KR20170082626A - 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

(식(I)중, R₁은 수소 또는 수산기이며, R₂, R₃, R₄, R₅ 및 R₆은 각각 독립해서 수소 또는 -OCH₃이고, R₇, R₈, R₉ 및 R₁₀은 각각 독립해서 수소 또는 -OCH₃이다)The long-term use of medicines may cause side effects, and medicines may not be effective enough. There is provided a pharmaceutical composition comprising a compound represented by the following formula (I).

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

Description

TECHNICAL FIELD [0001] The present invention relates to a medicinal composition for preventing or ameliorating dysuria, an antagonist for a dysuria-related receptor or a medicinal composition thereof or a method for preventing or improving dysuria using antagonistic medicines. METHOD FOR PREVENTING OR IMPROVING DYSURIA USING MEDICINAL COMPOSITION OR ANTAGONIST}

The present invention relates to a pharmaceutical composition for preventing or improving dysuria, an antagonist for dysuria-related receptors or a method for preventing or improving dysuria using antagonistic agents.

A healthy person's bladder can store up to 300-500 ml of urine. When an adult gets about 200ml of urine in the bladder, the signal about urinary tract goes to the brain and feels the first urination. When the brain receives the sent signal, the parasympathetic nerve becomes excited, and as a result, the muscarinic receptors of the bladder are stimulated to contract the smooth muscle (detrusor). On the other hand, in the urine collecting step, the sympathetic nerve becomes dominant and the detrusor becomes relaxed.

A healthy person can urinate properly if he or she feels urinary. However, if the urinary disturbance such as frequent urination or urinary incontinence occurs for various reasons, quality of life (QOL) is remarkably deteriorated. There are many people who neglect such dysuria. The reason is that, besides being ashamed to go to the hospital, it does not know that it has urination disorder.

In the International Continence Society held in 2002, symptoms associated with "frequent urination" or "nighttime urination", which are the main symptom of "urgency of urination", are referred to as "Overactive Bladder" (OAB) . Based on the proposal, the diagnosis, treatment and prevention of voiding dysfunction became active.

OAB is a disorder in which the urinary bladder overreacts, resulting in sudden strong urination. It has been reported that the prevalence increases with aging. In OAB patients, the sympathetic nerves and parasympathetic nerves are disrupted, and contractions and frequent urination occur because the contractions of the detrusor regardless of the urination are not known. In men, OAB has been reported to accompany enlarged prostate gland. The prostate gland exists beneath the bladder to surround the urethra. When the prostate gland is enlarged by aging, the urethra of the bladder outlet is depressed, causing " scarcity loss " and " scarcity ". Patients with OAB in women have a high incidence of "stress urinary incontinence" (ie, urine leaks if the abdomen temporarily contracts, such as sneezing, coughing, or exercise) due to slackening of the pelvic floor muscle due to aging and childbirth Reported. This is because the female urethra is shorter than the male urethra. Urinary dysfunction is an aging disease caused by various factors in a range including bladder to urethra. Therefore, prevention of voiding dysfunction is very important for a comfortable elderly person's life.

Anticholinergic drugs have been reported to be effective in the treatment of OBA (see, for example, Non-Patent Document 1).

"A comparison of the efficacy and tolerability of solifenacin in succinate and extended release tolterodine at treating overactive bladder syndrome: results of the STAR trial." Chapple et al., Eur Urol. 2005 Sep; 48 (3): 464-70.

However, the long-term use of a commercially available anticholinergic drug has been associated with side effects.

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a method for preventing and / or ameliorating dysuria using a drug or a medicament based on a compound present in nature.

The inventors of the present invention have conducted studies on prevention and / or improvement of urinary disturbance caused by highly safe food materials, and have found that the use of nobiletin, tangentetin, 3 ', 4', 5,7, - tetramethylquercetin, Has an antagonistic action on this urinary disturbance-related receptor and improves the urination disorder. Thus, the present invention has been completed.

According to the present invention,

There is provided a pharmaceutical composition for preventing or ameliorating dysuria comprising a compound represented by the following formula (I).

(In the formula (I), R ₁ is hydrogen or a hydroxyl group, and R ₂ , R _3,, R _4, R ₅ and R ₆ are each independently hydrogen or -OCH ₃ , and R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or -OCH ₃ )

According to the present invention,

Claims 1. A receptor antagonist comprising a compound represented by the formula (I), wherein said compound exhibits an antagonistic effect on a receptor associated with dysuria.

(Wherein 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 compounds represented by the above formula (I) inhibited the specific binding of [ ³ H] pirenzepine, a selective marker ligand for muscarinic receptors, to muscarinic receptors . Thus, the use of this antagonist can inhibit the specific binding of the ligand to a receptor associated with dysuria.

According to the present invention,

There is provided a therapeutic agent for dysuria, comprising a receptor antagonist comprising said compound. As demonstrated in Examples described later, urinary disorders can be treated by using a therapeutic agent comprising an antagonist comprising the compound represented by the above formula (I). Therefore, by administering this therapeutic agent to a patient suffering from a voiding disorder, it becomes possible to treat the voiding disorder.

According to the present invention,

A method for preventing or ameliorating dysuria, the method comprising:

Comprising administering to a subject suffering from the urination disorder a medicinal composition or a receptor antagonist comprising a compound represented by the following formula (I), wherein the compound exhibits an antagonistic action on a receptor associated with dysuria do.

(Wherein 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 Examples to be described later, the administration of a pharmaceutical composition or a receptor antagonist comprising the compound represented by the above-mentioned formula (I) improved micturition disorders. Therefore, it is possible to prevent or improve dysuria by administering this method to a patient suffering from a dysuria.

According to the present invention, it is possible to prevent or improve dysuria by administering a pharmaceutical composition or a receptor antagonist comprising the compound represented by the formula (I).

1 is a graph showing the antagonistic action of Shiikuwasha extract (Novartin-tangentetin mixture) on the specific binding of [ ³ H] pirenzepine in rat cerebellum cell membranes.
2A is a graph showing the antagonistic effect of nobiletin and tangentretin on the specific binding of [ ³ H] pirenzepine in rat cerebellum cell membranes.
Figure 2b shows the chemical formulas of Nobiletin, Tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and Sinensetin.
FIG. 2c is a graph showing the results of a comparison of the activity of muscarinic derivatives of Nobiletin, Tangeretin, 3 ', 4', 5,7-tetramethylquercetin and Sinensetin isolated from Shikuwa, M1-specific receptor binding activity (inhibitory effect on binding of [ ³ H] -Pirenzepin).
FIG. 2d shows a comparison of the results of a non-specific mousse with respect to Nobiletin, Tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and Sinensetin isolated from Shikuwa ([≪ ³ > H] -N-methyl scopolamine (NMS).
3 is a graph showing the antagonistic action of various extracts and compounds (muscarinic extract: 10 mu g / mL, compound: 10 mu M) on muscarinic receptors.
Fig. 4 is a graph showing the effect of Shikuwasa extract of acetic acid-induced frequent urine model rats to improve urination disorders. Fig. 4A shows the maximum bladder pressure of the model rat, Fig. 4B shows the basal pressure, Fig. 4C shows the threshold pressure, Fig. 4D shows the voiding interval, Fig. 4E shows the number of urination and Fig.
Figure 5 shows the effect of intravenous administration of Novartin on bladder pressure measurement of pelvic congestion-free rats.
Figure 6 shows the effect of intravenous administration of Novartin on the bladder contraction interval of pelvic congestion free rats. The left axis is the sham group, and the right axis is the pelvic congestion group.
Fig. 7 shows the effect of oral administration of novelletin on the bladder pressure measurement of pelvic congestion-free rats.
Figure 8 shows the effect of oral administration of novelletin on bladder contraction interval in pelvic congestion frequency-null rats.
Figure 9 shows the results of intravenous administration of Novartin on the bladder contraction interval of cerebral infarction rats. The left axis is the sham group and the right axis is the cerebral infarction group.
10 is a system schematic diagram for measuring the bladder internal pressure and the urination volume with time.
Fig. 11 shows the urination volume, number of urination, and maximum bladder pressure of cyclophosphamide (CYP) -induced cystitis model rat administered with Shikuwa extract (50 mg / kg) orally.
Figure 12 shows the action of Shikuwasa extract on calcium antagonists.
Fig. 13 shows the protocol of the parietal electrical stimulation (1 Hz) experiment.
Figure 14 shows the effect of atropine and alpha, beta methylene ATP on the contractile response of rat-extracted bladder by pneumatic electrical stimulation (1 Hz) in the presence of Novartin or tancretin. In the graph, the axis on the left is the control or sample (Novartin or tangentretin), the central axis is atropine, and the right axis is alpha, beta methylene ATP.
Figure 15 shows the action of nobiletin and tangentretin on cholinergic and purinergic contraction components.

Justice

Muscalin  Receptor

The muscarinic receptor, which is a type of acetylcholine receptor, is a receptor that has the same action as that of acetylcholine even by muscarinic, and exists as a receptor of the organ cell in the parasympathetic peripheral nerve. Muscarinic receptors are stimulated by parasympathetic nerves, and when acetylcholine is released from its periphery, muscarinic receptors are stimulated to excite or inhibit the cells of organs under control, but the organs of the organ are either excited or inhibited Depend on (ie, depending on the organ).

Antagonism

The term " antagonist " refers, for example, to a drug containing a substance that competitively inhibits the specific binding of a ligand to a receptor (i.e., antagonism). A substance having an antagonistic action is also called an antagonist. Antagonism may be caused by the antagonism of the antagonist by binding to the receptor and by the inability of the antagonist to bind to the receptor, and by the antagonist acting on the ligand (for example, by binding) .

summary

Hereinafter, embodiments of the present invention will be described in detail. In order to avoid complicating the same contents repeatedly, a detailed description thereof will be omitted.

≪ Embodiment 1: Pharmaceutical composition >

According to the present invention, there is provided a pharmaceutical composition for preventing or improving dysuria, comprising a compound represented by the following formula (I).

(Wherein 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 medicinal composition, it is possible to prevent or improve the urination disorder.

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 formula (I) may be -OCH ₃ . In the present specification, at least 1, 2, 3 or 4 of R ₂ to R ₆ in the formula (I) may be -OCH ₃ . In the present specification, at least one, two or three of R ₇ to R ₁₀ in the above formula (I) may be -OCH ₃ . In the present specification, -OCH ₃ and -OMe are mutually substitutable.

According to the present invention, there is provided a pharmaceutical composition for preventing or improving dysuria, wherein the compound represented by formula (I) is at least one compound selected from the group consisting of the following formulas (II) to (V).

And

By using this medicinal composition, it is possible to prevent or improve the urination disorder.

≪ Embodiment 2; Receptor antagonist related to urinary dysfunction>

According to the present invention there is provided a receptor antagonist comprising a compound represented by the following formula (I), wherein said compound exhibits antagonism to a receptor associated with dysuria.

(Wherein 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 , and R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or -OCH ₃ .

By using this antagonistic agent, it is possible to inhibit the specific binding of the ligand to the urinary disturbance-related receptor.

According to the present invention, a compound represented by the above-mentioned general formula (I) is a compound selected from the group consisting of compounds represented by the following formulas (II) to (V).

And

In the present specification, the urinary 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 above formulas (II) to (V) are referred to as nobiletin, tangeretin, 3 ', 4', 5,7-tetramethylquercetin and cinnencetin, respectively. (IV) and 3 ', 4', 5, 7-tetramethylquercetin can be substituted with each other, and the compound of formula (II) (V) and cinnencetin are substitutable with each other.

The compound may be a purified compound or a synthesized compound. In addition, the compound may be in the form contained in a crude extract (crude extract) of a plant. Novartin, tangretin, 3 ', 4', 5, 7-tetramethylquercetin and cinnencetin can be extracted from the citrus fruits and extraction methods described below. In addition, novilectin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and cinnensetin can be purified using a purification method described below. The compound may be a hydrate.

When the compound or the hydrate of the compound is used as a pharmaceutical composition or a pharmaceutical preparation (antagonist), the crystal may be treated according to a conventional method and mixed with an excipient or the like to be described later. Examples of the pharmaceutical preparation containing such a compound as an active ingredient include injections, suppositories, aerosols, transdermal absorbers, ointments, alerts, sprays, parenteral preparations, tablets, powders, granules, powders, capsules, troches, liquid preparations, and other oral preparations.

Herein, the tablets include sugar-coated tablets, coated tablets, and oral tablets, and the capsules include both hard capsules and soft capsules. Granules also include coated granules. In addition, the liquid preparation includes suspensions, emulsions, syrups, elixirs and the like, and the syrup includes dry syrup. In addition, each of the above-mentioned formulations includes not only a sustained-release formulation but also a sustained-release formulation. Such a preparation can be prepared by a known pharmaceutical preparation method using a base, a carrier, an excipient, a binder, a disintegrant, a lubricant, a colorant, etc. described in Japanese Pharmacopoeia (Japanese Pharmacopoeia) which is pharmacologically acceptable in the preparation of the preparation can do. Examples of such carriers and excipients include lactose, glucose, white sugar, mannitol, potato starch, corn starch, calcium carbonate, potassium phosphate, calcium sulfate, crystalline cellulose, licorice powder and Gentiana powder.

Examples of the pharmaceutical composition or the binder include starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, ethylcellulose, carboxymethylcellulose And the like. Examples of the disintegrant include starch, agar powder, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, methyl cellulose, ethyl cellulose and carboxymethyl cellulose. Examples of the lubricant include talc, magnesium stearate, and the like. The coloring agent may be any as long as it can be added to a pharmaceutical product, and is not particularly limited. In addition to these, a mating agent, a fodder, and the like may be suitably used as needed.

In the case of using a tablet or granule, if necessary, a tablet, gelatin, tablet cell, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, phthalic acid cellulose acetate, hydroxypropyl Methyl cellulose phthalate, methyl methacrylate, methacrylic acid polymer, or the like, or may be coated with a plurality of layers. The granules and the powder may be filled in capsules such as ethylcellulose or gelatin to form capsules.

When an injectable preparation is prepared using a pharmaceutical composition or antagonist comprising the compound or a pharmaceutical composition or antagonist comprising the hydrate of the compound, a pH adjusting agent, a buffering agent, a stabilizer, a solubilizing agent and the like may be added have.

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

In the present specification, the urination disorder may be overactive bladder and / or hypertrophy of the prostate. Urinary dysfunction (lower urinary tract dysfunction) can be divided into two groups. One is overactive bladder, the other is enlarged prostate. And activity The bladder is accompanied by a feeling of urgency of the urine, a state in which unbearable urination occurs suddenly. The overactive bladder may also be accompanied by one or more symptoms selected from the group consisting of frequency, nocturia and urinary incontinence. For the treatment of overactive bladder, anticholinergic drugs are used as first-line drugs. The enlarged prostate may be accompanied by increased urinary flow resistance and / or bladder blood flow obstruction due to lower urinary tract obstruction. For prostate hypertrophy, the alpha 1 blocking drug is used as the first drug of choice. In addition, the overactive bladder does not have to accompany the enlarged prostate, and the enlarged prostate does not have to accompany the overactive bladder. In this specification, the urination disorder may be an overactive bladder. In the present specification, the urination disorder may be prostatic hyperplasia.

In this specification, the urination disorder may be accompanied by one or more symptoms selected from the group consisting of urinary frequency, nocturia, urinary incontinence, restlessness and urinary incontinence. Frequent urination is a disease that increases the frequency of urination from mood to bedtime. Generally, when the number of urination per day is eight or more, diagnosis is made as frequency of urination, but the present invention is not limited thereto. Nighttime urinary frequency is a temporal mood disorder for temporary urination from sleep to the weather, and is usually diagnosed as nighttime urination if there is one or more urination, but is not limited thereto. In addition, urinary incontinence refers to a disease in which urinary leakage occurs involuntarily or unconsciously. Incontinence includes stress urinary incontinence, urge incontinence, first-degree incontinence, reflective incontinence, and incontinence. Persistent sensation is a disease accompanied by sensation that urine remains in urinary bladder (urine) after urination, and this sensation occurs irrespective of presence or absence of residual urine. Yoshinaga is a disease in which the intensity of urination is reduced and the voiding time is prolonged. The pathology may include urine tests, prostate-specific antigen (PSA) measurements, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), urinary bladder imaging, .

The antagonist of the pharmaceutical composition or receptor for dysuria of the present invention may contain a citrus extract containing the above formula (I). In the present specification, the citrus fruits are selected from the group consisting of Keraji (Citrus Keraji), Ponkan (C. reticulate), Dancy tangerine (C. tangerine), Jimikan (C. succosa) (Shikaikan; C. suhuiensis), Tachibana (C. tachibana), Kobenimikan (C. erythrosa), Kishumikan (C. kinokuni), Shiikuwasha (C. depressa) It may be Koji (C. leiocarpa). Among the citrus fruits, Shikuwa is preferred because it contains more nobiletin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and cinnencetin. In the present specification, the citrus extract is derived from one or a plurality of citrus fruits.

In the present specification, the pharmaceutical composition or antagonist is selected from the group consisting of nobiletin, tangeretin, 3 ', 4', 5,7-tetramethylquercetin and / , 4 ', 5, 7-tetramethylquercetin, and / or cinnencetin. In the present specification, the pharmaceutical composition or antagonist includes the extract containing the noviletin, tangeretin, 3 ', 4', 5,7-tetramethylquercetin, and / or cinnexetin, , Tangretin, 3 ', 4', 5, 7-tetramethylquercetin, and cinnencetin. The content of novilectin, tangeretin, 3 ', 4', 5,7-tetramethylquercetin and / or cinnenecetin in the extract was 10% by weight, 20% by weight, 30% by weight, 40% by weight, Even within a numerical range between any two points selected from the group consisting of weight%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 98 wt% and 99 wt% do.

The citrus portion which can be used in the present invention is not particularly limited as long as it is a site containing noviretin, tangentetin, 3 ', 4', 5,7-tetramethylquercetin and / or cinnensetin, In addition to the fruit, it can also be used as a raw material, such as peel, juice residue (dried residue), cut leaves and twigs, and dried products thereof. In the case of Shikuwasanaponkan and the like, it is preferable that the use of fruits, peel and juice residue is such that the content per unit weight of novirretin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and / Therefore, it is preferable. On the other hand, the contents of the leaves, twigs and the like obtained by the cell in a large amount, though not as large as the permafine, of the amounts of Novartin, Tangeretine, 3 ', 4', 5,7-tetramethylquercetin and / . Therefore, there is a possibility that the load on the environment can be reduced by using this.

The method of obtaining citrus juice residue is not limited, but, for example, a citrus fruit can be processed in a compactor to obtain a juice residue. The pressing machine is not particularly limited, but it is preferable to use a roll press machine, a filter press machine, a centrifugal filtration machine or the like in order to efficiently obtain a juicy rind.

The method of obtaining dried fruits of citrus fruit, peel and juice residue is not particularly limited, but citrus dried products can be obtained, for example, by heating and drying citrus fruits at 50-70 캜 for 15-45 minutes. At that time, if it is dried at less than 50 ° C, there is a possibility that it can not be sufficiently dried in a short time of 15-45 minutes, and when dried at a temperature exceeding 70 ° C, the components contained in citrus are likely to be denatured during drying. Therefore, when the citrus fruits are heated and dried, it is preferable to dry the citrus fruits at about 60 DEG C for about 30 minutes.

Sun dried can be used to obtain dried fruits of citrus fruit and skin and juice residue. The sun drying can reduce the electric energy of the drying machine and is suitable for drying work on an industrial scale from the viewpoint of energy cost. Further, since the temperature at the day of sun drying is lower than the temperature of the drying machine, it is possible to suppress the denaturation of the components of the dried material. When the dried material is obtained through sun-drying, the drying time can be appropriately adjusted according to the area, 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, 12 months. The drying place may be changed depending on weather conditions or degree of drying of citrus fruits. For example, at night, citrus may be preserved in a building (eg, a warehouse or factory).

In some embodiments, the citrus fruit can be obtained without drying the citrus fruit. If the citrus fruits are not dried, the drying cost and drying time can be reduced. When the citrus fruits are not dried, the citrus fruit extract may be obtained by using a site having a low water content (for example, peel).

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

First, Shikuwasa is washed in water, and then the squeezing process is performed with various squeezers capable of squeezing the fruit of Shikuwa as it is. As such a presser, for example, a roll (not shown) may be used in which a 10-20 mesh endless net is sandwiched between two compression rolls, and the fruit of a continuously supplied Sinkawa is compressed with these two compression rolls A press juice extractor, a foaming machine for wrapping the fruit of Shikuwa in a filter cloth and pressing the juice, and a centrifugal filtration type separation device.

The juice residue squeezed out of the juice is weighed in a predetermined amount, for example, about 300 to about 500 g (wet weight). Next, the residue is dried, for example, at about 50 to about 70 캜 using a dryer or the like. After weighing the weight of the dried juice residue (dry weight), the dried juice residue was placed in a suitable container, distilled water of 1-2 times the above-mentioned dry weight was added, and the container was immersed in a warm bath at about 60 to about 80 ° C And extracted 1-3 hours. After extraction, the beaker is taken out from the bath and naturally cooled. Thereafter, for example, a filter paper having a diameter of 5 μm or less is laid on a Buchner funnel, and the whole amount of the extract is suction filtered to separate the filtrate by filtration.

The obtained residue is again dried at about 50 to about 70 캜 using a dryer or the like and weighed. 2-3 times as much dry weight of ethanol is added and extraction is carried out at room temperature for 2-4 days. Next, as in the above, the entire amount of the extract is subjected to suction filtration, and the residue is separated by filtration. The filtrate is concentrated at about 30-40 < 0 > C for about 15-60 min.

To the obtained concentrate, 8% by weight of a 1% (w / v) aqueous sodium hydroxide solution of the concentrate is added and mixed well at room temperature. The mixture is continuously stirred at room temperature for 1 to 2 days, for example, using a stirrer. Thereafter, for example, a filter paper having a diameter of 5 μm or less is set in a funnel and subjected to natural filtration to separate a soluble component and an insoluble component. The obtained insoluble component is washed with 1-2 times the amount of water. The remaining insoluble components are eluted with 5-10 parts of ethanol. The ethanol-soluble portion is then concentrated at room temperature for about 15 to about 60 minutes. The amount of liquid after concentration is about 1 / 15-1 / 20 of the weight before concentration, and this liquid amount is measured.

To this concentrate, about 0.5-2% (w / v) aqueous sodium hydroxide solution of 8-10 times the liquid amount is added and mixed well. Next, the mixture is continuously stirred at room temperature for 4-8 days, for example, using a stirrer. Thereafter, for example, a filter paper having a diameter of 5 μm or less is set in a funnel, followed by natural filtration, and the residue is separated by filtration. In this way, fractions containing a high content of novilectin, tangeretin, 3 ', 4', 5, 7-tetramethylquercetin and / or cinnensetin can be obtained.

Another method for producing citrus extract using Shikuwa will be described.

The peel obtained from the fruits of citrus fruits is dried for 3 days by sun-drying, and the dried peel is obtained. Five times the amount of the solvent is added to a predetermined amount of dry skin, and the skin is extracted at a predetermined temperature for a predetermined period of time to obtain an extract. Examples of the extraction solvent include methanol, ethanol, acetone, ethyl acetate, water: methanol mixture of various mixing ratios, and water: ethanol mixture.

The obtained crude extract is filtered to remove solid components, and the whole amount of the filtrate is concentrated to obtain a concentrated liquid. To this concentrate is added a suitable amount of a water / organic phase mixture in an appropriate amount and subjected to liquid / liquid partitioning extraction. The distribution extraction is carried out several times while changing the composition of the mixed solution. The organic phase obtained through the partition extraction was concentrated and eluted by a step gradient method while increasing the solvent concentration stepwise by an open column. The desired fraction was added to the extract of perianth (hereinafter referred to as SPE) .

Next, a part of this fraction is concentrated, the solute concentration is adjusted, and the preparative column chromatography is carried out to carry out an adjustment agent (crude purification). The SPE is again supplied to column chromatography, and the amount of the Novartin and / or Tangeretine contained in the extract is quantified.

Concretely, 30-50 L of methanol is added to 6-10 kg of dried peels of citrus fruits such as Shikuwa and the mixture is extracted at 2-6 캜 for 7-21 days to obtain an extract. The extract is filtered to remove solid content, and the whole filtrate is concentrated using a flash evaporator. Next, 4L of water / ethyl acetate (1/1) was added and extraction was carried out. 4 L of 90% methanol / n-nucleic acid (1/1) was added to the obtained ethyl acetate phase, and liquid / liquid partitioning extraction was carried out again.

A portion of the 90% methanol phase is then concentrated, for example, by loading the sample onto an ODS silica gel column (Cosmosil 75 C ₁₈ -OPN, 50 mmφ × 500 mm) and increasing the methanol concentration by 20% To obtain a 60-100% methanol fraction. These fractions are dried on a rotary evaporator.

Then, for example, a portion of the above 80% methanol fraction is concentrated, methanol is added to adjust the solute concentration to 900 mg / ml, and the solution is loaded onto preparative column chromatography.

In the case of purifying novirretin, tangeretin, 3 ', 4', 5,7-tetramethylquercetin, and cinnenecetin from the above fraction, the purification can be performed by HPLC, although not limited thereto. Exemplary purification conditions are described below.

Sorting conditions: Column 5C _18- AR-II column (Nakarai Tesk Co., Ltd.)

Elution solvent: 70% methanol / water

Solute concentration: 900 mg / ml

Injection volume: 3 ml

Fraction volume: 10ml

Flow rate: 5 ml / min

Detection wavelength: UV215 nm

Range: 20mV

The elution times of novilectin, tangretin, 3 ', 4', 5, 7-tetramethylquercetin and cinnencetin were determined to be respectively novirretin, tangeretin, 3 ', 4', 5, 7, tetramethylquercetin and cinnensetin Can be determined by using the standard product of

Although the method for producing and purifying the extract containing noviletin, tangeretin, 3 ', 4', 5,7-tetramethylquercetin and cinnexetin has been exemplarily described, it has been found that, on an industrial scale, Can also be produced. In the case of an industrial scale, when it is possible to obtain the extract, it is possible to appropriately change the parameters of each manufacturing process, to add, change or repeat the manufacturing process, and to delete the manufacturing process.

≪ Embodiment 3: Therapeutic drug >

According to the present invention, there is provided a therapeutic agent for dysuria, comprising an antagonist for a receptor associated with dysuria related to said compound. It is possible to treat the urination disorder by using this therapeutic drug. Some urinary disturbance remedy can be used for preventing and / or improving urination disorders.

The therapeutic agent may be administered alone or in combination with another therapeutic agent, and administered in an effective amount to treat a voiding disorder. However, the total dose of the compound is determined by the attending physician within the scope of sound medical judgment. An effective amount for a patient suffering from a voiding disorder is a severity of voiding disorder; Age, weight, general health, sex and diet of the patient; Time of administration; The route of administration; The rate of excretion of the compound; Treatment period; And depends on the drug being used or co-administered with the therapeutic agent. The dose of the compound may not be a predetermined amount upon administration. For example, the dosage may be administered at a lower dosage than is necessary to achieve the desired therapeutic effect, and the dose may be increased until the desired effect is achieved.

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

The dose (daily dose) of the therapeutic agent per day is orally 0.01, 0.05, 0.1, 0.5 or 0.5 times as much as that of Novartin, tangeretin, 3 ', 4', 5,7-tetramethylquercetin and / , 1, 5, 10, 50, 100, 150 and 200 mg / kg body weight. The therapeutic agent is continuously administered over a period of time, that is, 3 days or more, preferably 1 week or more, more preferably 2 weeks or more, more preferably 1 month or more, for example, 6 months or 1 year or more . The above-mentioned therapeutic agent is preferably administered every day, but it is not necessary to administer it every day if it is continuously administered during the period. The above-mentioned therapeutic agent may be administered once a day or divided into several doses per day. The administration of the therapeutic drug may be terminated at the doctor's discretion or may be terminated at the patient's discretion.

≪ Embodiment 4: Functional food >

According to the present invention, a functional food for preventing or ameliorating dysuria comprising at least one compound selected from the group consisting of the compounds represented by formulas (II) to (V) or containing the compound represented by formula (I) / RTI > The functional food or the health product having the preventive effect or the improvement effect of the urination disorder can be provided by suitably adding the compound or the hydrate of the compound as necessary.

The compound or the hydrate of the compound can be used in the form of, for example, bread, cookies and biscuits, barley and rice grains for the addition of rice, udon, buckwheat noodles, pasta and other noodles, cheese, yogurt and other dairy products, jam, mayonnaise, , Other soy foods, tea, coffee and cocoa, soft drinks, fruit drinks, other non-alcoholic drinks, medicines, other alcoholic beverages, candy, chocolate, other snacks, chewing gum, Can be added to form a functional food. Or may be added to an animal feed to form a functional compound feed. In addition, when added to the above-mentioned yogurt, soy sauce, drinks, etc., a dissolution aid or a stabilizer may be appropriately added in order to prevent crystallization of the compound of the present invention and precipitation.

In addition, the compound or the hydrate of the compound may be used alone or as a mixture of two or more kinds thereof, and may be formulated into a powder, granule, tablet or capsule according to a conventional method. Here, in order to prepare the compound of the present invention as a powder, the extract obtained in the production process is concentrated and dried using a method such as freeze-drying, spray drying, or vacuum drying, and the dried solid is pulverized with a sample mill, a blender, . If necessary, corn starch, potato starch, dextrin, oyster shell powder, and the like may be added.

The powder obtained as described above may be appropriately tableted by adding the binder described above. After the tablets are formed, they may be coated with a coating agent such as the above-described saccharide or gelatin, or may be used as a tying agent by using other coating agents. The powder obtained as described above may be granulated in a conventional manner to prepare granules. The above powder or granules can also be formed into capsules by filling the aforementioned capsules with an appropriate amount. In the present invention, the functional food and the health food do not include plant fruits other than citrus fruits, which themselves contain the above-mentioned compounds in nature. However, the food or the like prepared by adding the compound to the citrus fruits and the like is not excluded from the present invention.

The amount of the nobilisetin contained in the functional food and the health food of one meal is between two points selected from the group consisting of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150 and 200 mg / To be within the range of.

≪ Embodiment 5: Method for preventing or improving urination disorder >

According to the present invention there is provided a method for preventing or ameliorating a dysuria disorder comprising administering to a subject suffering from the dysuria disorder a pharmaceutical composition or a receptor antagonist comprising a compound represented by the following formula And wherein said compound exhibits antagonism to a receptor associated with dysuria.

(In the formula (I), R ₁ is hydrogen or a hydroxyl group, and R _{2 ,} R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or -OCH ₃ , and R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or -OCH ₃ .

When the pharmaceutical composition or the receptor antagonist is administered to a patient, the dosage varies depending on conditions such as the severity of the patient's symptoms, age, weight, PSA level, urine flow rate and health condition. Generally, the dosage may be administered once or more times per day by the above-mentioned dose and dosage. The number and the amount of administration may be appropriately increased or decreased according to the above conditions.

The daily dose, the administration period and the administration frequency of the above pharmaceutical composition or antagonist may be the same as the aforementioned therapeutic drug. The administration of the above pharmaceutical composition or antagonist may be terminated according to the judgment of the physician or may be terminated at the judgment of the patient himself.

Although the embodiment of the present invention has been described above, it is an example of the present invention, and various configurations other than the above may be adopted.

Hereinafter, the present invention will be described with reference to the embodiments and drawings, but the present invention is not limited thereto. The operation of the apparatus and the use of the kit were in accordance with the manufacturer's protocol of each maker.

< Example  1>

500 g of the juice residue of Shikuwa Co., Ltd. from Higashimura, Okinawa was well washed, 1800 mL of distilled water at about 70 DEG C was added, and the plate was immersed in a bath at about 70 DEG C and heat-treated for 3 hours. The obtained heat-treated product was subjected to suction filtration using a Buchner funnel with a 3 μm diameter retention filter paper (ADVANTEC, No. 131) to obtain a residue. The residue was juice pressed.

300 g of the juice residue after the juice was weighed, and dried at 60 캜 for 30 minutes using a dryer (Panasonic Corporation, part number EH5101P Turbo Dry) to obtain 150 g of dried peel. 30% (v / v) ethanol of twice the weight of the juice residue was added thereto, and ethanol extraction was performed at room temperature for 3 days. Filter paper (ADVANTEC, No. 131) with a diameter of 3 μm was used for suction filtration using a Buchner funnel to obtain 3200 mg of a filtrate. The resulting filtrate was concentrated with a rotary evaporator to obtain a concentrated liquid.

The concentrate was added with a 10% aqueous solution of 1% (w / v) sodium hydroxide and stirred at room temperature for 1 day using a stirrer. The resulting insoluble component was washed with two times the amount of water. The remaining insoluble components were eluted with 5-10 parts of 100% (v / v) ethanol. The ethanol eluted components were then filtered using a Buchner funnel and the precipitate was filtered. The contents of novilectin and tangeretin were about 60% and 35%, respectively.

Novyletin , Tangerine , 3 ', 4', 5, 7- Tetramethylquercetin , Cynthian  refine

The obtained extract (filtrate) was subjected to HPLC analysis by a known method. The analysis conditions of HPLC are as follows.

HPLC apparatus: UV-8011 HPLC (manufactured by TOSOH)

Detector: UV Detector

250mm(Waters사제)Column: Cholester Waters inner diameter 4.6mm

250 mm (manufactured by Waters)

Elution solvent: 75% MeOH / water

Solute concentration: 0.1 mg / mL

Injection volume: 0.005 ml

Flow rate: 1.0 mL / min

Detection wavelength: UV 215 nm

Range: 1000mV

The Shikuwasa extract was recovered at a peak between about 8 and 12 minutes after injection. Novartin was recovered from a peak appearing at about 9 minutes after injection. Tangeretine was recovered from the peak appearing at about 11 minutes after injection. Nobiletin and tangietetine each had a purity of 95% or more. In addition, 3 ', 4', 5, 7-tetramethylquercetin and cinnencetine were also purified in the same manner using their respective products.

 < Example  2>

Muscalin Assessment of antagonism to the receptor

The crude cell membrane preparation prepared from the rat brain was used to determine the specific binding of [ ³ H] pirenzepine, which is a selective marker ligand for muscarinic receptors, in the presence of various plant extracts or component compounds at various concentrations, &Lt; / RTI &gt; These antagonistic activities were evaluated by radio receptor assay and calculated 50% inhibitory concentration (IC ₅₀ value), inhibition constant (Ki value) and Hill coefficient (nH).

SD rats (8-week-old male, purchased from SLC Co., Japan) were inoculated under ether anesthesia, and blood was collected from the abdominal descending aorta by heparinized syringes and slaughtered. After physiological saline cooled in the artery was refluxed, the brain was extracted. To the cerebellum-free brain was added a cooled 50 mM Tris buffer (pH 7.4) of 19 times the volume, homogenized, and centrifuged at 40,000 x g for 20 minutes at 4 ° C. The supernatant was removed, and the precipitate was again centrifuged at 40,000 x g for 20 minutes at 4 ° C after adding 50 mM Tris buffer (pH 7.4) cooled to 19 times the volume again. The supernatant was removed, and 50 mM Tris buffer (pH 7.4) cooled to 29 times the volume was added to the precipitate and suspended to prepare a receptor product.

As a tissue amount, 1 mg of the receptor product was added to 50 mM Tris buffer (pH 7.4), each concentration of the test sample and 1 nM [ ³ H] pirenzepine was added, and the final amount was adjusted to 0.5 mL. The reaction solution was incubated at 25 DEG C for 60 minutes.

After completion of the incubation, each reaction solution was subjected to rapid suction filtration on a glass fiber filter paper (Whatman GF / B) using a cell collector (cell harvester, Brandel). Immediately thereafter, the filter paper was washed with 3 mL of cooled 50 mM phosphate buffer (pH 7.4). The filter paper was prepared by adding toluene scintillator (2 L of toluene, 1 L of Triton-X, 15 g of 2,5-diphenylloxazole and 0.3 g of 1,4-bis [2- (5-phenyloxazolyl)] benzene) And thereafter, the radioactivity was measured using a liquid scintillation counter. 1 μM of atropine was used as a displacer and the radioactivity obtained in the absence and presence of the displacer was defined as a specific binding to the receptor, did.

The Shikuwasa extract (Novartin-Tanceretin mixture) inhibited [ ³ H] pirenzepine-specific binding in the rat cerebellum cell membrane in a concentration-dependent manner (FIG. 1). The IC ₅₀ value calculated from the inhibitory action was 13.5 ± 3.4 μg / mL (Table 1).

[ ³ H] pirenzepine (muscarinic receptor) IC ₅₀ nH
μg / mL Shikuwasa extract 13.5 ± 3.4 1.5 ± 0.5 The values are mean ± SE of 5 rats (mean ± SE).

From these results, it was suggested that Shikuwasa extract exhibits binding activity to muscarinic receptors.

< Example  3>

Muscalin  For the receptor Novyletin and Tangerine  Antagonism

The same tests as in Example 2 were carried out on purified nobiletine and tangeretine alone. The results are shown in Fig. As shown in FIG. 2 A, novilletine inhibits the specific binding of [ ³ H] pirenzepine to the rat cerebellum cell membrane in a concentration-dependent manner, while tangentin inhibits the binding of [ ³ H] pirenzepine to the rat cerebellum No concentration-dependent inhibitory effect was observed. The Ki value calculated from the inhibitory action of novirretin was 15.7 ± 2.8 μM (Table 2).

[ ³ H] pirenzepine (muscarinic receptor) K i n H
μM Novartin 15.7 ± 2.8 1.6 ± 0.3 The values are mean ± SE (mean ± SE) for 4-5 rats

This suggests that Novartin exhibits binding activity to muscarinic receptors. On the other hand, tangentin was found to be weak or absent in antagonism to muscarinic receptors.

Shikawa  Lt; RTI ID = 0.0 &gt; Muscalin  Receptor binding activity

( ³ H) -Pirenzepin (Fig. 2 (b)) for Novartin, Tangeretine, 3 ', 4', 5,7-tetramethylquercetin, the inhibitory action on the binding of), a non-specific alkaline mousse receptor binding activity ^{([3 H] -N-methyl} scopolamine (NMS) functional for binding) were compared.

The muscarinic M1 receptor-specific binding activity was stronger in the order of Novartin> 3 ', 4', 5, 7-tetramethylquercetin? Cinenecetin> tangeretin (Fig. 2C and Table 3).

[ ³ H] pirenzepine Log IC ₅₀ (M) IC ₅₀ ([mu] M) Ki (μM) Novyletin -4.915 + 0.124 12.16 12.10 Tangerine N.C. N.C. N.C. 3 ', 4', 5, 7-tetramethylquercetin -4.458 + 0.248 34.84 41.36 Sinensetin -4.480 + 0.252 33.08 57.19 Each value is mean ± standard error (means ± SE) for 3-4 rats.
NC: Not calculated.

Muscarinic nonspecific receptor binding activity was recognized in nobiletin and cinnencetin (Fig. 2d).

From these results, it has been found that Novartin, 3 ', 4', 5,7-tetramethylquercetin, cynesetin, and tangeretin from Shikuwa have muscarinic receptors, particularly muscarinic M1 receptor binding activity. In addition, tangietin showed weak binding activity (Fig. 2C). This indicates that these four compounds have a therapeutic effect on voiding dysfunction.

< Example  4>

Muscalin  Antagonism of various plant extracts and compounds on the receptor

The antagonistic action to muscarinic receptors in various plant extracts and compounds was studied. The plant extracts and compounds used are listed in Table 4.

Sample name Ingredient Name: Main Ingredient (Classification) Availability BGE (Butyl glycidyl ether) Black ginger extract: 5 7-dimethoxy flavone, etc. Orizai oil painting BE Cashews extract; Anthocyanins Extraction from Kashz EGCG Epigallocatechin gallate (catechin) Wako Ferulic acid Ferulic acid (phenylpropanoid) Sigma GBE Ginkgo Leaf Extract: Flavonoid, Kinko Ride, etc. Sigma GTE Green tea extract: Epigallocatechin gallate and others Taiyou Chemical Phenon BG-3 Naringenin Narinjenin (flavanone) Sigma Nobiletin Novartin (flavone) Purified from Shikuwasan's peel Quercetin Quercetin (flavonol) Sigma Resveratrol Resveratrol (stilbeneoid) Wako Theanine Theanine (amino acid) Wako

The results are shown in Fig. Antagonism to muscarinic receptors was confirmed by Novartin alone. However, antagonism to muscarinic receptors was hardly observed in various flavonoids or various flavonoids and polyphenol compounds containing various polyphenols. From these results, it was suggested that the antagonistic action on muscarinic receptors is not an action generally recognized in various polyphenol-containing plant extracts and polyphenol compounds, but a function specific to nobiletin.

< Example  5>

Acetic acid induced urinary frequency model Rat

The effect on urination disorders was evaluated using an acupuncture-induced urinary frequency model rat, which is a urinary model by cystitis. (0.8 g / kg ip and 0.4 g / kg sc) was injected continuously into the bladder at 3 mL / hr using a male SD rats (purchased from Japan SLC Co., Ltd.) at 8 weeks of age (MmHg), basal pressure (mmHg), threshold pressure (mmHg), and voiding interval (min) were measured by the bladder pressure measurement method after 1 hour from the administration of solvent or Shikuwa extract.

Acetic acid induced urinary frequency model In rats  About Shikawa  Action of extract administration

A significant increase in the maximum bladder pressure was observed when the extract of Shikuwa (50 mg / kg) was administered in acetic acid-induced urinary frequency rats (Fig. 4A). In addition, the voiding interval was significantly prolonged (FIG. 4D) and the number of voiding times was also significantly decreased (FIG. 4E). On the other hand, the extract had no significant effect on the base pressure (Fig. 4B), the threshold pressure (Fig. 4C), and the single void volume (Fig. 4F). These results showed that Shikuwasa extract improved urinary frequency.

< Example  6>

Pelvic congestion disorder model In rats  Action

Pelvic congestion disorder model Rat  making

Twenty-four Sprague-Dawley (SD) female rats (approximately 200 g) were anesthetized with 2% isoflurane inoculation and the laparotomies were ligated to the umbilical vein and uterine veins on both sides to cover the pelvic congestion. In addition, eight SD female rats (200 g or so) in which the bilateral sacral vein vein and uterine vein were exfoliated from surrounding tissues were used as Sham-gun. Three weeks after the operation, the following experiment was used.

Novyletin Intravenous  Administration experiment

In a group of 8 pelvic congestion and 8 sham group, a catheter for measurement of intrathoracic pressure with a diameter of about 1 mm was inserted into the bladder under a 2% isoflurane inhalation anesthesia, Physiological saline injection into the bladder and internal pressure measurement. A vein retention needle was inserted into the femoral vein of one side and used for administration of Novartin. The isoflurane inhalation anesthesia was stopped and continuous bladder pressure measurement was performed while physiological saline was injected into the bladder (3 ml / h) under urethane (0.6 mg / kg) light anesthesia restraint. When the bladder contraction reached a state where the bladder contractions appeared at regular intervals , Novartin (0.3, 1, 3, 10, 30 mg / kg) was administered intravenously every 15-20 minutes and the effect on bladder activity was examined by comparison with that before administration of Novarten (Figures 5 and 6 )

Novyletin  Oral administration experiment

Eight of the remaining 16 mice of the pelvic congested rats were used as a control group for pelvic congestion and 8 as novoletin group for pelvic congestion. Pelvic congestion Novartin (30 mg / kg) was dissolved in 1 mL of water for 1 week to 2 weeks after congestion and was administered once per day to the stomach with a sonde. Eight pelvic congestion control and eight Shamsamp were treated with 1 ml of water once daily for two weeks in the stomach. Two weeks after the intra-stomach administration, as in the intravenous administration of noviractin, continuous bladder pressure was measured under urethane (0.6 mg / kg) light anesthetic restraint and the difference in bladder activity between the three groups was examined 8)

Statistical processing

Results were expressed as mean ± standard deviation, and paired t- test or non-paired t- test was used, and p <0.05 was considered significant.

Novyletin Intravenous  Result of administration

The pelvic congestion group was significantly shorter than the bladder extraction interval (voiding interval) (9.3 ± 1.1 min, 12.4 ± 0.7 min, respectively) than the sham group and was frequency (Figure 6, before administration) The bladder contraction interval (16.9 ± 1.0 minutes at 30 mg / kg) was prolonged with -30 mg / kg intravenous administration and the bladder contraction interval (14.4 ± 0.9 minutes) was prolonged in the sham group at 10 mg / kg intravenous administration And Fig. 6). There was no change in the maximum contractive pressure or baseline pressure of the bladder.

Novyletin  Results of oral administration

The bladder contraction interval was shorter in the pelvic congestion control group (8.2 ± 0.7 min) and the pelvic congestion-free nonyletin group (10.6 ± 0.8 min) than in the sham group (13.2 ± 0.9 min) The gap was extended (Figs. 7 and 8). There was no difference in group 3 between the groups. The maximum bladder systolic pressure did not differ from the sham group (48.9 ± 2.0 cm H ₂ O), but the pelvic congestion level (46.3 ± 2.3 cm H ₂ O) was lower than the pelvic congestion control group (54.1 ± 3.0 cm H ₂ O) .

< Example  7>

Cerebral Infarction Model Rat Action on urination

Cerebral Infarction Model Rat  making

8 mm SD female rats (approximately 200 g) were implanted with 2% isoflurane inhalation anesthesia and the neck was incised in the midline. The 3-0 nylon thread extending from one internal carotid artery to the subepithelial artery was inserted and the internal carotid artery was ligated, And closed. In addition, 8 male SD rats (200 g or so) in which only one internal carotid artery was detached from the surrounding tissue were used as Sham-gun. On the first day after the operation, it was used for the following experiments.

Novyletin Intravenous  Administration experiment

In a group of 8 cerebral infarctions and 8 sham groups, a catheter for measuring bladder pressure of about 1 mm in diameter was inserted into the bladder under 2% isoflurane inhalation anesthesia, Injection furnace and internal pressure measurement furnace. A vein retention needle was inserted into the femoral vein of one side and used for administration of Novartin. The isoflurane anesthesia was stopped and the continuous bladder pressure was measured while physiological saline was injected into the bladder (3 mL / h) while restraining no anesthesia. When bladder contractions appeared at regular intervals, , 3, 10, and 30 mg / kg) were administered intravenously every 15-20 minutes, and the effects on bladder activity were compared with those before administration of Novarten.

Statistical processing

Results were expressed as mean ± standard deviation, and paired t- test or non-paired t- test was used, and p <0.05 was considered significant.

Novyletin Intravenous  Result of administration

In the cerebral infarction group, bladder contraction intervals (9.2 ± 0.6 min and 13.5 ± 0.9 min, respectively) were significantly shorter than those of Shamshin group, and they were frequency. In the cerebral infarction group, bladder contraction interval (14.4 ± 2.2 minutes at 3 mg / kg) was prolonged by intravenous administration of Novartin 3-10 mg / kg, and bladder contraction interval (17.1 ± 1.6 minutes) (Fig. 9). There was no change in the maximum contractive pressure and baseline pressure of the bladder.

< Example  8>

Drug-induced cystitis model Rat

Drug-induced cystitis model Rat  making

CYP-induced cystitis model rats followed the creation of methods such as Chow (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 , &Lt; / RTI &gt; 67 : 636641, 2006.). CYP (150 mg / kg) dissolved in physiological saline was intraperitoneally administered intraperitoneally, and 24 hours later, bladder pressure and urination volume were measured under urea anesthesia by the bladder pressure measurement method.

Urethane under anesthesia To measure bladder pressure  by Bladder pressure  And measurement of urination volume

The rats were incubated under the anesthesia with a thermostatic pad (The Deltaphase (R) Isothermal Pad: BRAINTREE SCIENTIFIC, INC.), Fixed with supine, and the lower abdomen was perfused with an urethane (0.8 g / kg, ip 0.4 g / I had an incision in the middle. The upper part of the bladder was slightly incised and a polyethylene tube (SP-45: Natsume Kogyo Co., Ltd.) was inserted into the bladder. The incision was ligated and the end of the tube inserted into the bladder was connected to a transducer (MLT0670 Disposable BP Transducer: ADINSTRUMENTS).

Using the apparatus shown in Fig. 10, the bladder pressure and the urination volume were measured with time. The rats were fixed with a bolt gauge (acrylic special production: Yamashita Kigen (Yu)) with supine. A dedicated syringe (10 mL) containing saline or 0.1% acetic acid solution was placed in a continuous injector (model 100 series: Kd scientific). (BP Amp: ADINSTRUMENTS) was continuously injected with physiological saline or 0.1% acetic acid solution into the bladder at 3 mL / hr through a water bath set at 37 ° C (THEAMO MINDER SJ-10R TYTECH) And recorded bladder pressure curves with data recording and interpretation software (Power Lab 4/26: ADINSTRUMENTS). In addition, the urine volume was measured by installing a chaarele on an upper dish electronic balance (GX-400, AD Co., Ltd., A & D), cumulatively collecting the liquid discharged from the urethral meatus, and recording the change in weight with Power Lab 4/26 . After the urination was stabilized, the urination parameters were measured before the administration for 30 minutes. Thereafter, 50 mg / kg of SE was orally administered, measurement was started again from 1 hour after administration, and data of 30 minutes thereafter was used as urination parameters after administration.

As shown in Fig. 11, oral administration of Shikuwasa extract (50 mg / kg) significantly increased urination volume and significantly decreased urination frequency in the CYP-induced cystitis model rat. In addition, the maximum bladder pressure decreased. From these results, it was revealed that Shikuwasa extract also regulates urination and improves urinary frequency in drug-induced cystitis model rats.

< Example 9 >

Shikawa  The Ca Antagonist drug  Binding activity to the receptor

Receptor Feature  pharmacy

SD rats were sacrificed under isoflurane anesthesia and blood was slaughtered from the abdominal descending aorta and the brain was harvested. The extracted brain removed the cerebellum. For measurement of (+) - [ ³ H] PN200-110, 30 mM Tris-HCl buffer (pH 7.2 or 7.5) was added to the brains after the extraction in an amount of 19 times and homogenized with a polytron homogenizer. The homogenate was centrifuged (4 ° C, 40,000 × g, 20 min), and the precipitate was suspended in a 32.3-fold buffer and used as a receptor film.

A 10-fold volume of homogenizing buffer (50 mM Tris, 1 mM EDTA, 2 μg / mL Soybean trypsin inhibitor, 10 μg / mL Bacitracin: pH 7.4) was added to the brain to measure [ ³ H] αβ-methylene ATP Homogenized with a Tron Homogenizer. The homogenate was centrifuged (4 ° C, 2,000 × g, 10 min), and the supernatant was collected. The precipitate was resuspended in a 10-fold volume of homogenizing buffer, centrifuged under the same conditions, and the supernatant was collected. The supernatant was collected and incubated at 37 ° C for 20 minutes, centrifuged (4 ° C, 40,000 × g, 20 min), and the precipitate was loaded in a 19-fold volume of incubation buffer (50 mM Tris, 3 mM CaCl _2, pH 7.5) Suspended, and made into a receptor membrane.

1,4- Dihydropyridine ( DHP ) Ca Antagonist drug  Measurement of receptor binding activity

Measurement of 1,4-DHP Ca antagonist receptor binding activity was followed by radio receptor assay using (+) - [ ³ H] PN200-110 as the labeled ligand. That is, the receptor product was incubated with Shikuwasa extract (SE) and 0.3 nM (+) - [ ³ H] PN200-110 in 50 mM Tris-HCl buffer (pH 7.4) at 25 ° C for 60 minutes under light shielding conditions. After incubation, the reaction solution was rapidly aspirated on glass fiber paper, and the filter paper was washed with 50 mM Na ⁺ / K ⁺ phosphate buffer (pH 7.5). The filter paper was dried, placed in a vial bottle, added with a toluene scintillator, allowed to stand at room temperature for about 6 hours, and its radioactivity was measured to calculate the specific binding amount. At this time, 1 μM nifedipine was used as a displacer. All measurements were carried out in duplicate.

result

Shikawa  Extract of calcium Antagonist drug  Binding to the receptor

The binding of (+) - [ ³ H] PN200-110, which is a ligand to the 1,4-DHP Ca antagonist drug receptor, was slightly inhibited by the Shikuwasa extract (Fig. 12). From these results, the Shikuwasa extract has activity of binding to the calcium receptor antagonist binding site, and calcium antagonistic action can be expected. Since calcium causes bladder smooth muscle contraction, it is suggested that Shikuwasa extract improves urinary frequency and urinary incontinence for overactive bladder through smooth muscle relaxation by calcium antagonism.

< Example  10>

Rat  For Extraction Bladder Novyletin  And Tangerine  Action

1. Reagents

Atropine and Tetrodotoxin were purchased from Sigma-Aldrich Co. (St Louis, MO, U.S.A.). Pyridoxylic acid phosphate-6'-azophenyl-2 ', 4'-disulfonic acid tetrasodium salt (PPADS; a selective P2X antagonist) was purchased from Tocris Bioscience (Minneapolis, , MN, USA). Novartin and tangeretin were extracted and purified from Shikuwa Co. 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 experiments. All animals were housed under constant temperature (22 ± 2 ℃), humidity (55 ± 2%) and normal illumination (7 ~ 19 hours). In addition, animal experiments were conducted in compliance with the animal care law and in accordance with the "Mukogawa Women's University Animal Experimental Regulations".

Pentobarbital (60 mg / kg, intraperitoneal injection) Wistar male rats under deep anesthesia were bled off and bladder was removed and oxygen (95% O ₂ and 5% CO ₂₎ in which the air passage 37 ℃ Krebs-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), 4 sections (3 x 5 mm) were made from the upper part of the bladder and used as a sample.

3. Shrinkage measurement by electrical stimulation

The specimen was mounted on a platinum electrode in a magnus tube filled with oxygen-permeated Krebs-Henseleit solution (5 mL, 37 ° C) and hanged under the condition of a static tension of 1 g. The specimens were stabilized at equilibrium for 30 min, washed 5 times with Krebs-Henselei solution every 3 min, and subjected to transmural electrical stimulation (10 Hz, 0.1 msec duration, 10 V, 10 sec) The resulting shrinkage response was measured.

A power transducer (T7-15-240, A & D Co. Ltd., Tokyo, Japan) was used for measurement, and Power Lab (registered trademark) software (ADInstruments Pty Ltd, CO, USA) was used for data acquisition and analysis . The wall electrical stimulation was applied to the specimen at 5 minute intervals. After 2 minutes of the parietal electrical stimulation, either Novartin (final concentration 10 ^-4 M) or tangentretin (final concentration 10 ^-4 M) or an equal volume of ethanol was administered as a control and 3 minutes later, The shrinkage reaction was measured by 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) , And the effect on the electrical stimulation of the wall. The flow of the wall electrical stimulation is shown in Fig. The contraction inhibited by atropine is shrinkage of cholinergic activity and desensitization by repetitive addition of?,? -Methylene ATP. Purine retraction is suppressed by purine retraction, retraction suppressed by tetrodotoxin is restrained by non-choline, , And the contraction not inhibited by tetrodotoxin was defined as the resistance to tetrodotoxin.

Statistical analysis method

The data obtained are shown as mean ± standard deviation. A significant difference test was used with Dunnett's test and a significance was determined at a risk of less than 5%. Also, GraphPad Prism 4.03 software (GraphPad Software, La Jolla, CA, USA) was used for data analysis.

5. Experimental results

For shrinkage by the parietal electrical stimulation, both Novartin (10 ^-4 M) and Tangeretin (10 ^-4 M) all exhibited inhibitory effects with inhibition rates of about 30% or about 19%, respectively (FIG. 14). In order to clarify the involvement pathway in the shrinkage reaction by this wall electrical stimulation, various compounds were used to further study. As shown in Fig. 14, when the contraction reaction was in control (control) of about 100% of the co-existence of a weak sense of to about 68%, α, β-methylene ATP (10 -6 M) in the presence atropine (10 ^-6 M) To about 5% (Fig. 14A). As shown in Fig. 14B, the shrinkage reaction observed in the presence of novilectin declined to about 70% and decreased to about 32% in the presence of atropine, to about 2% in the presence of a, b-methylene ATP. In the same order, about 81% in the presence of tancretin, about 42% in the presence of atropine, and about 3% in the presence of α, β-methylene ATP (FIG. In contrast, the contraction of tetrodotoxin resistance left in the presence of tetrodotoxin (10 ^-6 M) was only about 1% of the shrinkage response due to the electrical stimulation of the pore wall (data not shown).

Novartin and tangretin also showed significant inhibitory effects on shrinkage models due to parietal electrical stimulation in the excised bladder. From these results, it was suggested that Novartin and Tancretin inhibit the contraction of the bladder, thereby improving the urination disorder.

In addition, the shrinkage response due to the electrical wall stimulation was divided into cholinergic shrinkage inhibited by atropine and purine operative shrinkage stimulated by α, β-methylene ATP repeatedly. As a result, , The cholinergic shrinkage was about 32% and the purine operative shrinkage was about 63%, suggesting that the purine operative component is involved in the shrinkage reaction by the electrical wall stimulation.

On the other hand, in experiments conducted with novilectin, the cholinergic shrinkage was about 38% and the purine operative shrinkage was about 30%. In the tangentretin-treated experiment, the cholinergic shrinkage was about 39% % (Fig. 15). Taking the above results into consideration, it has been found as one possibility that the components inhibited by novirretin and tangretin during the contraction reaction by electrical stimulation of the parietal wall in the excised bladder are mainly purine operable components. From the above results, it has been suggested that the nobiletine and tangentetin also affect the purine operative shrinkage, thereby improving the urination disorder.

From the results of the present example, it has become clear that nobiletin, tangretin, 3 ', 4', 5, 7-tetramethylquercetin and cinnensetin have an antagonistic action on muscarinic receptors. In addition, since novilectin has an effect of improving urinary frequency, it is possible to prevent, ameliorate, or treat urination disorders by using nobiletin.

The present invention has been described above by way of examples. It is to be understood that the embodiments are merely illustrative and that various modifications are possible and that those skilled in the art understand the scope of the present invention.

Acknowledgments

"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."

Minor Redevelopment Project of Bio-formula of Agricultural Name (Korea Rural Development Administration)

Claims (13)

A pharmaceutical composition for preventing or ameliorating dysuria characterized by containing a compound represented by the following formula (I).

(Wherein (I), R1 is hydrogen or hydroxyl, R _2, R _3, R ₄ , R ₅ and R ₆ are each independently hydrogen or -OCH ₃ , and R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or -OCH ₃ , The method according to claim 1,
The compound represented by the formula (I) is a compound selected from the group consisting of the following formulas (II) to (V).

And

3. The method according to claim 1 or 2,
Wherein said urination disorder is overactive bladder and / or hyperplasia of the prostate. 4. The method according to any one of claims 1 to 3,
Wherein the urination disorder is accompanied by one or more symptoms selected from the group consisting of urinary frequency, nocturia, urinary incontinence, restlessness and urinary incontinence. A receptor antagonist comprising a compound represented by the formula (I)
Wherein said compound exhibits antagonism to a receptor associated with dysuria.

(In the formula (I), R ₁ is hydrogen or a hydroxyl group, and R ₂ , R ₃ , R ₄ , R ₅ and R ₆ is independently hydrogen or -OCH ₃ , and R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen or -OCH ₃ ) 6. The method of claim 5,
Wherein said dysuria-related receptor is selected from the group consisting of muscarinic receptors, calcium receptors and purine receptors. The method according to claim 5 or 6,
A receptor antagonist which is used to prevent or ameliorate dysuria. 8. The method according to any one of claims 5 to 7,
Wherein said urinary disturbance is overactive bladder and / or hypertrophy of the prostate. 9. The method according to any one of claims 5 to 8,
Wherein said urination disorder is accompanied by one or more symptoms selected from the group consisting of frequency, nocturia, urinary incontinence, restlessness and urinary incontinence. 10. The method according to any one of claims 5 to 9,
A receptor antagonist comprising citrus extract containing the above formula (I). 11. A therapeutic agent for urinary disturbance disorder, which comprises the receptor antagonist according to any one of claims 5 to 10. A functional food for preventing or ameliorating a dysuria characterized by containing the compound according to any one of claims 1 and 2. A method for preventing or ameliorating dysuria, the method comprising:
Comprising administering to a subject suffering from the dysuria, a pharmaceutical composition or a receptor antagonist comprising a compound represented by the following formula (I)
Wherein said compound exhibits an antagonistic effect on a receptor associated with dysuria.

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

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