WO2016075960A1 - Medicinal composition for preventing or improving dysuria, antagonist against dysuria-related receptor, and method for preventing or improving dysuria using medicinal composition or antagonist - Google Patents

Abstract

Long-term drug intake may cause a risk of adverse side effects and sufficient effects cannot be obtained in some cases by pharmacotherapy. Provided is a pharmaceutical composition that comprises a compound represented by chemical formula (I). In formula (I): R₁ represents a hydrogen atom or a hydroxyl group; R₂, R₃, R₄, R₅ and R₆ independently represent a hydrogen atom or -OCH₃; and R₇, R₈, R₉ and R₁₀ independently represent a hydrogen atom or -OCH₃.

Description

Pharmaceutical composition for preventing or ameliorating urination disorder, antagonist for urination disorder-related receptor or method for preventing or ameliorating urination disorder using the pharmaceutical composition or antagonist

The present invention relates to a pharmaceutical composition for preventing or ameliorating dysuria, an antagonist for a dysuria-related receptor, or a method for preventing or improving urinary dysfunction using the pharmaceutical composition or antagonist.

Healthy people's bladder can store up to 300-500ml of urine. In adults, when about 200 ml of urine accumulates in the bladder, a signal about urinary intention is sent to the brain, and a sense of initial urination is felt. When the brain receives such a signal, the paraswitchive nerve is excited, and as a result, the muscarinic receptor of the bladder is stimulated and the smooth muscle (detrusor muscle) contracts. On the other hand, in the urine accumulation stage, the sympathetic nerve is dominant and the detrusor muscle is in a relaxed state.

Healthy people can urinate properly when they feel urine, but quality of life (QOL) is significantly reduced when urination disorders such as frequent urination and urinary incontinence occur due to various causes. There are many people who have left such urination disorder. The reason for this is that, besides being embarrassed to go to the hospital, he cannot recognize that he has dysuria.

At the International Continence Society held in 2002, `` urinary urgency '' was the main symptom, and symptoms associated with `` frequent urination '' and `` night nocturia '' were called `` overactive bladder '' (Overactive Bladder: OAB) was proposed as a disease. Based on this proposal, efforts to diagnose, treat and prevent dysuria have become active.

OAB is a disease that causes sudden urinary urgency as a result of excessive reaction of the bladder at the urine storage stage, and it has been reported that the prevalence increases with aging. In patients with OAB, sympathetic nerves and parasympathetic nerves become unbalanced, and detrusor muscles involuntarily contract despite urine accumulation, resulting in urgency and frequent urination. In men, OAB has been reported to be associated with benign prostatic hyperplasia. The prostate gland exists to surround the urethra just below the bladder. When the prostate enlarges with age, the urethra at the outlet of the bladder is compressed, causing “weakness of urine” and “feeling of residual urine”. In female OAB patients, `` pressure stress urinary incontinence '' due to loosening of the pelvic floor muscles due to aging, childbirth, etc. (urinary leakage during sneezing, coughing, exercise, etc., which temporarily applies force to the abdomen) Have been reported to occur at a high frequency. This is because the female urethra is shorter than the male urethra. It can be said that dysuria is an age-related disease that develops due to various factors in the range from the bladder to the urethra. Therefore, prevention of urination disorder is extremely important in order to lead a comfortable elderly life.

It has been reported that anticholinergic drugs are effective for the treatment of OAB (for example, Non-Patent Document 1).

"A comparison of the efficacy and tolerability of solifenacin succinate and extended release tolterodine at treating overactive bladder syndrome: results of the STAR trial." Chapple et al., Eur Urol. 20053:

However, long-term use of commercially available anticholinergic drugs has the potential for side effects.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a drug based on a naturally occurring compound or a method for preventing and / or improving urination disorder using the drug.

The inventors of the present application conducted research on the prevention and / or improvement of dysuria using food materials with high safety, and nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and sinencetine It has been found that it has an antagonism against dysuria-related receptors and improves dysuria, thereby completing the present invention.

(式(I)中、
R₁は、水素又は水酸基であり、
R₂、R₃、R₄、R₅及びR₆は、それぞれ独立して、水素又は-OCH₃であり、
R₇、R₈、R₉及びR₁₀は、それぞれ独立して、水素又は-OCH₃である) According to the present invention,
There is provided a pharmaceutical composition containing a compound represented by the following chemical formula (I) for preventing or ameliorating dysuria.

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ )

(式(I)中、
R₁は、水素又は水酸基であり、
R₂、R₃、R₄、R₅及びR₆は、それぞれ独立して、水素又は-OCH₃であり、
R₇、R₈、R₉及びR₁₀は、それぞれ独立して、水素又は-OCH₃である)
　後述する実施例において実証されている通り、上記化学式(I)で示される化合物は、ムスカリン受容体の選択的標識リガンドである[³H]pirenzepineの、ムスカリン受容体に対する特異的結合を阻害した。従って、この拮抗剤を用いることで、排尿障害関連受容体に対するリガンドの特異的結合を阻害することができる。 According to the present invention,
Provided is a receptor antagonist comprising a compound represented by the following chemical formula (I), wherein the compound exhibits an antagonistic action on a dysuria-related receptor.

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ )
As demonstrated in the examples described below, the compound represented by the chemical formula (I) inhibited the specific binding of [ ³ H] pirenzepine, which is a selective labeled ligand of the muscarinic receptor, to the muscarinic receptor. Therefore, by using this antagonist, the specific binding of the ligand to the dysuria-related receptor can be inhibited.

According to the present invention,
There is provided a therapeutic agent for dysuria, comprising a receptor antagonist comprising the above compound. As demonstrated in the examples described later, dysuria can be treated by using a therapeutic agent containing an antagonist containing the compound represented by the chemical formula (I). Therefore, it is possible to treat dysuria by applying this therapeutic agent to patients suffering from dysuria.

(式(I)中、
R₁は、水素又は水酸基であり、
R₂、R₃、R₄、R₅及びR₆は、それぞれ独立して、水素又は-OCH₃であり、
R₇、R₈、R₉及びR₁₀は、それぞれ独立して、水素又は-OCH₃である)
　後述する実施例において実証されている通り、上記化学式(I)で示される化合物を含む医薬組成物又は受容体拮抗剤を投与することによって、排尿障害が改善した。従って、この方法を、排尿障害を患っている患者に施すことによって、排尿障害を予防又は改善させることが可能になる。 According to the present invention,
A method for preventing or ameliorating dysuria, the method comprising:
Including a step of administering a pharmaceutical composition containing a compound represented by the following chemical formula (I) or a receptor antagonist to a subject suffering from the dysuria, the compound having an antagonistic action on a dysuria-related receptor A method is provided that indicates

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ )
As demonstrated in the examples described later, dysuria was improved by administering a pharmaceutical composition or a receptor antagonist containing the compound represented by the chemical formula (I). Therefore, by applying this method to a patient suffering from urination disorder, it becomes possible to prevent or improve urination disorder.

FIG. 1 is a graph showing the antagonistic action of a seeker extract (nobiletin-tangeretin mixture) on the specific binding of [ ³ H] pirenzepine in rat brain crude cell membrane. FIG. 2a is a graph showing the antagonism of nobiletin and tangeretin on the specific binding of [ ³ H] pirenzepine in rat brain crude cell membrane. FIG. 2b shows the chemical formulas of nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and sinensetin. Fig. 2c shows the inhibitory effect on muscarinic M1-specific receptor binding activity ([ ³ H] -Pirenzepin binding of nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and sinencetin isolated from Sikhwaser ). Fig. 2d shows nonspecific muscarinic receptor binding activity ([ ³ H] -N-methyl scopolamine (NMS) for nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and synencetin isolated from Sikhwaser. ). FIG. 3 is a graph showing the antagonistic action of various extracts and Compound IV (extract: 10 μg / mL, compound: 10 μM) on muscarinic receptors. FIG. 4 is a graph showing the urination disorder improving action of the seeker extract in acetic acid-induced frequent urination model rats. 4a shows the maximum intravesical pressure in model rats, FIG. 4b shows the basal pressure, FIG. 4c shows the threshold pressure, FIG. 4d shows the urination interval, FIG. 4e shows the number of urinations, and FIG. Yes. FIG. 5 shows the effect of nobiletin intravenous administration on the measurement of intravesical pressure in pelvic congestive urinary rats. FIG. 6 shows the effect of nobiletin intravenous administration on the bladder contraction interval in pelvic congestive urinary rats. The left bar is the sham group (sham surgery group), and the right bar is the pelvic congestion group. FIG. 7 shows the effect of oral administration of nobiletin on measurement of intravesical pressure in pelvic congestive urinary rats. FIG. 8 shows the effect of oral administration of nobiletin on bladder contraction intervals in pelvic congestive urinary rats. FIG. 9 shows the results of intravenous administration of nobiletin on the bladder contraction interval in cerebral infarction frequent urination rats. The left bar is the Siamese group and the right bar is the cerebral infarction group. FIG. 10 schematically shows a system for measuring the intravesical pressure and the amount of urination over time. FIG. 11 shows the amount of urination, the number of urinations, and the maximum intravesical pressure in cyclophosphamide (CYP) -induced cystitis model rats orally administered with seeker extract (50 mg / kg). FIG. 12 shows the effect of seeker extract on calcium antagonist receptors. FIG. 13 shows the protocol for transmural electrical stimulation (1 Hz) experiment. FIG. 14 shows the effects of atropine and α, β methylene ATP on the contraction response of rat isolated bladder by transmural electrical stimulation (1 Hz) in the presence of nobiletin or tangeretin. In the graph, the left bar is a control or sample (nobiletin or tangeretin), the middle bar is atropine, and the right bar is α, β methylene ATP. FIG. 15 shows the effects of nobiletin and tangeretin on cholinergic and purinergic contractile components.

Definition Muscarinic Receptor Muscarinic Receptor, a kind of acetylcholine receptor, is a receptor that exhibits the same function as acetylcholine by muscarin, and exists as a receptor for organ-side cells in parasympathetic nerve extinction. When muscarinic receptors are stimulated by parasympathetic nerves and acetylcholine is released from the periphery, the muscarinic receptors are stimulated to excite and suppress the cells of the organs under their control. Whether it is excited or suppressed depends on the organ (ie, it depends on the organ).

Antagonism The term “antagonist” refers to an agent containing a substance having an action (ie, antagonism) that competitively inhibits specific binding of a ligand to a receptor, for example. A substance having an antagonistic action is also called an antagonist. Antagonism occurs when the antagonist binds to the receptor and becomes unable to bind to the receptor, or when the antagonist acts on the ligand (for example, binds) and the ligand cannot bind to the receptor. Cases where effects occur are included.

Overview Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description of omission is abbreviate | omitted.

(式(I)中、
R₁は、水素又は水酸基であり、
R₂、R₃、R₄、R₅及びR₆は、それぞれ独立して、水素又は-OCH₃であり、
R₇、R₈、R₉及びR₁₀は、それぞれ独立して、水素又は-OCH₃である)
　この医薬組成物を用いることで、排尿障害を予防又は改善させることができる。 <Embodiment 1: Pharmaceutical composition>
According to the present invention, there is provided a pharmaceutical composition containing a compound represented by the following chemical formula (I) for preventing or ameliorating dysuria.

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ )
By using this pharmaceutical composition, dysuria can be prevented or improved.

In the present specification, at least 1, 2, 3, 4, 5, 6, 7 or 8 or all of R ₂ to R _{10 in} the chemical formula (I) may be —OCH ₃ . In the present specification, at least 1, 2, 3 or 4 or all of R ₂ to R _{6 in} the chemical formula (I) may be —OCH ₃ . In the present specification, at least 1, 2 or 3 or all of R ₇ to R _{10 in} the chemical formula (I) may be —OCH ₃ . As used herein, —OCH ₃ and —OMe can be substituted for each other.

及び

　この医薬組成物を用いることで、排尿障害を予防又は改善させることができる。 According to the present invention, there is provided a pharmaceutical composition for preventing or improving urination disorder, wherein the compound represented by the chemical formula (I) is a compound selected from the group of the following chemical formulas (II) to (V): Provided.

as well as

By using this pharmaceutical composition, dysuria can be prevented or improved.

(式(I)中、
R₁は、水素又は水酸基であり、
R₂、R₃、R₄、R₅及びR₆は、それぞれ独立して、水素又は-OCH₃であり、
R₇、R₈、R₉及びR₁₀は、それぞれ独立して、水素又は-OCH₃である)
　この拮抗剤を用いることで、排尿障害関連受容体に対するリガンドの特異的結合を阻害することができる。 <Embodiment 2: Urinary dysfunction-related receptor antagonist>
According to the present invention, there is provided a receptor antagonist containing a compound represented by the following chemical formula (I), wherein the compound exhibits an antagonistic action on a dysuria-related receptor. .

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ )
By using this antagonist, specific binding of a ligand to a dysuria-related receptor can be inhibited.

及び

According to the present invention, there is provided a receptor antagonist, wherein the compound represented by the chemical formula (I) is a compound selected from the group of the following chemical formulas (II) to (V).

as well as

In the present specification, at least one urination disorder-related receptor is selected from the group consisting of a muscarinic receptor, a calcium receptor, and a purine receptor.

The compounds represented by the chemical formulas (II) to (V) are referred to as nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and sinencetin, respectively. In the present specification, the chemical formula (II) and nobiletin can be substituted with each other, the chemical formula (III) and the tangeretin can be substituted with each other, and the chemical formula (IV) and 3 ′, 4 ′, 5, 7-tetra Methyl quercetin can be substituted for each other, and chemical formula (V) and sinencetine can be substituted for each other.

The above compound may be a purified compound or a synthesized compound. Moreover, the form contained in the crude extract of a plant may be sufficient as the said compound. Nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and synencetin can be extracted from citrus and extraction methods described below. In addition, nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and sinencetin can be purified by using the purification method described later. The compound may be a hydrate.

When the above compound or a hydrate of the above compound is used as a pharmaceutical composition or pharmaceutical preparation (antagonist), the crystals may be treated according to a conventional method and mixed with excipients described later. Pharmaceutical preparations containing such compounds as active ingredients include injections, suppositories, aerosols, transdermal absorption agents, ointments, plasters, sprays and other parenterals, tablets, powders, granules, powders, capsules Agents, pills, troches, liquids and other oral preparations.

Here, the tablets include sugar-coated tablets, coated tablets, and buccals, and the capsules include both hard capsules and soft capsules. Granules also include coated granules. The above liquid preparations include suspensions, emulsions, syrups, elixirs and the like, and syrups include dry syrups. Each of the above-mentioned preparations includes not only those that are not sustained-released but also those that are sustained-released. These preparations are in accordance with known pharmacological manufacturing methods, such as bases, carriers, excipients, binders, disintegrants, lubricants, colorants, etc. described in the Japanese Pharmacopoeia that are pharmacologically acceptable when manufacturing the preparations. Can be used. Examples of such carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, and gentian powder.

Examples of the pharmaceutical composition or binder include starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like. Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethylcellulose, carboxymethylcellulose calcium, crystalline cellulose, calcium carbonate, methylcellulose, ethylcellulose, carboxymethylcellulose, and the like. Examples of the lubricant include talc and magnesium stearate. The colorant can be used as long as it is allowed to be added to pharmaceuticals, and is not particularly limited. In addition to these, flavoring agents, flavoring agents, and the like can be appropriately used as necessary.

For tablets or granules, sucrose, gelatin, purified shellac, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl as necessary Coating may be performed using a methacrylate, a methacrylic acid polymer, or the like, and coating may be performed with a plurality of layers. Furthermore, granules and powders can be packed into capsules such as ethyl cellulose and gelatin to form capsules.

When preparing an injection using a pharmaceutical composition or antagonist containing the above compound or a pharmaceutical composition or antagonist containing a hydrate of the above compound, a pH adjuster, buffer material, A solubilizer, solubilizer, and the like can also be added.

The antagonist related to the urination disorder-related receptor of the present embodiment may be used for preventing or improving urination disorder. In the present specification, dysuria refers to a condition in which urine output is difficult for some reason. The dysuria may be accompanied by subjective symptoms or not, or may be based on a doctor's diagnosis.

In the present specification, the dysuria may be overactive bladder and / or prostatic hypertrophy. Urination disorders (lower urinary tract dysfunction) can be divided into two groups. One is overactive bladder and the other is prostatic hypertrophy. Overactive bladder is accompanied by a sense of urgency, a symptom of sudden urinary urgency. Overactive bladder may also be accompanied by one or more symptoms selected from the group consisting of frequent urination, nocturia and urinary incontinence. In the treatment of overactive bladder, anticholine is used as the first-line drug. Prostatic hypertrophy may be associated with increased urinary flow resistance and / or impaired bladder blood flow due to lower urinary tract obstruction. In benign prostatic hyperplasia, an alpha 1 blocker is used as the first choice translation. In addition, overactive bladder may not be accompanied by prostatic hypertrophy, and prostatic hypertrophy may be accompanied by overactive bladder. In the present specification, the dysuria may be overactive bladder. In the present specification, the dysuria may be prostatic hypertrophy.

In the present specification, the dysuria may be accompanied by one or a plurality of symptoms selected from the group consisting of frequent urination, nocturia, urinary incontinence, residual urine sensation, and urinary dysfunction. Frequent urination is a symptom in which the number of urinations from getting up to bedtime increases. Usually, if the number of urinations per day is 8 or more, frequent urination is diagnosed, but this is not a limitation. Nocturia is a symptom of temporarily rising for urination from bedtime to waking up, and is usually diagnosed as nocturnal urination if there is at least one urination, but is not limited thereto. Urinary incontinence refers to a symptom of involuntary or unconscious urine leakage. Urinary incontinence includes stress urinary incontinence, urge incontinence, overflow urinary incontinence, reflex urinary incontinence, true urinary incontinence. Residual urine sensation is a symptom accompanied by the sensation of urine remaining in the bladder even after urination (residual urine), and this sensation occurs regardless of the presence or absence of residual urine. Urinary weakness is a symptom in which urination momentum decreases and urination time increases. Symptoms include urinalysis, prostate-specific antigen (PSA) measurement, urine flow measurement, ultrasonography, computed tomography (CT) examination, magnetic resonance imaging (MRI) examination, urinary bladder angiography, urodynamic examination or these It can be judged by the combination of

The antagonist for the pharmaceutical composition or dysuria-related receptor of the present specification may contain a citrus extract containing the above chemical formula (I). In the present specification, the citrus fruits include Keraji (CitrusCKeraji, Ponkan; C. reticulate), Dancy tangerine (C. tangerine), Jimikan (C. succosa), Shikaikan (Chi). . Suhuiensis), Tachibana (C. tachibana), Kobenimikan (C. erythrosa), Kishumikan (C. kinokuni), Shiikuwasha (C. depressa), Koji Preferably, the citrus fruit is a seeker because it contains more nobiletin, tangeretin, 3 ′, 4 ′, 5, -7-tetramethylquercetin and sinencetin. Is derived from one or more citrus fruits.

In the specification of the present application, the pharmaceutical composition or antagonist includes nobiletin, tangeretin synthesized in addition to nobiletin, tangeretin, 3 ′, 4 ′, 5, 7-tetramethylquercetin and / or synencetin derived from the extract, It may contain 3 ′, 4 ′, 5, 7-tetramethylquercetin and / or sinencetin. In the present specification, the pharmaceutical composition or antagonist includes the above-mentioned extract containing the above-mentioned nobiletin, tangeretin, 3 ′, 4 ′, 5, 7-tetramethylquercetin and / or synencetin, and synthesized nobiletin, tangeretin 3 ', 4', 5, 7-Tetramethylquercetin and sinencetin are not included. The content of nobiletin, tangeretin, 3 ′, 4 ′, 5, 7-tetramethylquercetin and / or sinencetin in the extract is 10% by weight, 20% by weight, 30% by weight, 40% in terms of dry weight of the extract. Within a numerical range between any two points selected from the group consisting of wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 98 wt% and 99 wt% It may be.

The citrus part that can be used in the present invention is not particularly limited as long as it contains nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and / or sinencetin, for example, in addition to fruits, Fruit peel, squeezed residue (squeezed lees), pruned leaves and twigs, dried products thereof and the like can also be used as raw materials. In the case of seekers, ponkans, etc., it is possible to use fruits, pericarp and juice residue, and the content per unit weight of nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and / or sinencetin It is preferable because there are many. On the other hand, pruned leaves, twigs, etc. contain nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and / or sinencetin per unit weight, but not as much as pericarp. To do. For this reason, there is a possibility that the load on the environment can be reduced by using these.

Although the method of obtaining the citrus juice residue is not particularly limited, for example, the juice residue can be obtained by treating the citrus fruit with a press. Although a press machine is not specifically limited, It is preferable to use a roll press machine, a filter cloth press machine, a centrifugal filtration type | mold separation apparatus, etc. in order to obtain the squeezed fruit skin efficiently.

The method for obtaining a dried product of citrus fruits and pericarp and juice residue is not particularly limited. For example, citrus fruits can be dried by heating at 50 to 70 ° C. for 15 to 45 minutes to obtain a dried product of citrus fruits. it can. At this time, if it is dried at less than 50 ° C, it may not be able to be sufficiently dried in a short time of 15 to 45 minutes. If it is dried at a temperature exceeding 70 ° C, the components contained in citrus fruits May denature during drying. Therefore, when the citrus fruits are dried by heating, it is preferable to dry them at about 60 ° C. for about 30 minutes.

It is also possible to obtain dried citrus fruits and pericarp and juice residue by sun drying. Since sun drying can reduce the electrical energy of a drying machine, it is suitable for drying operations on an industrial scale from the viewpoint of energy cost. Moreover, since the temperature in the sun drying is lower than the temperature of the drying machine, denaturation of the components of the dried product can be suppressed. When the dried product is obtained by sun drying, the drying time can be appropriately adjusted according to the region, season, climate, and amount of citrus fruits. For example, the drying time may be 3 days, 4 days, 5 days, 10 days, or 20 days, and may be 1 month, 2 months, 3 months, 6 months, or 12 months. You may change a drying place according to a weather condition or the degree of drying of citrus fruits. For example, at night, citrus fruits may be stored in a building (eg, warehouse or factory).

In some embodiments, citrus fruits can be obtained without being dried. When citrus fruits are not dried, the drying cost and drying time can be reduced. Moreover, when not drying citrus, you may acquire a citrus extract using a site | part (for example, peel) with little water content.

Hereinafter, an exemplary method for producing a citrus extract using a seeker will be described.

First, wash the sequwacer with water, and then perform squeezing with various presses that can squeeze the squeaker fruit as it is. As such a press, for example, a roll press press that rotates by rotating a 10-20 mesh endless net between two compression rolls and squeezes the continuously supplied seeker fruit with these two compression rolls. Examples include a soup machine, a filter cloth press machine that squeezes and squeezes the fruit of a seeker fruit wrapped in a filter cloth, a centrifugal filtration type separation device, and the like.

The juice residue obtained by pressing the fruit juice is weighed in a predetermined amount, for example, about 300 to about 500 g (wet weight). The residue is then dried using a dryer or the like, for example, at about 50 to about 70 ° C. After weighing the dried juice residue (dry weight), put the dried juice residue into a suitable container, and add 1 to 2 times the volume of the above-mentioned dry weight of distilled water to the container. Transfer to a hot bath at about 60 to about 80 ° C. and extract for 1-3 hours. After extraction, the beaker is taken out of the warm bath and allowed to cool naturally, and then, for example, a filter paper having a reserved particle diameter of 5 μm or less is laid on a Buchner funnel, the entire amount of the extract is suction filtered, and the residue is filtered off.

The obtained residue is again dried at about 50 to about 70 ° C. using a dryer or the like, and weighed. Add 2 to 3 times the dry weight of ethanol and extract at room temperature for 2 to 4 days. Next, as in the above, the whole extract is suction filtered and the residue is filtered off. Concentrate the filtrate at about 30-40 ° C. for about 15 to about 60 minutes.

To the obtained concentrate, add 8 to 10 times volume 1% (w / v) aqueous sodium hydroxide solution of this concentrate and mix well at room temperature. Continue stirring the mixture at room temperature for 1-2 days, eg, using a stirrer. Thereafter, for example, a filter paper having a retained particle diameter of 5 μm or less is set in a funnel and naturally filtered to separate a soluble component and an insoluble component. The insoluble component obtained is washed with 1 to 2 volumes of water. Elute the remaining insoluble components with 5-10 volumes of ethanol. The ethanol soluble portion is then concentrated at room temperature for about 15 to about 60 minutes. The liquid volume after concentration is about 1/15 to 1/20 of the weight before concentration, and this liquid volume is measured.

To this concentrated solution, add about 0.5 to 2% (w / v) aqueous sodium hydroxide, 8 to 10 times the volume, and mix well. The mixture is then kept stirred at room temperature for 4-8 days, for example using a stirrer. Thereafter, for example, a filter paper having a retained particle diameter of 5 μm or less is set in a funnel and naturally filtered, and the residue is separated by filtration. As described above, a fraction containing nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and sinencetin at a high content can be obtained.

Also, another exemplary method for producing a citrus extract using a seeker will be described.

The skin obtained from citrus fruits is naturally dried for 3 days in the sun to obtain dried skin. This 5-fold volume of solvent is added to a predetermined amount of dried peel and immersion extraction is performed at a predetermined temperature for a predetermined period to obtain an extract. Here, as the extraction solvent, for example, methanol, ethanol, acetone, ethyl acetate, water-methanol mixed solution of various mixing ratios, water-ethanol mixed solution and the like can be mentioned.
The obtained crude extract is filtered to remove solids, and the entire filtrate is concentrated to obtain a concentrated solution. An appropriate amount of an appropriate aqueous / organic phase mixture is added to this concentrated liquid, and liquid-liquid partition extraction is performed. The partition extraction is performed a plurality of times by changing the composition of the mixed solution. The organic phase obtained by partition extraction is concentrated, applied to an open column, and eluted by a step gradient method in which the solvent concentration is increased stepwise, and the desired fraction is obtained from the pericarp extract (hereinafter referred to as “SPE”). Use as

Subsequently, a part of this fraction is concentrated, the solute concentration is adjusted, and crude purification is performed by preparative column chromatography. The SPE is further subjected to column chromatography, and the amount of nobiletin and / or tangelitin contained in the extract is quantified.
Specifically, 30 to 50 L of methanol is added to 6 to 10 kg of dried citrus peel such as sequwercer and extracted at 2 to 6 ° C. for 7 to 21 days to obtain an extract. The extract is filtered to remove solids and the entire filtrate is concentrated using a flash evaporator. Next, 4 L of water / ethyl acetate (1/1) is added to perform partition extraction. 4 L of 90% methanol / n-hexane (1/1) is added to the obtained ethyl acetate phase, and liquid-liquid partition extraction is performed again.

Subsequently, a part of the 90% methanol phase is concentrated and applied to, for example, an ODS silica gel column (Cosmosil 75C ₁₈ -OPN, 50 mmφ × 500 mm), and step gradient elution is performed to increase the methanol concentration by 20%. A fraction of% -100% methanol is obtained. These fractions are dried on a large rotary evaporator.
Next, for example, among the above fractions, a part of the 80% methanol fraction is concentrated, and methanol is added to adjust the solute concentration to 900 mg / ml, followed by preparative column chromatography.

When purifying nobiletin, tangeretin, 3 ′, 4 ′, 5, 7-tetramethylquercetin and sinencetin from the above fraction, it is not limited, but can be purified using HPLC. Exemplary purification conditions are described below.

Preparative conditions: Column 5C ₁₈ -AR-II column (Nacalai Tesque)
Elution solvent: 70% methanol / water Solute concentration: 900 mg / ml
Injection volume: 3ml
Fraction volume: 10ml
Flow rate: 5ml / min Detection wavelength: UV215nm
Range: 20mV

The elution times for nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin, and sinencetin use the standard products of nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin, and sinencetin, respectively. Can be determined.

The production method and the purification method of the extract containing nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and sinencetin have been exemplarily described. It is also possible to produce things. In the case of an industrial scale, as long as it is possible to obtain the above extract, it is possible to appropriately change the parameters of each manufacturing process, and to add, change or repeat the manufacturing process, and to delete the manufacturing process. It is.

<Embodiment 3: Therapeutic>
According to the present invention, there is provided a therapeutic agent for dysuria including an antagonist relating to a dysuria-related receptor comprising the above compound. By using this therapeutic agent, it becomes possible to treat dysuria. Certain dysuria drugs can be used to prevent and / or ameliorate dysuria.

The therapeutic agent is administered in an effective amount to treat dysuria, whether administered alone or in combination with other therapeutic agents. However, the total dose of the compound is determined by the attending physician within the scope of sound medical judgment. Effective doses for patients suffering from dysuria are: severity of dysuria; patient age, weight, general health, sex and diet; administration time; route of administration; excretion rate of the compound; duration of treatment; Depends on the drug being used or co-administered. The dose of the compound does not have to be constant for each administration. For example, the dose may be administered at a dose lower than that required to achieve the desired therapeutic effect, and the dose may be gradually increased until the desired effect is obtained.

If necessary, the effective daily dose may be divided into multiple doses according to the purpose of administration. Those skilled in the art will be able to easily optimize the effective dose and the combined dosage regimen, depending on good medical practice and individual patient clinical symptoms.

Daily dose (daily dose) of the above therapeutic agents is 0.01, 0.05, 0.1, 0.5, 1 orally in terms of nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethyl quercetin and / or synencetin , 5, 10, 50, 100, 150 and 200 mg / kg body weight may be within a range between two points. The therapeutic agent is preferably administered continuously over a certain period of time, ie 3 days or longer, preferably 1 week or longer, more preferably 2 weeks or longer, even more preferably 1 month or longer, for example 6 months or 1 year or longer. The therapeutic agent is preferably administered daily, but may not be administered daily as long as it is continuously administered throughout the period. The above-mentioned therapeutic agent may be administered once a day, or may be administered in several divided daily doses. Administration of the therapeutic agent may be terminated by a doctor's judgment or may be terminated by the patient's self-judgment.

<Embodiment 4: Functional food>
According to the present invention, in order to prevent or ameliorate dysuria, containing a compound represented by the above chemical formula (I) or containing at least one compound selected from the group of the above chemical formulas (II) to (V) Functional foods are provided. By appropriately adding the above compound or a hydrate of the above compound as necessary, a functional food or health product having an effect of preventing or improving urination disorder can be provided.

The above compound or a hydrate of the above compound, for example, bread, cookies and biscuits, cooked wheat and millet, udon, buckwheat, pasta, other noodles, cheese, yogurt and other dairy products, jam, mayonnaise, miso, Made from soy sauce and other soy products, tea, coffee and cocoa, soft drinks, fruit drinks and other non-alcoholic drinks, medicinal liquors, other alcoholic drinks, candy, chocolate and other snacks, chewing gum, rice crackers, sheep's straw and other soybeans It can be added to confectionery and the like to make a functional food. Alternatively, it can be added to animal feed to make a functional mixed feed. In addition, when adding to said yogurt, soy sauce, a drink, etc., in order to prevent the compound of this invention from crystallizing and precipitating in these, a dissolution aid and a stabilizer can also be added suitably. .

In addition, health foods can be obtained by using the above compounds or hydrates of the above compounds alone, or by mixing two or more of them into powders, granules, tablets, and capsules according to conventional methods. Here, in order to make the compound of the present invention into a powder, the extract obtained in the production process is concentrated and dried using a method such as freeze drying, spray drying, vacuum drying, sample mill, blender, mixer The dry solid may be pulverized by, for example. Moreover, you may add corn starch, potato starch, dextrin, oyster shell powder, etc. as needed.

Further, the above-mentioned binder can be added to the powder obtained as described above as appropriate, and tableting can be performed. After making into tablets, the above-described coating agents such as sucrose or gelatin may be used to form sugar-coated tablets, or other coating agents may be used to make enteric solvents and the like. Further, the powder obtained as described above can be made into granules according to a conventional method to produce granules. Moreover, it can also be set as a capsule by filling the above-mentioned capsule with the above-mentioned powder and granule in an appropriate amount. In the present invention, the functional food and health food do not include citrus fruits or other plant fruits that naturally contain the compound. However, the said foodstuff etc. which are manufactured by adding the said compound to the said citrus fruits shall not be excluded from this invention.

The amount of nobiletin contained in the above functional foods and health foods is selected from the group consisting of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150, and 200 mg / kg body weight It can be adjusted to be within the range between two points.

(式(I)中、
R₁は、水素又は水酸基であり、
R₂、R₃、R₄、R₅及びR₆は、それぞれ独立して、水素又は-OCH₃であり、
R₇、R₈、R₉及びR₁₀は、それぞれ独立して、水素又は-OCH₃である)
　上記医薬組成物又は受容体拮抗剤を患者に投与する場合には、投与量は、患者の症状の重篤さ、年齢、体重、PSA値、尿流量及び健康状態等の諸条件によって異なる。一般的には、上述した用量及び用法で、1日1回若しくはそれ以上の回数にわたって投与すればよく、以上のような諸条件に応じて、投与の回数及び量を適宜増減すればよい。 <Embodiment 5: Method for preventing or ameliorating dysuria>
According to the present invention,
A method for preventing or ameliorating dysuria, the method comprising:
Administering a pharmaceutical composition or a receptor antagonist containing a compound represented by the following chemical formula (I) to a subject suffering from dysuria,
Methods are provided wherein the compounds exhibit antagonism against dysuria-related receptors.

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ )
When the above pharmaceutical composition or receptor antagonist is administered to a patient, the dosage varies depending on various conditions such as the severity of the patient's symptoms, age, weight, PSA value, urine flow rate, and health status. In general, the dosage and usage described above may be administered once or more times a day, and the number and amount of administration may be appropriately increased or decreased according to the above conditions.

The daily dose, administration period, and number of administrations of the pharmaceutical composition or antagonist may be the same as those of the therapeutic agent described above. Administration of the pharmaceutical composition or antagonist may be terminated by a doctor's judgment or may be terminated by the patient's self-judgment.

As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above can also be employ | adopted.

Hereinafter, the present invention will be further described with reference to examples and drawings, but the present invention is not limited thereto. The instrument operation and kit use were in accordance with the manufacturer's manufacturer's protocol.

<Example 1>
500 g of Shikuwasa squeezed rice cake from Higashimura, Okinawa Prefecture was thoroughly washed, 1800 mL of about 70 ° C distilled water was added, and the mixture was placed in a bath of about 70 ° C and heat-treated for 3 hours. The obtained heat-treated product was subjected to suction filtration using a Buchner funnel covered with a filter paper (manufactured by ADVANTEC, No. 131) having a retained particle diameter of 3 μm to obtain a residue. The residue was squeezed through a press.

After squeezing, 300 g (wet weight) of the squeezed residue was weighed and dried at about 60 ° C. for 30 minutes using a dryer (manufactured by Panasonic, product number EH5101P turbo dry) to obtain 150 g of dried peel. To this, 30% (v / v) ethanol, which is twice the weight of the juice residue, was added, and ethanol extraction was performed at room temperature for 3 days. Suction filtration was performed using a Buchner funnel on which filter paper (No. 131, manufactured by ADVANTEC) having a reserved particle diameter of 3 μm was spread to obtain 3200 mg of filtrate. The obtained filtrate was concentrated with a rotary evaporator to obtain a concentrated solution.

The concentrated solution was added with 10 volumes of 1% (w / v) aqueous sodium hydroxide solution and stirred with a stirrer for 1 day at room temperature. The insoluble component obtained was washed with 2 volumes of water. The remaining insoluble components were eluted with 5-10 volumes of 100% (v / v) ethanol. Next, the ethanol-eluted component was filtered off using a Buchner funnel. The contents of nobiletin and tangeretin were about 60% and about 35%, respectively.

Purification of nobiletin, tangeretin, tangeretin, 3 ′, 4 ′, 5, 7-tetramethylquercetin and sinencetin The obtained extract (filtrate) was subjected to HPLC analysis by a known method. The HPLC conditions are as follows.
HPLC apparatus: UV-8011 HPLC (manufactured by TOSOH)
Detector: UV Detector
Column: Cholester Waters ID 4.6mm x 250mm (made by Waters)
Elution solvent: 75% MeOH / water Concentration of solute: 0.1 mg / mL
Injection volume: 0.005ml
Flow rate: 1.0 mL / min Detection wavelength: UV 215 nm
Range: 1000 mV
The seeker extract was recovered from a peak occurring between 8 and 12 minutes after injection. Nobiletin was recovered from the peak occurring at about 9 minutes after injection. Tangeretin was recovered from a peak occurring at about 11 minutes after injection. Both nobiletin and tangeretin were more than 95% pure. In addition, 3 ′, 4 ′, 5, 7-tetramethylquercetin and sinencetin were similarly purified using these preparations.

<Example 2>
Evaluation of antagonism against muscarinic receptors Using a crude cell membrane preparation prepared from rat brain, it is a selective labeling ligand for muscarinic receptors in the presence of various plant extracts or component compounds at various concentrations [ ³ H The antagonism of muscarinic receptors was evaluated from the decrease rate of specific binding of pirenzepine. Such antagonism was evaluated by radioreceptor assay, and 50% inhibition concentration (IC ₅₀ value), inhibition constant (Ki value), and Hill coefficient (nH) were calculated.

SD rats (8 weeks old, male, purchased from Nippon SLC Co., Ltd.) were laparotomized under ether anesthesia, blood was collected from an abdominal descending aorta with a heparin-treated syringe and sacrificed. After refluxing the physiological saline cooled from the artery, the brain was removed. A 19-fold volume of cooled 50 mM Tris buffer (pH 7.4) was added to the brain from which the cerebellum had been removed, homogenized, and centrifuged at 40,000 × g for 20 minutes at 4 ° C. After removing the supernatant, 19-fold volume of cooled 50 mM Tris buffer (pH 7.4) was again added to the sediment, suspended, and centrifuged at 40,000 × g for 20 minutes at 4 ° C. After removing the supernatant, 29-fold volume of cooled 50 mM Tris buffer (pH 7.4) was added to the sediment and suspended to prepare a receptor preparation.

As a tissue amount, 1 mg of the receptor preparation was added to 50 mM Tris buffer, and each concentration of the test sample and 1 nM [ ³ H] pirenzepine were added to a final volume of 0.5 mL. This reaction solution was incubated at 25 ° C. for 60 minutes.

Each reaction solution was subjected to rapid suction filtration on glass fiber filter paper (Whatman GF / B) using Cell Harvester (manufactured by Brandel) after completion of incubation. The filter paper was immediately washed with 3 mL of a cooled 50 mM phosphate buffer (pH 7.4). Toluene scintillator (2 L of toluene, 1 L of Triton-X, 15 g of 2,5-diphenylloxazole, 0.3 g of 1,4-bis [2- (5-phenyloxazolyl)] benzene) was added to the filter paper, and the mixture was stirred at room temperature for 6 minutes. After standing for more than an hour, the radioactivity was measured using a liquid scintillation counter. As the displacer, 1 μM atropine was used, and the radioactivity obtained in the absence and presence of the displacer was defined as total binding and nonspecific binding, respectively, and the difference between them was defined as specific binding to the receptor. .

Sequwer extract (nobiletin-tangeretin mixture) inhibited specific binding of [ ³ H] pirenzepine in rat brain crude cell membrane in a concentration-dependent manner (FIG. 1). The IC ₅₀ value obtained from the inhibitory action was 13.5 ± 3.4 μg / mL (Table 1).

Thus, it was suggested that the seeker extract is capable of binding to the muscarinic receptor.

<Example 3>
Antagonistic effect of nobiletin and tangeretin on muscarinic receptors The same experiment as in Example 2 was carried out for purified nobiletin and tangeretin alone. The results are shown in FIG. As shown in Figure 2a, nobiletin inhibited the specific binding of [ ³ H] pirenzepine in rat brain crude cell membrane in a concentration-dependent manner, whereas tangeretin specifically bound [ ³ H] pirenzepine in rat brain crude cell membrane. No concentration-dependent inhibitory action was observed. The Ki value determined from the inhibitory action of nobiletin was 15.7 ± 2.8 μM (Table 2).

Thus, it was suggested that nobiletin exhibits a binding activity to the muscarinic receptor. On the other hand, it has been clarified that tangeretin has weak or no antagonism to muscarinic receptors.

Muscarinic receptor binding activity in four kinds of compounds derived from sequwers: Nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and synencetin (Fig. 2b) isolated from sequwers bind to muscarinic M1-specific receptors. The activity (inhibitory action on [ ³ H] -Pirenzepin binding) and nonspecific muscarinic receptor binding activity (action on [ ³ H] -N-methyl scopolamine (NMS) binding) were compared.

The muscarinic M1 receptor-specific binding activity was strong in the order of nobiletin> 3 ′, 4 ′, 5, 7-tetramethylquercetin≈sinensetine> tangeretin (FIG. 2c and Table 3).

Muscarin non-specific receptor binding activity was observed with nobiletin and synencetin (FIG. 2d).

These results revealed that nobiletin derived from nobiletin, 3 ', 4', 5, 7-tetramethylquercetin, synencetin, and tangeretin have muscarinic receptor, particularly muscarinic M1 receptor binding activity. Tangeretin also showed weak binding activity (FIG. 2c). This indicates that these four compounds have an effect of treating dysuria.

<Example 4>
Antagonism of various plant extracts and compounds against muscarinic receptors The antagonistic action against muscarinic receptors in various plant extracts and compounds was examined. The plant extracts and compounds used are listed in Table 4.

The result is shown in FIG. Nobiletin alone was confirmed to antagonize muscarinic receptors. However, in various extracts containing a large amount of other flavonoids and polyphenols and other flavonoids and polyphenol compounds, almost no antagonism against muscarinic receptors was observed. This suggests that the antagonistic action on muscarinic receptors is not an action generally observed in various polyphenol-containing plant extracts and polyphenol compounds, but an action specific to nobiletin.

<Example 5>
Acetic acid-induced frequent urinary model rat Using acetic acid-induced frequent urinary model rat, which is a frequent urinary model due to cystitis, the effect on dysuria was evaluated. 8-week-old female SD rats (purchased from Nippon SLC Co., Ltd.) under urethane anesthesia (0.8 g / kg ip, and 0.4 g / kg sc), 0.1% acetic acid at 3 mL / hr in the bladder Inducing frequent urination by continuous infusion, maximum intravesical pressure (mmHg), basal pressure (mmHg), threshold pressure (mmHg) and urination interval (min) for 30 minutes from 1 hour after administration of solvent or seeker extract Was measured by a cismetry method.

Effect of administration of seeker extract in acetic acid-induced frequent urination model rats In the acetic acid-induced frequent urination rats, administration of seeker extract (50 mg / kg) significantly increased the maximum intravesical pressure (Fig. 4a). In addition, the urination interval was significantly prolonged (FIG. 4d), and the number of urinations was also significantly decreased (FIG. 4e). On the other hand, this extract had no significant effect on basal pressure (FIG. 4b), threshold pressure (FIG. 4c), and single urine output (FIG. 4f). From these results, it was shown that the seeker extract can improve frequent urination.

<Example 6>
Action in pelvic congestive dysuria model rats Preparation of pelvic congestive dysuria model rats 24 Sprague-Dawley (SD) female rats (around 200 g) were incised in the midline of the lower abdomen under 2% isoflurane inhalation anesthesia. The uterine vein was ligated and the pelvis was congested. In addition, eight SD female rats (around 200 g) that had undergone only the operation of peeling the bilateral common iliac vein and uterine vein from surrounding tissues were used as the sham group. Three weeks after the surgery, it was used for the following experiment.

Intravenous administration experiment of nobiletin In 8 pelvic congestion groups and 8 sham groups, a catheter for measuring intravesical pressure with a diameter of about 1 mm transurethrally was inserted into the urinary bladder under 2% isoflurane inhalation anesthesia. Were used as a physiological saline injection path and an internal pressure measurement path into the bladder. A venous indwelling needle was inserted into the femoral vein on one side to administer nobiletin. Stop isoflurane inhalation anesthesia, urethane (0.6mg / kg) under superficial anesthesia restraint, injecting physiological saline into the bladder (3ml / h), continuous bladder pressure measurement, when bladder contraction appeared at regular intervals, Nobiletin (0.3, 1, 3, 10, 30 mg / kg) was intravenously administered at 15-20 minute intervals, and the effect on bladder activity was examined in comparison with before nobiletin administration (FIGS. 5 and 6).

Nobiletin Oral Administration Experiment Of the remaining 16 pelvic congestion rats, 8 were the pelvic congestion control group and 8 were the pelvic congestion nobiletin group. In the pelvic congestion nobiletin group, nobiletin (30 mg / kg) was dissolved in 1 ml of water once a day and administered into the stomach with a sonde for 2 weeks from the 1st week after the operation. Eight pelvic congestion control groups and eight sham groups were administered intragastrically with 1 ml of water once a day for 2 weeks. Two weeks after intragastric administration, as in the case of intravenous administration of nobiletin, continuous intravesical pressure measurement was performed under urethane (0.6 mg / kg) superficial anesthesia restraint, and the difference in bladder activity among the three groups was examined (Fig. 7 and 8).

Statistical processing results were expressed as mean ± standard error, and p <0.05 was considered significant using paired t-test or non-paired t-test.

Results of intravenous administration of nobiletin In the pelvic congestion group, the bladder contraction interval (urination interval) (9.3 ± 1.1 minutes and 12.4 ± 0.7 minutes, respectively) was significantly shorter than that in the sham group, and frequent urination (FIG. 6, before administration) ). In the pelvic congestion group, nobiletin 3-30 mg / kg intravenous administration increased the bladder contraction interval (16.9 ± 1.0 min at 30 mg / kg), and in the sham group 10 mg / kg intravenous administration bladder expansion interval (14.4 ± 0.9 min) ) Was extended (FIGS. 5 and 6). There was no change in the maximum contraction pressure or baseline pressure of the bladder.

Results of oral administration of nobiletin The bladder contraction interval was shorter in the pelvic congestion control group (control group) (8.2 ± 0.7 minutes) and the pelvic congestion nobiletin group (10.6 ± 0.8 minutes) than in the sham group (13.2 ± 0.9 minutes). In the pelvic congestion nobiletin group, the bladder contraction interval was longer than in the pelvic congestion control group (control group) (FIGS. 7 and 8). Bladder baseline pressure was not different between the three groups. Although the maximum bladder contraction pressure was not different from the sham group (48.9 ± 2.0 cmH ₂ O), the pelvic congestion nobiletin group (46.3) compared to the pelvic congestion control group (control group) (54.1 ± 3.0 cmH ₂ O) ± 2.3 cmH ₂ O).

<Example 7>
Effects on urination of cerebral infarction model rats Preparation of cerebral infarction model rats Eight SD female rats (around 200g) under midline incision of the neck under 2% isoflurane inhalation anesthesia and reach the basilar artery from the internal carotid artery on one side A 3-0 nylon thread was inserted and the internal carotid artery was ligated to create and close a cerebral infarction. In addition, 8 SD female rats (about 200 g) that were only subjected to the operation of peeling the internal carotid artery on one side from the surrounding tissue were used as a sham group. On the first day after surgery, it was used for the following experiments.

Nobiletin Intravenous Administration Experiment In 8 cerebral infarction groups and 8 sham groups, a catheter for measuring intravesical pressure with a diameter of about 1 mm transurethrally was inserted into the bladder under 2% isoflurane inhalation anesthesia. Were used as a physiological saline injection path and an internal pressure measurement path into the bladder. A venous indwelling needle was inserted into the femoral vein on one side to administer nobiletin. Stop anesthesia with inhalation of isoflurane, measure the continuous intravesical pressure while injecting physiological saline into the bladder under restraint without anesthesia (3 ml / h), and when bladder contractions appear at regular intervals, nobiletin (0.3, 1, 3) , 10, 30 mg / kg) were administered intravenously at 15-20 minute intervals, and the effect on bladder activity was examined in comparison with before nobiletin administration.

Statistical processing results were expressed as mean ± standard error, and p <0.05 was considered significant using paired t-test or non-paired t-test.

Results of intravenous administration of nobiletin In the cerebral infarction group, bladder contraction intervals (9.2 ± 0.6 minutes and 13.5 ± 0.9 minutes, respectively) were significantly shorter than in the sham group, and frequent urination occurred. In the cerebral infarction group, nobiletin 3-10 mg / kg intravenous administration extended the bladder contraction interval (14.4 ± 2.2 minutes at 3 mg / kg), and also in the sham group 3 mg / kg administration the bladder contraction interval (17.1 ± 1.6 minutes) Extended (Figure 9). There was no change in the maximum contraction pressure or baseline pressure of the bladder.

<Example 8>
Drug-induced cystitis model rats Preparation of drug-induced cystitis model rats CYP-induced cystitis model rats were prepared according to the method of Chow et al. (Chow YC, Yang S., Huang CJ, Tzen CY, Huang PL, Su YH and Wang PS: Epinephrine promotes hemostasis in rats with cyclophosphamide-induced hemorrhagic cystitis. Urology., 67: 636-641, 2006.). CYP (150 mg / kg) dissolved in physiological saline was administered once intraperitoneally, and after 24 hours, intravesical pressure and urination volume were measured under urethane anesthesia by cystometry.

Measurement of intravesical pressure and urinary output by cystometry method under urethane anesthesia The rat was kept warm and fixed in the supine position, and the lower abdomen was incised midline. A small incision was made at the top of the bladder, and a polyethylene tube (SP-45: Natsume Seisakusho Co., Ltd.) was inserted into the bladder. The incision was ligated, and the other end of the tube inserted into the bladder was connected to a pressure transducer (MLT0670 Disposable BP Transducer: ADINSTRUMENTS).

Using the apparatus shown in FIG. 10, the intravesical pressure and the amount of urination were measured over time. Rats were fixed in a prone position on a ball man gauge (acrylic custom-made item: Kengo Kashiyama). A dedicated syringe (Terumo syringe 10 mL) filled with physiological saline or 0.1% acetic acid solution was attached to a continuous injector (Model 100 Series: Kd Scientific). While continuously injecting physiological saline or 0.1% acetic acid solution into the bladder at 3 mL / hr through a water tank set at 37 ° C (THEAMO -10MINDER テ ッ ク SJ-10R Tytech Co., Ltd.), a pressure amplifier (BP Amp: ADINSTRUMENTS ), The intravesical pressure was measured over time, and cystometrograms were recorded with data recording and analysis software (Power 4/26: ADINSTRUMENTS). In addition, the amount of urination is set up on a dish top electronic balance (GX-400, A & D Co., Ltd.), and the fluid discharged from the urethral orifice is collected cumulatively, and the change in weight is measured by Power 尿 Lab 4 Recorded at / 26. After urination became stable, urination parameters were measured 30 minutes before administration. Thereafter, SE 50 mg / kg was orally administered, and measurement was started again 1 hour after the administration. Thereafter, 30 minutes of data was used as a urination parameter after administration.

As shown in FIG. 11, in CYP-induced cystitis model rats, oral administration of seeker extract (50 m / kg) significantly increased the amount of urination and significantly decreased the number of urinations. In addition, the maximum intravesical pressure decreased. From this, it was shown that sequwer extract extracts regulate urination function and improve frequent urination even in drug-induced cystitis model rats.

<Example 9>
Preparation of active receptor preparations that bind to Ca antagonist receptors of sequwer extract. SD rats were laparotomized under isoflurane anesthesia, blood was collected from the abdominal descending aorta and sacrificed, and the brain was removed. The cerebellum was removed from the removed brain. For the measurement of (+)-[ ³ H] PN200-110, 19 volumes of 30 mM Tris-HCl buffer (pH 7.2 or 7.5) was added to the isolated brain and homogenized with a Polytron homogenizer. The homogenate was centrifuged (4 ° C, 40,000 × g, 20 min), then 32.3 times the amount of buffer was added to the sediment and suspended to prepare a receptor membrane preparation.

[ ³ H] αβ-methylene ATP (MeATP) was measured in a 10-fold amount of homogenizing buffer (50 mM Tris, 1 mM EDTA, 2 μg / mL Soybean trypsin inhibitor, 10 μg / mL Bacitracin: pH 7.4) in the brain after extraction. In addition, it was homogenized with a Polytron homogenizer. The homogenate was centrifuged (4 ° C, 2,000 xg, 10 min), and the supernatant was collected. The sediment was resuspended in 10 times the amount of homogenizing buffer and centrifuged under the same conditions, and the supernatant was collected. Two supernatants are combined, incubated at 37 ° C for 20 minutes, centrifuged (4 ° C, 40,000 × g, 20 min), and the pellet is incubated with 19 volumes of incubation buffer (50 mM Tris, 3 mM CaCl ₂ : pH 7.5) was added and suspended to prepare a receptor membrane preparation.

Measurement of 1,4-Dihydropyridine (DHP) Ca antagonist receptor binding activity The measurement of 1,4-DHP Ca antagonist receptor binding activity was performed using (+)-[ ³ H] PN200-110 as the labeled ligand. Receptor assay was followed. In other words, the receptor sample was sought with Sequewacer extract (SE), 0.3 nM (+)-[ ³ H] PN200-110 in 50 mM Tris-HCl buffer (pH 7.4) at 25 ° C for 60 minutes under light-shielded conditions Incubated. After the incubation, the reaction solution was rapidly suction filtered onto a glass filter paper fiber, and the filter paper was immediately washed with 50 mM Na ⁺ / K ⁺ phosphate buffer (pH 7.5). The filter paper was dried, placed in a vial, added with toluene scintillator and allowed to stand at room temperature for about 6 hours, and then its radioactivity was measured to calculate the amount of specific binding. At this time, 1 μM nifedipine was used as the displacer. All measurements were done in duplicate.

Results Binding of the seeker extract to the calcium antagonist receptor Binding of the (+)-[ ³ H] PN200-110 receptor, which is a ligand for the 1,4-DHP Ca antagonist receptor, was slightly affected by the seeker extract. It was suppressed (FIG. 12). Therefore, the seeker extract also has an activity of binding to the calcium receptor antagonist binding site, and a calcium antagonistic action can be expected. Since calcium causes bladder smooth muscle contraction, it was suggested that sequwercer extract improves frequent urination and urinary incontinence in overactive bladder even through smooth muscle relaxation by calcium antagonism.

<Example 10>
Effects of nobiletin and tangeretin on rat isolated bladder
1. Reagents Atropine and Tetrodotoxin were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Pyridoxalphosphate-6-azophenyl-2 ', 4'-disulfonic acid tetrasodium salt (PPADS; a selective P2X antagonist) was purchased from Tocris Bioscience (Minneapolis, MN, USA). Nobiletin and Tangeretin were extracted and purified from Sikhwaser. Other reagents were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).

2. Experimental specimen Wistar male rats (300-350 g) purchased from Japan SLC, Inc. (Shizuoka, Japan) were used in the experiment. All experimental animals were kept under conditions of constant temperature (22 ± 2 ° C), constant humidity (55 ± 2%), and regular lighting (7-19 o'clock). Animal experiments were conducted based on the “Mukogawa Women's University Animal Experiment Regulations”, respecting the Animal Protection Act.

Pentobarbital (60 mg / kg, intraperitoneal administration) Wistar male rats under deep anesthesia were exsanguinated and the bladder was removed and oxygenated (95% O ₂ and 5% CO ₂ ) at 37 ° C Krebs- 4 sections (3 × 5) from the upper bladder in Henseleit solution (in mmol / L: NaCl 113, KCl 4.8, MgSO ₄ 1.2, KH ₂ PO ₄ 1.2, CaCl ₂ · 2H ₂ O 2.5, NaHCO ₃ 25, glucose 11.5) mm), and this was used as a specimen.

3. Measurement of contraction by transmural electrical stimulation The specimen was attached to a platinum electrode in a Magnus tube filled with oxygen-aerated Krebs-Henseleit solution (5 mL, 37 ° C) and suspended under a static tension of 1 g. After 30 minutes equilibration time to stabilize the specimen, wash out with Krebs-Henseleit solution 5 times every 3 minutes, transmural electrical stimulation (10 Hz, 0.1 msec duration, 10 V, 10 sec) and the resulting contraction response was measured. A transducer (T7-15-240, A & D Co. Ltd., Tokyo, Japan) is used for measurement, and PowerLab (registered trademark) software (ADInstruments Pty Ltd, CO, USA) is used for data acquisition and analysis. Using. Transmural electrical stimulation was added to the specimen at 5 minute intervals. 2 minutes after transmural electrical stimulation, nobiletin (final concentration 10 ^-4 M), tangeretin (final concentration 10 ^-4 M) or the same amount of solvent (Ethanol) was administered as a control, and 3 minutes later, the second transmural wall Contractile response was measured by applying electrical stimulation. In addition, atropine (final concentration: 10 ^-6 M), α, β-methylene ATP (final concentration: 10 ^-5 M) and tetrodotoxin (final concentration: 10 ^-6 M) were treated in order, and the effect on transmural electrical stimulation It was investigated. The flow of transmural electrical stimulation is as shown in FIG. Contraction suppressed by atropine is cholinergic contraction, and contraction desensitized by repeated addition of α, β-methylene ATP is purinergic contraction, contraction suppressed by tetrodotoxin is non-cholinergic, non-purinergic Contraction was defined as tetrodotoxin-resistant contraction that was not inhibited by tetrodotoxin.

4. Statistical analysis method The obtained data are shown as mean ± standard error. For the significance test, Dunnett's test was used, and a significance level of less than 5% was determined to be significant. For data analysis, GraphPad Prism 4.03 software (GraphPad Software, La Jolla, CA, USA) was used.

5. Experimental results Nobiletin (10 ^-4 M) and tangeretin (10 ^-4 M) both showed an inhibitory action against contraction caused by transmural electrical stimulation, and the inhibition rate was about 30% or 19%, respectively. (FIG. 14). In order to elucidate the pathway involved in the contractile response by transmural electrical stimulation, various compounds were further examined. As shown in FIG. 14, the contraction response which was about 100% in the control was attenuated to about 68% in the presence of atropy (10 ⁻⁶ M), and α, β-methylene ATP (10 ⁻⁶ M) was allowed to coexist. And decreased to about 5% (FIG. 14a). As shown in FIG. 14b, the contractile response observed in the presence of nobiletin is attenuated to about 70%, to about 32% in the presence of atropine, to about 2% in the presence of α, β-methylene ATP. Attenuated. In the same order, it was attenuated to about 81% in the presence of tangeretin, about 42% in the presence of atropine, and about 3% in the presence of α, β-methylene ATP (FIG. 14c). Of the contractile responses caused by transmural electrical stimulation, only about 1% of tetrodotoxin-resistant contractile responses remained in the presence of tetrodotoxin (10 ⁻⁶ M) (Data not shown).

Nobiletin and tangeretin showed a significant inhibitory action in a contraction model by transmural electrical stimulation in the isolated bladder. From this, it was suggested that nobiletin and tangeretin have the effect of improving dysuria by suppressing bladder contraction.

Furthermore, as a result of comparative analysis by dividing the contractile response by transmural electrical stimulation into a cholinergic contraction component suppressed by atropine and a purinergic contraction component desensitized by α, β-methylene ATP repeated treatment, In the control experiment, cholinergic contraction was about 32% and purinergic contraction was about 63%, suggesting that purinergic components are greatly involved in contraction response by transmural electrical stimulation. (FIG. 15).

On the other hand, in the experiment treated with nobiletin, cholinergic contraction was about 38% and purinergic contraction was about 30%, and in the experiment treated with tangeretin, cholinergic contraction was about 39% and purinergic contraction was about 39%. % (FIG. 15). Considering the above results, it is presumed as one possibility that the components that are suppressed by nobiletin and tangeretin in the contractile response by transmural electrical stimulation in the isolated bladder are mainly purinergic components. These results suggest that nobiletin and tangeretin also improve dysuria by affecting purinergic contraction.

From the results of this Example, it was revealed that nobiletin, tangeretin, 3 ', ’4', 5, 7-tetramethylquercetin and sinencetin have an antagonistic action on the muscarinic receptor. Furthermore, since nobiletin has the effect of improving frequent urination, it becomes possible to prevent, improve or treat urination disorder by using nobiletin.

In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Acknowledgment "This work was carried out with the support of" Cooperative Research Program for Agriculture Science & Technology Development (Project No.PJ009583) "Rural Development Administration, Republic of Korea."
Agricultural Life Bio Food and Drug Material Development Corporation (Korea Rural Development Agency)

Claims (13)

A pharmaceutical composition comprising a compound represented by the following chemical formula (I) for preventing or improving urination injury.

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ ) 2. The pharmaceutical composition according to claim 1, wherein the compound represented by the chemical formula (I) is a compound selected from the group of the following chemical formulas (II) to (V).

as well as

The pharmaceutical composition according to claim 1 or 2, wherein the dysuria is overactive bladder and / or prostatic hypertrophy. The pharmaceutical composition according to any one of claims 1 to 3, wherein the dysuria is accompanied by one or a plurality of symptoms selected from the group consisting of frequent urination, nocturia, urinary incontinence, residual urine sensation, and dysuria. object. A receptor antagonist containing a compound represented by the following chemical formula (I):
The said compound is a receptor antagonist which shows the antagonistic action with respect to a dysuria related receptor.

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ ) The receptor antagonist according to claim 5, wherein the dysuria-related receptor is selected from the group consisting of a muscarinic receptor, a calcium receptor and a purine receptor. The receptor antagonist according to claim 5 or 6, which is used for preventing or improving urination disorder. The receptor antagonist according to any one of claims 5 to 7, wherein the dysuria is overactive bladder and / or prostatic hypertrophy. The muscarinic reception according to any one of claims 5 to 8, wherein the dysuria is accompanied by one or a plurality of symptoms selected from the group consisting of frequent urination, nocturia, urinary incontinence, residual urinary sensation and dysuria. Body antagonist. The receptor antagonist according to any one of claims 5 to 9, comprising a citrus extract containing the chemical formula (I). A therapeutic agent for dysuria, comprising the receptor antagonist according to any one of claims 5 to 10. A functional food for preventing or improving urination disorder, comprising the compound according to claim 1 or 2. A method for preventing or ameliorating dysuria, said method comprising:
Administering to a subject suffering from dysuria, a pharmaceutical composition or a receptor antagonist containing a compound represented by the following chemical formula (I):
The method wherein the compound exhibits antagonism against a dysuria-related receptor.

(In formula (I),
R ₁ is hydrogen or a hydroxyl group,
R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or —OCH ₃ ;
R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or —OCH ₃ )

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