 to pH 3.1, it was clear that more than 97% of AcCh remained stable even after 12 days. At pH 2.6, AcCh stability decreased slightly, with only 88% remaining after 12 days.

[0062] Next, the stability of AcCh contained in the eggplant extract was examined under neutral and acidic conditions. A neutral solution (pH 7.0) was prepared using phosphate buffer, acidic solutions (pH 5.2, pH 5.0, pH 4.3, and pH 3.5) were prepared using acetic acid, and acidic solutions (pH 5.4 and pH 4.6) were prepared using ascorbic acid. Eggplant extract was added to each solution to achieve an AcCh concentration of 0.040 mg / mL. The solutions were allowed to stand at room temperature (23–25°C) for 0, 3, 6, and 12 days, after which the AcCh content was quantified. The results are shown in Table 8 and Figure 6.

[0063] [Table 8]

[0064] AcCh in eggplant extract was unstable under neutral conditions, with more than half of the AcCh decomposing after 3 days and almost all of it decomposing after 12 days. It was relatively stable under acidic conditions, with over 88% of the AcCh remaining after 12 days at pH 5.4-4.6, but in a solution adjusted to pH 3.5 with acetic acid, 24.6% of the AcCh was decomposed after 12 days. AcCh in eggplant was stable at room temperature under acidic conditions of pH 5.4-4.6. These results suggest that choline esters in agricultural products such as eggplant can be stabilized by adjusting the pH to 4.5 or 5.5 by adding an organic acid. If the pH of the suspension is within this range, the acid can be added at the appropriate time.

[0065] [Experiment 6] Changes in AcCh content in eggplant due to heating To investigate how heating affects AcCh content in eggplant, a representative long-oval eggplant variety, "Ryuma," was cut into 2-3 cm thick slices, wrapped in plastic wrap, and heated in a microwave oven (550 W) for varying periods of time. The slices were then allowed to cool at room temperature and freeze-dried, and the AcCh content of each slice was quantified. The change in AcCh content in eggplant was also investigated when the slices were placed in boiling water and boiled for 5 minutes. The temperature was measured and recorded immediately after each heating treatment. The results are shown in Table 9 and Figure 7.

[0066] [Table 9]

[0067] It was found that choline esters in eggplant increased when heated in a microwave oven. Five minutes of heating per 100g produced the greatest increase in AcCh, increasing the AcCh content 2.9-fold compared to unheated. Two minutes of heating per 100g increased AcCh content 2.6-fold, and 10 minutes of heating per 100g increased it 2.8-fold. The internal temperature reached 91.1°C after two minutes of heating per 100g, dropped to 92.6°C after five minutes, and dropped to 87.8°C after 10 minutes due to evaporation of water. Boiling per 100g for five minutes increased AcCh content 2.5-fold, with the internal temperature reaching 95.6°C. From the above results, it was inferred that under general heat processing conditions, the AcCh content increases and remains stable to a certain extent. Even if the processing conditions are different, if the temperature change is similar, the AcCh content is expected to increase. In addition, sterilization can be performed during this process.

[0068] The results of Experiments 4 to 6 revealed that acetylcholinesterase (EC number 3.1.1.7), a choline ester-degrading enzyme, affects choline ester content. As described in Non-Patent Document 3, acetylcholinesterase is widely present in plants. The decrease in AcCh shown in Table 8 is thought to be primarily due to decomposition by acetylcholinesterase. At neutral pH, close to the optimal pH of 8.0 to 8.5 for acetylcholinesterase, more AcCh was decomposed, whereas at low pH, where enzyme activity is reduced, AcCh decomposition was inhibited. Furthermore, heating inactivated acetylcholinesterase, preventing AcCh decomposition and resulting in the evaporation of water, resulting in concentration. Therefore, processing techniques that control acetylcholinesterase are important for eggplants and eggplant products. Therefore, we investigated the changes in AcCh content in frozen eggplants and eggplant products, which inhibit the function of eggplant acetylcholinesterase.

[0069] [Experiment 7] Changes in AcCh content in frozen fresh eggplant and frozen-heated eggplant Fresh eggplant (cut into approximately 10g pieces) and cooked eggplant (cut into approximately 10g pieces and microwaved (550W) for 5 minutes per 100g) were frozen at -20°C. Samples were taken every month, freeze-dried, and the AcCh content was quantified to examine changes in AcCh content over a 6-month period. Fresh eggplant and cooked eggplant were each cut from the same eggplant. The results are shown in Table 10.

[0070] [Table 10]

[0071] The AcCh content in fresh and cooked eggplants remained almost unchanged by freezing, demonstrating that freezing can stably preserve the choline esters in eggplants for long periods of time. This is the result of freezing fresh eggplants, which suppresses the activity of cholinesterase, a factor that breaks down choline esters in eggplants. The change in AcCh content was almost the same as that observed in cooked eggplants, which had been heated beforehand to inactivate the cholinesterase, suggesting that freezing sufficiently suppressed cholinesterase activity.

[0072] [Experiment 8] Eggplant extract preparation test Eggplant extracts were prepared using freeze-dried eggplant powder and fresh eggplant. The method for preparing the extract using freeze-dried eggplant powder is as follows. Freeze-dried eggplant powder (1 g) was weighed into a 50 mL centrifuge tube and 10 times the weight (10 g) of a solvent containing 10% EtOH increments (from 10% to 100%) was added. The mixture was shaken at 3000 rpm for 30 minutes at room temperature and then filtered to obtain the supernatant. The supernatant was transferred to a 200 mL eggplant flask, and purified water (10 g) was added and concentrated using an evaporator. When the liquid volume was reduced to approximately 1 / 5 of its original volume, the contents were completely transferred to a 15 mL centrifuge tube and freeze-dried. The freeze-dried product was used as the eggplant extract. The yield was calculated and quantified using the methods described in "2. Extraction Method" and "3. LC / MS / MS Analysis" above. For extractions using 50% EtOH and EtOH, L-ascorbic acid was added to prepare the extract. The results are shown in Table 11.

[0073] [Table 11]

[0074] When using fresh eggplant, various concentrations of EtOH were added to cut fresh eggplant (10 g) and crushed in a mill mixer. The same method was used to prepare freeze-dried eggplant powder, and the yield and choline ester content were determined in the same manner. The weight of EtOH added was 0.5, the same amount, or twice the weight of the fresh eggplant. When using the same amount of EtOH, L-ascorbic acid was added to prepare the extract. The results are shown in Table 12.

[0075] [Table 12]

[0076] Eggplant extract is a water-soluble semi-solid product that is more concentrated in choline esters than freeze-dried and / or hot-air-dried powders. The addition of L-ascorbic acid to acidify the solution and inhibit oxidation stabilizes the choline esters and prevents color change during the extraction process.

[0077] [Experiment 9] Single oral administration test of freeze-dried eggplant to spontaneously hypertensive rats Freeze-dried eggplant was orally administered in a single dose to spontaneously hypertensive rats (SHRs) to examine its antihypertensive effect. Male 14-week-old SHRs (body weight 324-368 g) were fasted for 12 hours after one week of acclimation, and then a single dose of freeze-dried eggplant powder suspended in water was orally administered using a probe. Freeze-dried eggplant powder was prepared by washing Senshu water eggplants produced in Hannan City, Osaka Prefecture, sterilizing them with hypochlorite, freeze-drying the edible portion, and then finely grinding it. Over 99% of the choline esters in the freeze-dried eggplant powder were AcCh, and the AcCh content was 2.25 mg / g D.W. The dose of AcCh administered was 1.00 × 10 -9 The freeze-dried eggplant powder was administered orally in a single dose of 0.065 mg / kg, equivalent to 0.146 μg / kg mol. To compare the antihypertensive effect, 1.00 × 10 AcCh preparation was administered orally. -9 A single oral administration test of 1000 mol / kg was conducted in the same manner, and systolic blood pressure was measured. A control group received only water under the same conditions. At 0, 3, 6, 9, and 24 hours after oral administration, systolic blood pressure, diastolic blood pressure, and heart rate were measured by the tail cuff method using a non-invasive blood pressure monitor, Softron BP-98A (Softron Co., Ltd., Tokyo). The results of the single oral administration test of eggplant freeze-dried powder are shown in Figures 8 to 10.

[0078] The eggplant freeze-dried powder group showed a significant decrease in systolic blood pressure (p<0.05) compared to the control group at 3 and 9 hours after administration. The maximum blood pressure decrease was -17.8 mmHg at 9 hours after administration, -10.0 mmHg compared to the control group. -9 The blood pressure lowering effect was almost the same as that of the control group when administered 100 mol / kg. Diastolic blood pressure also tended to decrease, with a maximum blood pressure decrease of -11.3 mmHg 9 hours after administration, -8.3 mmHg compared to the control group. Heart rate tended to be lower than the control group 3 hours after administration, but then tended to increase until 9 hours after administration. Thus, the blood pressure lowering effect of oral administration of eggplant was confirmed.

[0079] The effective daily dose of AcCh in SHR after a 12-hour fast is 1.00 × 10 -9 mol (0.146 μg) / kg, the effective daily dose of AcCh in non-fasting SHR was 1.00 × 10 -8 mol (1.46 μg) / kg (Non-Patent Document 21). The AcCh dose that causes a hypotensive effect when orally ingested by SHR under normal conditions was extrapolated to humans using Cleber's law (Non-Patent Document 22). Cleber's law states that the metabolic rate in mammals is proportional to the 3 / 4 power of body weight. Assuming that the body weight of an SHR is 370 g and that of a human is 60 kg, the effective dose for humans is estimated to be 45.4 times the effective dose for SHR. In other words, the daily AcCh intake by an SHR that causes a hypotensive effect is 3.70 × 10 -9 mol (0.540 μg), and the effective daily human dose is 1.68 × 10 -7 mol (24.5 μg).

[0080] The present inventors have also previously discovered the antihypertensive and vasodilatory effects of fermented osmanthus (a product of lactic acid fermentation of buckwheat plants), which contains choline esters AcCh, BuCh, LaCh, and PrCh as active ingredients, and have demonstrated that purified AcCh, BuCh, and PrCh exert antihypertensive effects when administered orally in a single dose to SHRs (Patent Documents 1 and 2). Further investigations were conducted to examine the antihypertensive effects of oral administration of choline esters in humans. Subjects with high-normal blood pressure and stage 1 hypertension (6 men and 6 women, aged 33-63 years) were asked to consume one bottle of a fermented Kyobaku-containing beverage (containing 25 μg of choline ester per bottle) daily for four weeks. As a result, the subjects' systolic blood pressure decreased by -11.8 mmHg (P=0.0080, t-test) compared to before ingestion. In other words, in humans, a significant decrease in blood pressure was observed after four weeks of continuous ingestion of a fermented Kyobaku-containing drink containing 25 μg of choline ester as a blood pressure lowering component. Thus, the blood pressure lowering effect was confirmed at a dose very close to the effective dose of AcCh that caused a blood pressure lowering effect in SHRs, calculated by extrapolating it to humans using Kleber's law.

[0081] In a repeated oral administration study to non-fasting SHR, -9 The hypotensive effect was significantly attenuated at 0.292 μg / kg mol. This AcCh dose was extrapolated to humans using Kleber's law (Non-Patent Document 22), and the lower limit of the effective daily dose in humans was 3.36 × 10 -8 mol (4.91 μg). In a similar test, 2.00 × 10 -7 When this AcCh dose was extrapolated to humans using Kleber's law (Non-Patent Document 22), the upper limit of the effective daily dose in humans was 3.36 × 10 -6 Therefore, the effective daily human dose for hypertensive individuals with genetic traits, such as SHR, is estimated to be in the range of 5 to 500 μg. The dose of choline ester that induces a blood pressure lowering effect can be finally determined by taking into consideration the effective human dose estimated as above, as well as the amount of choline ester derived from food other than the composition of the present invention.

[0082] [Experiment 10] Single oral administration test of freeze-dried eggplant to normotensive rats The antihypertensive effect of freeze-dried eggplant was examined by single oral administration to Wistar Kyoto rats (WKY rats) using the same method as in Experiment 9. WKY rats are the parent strain of SHR and are normotensive rats with genetic factors that cause hypertension, and are used as test controls. The animals used were male 14-week-old WKY rats (body weight 320-362 g), and the same freeze-dried eggplant powder as in Experiment 9 was used. The dose was 0.065 mg / kg (AcCh 1.00 × 10 -9 mol (equivalent to 0.146 μg) / kg) and 6.5 mg / kg (AcCh 1.00 × 10 -7 mol (equivalent to 14.6 μg / kg). The results of a single oral administration test of freeze-dried eggplant powder to WKY rats are shown in Figure 11.

[0083] AcCh caused hypotensive effects in SHRs at 1.00 × 10 -9 Administration of freeze-dried eggplant powder equivalent to 1.00×10 mol / kg did not change the blood pressure of WKY rats. -7 mol / kg, the administration of freeze-dried eggplant powder resulted in a 1.00×10 -9 The antihypertensive effect was equivalent to that of the freeze-dried eggplant powder administered at a dose equivalent to 1000 mol / kg. This indicates that a larger amount of freeze-dried eggplant powder is required to induce antihypertensive effects in normotensive WKY rats.

[0084] [Experiment 11] Measurement of vascular isometric tension using crushed eggplant samples The crushed eggplant samples used in the tests were prepared as follows. Senshu Mizunasu eggplants from Hannan City, Osaka Prefecture were washed with water and sterilized with hypochlorite. The edible portions were sliced ​​into 1-3 cm widths and ground into a paste using a commercially available food processor (Crush Mill IFM-C20G, Iwatani Corporation). Eggplant slices were heat-sterilized (approximately 90°C for approximately 5 minutes) and freeze-dried to obtain freeze-dried eggplant fruit. Alternatively, eggplant hot-air dried eggplant fruit was obtained by hot-air drying (approximately 90°C for approximately 1-2 hours). These were then ground into powder using a mill mixer (MASTER, Tokyo Unicom Corporation). Each pulverized sample was passed through a stainless steel sieve (JIS standard, 20 mesh), and those that passed through were designated as 20 mesh pass, while those that did not were designated as 20 mesh on. As a result, fresh eggplant fruit pulverized material 20 mesh pass (sample 1), fresh eggplant fruit pulverized material 20 mesh on (sample 2), freeze-dried powder 20 mesh pass (sample 3), freeze-dried powder 20 mesh on (sample 4), hot-air dried powder 20 mesh pass (sample 5), and hot-air dried powder 20 mesh on (sample 6) were prepared.

[0085] All samples were stored at -98°C until use in testing. The choline ester content in Samples 1 and 2 was quantified by freeze-drying, finely pulverizing, and then using the methods described above in "2. Extraction Method" and "3. LC / MS / MS Analysis." Samples 3 and 5 were used as powders and quantified by the methods described above in "2. Extraction Method" and "3. LC / MS / MS Analysis." Samples 4 and 6 were pulverized and then quantified by the methods described above in "2. Extraction Method" and "3. LC / MS / MS Analysis."

[0086] More than 99% of the choline esters in the pulverized eggplant sample were AcCh, and the AcCh content was as shown in Table 13. [Table 13]

[0087] The vascular isometric tension was measured as follows. Male 14-week-old SHR rats (weight: 320-346 g) were anesthetized with ether and sacrificed by laparotomy. The rats were then exsanguinated and the thoracic aorta was rapidly excised. The excised aorta was immersed in saline to thoroughly flush out the blood, and then the connective tissue and adipose tissue attached to the blood vessels were removed in Krebs-Henseleit solution to prepare ring specimens approximately 2-3 mm wide. The ring preparations were placed in the organ bath of an isometric tension tester (UFER UC-05A, Iwashiya Kishimoto Medical Industries, Kyoto, Japan) filled with Krebs-Henseleit solution (119 mM NaCl, 4.7 mM KCl, 1.1 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, pH 7.4, 37°C) aerated with a mixed gas (95% O2, 5% CO2). A resting tension of 1.0 g was applied to the ring preparations for 60 minutes. Phenylephrine (PE, 0.3 μM) was then added to the organ bath. Once the vessels contracted and stabilized, AcCh (final concentration 100 μM) was added. After confirming the integrity of the endothelium, the ring preparations were washed with Krebs-Henseleit solution and returned to the resting tension. After 15 minutes, the vessels were contracted again with the contractile agent PE (0.3 μM) to confirm that the contraction reached its maximum. -9 , 10 -8 , 10 -7 , 10 -6.5 , 10 -6 , 10 -5.5 , 10 -5 Each pulverized eggplant sample suspended in Krebs-Henseleit solution was added to the vessel so that the EC was 1.0 M. The tension change was measured and recorded using a transducer (UFER UM-20, Iwashiya Kishimoto Medical Industry Co., Ltd.), and the expansion rate (%) was calculated using the following formula (1). The mean ± standard error (Mean ± SE) of the results of three repeated tests was calculated. The results of the vascular isometric tension test for the pulverized eggplant samples are shown in Figure 12, and the results and the EC calculated by the probit method are also shown. 50 (50% effective concentration), and the statistical analysis results are shown in Table 14.

[0088] Formula (1) Expansion rate (%) = {maximum tension (g) - tension when sample is added (g)} / maximum tension (g)

[0089] [Table 14]

[0090] AcCh final concentration is 10 -9 , 10 -8 , 10 -7 , 10 -6.5 , 10 -6 , 10 -5.5 , 10 -5 The concentration of the eggplant pulverized sample added to achieve M is shown in Table 14. Since the freeze-drying yield of the fresh eggplant pulverized material used in this test was 6.0%, the eggplant pulverized material concentration in the vascular isometric tension test using 20-mesh fresh eggplant pulverized material was the highest, at 19 mg / mL. The contents of chlorogenic acids and gamma-aminobutyric acid (GABA), which may affect vasodilation other than choline esters, were 0.56 mg / gFW and 0.24 mg / gFW, respectively. The concentrations in this test system were 11 μg / mL and 4.8 μg / mL, respectively. In tests using the respective preparations, no effect on blood vessels was observed at these concentrations. In test systems using other crushed eggplant, the concentrations of these compounds were lower, so their involvement in vasodilation was extremely limited, and choline esters were considered to be the main vasodilator components.

[0091] All the eggplant powder samples showed a dose-dependent vasodilatory effect. The freeze-dried powder (20 mesh pass) (sample 3) showed the strongest vasodilatory effect when the AcCh concentration in the chamber was 10 -8 The eggplant crushed sample (6.2 × 10 -4 The addition of eggplant freeze-dried powder (mg / mL) showed a significant 19% vasodilation (p<0.05) compared to before the addition of eggplant freeze-dried powder. -6 M (eggplant crushed sample 6.2 × 10 -2 At higher concentrations, although there was some contraction, the vasodilation was still significant. The maximum dilation rate was observed at an AcCh concentration of 10 -6 The eggplant crushed sample (6.2 × 10 -2The next strongest vasodilatory effect was observed in the hot air dried powder (20 mesh pass) when the AcCh concentration in the chamber was 10 -8 The eggplant crushed sample (7.2 × 10 -4 The addition of eggplant freeze-dried powder (mg / mL) showed a significant 15% vasodilation (p<0.05) compared to before the addition of eggplant freeze-dried powder. -6 M (eggplant crushed sample 7.2 × 10 -2 At higher concentrations, although there was some contraction, the vasodilation was still significant. The maximum dilation rate was observed at an AcCh concentration of 10 -6 The eggplant crushed sample (7.2 × 10 -2 These results confirmed the strong vasodilatory effect of eggplant dried powder.

[0092] On the other hand, samples 4 and 6 had an AcCh concentration of 10 -7 M (lyophilized powder 8.9×10 -3 mg / mL, hot air dried powder 9.8×10 -3 The maximum dilation rate was observed at AcCh concentrations of 10 mg / mL. -5 When the eggplant pulverized sample was added, the vasodilatory effect of the freeze-dried powder was 57% and that of the hot-air dried powder was 50%. -8 A significantly stronger vasodilatory effect was observed in M, and -8 Similarly, the vasodilatory effect of sample 5 was 6.5 times greater than that of sample 6 at an AcCh concentration of 10 -8 Significantly stronger than M, 10 -8 The vasodilatory effect of M was 7.8 times greater. From the above results, it was confirmed that Samples 3 and 5 had stronger vasodilatory effects than Samples 4 and 6, respectively.

[0093] In addition, samples 1 and 2 each had an AcCh concentration of 10 -6 M (1.4 mg / mL), 10 -6.5Although significant vasodilation (p<0.05) was observed from M (0.59 mg / mL), the vasodilation effect was weaker than that of the dry powder, and it was revealed that the 20-mesh pass dry powder exerted a higher effect in vivo.

[0094] EC 50 is an index showing the strength of the biological effect of the test sample. 50 The EC value of sample 5 was 0.015 mg / mL, which was significantly stronger than the eggplant powders other than sample 5. The activity was 467 times stronger than sample 1, 660 times stronger than sample 2, 16 times stronger than sample 4, and 26 times stronger than sample 6. The EC value of sample 5, which showed the next strongest vasodilatory effect, was 0.015 mg / mL. 50 The activity was 0.027 mg / mL, which was significantly stronger than that of the eggplant powders other than sample 3, and the activity was 259 times that of sample 1, 367 times that of sample 2, 9 times that of sample 4, and 14 times that of sample 6. From these results, it was concluded that dried eggplant powder finer than 20 mesh has a strong vasodilatory effect.

[0095] [Experiment 12] Repeated oral administration test using freeze-dried eggplant Freeze-dried eggplant was orally administered daily to spontaneously hypertensive rats (SHR) for 4 weeks to confirm its antihypertensive effect and to evaluate its anti-stress effect by examining changes in urinary catecholamine levels. After 6 days of acclimation, 6-week-old male SHRs (body weight 263-286g) were divided into two groups: a control group (administered pure water, n=6) and a test group (administered freeze-dried eggplant powder, n=6). They were housed in individual metabolic cages at a room temperature of 23±4°C, humidity of 50±20%, and a 12-hour light-dark cycle (light period 5:30-17:30). They were fed a standard feed (MF, Oriental Yeast Co., Ltd.) and tap water ad libitum. Tosa Taka eggplants (harvested in February, Kochi Prefecture) were used as raw materials, and freeze-dried eggplant powder was prepared using the method described in [Experiment 9]. The dose of freeze-dried eggplant powder was 0.82 mg / kg (bw) per rat (equivalent to 100g AcCh). -8The test animals were orally administered with a stainless steel oral gastric tube at a concentration of 0.1 mmol / kg for 30 days. During the test period, food and water intake were measured twice a week, body weight was measured once a week, and urine volume was measured daily. Blood pressure (systolic and diastolic) was measured before the start of the test and on days 7, 14, 21, and 28 after the start of the test, as described above in [Experiment 9]. Each measurement was repeated three times, and the mean ± standard error (Mean ± SE) was calculated.

[0096] Urinary catecholamines were quantified using a urine sample taken the day before blood pressure measurement. Collection of 24-hour urine samples began the day before the start of repeated oral administration. Urine was collected once daily using a urine collection container containing 1 mL of 5N hydrochloric acid. After measuring the urination volume, the collected urine was frozen and stored at -80°C until analysis. At the time of analysis, the urine sample was thawed and centrifuged (4°C, 1000 × g, 3 minutes). The resulting supernatant (200 μL) was applied to a MonoSpin® PBA (GL Sciences, Inc.), washed, and then eluted with 400 μL of 1% aqueous acetic acid. The concentrated urinary catecholamines were used as the analytical sample. Urinary catecholamines were quantified three times using an HPLC system (Prominence HPLC system, Shimadzu Corporation, Kyoto: system controller: CBM-20A, liquid delivery unit: LC-20AD, column oven: CTO-10A) and an electrochemical detector (ECD, GL Sciences Inc., ED723 diamond electrode) under the following conditions, and the mean ± standard error (Mean ± SE) was calculated. Column: Inertsil ODS-4 (4.6 x 250 mm, GL Sciences, Inc.) Flow rate: 0.8mL / min, separation temperature: 35℃, standard injection volume: 20μL Mobile phase: Acetate-citrate buffer / CH3CN (100 / 16 v / v) Detection conditions: ECD 800mV (reference electrode Ag / AgCl)

[0097] As shown in Tables 15, 16 and 17, no significant differences were observed between the two groups in body weight, urine volume, water intake and total food intake throughout the feeding period.

[0098] [Table 15]

[0099] [Table 16]

[0100] [Table 17]

[0101] As shown in Figures 13 and 14, the test group showed significantly lower systolic blood pressure than the control group on days 14, 21, and 28 after the start of the test, and significantly lower diastolic blood pressure on day 28. This confirms that the blood pressure lowering effect of ingesting freeze-dried eggplant powder, which was evident in the single oral administration test, was also confirmed in the repeated oral administration test.

[0102] As shown in Figures 15 and 16, the amounts of adrenaline and noradrenaline, which are typical catecholamines found in urine, were significantly lower in the test group compared to the control group. Urinary adrenaline levels were significantly lower in the test group than in the control group on days 20 and 27 after the start of the test, and urinary noradrenaline levels were significantly lower in the test group than in the control group on days 6 and 27 after the start of the test.

[0103] Norepinephrine is a neurotransmitter released from sympathetic nerve endings, and adrenaline is produced by the conversion of norepinephrine in the adrenal glands. These catecholamines act on adrenaline receptors to regulate various organs and metabolic systems. The released catecholamines act on the effector organs reached by the nerves, and some enter the bloodstream and affect the entire body. While blood catecholamines are metabolized and inactivated, some are excreted unchanged in urine. Therefore, urinary catecholamine levels serve as an indicator of sympathetic nervous activity in the body. Increased sympathetic nervous activity results in increased urinary catecholamine levels. The sympathetic nervous system promotes arousal, attack, defense, and escape behaviors. Catecholamines released by increased sympathetic nervous activity act on vascular adrenaline α-receptors, constricting blood vessels and causing an increase in blood pressure. Therefore, the blood pressure-lowering mechanism of orally administered choline esters is thought to be closely related to the reduction in catecholamines due to the inhibition of sympathetic nervous activity.

[0104] In addition, it is known that the body responds to stress by increasing sympathetic nervous activity, and stress increases the amount of catecholamines, so catecholamines are known as stress indicators. Catecholamines (adrenaline, noradrenaline, and dopamine) were used as an indicator of the in vivo anti-stress effect of an anti-stress composition containing β-carotene as an active ingredient, and a significant decrease in catecholamines following ingestion of the composition demonstrated the anti-stress effect (Patent Document 3). Furthermore, the anti-stress effect was confirmed by significantly lower blood adrenaline (referred to as epinephrine in the literature) and noradrenaline (referred to as norepinephrine in the literature) levels following restraint stress following administration of yeast hydrolysate (Non-Patent Document 23). In assessing the anti-stress effect of candesartan (an angiotensin II type 1 receptor antagonist), urinary catecholamines (adrenaline and noradrenaline) were used as indicators of stress from individual rearing in metabolic cages, and significantly lower levels of each were considered to support the anti-stress effect.

[0105] In Experiment 12, the significant decrease in urinary catecholamine levels following ingestion of freeze-dried eggplant powder was attributed to the suppression of sympathetic nervous activity and its anti-stress effects. In other words, the anti-stress effects of freeze-dried eggplant powder were confirmed. The vasodilatory effect in Experiment 11 was caused by the action of choline esters contained in eggplant on muscarinic acetylcholine receptors (Non-Patent Document 25). This anti-stress effect is also thought to be the result of the choline esters contained in eggplant acting on muscarinic acetylcholine receptors to suppress sympathetic nervous activity and catecholamine release from peripheral nerves. Therefore, eggplant extracts and processed foods containing a certain amount of choline ester are thought to have anti-stress effects. The antihypertensive and anti-stress effects caused by the suppression of sympathetic nervous activity by orally ingested choline esters are closely related. Therefore, the difference in choline ester doses between SHR and WKY rats, which showed the antihypertensive effect, also applies to the anti-stress effect.

[0106] 4. Consideration of implementation Research by the present inventors has shown that choline esters can lower blood pressure at extremely low doses of 25 μg per day. The weight of agricultural produce required to ingest 25 μg of AcCh was set as the recommended daily intake, and the recommended daily intakes of the 20 types of fresh agricultural produce quantified in Experiments 1 and 2 were calculated. The results are shown in Table 18.

[0107] [Table 18]

[0108] As shown in Table 18, consuming 25 μg of AcCh requires extremely large amounts of the 18 agricultural products other than eggplant and bamboo shoots. For example, lettuce requires a daily intake of more than 7.5 kg, which is unrealistic for daily intake and impossible to achieve on a continuous basis. On the other hand, eggplant and bamboo shoots have recommended daily intakes of only 0.41 g and 0.25 g, respectively, suggesting that they may exhibit antihypertensive effects even with daily intakes that are sustainable. Furthermore, of the seven eggplant varieties used in this study, five varieties—Senshu Mizunasu, Batten Nasu, Koryo Salad Nasu, Higo Murasaki, and Ryuma—have soft skin and little astringency, making them suitable for raw consumption. Raw consumption eliminates the need to consider the loss of choline esters caused by cooking. [Industrial Applicability]

[0109] The composition of the present invention has a significant antihypertensive effect, and by incorporating it as an active ingredient, it is possible to produce a food with functional claims or a pharmaceutical for treating hypertension or the like.

Claims

[Claim 1] The invention described herein.

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