Choline ester-containing compositions for oral administration

Abstract

The objective of this invention is to provide a composition having antihypertensive and / or anti-stress effects, incorporating choline esters such as acetylcholine as active ingredients and readily orally ingested by humans. The objective of this invention is to provide novel oral compositions having antihypertensive and vasodilatory effects, incorporating choline esters such as acetylcholine and readily orally ingested by humans, and to provide foods that serve as useful sources of these compositions.

Description

[0001] This application is a divisional application of the invention patent application filed on October 16, 2017, with application number 201780063705X and invention title "Composition Containing Choline Esters for Oral Ingestion". Technical Field

[0002] This invention relates to compositions having antihypertensive and / or anti-stress effects, in which choline esters, compounds obtained by forming ester bonds between choline and organic acids, are used as active ingredients, and to methods thereof. Background Technology

[0003] Among choline esters, acetylcholine is known to be an essential neurotransmitter for mammalian life. Furthermore, in 1929, a blood pressure-lowering substance, distinct from histamine, was isolated from the spleen of a horse; this active substance was chemically identified as acetylcholine (Non-Patent Literature 1). Additionally, acetylcholine is known to be the blood pressure-lowering substance in ergot, and it has been confirmed that fungi produce acetylcholine (Non-Patent Literature 2). It has been reported that acetylcholine is found in edible plants, edible fungi, royal jelly, and milk; it is also present in Bacillus subtilis and yeast. Among edible plants, eggplant and bamboo shoots have high acetylcholine content (Non-Patent Literature 3-7).

[0004] In addition to acetylcholine, a variety of choline esters were also found.

[0005] Propionylcholine, with a propionyl group having a carbon chain one carbon longer than the acetyl group, was discovered in the spleen of cattle in 1953 (Non-Patent Literature 8, 9). Subsequently, propionylcholine was confirmed to be produced in bull semen, in the hemolymph and smooth muscle of European crayfish, horseshoe crab, European bird-tailed clam, giant mantis clam, Mediterranean mussel, freshwater toothless mussel, Roman snail, in the electrogenic tissue culture of Japanese electric ray, and in plants such as croton, mung bean, plantain, poplar, and birch (Non-Patent Literature 10-13).

[0006] In addition, butyrylcholine was discovered in brain extracts in 1954 (Non-Patent Document 14), and has been shown to coexist with acetylcholine and propionylcholine in arthropods and mollusks (Non-Patent Document 11).

[0007] In addition to propionylcholine and butyrylcholine, several other choline esters have been identified in mollusks. For example, urocanoylcholine was identified in a species of *Sinonovacula*, β,β-dimethylacrylcholine (isopreneylcholine) was identified in a species of *Lycopodium*, acrylcholine was identified in a species of *Moth*, and imidazole propionylcholine was identified in a species of *Lycopodium* (Non-Patent Literature 15–18).

[0008] The inventors have studied the active ingredients containing antihypertensive and vasodilatory effects in fermented buckwheat (a lactic acid fermentation product of buckwheat plant), and provided an extract composition containing an alkyl quaternary ammonium compound mainly composed of multiple choline esters, including at least acetylcholine and propionylcholine. They also found that a single oral administration of purified acetylcholine, propionylcholine, and butyrylcholine to spontaneously hypertensive rats (SHR) showed an antihypertensive effect (Patent Documents 1 and 2). On the other hand, as stated in the comprehensive information form (Interview Form) of pharmaceuticals using acetylcholine chloride as an active ingredient, "acetylcholine is almost not absorbed after oral administration and is therefore formulated as an injection." To date, the applicability of choline esters to humans has been based on injections, and there has been no discussion regarding their effects through oral intake.

[0009] Existing technical documents Patent documents Patent Document 1: International Publication No. 2015 / 147251 Patent Document 2: Japanese Patent Application Publication No. 2015-189745 Patent Document 3: Japanese Patent Application Publication No. 06-065068 Non-patent literature Non-patent literature 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 literature 2: Ewins AJ. Acetylcholine, a new active principle of ergot. Biochem J 8:44-49, 1914. Non-patent literature 3: Momoki Yoshie, "Acetylcholine in Plants", Chemical Regulation of Plants, 30(1), 49-61 (1995) Non-patent document 4: Kawashima Koichiro, "The Source of Acetylcholine and Non-Nervous Acetylcholine", "Basic Aging Research (Basic Aging Research)", 34(4), 12-24 (2010) Non-patent literature 5: Masato Shinoda et al., “On the blood flow increasing factor in royal jelly”, Journal of Pharmacy, 98(2), 139-145 (1978) Non-patent literature 6: Whittaker VP. Acetylcholine in Milk. Nature 181:856-857, 1958. Non-patent literature 7: Horiuchi Y, Kimura R, Kato N, Fujii T, Seki M, Endo T, Kato T, Kawashima K. "Evolutionary study on acetylcholine expression". Life Sci 72:1745-1756, 2003. Non-patent literature 8: Banister J, Whittaker VP, Wijesundera S. "The occurrence of homologues of acetylcholine in oxspleen". J Physiol 121(1):55-71, 1953. Non-patent literature 9: Gardiner JE, Whittaker VP. Identification of propionylcholine as a constituent of oxspleen. Biochem J 58(1):24-29, 1954. Non-patent literature 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 literature 11: Wolfgang W, Jutta N, Dettmar W. Distribution of cholinesters and cholinesterases in hemolymphs and smooth muscles of molluscs. Comp Biochem Phys C 61(1):121-131, 1978. Non-patent literature 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 literature 13: Miural GA, Shin TM. Identification of proprionylcholine in higher plants. Physiol Plant 62:341-343, 1984. Non-patent literature 14: Holtz P, Schumann HJ. Butyrylcholine in brain extracts. Naturwissenschaften 41:306, 1954. Non-patent literature 15: Erspamer V, Benati O. Identification of murexine as beta-[imidazolyl-(4)]acrylcholine. Science 117:161-162, 1953. Non-patent literature 16: Keyl MJ, Michaelson IA, Whittaker VP. Physiologically active choline esters in certain marine gastropods and other invertebrates. J Physiol 139:434, 1957. Non-patent literature 17: Whittaker VP. Acrylylcholine: a new naturally occurring pharmacologically active choline ester from Buccinum undatum. BiochemPharmacol 1(4):342-346, 1959. Non-patent literature 18: Roseghini M. Occurrence of dihydromurexine (imidazole propionylcholine) in the hypobranchial gland of Thais (purpura) haemastoma. Experientia 27(9):1008-1009, 1971. Non-Patent Literature 19: Comprehensive Information Sheet for OVISOT (Registered Trademark) 0.1g Injection, 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: Proceedings of the 2016 Annual Meeting of the Japanese Society for Agricultural Chemistry, 4E076, published on March 5, 2016. Non-Patent Document 22: Kleiber, M. The fire of life: An introduction to animal energetics. New York: Wiley, 1961. Non-Patent Literature 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 literature 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 literature 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 The technical problem that the invention aims to solve The inventors believe it is important to clarify the fact that although choline esters can be taken orally to lower blood pressure safely and easily without putting a burden on the body, there is currently no technology for the appropriate oral administration of acetylcholine to humans, nor a suitable source of supply for such technology.

[0010] Therefore, the objective of this invention is to provide a novel oral composition having antihypertensive and vasodilatory effects, incorporating choline esters such as acetylcholine as active ingredients and which can be easily orally ingested by humans, as well as to provide a food source that is useful for it.

[0011] Technical solutions adopted to solve technical problems In the course of repeated and careful research to solve the above-mentioned problems, the inventors were surprised to find that choline esters exist in appropriate amounts for oral administration to exhibit antihypertensive and vasodilatory effects, and at said amounts they also exert anti-stress effects. Furthermore, they discovered suitable edible plants as raw materials for efficiently supplying choline esters for this purpose. After further research, the present invention was completed.

[0012] Therefore, the present invention relates to the following.

[0013] [1] A composition having antihypertensive and / or anti-stress effects with choline ester as the active ingredient, wherein the choline ester content is 5 μg to 50 mg, for oral administration.

[0014] [2] The composition described in [1] above is a food composition.

[0015] [3] The composition according to [1] above, wherein the composition is a pharmaceutical composition for lowering blood pressure and / or relieving stress.

[0016] [4] The composition according to any one of [1] to [3] above, wherein the choline ester is derived from edible plants.

[0017] [5] The composition according to any one of [1] to [4] above, wherein the composition is formed from freeze-dried powder and / or hot-air dried powder of edible plants.

[0018] [6] The composition according to [5] above, wherein the composition is formed from freeze-dried powder and / or hot-air dried powder that can pass through a 20-mesh sieve.

[0019] [7] The composition according to any one of [1] to [4] above, wherein the composition is an extract obtained by extracting edible plants with ethanol or aqueous ethanol.

[0020] [8] The composition according to any one of [4] to [7] above, wherein the edible plant is a species of the genus *Solanum* of the family Solanaceae (Solanaceae). Solanum melongena The fruit of the bamboo and / or the new shoots of the Bambuseae tribe of the Bambusoideae subfamily of the Poaceae family.

[0021] [9] The composition according to any one of [1] to [3], wherein the choline ester includes one or more selected from acetylcholine, butyrylcholine and propionylcholine.

[0022]

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

[0023]

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

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

[0024]

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

[11] above, wherein the composition is prepared by freeze processing.

[0025]

[13] The composition according to any one of [1] to [3] above, wherein the composition is a frozen-processed eggplant species of the Solanaceae family (Solanum genus). Solanum melongena (a portion or all of the fruit)

[0026]

[14] A method for manufacturing a composition for oral administration containing choline esters as an active ingredient, having antihypertensive and / or anti-stress effects, comprising: a step of preparing edible plants into freeze-dried powder and / or hot-air dried powder; and a step of distributing the freeze-dried powder and / or hot-air dried powder according to conditions where the choline ester content reaches 5 μg to 50 mg.

[0027]

[15] According to the method described in

[14] above, the edible plant is the solanaceae family, solanaceae genus, solanum species ( Solanum melongena The fruit of the bamboo and / or the new shoots of the tribe Bambuseae (Poaceae, Bambusoideae, Bambuseae) of the subfamily Bambusoideae in the family Poaceae.

[0028]

[16] The method described in

[14] or

[15] above further includes a step of heating the edible plant.

[0029]

[17] The method according to any one of

[14] to

[16] above further includes the step of suspending freeze-dried powder and / or hot-air dried powder of edible plants in water and adding acid to the resulting suspension.

[0030]

[18] The method described in

[17] above further includes the step of adjusting the pH of the acid-added suspension to 5.5 to 4.5.

[0031]

[19] A composition for oral administration containing choline esters as an active ingredient, having a hypotensive effect and / or an anti-stress effect, wherein the composition is manufactured by the method described in any one of

[14] to

[18] above.

[0032]

[20] A method for manufacturing an extract with choline esters as the active ingredient for oral administration, having antihypertensive and / or anti-stress effects, comprising: a step of extracting edible plants or freeze-dried powder and / or hot-air dried powder of edible plants with ethanol or aqueous ethanol.

[0033]

[21] The method described in

[20] above includes the step of extracting edible plants or freeze-dried powder and / or hot-air dried powder of edible plants with ethanol.

[0034]

[22] The method described in

[20] above includes the step of extracting edible plants or freeze-dried powder and / or hot-air dried powder of edible plants with aqueous ethanol; wherein the ethanol concentration of aqueous ethanol is 25-60% (w / w) or 95% (w / w) or more.

[0035]

[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.

[0036]

[24] The method according to any one of

[20] to

[23] above, wherein the method includes the step of adjusting the choline ester content of the extract to 5 μg to 50 mg.

[0037]

[25] An extract for oral administration containing choline esters as an active ingredient, having antihypertensive and / or anti-stress effects, wherein the composition is manufactured by the method described in any one of

[20] to

[24] above.

[0038] The effects of the invention This invention eliminates the need to formulate choline esters, the active ingredient, into an injectable form, providing a composition with antihypertensive effects that is easily taken orally over a long period. Particularly in the case of compositions formed from freeze-dried powders of edible plants, extraction and purification processes are unnecessary, allowing for very low-cost processing. Furthermore, to increase choline ester levels while simultaneously sterilizing, heating under certain conditions can be performed before freeze-drying; to increase the stability of choline esters, acid can be added before or after freeze-drying. Choline esters are widely consumed, therefore the compositions of this invention are highly safe and can be used in both oral formulations and antihypertensive pharmaceutical compositions.

[0039] Furthermore, if the composition of the present invention is used, it can be used in various forms such as products obtained by cutting edible plants (fresh) according to conditions containing a specified amount of choline esters, products obtained by heating or freezing the edible plants, dried powder of edible plants (freeze-dried powder, hot air-dried powder), suspension of the dried powder, extract obtained by extracting choline esters from fresh plants, and concentrated extract obtained by concentrating the extract, in beverages, food or pharmaceuticals.

[0040] When cholinesterols act on cholinergic receptors (muscarinic acetylcholine receptors and nicotinic acetylcholine receptors), stimulation of nicotinic acetylcholine receptors requires a higher concentration of acetylcholine than stimulation of muscarinic acetylcholine receptors (Non-Patent Literature 20). Furthermore, blood pressure decreases due to the action of muscarinic acetylcholine receptors, but this effect is thought to be counteracted by the dominance of sympathetic nerve activity through the action of nicotinic acetylcholine receptors. This invention is based on the finding that oral ingestion of cholinesterols in very small amounts exhibits a different effect than that of large oral ingestion. That is, the cholinesterols in the compositions of this invention are present in very small amounts compared to 0.1 g / dose of OVISOT (registered trademark) for injection, but act sufficiently on muscarinic acetylcholine receptors to exert a blood pressure-lowering effect, without acting on nicotinic acetylcholine receptors, and are contained in an amount that does not counteract the blood pressure-lowering effect. In the composition of the present invention, due to the balanced choline ester content that conforms to the effects of the two receptors, acetylcholine exerts a blood pressure-lowering effect based on oral intake, contrary to the common technical knowledge that it is considered impossible to administer orally.

[0041] In this invention, when using edible plants high in cholinesterol, such as eggplant and bamboo shoots, a smaller quantity of plant matter is sufficient, making it suitable for oral consumption. In contrast, when choosing plants low in cholinesterol, such as lettuce, a daily intake of approximately 7.5 kg or more based on fresh weight is required, which is impractical for a single day. Even with freeze-drying lettuce, the yield is only about 3.60%, requiring a daily intake of approximately 270 g. On the other hand, for eggplant, the target daily intake based on fresh weight, determined using animal studies using SHR (Solar Human Respiratory Health), is only 0.41 g, thus providing a sustainable daily intake for lowering blood pressure. Furthermore, directly freeze-drying or freeze-drying eggplant after heating further reduces the target daily intake. Therefore, using processed eggplant products allows for the creation of a very reasonable and economical oral food.

[0042] Brief description of the attached diagram Figure 1 It is a chart showing the yield of various fresh agricultural products after freeze-drying.

[0043] Figure 2This is a chart showing the content of various choline esters and choline in different fresh agricultural products. The vertical axis is logarithmic. AcCh: Acetylcholine, BuCh: Butyrylcholine, PrCh: Propionylcholine, Ch: Choline.

[0044] Figure 3 This is a chart showing the changes in the content of various choline esters and choline in fresh eggplant fruits stored at room temperature from day 1 to day 5 over time (p* < 0.05, p** < 0.01 relative to one day). AcCh: Acetylcholine, BuCh: Butyrylcholine, PrCh: Propionylcholine, Ch: Choline.

[0045] Figure 4 This is a chart showing the choline ester content in fresh eggplant fruits stored at room temperature and refrigerated for 6 days (p* < 0.05, p** < 0.01). The vertical axis is logarithmic. AcCh: Acetylcholine, BuCh: Butyrylcholine, PrCh: Propionylcholine.

[0046] Figure 5 This is a graph showing the change in acetylcholine levels over time with pH when using standard samples. AcCh: Acetylcholine.

[0047] Figure 6 This is a graph showing the change in acetylcholine levels over time with pH when using eggplant extract. AcCh: Acetylcholine.

[0048] Figure 7 This is a graph showing the change in the amount of acetylcholine in eggplant caused by heating. AcCh: Acetylcholine.

[0049] Figure 8 This indicates that the SHR contains an equal amount of acetylcholine 1.00 × 10⁻⁶. -9 A graph showing the results of a single oral administration trial (systolic blood pressure) of mol / kg eggplant freeze-dried powder (p* < 0.05, relative to water administration).

[0050] Figure 9 This indicates that the SHR contains an equal amount of acetylcholine 1.00 × 10⁻⁶. -9 A graph showing the results (diastolic blood pressure) of a single oral administration trial of mol / kg freeze-dried eggplant powder.

[0051] Figure 10 This indicates that the SHR contains an equal amount of acetylcholine 1.00 × 10⁻⁶. -9 A graph showing the results (heart rate) of a single oral administration trial of mol / kg freeze-dried eggplant powder.

[0052] Figure 11This 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, relative to water administration).

[0053] Figure 12 This is a graph showing the results of the isometric tension test of freeze-dried eggplant powder (relative to freeze-drying through a 20-mesh sieve, p* < 0.05, p** < 0.01; relative to hot-air drying through a 20-mesh sieve, p# < 0.05, p## < 0.01).

[0054] Figure 13 This is a graph showing the results (systolic blood pressure) of repeated oral administration of SHR eggplant freeze-dried powder (p* < 0.05, p** < 0.01, relative to water administration).

[0055] Figure 14 This is a graph showing the results (diastolic blood pressure) of repeated oral administration of SHR eggplant freeze-dried powder (p* < 0.05, relative to water administration).

[0056] Figure 15 This is a graph showing the results of repeated oral administration of SHR eggplant freeze-dried powder (changes in urinary adrenaline levels) (p* < 0.05, p** < 0.01, relative to water administration).

[0057] Figure 16 This is a graph showing the results of repeated oral administration of SHR eggplant freeze-dried powder (changes in urinary norepinephrine levels) (p* < 0.05, p** < 0.01, relative to water administration).

[0058] Methods of implementing the invention This invention relates to compositions having a blood pressure-lowering effect with choline esters as the active ingredient, and to the same compositions having a further anti-stress effect.

[0059] The composition of the present invention is for oral administration, and the choline ester content may be 5 μg to 50 mg.

[0060] In the compositions of the present invention, the choline ester content is 5-500 μg, preferably 5-250 μg, more preferably 10-50 μg, and particularly preferably 25 μg. This choline ester content is preferred, for example, in cases used for patients with hypertension.

[0061] Furthermore, the choline ester content can be 5 μg to 50 mg, preferably 500 μg to 50 mg, and even more preferably 500 μg to 5 mg. This choline ester content is preferred, for example, in healthy individuals (not hypertensive) but with elevated blood pressure.

[0062] The compositions of the present invention can be food compositions or pharmaceutical compositions. When the compositions of the present invention are pharmaceutical compositions, they can be pharmaceutical compositions for lowering blood pressure and / or relieving stress.

[0063] Choline esters, which are the active ingredients in the compositions of the present invention, can be any of those derived from animals, plants, or microorganisms, but are preferably derived from organisms consumed by humans, and particularly preferably from edible plants.

[0064] As edible plants, any edible plant containing cholinesterol is acceptable; there are no specific limitations. Examples of edible plants include cucumbers, tomatoes, red peppers, green peppers, eggplants, asparagus, yams, cabbage, lettuce, carrots, apples, green peppers, Japanese eggplants, grapes, long-haired radishes, broccoli, alfalfa, bean sprouts, buckwheat, and bamboo shoots. From the perspective of acetylcholine content, solanum species (of the Solanaceae family, genus Solanum) are preferable. Solanum melongena ), Poaceae, Bambusoideae, Bambuseae, especially the Solanaceae family, Solanum genus (sowary). Solanum melongena The fruit of the bamboo and / or the new shoots of the tribe Bambuseae (Poaceae, Bambusoideae, Bambuseae) of the subfamily Bambusoideae in the family Poaceae.

[0065] Solanaceae family, Solanum genus, Solanum species ( Solanum melongena Among the varieties, the better ones are Quanzhou water eggplant, Batten eggplant, Guangling salad eggplant (also known as: Meinan), Feihou purple, Dachang eggplant, Zhuyang, etc., because they can be eaten raw, Quanzhou water eggplant, Batten eggplant, Guangling salad eggplant, and Feihou purple are even better. Feihou purple is especially good.

[0066] Examples of choline esters that may be included in the compositions of the present invention include acetylcholine, butyrylcholine, propionylcholine, lactylcholine, etc., and a composition may contain one or more of these. In particular, when the choline esters are derived from plants, the compositions of the present invention contain one or more of acetylcholine, butyrylcholine, and propionylcholine, but do not contain lactylcholine.

[0067] In the compositions of the present invention, the daily intake of choline esters is adjusted to a specified range.

[0068] For example, in one package, the cholinesterol content is adjusted to the range of 5–125 μg, preferably 10–75 μg, and particularly preferably 15–50 μg, for oral administration once to several times a day (preferably about three times). Furthermore, for example, the total cholinesterol content can be adjusted to be within the aforementioned range through multiple packages. In this case, the cholinesterol concentration reaches 5–2500 μg / g.

[0069] Furthermore, if the daily choline ester concentration is adjusted to a value higher than the aforementioned range, 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 a higher level using the method described above. In this case, the choline ester concentration can be 500 to 250 mg / g.

[0070] The compositions of the present invention are adjusted to a specified choline ester content. In the compositions of the present invention, for example, the choline ester content can be adjusted by using products obtained by cutting fresh agricultural products, frozen products, freeze-dried products, or extracts, etc. The compositions of the present invention are preferably compositions formed from freeze-dried powders and / or extracts of edible plants.

[0071] The composition of the present invention can be a product cut into one-day servings to achieve the required choline ester content for one day, and can be individually packaged in vacuum bags or the like to prevent quality deterioration or browning of the pulp.

[0072] Furthermore, the composition of the present invention is preferably freeze-processed. Freezing inhibits the activity of cholinesterase mixed into the composition, thus preserving cholinesterase for a longer period. The composition of the present invention is preferably a freeze-processed *Solanum* species (Solanaceae family). Solanum melongena (a portion or all of the fruit)

[0073] In one embodiment, the present invention relates to a method for manufacturing a composition having antihypertensive and / or anti-stress effects for oral administration, with choline esters as the active ingredient.

[0074] The method includes: a step of preparing edible plants into freeze-dried powder and / or hot-air dried powder or extract; and a step of dispensing the freeze-dried powder and / or hot-air dried powder or extract according to the condition that the choline ester content reaches a specified amount.

[0075] Here, the specified amount can be 5 μg to 250 mg, preferably 5 μg to 50 mg, and more preferably 10 μg to 50 mg. In addition, in one form, the specified amount can be, for example, 5 to 500 μg, 5 to 250 μg, 10 to 50 μg, or 25 μg.

[0076] The method of the present invention may further include a step of heating the edible plants. Heating can be performed by microwave oven or by blanching in hot water. For example, when heating in a 550W microwave oven, 100g of edible plants should be heated for 1-15 minutes, preferably 2-10 minutes, and more preferably 4-6 minutes. Furthermore, when blanching in hot water, it is preferable to heat in water at 90-100°C. Heating the edible plants in this way can sterilize them and also increase the choline ester content within the edible plants.

[0077] In one embodiment, the method of the present invention is characterized by further heat treatment (for sterilization and choline ester enhancement) of the edible plants, while generally avoiding microbial spoilage by freeze-drying or hot-air drying.

[0078] The method of the present invention may further include: suspending freeze-dried powder and / or hot-air dried powder of edible plants in water, and adding acid to the resulting suspension. The pH of the acid-added suspension is adjusted to, for example, 5.5–4.5, preferably 5.4–4.6. By adjusting the pH in this way, choline esters are stabilized, and a composition (suspension) with good long-term shelf life can be prepared.

[0079] The present invention also relates to a method for manufacturing an extract containing choline esters as the active ingredient, which has antihypertensive and / or anti-stress effects for oral administration.

[0080] The method of the present invention includes the step of extracting edible plants or freeze-dried powder and / or hot-air dried powder of edible plants with ethanol or aqueous ethanol.

[0081] More specifically, the method of the present invention includes adding ethanol or aqueous ethanol to freeze-dried powder and / or hot-air dried powder of edible plants, or adding ethanol to fresh edible plants, pulverizing and removing residue to obtain an extract obtained by concentrating choline esters.

[0082] When using aqueous ethanol for extraction, the concentration of the aqueous ethanol is not particularly limited and can be appropriately selected based on the extraction rate and concentration rate of choline esters. For example, the concentration of aqueous ethanol can be 10% (w / w), preferably 10–99% (w / w), more preferably 25–60% (w / w) or above 95% (w / w), and particularly preferably 30–60% (w / w) or above 99% (w / w).

[0083] L-ascorbic acid may be added to the ethanol or aqueous ethanol used for extraction, for example, at 1 to 5 wt%, preferably 3 wt%.

[0084] The method of the present invention may include the step of adjusting the choline ester content of the extract to 5 μg to 50 mg.

[0085] The present invention also relates to a composition for oral administration containing choline esters as an active ingredient, which has antihypertensive and / or anti-stress effects and is manufactured by the method described above.

[0086] The dried powder in the compositions of the present invention is preferably a powder that has passed through a sieve with an appropriate aperture. The compositions of the present invention are preferably formed from freeze-dried powder and / or hot-air dried powder that can pass through, for example, a 20-mesh sieve.

[0087] Here, hot-air dried powder can be prepared, for example, by exposing edible plants to hot air at about 90°C for about 1 to 2 hours to dry them and then pulverizing them into powder.

[0088] The compositions of the present invention can be used as active ingredients in various functional health food or pharmaceutical compositions.

[0089] In the case of food, it can be combined with appropriate food additives to be used as a food composition. Furthermore, it is not limited to such food compositions; it can also be incorporated into green tea, black tea, oolong tea, whole grain tea, etc., as a beverage, or into biscuits, bread, sugar, etc., as a food, provided in a form suitable for daily consumption. In addition, it can be formulated into dosage forms conforming to the formulation methods of pharmaceuticals below, and used as a so-called nutritional supplement.

[0090] When manufactured into pharmaceutical products, it can be combined with appropriate pharmaceutical additives and formulated into various dosage forms according to conventional preparation methods. Examples of such dosage forms include solid dosage forms such as powders, granules, capsules, pills, and tablets, and oral dosage forms such as aqueous solutions, suspensions, and emulsions.

[0091] When the compositions of the present invention are used as food, they are not limited to general beverages and foods, but can also be used as functional health foods that enhance health by performing specific functions.

[0092] Specific examples of this situation include nutritional supplements such as capsules, tablets, powders, and granules containing the composition of the present invention as an active ingredient; baked goods such as bread, cakes, and cookies; condiments such as sauces, soups, gravy, and mayonnaise; dairy products such as milk, yogurt, and cream; snacks such as chocolate and candy; and various beverages such as green tea, black tea, oolong tea, barley tea, whole grain tea, fruit juice, vegetable drinks, dairy drinks, soft drinks, and carbonated drinks.

[0093] The dosage when using the composition of the present invention as the active ingredient in a pharmaceutical composition varies depending on the proportions of each ingredient, and also on various factors such as the patient's age, weight, sex, symptoms, and method of administration. For adults, the dosage can be selected daily orally in the range of 5 μg to 50 mg of choline esters, or, as an alternative form, in the range of 5 μg to 500 μm. Furthermore, the dosage can be adjusted appropriately according to the degree of symptom improvement. As for the frequency of administration, it can be administered once daily or several times daily.

[0094] When the composition of the present invention is used as food, the intake amount can be selected with reference to the oral administration of the above-mentioned pharmaceutical products. However, the situation with beverages and food is different from that with pharmaceutical products. There are no particular limitations on the dosage and frequency of administration. As long as no particularly serious symptoms occur, the intake amount can be selected for the purpose of maintaining health and taking into account taste and preferences, and is not limited to the above-mentioned range.

[0095] Example The following examples and experimental examples illustrate embodiments of the present invention, but the present invention is not limited to these examples. Furthermore, the meanings of the abbreviations used in the examples are as follows: EN: (2-aminoethyl)trimethylammonium neopentylamide, AcCh: acetylcholine, BuCh: butyrylcholine, Ch: choline, LaCh: lactylcholine, PrCh: propionylcholine. Additionally, EN, AcCh, BuCh, Ch, LaCh, and PrCh are collectively referred to as choline compounds below. EtOH: ethanol.

[0096] [Experimental Materials and Methods] 1. Analyze the sample As samples for analysis, various agricultural products listed in Table 1 were obtained.

[0097] [Table 1] 2. Extraction method (1) Sample preparation Fresh agricultural products (analytical samples) were immediately washed with tap water upon acquisition. After wiping off the moisture, only the edible parts were sliced ​​into 1-3 cm wide pieces as needed. The edible parts were freeze-dried using a freeze dryer (FDU-2000, Tokyo Rika Kiki Co., Ltd.). The freeze-dried material was then pulverized into powder using a grinder / mixer (MASTER, Tokyo Unicom Co., Ltd.).

[0098] (2) Preparation of reagents Weigh out 59.99 mg of sodium dihydrogen phosphate and 70.98 mg of disodium hydrogen phosphate, dissolve them in pure water (100 mL), and prepare a 10 mM phosphate buffer.

[0099] EN (0.80 mg) was dissolved in 10 mM phosphate buffer (1 mL) to prepare 800.00 μg / mL, and then diluted 100 times to prepare 8.00 μg / mL, which was used as the internal standard for EN.

[0100] (3) Oscillation extraction Weigh 10 mg of the lyophilized material into a 2 mL test tube and add 10 μL of EN internal standard. Add 190 μL of 10 mM phosphate buffer, vortex for 3 minutes, then centrifuge (1000 × g, room temperature, 3 minutes) using a CFM-200 centrifuge (Eway Corporation) to obtain the supernatant. Add 200 μL of 10 mM phosphate buffer to the residue again, and repeat the stirring, centrifugation, and supernatant collection process twice. Combine all the collected supernatants (approximately 600 μL) to prepare the extraction sample.

[0101] (4) Solid phase extraction The solid-phase extraction tubes used were weakly acidic cation exchange tubes, Inert Sep CBA 100 mg / 1 mL (Agilent Technologies). After equilibrating the solid-phase extraction tubes activated with methanol (1 mL) and pure water (1 mL) with 10 mM phosphate buffer (8 mL), the extracted sample prepared in step (3) above (approximately 600 μL) was added. The tubes were stabilized with 10 mM phosphate buffer (600 μL), washed with pure water (2.5 mL), and eluted with hydrochloric acid (500 μL).

[0102] (5) Quantitative sample preparation The eluent (500 μL) from the solid-phase extraction elution with hydrochloric acid was precisely diluted to 1 mL in a 1 mL volumetric flask using LC / MS / MS analytical solvent. This volumetric flask was then divided into three 300 μL portions. A choline compound mixture was added to each portion, and the eluent was diluted two-fold with LC / MS / MS analytical solvent (Table 2) to prepare quantitative samples.

[0103] [Table 2] The choline compound mixed solutions were prepared as shown in Table 3. The concentration of each choline compound stock solution was determined based on the analytical results of the unadded choline compound mixed solutions (Table 2-A). The choline compound stock solutions were prepared to their respective concentrations by diluting them with LC / MS / MS analytical solvent. In the presence of undetected choline compounds, only an equal volume of LC / MS / MS analytical solvent free of that compound was added.

[0104] [Table 3] 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 a solution containing 0.01% formic acid and 33% methanol. LC / MS / MS analysis was performed using an ACQUITY UPLC (Waters Corporation)-Quattromicro API (Waters Corporation) system at the following conditions: flow rate 0.5 mL / min (LC), 0.3 mL / min (MS), injection volume 50 μL, separation temperature 40 °C, analysis time 30 min, ionization mode ESI+·MRM, capillary voltage 3500 V, cone voltage 10 V, collision voltage 10 V, nitrogen flow (desolventization) 600 L / h, nitrogen flow (cone) 50 L / h, nitrogen source temperature 120 °C, and nitrogen desolventization temperature 350 °C. The m / z values ​​for each choline compound in the MRM mode are shown in Table 4.

[0105] [Table 4] (2) Standard addition method A calibration curve was constructed based on the peak area values ​​of the chromatograms obtained by LC / MS / MS analysis, and choline compounds were quantified by the standard spiking method. The concentrations of each choline compound were corrected using the recovery rates calculated according to the calibration curve based on EN internal standards, and the accurate concentrations of choline compounds in the quantitative sample were calculated. After converting the obtained concentrations to the content in the freeze-dried product (mg / g D.W.), the amount of each choline compound per 100g fresh weight (μg / 100g F.W.) was calculated based on the yields before and after freeze-drying.

[0106] [Experiment 1] Fresh weight and dry weight Calculate the fresh and dry weights of various fresh agricultural products, and then calculate the yields before and after freeze-drying. The results are shown below. Figure 1 .

[0107] like Figure 1 As shown, lettuce (Shinano Hope) (fresh weight: 79.19g, dry weight: 2.85g) had the lowest yield at 3.60%, while yam (long yam) (fresh weight: 79.76g, dry weight: 21.23g) had the highest yield at 26.62%. In most agricultural products, the yield is approximately 5% to 10%.

[0108] [Experiment 2] Quantitative analysis of choline compounds in various fresh agricultural products Choline compounds were extracted from 20 kinds of fresh agricultural products using the above method. Five choline compounds (AcCh, Ch, BuCh, PrCh, and LaCh) were analyzed by LC / MS / MS with n=3. The results are shown below. Figure 2 .

[0109] AcCh and Ch were detected in all tested agricultural products. On the other hand, LaCh was not detected in any tested agricultural products. Furthermore, BuCh was detected in agricultural products other than tomatoes, lettuce, alfalfa sprouts, bean sprouts, buckwheat sprouts, apples, Japanese eggplants, grapes, and bamboo shoots. PrCh was detected in agricultural products other than tomatoes and bean sprouts.

[0110] The quantitative results of this experiment showed that bamboo shoots and eggplants contained more than 1,000 times the amount of AcCh found in 18 other fresh agricultural products. Bamboo shoots had the highest AcCh content.

[0111] [Experiment 3] Quantitative analysis of choline compounds in six eggplant varieties For the year-round edible eggplants containing a large amount of AcCh in Experiment 2, the differences in choline compound content among varieties were investigated. Choline compounds were extracted from six eggplant varieties using the methods described above, and five choline compounds (AcCh, Ch, BuCh, PrCh, and LaCh) were analyzed by LC / MS / MS with n=3. The results are shown in Table 5.

[0112] LaCh was not detected in any of the varieties. In addition, among the six varieties, Higuchi Purple had the highest AcCh content.

[0113] [Table 5] To date, AcCh in eggplant has been quantified using high-performance liquid chromatography-electrochemical detection (HPLC-ECD), with a reported concentration of 6.09 × 10⁻⁶. 3 μg / 100g F.W. (Non-Patent Literature 7), and the quantitative results obtained by LC / MS / MS analysis in this experiment are also the same AcCh content.

[0114] [Experiment 4] Changes in the content of choline compounds in eggplant over time Eggplants are generally considered to have a shelf life of 2-3 days at room temperature and about 1 week at 7-10℃. However, due to the potential for low-temperature damage such as crater-like depressions, browning, or blackening of the peel, storage below 6℃ is not recommended. The ideal storage temperature for eggplants is considered to be above 10℃ and below 20℃.

[0115] In this experiment, to investigate the changes in choline compound levels over time in the eggplant variety "Feihou Purple" stored at room temperature, samples were taken from the same individual (approximately 10g each time) wrapped in newspaper and stored at room temperature in a dark place. Another individual was divided into two parts, and one part was sampled on day 6. Additionally, another part of the divided sample was wrapped in newspaper and refrigerated for 6 days. Choline compounds were extracted using the same method, and five choline compounds (AcCh, Ch, BuCh, PrCh, and LaCh) were analyzed by LC / MS / MS at n=3. The results are shown in Table 6. Figure 3 and Figure 4 .

[0116] [Table 6] LaCh was not detected in any of the samples. The content of each choline compound tended to decrease slowly with increasing retention time. Figure 3 Furthermore, in this experiment, no low-temperature damage such as dents occurred in the refrigerated individuals, but the residual cholinesterase levels were higher in the refrigerated individuals compared to those stored at room temperature. Figure 4 ).

[0117] Based on the results of this experiment, if eggplant is stored at room temperature for 5 days, the choline ester content tends to decrease slowly with increasing days. However, more than 80% of the AcCh from the first day remains in individuals stored at room temperature for 5 days. Therefore, it can be considered that the amount of AcCh in eggplant stored at room temperature does not change drastically and has high stability.

[0118] [Experiment 5] Stability of AcCh under acidic conditions A method for further stabilizing choline esters in eggplant was explored. Generally, ester compounds are easily hydrolyzed under alkaline conditions and are relatively stable under acidic conditions. Furthermore, over 90% of the choline esters in eggplant are AcCh. Therefore, the concentration changes of AcCh standards under neutral and acidic conditions were first investigated over time. Neutral solutions (pH 7.0) were prepared using phosphate buffer, and acidic solutions (pH 4.1, pH 3.6, pH 3.1, pH 2.6) were prepared by adding acetic acid. The AcCh content was quantified after each pH solution with an AcCh concentration of 1.9 mg / mL was allowed to stand at room temperature (23–25 °C) for 0, 3, 6, and 12 days. The results are shown in Table 7. Figure 5 .

[0119] [Table 7] Under neutral conditions, AcCh decomposes over time, decreasing to approximately half after 12 days. On the other hand, at pH 4.1 (0.0015% acetic acid), AcCh is stable, but an increase in AcCh content is observed after 12 days. Between pH 4.1 and pH 3.1, AcCh remains stable at over 97% after 12 days. At pH 2.6, the stability of AcCh decreases slightly, with a retention rate of 88% after 12 days.

[0120] Next, the stability of AcCh contained in eggplant extract was investigated under neutral and acidic conditions. Neutral solutions (pH 7.0) were prepared with phosphate buffer, acidic solutions (pH 5.2, pH 5.0, pH 4.3, pH 3.5) were prepared with added acetic acid, and acidic solutions (pH 5.4, pH 4.6) were prepared with added ascorbic acid. Solutions of each pH containing eggplant extract at an AcCh concentration of 0.040 mg / mL were allowed to stand at room temperature (23–25 °C) for 0, 3, 6, and 12 days, and the AcCh content was then quantified. The results are shown in Table 8. Figure 6 .

[0121] [Table 8] AcCh in eggplant extract is unstable under neutral conditions; more than half of the AcCh decomposes after 3 days, and almost all of it decomposes after 12 days. It is relatively stable under acidic conditions; at pH 5.4–4.6, more than 88% of AcCh remains after 12 days. However, in a solution adjusted to pH 3.5 with acetic acid, 24.6% of the AcCh decomposes after 12 days. AcCh in eggplant is stable at room temperature under acidic conditions at pH 5.4–4.6.

[0122] Based on the above results, adding organic acids to adjust the pH to 5.5–4.5 can further stabilize choline esters in agricultural products such as eggplant. As long as the suspension is prepared within this range, the acid can be added at the appropriate time.

[0123] [Experiment 6] Changes in AcCh content in eggplant caused by heating To investigate the effect of heating on the AcCh content in eggplant, the representative long-oval eggplant variety "Longma" was cut into 2-3 cm thick slices, wrapped in plastic wrap, and heated in a microwave oven (550W) for different times. The AcCh content in the samples obtained after cooling at room temperature and freeze-drying was then quantified. In addition, the change in AcCh content in eggplant blanched in boiling water for 5 minutes was also examined. Temperatures were measured and recorded immediately after these heat treatments. The results are shown in Table 9. Figure 7 .

[0124] [Table 9] It was found that cholinesterol content in eggplant increased after microwave heating. Heating for 5 minutes per 100g resulted in the greatest increase in AcCh content, which increased to 2.9 times that of unheated eggplant. The increase in AcCh content was 2.6 times that of unheated eggplant when heated for 2 minutes per 100g, and 2.8 times that of unheated eggplant when heated for 10 minutes per 100g. The internal temperature reached 91.1℃ when heated for 2 minutes per 100g, 92.6℃ when heated for 5 minutes per 100g, and decreased to 87.8℃ due to water evaporation when heated for 10 minutes per 100g. Blanching for 5 minutes per 100g resulted in a 2.5-fold increase in AcCh content, with an internal temperature of 95.6℃.

[0125] Based on the above results, it can be inferred that the AcCh content increases under general heating processing conditions and remains relatively stable. It can be further inferred that even with different processing conditions, as long as the temperature change is the same, the AcCh content will increase. Furthermore, this process can be used for sterilization.

[0126] The results of experiments 4–6 show that acetylcholinesterase (EC number 3.1.1.7), an enzyme that breaks down cholinesterase, affects cholinesterase content. As described in Non-Patent Literature 3, acetylcholinesterase is widely distributed in plants. The reduction of AcCh shown in Table 8 is considered to be the main reason for acetylcholinesterase-induced decomposition. It can be assumed that more AcCh is decomposed at neutral pH (8.0–8.5), close to the optimal pH for acetylcholinesterase, while decomposition of AcCh is inhibited at low pH where enzyme activity decreases. Furthermore, it can be assumed that heating inactivates acetylcholinesterase, thus preventing the decomposition of AcCh and concentrating it through water evaporation. Therefore, controlling the processing technology of acetylcholinesterase is important for eggplant and eggplant products. Thus, the changes in AcCh content in frozen eggplant and eggplant products, where acetylcholinesterase inactivation in eggplant were investigated.

[0127] [Experiment 7] Experiment on the changes in AcCh content in frozen fresh eggplant and frozen heated eggplant Fresh eggplant (cut into approximately 10g pieces) and heated eggplant (samples cut into approximately 10g pieces were microwaved for 5 minutes at 550W per 100g piece) were frozen at -20℃. Samples were taken out every month, freeze-dried, and the AcCh content was quantified. The changes in AcCh content over 6 months were investigated. Furthermore, samples from the same eggplant fruit were used for both fresh and heated eggplant samples. The results are shown in Table 10.

[0128] [Table 10] It was found that AcCh content in both fresh and cooked eggplant remained almost unchanged after freezing, indicating that freezing processing can stably preserve cholinesterase in eggplant over a long period. In the case of fresh eggplant, this is a result of freezing inhibiting the activity of cholinesterase, the main cause of cholinesterase breakdown in eggplant. The change in AcCh content was almost identical to that in cooked eggplant, where pre-heating inactivates cholinesterase, suggesting that freezing-based inhibition of cholinesterase activity is sufficient.

[0129] [Experiment 8] Preparation of Eggplant Extract Eggplant extract was prepared using freeze-dried eggplant powder and fresh eggplant. The method for preparing the extract using freeze-dried eggplant powder is as follows.

[0130] 1 g of freeze-dried eggplant powder was weighed into a centrifuge tube (50 mL capacity). Ten times its weight (10 g) of solvent was added, with the EtOH concentration varying from 10% to 100% in 10% increments. The mixture was stirred at 3000 rpm for 30 minutes at room temperature, and then filtered to obtain the supernatant. The supernatant was transferred to an eggplant-shaped flask (200 mL), and 10 g of pure water was added. The mixture was concentrated using an evaporator. When the volume reached approximately 1 / 5, the contents were completely transferred to a centrifuge tube (15 mL) and freeze-dried. The freeze-dried product was used as the eggplant extract, and the yield was determined. Quantification was performed using the methods described in "2. Extraction Method" and "3. LC / MS / MS Analysis" above. Extracts were prepared using 50% EtOH and EtOH extraction with the addition of L-ascorbic acid. The results are shown in Table 11.

[0131] [Table 11] When using fresh eggplant, EtOH of various concentrations was added to 10g of chopped fresh eggplant and the mixture was pulverized using a grinder. The resulting extract was prepared using the same method as freeze-dried eggplant powder, and the yield and choline ester content were calculated accordingly. The weight of EtOH added was set at 0.5 times, equal to, and twice the weight of the fresh eggplant. Furthermore, L-ascorbic acid was added to the extract using an equal amount of EtOH to prepare the extract. The results are shown in Table 12.

[0132] [Table 12] Eggplant extract is a water-soluble semi-solid product in which choline esters are concentrated compared to freeze-dried and / or hot-air-dried powders. Stabilization of choline esters during extraction and prevention of color changes can be achieved by adding L-ascorbic acid to acidify the solution while controlling oxidation.

[0133] [Experiment 9] Single oral administration test of freeze-dried eggplant to spontaneously hypertensive rats The antihypertensive effect of freeze-dried eggplant was investigated by single oral administration to spontaneously hypertensive rats (SHR). Male 14-week-old SHR (weighing 324–368 g) were acclimatized for one week, then fasted for 12 hours before being administered a single oral dose of freeze-dried eggplant powder suspended in water using a probe. The freeze-dried eggplant powder was prepared by washing Izumizu water eggplant from Hannan City, Osaka Prefecture, sterilizing with hypochlorous acid, freeze-drying the edible portion of the sample, and then finely pulverizing it. Over 99% of the choline esters in the freeze-dried eggplant powder were AcCh, with an AcCh content of 2.25 mg / g D.W. The dosage was set at the equivalent of 1.00 × 10⁻⁶ AcCh. -9 Six SHRs were administered an oral dose of 0.065 mg / kg of freeze-dried eggplant powder (0.146 μg / kg). To compare the antihypertensive effect, 1.00 × 10⁻⁶ mg / kg of AcCh standard was also administered. -9 In a single-dose administration trial of eggplant freeze-dried powder, systolic blood pressure was measured. In the control group, water was administered only under the same conditions. Systolic blood pressure, diastolic blood pressure, and heart rate were measured at 0, 3, 6, 9, and 24 hours after oral administration using a non-invasive Softron BP-98A blood pressure monitor (Softron Co., Ltd., Tokyo) via the tailcuff method. Results of the single-dose oral administration trial of eggplant freeze-dried powder are shown in... Figures 8-10 .

[0134] The eggplant freeze-dried powder administration group induced a significant decrease in systolic blood pressure compared to the control group at 3 and 9 hours after administration (p < 0.05). The maximum blood pressure decrease was -17.8 mmHg at 9 hours after administration, a decrease of 10.0 mmHg compared to the control group, indicating a correlation with acetylcholine levels of 1.00 × 10⁻⁶. -9 The mol / kg dosage showed almost the same antihypertensive effect. Furthermore, diastolic blood pressure also showed a decreasing trend, with a maximum reduction to -11.3 mmHg 9 hours after administration, a decrease of 8.3 mmHg compared to the control group. Heart rate showed a decreasing trend compared to the control group 3 hours after administration, followed by an increasing trend up to 9 hours after administration. This confirms the antihypertensive effect of eggplant administered orally.

[0135] The effective daily dose of AcCh for SHR after a 12-hour fast is 1.00 × 10⁻⁶. -9 The effective daily dose of AcCh in unefast SHR was 1.00 × 10⁻⁶ mol (0.146 μg) / kg, while the effective daily dose in unefast SHR was 1.00 × 10⁻⁶ mol (0.146 μg) / kg (which showed a significant inhibitory effect on blood pressure elevation in repeated oral administration trials). -8The effective dose of SHR (1.46 μg / kg) was calculated using Kleiber's Law (Non-Patent Literature 21). The amount of AcCh required to induce a blood pressure-lowering effect in humans through oral intake of this normally controlled SHR was extrapolated to the human level using Kleiber's Law (Non-Patent Literature 22). Kleiber's Law states that mammalian metabolic rate is proportional to the 3 / 4 power of body weight. Assuming the SHR's weight is 370 g and a human's weight is 60 kg, the effective dose of SHR can be estimated to be 45.4 times the effective dose for humans. That is, the effective AcCh intake for inducing a blood pressure-lowering effect in SHR is 3.70 × 10⁻⁶ mol / kg. -9 If the effective daily dose is 1.68 × 10⁻⁶ mol (0.540 μg), then the effective daily dose for a person is 1.68 × 10⁻⁶ mol. -7 mol (24.5 μg).

[0136] Furthermore, the inventors have discovered the hypotensive and vasodilatory effects of fermented buckwheat (a lactic acid fermentation product of buckwheat plant). As active ingredients, choline esters including AcCh, BuCh, LaCh, and PrCh, when purified AcCh, BuCh, and PrCh are administered orally to an SHR (Single-Heat Rinse-Off) product, exhibit a hypotensive effect (Patent Documents 1 and 2). Further in-depth research has explored the hypotensive effect of oral administration of choline esters to humans.

[0137] Six men and six women aged 33–63 years with high-normal blood pressure and those with stage 1 hypertension were given one serving of a beverage containing fermented buckwheat (each serving contained 25 μg of choline esters) daily for four weeks.

[0138] The results showed that the subjects' systolic blood pressure decreased by -11.8 mmHg compared to pre-ingestion levels (p = 0.0080, t-test). That is, for humans, a significant decrease in blood pressure was confirmed by consuming a fermented buckwheat beverage containing 25 μg of cholinesterol as a blood pressure-lowering component for four consecutive weeks. This blood pressure-lowering effect was confirmed at a dosage very close to the effective dosage of AcCh, which induces a blood pressure-lowering effect on systolic blood pressure, obtained by extrapolating to humans using Kleiber's law.

[0139] In repeated oral administration trials to unfasted SHRs, 2.00 × 10 -9 The antihypertensive effect was significantly reduced at 0.292 μg / kg. Using Kleiber's law (non-patent literature 22) to extrapolate the dosage of this AcCh to humans, the lower limit of the effective daily human dose was found to be 3.36 × 10⁻⁶. -8 mol (4.91 μg) / kg. In the same experiment, 2.00 × 10 -7 The blood pressure-lowering effect was significantly reduced at 29.2 μg / kg. When this AcCh dosage was extrapolated to human levels using Kleiber's Law (Non-Patent Document 22), the upper limit of the effective daily dose for humans was 3.36 × 10⁻⁶.-6 mol (491 μg) / kg. Therefore, the effective daily dose for hypertensive individuals with genetic traits like SHR is estimated to be in the range of 5–500 μg.

[0140] The amount of choline esters that induces the blood pressure-lowering effect can be ultimately determined by taking into account the effective dosage as described above, as well as the amount of choline esters derived from diets other than the compositions of the present invention.

[0141] [Experiment 10] Single oral administration test of freeze-dried eggplant to normotensive rats The hypotensive effect was investigated by a single oral administration of freeze-dried eggplant powder to Wistar Kyoto rats (WKY rats) using the same method as in Experiment 9. WKY rats, parents of SHR, were normotensive rats with genetic predisposition to hypertension and were used as controls. Male 14-week-old WKY rats (320–362 g) were used, and the same freeze-dried eggplant powder as in Experiment 9 was administered. The dosage was set at 0.065 mg / kg of freeze-dried eggplant powder (equivalent to AcCh 1.00 × 10⁻¹⁰). -9 mol (0.146 μg) / kg) and 6.5 mg / kg (equivalent to AcCh 1.00 × 10 -7 mol (14.6 μg) / kg).

[0142] Results of a single oral administration study of freeze-dried eggplant powder to WKY rats are shown in Figure 11 .

[0143] AcCh 1.00×10 with SHR trigger operation -9 No change in blood pressure was observed in WKY rats when administered an equivalent mol / kg dose of freeze-dried eggplant powder. (Compared to AcCh 1.00×10) -7 When eggplant freeze-dried powder equivalent to mol / kg was administered, it triggered an AcCh reaction equivalent to 1.00 × 10⁻⁶ mol / kg in SHR. -9 A dose of mol / kg of freeze-dried eggplant powder had the same antihypertensive effect. Therefore, it is known that a greater amount of freeze-dried eggplant powder is needed to induce an antihypertensive effect in normotensive WKY rats.

[0144] [Experiment 11] Measurement of isotensive tension of blood vessels using eggplant pulverized samples The eggplant pulverized samples used in the experiment were prepared as follows: Izumi water eggplants from Hannan City, Osaka Prefecture, were washed and sterilized with hypochlorous acid. The edible portion of the resulting sample was sliced ​​into 1-3 cm wide pieces and pulverized using a commercially available food processor (CrushMillser IFM-C20G, Iwatani Sangyo Co., Ltd.) to form a paste. Alternatively, freeze-dried eggplant fruit slices (1-3 cm wide) obtained by heat sterilization (approximately 90°C, approximately 5 minutes) or hot-air dried eggplant fruit obtained by hot air drying (approximately 90°C, approximately 1-2 hours) were pulverized using a grinding mixer (MASTER, Tokyo Unicom Co., Ltd.) to form a powder. Each pulverized sample was passed through a stainless steel sieve (JIS standard, 20 mesh). The samples that passed through the sieve were considered as having passed through the 20 mesh sieve, and the samples that did not pass through the sieve were considered as having passed through the 20 mesh sieve. This process was used to prepare the following samples: fresh eggplant fruit pulverized material passed through the 20 mesh sieve (sample 1), fresh eggplant fruit pulverized material passed through the 20 mesh sieve (sample 2), freeze-dried powder passed through the 20 mesh sieve (sample 3), freeze-dried powder passed through the 20 mesh sieve (sample 4), hot air dried powder passed through the 20 mesh sieve (sample 5), and hot air dried powder passed through the 20 mesh sieve (sample 6).

[0145] All samples were stored at -98°C until used for testing. For the choline ester content in samples 1 and 2, the samples were freeze-dried, finely pulverized, and quantified using the methods described in "2. Extraction Method" and "3. LC / MS / MS Analysis" above. For samples 3 and 5, the powders were used directly, and quantification was performed using the methods described in "2. Extraction Method" and "3. LC / MS / MS Analysis" above. For samples 4 and 6, the samples were finely pulverized, and quantification was performed using the methods described in "2. Extraction Method" and "3. LC / MS / MS Analysis" above.

[0146] More than 99% of the choline esters in the eggplant pulverized sample were AcCh, and the AcCh content is shown in Table 13.

[0147] [Table 13] The isometric tension of blood vessels was measured as follows.

[0148] For male 14-week-old SHRs (weighing 320–346 g), the rats were euthanized by abdominal dissection and exsanguination under ether anesthesia, and the thoracic aorta was quickly removed. The removed aorta was immersed in physiological saline to thoroughly wash away the blood, and then the connective tissue and adipose tissue attached to the blood vessels were removed in Krebs-Henseleit solution to prepare a ring specimen approximately 2–3 mm wide. The prepared ring specimens were placed in an organ bath of an isometric tension testing apparatus (UFER UC05A, Kishimoto Medical Industry Co., Ltd.) filled with a Kreber-Hensleit solution (119 mM NaCl / 4.7 mM KCl / 1.1 mM KH2PO4 / 1.2 mM MgSO4 / 25 mM NaHCO3, pH 7.4, 37°C) containing a mixed gas (95% O2, 5% CO2). The optimal static tension of 1.0 g was applied to stabilize the ring specimens for 60 minutes. Then, phenylephrine (PE, 0.3 μM) was added to the organ bath. After vasoconstriction and stabilization, AcCh (final concentration 100 μM) was added. Once the endothelial condition was confirmed to be normal, the specimens were washed with Kreber-Hensleit solution to restore the static tension. After 15 minutes, the blood vessels were constricted again using the vasoconstrictor PE (0.3 μM) to confirm that the constriction had reached its maximum. Then, the final concentration of AcCh was increased to 10. -9 10 -8 10 -7 10 -6.5 10 -6 10 -5.5 10 -5 Eggplant pulverized samples were suspended in a Kreber-Hensleit solution under M conditions. Tension changes were measured and recorded using a transducer (UFER UM-20, Kishimoto Medical Industry Co., Ltd.), and the diastolic rate (%) was calculated using the following formula (1). The mean ± standard deviation (Mean ± SE) of three repeated tests was then calculated. The results of the isometric tension test of the eggplant pulverized samples are shown in... Figure 12 The results and EC obtained by the probability unit method 50 (50% effective concentration) Statistical analysis results are shown in Table 14.

[0149] Equation (1) Relaxation rate (%) = {maximum tension (g) - tension at sample addition (g)} / maximum tension (g) [Table 14] According to the final concentration of AcCh reaching 10 -9 10 -8 10 -7 10 -6.5 10 -6 10 -5.5 10-5 The concentration of eggplant pulverized sample added under condition M is shown in Table 14. The freeze-drying yield of the fresh eggplant fruit pulverized material used in this experiment was 6.0%. Therefore, the concentration of eggplant pulverized material in the isometric tension test of fresh eggplant fruit pulverized material sieved through 20 mesh was the highest, at 19 mg / mL.

[0150] Besides cholinesterol, chlorogenic acid and γ-aminobutyric acid (GABA), which may affect vasodilation, were present at concentrations of 0.56 mg / gFW and 0.24 mg / gFW, respectively, in this experimental system at concentrations of 11 μg / mL and 4.8 μg / mL. No effect on blood vessels was observed at these concentrations in experiments using their respective standards. In experimental systems using other eggplant pulverized materials, the concentrations of these compounds were even lower, thus their intervention on vasodilation was extremely limited; therefore, cholinesterol can be considered the main vasodilatory component.

[0151] In all eggplant pulverized samples, a dose-dependent vasodilatory effect was observed. The freeze-dried powder exhibiting the strongest vasodilatory effect, sieved through a 20-mesh sieve (sample 3), showed an AcCh concentration of 10 in the laboratory. -8 M condition added eggplant pulverized sample (6.2×10 -4 After adding (mg / mL), a significant vasodilation of 19% was observed compared to before adding freeze-dried eggplant powder (p<0.05), subsequently reaching 10 mg / mL. -6 M (eggplant pulverized sample 6.2×10) -2 (mg / mL) showed significant vasodilation depending on the dosage, although slight vasoconstriction occurred at higher concentrations, but significant vasodilation was still observed. Maximum vasodilation was achieved at AcCh concentrations reaching 10. -6 M's eggplant pulverized sample (6.2×10) -2 At 80% (mg / mL), the hot-air-dried powder exhibiting a secondary vasodilatory effect was sieved through a 20-mesh sieve at an AcCh concentration of 10 mg / mL. -8 M condition added eggplant pulverized sample (7.2×10 - 4 After adding (mg / mL), a significant vasodilation of 15% was observed compared to before adding freeze-dried eggplant powder (p<0.05), subsequently reaching 10 mg / mL. -6 M (eggplant pulverized sample 7.2×10) -2 (mg / mL) showed significant vasodilation depending on the dosage, although slight vasoconstriction occurred at higher concentrations, but significant vasodilation was still observed. Maximum vasodilation was achieved at AcCh concentrations reaching 10. -6 M's eggplant pulverized sample (7.2 × 10⁻⁶) -2 The concentration of the eggplant powder was 78% at 1 mg / mL. Based on these results, the strong vasodilatory effect of dried eggplant powder was confirmed.

[0152] On the other hand, samples 4 and 6 reached an AcCh concentration of 10. -7 M (freeze-dried powder 8.9×10 -3 mg / mL, hot-air dried powder 9.8 × 10 -3 Significant vasodilation was observed from the addition of AcCh at a concentration of 10 mg / mL (p < 0.05), with the maximum vasodilation rate reaching 10 mg / mL. -5 The eggplant powder in sample M was 57% freeze-dried and 50% hot-air dried. Comparing the vasodilatory effects of samples 3 and 4, the concentration of AcCh reached 10% with the addition of sample 3. -8 M confirmed a significantly stronger vasodilatory effect, 10 -8 The vasodilatory effect of M was 6.5 times greater. Similarly, the vasodilatory effect of sample 5 compared to sample 6 increased by 10% from the AcCh concentration. -8 M is significantly stronger, 10 -8 The vasodilatory effect of M was 7.8 times greater. Based on the above results, it is confirmed that the vasodilatory effects of samples 3 and 5 are stronger than those of samples 4 and 6, respectively.

[0153] Furthermore, it is known that samples 1 and 2 reached an AcCh concentration of 10. -6 M (1.4 mg / mL), 10 -6.5 Significant vasodilatory effects were observed starting at M (0.59 mg / mL) (p < 0.05), but the vasodilatory effect was weaker than that of the dried powder. The dried powder sieved through a 20-mesh sieve had a high effect on organisms.

[0154] EC 50 An indicator of the intensity of the biological effects of the test specimen. The vasodilatory effect (EC5) of specimen 3 in this test. 50 The concentration was 0.015 mg / mL, confirming significantly stronger activity than eggplant powder other than sample 5, with an activity intensity 467 times that of sample 1, 660 times that of sample 2, 16 times that of sample 4, and 26 times that of sample 6. Furthermore, sample 5, which showed a secondary vasodilatory effect, had an EC50 of 0.015 mg / mL. 50 The concentration was 0.027 mg / mL, confirming a significantly stronger activity than eggplant pulverized materials other than sample 3. The activity intensity 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.

[0155] Based on the above results, it is concluded that dried eggplant powder finer than 20 mesh has a stronger vasodilatory effect.

[0156] [Experiment 12] Repeated oral administration test using freeze-dried eggplant Freeze-dried eggplant was administered orally to spontaneously hypertensive rats (SHR) daily for 4 weeks to confirm its antihypertensive effect. Changes in urinary catecholamine levels were also examined to evaluate its anti-stress effect.

[0157] Six-week-old male SHRs (weighing 263–286 g) were domesticated and fed for six days before being divided into a control group (administered with pure water, n=6) and an experimental group (administered with freeze-dried eggplant powder, n=6). Both groups were housed in separate metabolic cages under conditions of 23±4℃, 50±20% humidity, and a 12-hour light-dark cycle (5:30 AM–5:30 PM). Standard feed (MF, Oriental Yeast Industry Co., Ltd.) was used, and tap water was provided freely. The eggplant used was Tosa Taka eggplant (harvested in February, from Kochi Prefecture), and freeze-dried eggplant powder was prepared using the method described in [Experiment 9]. The dosage of the freeze-dried eggplant powder was set at 0.82 mg / kg (bw) per animal (AcCh conversion: 10). -8 The drug was administered orally for 30 days via a stainless steel oral nasogastric tube (mol / kg). During the experiment, feed intake and water intake were measured twice weekly, body weight once weekly, and urine output daily. Blood pressure (systolic and diastolic) was measured before the start of the experiment and on days 7, 14, 21, and 28 after the start of the experiment, as described in [Experiment 9]. Each measurement was repeated three times, and the mean ± standard deviation (Mean ± SE) was calculated.

[0158] Urinary catecholamines were quantified using urine samples taken the day before blood pressure measurement. All urine samples were collected within 24 hours starting the day before the commencement of repeated oral administration. Urine was collected once daily using a collection container containing 1 mL of 5N hydrochloric acid. After measuring the urine volume, the collected urine was frozen at -80°C until analysis. For analysis, the urine samples were thawed, and the supernatant obtained by centrifugation (4°C, 1000×g, 3 min) was washed with MonoSpin PBA (Agilent Technologies), dissolved in 400 μL of 1% acetic acid aqueous solution, and the concentrated urinary catecholamine sample was 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, delivery unit: LC-20AD, column furnace: CTO-10A) and an electrochemical detector (ECD, Agilent Technologies, ED723 diamond electrode) under the following conditions, and the mean ± standard deviation (Mean ± SE) was obtained.

[0159] Column: Inertsil ODS-4 (4.6×250mm, Agilent Technologies) Flow rate: 0.8 mL / min, separation temperature: 35℃, standard injection volume: 20 μL Mobile phase: Acetate-citrate buffer / CH3CN (100 / 16, v / v) Detection conditions: ECD 800mV (reference electrode Ag / AgCl) As shown in Tables 15, 16 and 17, there were no significant differences in body weight, urine output, water intake and total feed intake between the two groups throughout the feeding period.

[0160] [Table 15] [Table 16] [Table 17] like Figure 13 and Figure 14 As shown, compared with the control group, the test group showed significantly lower systolic blood pressure on days 14, 21, and 28 of the trial, and significantly lower diastolic blood pressure on day 28. The blood pressure-lowering effect of eggplant freeze-dried powder intake, which was found in the single oral administration trial, was also confirmed in the repeated oral administration trial.

[0161] like Figure 15 and Figure 16 As shown, the levels of epinephrine and norepinephrine, representative catecholamines in urine, were significantly lower in the test group compared to the control group. Urinary epinephrine levels were significantly lower in the test group on days 20 and 27 after the start of the experiment, and urinary norepinephrine levels were significantly lower in the test group compared to the control group on days 6 and 27 after the start of the experiment.

[0162] Norepinephrine is a neurotransmitter released from sympathetic nerve endings, while adrenaline is produced by the conversion of norepinephrine in the adrenal glands. These catecholamines act on adrenaline receptors and participate in the control of various organs and metabolic systems. While the released catecholamines act on effectors reached by the nerves, some migrate into the bloodstream to affect the whole body. Blood catecholamines are metabolized and inactivated, and sometimes they are directly excreted into the urine. Therefore, the amount of catecholamines in urine becomes an indicator of sympathetic nervous system activity in the organism. That is, hyperactive sympathetic nervous system activity results in increased urinary catecholamine levels. The sympathetic nervous system promotes arousal, aggression, defense, and escape behaviors. Catecholamines released due to hyperactive sympathetic nervous system activity act on adrenaline α receptors in blood vessels, causing vasoconstriction and raising blood pressure. Therefore, the mechanism of the hypotensive effect of orally ingested choline esters is believed to be closely related to the reduction of catecholamines produced by the inhibition of sympathetic nervous system activity.

[0163] Furthermore, it is known that organisms respond to stress by increasing sympathetic nerve activity, and catecholamine levels increase due to stress. Therefore, catecholamines are known as stress indicators.

[0164] As an indicator of the anti-stress effect of an anti-stress composition with β-carotene as the active ingredient in organisms, catecholamines (adrenaline, norepinephrine, and dopamine) were used. A significant reduction in catecholamines due to the composition's uptake demonstrated an anti-stress effect (Patent Document 3). Furthermore, significantly low levels of serum epinephrine (described in the literature) and norepinephrine (described in the literature) levels caused by restraint stress after administration of yeast hydrolysate corroborated the anti-stress effect (Non-Patent Document 23). In the evaluation of the anti-stress effect based on candesartan cetirizine (an angiotensin II receptor 1 antagonist), urinary catecholamines (adrenaline and norepinephrine) were used as an indicator of solitary feeding stress in metabolic cages, and significantly low levels of each were considered evidence of the anti-stress effect.

[0165] In Experiment 12, the significant decrease in urinary catecholamine levels due to the intake of freeze-dried eggplant powder can be attributed to the anti-stress effect resulting from the inhibition of sympathetic nerve activity by the freeze-dried eggplant powder. This confirms the anti-stress effect of freeze-dried eggplant powder intake. The vasodilatory effect in Experiment 11 was induced by the action of cholinesterol contained in eggplant on muscarinic acetylcholine receptors (Non-Patent Literature 25). This anti-stress effect is also considered to be the result of the action of cholinesterol contained in eggplant on muscarinic acetylcholine receptors, inhibiting sympathetic nerve activity and thus inhibiting the release of catecholamines from nerve endings. It can be concluded that extracts and processed foods made from eggplant containing a certain amount of cholinesterol have anti-stress effects. The blood pressure-lowering effect induced by the inhibition of sympathetic nerve activity from oral intake of cholinesterol is closely related to the anti-stress effect. Therefore, the difference in cholinesterol dosage between SHR and WKY rats exhibiting blood pressure-lowering effects can also be applied to anti-stress effects.

[0166] 4. Implementation Discussion Through the inventors' research, a blood pressure-lowering effect was confirmed with an intake of a very small amount of 25 μg per day for cholinesterol. The weight of agricultural products required to ingest 25 μg of AcCh was set as the daily intake target, and the daily intake targets for 20 kinds of fresh agricultural products quantified in Experiments 1 and 2 were calculated. The results are shown in Table 18.

[0167] [Table 18] As shown in Table 18, to obtain 25 μg of AcCh, a very large amount of the 18 agricultural products other than eggplant and bamboo shoots would be required. For example, for lettuce, approximately 7.5 kg or more would be needed daily, which is unrealistic for a single day and impossible to maintain consistently. On the other hand, the target daily intake for eggplant and bamboo shoots is only 0.41 g and 0.25 g, respectively, so a blood pressure-lowering effect can be expected with a sustainable daily intake. Furthermore, among the seven eggplant varieties used in this experiment, five varieties—Quanzhou water eggplant, Batten eggplant, Guangling salad eggplant, Feihou purple, and Longma—have soft skin and low astringency, making them suitable for raw consumption. When consumed raw, the reduction of cholinesterol due to cooking need not be considered.

[0168] Industrial applications The composition of the present invention has a significant blood pressure lowering effect, and by containing it as an active ingredient, it can be used to manufacture functional labeled foods or pharmaceuticals for the treatment of hypertension.

Claims

1. A composition having antihypertensive and / or anti-stress effects, with choline esters as the active ingredient, wherein, The choline ester content is 5μg to 50mg, and it is intended for oral administration.

2. A method for manufacturing a composition having antihypertensive and / or anti-stress effects for oral administration, with choline esters as the active ingredient, wherein, include: The process of preparing edible plants into freeze-dried powder and / or hot-air dried powder; the process of distributing freeze-dried powder and / or hot-air dried powder according to the condition that the choline ester content reaches 5μg to 50mg.

3. A composition for oral administration containing choline esters as the active ingredient, having antihypertensive and / or anti-stress effects, wherein, The composition is manufactured by the method of claim 2.

4. A method for manufacturing an extract containing choline esters as the active ingredient, for oral administration, having antihypertensive and / or anti-stress effects, wherein, include: The process of extracting edible plants or freeze-dried and / or hot-air dried powders of edible plants with ethanol or aqueous ethanol.

5. An extract containing choline esters as the active ingredient, for oral administration, possessing antihypertensive and / or anti-stress effects, wherein, The extract is produced by the method described in claim 4.

Images