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 causative molecule of vasospasm. Furthermore, it has been revealed that a key mechanism in vasospasm is that SPC activates Fyn, a protein belonging to the Src family of tyrosine kinases, by irreversibly myristoylating the second glycine molecule at the N-terminus and reversibly palmitoylating the third and sixth cysteine ​​molecules. This activates Fyn by binding to local structures called membrane rafts on the vascular smooth muscle cell membrane, and subsequently activates Rho kinase by binding to the membrane rafts.

[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 a salt thereof, 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. [Effect of the Invention]

[0013] The vasospasm inhibitor and the oral composition for suppressing vasospasm of the present invention have an action of preventing and suppressing abnormal contraction (vasospasm) of blood vessels, and are useful for preventing or treating various diseases caused by vasospasm by ingesting them as pharmaceuticals, foods, or functional foods (including foods for specified health use, foods with functional claims, nutrient-functional foods, etc.). [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 collina on the abnormal contraction of vascular smooth muscle cells. [Figure 2] It is a graph showing the inhibitory effect of the HPLC fractions (Fr.A to F) of the aerial part extract of Salsola collina 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 fractions (Fr.A and Fr.B) of the aerial part extract of Salsola collina 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 a salt thereof, 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 a salt thereof, 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, and examples include Salacia oblonga, Salacia reticulata, Salacia chinensis, Salacia prinoides, Salacia latifolia, Salacia burunoniana, Salacia grandiflora, and Salacia macrosperma. In addition, the above Salacia plants can 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 may be coated with a conventional coating as needed, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, or double-coated or multi-layered tablets.

[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 administration frequency is once or several times a day (for example, 2 to 3 times).

[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 the 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 can be administered before or after abnormal vasoconstriction or vasospasm occurs.

[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 of the above-ground parts (stem, trunk) 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, 1,000 mL of hot water was added to 100 g of dried shredded Salacia material. The mixture was boiled for 1 hour, during which time it was stirred periodically with a spatula. The extract was filtered through filter paper, and the filtrate was collected to obtain the above-ground Salacia extract and the underground Salacia extract, 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). After gently agitating the cells to homogenize them within the wells, culturing was initiated in a CO2 incubator (37°C, 5% CO2). 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). After gently agitating the cells to homogenize them within the well, they were cultured again in a CO2 incubator. 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, the plate was removed, and the absorbance of only the viable cells that had turned orange due to the water-soluble formazan was measured at 450 nm using a microplate reader, and the cell viability was calculated. Cytotoxicity experiments were conducted on extracts from the aerial and underground parts of Salacia, and 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, followed by UV sterilization. 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 cultures were incubated in a CO2 incubator for approximately 12 hours. The supernatant was removed, 200 μL of membrane staining reagent was added to the center, and the sample was incubated in a CO2 incubator for approximately 5 minutes.

[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. A 200 μL condensate (60 μM sphingosylphosphorylcholine; SPC) was added to the center, and images were acquired after 5 minutes (fixed-point imaging). The relative value was calculated by setting the cell area before contraction induction (untreated) to 100, and the rate of change compared to before addition was evaluated to quantify whether abnormal contraction occurred.

[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 by setting the cell area before contraction induction (untreated) to 100, and the rate of change compared to before addition was evaluated to quantify the suppression of the abnormal contraction that occurred.

[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 was subjected to an HP-20 column, and the liquid that flowed out without adsorption was collected as the "pass-through 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) (Nacalai Tesque Co., Ltd.) was set. The void volume that flows out after adding the developing solvent (methanol:water = 60:40) was collected as Fraction (Fr.) 1, Fr. 2 from 0 to 9 minutes, Fr. 3 from 10 to 19 minutes, Fr. 4 from 20 to 29 minutes, Fr. 5 from 30 to 39 minutes, Fr. 6 from 40 to 49 minutes, and Fr. 7 from 50 to 59 minutes. The washing portion from 60 to 69 minutes was collected as Fr. 8.

[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. Since Fr.2-6 showed both preventive and inhibitory effects, and their effects were similar, and all exhibited identical absorption spectra, Fr.2-6 were mixed together.

[0061] 5. Fractionation A mixture of Fr.2-6 was subjected to HPLC and fractionated into Fr.A-F. Of these, Fr.A was a broad peak with absorption at UV 270 nm and a retention time of 8-10 minutes, Fr.B was a broad peak with three peak tops with absorption at UV 270 nm and a retention time of 13-20 minutes, and Fr.C-F did not have UV absorption, so they were fractionated at 10-minute intervals starting from 20 minutes, yielding a total of 6 fractions. Fr.B, which has three peak tops, was further fractionated and purified into Fr.B-1 (retention time 13.8 mins), Fr.B-2 (retention time 19.6 mins), and Fr.B-3 (retention time 18.0 mins). 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 The effects of fractions Fr.A to F 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. Statistical significance was tested 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. In addition, for the fractionated Fr.7, Fr.7a, and Fr.7b, the effects on the sustained contraction of vascular smooth muscle cells (vascular abnormal contraction) were confirmed by the preventive experiment and inhibitory experiment described in Example 1. The concentration of each test sample was 10 μg / mL. The results are shown in Fig. 4. The significance test was performed by ANOVA and Dunnet test. Weak preventive and inhibitory effects were observed for Fr.7 and Fr.7b, and significant preventive and inhibitory effects were observed for Fr.7a.

[0063] 7. Mass Spectrometry and Database Search Distilled water: methanol = 1:1 was added to the concentrated dried product of each fractionated fraction and dissolved. The measurement conditions for mass spectrometry were ionization ESI, Scan Type EPI, Curtain Gas 20 psi, Collision Gas High, Ion Spray Voltage 5500 V, Temperature 500 °C, and Collision Energy 40 eV. For data analysis, the free software MassBank was used.

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

[0065] Fr.7a 1 H NMR (TMS) OH 9.68, 12.71 ppm CH 6.91, 6.91, 7.93, 7.93 ppm 1313C NMR (TMS) C 122.8, 163.7, 169.3 ppm CH 115.8, 115.8, 131.7, 131.7 ppm

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

[0067] Fr.A was estimated to be 2,5-dihydroxybenzoic acid based on its molecular weight of 154.12 determined by mass spectrometry and NMR analysis. It was then identified as 2,5-dihydroxybenzoic acid by confirming its retention time using HPLC with a standard sample. Fr.A 2,5-Dihydroxybenzoic acid

[0068] [ka]

[0069] Fr.B was identified by mass spectrometry with a molecular weight of 354.31. Since separation and purification were difficult using NMR, Fr.B-1 (retention time 13.8 min) was identified as trans-3-caffeoylquinic acid, Fr.B-2 (retention time 19.6 min) as trans-4-caffeoylquinic acid, and Fr.B-3 (retention time 18.0 min) as trans-5-caffeoylquinic acid by confirming the retention time using standard samples via HPLC. 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] Mass spectrometry determined that Fr.7 has a molecular weight of 138.12. NMR analysis suggested that Fr.7a is 2-hydroxybenzoic acid and Fr.7b is 4-hydroxybenzoic acid. HPLC retention time analysis using standard samples confirmed that Fr.7a is 2-hydroxybenzoic acid and Fr.7b is 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, 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 claim 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 claim 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 claim 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, 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.

6. The oral composition for inhibiting vasospasm according to claim 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 inhibiting vasospasm according to claim 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 its salt, and (B) The oral composition for inhibiting vasospasm according to claim 5, wherein the composition 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.

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