JP2018177738A - Trpv4 activity inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a TRPV4 activity inhibitor that inhibits TRPV4 activity and is useful for IBS prevention or improvement, and an agent for preventing or improving IBS.SOLUTION: A TRPV4 activity inhibitor contains at least one selected from rosmarinic acid, dimethyl rosmarinic acid and a salt or solvate thereof as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a TRPV4 activity inhibitor and an agent for preventing or improving irritable bowel syndrome.

  TRPV4 (Transient receptor potential cation channel subfamily V member 4) is one of the proteins constituting the temperature sensitive TRP channel. TRPV4 is expressed in a wide range of tissues such as kidney, lung, bladder, heart, skin, brain, gastrointestinal tract, and is considered to play different and broad physiological roles.

  Irritable bowel syndrome (Irritable Bowel Syndrome (IBS)) is different from enteritis due to infection with bacteria and virus, and diseases such as ulcerative colitis and colon cancer, and organic diseases such as inflammation and ulcers can be examined It is a disease that causes abdominal discomfort such as diarrhea, constipation, abdominal pain, and pressure on the lower abdomen due to excess gas, though it is not recognized.

As the cause, abnormalities in digestive tract movement around the large intestine due to abnormalities in the autonomic nerve that controls the movement of the intestine, reduction in digestive tract perception threshold, stress caused by mental anxiety or excessive tension, etc., distortion of lifestyle Such complex factors are pointed out. In patients with IBS, the amount of TRPV4 agonist polyunsaturated fatty acid metabolite (5,6-epoxyeicosatrienoic acid; 5,6-EET) is high in the colon, and the colon is broken including the 5,6-EET When the liquid is administered to the mouse colon, the intestinal motility reaction increases and the intestine overreacts. In this case, it is reported that suppressing the expression of TRPV4 suppresses the overreactivity (Non-patent Document 1). ).
Therefore, it is expected to prevent or improve IBS by suppressing TRPV4 activity.

Rosmarinic acid, which is a kind of tannin contained in a large number of plants of the family Lameraceae, has an action to suppress the onset and progression of amyotrophic lateral sclerosis (ALS) (Patent Document 1), and an insulin secretion promoting action It has been reported that there is a certain thing (patent document 2), the improvement effect of hay fever (non-patent document 2), and the like.
Moreover, dimethyl rosmarinic acid excreted in human urine as a metabolite of rosmarinic acid has been reported to have an inhibitory activity on induction of cyclooxygenase 2 and to have an anti-inflammatory activity (Patent Document 3).
However, it is not known at all that rosmarinic acid or dimethylrozumarinic acid has an inhibitory activity on TRPV4 activity, or is useful for preventing or improving IBS.

JP, 2009-256282, A JP, 2005-139136, A JP, 2005-272362, A

Nicolas Cenac et al., Gastroenterology 2015 149, 433-444 Osakabe N et al., Biofactors. 2004 21, 127-31

  The present invention relates to providing a TRPV4 activity inhibitor which suppresses TRPV4 activity and is useful for the prevention or improvement of IBS, and a preventive or improvement agent of IBS.

  The inventors of the present invention conducted intensive studies on a material that suppresses TRPV4 activity, and as a result, dimethylrhomarinic acid, which is a human metabolite of rosmarinic acid, has an activity of suppressing TRPV4 activity and is useful for preventing or improving IBS. I found it to be.

That is, the present invention relates to the following 1) to 4).
1) A TRPV 4 activity inhibitor comprising, as an active ingredient, at least one selected from rosmarinic acid, dimethylrozumarinic acid and salts or solvates thereof.
2) An agent for the prophylaxis or amelioration of irritable bowel syndrome, which comprises, as an active ingredient, at least one selected from rosmarinic acid, dimethylrozumarinic acid and salts or solvates thereof.
3) A TRPV 4 activity suppressing food comprising, as an active ingredient, at least one selected from rosmarinic acid, dimethylrozumarinic acid and salts or solvates thereof.
4) A food for preventing or ameliorating irritable bowel syndrome, which comprises, as an active ingredient, at least one selected from rosmarinic acid, dimethylrozumarinic acid and salts or solvates thereof.

  The TRPV4 activity inhibitor of the present invention can effectively suppress TRPV4 activity, and is useful for the prevention or amelioration of diseases caused by activation of TRPV4 channel, such as irritable bowel syndrome.

  As used herein, “TRPV4” means “Transient receptor potential cation channel subfamily V member 4”. TRPV4 is a protein encoded by the TRPV4 gene in humans.

  The "rosmarinic acid" of the present invention is a kind of tannin which is abundantly contained in plants of the family Lamella, and is represented by the following formula (A). The "dimethyl rosmarinic acid" of the present invention is a human metabolite of rosmarinic acid (the above-mentioned patent document 3) and means 3,3'-dimethyl rosmarinic acid represented by the following formula (B).

In the present invention, rosmarinic acid may be a commercially available product or may be chemically synthesized based on a conventional method (SYNTHESIS, 755-762 (1996), Can. J. Chem., 75, 1783-1794 (1997) Or rosmarinic acid-containing plants or plants obtained by extraction and purification from cultured cells (JP-A-62-32889).
Dimethyl rosmarinic acid can be synthesized, for example, according to the following reaction scheme, as shown in the below-mentioned Preparation Examples.

  Optical isomers exist in rosmarinic acid and dimethyl rosmarinic acid of the present invention. The rosmarinic acid and dimethylrozumarinic acid of the present invention may be any of these optical isomers or a mixture of optical isomers. In the present invention, R form or a mixture of optical isomers is preferred.

  Rosmarinic acid and dimethylrozumarinic acid of the present invention can be pharmaceutically acceptable salts or solvates thereof. Preferred examples of such salts include sodium salts, potassium salts, alkali metal and alkaline earth metal salts such as calcium salts, or acid addition salts. Preferred examples of the solvate include hydrate, alcoholate or acetone solvate.

As shown in the Examples below, dimethyl rosmarinic acid, which is a metabolite of rosmarinic acid, suppresses TRPV4 activity. That is, when the transformed cells (TRPV4 expressing cells) functionally expressing TRPV4 are brought into contact with dimethyl rosmarinic acid by the introduction of TRPV4 gene in the presence of the TRPV4 stimulator (TRPV4 agonist, agonist), It has the effect of suppressing TRPV4 activity, that is, suppressing the influx of the amount of cations in cells by the TRPV4 stimulant.
Therefore, dimethyl rosumarinic acid, its precursor rosumarinic acid, and a salt or solvate thereof (hereinafter also referred to as "the compound of the present invention") become a TRPV4 activity inhibitor, for suppressing the TRPV4 activity, or TRPV4 activity It can be used to make inhibitors.

As described above, in the colon of an IBS patient, the amount of 5,6-EET (TRPV4 agonist), which is a metabolite of polyunsaturated fatty acids, is high, and the disrupted colon containing the 5,6-EET is used as a mouse. It has been reported that when administered to the colon, the intestine overreacts, and the overreactivity is suppressed by suppressing the expression of TRPV4 (the above non-patent document 1).
Therefore, it is considered that the TRPV4 activity inhibitor can be used for preventing or improving IBS as an agent for preventing or improving IBS.

  In the present invention, "suppressing the activity of TRPV4" refers to suppressing the activity of the receptor TRPV4. Specifically, an activity expressed by binding a TRPV4 stimulant to TRPV4, for example, the ability to modulate ion flux (eg, ability to transport calcium ion, cation such as sodium ion from extracellular to intracellular, etc.) And means to suppress or inhibit the ability to control membrane potential (eg, the ability to generate current).

  In the present invention, “IBS (Irritable Bowel Syndrome)” is a generic term for diseases caused mainly by abnormalities in the motor and secretory function of the large intestine, such as enteritis due to bacterial or viral infection, ulcerative colitis, colon cancer, etc. Unlike illness, it means a disorder that is diagnosed when the test shows no abnormalities but symptoms that repeat diarrhea and constipation persist.

  In the present invention, the "use" for suppressing TRPV4 activity or for preventing or improving IBS may be administration or intake to animals including humans, and therapeutic use even for non-therapeutic use. It may be. "Non-therapeutic" is a concept that does not include medical practice, that is, a concept that does not include a method for surgery, treatment or diagnosis of a human being, more specifically, a doctor or a person who receives instructions from a doctor who operates a human , It is a concept that does not include a method of performing treatment or diagnosis.

  As used herein, “prevention” refers to preventing or delaying the onset of a disease or condition in an individual, or reducing the risk of developing an individual's disease or condition. Also, “improvement” refers to the improvement of disease, symptom or condition, prevention or delay of deterioration of disease, symptom or condition, or reversal, prevention or delay of progression of disease, symptom or condition.

The agent for suppressing or improving the activity of TRPV4 of the present invention and the agent for preventing or improving IBS are pharmaceuticals, quasi-drugs and supplements that exert the effect of suppressing or improving the activity of TRPV4 and IBS when ingested or administered to animals including humans. Alternatively, it can be a food, or can be a material or a preparation for blending into these.
Further, the food of the present invention includes, in addition to general food and drink, food where indicated as necessary, functional food, food for sick persons, food for specified health, functional food for indication, supplement .

  The above-mentioned medicament (including quasi-drugs) containing the compound of the present invention may be administered in any dosage form, although oral administration is preferred. At the time of administration, the active ingredient can be administered in the form of a conventional pharmaceutical preparation by mixing it with a solid or liquid nontoxic pharmaceutical carrier suitable for oral administration, rectal administration, injection and the like.

  As such a preparation, for example, solid agents such as tablets, granules, powders, capsules and the like, solutions such as solutions, suspensions, emulsions and the like, lyophilizers and the like can be mentioned. These formulations can be prepared by conventional means on formulation. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents, excipients and the like can be added as appropriate.

In addition, the form of the above-mentioned food compounded with the compound of the present invention is a drink, tea-based beverage, coffee beverage, fruit juice beverage, carbonated beverage, jelly, wafer, biscuit, bread, noodle, sausage, etc. In addition to various foods such as those described above, compositions for supplementing nutrition in the same form (solid preparations such as tablets, capsules, troches and the like) as the above-mentioned orally administered preparations can be mentioned. Among them, tablets are preferred, and chewable tablets are more preferred.
In order to prepare various forms of food, the compounds of the present invention may be used alone or in combination with other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, coloring agents, antioxidants. A moisturizer, a thickener, etc. can be used in combination as appropriate.

  The content of the compound of the present invention in the above-mentioned pharmaceuticals (including quasi-drugs) and foods varies depending on the use form, but usually, it is preferably 0.01% by mass or more, more preferably 0.1% by mass The content is more preferably 1% by mass or more, preferably 95% by mass or less, more preferably 90% by mass or less, and still more preferably 70% by mass or less. Moreover, Preferably it is 0.01-95 mass%, More preferably, it is 0.1-90 mass%, More preferably, it is 1-70 mass%.

When the compound of the present invention is used as a medicine or food, or mixed with medicine or food, the dose to human may vary according to the subject's condition, body weight, sex, age or other factors, but orally The daily dose per adult in the case of administration is usually 0.01 mg or more, more preferably 1 mg or more, still more preferably 100 mg or more, preferably as dimethyl rosmarinic acid or rosmarinic acid. It is 80000 mg or less, more preferably 15000 mg or less, still more preferably 5000 mg or less. Moreover, Preferably it is 0.01-80,000 mg, More preferably, it is 1-15,000 mg, More preferably, it is 100-5000 mg.
Moreover, although the said formulation may be administered according to arbitrary administration plans, it is preferable to divide into once to several times a day, and to administer continuously for several weeks to several months.

  The subject for administration or intake is not particularly limited as long as it is an animal including human who needs or desires to suppress or improve TRPV4 activity and IBS, but stress or the like may cause diarrhea, constipation, abdominal pain, and lower abdominal region due to excessive gas. Administration or intake to a human who exhibits symptoms such as tension is effective.

With regard to the embodiments described above, the following aspects are disclosed in the present invention.
The TRPV4 activity inhibitor which uses as an active ingredient 1 or more types selected from <1> rosmarinic acid, a dimethyl rosmarinic acid, and those salts or solvates.
<2> An agent for the prophylaxis or amelioration of irritable bowel syndrome, which comprises, as an active ingredient, one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof.
The food | food for TRPV4 activity suppression which uses as an active ingredient 1 or more types selected from <3> rosmarinic acid, dimethyl rosmarinic acid, and those salts or solvates.
The food for prevention or improvement of the irritable bowel syndrome which uses as an active ingredient 1 or more types selected from <4> rosmarinic acid, dimethyl rosmarinic acid, and those salts or solvates.

<5> Use of one or more kinds selected from rosmarinic acid, dimethyl rosmarinic acid and salts or solvates thereof for producing TRPV4 activity inhibitor.
Use of 1 or more types selected from rosmarinic acid, dimethyl rosmarinic acid, and those salts or solvates for manufacturing the preventive or improving agent of <6> irritable bowel syndrome.
Use of 1 or more types selected from rosmarinic acid, dimethyl rosmarinic acid, and those salts or solvates for manufacturing the foodstuff for <7> TRPV4 activity suppression.
Use of 1 or more types selected from rosmarinic acid, dimethyl rosmarinic acid, and those salts or solvates for manufacturing the foodstuff for prevention or amelioration of <8> irritable bowel syndrome.

<9> One or more types selected from rosmarinic acid, dimethyl rosmarinic acid, and salts or solvates thereof for use in suppressing TRPV4 activity.
<10> One or more types selected from rosmarinic acid, dimethyl rosmarinic acid, and salts or solvates thereof for use in the prevention or amelioration of irritable bowel syndrome.

The TRPV4 activity suppression method which administers or ingests 1 or more types selected from <11> rosmarinic acid, a dimethyl rosmarinic acid, and those salts or solvates in the effective dose to the subject which requires them.
<12> A method for preventing or ameliorating irritable bowel syndrome, which comprises administering or ingesting, in an effective amount, one or more selected from rosmarinic acid, dimethylrozumarinic acid and salts or solvates thereof to a subject in need thereof.

<13> <1> and <5> TRPV4 activity inhibitors, <2> and <6> prophylactic or ameliorating agents for irritable bowel syndrome, <3> and <7> foods for suppressing TRPV4 activity, <4> The content of the active ingredient in the food for preventing or ameliorating irritable bowel syndrome of &lt; 8 &gt; is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and still more preferably in the total amount. It is 1% by mass or more, preferably 95% by mass or less, more preferably 90% by mass or less, and still more preferably 70% by mass or less. Moreover, Preferably it is 0.01-95 mass%, More preferably, it is 0.1-90 mass%, More preferably, it is 1-70 mass%.

In <14> <11> or <12>, the daily dose per adult is preferably 0.01 mg or more, more preferably 1 mg or more, still more preferably 100 mg or more as rosmarinic acid or dimethyl rosumarinic acid Also, it is preferably 80000 mg or less, more preferably 15000 mg or less, and still more preferably 5000 mg or less. Moreover, Preferably it is 0.01-80,000 mg, More preferably, it is 1-15,000 mg, More preferably, it is 100-5000 mg.

EXAMPLES The present invention will be described in more detail based on examples given below, but the invention is not meant to be limited by these.
Production Example Synthesis of dimethyl rosumarinic acid (1) Reagents Sodium 3- (3-methoxy-4-hydroxyphenyl) lactate (1) (racemic mixture) from Tokyo Kasei, ferulic acid (3) from Wako Pure Chemical Industries, Ltd. I purchased and used it.

(2) Synthesis of α-hydroxy-3-methoxy-4- (2-propyrenyloxy) benzenepropanoic acid 2-propynyl ester (2)

7. Sodium 3- (3-methoxy-4-hydroxyphenyl) lactate (500 mg, 2.14 mmol) and potassium carbonate (885 mg, 6.41 mmol) were placed in a 50 mL recovery flask and purged with argon, and then super dehydrated acetone 7. 1 mL was added and dispersed. After that, allyl bromide (2.77 mL, 32.0 mmol) was added, and after N ₂ substitution, stirred under reflux conditions for 20 hours.
After completion of the reaction, filtration, the filter residue was washed with methylene chloride (25 mL), the filtrate and the washing solution were combined, and concentrated under reduced pressure. To this was added 20 mL of methylene chloride and 20 mL of H ₂ O, and after separation, the methylene chloride layer was recovered. The aqueous layer was further extracted three times with 5 mL of methylene chloride and recovered. The collected methylene chloride layers were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure (988.2 mg).
The product was purified by silica gel column chromatography to obtain 513 mg (colorless, oily, 82% yield) of the purified product of the title compound (2). The conditions for silica gel column chromatography are as follows.

<Conditions of silica gel column chromatography>
Column: Universal Premium L (30μm, 60Å, 3.0x16.5cm, 40g) [YAMAZEN]
Injection column: INJECT COLUMN S [YAMAZEN]
Eluent: hexane / ethyl acetate = 95/5 (4 min) 95/5 (11 min) 74/26 (14 min) 74/26
Flow rate: 20 mL / min, preparation time: 29 min, preparation volume: 12 ml / piece, 48 aliquots After confirmation of each Fr. by TLC, target Fr. (fr. 43-53) is concentrated under reduced pressure, Dry.

(3) Synthesis of 3-methoxy-4- (2-propyrenyloxy) phenyl-2-propylene acid (4)

To ferulic acid (3) (500 mg, 2.58 mmol), 17 mL of super dehydrated ethanol was added and dissolved. Thereafter, allyl bromide (267 μL, 3.09 mmol) and potassium carbonate (427 mg, 3.09 mmol) were sequentially added, and after N ₂ substitution, the mixture was stirred under reflux conditions for 5.5 hours. The reaction mixture was allowed to cool to room temperature while confirming the reaction progress by TLC, potassium hydroxide (434 mg, 7.74 mmol) was added, and the mixture was further stirred for 16 hours.
After completion of the reaction, the reaction solution was concentrated under reduced pressure, dissolved again in 25 mL of H ₂ O, and 2 N hydrochloric acid and concentrated hydrochloric acid were added to adjust to pH = 0. The mixture was extracted 5 times with ethyl acetate (25 mL), dried over anhydrous magnesium sulfate and concentrated under reduced pressure (687.5 mg).
The product was purified by silica gel column chromatography to obtain 221.4 mg (colorless, oily, yield 37%) of a purified product of the title compound (4). The conditions for silica gel column chromatography are as follows.

<Conditions of silica gel column chromatography>
Column: Universal Premium L (30μm, 60Å, 3.0x16.5cm, 40g) [YAMAZEN]
Injection column: INJECT COLUMN M [YAMAZEN]
Eluent:
1% acetic acid in hexane / 1% acetic acid in ethyl acetate = 100/0 (4 min) 100/0 (11 min) 84/16 (24 min) 84/16 (0 min) 80/20 (7 min) 80/20
Flow rate: 20 mL / min, preparation time: 46 min, preparation volume: 12 ml / 7, 77 aliquots Preparation Each Fr. is confirmed by TLC, then target Fr. (fr. 48-75) is concentrated under reduced pressure, Dry.

(4) α-[[3- [3-methoxy-4- (2-propyrenyloxy) phenyl] -1-oxo-2-propynyl] oxy] -3-methoxy-4- (2-propyrenyloxy) benzenepropanoic acid 2 -Synthesis of propynyl ester (5)

To Compound (2) (100 mg, 0.34 mmol), 1.7 mL of methylene chloride was added and dissolved. After that, Compound (4) (104 mg, 0.44 mmol) and dimethylaminopyridine (6.3 mg, 0.051 mmol) were sequentially added, and after cooling to -20 ° C, N, N'-dicyclohexylcarbodiimide (88.2 mg, 0.43 mmol) was added. After N ₂ substitution and stirring at room temperature for 24 hours, the precipitated urea was removed by filtration, washed with ethyl acetate and concentrated under reduced pressure (288.5 mg).
This was purified by silica gel column chromatography to obtain 151.8 mg (white, waxy) of the purified product of the title compound (5). The conditions for silica gel column chromatography are as follows.

<Conditions of silica gel column chromatography>
Column: Universal Premium L (30μm, 60Å, 3.0x16.5cm, 40g) [YAMAZEN]
Injection column: INJECT COLUMN S [YAMAZEN]
Eluent:
Hexane / Ethyl acetate = 87/13 (4 min) 87/13 (11 min) 66/34 (14 min) 66/34
Flow rate: 20 mL / min, preparation time: 29 min, preparation volume: 12 ml / tube, 48 fractions After confirmation of each Fr. by TLC, target Fr. (fr. 34-36) is concentrated under reduced pressure, Dry.

(5) Synthesis of 3,3'-dimethyl rosmarinic acid (B)

Compound (5) (87.3 mg, 0.172 mmol) is taken in a 50 mL eggplant flask in a glove box under an atmosphere of N ₂ , 8.6 mL of super-dehydrated tetrahyfdrofuran and tetrakis (triphenylphosphine) palladium (0) complex (Pd (PPh ₃ ) ₄ ) (19.8 mg, 17.2 μmol) and morpholine (450 μL, 5.15 mmol) were sequentially added. The reaction mixture was stirred at room temperature for 18 hours under N ₂ atmosphere.
After completion of the reaction, the reaction solution was concentrated under reduced pressure, then acidified with 20 mL of 1 N hydrochloric acid, and this was extracted three times with ethyl acetate (20 mL). The ethyl acetate layers were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure (117.0 mg). This was purified by preparative TLC to obtain 61.4 mg (white, powdery, 92% yield) of the purified product of the title compound (B). The conditions for preparative TLC are as follows.
In the following examples, the synthesized dimethylrozmarinic acid was appropriately diluted with dimethyl sulfoxide (DMSO) and used for evaluation.
<Conditions of preparative TLC>
TLC: PLC Silica gel 60 F ₂₅₄ (1 mm, 20 × 20 cm, # 1.13895.0001) [Merck]
Developing solvent: chloroform / acetone / 88% formic acid = 75 / 16.5 / 8.5 200 mL
Desorbent: chloroform / acetone / 88% formic acid = 75 / 16.5 / 8.5 15 mL

Example 1 Evaluation of TRPV4 Activity Inhibitory Action (1) Preparation of Human TRPV4 Gene Expression Vector A cDNA obtained by reverse transcription of total RNA extracted from human duodenal cells (Hutu-80 cells, purchased from American Type Culture Collection) is used as a template The polymerase chain reaction (PCR) was performed under the following conditions using a primer set consisting of oligonucleotides represented by the base sequences shown below, which were synthesized with reference to the published human TRPV4 gene sequence. .

<Primer set>
Forward primer; 5'-CACCATGGCGGATTCCAGCGAAGGCCC-3 '(SEQ ID NO: 1)
Reverse primer; 5'-CTAGAGCGGGGCGTCATCAGTCC-3 '(SEQ ID NO: 2)

<PCR conditions>
a) PCR solution composition
cDNA (Template) 15 μL
5x PrimeStar GXL Buffer 10 μL
dNTPs mixture (2.5mM) 4μL
1 μl PrimeStar GXL DNA Polymerase (Takara Bio)
Forward Primer (10 μM) 1 μL
Reverse Primer (10 μM) 1 μL
Water 18 μL
b) Temperature and cycle conditions
95 ° C 2 min
↓
98 ° C 10sec 33 cycles
70 ° C 2 min

  The obtained PCR product was purified using High Pure PCR Product Purification Kit (trade name, manufactured by Roche). A human TRPV4 gene expression vector was produced using the purified PCR product and pcDNA3.1 Directional TOPO Expression Kit (trade name, manufactured by Invitrogen Corporation).

(2) Preparation of human TRPV4 expressing cells Culture of a human cervical cancer cell line (HeLa cells, purchased from American Type Culture Collection) using DMEM / F12 medium (manufactured by Invitrogen) containing 10% fetal bovine serum went. HeLa cells were seeded at 5 × 10 ⁵ cells / flask in T-75 cell culture flasks. After 3 days of culture, cells for the human TRPV4 gene expression vector (8 μg) prepared in (1) above were transfected using TransIT-HeLaMONSTER Transfection Kit (manufactured by Mirus), and cultured for 1 day.
Detach cells with Detachin (Genlantis), and seed in 96 well Optical bottom plate (Nunc) with DMEM / F12 medium containing 10% fetal bovine serum at a cell density of 1.5 × 10 ⁴ cells / 90 μL / well, and further It was cultured for one day.

(3) Measurement of intracellular calcium ion influx activity Measurement of intracellular calcium ion influx activity was performed using Calcium Kit II-fluo 4 (trade name, DOJINDO). 90 μL / well of Loading buffer containing Fluo 4-AM was added to the human TRPV4 expressing cells prepared in (2) above, and incubated at 37 ° C. for 1 hour. Thereafter, the fluorescence intensity (excitation wavelength: 488 nm, fluorescence wavelength: 524 nm) was measured every two seconds using a fluorescence plate reader FDSS 3000 (trade name, manufactured by Hamamatsu Photonics K. K.) at 37 ° C. Thirty seconds after the start of measurement, the TRPV4 agonist GSK1016790a (manufactured by Sigma) and the dimethyl rosmarinic acid solution prepared in the above preparation example as a sample are respectively diluted with the dilution buffer of the measurement kit, and their mixed solution (mixed just before addition) Was added at 20 μL / well, and changes in fluorescence intensity were measured every 2 seconds up to 300 seconds. GSK1016790a was added to a final concentration of 3 nM.
In addition, as a positive control of TRPV4 activity suppression, an antagonist of TRPV4 HC067047 was added at a final concentration of 100 μM.
The TRPV4 activity of the sample was calculated by the following equation, with the calcium ion inflow rate by the treatment of the TRPV4 agonist GSK1016790a as 100%.

(1)
Calcium ion influx ratio (%) = [(F300 / F0)-(F300C2 / F0C2)] / [(F300C1 / F0C1)-(F300C2 / F0C2)] × 100
F300: fluorescence intensity of GSK1016790a and sample added at 300 seconds after measurement start F300C1: fluorescence intensity of GSK1016790a and solvent added at 300 seconds after measurement start F300C2: well added only solvent after 300 seconds of measurement start Fluorescence intensity F0: fluorescence intensity of the same well as F300 immediately after the start of measurement F0C1: fluorescence intensity of the same well as F300 C1 immediately after start of measurement F0C2: fluorescence intensity of a well to which only solvent immediately after start of measurement was added

  The calcium ion influx rate when the sample was added was assayed using Dunnett's test in comparison with GSK1016790a + solvent addition. The results when dimethyl rosmarinic acid is added are shown in Table 2 (in the table, GSK1016790a and dimethyl rosmarinic acid are abbreviated as GSK and MeRA, respectively).

  From Table 2, similar to HC067047 (antagonist of TRPV4) which is a positive control, dimethyl rosmarinic acid significantly reduced calcium ion influx in a concentration-dependent manner.

From the above results, application of dimethylrozumarinic acid significantly suppresses TRPV4 activity. This indicates that dimethylrosmarinic acid is effective in suppressing TRPV4 activity. Furthermore, it has been shown that dimethyl rosmarinic acid, which has an activity of suppressing TRPV4 activity, is effective for the prevention or amelioration of irritable bowel syndrome.
As described above, dimethylrozumarinic acid is an internal metabolite of rosmarinic acid, so it is considered that rosmarinic acid, which is a precursor, is also effective in suppressing TRPV4 activity and preventing or ameliorating irritable bowel syndrome.

Claims (4)

  A TRPV 4 activity inhibitor comprising, as an active ingredient, at least one selected from rosmarinic acid, dimethylrozumarinic acid and salts or solvates thereof.   An agent for the prophylaxis or amelioration of irritable bowel syndrome, which comprises, as an active ingredient, at least one selected from rosmarinic acid, dimethyl lozumarinic acid and salts or solvates thereof.   A food for suppressing the activity of TRPV 4 which comprises, as an active ingredient, at least one selected from rosmarinic acid, dimethyl rosumarinic acid and salts or solvates thereof.   A food for preventing or ameliorating irritable bowel syndrome, which comprises, as an active ingredient, at least one selected from rosmarinic acid, dimethyl lozumarinic acid and salts or solvates thereof.