KR100675819B1 - Composition containing extract from cimicifugae rhizoma or derivatives thereof as inhibitor for nicotinic acetylcholine receptor - Google Patents

Abstract

 The present invention relates to a composition for inhibiting the activity of the nicotine acetylcholine receptor comprising a horse riding extract or a compound derived therefrom. In particular, the present invention specifically inhibits nicotine-acetylcholine receptor activity by acting specifically on nicotine-acetylcholine receptor activity, and thus can be usefully used as a therapeutic agent for neurological diseases related to catecholamine activity and secretion of nicotine-acetylcholine receptor. Providing equestrian extracts and equestrian derived compounds.

Description

COMPOSITION CONTAINING EXTRACT FROM CIMICIFUGAE RHIZOMA OR DERIVATIVES THEREOF AS INHIBITOR FOR NICOTINIC ACETYLCHOLINE RECEPTOR}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a composition for inhibiting the activity of the nicotine acetylcholine receptor comprising a horse riding extract or a compound derived therefrom, and more specifically, nicotine-acetylcholine receptor activity by specifically acting on the nicotine-acetylcholine receptor activity Inhibition of the present invention relates to a cymifufuside which can be usefully used as a therapeutic agent for neurological diseases related to the catecholamine activity and secretion of the nicotine-acetylcholine receptor.

[Private Technology]

Menopausal symptoms are found in 40 to 60 percent of menopausal women. Menopause symptoms are mainly caused by anxiety, depression, sleep disorders, hot flashes and headaches. Hormonal treatments have been performed for the purpose of alleviating these symptoms, but recently, side effects of hormonal therapy have been reported.

As a herbal medicine, horseback riding is known to be effective in alleviating gynecological symptoms and analgesic effects such as chronic ovarian infection, endometritis, dysmenorrhea, dysmenorrhea, excessive menstruation and infertility. Therefore, phytoestrogens contained in horse riding may have an effect as an estrogen substitute in postmenopausal women, but no specific studies have been conducted. According to a recent report, the components of horse riding, which are effective as a substitute for estrogen in alleviating menopausal symptoms, interact with cell surface receptors rather than through estrogen's unique mechanism of action by binding to estrogen receptors in cells. It is believed to result in mitigation (Borrelli F, Izzo AA, Ernst E, Life Sci. 2003 25; 73 (10): 1215-29). However, a substance having a specific menopausal symptom relieving effect has not been identified, and studies on its mechanism of action have been insufficient.

BD-xylopyranoside, (3B, 15a, 23R, 24S) -16,23: 16,24-diepoxy-15,25-diphdroxy-9,19-cyclolanostan-3-yl), is isolated from horse riding. It is one of the ingredients. Simicifugoside is a triterpeneglycoside-based compound, and its basic structure is called phytoestrogens because it has a steroid skeleton, but there are no specific studies.

Adrenal medulla chromaffin cells, on the other hand, are cells derived from the nervous system and function as part of the sympathetic nervous system after adulthood. The adrenal glands secrete stress hormones, such as cortisol, in the cortex and catecholamines and several neuropeptides in the blood, depending on various stimuli in the body. The catecholamines secreted by the adrenal glands include dopamine, norepinephrine and epinephrine, which are known to play important roles in stress and emotional behavior. They act on both the nervous and central nervous system. Catecholamines have already been studied for their importance and role in models where abnormalities occur in the central nervous system. Unbalanced secretion of catecholamines in synapses can lead to diseases of the nervous system, such as manic depression or schizophrenia. In addition, when an external signal is transmitted to nerve cells, intracellular calcium concentration increases, which acts as a signal for neurotransmitter secretion through nerve gaps, and regulates metabolism through heart rate, respiration, muscle contraction, and insulin secretion control. It causes physiological symptoms. Thus, therapeutic agents in the psychiatric lineage are reported to act primarily on catecholamine secretion mechanisms (Seeman P. and Van Tol HH 1994. Trends Pharmacol. Sci. 15, 264-270; Kilpatrik DL, Ledbetter FH, Carson KA, Kirshner AG , Slepetis RJ and Kirshner N. 1980 J. Neurochem 35, 679-692;. Jarry H., Metten M., Spengler B., Christoffel V., and Wuttke W. 2003 Maturitas 44 (Suppl.1), S31-S38 .).

Signaling receptors that play an important role in catecholamine secretion include acetylcholine receptors. Acetylcholine receptors are classified into muscarinic acetylcholine receptors and nicotine acetylcholine receptors depending on the type of ligand and intracellular signal transduction mechanism. The double nicotine acetylcholine receptor binds to nicotine in addition to acetylcholine and mediates a sustained excitatory response of about 1/1000 seconds. Nicotine acetylcholine receptors form potassium and sodium ion channels that are regulated by ligand binding. Nicotine acetylcholine receptors are known to be associated with neurological disorders, in particular nicotinic acetylcholine receptor activity inhibitors can be used as treatments for analgesia, analgesics and anesthesia for increased blood pressure, vasoconstriction, cold and hot hands, insomnia, depression and hot flashes (Carroll FI, Lee JR, Navarro HA, Ma W, Brieaddy LE, Abraham P, Damaj MI, Martin BR. J Med Chem. 2002 Oct 10; 45 (21): 4755-61 .; Hama A, Menzaghi F. Brain Res. 2001 Jan 5; 888 (1): 102-106 .; Gentry CL, and Lukas RJ 2001 J pharmacol Exp Ther. 299 (3), 1038-1048).

Accordingly, an object of the present invention is to provide a composition for inhibiting nicotine acetylcholine receptor activity, which specifically acts on the nicotine acetylcholine receptor and inhibits its activity.

It is another object of the present invention to provide a composition for treating a disease related to nicotine acetylcholine receptor.

In order to achieve the above object, the present invention provides a composition for inhibiting nicotine-acetylcholine receptor activity comprising a horse riding extract as an active ingredient.

In another aspect, the present invention provides a composition for inhibiting nicotine-acetylcholine receptor activity comprising a compound selected from the group consisting of compounds of Formulas 1 to 3 as an active ingredient.

(Formula 1)

(Formula 2)

(Formula 3)

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, while searching for an antagonist capable of specifically acting on the nicotine acetylcholine receptor to inhibit its activity, confirmed that the equestrian extract and the compound derived therefrom inhibited the nicotine acetylcholine receptor activity. On the basis of this, the present invention has been completed.

The present invention relates to a composition for inhibiting the activity of the nicotine acetylcholine receptor comprising a horse riding extract or a compound derived therefrom.

The horseback riding (Cimicifugae rhizoma) of the present invention may be horseback riding (C. davurica MAX.), Stork riding (C. foetida L.) or candlestick riding (C.simplex WOR.MSK). Horse riding extracts can be extracted using an extractant from the group consisting of horse riding outposts, leaves, roots, stems, seeds, flowers and mixtures thereof. For example, horse riding roots or pulverized products thereof may be extracted or fractionated by adding water or an organic solvent in a weight ratio of 1: 1 to 1: 100 to prepare a horse riding extract. The organic solvent includes an alcohol having 1 to 5 carbon atoms, hexane, acetone, ethyl acetate, methylene chloride or chloroform organic solvent, and alcohols include methanol, ethanol, butanol or isopropanol, but are not limited thereto. The alcohol may be diluted alcohol such as 50-95% alcohol water. The extraction method may be an immersion method or a layer separation used in preparing a conventional plant extract, for example, a methanol or ethanol crude extract.

Equestrian-derived compounds according to the present invention may be extracted from plants or prepared by chemical synthesis, preferably from the roots of equestrian by known methods.

The equestrian-derived compound of the present invention is represented by the chemical formula 1, 26-deoxyactin (Deoxyactein, Formula ₃₇ C ₅₆ H ₁₀ O, Molecular Weight: 660.8) and Formula 3 Cymiramoside A (C ₃₇ H ₅₆ O ₁₁ , molecular weight: 676.8).

The equestrian extract of the present invention and its derived compounds significantly inhibit catecholamines through nicotine acetylcholine receptors (FIGS. 1A-1C and 2A-2C), in particular simimifugoside exhibits about 50% inhibitory effect at 20 μM. . The nicotinic acetylcholine receptor-specific inhibitory activity of the mycifufugoside significantly reduced the influx of intracellular calcium and sodium ions during DMPP treatment of adipocytes pretreated with the cymifufuside. 3 to 4), in the case of increasing the intracellular calcium ion concentration through a route other than DMPP can be confirmed through the experimental results do not have a significant effect (Fig. 5). In addition, 26-deoxyactin and cymirasemoside also significantly inhibited intracellular calcium ion increase by DMPP (FIG. 6).

Therefore, the equestrian extract of the present invention and the compounds derived therefrom respectively act specifically on the nicotine acetylcholine receptor and inhibit its activity, and thus can be used as nicotine acetylcholine receptor antagonists, and also due to the excessive secretion of catecholamines. It can be used as a therapeutic agent. Applicable diseases include sympathetic neuropathy and menopausal symptoms. Diseases caused by sympathetic hyperactivity include increased blood pressure, vasoconstriction, cold hands and feet, and hyperhidrosis. Menopausal symptoms include insomnia, depression, and hot flashes.

The composition for inhibiting nicotine acetylcholine receptor activity of the present invention may include, as an active ingredient, each of a horse riding extract and a compound derived therefrom alone, and may be a pharmacologically acceptable carrier according to the formulation, method of use, and purpose of use. It may further comprise excipients. When provided in a mixture, an active horse riding extract or a compound derived therefrom may be included in the composition in an amount of 0.1 to 99% by weight, but is usually included in an amount of 1 to 20% by weight.

The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, which may be used one or more, but are not limited to these, and are common carriers And excipients are both available. In addition, when pharmaceuticalizing a composition for inhibiting nicotine acetylcholine receptor activity, it may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

The composition for inhibiting nicotine acetylcholine receptor activity can be used orally or parenterally. Since the formulation of the composition may vary depending on the method of use, it is not limited thereto. Examples of formulations include Plasters, Granules, Lotions, Powders, Syrups, Liquids and Solutions, Aerosols, Ointments, Fluids Fluxextracts, emulsions, suspensions, infusions, tablets, injections, capsules and pills. The nicotine acetylcholine receptor antagonist compositions may also be in individual dosage forms, such as tablets, coated tablets, capsules, pills, suppositories, and ampoules. The content of its active compound in individual dosage forms can be in one dose, for example, usually in whole, 1/2, 1/3 or 1/4 times the daily dose.

The dosage of the composition for inhibiting nicotine acetylcholine receptor activity may be determined in consideration of the age, sex, condition, absorbency of the active ingredient in the body, inactivation rate, and the drug used in combination, for example, based on a single active ingredient. 0.1 to 1 M, may be administered 1 to 5 times a day.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1: Horse Riding Extract

1-1. Riding extract manufacturer

50 g of riding roots were ground with a mixer and extracted in 200 ml of 80% ethanol for one day, ethanol was blown off at 80 ° C of hot water, and the remaining one was lyophilized to prepare a horse riding extract.

1-2. Validation of Horse Riding Extracts

end. Cell preparation

Adrenal medulla cells were isolated through two stages of collagenase treatment slightly modified in known methods. In other words, the adrenal gland was removed from the local slaughterhouse and the surrounding adipose tissue was removed, and then the internal blood cell cells were washed through the vein with Locke's solution without calcium and magnesium and then treated with 0.2% collagenase for 20 minutes. Thereafter, only the adrenal medulla was separated and finely chopped with a surgical knife, and then again treated with collagenase solution for 1 hour, filtered through a thin network, and only medullary cells were obtained through centrifugation.

Cells were suspended in DMEM / F12 medium in liquid medium supplemented with 10% serum (FBS) and 1% antibiotics extracted from bovine fetuses, and dispensed 500.000 cells / well in 24 well plates to measure catecholamine secretion, followed by 95% After 2-3 days in air / 5% CO ₂ incubator was used for the experiment.

I. Effect of Horse Riding Extract on Catecholamine Secretion

Riding extract was used by dissolving in DMSO (Dimethylsulfoxide) solvent.

To measure the catecholamines secreted from the adrenal medulla cells, cells cultured in 24 well plates were washed twice with Locke's solution containing calcium and magnesium, pretreated horse riding extract for 5 minutes by concentration, and then DMPP for 10 minutes. (1,1-dimethyl-4-phenylpiperazinium iodide) and KCl were treated with 10 μM and 60 mM, respectively. Cell culture supernatants were then obtained and analyzed by high performance liquid chromatography (HPLC) to determine the catecholamine content. Catecholamine secretion was measured in one chromatin cell in an amine-free solution and used for the experiment. The carbon fiber electrode was attached to the cell membrane of a single cell and detected by electrochemical detection.

In the case of HPLC, norepinephrine is usually separated at around 2 minutes and 20 seconds, followed by detection of epinephrine at about 2 minutes and 35 seconds and dopamine at about 3 minutes and 20 seconds.

The experimental results are shown in Figures 1a, 1b and 1c.

Figure 1a shows the catecholamine secretion according to the equestrian extract treatment concentration, the empty bar means norepinephrine (noradrenaline) in the catecholamine, gray bar means epinephrine (adrenaline). From the results in FIG. 1A, catecholamine secretion by DMPP was inhibited according to the concentration of the equestrian extract.

Figure 1b shows the catecholamine secretion by KCl of 60 mM after pretreatment of the horse riding extract for 5 minutes by concentration in a single cell, it can be seen that the equestrian extract does not inhibit the catecholamine secretion by KCl.

Figure 1c shows the catecholamine secretion rate by DMPP of 10 μM after pretreatment of horse riding extract at 300 ㎍ / ㎖ in single cells for 5 minutes, the catecholamine secretion was significantly reduced when pre-treatment of the mysifufugoside, DMPP after cell washing When reprocessing was confirmed that the catecholamine secretion ratio is recovered.

Example 2: Verification of the inhibitory effect of catecholamine secretion

It was carried out in the same manner as in Example 1, instead of the horse riding extract was used for the mycifufugoside.

Simicyfugoside was purchased from Chromadex Inc. and dissolved in DMSO (dimethylsulfoxide) solvent.

Experimental results are shown in Figures 2a, 2b and 2c.

Figure 2a shows the amount of catecholamine secretion according to the concentration of the mysifufugoside treatment, catecholamine secretion by DMPP was suppressed according to the concentration of the mysifufugoside. In addition, when the concentration of cysifufugoside was 20 μM, the catecholamine inhibitory effect was about 50%, and at 100 μM, the inhibitory effect was over 85%.

Figure 2b shows the catecholamine secretion ratio by DMPP of 10 μM after pretreatment of simicifufuside at 60 μM for 5 minutes in a single cell, the catecholamine secretion is significantly reduced when pre-treatment with simifufuside, When the catecholamine secretion ratio of the non-pretreated control group was set at 100%, the secretion ratio of the treated group was only 33%.

Figure 2c shows the catecholamine secretion ratio by 60 mM KCl after pretreatment of simifufugoside to 60 μM for 5 minutes in a single cell, it can be seen that the simimifugoside does not inhibit the catecholamine secretion by KCl. .

Example 3: verification of the inhibitory effect of intracellular calcium ion increase by treatment

Chromatin cells, put a fluorescent dye (Fura2 / AM) that binds to calcium intracellularly, was added to the treatment of simifufugoside to observe the change in calcium increase due to DMPP or KCl treatment. The results are shown in Figures 3a to 3c.

Figure 3a is a graph showing the intracellular calcium influx according to the concentration of simifufugoside treatment. In FIG. 3A, the increased intracellular calcium influx induced by catecholamines was significantly inhibited with the concentration of pre-treated cysifufuside. In particular, the inhibitory effect of 50% intracellular calcium influx was observed at the treatment concentration of 20 μM of simicifufuside, and at least 98% was inhibited at 300 μM.

Figure 3b is a photograph showing the degree of intracellular calcium ion influx according to the pre-treatment and DMPP treatment, i) is both untreated cells of simifufuside and DMPP, ii) cells treated with DMPP only , iii) are cells treated with only simifufugoside, iv) are cells treated with DMPP after pretreatment with simifufugoside, the lower left photograph is a cell photograph, and the lower right photograph is Fura2 / AM bound to calcium ions. Fluorescence is shown graphically. In the above, DMPP acts to increase the intracellular calcium ion influx, but it can be seen that Shimishifugoside significantly inhibits the calcium ion influx.

Figure 3c is a picture showing the degree of intracellular calcium ion influx according to the pre-treatment and KCl treatment, i) is both untreated cells of simifufuside and KCl, ii) cells treated with KCl only , iii) are cells treated with only the simifufugoside, and iv) are cells treated with KCl after pretreatment with the simifufugoside, the lower left photograph is a cell photograph, and the lower right photograph is Fura2 / AM bound to calcium ions. Fluorescence is shown graphically. In FIG. 3c, even when KCl was treated after the pre-treatment of simifufugoside, a similar level of calcium ion influx was observed as in the case of KCl alone treatment, so that the simifufuside had no effect on intracellular calcium ion concentration by KCl. It can be seen.

Example 4: verification of the inhibitory effect of intracellular sodium ion increase by treatment

After punching one cell into one cell with a microglass tube and forcibly depolarizing the cell with an electrode connected to the tube, the amount of sodium introduced was measured. It was. The results are shown in FIG.

Figure 4 shows the influx of sodium ions in depolarized chromaffin cells, A is DMPP after treatment with 10 μM of DMPP 10 μM pretreated with DMPP 10 μM alone, DMPP after 60 μM pretreatment Intracellular sodium ions were treated at 10 μM treatment and after DMPP 10 μM treatment alone, and B represents intracellular sodium ion influx with increasing concentration of pre-treated cysifufuside. In Figure 4, it can be seen that the simycifugoside effectively inhibits the influx of intracellular sodium ions, in particular, the simifufugoside can be seen that the inhibitory effect increases with concentration. Simicifugoside inhibits intracellular sodium influx by 50% at 3 μM and at least 87% at 60 μM.

Thus, the mycifufugoside significantly inhibits the influx of sodium ions generated during cell depolarization.

Example 5: Verification of DMPP Specific Action of Simycifufuside

It was confirmed in Example 2 that the mycifufugoside inhibits the increase in intracellular calcium ions generated by DMPP stimulation, and to determine whether the above-described inhibitory effect of calcium ion increase specifically occurs only in DMPP stimulation. Therefore, in addition to DMPP, a stimulant of nicotine-acetylcholine receptor, KCl, BK (Bradykynin) and VT (veratridine), which are substances that increase the concentration of calcium ions in the cells of chromatin, were treated to the cells treated with the mycifufugoside. Afterwards, intracellular calcium ion changes were measured. The results are shown in FIG.

Figure 5 shows the intracellular calcium ion changes when stimulated with DMPP, KCl, BK and VT in the pre-treated cells, respectively, Shimishifugo that inhibits the intracellular calcium increase by DMPP about 60% or more At the side 20 μM concentration, intracellular calcium increase by KCl, BK and VT was hardly inhibited.

Thus, it can be seen that the mycicifugoside of the present invention specifically acts only on the nicotine-acetylcholine receptor to inhibit its activity.

Example 6: Verification of inhibitory effect of intracellular calcium ion increase by treatment

26-deoxyactin and cymirasemoside were purchased from Chromadex Inc. as a cymifufuside derivative, and experimented in the same manner as in Example 3, to increase intracellular calcium ion by the compounds. The inhibitory effect was observed.

FIG. 6 shows intracellular calcium influx when DMPP was treated with 10 μM after pretreatment with 60 μM of each of cysifufuside (CF), 26-deoxyactin (26D-Actein), and simirasemoside (CR). Is a graph. In FIG. 6, it can be seen that the mycifufuside, 26-deoxyactin, and the simirasemoside significantly inhibit the increase of intracellular calcium influx induced by the catecholamine.

As described above, the equestrian extract of the present invention and the compound derived therefrom specifically acts on the nicotine-acetylcholine receptor activity and inhibits the nicotine-acetylcholine receptor activity, thus the activity and secretion of the nicotine-acetylcholine receptor It can be usefully used as a therapeutic agent for neurological diseases related to catecholamines.

Figure 1a shows the amount of catecholamine secretion according to the horse riding extract treatment concentration.

Figure 1b shows the catecholamine secretion rate by KCl of 60 mM after pretreatment of horse riding extract for 5 minutes by concentration in a single cell.

Figure 1c shows the catecholamine secretion rate by DMPP of 10 μM after pretreatment of horse riding extract at 300 ㎍ / ㎖ in single cells for 5 minutes.

Figure 2a shows the rate of inhibition of catecholamine secretion according to the concentration of the mysifufugoside treatment.

Figure 2b shows the rate of inhibition of catecholamine secretion by DMPP (1,1-dimethyl-4-phenylpiperazinium iodide) after pretreatment with simifufugoside to 60 μM in single cells.

Figure 2c shows the rate of inhibition of catecholamine secretion by KCl after pretreatment with 60 μM of mycifufugoside in a single cell.

Figure 3a shows the rate of inhibition of intracellular calcium ion influx according to the concentration of mycifufugoside treatment.

Figure 3b is a photograph showing the degree of intracellular calcium ion influx according to whether or not the pre-treated with the mysifufugoside DMPP.

Figure 3c is a photograph showing the degree of intracellular calcium ion influx according to whether or not the pre-treated with Shimishifugoside.

Figure 4 shows the influx of sodium ions in depolarized chromaffin cells, A is DMPP after treatment with 10 μM of DMPP 10 μM pretreated with DMPP 10 μM alone, DMPP after 60 μM pretreatment Intracellular sodium ions were treated at 10 μM treatment and after DMPP 10 μM treatment alone, and B represents intracellular sodium ion influx with increasing concentration of pre-treated cysifufuside.

FIG. 5 compares intracellular calcium ion changes when simifufugosides were stimulated with DMPP, KCl, Bradykynin (BK) and veratridine (VT), respectively.

FIG. 6 is a graph showing intracellular calcium influx when DMPP treatment was performed after pretreatment of each of cysifufuside (CF), 26-deoxyactin (26D-Actein), and simirasemoside (CR).

Claims (9)

For inhibiting nicotine-acetylcholine receptor activity for the treatment of sympathetic hyperalgesia-related diseases selected from the group consisting of horseshoe coldness, and hyperhidrosis comprising an equestrian extract obtained by using alcohol or dilute alcohol water thereof as an extractant as an active ingredient. Composition. The composition of claim 1, wherein the horse riding extract is extracted from a horse riding root with a solvent selected from the group consisting of ethanol, methanol and dilute alcohol water. delete delete delete To a sympathetic hyperactivity-related disease or insomnia, hot flashes and depression selected from the group consisting of blood pressure increase, vasoconstriction, cold hands and legs, and hyperhidrosis comprising a compound selected from the group consisting of compounds of Formulas 1 to 3 as an active ingredient Composition for inhibiting nicotine-acetylcholine receptor activity for treating menopausal symptoms selected from the group consisting of: (Formula 1) (Formula 2) (Formula 3) delete delete delete