WO2017160128A1 - Composition for activation of bkca channels - Google Patents

Abstract

The present invention relates to a composition capable of activating BK_Ca channels, containing an extract of Sophora flavescens as an active ingredient. The composition of the present invention can effectively activate BK_Ca channels and can be used in the prevention or treatment of various diseases caused by an inactivation or an activation decrease of BK_Ca channels.

Description

Composition for activating BKCA channel

The present invention relates to a composition capable of activating a BK _Ca channel comprising an extract of Sophora flavescens as an active ingredient.

Large conductance Ca ^{2 +} - activity K ⁺ channels (BK _Ca channels) are membrane activated by polarization and / or intracellular Ca ^{2 +,} to pass across the cell to K ⁺ ion film (Cui et al 2009; Yang et . al. 2015). BK _Ca channels are widely expressed in various types of excitable and nonexcitable cells, neurotransmitter release (Raffaelli et al. 2006), smooth muscle contraction (Brenner et al. 2000; Herrera et. al. 2000), and the regulation of some important physiological processes, including periodic behavioral rhythms (Meredith et al. 2006). Dysfunction of the BK _Ca channel is epilepsy (Lorenz et al. 2007; Du et al. 2005), erectile dysfunction (Werner et al. 2005) and overactive bladder (OAB) (Meredith et. al. 2004).

OABs are generally characterized by the presence of urinary urgency with increased day or night time frequency (Cerruto et al. 2012). OAB affects approximately 17% of the Western population, both men and women, and increases with age (Coyne et al. 2013). There are several classes of drugs for OAB targeting different receptors in the bladder, including anti muscarinic agents, mixed-action drugs and β-adrenergic receptor agonists (Abraham et al. 2015). Although the center of OAB treatment is anti-muscarin pharmacology, side effects and decreasing efficacy are responsible for the long-term compliance problem (Jayarajan et al. 2013). Therefore, there is a great need for new therapeutic treatments for OAB that directly target urinary bladder smooth muscle (UBSM) and have fewer side effects. Among therapeutic targets injured for OAB, bladder K ⁺ channels showed great potential in preclinical experiments. However, until now activators or initiators of clinically studied K ⁺ channels have yielded disappointing results (Andersson et al. 2013).

The BK _Ca channel is one of the most physiologically important K + channels and regulates UBSM function in health and disease (Petkov et al. 2014). BK _Ca channels are highly expressed in UBSM (Hristov et al. 2011). By a membrane polarization and intracellular Ca ^{2 +} by both the unique activation, BK _Ca channels activate is the base of the first repolarization phase of the spontaneous action potential to trigger a maintenance and UBSM homology shrinkage (phasic contraction) of the stabilizing membrane potential . Moreover, a series of studies the BK _Ca channel cholinergic and purinergic - showed that there were reports that an important role in reducing the induced contractility (contractility), BK _Ca channel expression or changes the function can contribute to OAB generation ( Werner ME et al 2007). Therefore, induction of UBSM relaxation by chemical activation of endogenous BK _Ca channels is possible. In fact, several activator compounds of the BK _Ca channel have been reported for their bladder relaxation effects (dela Pena et al. 2009; Layne et al. 2010; Ahn et al. 2011; La Fuente et al; Park et al. 2014) Furthermore, the possibility has been suggested as a therapeutic target for OAB syndrome. However, the efficacy and specificity of BK _Ca channel activators remained a problem for their clinical use (Nardi et al. 2006; Bentzen et al. 2014). Therefore, there is a need to discover novel BK _Ca channel activators with high specificity and better efficacy.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors earnestly researched to find a composition for activating BK _Ca channel including a natural extract component. As a result, by identifying that the ginseng extract has BK _Ca channel activation ability, the present invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating BKCa channel activity lowering-related conditions, diseases or diseases, comprising high ginseng extract as an active ingredient.

Another object of the present invention is to provide a functional food composition for improving the symptoms of incontinence, bladder overactivity and erectile dysfunction.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a BKCa channel activity lowering-related condition, disease or condition, comprising high ginseng extract as an active ingredient.

The present inventors earnestly researched to find a composition for activating BK _Ca channel including a natural extract component. As a result, it was confirmed that the ginseng extract has BK _Ca channel activation ability.

We explored novel natural activators of BK _Ca channels using newly developed cell-based assays. Since the mutant BK _Ca channels used in this assay exhibited significantly improved Ca ²⁺ sensitivity, it was not necessary to increase intracellular Ca ²⁺ additionally for channel activation (Lee et al. 2013). Therefore, we were able to use commercially available thallium (Tl ⁺ ) -based fluorescence analysis for voltage active K ⁺ channels. By exploring the natural extracts, we were able to discover novel BK _Ca channel activators including the red ginseng extract.

"Sophora flavescens" of the present invention is a perennial grass belonging to the legumes, although not particularly limited, may be used in a dried form.

The term ginseng extract herein is used in various extraction solvents, such as (a) water, (b) anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohols; A mixed solvent with water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) 1,3-butylene glycol and (h) butyl acetate can be obtained as an extraction solvent. Preferably, the extract of the present invention is obtained by using water and / or ethanol as the extraction solvent, and as a specific example, 70% ethanol can be obtained as the extraction solvent. On the other hand, it will be apparent to those skilled in the art that the extract of the present invention can obtain an extract having substantially the same effect using not only the extraction solvent but also other extraction solvents.

In one embodiment of the present invention, the ginseng extract of the present invention is a polar organic solvent extract.

In one embodiment of the invention, the polar organic solvent of the invention is (a) water, (b) anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, or mixtures thereof.

As used herein, the term "extract" has a meaning commonly used as a crude extract in the art as described above, but broadly includes a fraction additionally fractionating the extract. That is, the ginseng extract of the present invention includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the extract of the present invention. In addition, the extract of the present invention includes those prepared in powder form by an additional process such as vacuum distillation and freeze drying or spray drying.

As used herein, the term “decreased BK _Ca channel activity-related condition, disease or disease” means that the activity of the BK _Ca channel is lowered or substantially inactivated relative to normal levels, and thus controlled by BK _Ca channel activity. Degradation of important physiological functions such as neuronal excitability, neurotransmitter secretion, smooth muscle cell contraction, and frequency tuning of hair cells, resulting in a disease or condition do.

In one embodiment of the present invention, the BK _Ca channel activity lowering-related condition, disease or condition of the present invention is cardiovascular disease, obstructive or inflammatory airway disease, lower urinary tract disorders, erectile dysfunction , Anxiety and anxiety-related conditions, epilepsy or pain.

Membrane depolarization (membrane depolarization) and intracellular Ca ^{2 +} concentrations increase BK or calcium having a large conductive known as Maxi-K channels of the - activated potassium channel (large-conductance calcium-activated potassium (BK Ca) channels) to activate Salkoff, et al., 2006; Cui, et al., 2009). These channels are known to perform important physiological functions in neuronal excitability, neurotransmitter secretion, smooth muscle cell contraction, and frequency tuning of hair cells (Brenner, et al., 2000; Nelson, et al., 1995; Fettiplace and Fuchs, 1999). BK _Ca channels consist of a pore-forming α-subunit and a regulatory β-subunit. Α- subunit of the BK _Ca channel is composed of seven-pass membrane domain (Catterall, 1995) C- terminus K ⁺ conductivity; to include two regulatory elements for controlling the (K ⁺ conductance domain RCK) domain it forms a ring gate (gating ring) responsive to the Ca ^{2 +} concentrations within cells (Jiang, et al., 2001 ). BK _Ca channels are known to be therapeutic targets closely related to hypertension, coronary artery spasm, urinary incontinence and many neurological diseases (Ghatta, et al., 2006). Mice lacking BK _Ca channels are known to exhibit symptoms such as incontinence, bladder overactivity and erectile dysfunction (Meredith, et al., 2004; Werner, et al., 2005). It is known that these diseases can be prevented or treated by restoring or activating them. It is also known that dysfunction of the BK _Ca channel can lead to cerebellar ataxia and paroxysmal movement disorders, and can be a therapeutic target for these diseases (Lee and Cui, 2010). Activation of BK _Ca channels stabilizes cells by increasing K ⁺ efflux and causing hyperpolarization. Thus, substances that open or enhance the activity of BK _Ca channels may confer therapeutic benefits that reduce intracellular excitability and relieve tension in smooth muscle cells.

As used herein, the term "cardiovascular disease" is a general term used to classify a number of conditions that affect the vasculature of the heart, heart valves, blood, and the body, and diseases affecting the heart or blood vessels. Include them.

Cardiovascular diseases of the present invention are atherosclerosis, atherothrombosis, atherosclerosis, coronary artery disease, ischemia, reperfusion injury, hypertension, restenosis, arterial inflammation, myocardial ischemia or ischemic heart disease, stable and unstable angina, stroke, congestive heart failure Aortic diseases such as aortic stenosis or aortic aneurysm and peripheral vascular disease. The term “peripheral vascular disease (PVD)” herein refers to diseases of the heart and central nervous system vessels often encountered upon narrowing of the vessels of the extremities, for example, blood vessels are free of defects, but not cold, stress or Functional diseases resulting from stimuli such as smoking, and organic diseases resulting from structural defects of the vascular system such as atherosclerosis lesions, local inflammation or traumatic injury.

Obstructive or inflammatory airway diseases of the present invention include airway hyperresponsiveness, pneumoconiosis, aluminumemia, carbonosis, asbestosis, septicemia, ptilosis, siderosis, silicosis, tobacco poisoning, byssinosis Sarcoidosis, beryllium disease, emphysema, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), acute or chronic infectious lung disease, chronic obstructive pulmonary disease (chronic) obstructive pulmonary disease (COPD), bronchitis, chronic bronchitis, whiskey bronchitis, exacerbation of airway hyperreactivity or cystic fibrosis, or cough including chronic cough, exacerbation of airway hyperreactivity, pulmonary fibrosis, pulmonary hypertension, inflammatory lung disease And diseases of acute or chronic respiratory infections.

The term "lower urinary tract disease" herein includes all lower urinary tract diseases characterized by irritable bladder with or without urinary, frequent, urinary, and nocturia. Thus, the lower urinary tract disorders of the present invention are hypersensitive bladder such as overactive bladder, overactive detrusor, unstable bladder, detrusor hyperreflexia, sensory urgency, and symptoms of detrusor hyperactivity. urinary bladder, urinary incontinence or urge urinary incontinence, urinary stress incontinence, slow urination, dribbling, urination, and / or pressure to urinate at an acceptable rate Lower urinary tract disease symptoms, including obstructive urination as needed, and symptoms such as frequent urination and / or urination. In addition, lower urinary tract diseases may include neurogenic bladder resulting from neurological damage, including but not limited to stroke, Parkinson's disease, diabetes, multiple sclerosis, peripheral neuropathy, or spinal cord injury. have. Lower urinary tract diseases may also include spastic bladder in patients with prostatitis, interstitial cystitis, prostatic hyperplasia, and spinal cord injury. According to certain embodiments of the present invention, the lower urinary tract disease of the present invention may include: an overactive bladder, an unstable bladder, overactive detrusor muscle, detrusor instability, detrusor hyperreflexion, sensory urgency, urinary incontinence, urinary incontinence, stress incontinence, nerves Relfex urinary incontinence, slow urination, late dropping, dysuria and spastic bladder.

The term “erectile dysfunction” herein is a persistent inability to acquire or maintain an erection sufficient to perform sexual intercourse, which is closely associated with endothelial cell dysfunctions.

Diseases, disorders or conditions associated with the modulation of BK _Ca channels of the invention include pain disorders; Generalized anxiety disorder, anxiety panic, obsessive compulsive disorder, social phobia, performance anxiety, posttraumatic stress disorder, acute stress response, adjustment disorder, hypochondriacal disorder, Anxiety and anxiety-related conditions such as separation anxiety disorders, agoraphobia, and certain phobias; And simple partial seizures, complex partial seizures, secondary generalized seizures, absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic seizures epilepsy such as generalized seizure, including but not limited to tonic clonicseizure and atonic seizure.

In addition, anxiety associated with certain phobias includes, but is not limited to, animals, insects, storms, driving, flying, height or leg crossings, closed or confined spaces, water, blood or wounds, as well as injection or surgical medical and dental processes It doesn't happen.

Pain disorders are also disorders with pain, for example acute pain such as musculoskeletal pain, post operative pain and surgical pain; Chronic inflammatory pain (eg, rheumatoid arthritis and osteoarthritis), neuropathic pain (eg, post herpetic neuralgia, trigeminal neuralgia and sympathetically maintained pain) and cancer Chronic pain such as associated pain and fibromyalgia; Pain associated with migraine headaches; Pain (both chronic and acute) and / or fever and / or infections in conditions such as rheumatic fever; Symptoms associated with other viral infections, such as influenza or the common cold; Lower back pain and neck pain; headache; toothache; Sprains and strains; Myositis; Neuralgia; Fasciitis (synovitis); Arthritis, including rheumatoid arthritis; Degenerative joint diseases, including osteoarthritis; Gout and ankylosing spondylitis; Tendinitis; Bursitis; Skin related conditions such as psoriasis, eczema, burns and dermatitis; Sports injuries; And injuries such as those caused by surgical and dental procedures.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered topically to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like. .

The pharmaceutical compositions of the present invention may be administered orally, and solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, which solid preparations contain at least one compound of the present invention. One or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions or syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.

Formulations for oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. Conventional formulations include, for example, oral (tablets, capsules, powders), oral cavity, sublingual, intrarectal, intravaginal, intranasal, topical or parenteral (intravenous, cavernous, intramuscular, subcutaneous). And dosage forms). For example, the compounds according to the invention may be in the form of tablets containing starch or lactose, in the form of capsules alone or in the form of excipients, or in the form of elixirs or suspensions containing chemicals which flavor or color. It can be administered orally, orally or sublingually. Liquid formulations may be suspending agents (eg, methyl cellulose, semisynthetic glycerides such as witepsol or mixtures of apricot kernel oil with PEG-6 esters or PEG-8 with caprylic / capric glycerol). Pharmaceutically acceptable additives such as glyceride mixtures such as mixtures of lides). It is also most preferred to use it as a sterile aqueous solution when injected parenterally, for example, intravenously, intracavernosally, intramuscularly, subcutaneously, and intratracheally, wherein the solution is to have isotonicity with blood. It may also contain other substances (eg salts or monosaccharides such as mannitol, glucose).

In one embodiment of the invention, the composition of the present invention shifts the conductivity-voltage (GV) correlation of the BK _Ca channel in the negative voltage direction. Moving the conductivity-voltage correlation in the negative voltage direction indicates that the BK _Ca channel is activated by stabilizing the open shape of the channel.

According to another aspect of the present invention, the present invention provides a functional food composition for improving the symptoms of incontinence, bladder overactivity, and erectile dysfunction including high ginseng extract as an active ingredient.

Functional food compositions of the present invention include ingredients that are commonly added in the manufacture of food, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating BKCa channel activity lowering-related conditions, diseases or diseases, comprising high ginseng extract as an active ingredient.

(b) The present invention provides a functional food composition for improving the symptoms of incontinence, bladder overactivity and erectile dysfunction.

(c) With the composition of the present invention, it is possible to effectively activate the BK _Ca channel, it can be used for the prevention or treatment of improving, or a variety of diseases of various symptoms caused by the BK _Ca channel is inactive or deactivation.

Figure 1 shows the effect of BK _Ca channel activity of the extract of the ginseng extracted with 70% ethanol using a cell-based fluorescence assay. BK _Ca channel activity of the Ginseng extract was used for Tl ⁺ -based fluorescence (FluxOR ^™ ) analysis of AD293 cells stably expressing overactive mutant BKCa channels (G803D / N806K). Fluorescence change in the treatment of 10-1000 μg / mL Ginseng extract was expressed as a relative fluorescence unit (RFU). Extracts of each concentration were transferred to each test-well at 5 μM final concentration before the experiment and stimulus buffer was added at 120 seconds. DMSO (1%, vehicle) and CTBIC, a BK _Ca channel activator, were used as comparators.

Figure 2 shows the concentration-dependent increase in the fluorescence signal after treatment with curarinone, the active ingredient of red ginseng extract. AD293 cells stably expressing mutant BK _Ca channels were treated with different concentrations of curarinone. 3A shows representative fluorescent traces. After baseline was obtained for 20 seconds, the Tl ⁺ -containing stimulation buffer was treated. Cells were cultured in the presence of 1% DMSO (□) or other concentrations of curarinone (■: 3 μΜ, ●: 5 μΜ, ▲: 10 μΜ, ▼: 30 μΜ) as vehicle. Cells were also incubated with 1 μM of paxilline, a BK _Ca channel blocker, with curarinone (5 μM) (◆). 3B shows the initial increase in RFU at different concentrations of curarinone. Error bars (SEM) are shown. Inset shows the chemical structure of curarinone.

Figure 3 shows the results of comparing the activity of the ginseng extract and the curarinone single compound. FluxOR ^™ analysis was performed on 50 μg / mL and 100 μg / mL 70% ethanol extracts and 10 μM curarinone, respectively, to compare fluorescence increase. The concentration of the ginseng extract showing a fluorescence increase similar to 10 μM curarinone was confirmed.

4 shows the reversible enhancement of curarinone of macroscopic BK _Ca channel currents. Representative diary-plots of tail currents induced by BKCa channels are shown as continuous recordings. Ion currents were recorded every second with a 50-ms step-pulse of 100 mV from a holding voltage of -100 mV. Current was obtained at a specific point (0.8 ms after 100 mV voltage pulse). Each representative current trace ad represents the current at the point indicated by the arrow.

5 shows the effect of curarinone on the current-voltage and conductivity-voltage relationships of the macroscopic BK _Ca channel current. FIG. 5A shows a representative trace of BK _Ca channel current at 3 μM [Ca ²⁺ ] _i at different curarinone concentrations. Ion currents were induced with 100-ms voltage step-pulses. The current was recorded in 10 mV increments from -80 mV to 200 mV. The holding voltage was -100 mV. 5B shows the effect of curarinone on the conductivity-voltage (GV) relationship. Conductivity was obtained from peak-tail current. The current was normalized by the maximum current of the vehicle trace. Each symbol represents conductivity at different curarinone concentrations: vehicle (■, n = 12), 3 μM (•, n = 8), 5 μM (▲, n = 12), 10 μM (▼, n = 8) and 20 μΜ (◆, n = 4). 5C shows the effect of curarinone on half-maximum voltage (V _1/2 ). Each symbol on the graph represents the SEM and mean of V _1/2 . Results were obtained by fitting all independent data sets using the Boltzmann function (P ₀ = [1 / (1 + exp {(V _1/2 -V) / k}]).

6 shows the effect of curarinone on activation and deactivation of macroscopic BKCa currents. 6A and 6B represent representative traces of activation (A) and inactivation (B) when treated with vehicle (black) or 20 μM of curinone (grey). The current traces obtained at 100 mV were compared. 6 C and D show the activation and deactivation time-constant values τ at different concentrations of curarinone. Symbols are vehicle (□, n = 12), 3 μM (■, n = 8), 5 μM (●, n = 12), 10 μM (▲, n = 8) and 20 μM (◆, n = 4) Indicates. Time-constant values were obtained from fitting all independent data sets using the exponential standard function (y (t) = A ₁ exp (−t / τ ₁ ) + C) using the Clampfit program.

7 shows the effect of curarinone on a single BK _Ca channel. Each graph in A of FIG. 7 shows a typical single-channel current recording of BK _Ca channels at different film voltages. The concentration of intracellular Ca ^{2 +} (pipette Ca ^{2 +} concentration) was fixed at 10 μM. The current was initially recorded in the absence of curarinone at different voltages and then in the presence of curarinone (5 μM). Curarinone solution was spread on the extracellular side. Solid lines represent closed levels of single BK _Ca channels and dashed lines represent open levels. 7B shows the effect of curarinone on single-channel conductivity. The urinary current-amplitude of the channel was measured in Ca ²⁺ solution in 10 μM cells. After recording the channel current for the first time in the absence of Curarinone (○), 5 μM of Curarinone (●) was sparged. The film voltages were 75 mV (n = 4), 50 mV (n = 5), 25 mV (n = 5), -25 mV (n = 3), and -50 mV (n = 2), respectively. Each data preset on the graph was obtained from all point amplitude histograms fitted using a Gaussian function. The conductivity of a single channel was estimated using the slope fitted as a linear function. FIG. 7C shows the effect of curarinone on the voltage-dependent open-pore (P ₀ ) of a single BK _Ca channel. Opening probability was measured using a trace as in B of FIG. 7. The increase in cell membrane dependent open probability of a single BK _Ca channel was measured in the absence of curarinon (○) and 5 μM curarinone treatment (●), and the data points were measured by the Boltzmann function (P ₀ =). [1 / (1 + exp {(V _1/2 -V) / k}]) The D in FIG. 7 shows the absence (top trace) and the presence (bottom trace) of 5 μM Curranone at 50 mV. Representative single-channel currents of BK _Ca channels on C. Figure 7 E shows the average open-time of a single BK _Ca channel on the absence (empty bars) and presence (filled bars) of 5 μΜ curranone at 50 mV. And the effect of curarinone on mean closure time, each bar graph shows mean ± SEM (n = 5).

8 shows the effect of curarinone on ACh induced contraction in isolated rat bladder strips. FIG. 8A shows representative contraction traces induced by ACh with and without preincubation of curarinone. FIG. 8B shows the percent percent relaxation induced by curinone on ACh-induced contraction. Each bar graph represents the mean ± SEM of 6 experiments. Black represents control, white represents curarinone treatment experiment.

9 shows the effect of curarinone on urination behavior in rats. 9A shows the effect of curarinone on the frequency of urination of WKY and SHR after intraperitoneal injection of curarinone (0.5 and 5 mg / kg). The frequency of urination was observed for 3 hours. 9B shows the total number of urinations. Each symbol or bar represents the mean ± SEM of 5 (WKY) or 7 (SHR) animals ( ^*** P <0.001).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example 1: Experimental Materials

Water or 70% ethanol was added to 200 g of dried ginseng (Sophora flavescens), and the mixture was extracted in a flask with a reflux condenser and extracted at 90 ° C. for 3 hours in a water bath. The extract was filtered using filter paper and concentrated using a rotary vacuum evaporator. The above method was repeated two more times, and the solvent was sufficiently removed and lyophilized to obtain 20 g of red ginseng water extract and 24 g of 70% EtOH extract.

Curarinone and its derivatives, the active ingredient of the ginseng extract, were purified from dried ginseng (Kushen) of Sophora Flavescens (Jung et al. 2008). Curarinone and other compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) as stock solution. 4-Chloro-7- (trifluoromethyl) -10H-benzofuro [3,2-b] indole-1-carboxylic acid (CTBIC) was also dissolved in DMSO.

Example 2: Cell Culture

Modified HEK293 cells expressing mutant BK _Ca channels (Lee et al., 2013) were supplemented with DMEM (Dulbecco's) supplemented with 10% fetal bovine serum and antibiotic 1 mg / ml Geneticin (Gibco). Modified Eagle's medium. Cells were incubated at 37 ° C., 5% CO ₂ humidified conditions.

Example 3: Fluorescence Analysis and Data Analysis

AD-293 cells stably expressing the mutant BK _Ca channel (G803D / N806K) were used for cell-based analysis (Lee et al., 2013). Approximately 20000 cells / well were placed on a black-wall assay plate (Corning Incorporated) coated with poly-D-lysine (Sigma-Aldrich), a 96-well clear-bottom. Inoculation. FluxOR ^™ calcium channel analysis (Invitrogen) was used to analyze the efficacy of gourd extract and curarinone, the active ingredient of the extract. Experiments were performed according to the manufacturer's following instructions: Growth medium was replaced with 80 μl / well of loading buffer containing FluxOR ^™ fluorescent dye and incubated for 1 hour in the absence of light. After incubation, the loading buffer was replaced with 100 μl / well of assay buffer containing various concentrations of compounds of interest and incubated for 20-30 minutes.

100 mg of 70% ethanol extracts of red ginseng were dissolved in 1 ml of DMSO to a concentration of 100 mg / ml. The solution was further diluted in DMSO and used to ensure that the ratio of DMSO to the final buffer did not exceed 1% of the final buffer. For example, for 1000 μg / ml samples, 100 mg / ml extract solution was diluted 1/100 by adding 4 μl to 400 μl final buffer. For 100 μg / ml samples, 100 mg / ml extract solution was used. A 10 mg / ml extract solution diluted 1/10 with additional DMSO was added by diluting 1/100 by adding 4 μl to 400 μl of the final buffer. DMSO (1%) is the vehicle and used for all test compounds. CTBIC (Cormemis et al. 2005; Lee et al. 2012) previously identified as an activator of BK _Ca channel was used as a positive control. For fluorescence measurements, SynergyTM H1 hybrid multi-mode microplate reader (BioTek Instrument Inc., Winnoski, VT) and Cen5 software were used for initial screening and Flexstation 3 multi-mode microplate reader (Molecular Devices) for further analysis. and it was used softMax ^® Pro software, respectively. Fluorescent signals were obtained at excitation wavelength of 485 nm and emission wavelength of 528 nm. Membrane polarization occurred with a stimulus buffer containing thallium ions. The fluorescence signal was measured in two states: every 10 seconds for 2 minutes before the stimulation buffer and then every 10 seconds for 3 minutes after the addition of the stimulation buffer for synergy H1 and every 2 seconds for 20 seconds before the stimulation buffer. Seconds and then every 2 seconds for 160 seconds after adding stimulation buffer for FlexStation 3.

The change in fluorescence signal is expressed in relative fluorescence units (RFU or F / F ₀ , where F ₀ is the minimum fluorescence value of each fluorescence trace). To quantitatively compare the effect of BK _Ca channel activation of curarinone and its derivatives, the initial fluorescence increase was calculated using the first three points after treatment with the stimulation buffer, and the linear slope was OriginPro 9.1 (OriginLab Corp., Northampton, MA). Predicted using.

[Example 4: Comparison of FluxOR ^™ Analysis Results between Ginseng Extract and Active Ingredient Curarone Single Extract]

In order to compare the efficacy of Ginseng extract and single active extract of Curarinone, the concentrations of the extracts of Red Ginseng showing similar fluorescence increase as 10 μM Curarinone were identified. FluxOR ^™ analysis was performed on 50 μg / mL and 100 μg / mL 70% ethanol extracts and 10 μM curarinone, respectively, to compare fluorescence increase.

Example 4 Functional Expression of Replicated BK _Ca Channels in Xenopus Oocytes

Xenopus laevis oocytes heterologously expressing BK _Ca channel α-subunit (Slo1) were used for electrophysiological recordings. Subcloning and functional expression of rat BK _Ca channel α-subunits using oocyte expression vector pNBC1.0 has been previously reported (Ha et al., 2000). Sequence information of Slo1 used in the present invention is disclosed in GenBank as expression number AF135265. Plasmid DNA was linearized using NotI restriction enzymes and complementary RNA (cRNA) from the linear form of DNA using the mMessage Machine (Ambion) of nucleoside triphosphate and cap analog m7G (5 ') ppp (5') G. Synthesis was carried out using T7 RNA polymerase in the presence.

Oocytes from stages V to VI were surgically removed from ovarian lobes of anesthetized X. laevis (Xenopus I, Dexter, MI). The removed oocytes were transferred to Ca ²⁺ -free oocyte Ringer's (OR) culture medium (86 mM NaCl, 1.5 mM KCl, 2 mM MgCl ₂ and 10 mM HEPES, pH 7.6). Oocytes are incubated for 1 hour 30 minutes to 2 hours in Ca ^{2+ -free} OR medium containing 3 mg / ml Collagen Degrading Enzyme (Worthington Biochemicals). It was. The oocytes were then washed with Ca ^{2+ -free} OR medium and ND-96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM HEPES, and 50 g / ml gentamycin, pH 7.6). Washing extensively). Washed oocytes were stored at 18 ° C. in ND-96 medium. Oocytes were stabilized for at least one day before use. After stabilization, for each oocyte, approximately 50 ng of synthesized cRNA in 50 nl nuclease-free water for macroscopic current recording and 1 for single-channel recording with microdispenser (Drummond Scientific, Broomall, PA) ng (in 50 nl nuclease-free water) was injected. The cRNA injected oocytes were incubated in ND-96 medium for 1 to 3 days at 18 ° C. Immediately before the patch-clamp experiment, the oocyte's vitreous membrane was manually removed with fine forceps.

Example 5: Electrophysiological Records and Data Analysis

All macroscopic current recordings and single-channel recordings were performed using the gigaohm-seal patch-clamp method in an outside-out arrangement as is known (Ha et al., 2000). Patch pipettes were prepared from borosilicate glass (WPI, Sarasota, FL) and fire-polished with a resistance of 2-4 MΩ for patch recording. For single-channel recording, patch pipettes were also non-polished with 4-8 MΩ resistors and coated with beeswax to reduce electrical noise. Channel current was amplified using an Axopatch 200B amplifier (Axon Instruments), low-pass filtered at 1 kHz using a four-pole Bessel filter, Digidata 1200A ( Axon Instruments) was used to digitize at a 10 point / ms rate.

At high concentrations of intracellular Ca ²⁺ , a single BK _Ca channel was readily activated by membrane translocation simply delivered at 100 mV. For single-channel analysis, the transition between closed and open states was determined by setting the titer at half unitary current amplitude. To determine the single-channel conductivity of the expressed channel, the average amplitude of the channel current was obtained from the histogram fitted with Gaussian distributions and the average current is plotted against the transmembrane voltage. Slope-conductivity values were obtained from linear regression. The macroscopic current of the expressed BK _Ca channel was activated to a membrane potential, usually in the range of -80 to 200 mV, in 10 mV increments by voltage-clamp pulses delivered from a holding potential of -100 mV. The dwell-time of the open and closed events recorded for a single BKCa channel was analyzed using the linear histogram method. The dwell-time distribution was fitted to a single index using the simplex-least-squares fitting methods (Clampfit, Axon Instruments). The peak in the dwell-time distribution was located at the time-constant of the exponential component.

To prevent the activation of endogenous calcium-activated chloride channels, the solutions for single and macroscopic channel recordings contained gluconate as a nenpermeant anion. Intracellular and extracellular solutions contained the following components unless otherwise specified: 120 mM calcium gluconate, 10 mM MHEPES, 4 mM KCl, and 5 mM MEGTA, pH 7.2. To provide the required free [Ca ²⁺ ] _i , the appropriate amount of total Ca ²⁺ to be added into the intracellular solution was determined by the program MaxChelator (Patton et al., 2004; http://maxchelator.stanford.edu/). Calculated using. To accurately compare channel characteristics, the same set of intracellular solutions was used throughout the experiments. Commercial software packages such as Clampex 8.0 or 8.1 (Axon Instruments) and Origin 9.1 (OriginLab Corp., Northampton, Mass.) Were used to obtain and analyze both single-channel and macroscopic recording data. The data were summarized as mean ± SE (n = number of independent records) and compared using a paired Student's t-test. P-values less than 0.05 were considered statistically significant.

Example 6 Recording Isometric Tension of Bladder Smooth Muscle

Isometric tension recordings of UBSM experiments were performed by conventionally known methods (dela Pena et al. 2009; Kullmann et al. 2014). In short, male SpragueDawley rats (300-350 g) were euthanized with CO ₂ asphyxiation. The bladder was then excised and split longitudinally into four strips (approximately 2 × 8 mm). The separated strip was clipped between the static mount and the force-displacement transducer and 10 ml of Krebs solution ((mM): 118.4 NaCl, 4.7 KCl, 1.2 KH ₂ PO ₄ , 1.2 MgSO _It was suspended in a temperature-controlled (37 ° C.) organ bath containing ₄ , 25.0 NaHCO ₃ , 2.5 CaCl ₂ , and 12.2 glucose; pH 7.35-7.40). The long term bath was continuously bubbled using a mixture of 95% O ₂ and 5% CO ₂ . Each UBSM strip was stretched to an optimal isometric tension of 1.0 g and equilibrated for 60 minutes. During equilibration, the tissue was washed every 15 minutes with fresh Krebs solution and the base tension was adjusted to 1.0 g. After equilibration the strips were stabilized by repeated application of acetylchlorine (1 μM) until a sustained reaction was recorded. To investigate the relieving effect of curinone, the active ingredient of the ginseng extract, the tissue was pre-incubated with curarinone for 30 minutes prior to the addition of acetylcholine, and then acetylcholine-induced in the presence of curarinone. The contraction reaction of was repeated. Relaxation was expressed as a percent decrease in tension resulting from contraction of acetylcholine-induced. One strip in each series was assigned as a time control. Changes in isometric tension were recorded using a Power Lab Data Acquisition System (ADInstruments) attached to a computer installed with Lab Chart Software (Version 7, AD Instruments). Data was summarized as mean ± SE (n = number of DSM strips) and compared using a paired Student's t-test. P-values less than or equal to 0.05 were considered statistically significant.

[result]

1. Confirmation of BK _Ca Channel Activity of Dead Apple Extract Using Thallium-Fluorescence Analysis

BK _Ca channel activity by Ginseng extract was confirmed using a cell-based assay employing Tl ⁺ fluorescence. FluxOR ^™ analysis results are shown in FIG. 1. 1% of DMSO in FIG. 1 is a DMSO control of 1% of the total buffer, and CTBIC was used as a positive control as a channel activation substance already known. At 50 μg / ml final concentration, Tl ⁺ and fluorescence begins to increase, a confirmation, it was confirmed that the fluorescent Tl ⁺ to increase in a concentration-dependent manner up to 1000 μg / ml (see FIG. 1).

The strongest fluorescence increase among the single components included in the alpine extract was obtained from the treatment of curarinone (Fig. 2). Curarinone increased Tl ⁺ fluorescence in a dose-dependent manner (see Figure 2A) and the fluorescence increase was completely blocked by co-treatment with 1 μM paxillin, a selective BK _Ca channel inhibitor. The initial increase in RFU caused by different concentrations of curarinone was quantified in FIG. 2B (n = 4).

2. Confirmation of BK _Ca Channel Activation Patterns of Single Ingredient Curarinone in Ginseng Extract and Ginseng Extract

Sophora active ingredient of BK _Ca of Sophora flavescens extract of 100 μg / ml via the check channel activity BK _Ca channel activating effect of Kurashiki rinon 10 μM flavanone compounds containing large amounts in Sophora flavescens extract BK _Ca channels to enable aspects of the extract and Almost identical (see FIG. 3), the increase in Tl ⁺ fluorescence of the ginseng extract was predicted to be caused by curarinone, and subsequent experiments were carried out with curarinone.

3. Effect of Ginseng Extract on Macroscopic Current of BK _Ca Channel

Although cell-based Tl ⁺ -fluorescence assays have been adapted for high-throughput screening and provided the first BK _Ca channel activator candidates, the activity of each compound in electrophysiology using wild-type BK _Ca channels There was a need to demonstrate and characterize. Therefore, we have characterized the effect of curarinone on the α subunit of rat BK _Ca channel (rSlo1) heterologously expressed on Xenopus oocytes. Macroscopic current time on the channel-dependent effect, in the presence of an outside-in Ca ^{2 +} 3 μM Cells were examined using excised (excised) membrane patch out of the array (Fig. 4). While small tail currents are induced by each test pulse (see Figure 4a), treatment of 5 μg / ml of curinone on the extracellular side results in time-dependent tail-currents. Greatly enhanced in a manner (see b and c in FIG. 4). On removal of the curarinone, the channel current was gradually de-potentiated relative to the basal level (see FIG. 4 d). It is noteworthy that both the strengthening and de-enhancing of the BK _Ca channel by the curarinone shows two phases: the first rapid increase in seconds, and the slow and gentle increase in minutes. When fitted as a double-exponential function, the association time-constant values are 1.47 ± 0.34 seconds for the fast phase (τfast) and 64.9 ± 8.8 seconds for the _slow phase (τ _slow ), respectively. It was expected. Although two different phases are apparent, the de-intensification of the BK _Ca channel was longer than that with dissociation time-constants measured at 2.78 ± 0.95 seconds and 90.06 ± 12.21 seconds, respectively. No significant changes in BK _Ca channel currents were observed when curarinone was treated from the inside of cells using excised inside-out patch recordings. Therefore, these results indicate that curarinone can enhance the activity of BK _Ca channel directly and reversibly from outside the cell.

4. Concentration-dependent Effect of Ginseng Extract on Macroscopic Current of BK _Ca Channel

Next, we studied the mechanism of strengthening Ginseng extract-induced BK _Ca channel. BK _Ca channels were activated by a series of voltage pulses, and macroscopic currents were recorded under increased concentrations of activated extracellular curarinone. It is evident that as the curarinon concentration increases, the channel current is activated at lower voltages and deactivated more slowly (see Figure 5A). In FIG. 4B, the voltage-dependent activation of the macroscopic BK _Ca channel current is shown as the conductivity-voltage (GV) relationship. Curarinon gradually shifts the GV curve to the left and increases the maximum conductivity (G _max ) in a dose-dependent manner. Curarinon-dependent migration in the GV relationship was further quantified and shown in FIG. 5C. In the presence of 20 μM Curarinone, the half-active voltage (V _1/2 ) is shifted approximately 80 mV in the negative direction from 107.4 ± 2.2 mV to 27.7 ± 3.0 mV. Curarinone also gradually increased the channel's maximum conductivity (G / G _max ). G / Gmax was predicted to be 1.35 at 20 μM curarinone, which is about 1.8 times higher than the vehicle control. Therefore, these results show that curarinone enhances the BK _Ca channel and increases the channel's maximum opening potential by activating the channel at more negative membrane voltages.

5. Activation and Deactivation Kinetics Effect of Ginseng Extract on BK _Ca Channel

As shown in the macroscopic current trace of FIG. 5A, inactivation of BK _Ca channels was shown to be significantly affected by the ginseng extract of the present invention. Therefore, the effect of curinone, an active ingredient of ginseng extract, on the opening and closing movement of BK _Ca channel was investigated. In FIG. 6, the activation (or opening) and inactivation (or closure) procedures of the BK _Ca channel were analyzed. While the current level increased in the presence of 20 μM, the activation rate did not vary considerably (see FIG. 6A). At four increased concentrations of curarinone (0, 5, 10, 20 μM) the activation time-constant (τ _activation ) was not significantly increased (see FIG. 6C). On the other hand, the deactivation rate was dramatically reduced by curarinone. Interestingly, the slowing down of inactivation by the Kurarinon was voltage-dependent in that when the channel has been activated by a higher amount of voltage, the Kurarinon makes the closure of the BK _Ca channel more noticeably slower. . Collectively, these results show that curarinone stabilizes the open form of the BK _Ca channel and that the binding affinity of the curarinone can be stronger with channel form at higher voltages.

6. Effect of Ginseng Extract on Single-Channel Current of BK _Ca Channel

To further understand the mechanism of ginseng extract action, the effect was investigated at the single-channel level. It was recorded at-out patches - a single BK _Ca channel stand out under the presence of 10 μM Ca ^{2 +} cells. To activate the BK _Ca channels, the membrane voltage was initially depolarized until higher than 80 mV and the number of channels in the patch membrane was counted. Only patches containing a single channel were used for subsequent experiments. Representative traces of a single channel in the absence or presence of 5 μM of curarinone are shown in FIG. 7A. The opening of the channel was highly dependent on the membrane voltage as expected. However, the gated behavior was dramatically altered by the application of 5 μM Kurarinon to the outside of the cell. While the BK _Ca channel was hardly open in the control solution on -25 mV, the more frequent opening of the channel was evident. At 50 mV, the channel remained open for an extended period of time in the presence of curarinone. To test the effect of curarinone on channel conductivity, the unitary current amplitude of each BK _Ca channel was measured at various film voltages in the presence or absence of a compound and the single-channel current-voltage (IV) relationships were measured. Shown (FIG. 7B). Single-channel conductivity was expected to be 221.1 ± 16.4 in the control and 238.3 ± 8.7 pS in the curarinone, respectively, indicating that the compound does not significantly modify the single-channel conductivity of the channel. The effect of curarinone on P _{0 of} a single channel was then analyzed. P ₀ was measured at several different voltages in the presence and absence of curarinone and fitted to the Boltzmann function (see FIG. 7C). The voltage V _1/2 required for half the maximum opening of the half-was 43.7 ± 2.1 mV under the activation voltage (V _1/2) was 68.7 ± 3.7 mV, 5 μM in the control Kurashiki Lee discuss the presence (Fig. 7D) . These results are in good agreement with the already mentioned findings in macroscopic channel currents, indicating that curarinone can enhance BK _Ca channels by increasing the likelihood of channel opening without affecting single channel conductivity. The opening and closing behavior of single BK _Ca channels on the presence of curarinone was then analyzed. Since curarinone increases the P0 of the Dail BK _Ca channel very dramatically, the analyzes of the present invention were limited to single-channel recordings at 50 mV where open-closed conversion can be compared at reasonable time-scales ( See FIG. 7D). Mean closure time was measured to be 3.69 ± 0.80 ms in the absence of curarinone and 3.60 ± 4.1 ms in the presence of 5 μM of curinone, whereas average open-time was measured to be 2.98 ± 0.34 ms and 5.77 ± 0.90 ms, respectively. (See FIG. 7E). These results indicate that the binding of the curarinone stabilizes the open form, thereby reducing the closure rate of the channel without affecting the open transition of the channel, further confirming the results of the macrocurrent recording.

7. Effect of Ginseng Extract on Rat Bladder Tissue

Since ginseng extract strongly potentiates the replication BK _Ca channel expressed on the heterologous system, we tried to determine if ginseng extract could also relax bladder smooth muscle in vivo. In order to confirm the efficacy of Kurarinon, the active ingredient of Ginseng extract on acetylcholine (ACh) -induced contraction of UBSM, isometric tension of rat detrusor strips was recorded. Whereas 1 μM ACh caused a decrease in peak tension and thus relatively stable plateau levles (see FIG. 8A), pretreatment of tissue with curarinone significantly inhibited ACh-induced contraction. The relaxation effect was 58.2 ± 6.2% in 100 μM Curarinone (p <0.05, n = 6) compared to vehicle treatment (see FIG. 8B). In contrast, there was no significant change in contractile response in vehicle treated time matched control tissue.

8. Effect of Ginseng Extract on Urination Behavior of WKY and SHR

To further demonstrate the effect of ginseng extract on bladder relaxation and urination activity, the urination behavior of Wistar Kyoto rat (WKY) and spontaneous hypertensive rat (SHR) was investigated. The cumulative frequency of urination is shown in FIG. 9A for WKY and SHR administered orally with curarinone, an active ingredient of Ginseng extract. By administration of curarinone, a distinct difference in the frequency of urination was evident between WKY and SHR. While the frequency of urination of control WKY was not affected by curarinone above 5 mg / kg dose, the compound reduced the frequency of urination for SHR in a dose-dependent manner. The total urination frequency for 3 hours is shown in Figure 9B. When 5 mg / kg of curinone was administered, a significant decrease in the frequency of urination was observed in SHR (10.9 ± 1.4 for vehicle and 6.9 ± 0.8 for curarinone). No such reduction in urination frequency was observed in WKY control rats. Together with ex vivo isometric tension recordings, the results show that curarinone and red ginseng extract containing it are effective candidate compounds that have bladder relaxation activity by additionally enhancing the detrusor muscle BK _Ca channel and improve OAB syndrome.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

references

Abraham N, Goldman HB (2015) An update on the pharmacotherapy for lower urinary tract dysfunction. Expert Opin Pharmacother 16 (1): 79-93

2.Ahn HS, dela Pena I, Kim YC, Cheong JH (2011) 4-Chloro-7-trifluoromethyl-10H-benzo [4,5] furo [3,2-b] indole-1 carboxylic acid (TBIC), a putative BKCa channel opener with uterine relaxant activities. Pharmacology, 87 (5-6): 331-40

3.Andersson KE (2004) Antimuscarinics for treatment of overactive bladder. Lancel Neruol 3 (1): 46-53

4.Andersson KE (1997) The overactive bladder: pharmacologic basis of drug treatment. Urol 50 (6A suppl): 74-84

5. Andersson KE, Chapple CR, Cardozo L, Cruz F, Hashim H, Michel MC, et al. (2013) Pharmacological treatment of urinary incontinence. In: Abrams P, Cardozo L, Khoury S, Wein AJ, editors. Incontinence, 5th International Consultation on Incontinence. [Paris]: ICUD-EAU; pp. 623728

6.Brading AF (1997) A myogenic basis for the overactive bladder. Urol 50 (suppl6A): 57-67

7.Bentzen BH, Olesen SP, Rønn LC, Grunnet M (2014) BK channel activators and their therapeutic perspectives. Front Physiol 5: 389

8.Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT and Aldrich RW (2000) Vasoregulation by the β1 subunit of the calcium-activated potassium channel. Nature 407, 870 876.

9.Cvia-Saiz M, Busto MD, Pilar-Izquierdo MC, Ortega N, Perez-Mateos M, Muniz P (2010) Antioxidant properties, radical scavenging activity and biomolecule protection capacity of flavonoid naringenin and its glycoside naringin: a comparative study. J Sci Food Agric 90 (7): 1238-44.

10. Cerruto MA, Asimakopoulos AD, Artibani W, Del Popolo G, La Martina M, Carone R, Finazzi-Agro E (2012) Insight into new potential targets for the treatment of overactive bladder and detrusor overactivity. Urol Int. 89 (1): 1-8.

11. Coyne KS, Sexton CC, Bell JA, Thompson CL, Dmochowski R, Bavendam T, et al. (2013) The prevalence of lower urinary tract symptoms (LUTS) and overactive bladder (OAB) by racial / ethnic group and age: results from OABPOLL. Neurourol Urodyn. 32: 230237.

12. Cui J, Yang H and Lee US (2009) Molecular mechanisms of BK channel activation. Cell Mol Life Sci 66, 852-75.

13.De Groat WC (1997) A neurologic basis for the overactive bladder, Urol, 50 (6A Suppl): 36-52

14.De Naeyer A, Vanden Berghe W, Pocock V, Milligan S, Haegeman G, De Keukeleire D (2004) Estrogenic and anticarcinogenic properties of kurarinone, a lavandulyl flavanone from the roots of Sophora flavescens. J Nat Prod. 67 (11): 1829-32.

15. dela Pena IC, Yoon SY, Kim SM, Lee GS, Ryu JH, Park CS, Kim YC, Cheong JH (2009) Bladder-relaxant properties of the novel benzofuroindole analogue LDD175. Pharmacology 83 (6): 367-78

16. Du G, Jin L, Han X, Song Z, Zhang H, Liang W (2009) Naringenin: a potential immunomodulator for inhibiting lung fibrosis and metastasis. Cancer Res 69 (7): 3205-12.

17.Du W, Bautista JF, Yang H, Diez-Sampedro A, You SA, Wang L, Kotagal P, Luders HO, Shi J, Cui J, Richerson GB and Wang QK (2005) Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder. Nat Genet 37, 733738.

18. Ghatta S, Nimmagadda D, Xu X and O'Rourke ST (2006) Large-conductance, calcium activated potassium channels: structural and functional implications. Pharmacol Ther 110, 103-16.

19. Gormemis AE, Ha TS, Im I, Jung KY, Lee JY, Park CS, and Kim YC, 2005. Benzofuroindole analogues as potent BKCa channel openers. Chembiochem 6, 17451748.

20. Heppner TJ, Bonev AD, Nelson MT (1997) Ca2 + -activated K + channels regulate action potential repolarization in urinary bladder smooth muscle. Am J Physiol Cell Physiol 273: C110C117

21.Herrera GM, Heppner TJ and Nelson MT (2000) Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. Am. J. Physiol Regul. Integr. Comp Physiol 279: R60-R68

22.Herrera GM, Etherton B, Nausch B, Nelson MT (2005) Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle. Am J Physiol Regul Integr Comp Physiol 289: R402R409

23.Hristov KL, Chen M, Kellett WF, Rovner ES, Petkov GV (2011) Large-conductance voltage- and Ca ²⁺ -activated K ⁺ channels regulate human detrusor smooth muscle function. Am J Physiol Cell Physiol 301: C903 C912

24. Jayarajan J, Radomski SB (2013) Pharmacotherapy of overactive bladder in adults: a review of efficacy, tolerability, and quality of life. Res Rep Urol. 6: 1-16

25. Jensen, BS (2002) BMS-204352: a potassium channel opener developed for the treatment of stroke. CNS Drug Rev 8 (4), 353 360.

26. Jin JH, Kim JS, Kang SS, Son KH, Chang HW, Kim HP (2010) Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens. J Ethnopharmacol 127 (3): 589-95.

27. Jung HA, Jeong DM, Chung HY, Lim HA, Kim JY, Yoon NY, Choi JS (2008) Re-evaluation of the antioxidant prenylated flavonoids from the roots of Sophora flavescens. Biol Pharm Bull 31 (5): 908-15

28.Kim BH, Na KM, Oh I, Song IH, Lee YS, Shin J, Kim TY (2013) Kurarinone regulates immune responses through regulation of the JAK / STAT and TCR-mediated signaling pathways. Biochem Pharmacol 85 (8): 1134-44.

29.Knaus HG, McManus OB, Lee SH, Schmalhofer WA, Garcia-Calvo M, Helms LM, Sanchez M, Giangiacomo K, Reuben JP, Smith AB 3rd, et al. (1994) Tremorgenic indole alkaloids potently inhibit smooth muscle high- conductance calcium-activated potassium channels. Biochemistry 33 (19), 5819-5828.

30.Kullmann FA, Daugherty SL, de Groat WC, Birder LA (2014) Bladder smooth muscle strip contractility as a method to evaluate lower urinary tract pharmacology. J Vis Exp 18; (90): e51807.

31.Layne JJ, Nausch B, Olesen SP, Nelson MT (2011) BK channel activation by NS11021 decreases excitability and contractility of urinary bladder smooth muscle.Am J Physiol Regul Integr Comp Physiol 298 (2): R378-84

32. Lee BC, Lim HH, Kim S, Youn HS, Lee Y, Kim YC, Eom SH, Lee KW, Park CS (2012) Localization of a site of action for benzofuroindole-induced potentiation of BKCa channels. Mol Pharmacol 82 (2): 143-55

33. Lee BC, Kim HJ, Park SH, Phuong TT, Kang TM and Park CS (2013) Development of cell based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BKCa channel modulators. J Biotechol 167 (1), 41-46

Lee SW, Lee HS, Nam JY, Kwon OE, Baek JA, Chang JS, Rho MC, and Kim YK (2005) Kurarinone isolated from Sophora flavescens Ait inhibited MCP-1-induced chemotaxis.J Ethnopharmacol 97 (3): 515-9.

35. Lorenz S, Heils A, Kasper JM, and Sander T (2007) Allelic association of a truncation mutation of the KCNMB3 gene with idiopathic generalized epilepsy. Am J Med Genet B Neuropsychiatr Genet 144B, 1013.

36. Marty A (1981) Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature 291, 497 500.

37. Matsui M, Motomura D, Karasawa H, Fujikawa T, Jiang J, Komiya Y, Takahashi S, Taketo M (2000) Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype. Proc Natl Acad Sci USA 97: 95799584.

38. McMurry G, Casey JH, Naylor AM (2006) Animal models in urological disease and sexual dysfunction. Br J Pharmacol 147: S62-S79.

39. McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, and Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14, 645 650.

40. Meredith AL, Thorneloe KS, Werner ME, Nelson MT, and Aldrich RW (2004) Overactive bladder and incontinence in the absence of the BK large conductance Ca2 + -activated K + channel. J Biol Chem 279, 36746-52.

41. Meredith AL, Wiler SW, Miller BH, Takahashi JS, Fodor AA, Ruby NF and Aldrich RW (2006) BK calcium-activated potassium channels regulate circadian behavioral rhythms and pacemaker output. Nat Neurosci 9, 10411049.

42.Nardi VC and Olesen SP (2006) Potassium channel openers: the case of BK channel activators. Lett Drug Des Discov 3: 210218

43.Park S, Regmi SC, Park SY, Lee EK, Chang JH, Ku SK, Kim DH, Kim JA (2014) Protective effect of 7-O-succinyl macrolactin A against intestinal inflammation is mediated through PI3-kinase / Akt / mTOR and NF-kappaB signaling pathways. Eur J Pharmacol 735C: 184-192

44.Petkov GV (2014) Central role of the BK channel in urinary bladder smooth muscle physiology and pathophysiology.Am J Physiology 307 (6): R571-R584

45. Raffaelli G, Saviane C, Mohajerani MH, Pedarzani P and Cherubini E (2004) BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus. J Gen Physiol 557, 147157.

46. Sanchez M, McManus OB (1996) Paxilline inhibition of the alpha-subunit of the high-conductance calcium-activated potassium channel. Neuropharmacology 35 (7), 963-968

47. Seo OW, Kim JH, Lee KS, Lee KS, Kim JH, Won MH, Ha KS, Kwon YG, Kim YM (2012) Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-κB-dependent cFLIP expression in HeLa cells. Exp Mol Med 44 (11): 653-64

48. Shieh CC, Coghlan M, Sullivan JP, Gopalakrishnan M (2000) Potassium channels: molecular defects, diseases and therapeutic opportunities. Pharmacol Rev 52: 557594.

49.Sun, M., Han J, Duan J, Cui Y, Wang T, Zhang W, Liu W, Hong J, Yao M, Xiong S and Yan X. 2007, Novel antitumor activities of Kushen flavonoids in vitro and in vivo . Phytother. Res. 21 (3): 269-277

50. Tang W, Eisenbrand G (1992) Chinese Drugs of Plant Origin. Springer-Verkag, pp. 931943.

51. Werner ME, Zvara P, Meredith AL, Aldrich RW and Nelson MT (2005) Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel. J Physiol 567, 545-56.

52.Yang H, Zhang G, Cui J (2015) BK channels: multiple sensors, one activation gate, Front. Physiol. 6:29.

53.Yilma AN, Singh SR, Morici L, Dennis VA (2013) Flavonoid naringenin: a potential immunomodulator for Chlamydia trachomatis inflammation. Mediators Inflamm. 2013: 102457.

54. Zhou H, Lutterodt H, Cheng Z, Yu LL (2009) Anti-Inflammatory and antiproliferative activities of trifolirhizin, a flavonoid from Sophora flavescens roots. J Agric Food Chem. 57 (11): 4580-5.

Claims (6)

1. Pharmaceutical composition for the prevention or treatment of BK _Ca channel activity lowering-related conditions, diseases or diseases comprising a ginseng extract as an active ingredient.
2. The composition of claim 1, wherein the ginseng extract is a polar organic solvent extract.
3. The composition of claim 1, wherein the polar organic solvent is (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, or a mixture thereof.
4. The method of claim 1, wherein the BK _Ca channel activity lowering-related condition, disease or condition is cardiovascular disease, obstructive or inflammatory airway disease, lower urinary tract disorders, erectile dysfunction, anxiety and anxiety- A composition characterized in that of a related condition, epilepsy or pain.
5. The composition of claim 1, wherein the composition shifts the conductivity-voltage (GV) correlation of the BK _Ca channel in the negative voltage direction.
6. Functional food composition for improving the symptoms of incontinence, bladder overactivity and erectile dysfunction comprising high ginseng extract as an active ingredient.

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