Vasospasm inhibitors

Abstract

The present invention provides a vasospasm inhibitor and an oral composition for inhibiting vasospasm that can suppress abnormal vasoconstriction (vasospasm). [Solution] A vasospasm inhibitor and an oral composition for inhibiting vasospasm, containing as an active ingredient at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, trans-5-caffeoylquinic acid, and 2-hydroxybenzoic acid, or a salt thereof.

Description

[Technical Field]

[0001] The present invention relates to a vasospasm inhibitor and an oral composition for inhibiting vasospasm. [Background technology]

[0002] Ca is needed for vasoconstriction. 2+ Concentration-dependent "normal contraction" and Ca 2+ Vasoconstriction (vasospasm), in which vascular smooth muscle contracts independently of blood pressure, is known, and a considerable portion of normal contraction, including its mechanism, has been elucidated, which is responsible for the normal function of blood vessels such as maintaining blood pressure. On the other hand, vasospasm, which is a sustained contraction of vascular smooth muscle, occurs suddenly without any warning, and the blood vessels remain constricted and do not return to their normal state, causing ischemia. When vasoconstriction (vasospasm) occurs in the brain, it causes fatal diseases such as "cerebral infarction," and when it occurs in the heart, it causes fatal diseases such as "myocardial infarction" and "angina pectoris."

[0003] To date, it has been revealed that sphingosylphosphorylcholine (SPC), a type of sphingolipid, is a molecule that causes vasospasm. Furthermore, SPC activates Fyn by performing irreversible myristoylation to the second glycine at the N-terminus of Fyn, a protein belonging to the Src family tyrosine kinase, and reversible palmitoylation to the third and sixth cysteine. This allows it to bind to local structures called membrane rafts on the vascular smooth muscle cell membrane, and subsequently activates Rho ki It has been revealed that the binding and activation of NASE to membrane rafts is an important mechanism in vasospasm.

[0004] Substances that do not affect normal vasoconstriction but specifically inhibit abnormal vasoconstriction are useful in preventing or treating diseases involving vasospasm (such as cerebral infarction, myocardial infarction, and angina pectoris). In recent years, substances with vasospasmodic effects have been reported.

[0005] Patent Document 1 discloses a vasospasm inhibitor containing a hot water extract of a plant of the genus Salacia.

[0006] Patent Document 2 identifies 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid as compounds containing extracts of Salacia plants that have antivasoconstrictive effects.

[0007] On the other hand, chlorogenic acids are polyphenols found in plants such as coffee beans, and have been reported to have physiological functions such as antioxidant effects, blood pressure lowering effects, and lipid burning promoting effects. Caffeoylquinic acids such as 3-caffeoylquinic acid, 4-caffeoylquinic acid, and 5-caffeoylquinic acid are known as chlorogenic acids (Patent Document 3).

[0008] Patent Document 4 discloses that an ester compound of a chlorogenic acid derivative and arginine exhibits vasodilatory effects. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2016-56138 [Patent Document 2] Japanese Patent Publication No. 2019-104695 [Patent Document 3] Japanese Patent Publication No. 2024-41656 [Patent Document 4] Japanese Patent Publication No. 2006-298802 [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a novel vasospasm inhibitor and an oral composition for inhibiting vasospasm that can suppress abnormal vasoconstriction (vasospasm). [Means for solving the problem]

[0011] In order to solve the above problems, the inventors conducted further studies on extracts of Salacia plants and, as a result, identified several compounds from Salacia plant extracts that have antivasoconstrictive effects. Furthermore, they found that combining these newly identified compounds synergistically enhances the antivasoconstrictive effect. The present invention is based on these findings.

[0012] In other words, the present invention is as follows. [1] A vasospasm inhibitor containing, as an active ingredient, at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, trans-5-caffeoylquinic acid, and 2-hydroxybenzoic acid, or a salt thereof. [2] The vasospasm inhibitor according to [1], wherein the active ingredient is at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof. [3] The vasospasm inhibitor according to [1], wherein the active ingredient is at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof. [4] The active ingredient is, (A) 2,5-dihydroxybenzoic acid or its salt, and (B) The vasospasm inhibitor according to [1], which is at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof. [5] An oral composition for inhibiting vasospasm, comprising as an active ingredient at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, trans-5-caffeoylquinic acid, and 2-hydroxybenzoic acid, or a salt thereof. [6] The oral composition for suppressing vasospasm according to [5], wherein the active ingredient is at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid or a salt thereof. [7] The oral composition for suppressing vasospasm according to [5], wherein the active ingredient is at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid or a salt thereof. [8] The active ingredient is (A) 2,5-dihydroxybenzoic acid or a salt thereof, and (B) The oral composition for suppressing vasospasm according to [5], which is at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid or a salt thereof.

Effects of the Invention

[0013] The vasospasm inhibitor and the oral composition for suppressing vasospasm of the present invention have an effect of preventing and suppressing abnormal contraction (vasospasm) of blood vessels. By ingesting them as pharmaceuticals, foods, or functional foods (including foods for specified health uses, foods with functional claims, foods with nutritional functions, etc.), they are useful for preventing or treating various diseases caused by vasospasm.

Brief Description of the Drawings

[0014] [Figure 1] It is a graph showing the preventive effect and inhibitory effect of the aerial part extract and the underground part extract of Salsola on the abnormal contraction of vascular smooth muscle cells. [Figure 2] It is a graph showing the inhibitory effect of the HPLC fraction (Fr.A~F) of the aerial part extract of Salsola on the abnormal contraction of vascular smooth muscle cells. [Figure 3] It is a graph showing the preventive effect and inhibitory effect of the HPLC fraction (Fr.A and Fr.B) of the aerial part extract of Salsola on the abnormal contraction of vascular smooth muscle cells. [Figure 4] This graph shows the preventive and inhibitory effects of HPLC fractions (Fr.7, Fr.7a, and Fr.7b) of Salacia aerial extract on abnormal contraction of vascular smooth muscle cells. [Modes for carrying out the invention]

[0015] The compounds used in the present invention are at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, trans-5-caffeoylquinic acid, and 2-hydroxybenzoic acid, or a salt thereof. Each compound is represented by the following formula.

[0016] 2,5-Dihydroxybenzoic acid (compound of formula (I))

[0017] [ka]

[0018] trans-3-caffeoylquinic acid (compound of formula (II))

[0019] [ka]

[0020] trans-4-caffeoylquinic acid (compound of formula (III))

[0021] [ka]

[0022] trans-5-caffeoylquinic acid (compound of formula (IV))

[0023] [ka]

[0024] 2-Hydroxybenzoic acid (compound of formula (V))

[0025] [ka]

[0026] The active ingredient used in the present invention is preferably at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof, and more preferably at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof.

[0027] In the present invention, it is preferable to use only at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, trans-5-caffeoylquinic acid, and 2-hydroxybenzoic acid, or a salt thereof, as the active ingredient. Furthermore, it is more preferable to use only at least one compound selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof, as the active ingredient, and even more preferable to use only at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof, as the active ingredient.

[0028] Furthermore, in the present invention, as an active ingredient, (A) 2,5-dihydroxybenzoic acid or its salt, and (B) It is also preferable to use a combination of at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof.

[0029] In the present invention, as an active ingredient, (A) 2,5-dihydroxybenzoic acid or its salt, and (B) It is also preferable to use only a combination of at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof.

[0030] The following combinations of (A) and (B) are possible: (1) A combination of 2,5-dihydroxybenzoic acid or a salt thereof, and trans-3-caffeoylquinic acid or a salt thereof. (2) A combination of 2,5-dihydroxybenzoic acid or a salt thereof, and trans-4-caffeoylquinic acid or a salt thereof. (3) A combination of 2,5-dihydroxybenzoic acid or a salt thereof, and trans-5-caffeoylquinic acid or a salt thereof.

[0031] In the present invention, the molar ratio of (A):(B) can be in the range of 0.5:1 to 5:1, 1:1 to 4:1, 1.5:1 to 3.5:1, 1.8:1 to 3.2:1, 0.5:1 to 3:1, 1:1 to 3:1, or 2:1 to 3:1. It is preferable that the amount of (A) is greater than the amount of (B). By increasing the amount of (A) compared to (B), the vasospasm inhibitory effect is synergistically enhanced.

[0032] The salt of the compound that is the active ingredient of the present invention is not particularly limited as long as it is a pharmaceutically acceptable salt. Examples of such salts of the compound include alkali metal salts and alkaline earth metal salts. Examples of alkali metal salts include lithium salts, sodium salts, potassium salts, etc. Examples of alkaline earth metal salts include magnesium salts, calcium salts, etc.

[0033] The active ingredient compound of the present invention can be synthesized by known methods. Alternatively, commercially available products can be used. Furthermore, it can be obtained by extraction and purification from the roots, trunks, branches, stems, or leaves of plants of the genus Salacia. The plants of the genus Salacia are not particularly limited as long as they belong to the genus Salacia of the family Salaciaaceae, including Salacia oblonga, etc. Salacia reticulata, Salacia chinensis Salacia prinoides, Salacia latifolia ), Salacia burunoniana, Salacia grandiflora (S. Examples include *Salacia grandiflora* and *Salacia macrosperma*. Furthermore, the above-mentioned Salacia species may be used individually or in combination of two or more species. Extraction and purification can be carried out according to the methods described in the examples below.

[0034] In this invention, vasospasm refers to cytoplasmic Ca 2+ This refers to abnormal contraction of vascular smooth muscle that is independent of Ca concentration. 2+ This refers to contractions that do not fall under the category of "normal contractions" which are dependent on concentration.

[0035] The effect on sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction) can be predicted, for example, by the same method as the "contraction experiment" and / or "prevention experiment" described in the examples below.

[0036] The vasospasm inhibitor and oral composition for inhibiting vasospasm of the present invention have effects such as preventing cerebral infarction, preventing myocardial infarction, inhibiting cerebral vasospasm after subarachnoid hemorrhage, and preventing angina pectoris. Therefore, they can be advantageously used as pharmaceuticals such as agents for treating cerebral infarction-related diseases, agents for treating myocardial infarction-related diseases, agents for inhibiting cerebral vasospasm, agents for inhibiting cardiovascular vasospasm, and other agents for preventing or treating vasospasm. They can also be used as preventive agents or symptom-improving agents for conditions involving vasospasm, and as pharmacological composition materials for producing supplements and functional foods (including Foods for Specified Health Uses, Foods with Function Claims, and Nutrient Function Foods) that have preventive and ameliorative effects on conditions involving vasospasm.

[0037] The vasospasm inhibitor and oral composition for inhibiting vasospasm of the present invention are manufactured by known methods and contain an active ingredient compound (hereinafter, the compound used as an active ingredient in the present invention is also referred to as "the compound of the present invention") in amounts of 0.000001 to 99.9% by weight, 0.00001 to 99.8% by weight, 0.0001 to 99.7% by weight, 0.001 to 99.6% by weight, 0.01 to 99.5% by weight, 0.1 to 99% by weight, 0.5 to 60% by weight, 1 to 50% by weight, or 1 to 20% by weight. The vasospasm inhibitor may be a solid dosage form or a liquid dosage form.

[0038] The vasospasm inhibitor and oral composition for inhibiting vasospasm of the present invention may contain pharmaceutically acceptable carriers widely used in the field of pharmaceutical manufacturing. For example, such pharmaceutically acceptable carriers include solvents (e.g., water, physiological saline, buffer, glycerin, organic solvents), emulsifiers, suspending agents, disintegrants, binders, solubilizers, excipients, diluents, pH buffers, solubilizers, isotonic agents, stabilizers, preservatives, lubricants, flavoring agents, sweeteners, gelling agents, absorption retarders, liposomes, and the like. Furthermore, the above-mentioned vasospasm inhibitor can be formulated in a dosage form suitable for parenteral administration, topical administration, or oral administration. These dosage forms are not particularly limited and include tablets, pills, lozenges, capsules, granules, injectable preparations by subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc. (e.g., sterile aqueous solutions or dispersions), sterile powders, and the like.

[0039] When forming the tablets, a wide range of carriers conventionally known in this field can be used, such as excipients like lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid; binders like water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone; and dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, and charcoal. Examples of disintegrants include calcium sulfate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose, etc.; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers such as quaternary ammonium bases, sodium lauryl sulfate, etc.; humectants such as glycerin, starch, etc.; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.; stearates such as refined talc, magnesium stearate, etc.; lubricants such as boric acid powder, polyethylene glycol, etc. Furthermore, tablets can be coated with conventional coatings as needed, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, and film-coated tablets. It can be a coating tablet, a double tablet, or a multi-layer tablet.

[0040] When forming the product into pill form, a wide range of conventionally known carriers can be used, such as excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc; binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol; and disintegrants such as laminaran and agar. When forming the product into suppositories, a wide range of conventionally known carriers can be used, such as polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.

[0041] When prepared as an injectable preparation, the liquid and suspension preparations are preferably sterilized and isotonic with blood. When forming these liquid, emulsion, and suspension preparations, all diluents commonly used in this art can be used, such as water, physiological saline, vegetable oil, solubilizers, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of sodium chloride, glucose, or glycerin to prepare an isotonic solution may be included in the preparation, and ordinary solubilizers, buffers, analgesics, etc. may also be added. Furthermore, colorants, preservatives, fragrances, flavorings, sweeteners, and other pharmaceuticals may be included in the preparation as needed.

[0042] The effective dose of the vasospasm inhibitor and oral composition for vasospasm inhibition of the present invention depends on the sex, age, symptoms, etc. of the recipient, but the amount of the compound of the present invention contained in such vasospasm inhibitor and oral composition for vasospasm inhibition is in the range of 0.005 mg to 50 mg / kg body weight / day per adult, preferably 0.001 mg to 10 mg / kg body weight / day, and more preferably 0.01 mg to 5 mg / kg body weight / day. The number of doses is once to several times a day. For example, administer it in 2-3 divided doses.

[0043] Furthermore, the compounds of the present invention can be used as foods, food compositions, food materials, or food additives to prevent vasospasm in healthy individuals. For example, the compounds of the present invention can be used in the manufacture of functional foods, health foods, supplements, etc., for alleviating mild vasospasm.

[0044] For example, the compound of the present invention may be mixed with appropriate additives such as rice flour, oils and fats, starch, lactose, maltose, vegetable oil powder, cocoa butter powder, and stearic acid, and then molded into an edible form, such as a paste, drink, soft capsule, seamless capsule, hard capsule, granules, tablets, or pills, using conventional means, for consumption. It may also be added to various foods, such as bread (white bread, sweet bread, etc.), jam, biscuits, cookies, rice crackers and other confectionery, cakes, gum, instant foods (instant ramen, instant miso soup, instant soup, etc.), ice cream products, and beverages such as yogurt, milk, drinks, and soft drinks (tea, coffee, black tea, juice, etc.). The amount of the compound of the present invention is appropriately determined depending on the type and state of the edible composition.

[0045] Mammals are preferred as the target subjects for administration of vasospasm inhibitors and oral compositions for inhibiting vasospasm. Examples of mammals include humans, dogs, cats, rabbits, horses, sheep, goats, cattle, pigs, monkeys, rats, mice, and guinea pigs. Humans are particularly preferred as the mammal.

[0046] The compounds of the present invention suppress abnormal contraction of vascular smooth muscle cells that form blood vessels. Therefore, the vasospasm inhibitor and the oral composition for inhibiting vasospasm can suppress vasospasm. The vasospasm inhibitor and the oral composition for inhibiting vasospasm are used before abnormal vasoconstriction or vasospasm occurs. Alternatively, it can be administered later.

[0047] The compounds of the present invention prevent abnormal contraction of vascular smooth muscle cells that form blood vessels. For this reason, the vasospasm inhibitor and the oral composition for inhibiting vasospasm may also be used as a vasospasm preventive agent or an oral composition for preventing vasospasm. Preferably, the vasospasm preventive agent and the oral composition for preventing vasospasm are administered before abnormal vasoconstriction or vasospasm occurs.

[0048] In another embodiment, an oral composition for inhibiting vasospasm or for preventing vasospasm is provided, containing the compound of the present invention as an active ingredient. Examples of oral compositions include supplements, food compositions, functional foods (including foods for specified health uses, foods with functional claims, foods with nutritional function claims, etc.), and food additives. [Examples]

[0049] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples.

[0050] Example 1 The preventive / inhibitory / improving effects of Salacia extract on sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction). 1. Extraction Dried shredded material, consisting of the above-ground parts (trunk, stem) and underground parts (roots) of Salacia plants, each cut into 6 mm blocks, was mixed with 10 times its volume of hot water. For example, 100 g of dried shredded Salacia material was mixed with hot water. 1,000 mL of water was added. It was then boiled for 1 hour to extract the liquid, and during that time, spatula was used to remove excess liquid. The mixture was stirred with a tumbler. The extract was filtered through filter paper, and the filtrate was collected to obtain the above-ground extract of Salacia and the underground extract of Salacia, respectively.

[0051] 2. Confirmation of effectiveness The effects of extracts from the aerial and underground parts of Salacia on the sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction) were confirmed. The experiment was conducted using the method described in the cell experiment below.

[0052] 3. Cell experiments 3-1.Cytotoxicity Normal human coronary artery smooth muscle cells (HCASMCs) were treated with growth medium to reach 1.0 × 10⁻⁶ cells. 5 A cell suspension was prepared at a concentration of cells / mL. 100 μL of growth medium was added to a 96-well plate, and 100 μL / well (10,000 cells / well) of cell suspension was seeded (100 μL of growth medium was added to the blanks instead of cell suspension). Gently agitate to homogenize the cells in the well, then place in a CO2 incubator (37°C, 5% CO2). Culturing was started within the ). After 24 hours from the start of culture, the plates were removed, the supernatant was removed, and 100 μL / well of the test sample was added (100 μL / well of growth medium was added to the control). Gently agitate to homogenize the cells in the well, then culture again in a CO2 incubator. Ta. 48 hours after the start of culture, the plate was removed, 10 μL / well of succinate tetrazolium reductase chromogenic reagent was added, and after gentle stirring, the mixture was reacted in a CO2 incubator. Three hours after the start of the reaction, remove the plate and use a microplate reader to analyze the water-soluble form. Only the number of viable cells that produced Mazan's orange color were measured by absorbance at 450 nm, and the cell viability was calculated. Cytotoxicity experiments were conducted on extracts from the aerial and underground parts of Salacia. As a result, no cytotoxicity was observed in the concentration range of 0 to 100 μg / mL.

[0053] 3-2. Contraction Experiment 30 μL of the collagen mixture was added to the center of a glass-bottom dish, spread thinly, and left to stand in a clean bench for 2 hours before being sterilized with ultraviolet light. After adding 1.8 mL of growth medium and seeding 120 μL of HCASMC cell suspension (12,000 cells / dish), the cells were incubated in a CO2 incubator for approximately 48 hours. The supernatant was removed, 2 mL of basal medium was added, and the mixture was incubated in a CO2 incubator for approximately 12 hours. Remove the supernatant, add 200 μL of membrane staining reagent to the center, and incubate in a CO2 incubator for approximately 5 minutes. It was cultured.

[0054] (1) Prevention experiment The supernatant was removed, 200 μL of the test sample was added to the center, and the mixture was incubated in a CO2 incubator for approximately 30 minutes. The glass-bottom dish was removed, and an image was obtained using a fluorescence microscope before the addition of SPC. Add 200 μL of shrinkage agent (60 μM sphingosylphosphorylcholine; SPC) to the center and wait 5 minutes. Images were later acquired (fixed-point photography). The relative value was calculated with the cell area before contraction induction (untreated) set to 100, and the percentage change compared to before addition was evaluated. The value and the absence of abnormal contractions were quantified.

[0055] (2) Inhibition experiment The glass-bottom dish was removed, and an image was obtained using a fluorescence microscope before the addition of SPC. 200 μL of a shrinking agent (60 μM SPC) was added to the center, and 5 minutes later, 200 μL of the test sample was added to the center and images were acquired (fixed-point imaging). The relative value was calculated with the cell area before contraction induction (untreated) set to 100, and the percentage change compared to before addition was evaluated. The value and the ability to suppress the abnormal contractions that occurred were quantified.

[0056] The results are shown in Figure 1. The concentration of each test sample was 10 μg / mL. Significant difference testing was performed using ANOVA and Dunnett's test. When the cell area of ​​untreated cells was set to 100, the cells contracted due to the shrinkage agent, reducing the cell area to approximately 45%. However, prophylactic co-culturing with Salacia aerial and underground extracts suppressed (prevented) abnormal contraction. Furthermore, investigations into the inhibitory effects of Salacia aerial and underground extracts revealed that both were able to suppress abnormal contraction.

[0057] Example 2 1. Fractionation A Diaion HP-20 (Mitsubishi Chemical Corporation) was set up in an open column. Salacia aerial extract is passed through an HP-20 column, and the liquid that flows out without being adsorbed is "passed through". It was recovered as a "fraction." Methanol was applied to the HP-20 column to elute the components adsorbed on the HP-20, and the resulting liquid was collected as the "elution fraction."

[0058] 2. Confirmation of effectiveness The effects of the fractionated "pass-through fraction" and "elution fraction" on sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction) were confirmed by the preventive and inhibitory experiments described in Example 1. The concentration of each test sample was 20 μg / mL. The "pass-through fraction" showed neither preventive nor inhibitory effects. The "eluted fraction" showed both preventive and inhibitory effects.

[0059] 3. Fractionation To further fractionate the "elution fraction," silica gel (C) is placed on an open column. 18 -OPN)(Naka I set up (Litesk Co., Ltd.). Add the developing solvent (methanol:water = 60:40) and record the void volume that flows out as Fraction (Fr.). 0-9 minutes is Fr.2, 10-19 minutes is Fr.3, 20-29 minutes is Fr.4, 30-39 minutes is Fr.5, 40-49 minutes is Fr.5. The first minute was collected as Fr.6, the 50-59 minute portion as Fr.7, and the 60-69 minute portion as Fr.8 for washing.

[0060] 4. Confirmation of effectiveness The effects of fractions Fr.1 to Fr.8 on sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction) were confirmed by the preventive and inhibitory experiments described in Example 1. The concentration of each test sample was 10 μg / mL. No preventive or inhibitory effects were observed in Fr.1 and Fr.8. Fr.7 showed weak prophylactic and inhibitory effects, and since two peaks were observed, it was re-fractionated into Fr.7a and Fr.7b and purified. Fr.2-6 showed both preventive and inhibitory effects, and their effects were similar; all were administered by the same inhaler. Based on the yield spectrum observed, Fr.2-6 were mixed.

[0061] 5. Fractionation A mixture of Fr.2-6 was subjected to HPLC and fractionated into Fr.A-F. Of these, Fr.A absorbed UV light at 270 nm. It is a broad peak with absorption and retention time of 8-10 minutes, and Fr.B has absorption at UV270 nm. The sample showed a broad peak with three peak tops at retention times of 13-20 minutes. Since Fr.C-F did not exhibit UV absorption, the sample was fractionated at 10-minute intervals starting from a retention time of 20 minutes, yielding a total of six fractions. Fr.B, which has three peak tops, is divided into Fr.B-1 (holding time 13.8 minutes) and Fr.B-2 (holding time 19.6 minutes). The sample was then re-fractionated into Fr.B-3 (retention time 18.0 minutes) and purified. HPLC conditions Column: CAPCELL PAK C18 (4.6φ × 250 mm) (Osaka Soda Co., Ltd.) Mobile phase: 0.05 vol% H3PO4 / CH3CN=90 / 10 Temperature: 25℃ Flow rate: 1 mL / min Detection: Photodiode array Analysis time: 1 hour

[0062] 6. Confirmation of effectiveness Regarding the fractions Fr.A-F, the effects of sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction) The effects were confirmed by the preventive and inhibitory experiments described in Example 1. The concentration of each test sample was 10 μg / mL. Significant difference testing was performed using ANOVA and Dunnett's test. Figure 2 shows the results of the inhibition experiments for Fr.A to F. Significant inhibitory effects were observed in Fr.A and Fr.B. . Next, prevention and inhibition experiments were conducted for Fr.A and Fr.B. The results are shown in Figure 3. Significant preventive and inhibitory effects were observed only in the two fractions, Fr.A and Fr.B. Furthermore, the effects of the fractionated Fr.7, Fr.7a, and Fr.7b on sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction) were confirmed by the preventive and inhibitory experiments described in Example 1. The concentration of each test sample was 10 μg / mL. The results are shown in Figure 4. Statistical significance tests were performed using ANOVA and Dunnett's test. Weak preventive and inhibitory effects were observed in Fr.7 and Fr.7b, while significant preventive and inhibitory effects were observed in Fr.7a.

[0063] 7. Mass Spectrometry and Database Search To the concentrated dry matter of each fraction, distilled water and methanol in a 1:1 ratio were added and dissolved. The mass spectrometry measurement conditions were: ionization ESI, Scan Type EPI, Curtain Gas 20 psi, Collisio n Gas High, Ion Spray Voltage 5500 V, Temperature 500℃, Collision Energy 40 eV That's what I decided. For data analysis, we used the free software MassBank.

[0064] The NMR measurement data for Fr.A, Fr.7a, and Fr.7b are shown below. Fr.A 1 1H NMR (TMS) OH 9.45, 12.04, 15.2 ppm CH 7.28, 7.29, 7.35 ppm 13 13C NMR (TMS) C 115.6, 151.0, 154.8, 171.8 ppm CH 115.6, 118.7, 122.5 ppm

[0065] Fr.7a 1 1H NMR (TMS) OH at 9.68, 12.71 ppm CH at 6.91, 6.91, 7.93, 7.93 ppm 13 13C NMR (TMS) C at 122.8, 163.7, 169.3 ppm CH at 115.8, 115.8, 131.7, 131.7 ppm

[0066] Fr.7b 1 1H NMR (TMS) OH at 12.04, 15.20 ppm CH at 7.08, 7.51, 7.58, 8.07 ppm 13 13C NMR (TMS) C at 113.1, 162.2, 171.8 ppm CH at 117.6, 121.2, 131.7, 135.3 ppm

[0067] Fr.A was determined to have a molecular weight of 154.12 by mass spectrometry and was deduced to be 2,5-dihydroxybenzoic acid from NMR confirmation. It was identified as 2,5-dihydroxybenzoic acid by confirming the retention time by HPLC using a standard product. Fr.A 2,5-dihydroxybenzoic acid

[0068]

Chemical formula

[0069] Fr.B had a molecular weight of 354.31 by mass spectrometry. Since NMR separation and purification were difficult, a standard product was used. By confirming the retention time using HPLC, Fr.B-1 (retention time 13.8 mins) was identified as trans-3-caffeoylquinic acid, Fr.B-2 (retention time 19.6 mins) as trans-4-caffeoylquinic acid, and Fr.B-3 (retention time 18.0 mins) as trans-5-caffeoylquinic acid. Fr.B-1 trans-3-caffeoylquinic acid

[0070] [ka]

[0071] Fr.B-2 trans-4-caffeoylquinic acid

[0072] [ka]

[0073] Fr.B-3 trans-5-caffeoylquinic acid

[0074] [ka]

[0075] Fr.7 was determined to have a molecular weight of 138.12 by mass spectrometry, and NMR analysis suggested that Fr.7a is 2-hydroxybenzoic acid and Fr.7b is 4-hydroxybenzoic acid. Retention time was then confirmed by HPLC using the standard. Based on this, Fr.7a was identified as 2-hydroxybenzoic acid and Fr.7b as 4-hydroxybenzoic acid. Fr.7a 2-Hydroxybenzoic acid

[0076] [ka]

[0077] Fr.7b 4-Hydroxybenzoic acid

[0078] [ka]

[0079] Cytotoxicity experiments described in Example 1 were performed on the standards of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, trans-5-caffeoylquinic acid, and 2-hydroxybenzoic acid. The results showed no cytotoxicity in the concentration range of 0 to 100 μM.

[0080] Example 3 Confirmation of synergistic effects In the shrinkage experiment, when co-culturing with HCASMC, standards containing 2,5-dihydroxybenzoic acid and caffeoylquinic acid, which have strong abnormal shrinkage prevention and inhibitory effects, were added simultaneously. The effect on the preventive effect was confirmed by the preventive experiment described in Example 1. The ratio of standards in the test samples is shown as the molar ratio. A significant preventive effect was observed when the amount of 2,5-dihydroxybenzoic acid added was greater than the amount of caffeoylquinic acid added.

[0081] [Table 1] [Industrial applicability]

[0082] The vasospasm inhibitor and oral composition for inhibiting vasospasm of the present invention are useful as pharmaceutical or food compositions for preventing or suppressing vasospasm (abnormal vasoconstriction).

Claims

1. A vasospasm inhibitor containing only one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof, as an active ingredient.

2. An oral composition for inhibiting vasospasm, containing only at least one compound selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid, or a salt thereof, as an active ingredient.

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