vasospasm inhibitors

Abstract

The present invention provides a vasospasm inhibitor and an oral composition for inhibiting vasospasm, which can inhibit abnormal contraction of blood vessels (vasospasm). [Solution] A vasospasm inhibitor and an oral composition for inhibiting vasospasm, which contain as an active ingredient at least one compound or a salt thereof 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.

Description

[Technical Field]

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

[0002] Ca is required for vascular contraction 2+ "Normal contraction" dependent on Ca concentration and 2+ "Abnormal vasoconstriction (vasospasm)" is known as a concentration-independent contraction of vascular smooth muscle, and much of the mechanism behind normal contraction, which is responsible for normal vascular functions such as maintaining blood pressure, has been clarified. On the other hand, vasospasm, which is a sustained contraction of vascular smooth muscle, occurs suddenly without warning, causing ischemia as blood vessels remain constricted and do not return to their original state. Abnormal vasoconstriction (vasospasm) in the brain can cause cerebral infarction, and in the heart can cause fatal diseases such as myocardial infarction and angina pectoris.

[0003] It has been revealed that sphingosylphosphorylcholine (SPC), a type of sphingolipid, is the molecule responsible for vasospasm. Furthermore, SPC irreversibly myristoylates the second glycine residue of the N-terminus of Fyn, a member of the Src family of tyrosine kinases, and reversibly palmitoylates the third and sixth cysteines. 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 membrane rafts. This is an important mechanism in vasospasm.

[0004] Substances that specifically inhibit abnormal vascular contraction without affecting normal vascular contraction are useful for the prevention or treatment of diseases involving vasospasm (such as cerebral infarction, myocardial infarction, and angina pectoris). In recent years, substances with vasospasm inhibitory 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] In Patent Document 2, 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid are identified as compounds contained in extracts of Salacia plants that have an inhibitory effect on vasospasm.

[0007] Meanwhile, chlorogenic acids are polyphenols present in plants such as coffee beans, and have been reported to have physiological functions such as antioxidant activity, blood pressure lowering activity, and lipid burning promotion activity. Known chlorogenic acids include caffeoylquinic acids such as 3-caffeoylquinic acid, 4-caffeoylquinic acid, and 5-caffeoylquinic acid (Patent Document 3).

[0008] Patent Document 4 discloses that an ester conjugate of a chlorogenic acid derivative and arginine exhibits a vasorelaxing effect. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-56138 [Patent Document 2] Japanese Patent Application Publication No. 2019-104695 [Patent Document 3] Japanese Patent Application Publication No. 2024-41656 [Patent Document 4] Japanese Patent Application Laid-Open No. 2006-298802 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a novel vasospasm inhibitor and an oral composition for inhibiting vasospasm, which are capable of inhibiting abnormal contraction of blood vessels (vasospasm). [Means for solving the problem]

[0011] In order to solve the above-mentioned problems, the present inventors further investigated extracts of Salacia plants and, as a result, identified several compounds having antivasospasm activity from extracts of Salacia plants. Furthermore, they found that the antivasospasm activity is synergistically enhanced by combining these newly identified compounds. The present invention was made based on these findings.

[0012] That is, the present invention is as follows. [1] A vasospasm inhibitor containing, as an active ingredient, at least one compound or a salt thereof 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. [2] The vasospasm inhibitor described in [1], wherein the active ingredient is at least one compound or a salt thereof selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid. [3] The vasospasm inhibitor described in [1], wherein the active ingredient is at least one compound or a salt thereof selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid. [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, containing as an active ingredient at least one compound or a salt thereof 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. [6] The oral composition for inhibiting vasospasm described in [5], wherein the active ingredient is at least one compound or a salt thereof selected from the group consisting of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid. [7] The oral composition for inhibiting vasospasm described in [5], wherein the active ingredient is at least one compound or a salt thereof selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid. [8] The active ingredient is (A) 2,5-dihydroxybenzoic acid or a salt thereof, and (B) The oral composition for inhibiting vasospasm described in [5], which is at least one compound or a salt thereof selected from the group consisting of trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, and trans-5-caffeoylquinic acid. [Effects of the Invention]

[0013] The vasospasm inhibitor and oral composition for inhibiting vasospasm of the present invention have the effect of preventing and inhibiting abnormal contraction of blood vessels (vasospasm), and when ingested as a medicine, food, or functional food (including specified health foods, functional foods, nutritional functional foods, etc.), they are useful for preventing or treating various diseases caused by vasospasm. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a graph showing the preventive and inhibitory effects of an extract from the aerial parts of Salacia and an extract from the underground parts of Salacia on abnormal contraction of vascular smooth muscle cells. [Figure 2] 1 is a graph showing the inhibitory effect of HPLC fractions (Fr. A to F) of an extract from the aerial parts of Salacia on abnormal contraction of vascular smooth muscle cells. [Figure 3] 1 is a graph showing the preventive and inhibitory effects of HPLC fractions (Fr. A and Fr. B) of an extract from the aerial parts of Salacia on abnormal contraction of vascular smooth muscle cells. [Figure 4] 1 is a graph showing the preventive and inhibitory effects of HPLC fractions (Fr. 7, Fr. 7a, and Fr. 7b) of an extract from the aerial parts of Salacia on abnormal contraction of vascular smooth muscle cells. DETAILED DESCRIPTION OF THE INVENTION

[0015] The compound used in the present invention is 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 of which 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 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. It is more preferable to use 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 it is even more preferable to use 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] In addition, in the present invention, as an active ingredient, (A) 2,5-dihydroxybenzoic acid or a salt thereof, and (B) It is also preferred 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, the active ingredient is (A) 2,5-dihydroxybenzoic acid or a salt thereof, and (B) It is also preferred 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 combinations of (A) and (B) include the following: (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) may 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. The molar ratio of (A) to (B) is preferably such that the amount of (A) is greater than the amount of (B). By increasing the amount of (A) compared to the amount of (B), the vasospasm inhibitory effect is synergistically enhanced.

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

[0033] The compounds serving as active ingredients of the present invention can be synthesized by known methods. Alternatively, commercially available products can be used. Furthermore, they can be obtained by extraction and purification from the roots, trunks, branches, stems, or leaves of Salacia plants. The Salacia plants are not particularly limited as long as they belong to the genus Salacia in the family Salaciaaceae. Examples of Salacia plants include Salacia oblonga, Salacia reticulata, Salacia chinensis, Salacia prinoides, Salacia latifolia, Salacia brunoniana, Salacia grandiflora, and Salacia macrosperma. The above Salacia plants can be used alone or in combination. Extraction and purification can be performed according to the methods described in the Examples below.

[0034] In the present invention, vasospasm refers to cytoplasmic Ca 2+ It means abnormal contraction of vascular smooth muscle that is not dependent on Ca concentration. 2+ This refers to contraction that does not fall under the "normal contraction" that is concentration-dependent.

[0035] The effect on sustained contraction of vascular smooth muscle cells (abnormal vascular contraction) can be predicted, for example, by methods similar to 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, and can therefore be advantageously used as pharmaceuticals such as therapeutic agents for cerebral infarction-related diseases, therapeutic agents for myocardial infarction-related diseases, cerebral vasospasm inhibitors, cardiac vasospasm inhibitors, and other preventive or therapeutic agents for vasospasm, as well as prophylactic agents and 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 functional claims, foods with nutrient claims, etc.) that have the effect of preventing or improving conditions involving vasospasm.

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

[0038] The vasospasm inhibitor and oral composition for vasospasm inhibition of the present invention may contain a pharmaceutically acceptable carrier widely used in the field of pharmaceutical manufacturing. Examples of pharmaceutically acceptable carriers include solvents (e.g., water, physiological saline, buffer solutions, glycerin, organic solvents), emulsifiers, suspending agents, disintegrants, binders, solubilizers, excipients, diluents, pH buffers, solubilizers, isotonicity agents, stabilizers, preservatives, lubricants, flavoring agents, sweeteners, gelling agents, absorption retardants, liposomes, and the like. The vasospasm inhibitor can also be formulated into dosage forms suitable for parenteral, topical, or oral administration. These dosage forms include, but are not limited to, tablets, pills, lozenges, capsules, granules, subcutaneous, intravenous, intramuscular, or intraperitoneal injections (e.g., sterile aqueous solutions or dispersions), and sterile powders.

[0039] When forming into tablets, a wide variety of carriers conventionally known in this field can be used, including excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone; dry starch, sodium alginate, powdered agar, powdered laminaran, sodium bicarbonate, and carbon dioxide; Examples of suitable disintegrants include disintegrants such as calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc.; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc.; absorption promoters 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 purified talc, magnesium stearate, etc.; boric acid powder, lubricants such as polyethylene glycol, etc. Furthermore, tablets can be coated with a conventional coating, as needed, for example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, or double-layered or multi-layered tablets.

[0040] When forming into pills, a wide variety of carriers conventionally known in this field can be used, such as excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oils, kaolin, talc, etc., binders such as powdered gum arabic, powdered tragacanth, gelatin, ethanol, etc., disintegrants such as laminaran, agar, etc. When forming into suppositories, a wide variety of carriers conventionally known can be used, such as polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.

[0041] When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood. When forming these solutions, emulsions, and suspensions, any diluent commonly used in this field can be used, such as water, physiological saline, vegetable oil, solubilizer, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In this case, the preparation may contain sufficient amounts of salt, glucose, or glycerin to prepare an isotonic solution, and conventional solubilizers, buffers, soothing agents, etc. Furthermore, the preparation may contain coloring agents, preservatives, fragrances, flavors, sweeteners, and other pharmaceuticals, as needed.

[0042] The effective dose of the vasospasm inhibitor and oral composition for inhibiting vasospasm of the present invention depends on the sex, age, symptoms, etc. of the subject, but the amount of the compound of the present invention contained in such vasospasm inhibitor and oral composition for inhibiting vasospasm 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 a day or in divided doses several times (for example, 2 to 3 times).

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

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

[0045] The subjects to which the vasospasm inhibitor and oral composition for inhibiting vasospasm are administered are preferably mammals. Examples of mammals include humans, dogs, cats, rabbits, horses, sheep, goats, cows, pigs, monkeys, rats, mice, and guinea pigs. Humans are particularly preferred.

[0046] The compounds of the present invention inhibit abnormal contraction of vascular smooth muscle cells that form blood vessels. Therefore, the vasospasm inhibitor and oral composition for inhibiting vasospasm can inhibit vasospasm. The vasospasm inhibitor and oral composition for inhibiting vasospasm can be administered before or after the onset of abnormal vascular contraction or vasospasm.

[0047] The compound of the present invention prevents abnormal contraction of vascular smooth muscle cells that form blood vessels. Therefore, the vasospasm inhibitor and the oral composition for inhibiting vasospasm may be used as a vasospasm preventive agent or oral composition for preventing vasospasm. Preferably, the vasospasm preventive agent and oral composition for preventing vasospasm are administered before the occurrence of abnormal vascular contraction or vasospasm.

[0048] In another embodiment, there is provided an oral composition for inhibiting or preventing vasospasm, which comprises 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 nutrient functions, etc.), food additives, etc. [Example]

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

[0050] Example 1 Preventive / inhibitory / ameliorative effects of Salacia extract on sustained contraction of vascular smooth muscle cells (abnormal vascular contraction) 1. Extraction The above-ground parts (stem, stalk) and underground parts (roots) of a Salacia plant were each shredded into 6 mm blocks, and 10 volumes of hot water were added to the shredded material. For example, 100 g of dried shredded Salacia was added to 1,000 mL of hot water. The mixture was boiled for 1 hour to extract, with periodic stirring using a spatula. The extract was filtered through filter paper, and the filtrate was collected and used as the Salacia above-ground part extract and the Salacia underground part extract, respectively.

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

[0052] 3. Cell Experiments 3-1.Cytotoxicity Normal human coronary artery smooth muscle cells (HCASMC) were cultured in a growth medium at a concentration of 1.0 × 10 5 A cell suspension of 100 cells / mL was prepared. 100 μL of growth medium was added to a 96-well plate, and the cell suspension was seeded at 100 μL / well (10,000 cells / well) (100 μL of growth medium was added to the blank instead of the cell suspension). After gentle stirring to homogenize the cells in the wells, culturing was initiated in a CO2 incubator (37°C, 5% CO2). After 24 hours from the start of culture, the plate was removed, the supernatant was removed, and the test sample was added at 100 μL / well (for the control, growth medium was added at 100 μL / well). The cells were gently stirred to uniformly distribute them in the wells, and then cultured again in a CO2 incubator. After 48 hours of incubation, the plate was removed, and 10 μL / well of a color-developing reagent containing succinate tetrazolium reductase was added. After gentle stirring, the plate was allowed to react in a CO 2 incubator. Three hours after the start of the reaction, the plate was removed, and the number of viable cells that had developed the orange color of water-soluble formazan was measured by a microplate reader at 450 nm for absorbance, and the cell viability was calculated. Cytotoxicity tests were conducted on extracts from the aboveground parts and underground parts of Salacia, and no cytotoxicity was observed within the concentration range of 0 to 100 μg / mL.

[0053] 3-2. Contraction experiment 30 μL of the collagen mixed solution was added to the center of the glass-bottom dish, spread thinly, and left to stand in a clean bench for 2 hours, followed by ultraviolet sterilization. 1.8 mL of growth medium was added, and 120 μL of HCASMC cell suspension was seeded (12,000 cells / dish), followed by culturing in a CO 2 incubator for approximately 48 hours. The supernatant was removed, and 2 mL of basal medium was added, followed by culturing in a CO2 incubator for approximately 12 hours. The supernatant was removed, and 200 μL of membrane staining reagent was added to the center, followed by incubation in a CO2 incubator for approximately 5 minutes.

[0054] (1) Preventive Experiments The supernatant was removed, and 200 μL of the test sample was added to the center and cultured in a CO incubator for approximately 30 minutes. The glass-bottom dish was removed and images were taken under a fluorescence microscope before the addition of SPC. 200 μL of a contractile agent (60 μM sphingosylphosphorylcholine; SPC) was added to the center, and images were taken 5 minutes later (fixed-point imaging). The relative value was calculated by setting the cell area before contraction induction (untreated) as 100, and the rate of change from before addition was evaluated to quantify the absence of abnormal contraction.

[0055] (2) Inhibition experiment The glass-bottom dish was removed and images were taken under a fluorescence microscope before the addition of SPC. 200 μL of a contractile agent (60 μM SPC) was added to the center, and 5 minutes later, 200 μL of a test sample was added to the center and images were taken (fixed-point photography). The relative value was calculated with the cell area before contraction induction (untreated) set at 100, and the rate of change from before addition was evaluated to quantify the ability to suppress the abnormal contraction that occurred.

[0056] The results are shown in Figure 1. The concentration of each test sample was 10 μg / mL. Significance tests were performed using ANOVA and Dunnett's test. When the cell area of ​​untreated cells was taken as 100, the contractile agent caused the cells to shrink, reducing the cell area to approximately 45%. However, when a Salacia aerial part extract and a Salacia underground part extract were co-cultured as a preventative measure, the abnormal shrinkage was suppressed (prevented). Furthermore, when the inhibitory effects of the Salacia aerial part extract and the Salacia underground part extract were examined, it was found that both extracts were able to suppress the abnormal shrinkage.

[0057] Example 2 1. Fractionation A Diaion HP-20 (Mitsubishi Chemical Corporation) was set in the open column. The extract from the aerial parts of Salacia was applied to an HP-20 column, and the liquid that flowed through without adsorption was collected as the "flow-through fraction." To elute the components adsorbed to the HP-20, methanol was applied to the HP-20 column, and the liquid that came out was collected as the "eluted fraction."

[0058] 2. Confirmation of effectiveness The effects of the fractionated "flow-through fraction" and "eluted fraction" on sustained contraction of vascular smooth muscle cells (abnormal vascular contraction) were confirmed by the prevention experiment and the inhibition experiment 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" was confirmed to have both preventive and inhibitory effects.

[0059] 3. Fractionation To further fractionate the eluted fraction, silica gel (C 18 -OPN) (Nacalai Tesque Inc.) was set. The void volume that flowed out after adding the developing solvent (methanol:water = 60:40) was collected as Fraction (Fr.) 1, 0-9 min as Fr. 2, 10-19 min as Fr. 3, 20-29 min as Fr. 4, 30-39 min as Fr. 5, 40-49 min as Fr. 6, 50-59 min as Fr. 7, and the wash fraction from 60-69 min 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 vascular contraction) were confirmed by the prevention experiment and the inhibition experiment described in Example 1. The concentration of each test sample was 10 μg / mL. No preventive or inhibitory effect was observed in Fr.1 and Fr.8. Fr. 7 showed weak preventive and inhibitory effects, and since two peaks were observed, it was further fractionated into Fr. 7a and Fr. 7b and purified. Fr. 2 to 6 were confirmed to have both preventive and inhibitory effects, and the effects were comparable. Since all of them showed the same absorption spectrum, Fr. 2 to 6 were mixed.

[0061] 5. Fractionation The mixture of fractions 2 to 6 was subjected to HPLC and fractionated into fractions A to F. Of these, fraction A is a broad peak with UV absorption at 270 nm and a retention time of 8 to 10 minutes, fraction B is a broad peak with three peak tops with UV absorption at 270 nm and a retention time of 13 to 20 minutes, and fractions C to F have no UV absorption and were fractionated at 10-minute intervals starting from the retention time of 20 minutes, yielding a total of six fractions. Fraction B, which had three peak tops, was further fractionated and purified into Fraction B-1 (retention time 13.8 min), Fraction B-2 (retention time 19.6 min), and Fraction B-3 (retention time 18.0 min). 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 A to F on sustained contraction of vascular smooth muscle cells (abnormal vasoconstriction) were confirmed by the prevention and inhibition experiments described in Example 1. The concentration of each test sample was 10 μg / mL. Significance tests were performed using ANOVA and Dunnett's test. The results of the inhibitory experiment for Fr. A to F are shown in Figure 2. Significant inhibitory effects were observed in Fr. A and Fr. B. Next, prevention and suppression experiments were carried out for Fr. A and Fr. B. The results are shown in Figure 3. Only two fractions, Fr.A and Fr.B, showed significant preventive and inhibitory effects. Furthermore, the effects of fractions Fr. 7, Fr. 7a, and Fr. 7b on sustained contraction of vascular smooth muscle cells (abnormal vascular contraction) 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. Significance tests were performed using ANOVA and Dunnett's test. Weak preventive and inhibitory effects were observed for Fr. 7 and Fr. 7b, while significant preventive and inhibitory effects were observed for Fr. 7a.

[0063] 7. Mass Spectrometry and Database Searching Distilled water:methanol=1:1 was added to the concentrated dry product of each fraction, and the mixture was dissolved. The mass spectrometry conditions were as follows: ESI ionization, EPI scan type, Curtain gas 20 psi, Collision gas high, Ion spray voltage 5500 V, Temperature 500°C, Collision energy 40 eV. The free software MassBank was used for data analysis.

[0064] The NMR measurement data for 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 13C NMR (TMS) C 122.8, 163.7, 169.3 ppm CH 115.8, 115.8, 131.7, 131.7 ppm

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

[0067] Fraction A had a molecular weight of 154.12 by mass spectrometry, and was estimated to be 2,5-dihydroxybenzoic acid by NMR confirmation. It was also identified as 2,5-dihydroxybenzoic acid by confirmation of the retention time by HPLC using an authentic sample. Fr.A 2,5-dihydroxybenzoic acid

[0068] [ka]

[0069] Mass spectrometry revealed that Fr. B had a molecular weight of 354.31. However, because separation and purification by NMR was difficult, 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 HPLC using authentic samples. 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 revealed that Fr. 7 had a molecular weight of 138.12, and NMR confirmed that Fr. 7a was estimated to be 2-hydroxybenzoic acid and Fr. 7b was estimated to be 4-hydroxybenzoic acid. HPLC confirmation of the retention times using authentic samples identified Fr. 7a 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 preparations of 2,5-dihydroxybenzoic acid, trans-3-caffeoylquinic acid, trans-4-caffeoylquinic acid, trans-5-caffeoylquinic acid, and 2-hydroxybenzoic acid. Results showed no cytotoxicity at concentrations ranging from 0 to 100 μM.

[0080] Example 3 Confirmation of synergistic effects In the contraction experiment, when co-cultured with HCASMC, preparations of 2,5-dihydroxybenzoic acid and caffeoylquinic acids, which have strong preventive and inhibitory effects on abnormal contraction, were simultaneously added, and the influence on the preventive effect was confirmed by the prevention experiment described in Example 1. The ratio of preparations in the test sample indicates the molar ratio. When the amount of 2,5-dihydroxybenzoic acid added was greater than that of caffeoylquinic acid, a significant preventive effect was achieved.

[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 inhibiting vasospasm (abnormal vascular contraction).

Claims

1. As an active ingredient, (A) 2,5-dihydroxybenzoic acid or a salt thereof, and (B) containing 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; A vasospasm inhibitor, wherein the molar ratio of (A):(B) is 2:1 to 3:

1.

2. As an active ingredient, (A) 2,5-dihydroxybenzoic acid or a salt thereof, and (B) containing 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; An oral composition for inhibiting vasospasm, wherein the molar ratio of (A):(B) is 2:1 to 3:1.

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