KR101349142B1 - Compositions for Activating hTRPA1 Comprising Methyl Syringate and Uses thereof - Google Patents

Abstract

The present invention provides a variety of uses associated with increasing hTRPA1 activity of methyl syringes, eg, for screening irritant flavours, diabetes or obesity agents and hTRPA1 antagonists. The methyl syringe of the present invention provides a variety of uses associated with hTRPA1 activation, and is a natural compound with relatively low side effects and high industrial applicability to the human body. The methyl syringe of the present invention is not only a novel drug or food material that helps maintain homeostasis in humans associated with the activation of hTRPA1, but can be usefully used for screening antagonists of hTRPA1.

Description

Compositions for Activating hTRPA1 Comprising Methyl Syringate and Uses

FIELD OF THE INVENTION The present invention relates to a TALTRPA1 activating composition comprising methyl syringe gate and use thereof.

TRPA1 (Transient Receptor Potential cation channel subfamily, member 1) is a non-selective cation channel belonging to the superfamily of TRP ion channels. Similar to other family members, the TRPA1 channel is formed by tetratization of four subunits, each subunit containing six transmembrane domains, a pupil loop and intracellular N-terminal and C-terminal . TRPA1 is expressed in sensory neurons and is localized with pain markers such as TRPV1, calcitonin gene-related peptides and bradykinin receptors (Nagata, K. et al., Journal of Neuroscience 2005, 25, 4052-4061; Story, GM Bautista, DM et al., Proceedings of the National Academy of Science, 2005, 102, 12248- 12252; Jaquemar, D. et al., Journal of Biological Chemistry 1999, 274, 7325-7333).

In the pain model, knockdown of TRPA1 suppressed cooling hyperalgesia induced by inflammation and nerve injury (Obata, K. et al., Journal of Clinical Investigation 2005, 115, 2393-2401; Jordt, SE et al. Nature 2004, 427, 260-265; Katsura, H. et al., Exploratory Neurology 2006, 200, 112-123). In addition, TRPA1 gene knockdown causes impairment of sensory function and defects in bradykinin-induced pain hypersensitivity (Kwan, KY et al. Neuron 2006, 50, 277-289; Bautista, DM et al. , 1269-1282).

These results show that TRPA1 plays a very important role in sensory and pain states.

Recently, however, experimental results have shown that the TRPA1 agonist interacts directly with TRPA1. AITC and Shinnam aldehyde have been reported to covalently transform cysteine and lysine residues at the N-terminus of TRPA1 and activate this channel (Hinman, A., Chuang, HH, Bautista, DM, and Julius, D. MacFerson, LJ, Dubin, AE, Evans, MJ, Marr, F., Schultz, PG, Cravatt, BF, and Patapoutian, A., Nature 2007, Proc. Of the National Academy of Science USA 2006, 103, 19564-19568; 445, 541-545).

On the other hand, it is a native plant which is widely used for edible purposes such as wild vegetables and seasoning among Korean oriental plants having a stimulant chemical sense such as spicy taste, tingling and shoot taste, pictu s, English name: Carstor Araila) (sprout, hereinafter referred to as KPs). The chestnut is a plant belonging to the Acanthopanax acacia tree and has been used for neuralgia, arthritis and diabetes by the bark of the bark. Saponin is the major component of the sea tangle, and syringin, coniferyl aldehyde glucoside, and liriodendrin are known as phenolic materials. have. Saponins are mainly Kalopanaxsaponin-based materials composed of monodesmoside and bisdesmoside as glycosides of hederagenin. On the other hand, the seeds of chestnut are collectively used as herbs in Korea, and the leaves of the chestnut are widely used for edible purposes because they are not edible because their leaves are rough and their taste changes as the season passes.

Research on "taste", the most important factor determining the value of food, involves a wide variety of disciplines ranging from basic research at the molecular level to consumer and industrial application studies. The taste substance is "receptive" to receptors in the taste bud cells of the taste buds and transmits the signals to the central nervous system via the taste nerve conduction path, That is, through a series of dynamic mechanisms, from peripheral to central. Recently, molecular genetics of taste receptors have been clarified with the development of related biochemistry such as molecular biochemistry, and some G-protein coupled receptors (GPCR) and TRP (cationic receptor potential) cation channel receptors have been cloned and recently cloned receptors And it became possible to study the cell level used. The cloned receptor research technique using the new taste of the active substance in vitro (in vitro screening and sensory analysis techniques, and at the same time, the most advanced and new methods used for the development of new drugs have been applied to the study of the taste of foods. Food studies at the receptor level for taste components are rarely present in Korea at present and are also in the beginning stages in the world.

Recently, it has been reported that the irritating taste substance, which is represented by spicy taste, cool taste, tingling and stinging taste among taste components, activates TRP-based cation channels. TRP-based receptor activators are also important from this point of view because TRP-based receptors play an important role in other biological mechanisms as a typical cellular sensor for external stimulation. Capsaicin, a major component of the hot pepper, causes pain by activating the hTRPV1 receptor (Transient Receptor Potential cation channel subfamily V member 1) receptor. hTRPV1 is classically classified as a non-selective cation channel with heat-sensitive and ligand-gated features and is known to integrate nociceptive stimuli in sensory neurons. hTRPV1 is present in the membrane of neurons that sense pain and temperature. The reason we feel burning immediately when we eat pepper is because capsaicin, a component of pepper, activates hTRPV1 in pain neurons. High concentrations of capsaicin may cause burning sensation in other sensitive areas of the skin as well as the tongue. As a plant-derived component that activates this hTRPA1, allicin, a component of garlic, can stimulate both hTRPA1 and hTRPV1.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a new material that helps maintain homeostasis in the body associated with activation of hTRPA1. As a result, the present inventors have kalopanax (Kalopanax pictus ) Net extracts and fractions are related to the activation of hTRPA1, and the present invention was completed by elucidating that methyl serine gate included in the extracts of lacquer tree is the most important active ingredient contributing to the activation of hTRPA1.

Accordingly, an object of the present invention is to provide a composition for activating hTRPA1.

Another object of the present invention is to provide a stimulating flavor composition that activates hTRPA1.

It is another object of the present invention to provide a composition for treating diabetes or obesity that activates hTRPA1.

Another object of the present invention is to provide a method of screening an antagonist for hTRPA1.

Another object of the present invention is to provide a method for activating hTRPA1.

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

According to one aspect of the present invention, the present invention provides a composition for activating hTRPA1 (human Transient Receptor Potential A1) containing methyl syringe as an active ingredient.

The present inventors have sought to develop a new material that helps maintain homeostasis in the body associated with activation of hTRPA1. As a result, the present inventors have found that Kalopanax pictus net extracts and fractions are related to the activation of hTRPA1, and that methyl syringes contained in the net extracts and fractions are important active ingredients that contribute to the activation of hTRPA1.

Methyl syringe (methyl 4-hydroxy-3,5-dime-thoxybenzoate) is found in the leaves of vines and Cestrum parquis. Methyl syringes have also been identified in robinia, rape, chestnuts, clover, linden blossoms, dandelions, sunflowers and fir honeys. Methyl syringe is mainly contained in honeys such as Manuka honey, and has been reported as a phenolic compound of the genus Ogalpi, such as Yin. The functional role of the methyl syringe has been confirmed for its antimicrobial activity against Staphylococcus aureus, but there are no studies on TRP-based cation channel receptors, especially hTRPA1.

According to a preferred embodiment of the present invention, the composition of the present invention activates hTRPA1 to induce irritating flavor. Therefore, according to another aspect of the present invention, the present invention provides an irritating flavor composition comprising methyl syringe gate as an active ingredient.

Since the link between the activation of hTRPA1 and the stimulatory flavor has been described in many literatures, the data in the following examples that methyl syringes strongly activate hTRPA1 fully support the use of methyl syringes as stimulating flavors. Allyl isothiocyanate (AITC), which is a mustard oil component as a stimulative flavoring agent (Bandell M, et al., Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41: 849857 (2004)); Jordt SE et al., Mustard oils and cannabinoids excite sensory nerve fibers through the TRP channel ANKTM1. Nature 427: 260265 (2004)) are well known as TRPA1 agonists. Also, cinnamon aldehyde in cinnamon, alicin and diallyl sulfide in garlic and onion, carbacol, isovelleral and water pepper and Tasmanian pepper in oregano, Polygodial, etc., are known as TRPA1 agonists as a stimulant flavor component (Bandell et al., 2004); Bautista DM (2004), a noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin et al., Pungent products from garlic activate the sensory ion channel TRPA1. Proc Natl Acad Sci USA 102: 1224812252 (2005); Escalera J et al., TRPA1 mediates the noxious effects of natural sesquiterpene deterrents. J Biol Chem 283: 2413624144 2008); Macpherson LJ et al., The pungency of garlic: activation of TRPA1 and TRPV1 in response to allicin. Curr Biol 15: 929934 (2005); Xu H et al., Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channe Nat Neurosci 9: 628635 (2006)).

Stimulating flavor by the methyl syringe of the present invention is a 'taste' sensitized through taste, taste is the taste by the action of the chemical receptors of the tongue, oral mucosa and larynx. These chemical receptors contain taste buds, taste organs that taste. Microscopic villi appear on the surface of taste buds, and ions or molecules that taste through the tiny pores called micropores pass through ion channels in the cell membrane. Sweetness, bitterness, salty taste, sourness and umami are recognized by the above-mentioned receptors in the raw rice which is the basis of taste, and spicy, astringent taste, etc. are a sensation which is complexly felt through the receptors and free nerves in the mouth and the mucous membrane of the tongue. For example, irritating flavor (pungent, tingling or stinging taste) is a kind of sick feeling that is felt in the free nerves in the mouth.

It is reported that the irritating taste substance represented by the pungent taste, cool taste, tingling taste or stinging taste among the taste components activates a TRP-based cation channel such as hTRPA1. TRP receptors are also important in this regard because TRP receptors play an important role in other biological mechanisms as typical cellular sensors for external stimuli.

Preferably, the irritating flavor composition of the present invention is a composition characterized by causing a spicy taste (surname), a stinging taste (smut) or a combination taste thereof.

According to a preferred embodiment of the invention, methyl syringe activates hTRPA1 and promotes the secretion of glucagon-like peptide-1 (GLP-1) to prevent or treat diabetes, obesity, hypertension, atherosclerosis or myocardial infarction. Has Accordingly, according to another aspect of the present invention, the present invention provides a prophylactic, ameliorating or treating composition of diabetes mellitus, obesity, hypertension, atherosclerosis or myocardial infarction comprising methyl syringe as an active ingredient.

Since the link between activation of hTRPA1 and inhibition of obesity has been described in several literatures, the data in the following examples that methyl syringes strongly activate hTRPA1 suggest that methyl syringes induce gastric excretion delay and inhibit obesity through appetite suppression. Support the action.

Tatsuo Watanabe et al., Food components activating TRPA1, Recent Researches in Modern Medicine , ISBN: 978-960-474-278-3, p. 460-462 (2011) suggest that TRPA1 activation of agonists of TRPA1 (eg, pepper extract, piperine, or cinnamicaldehyde) enhances energy metabolism in vivo and inhibits abdominal fat accumulation. Also, Doihara H et al., TRPA1 agonists delay gastric emptying in rats through serotonergic pathways, Naunyn Schmiedebergs Arch Pharmacol . 380 (4): 353-7 (2009) disclose that agonists of TRPA1 delay gastric emptying and induce appetite suppression.

On the other hand, GLP-1 (Glucagon-like-peptide-1) is a proglucagon-derived peptide that is separated from intestinal L-cells in response to nutrient ingestion (Drucker, DJ: The Glucagon-Like Peptides. Diabetes 47: 159- 169 (1998)). GLP-1 acts to promote secretion of insulin from beta cells of the pancreas. GLP-1 also acts to inhibit gastric emptying and food intake. Thus, GLP-1 and its agonists (such as GLP-1 secretion promoters) have been shown to be effective in the treatment of diabetes (especially type 2 diabetes) (Toft-Nielsen M, et al., "Determinants of the effectiveness of glucagon- type 2 diabetes ". J Clin Endocrinol Metab 86 (8): 385360 (2001)), obesity (Hayes MR et al., Comparative effects of the long-acting GLP-1 receptor ligands, liraglutide and exendin-4, on food intake and body weight suppression in rats. Obesity ... (Silver Spring) Jul ; 19 (7): 1342-9 (2011), hypertension (Tanaka T et al, The role of incretins in salt-sensitive hypertension: the potential use of dipeptidyl peptidase-IV inhibitors Curr Opin Nephrol Hypertens . Sep; 20 (5): 476-81 (2011)); Inhibition of monocyte adhesion to endothelial cells and attenuation of atherosclerotic lesions by a glucagon-like peptide-1 receptor agonist, exendin-4; Diabetes . Apr; 59 (4): 1030-7 2010); Rizzo M et al, Glucose lowering and anti-atherogenic effects of incretin-based therapies: GLP-1 analogues and DPP-4-inhibitors. Expert Opin Investig Drugs . Oct; 18 (10): 1495-503 (2009)); Myocardial infarction (Ku HC et al., DPP4 deficiency preserving cardiac function via GLP-1 signaling in rats induced to myocardial ischemia / reperfusion. Naunyn Schmiedebergs Arch Pharmacol. Aug; 384 (2): 197-207 al., Glucagon-like peptide- 1 (GLP-1), immediately prior to reperfusion, decreases neutrophil activation and reduces myocardial infarct size in rodents. Horm Metab Res . May; 43 (5): 300-5 (2011)).

According to another aspect of the invention, the invention provides a method for screening an antagonist against human Transient Receptor Potential A1 (hTRPA1) comprising the following steps:

(a) treating hTRPA1 with methyl syringe as a hTRPA1 agonist and a candidate substance to be analyzed; And

(b) measuring the activity of the hTRPA1; When the activity of the hTRPA1 is low compared to the control group not treated with the candidate, the candidate is determined as an antagonist against hTRPA1.

As demonstrated in the examples below, the methyl syringe of the present invention induces activation of hTRPA1. Examples in which activators of hTRPA1 are used for screening hTRPA1 antagonists are described in many literatures. For example, US Patent Application Publication Nos. 20070196866, 20080050750, 20090269280, and 20100273773 describe methods for screening hTRPA1 antagonists that inhibit or reduce the activity of hTRPA1 activated by an activator of hTRPA1. .

According to the method of the present invention, mTR as a hTRPA1 agonist and the candidate substance to be analyzed are first treated with hTRPA1. The methyl syringe of the present invention activates hTRPA1, and a substance capable of inhibiting this activation becomes an antagonist of hTRPA1.

HTRPA1 used in the present invention is a cell expressing hTRPA1 itself or hTRPA1. Preferably, hTRPA1 used in the present invention is a cell stably expressing hTRPA1. The hTRPA1-expressing cells are preferably animal cells, including but not limited to HEK293 cells, neurons, Chinese Hamster Ovary (CHO) cells, COS-7, HeLa, PC-12 and BAF.

Candidates to be analyzed in the present invention include various materials. For example, the candidates include, but are not limited to, chemicals, proteins, peptides, antibodies, nucleic acids, and natural extracts. Candidates analyzed by the screening methods of the invention may be a single compound or a mixture of compounds (eg, a natural extract or a cell or tissue culture). Candidates can be obtained from libraries of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc . Natl. Acad . Sci . USA 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci . USA 91, 11422, 1994; Zuckermann et al., J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int . Ed . Engl . 33,2059,1994; Carell et al., Angew . Chem . Int . Ed. Engl . 33, 2061; Gallop et al., J. Med . Chem . 37, 1233, 1994, and the like.

Step (b) in the method of the present invention can measure the activity of hTRPA1 in a variety of ways.

Since hTRPA1 is a cation channel receptor, the potential change of the cell membrane is generated by the activity of hTRPA1. Therefore, the activity of hTRPA1 can be measured by measuring the change of the cell membrane potential. For example, the activity of hTRPA1 can be measured using a fluorescent agent that emits fluorescence in response to a change in cell membrane potential.

Representative cations introduced into cells by activation of hTRPA1 are calcium ions. Calcium-sensitive fluorescent agents used to measure intracellular influx of calcium ions include Fluo3, Fluo4, Fluo5, Calcium Green, Calcium Orange, Calcium Yellow, Fura-2, Fura-4, Fura-5, Fura-6, Fura -FF, Fura Red, indo-1, indo-5, BTC (Molecular Probes) and FLIPR Calcium3 wash-free die (Molecular Devices).

When the activity of hTRPA1 is measured using a fluorescent agent, it can be carried out using various fluorimeters known in the art.

As a result of measuring the activity of hTRPA1, it is determined that the candidate is an antagonist to hTRPA1 when compared to a control that has not been treated with the candidate. The term "low" as used herein referring to the term "control" means that the relative fluorescence unit (RFU) is 2-100 when measuring the activity of hTRPA1 as a fluorescence change using, for example, a calcium-sensitive fluorescent agent. Means pear low.

The hTRPA1 antagonist screened by the method of the present invention can be used as a therapeutic agent for pain (for example, chronic pain, acute pain, neuropathic pain, seborrheic pain, abdominal pain and neuralgia), acute cerebral ischemia, inflammation, neurodegenerative disease, Can be used as a therapeutic agent for diseases and a therapeutic agent for vomiting.

According to another aspect of the present invention, the present invention provides a method for activating human Transient Receptor Potential A1 (hTRPA1) comprising treating isolated cells with methyl syringe as hTRPA1 agonist.

Cells used in the present invention include cells endogenously containing the hTRPA1 gene or cells transformed with the hTRPA1 gene.

According to a preferred embodiment of the present invention, the methyl syringe used in the present invention is contained in the extract of the Rhus japonica or the Rhus saponum.

The term " extract " used herein to refer to the order of the Chinese cabbage includes not only the extract obtained by treating the extract in the order of the cabbage, but also the resultant product obtained by treating the Chinese cabbage (e.g., powdered) . ≪ / RTI >

When extracting the net extract of the genus Xanthium used in the composition of the present invention in the order of extracting the extract, various extracting solvents can be used. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents include (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable as nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF (Tetrahydrofuran).

More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) (E) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. . Even more preferably, the extract of the present invention is obtained by treating water or ethanol, most preferably ethanol, in the order of the beech.

The term " fraction " used herein to refer to the chestnut tree means a fraction enriched with the hTRPA1 activating component by further performing a solvent treatment on the extract of chestnut tree. The solvent used to obtain the pure water fraction may be any extraction solvent conventionally used in the art, preferably (a) an anhydrous or a lower alcohol having 1 to 4 carbon atoms (e.g., methanol, ethanol, propanol, (C) acetone, (d) ethyl acetate, (e) chloroform, (f) 1, 2-propanol, 3-butylene glycol, (g) hexane, (h) diethyl ether, or (i) butyl acetate.

Most preferably, the net fraction of the muniskill is a fraction of the water layer obtained by adding ethyl acetate to the pure ethanol extract of the oak tree.

The net extracts or the solvent fractions obtained by the method of the present invention include not only those obtained by using the above-mentioned solvents, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the net extract or fraction.

The net extract or fraction may be prepared in powder form by an additional process such as vacuum distillation and freeze drying or spray drying.

As used herein, the term "comprising as an active ingredient" is meant to include an amount sufficient to achieve the efficacy or activity of the methyl syringe gate described herein.

Among the compositions of the present invention, a composition for preventing, ameliorating or treating obesity can be provided as a pharmaceutical composition.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. 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. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered orally.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

Among the compositions of the present invention, irritating flavor compositions and compositions for preventing, improving or treating obesity can be provided as food compositions.

When the composition of the present invention is made of a food composition, it contains not only methyl syringe as an active ingredient, but also components commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Includes the first. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared with a drink, the citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc. Can be.

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

(a) The present invention provides a variety of uses associated with increasing hTRPA1 activity of methyl syringes, such as irritant flavours, diabetes or obesity agents and hTRPA1 antagonist screening.

(b) The methyl syringe of the present invention provides a variety of uses related to hTRPA1 activation and is a natural compound with relatively low side effects and high industrial applicability to the human body.

(c) The methyl syringe of the present invention is not only a novel drug or food material that helps maintain homeostasis in humans associated with the activation of hTRPA1, but can be usefully used for screening antagonists of hTRPA1.

Fig. 1 shows the production process of a water-soluble crude extract (KPsx) and an alcoholic extract (KPsEtx) in the order of a pine tree.
Figure 2 shows a fractionation process to obtain a water fraction (DW) and an acetic acid fraction (EA) from a crude extract. 2A is a process of obtaining KPsx-DW and KPsx-EA from KPsx and FIG. 2b is a process of obtaining KPsEtx-DW and KPsEtx-EA from KPsEtx.
Figure 3 shows the chemical structure of KPs standards and phenolic compounds. The chemical structure of the left side of the structure of the saponin-based compounds, depending on the R group knife Panax _{saponin-A (R 1 = α-L} -Rha- (1 → 2) -α-L-Ara, R 2 = OH, R 3 = H) and calopanax saponin B (R ₁ = α-L-Rha- (1 → 2) -α-L-Ara, R ₂ ═OH, R ₃ = α-L- -D-Glc- (1 → 6) -β-D-Glc). The chemical structure on the right is the structure of a phenolic compound, a being a syringe (R ₁ = CH ₂ OH, R ₂ = OMe, R ₃ = H) and methyl syringe (R ₁ = CH ₂ OH, R ₂ = H, R ₃ = Api), b is protocatecuic acid (R ₁ = -H, R ₂ = H, R ₃ = Me), Chlrogenic acid ^B (R ₁ = -H, R ₂ = OMe, R ₃ = Glc) and The chemical structure of methyl syringe (R ₁ = -Me, R ₂ = OMe, R ₃ = Rha) is shown.
Figure 4 shows HPLC standard chromatograms of saponin compounds and phenolic compounds.
Figure 5 shows the chromatogram of KPsEx.
Figure 6 shows the chromatogram of KPsEtx-DW.
Figure 7 shows the chromatogram of KPsEtx-EA.
Figure 8 shows the expression of hTRPA1 channel cellular response to KPs extract through a Ca ^{2 +} imaging method.
Figure 9 is a photograph showing the expression of hTRPA1 channel cellular response to the gate ache methyl through Ca ^{2 +} imaging method. RR (-) is the case where RR (+) is processed when the block kernel RR (Ruthenium Red) of the TRP channel is not processed. AITC (Allyl isothiocyanate) was used as a positive control.
10 shows the results of measuring the response of hTRPA1 channel expressing cells to methyl syringe using a cell based assay. Allyl isothiocyanate (AITC) and (Cinammaldehyde) are positive controls of hTRPA1.
Figure 11 is a graph showing the effects of the addition of HC-030031 of the antagonist of hTRPA1 for the Ca ^{2 +} responses induced by the gate ache methyl. Allyl isothiocyanate (AITC) and (Cinammaldehyde) are positive controls of hTRPA1. The Ca ^{2 +} responses induced by methyl ache gate was inhibited by the cation channel blocker RR (30 μM) and TRPA1 antagonist HC-030031 (100 μM). Black column shows hTRPA1 activity by AITC (0.01, 0.03 mM), CALD (0.03, 0.3 mM) and methyl syringe (0.3, 3 mM). Gray and white columns show hTRPA1 activity when treated with RR and HC-030031, respectively. Each column is described as mean ± SEM. *, ** and *** represent p <0.05, 0.01 and 0.001, respectively (unpaired t -test).
12 is a graph showing the results of GLP-1 secretion response of NCI-H716 cells to methyl syringe. Data are expressed as mean ± SD ( n = 3). ** P < 0.01; *** P < 0.001 vs. Untreated control.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental material

(Sprout, hornbeam, and pearl) of Kalopanax pictus (English name: Carstor Araila), widely used for edible wild plants and seasonings among Korean oriental plants with spicy taste, tingling and stinging taste, Hereinafter referred to as KPs) was used. Methyl syringe (Formula 1) was purchased from Sigma-Aldrich.

Formula 1

Experimental Method

Establishment of extract titration conditions

The outposts of KPs were collected from the mountainous area, washed, lyophilized, pulverized and stored at -80 ° C. Water-soluble crude extracts (KPsx) and alcoholic extracts (KPsEtx) of each sample were prepared according to the extraction method of FIG. 1 and each was stored at 4 ° C under reduced pressure drying and freeze drying.

(KPsx-DW and KPsEtx-DW) and EA (ethyl acetate) water-soluble fractions (KPsx-EA and KPsEtx-EA) were fractionated by KPsx and KPsEtx according to the method of FIG. .

HPLC Analysis of Active Ingredients of KPs Extracts

Kalopanaxsaponin A (α-heatherin) and Carlopanax saponin B (heatherracoside) for six fractions of KPs (KPsx, KPsEtx, KPsx-DW, KPsEtx-DW, KPsx-EA and KPsEtx-EA) C) and other phenolic compounds such as syringin, methyl syringate, chlorogenic acid, coniferyl alcohol, and protocatechuic acid. Quantification by HPLC analysis using (Figure 3).

The phenolic components were analyzed by using Bondpack C18 (Waters, USA, 10, 300 × 3.9 mm) column, distilled water containing 2% acetic acid (solvent A) and 0.5% acetic acid (50% acetonitrile, solvent B) was eluted at a rate of 0.8 ml / min with an elution gradient of 80% after 60 minutes from the initial solvent B of 10%. The temperature of the column was maintained at 40 ° C and the detection of the sample was measured at 280 nm.

Saponin components were analyzed using a Bond pack C18 column (Waters, USA, 10 μm, 300 × 3.9 mm) and the mobile phase was distilled water (solvent A) and acetonitrile (solvent B) And eluted with a gradient of 35%. The elution rate was maintained at 1.6 ml / min and the temperature of the column was maintained at 25 ° C. The detection of the sample was carried out at 203 nm.

hTRPA1  Stable expressing cell line

The coding region of hTRPA1 was cloned into the pcDNA5 / FRT Complete System (Invitrogen) according to the manufacturer's protocol. To confirm whether or not the hTRPA1 gene was inserted into the vector, the vector was sequenced using ABI 130 or 310 DNA genetic analyzer (Applied Biosystems), and the constructed vector and pOG44 (Invitrogen) were transferred to HEK 293 cells using Lipofectamine 2000 ). After 24 hours, 100 μg / ml of hygromycin B (Invitrogen) was selectively treated and cultured for another 2 to 3 weeks. After that, only the antibiotic resistant cells were collected and the reaction of hTRPA1 was measured and then cultured again. The cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum.

Ca ²⁺  Evaluation of hTRPA1 Action Using Imaging

Ca ²⁺ imaging was performed to measure hTRPA1 activation. Fura-2 / AM (Molecular Probes, USA) was used as a marker for measuring changes in intracellular calcium. Twenty to twenty-two hours prior to conducting the experiment, cell lines stably expressing hTRPA1 were dispensed into 96-well plates. Incubated at 27 ° C. for 30 minutes in a solution to which Pura-2 / AM (5 μM) was added. Cells were washed with assay buffer (140 mM NaCl, 10 mM HEPES, 2 mM CaCl ₂ 2H ₂ O, 2 mM EGTA, 1 mM MgCl ₂ 6H ₂ O, 10 mM glucose and 5 mM KCl) And the mixture was allowed to stand at room temperature for 15 minutes. Fura - 2 / AM - loaded cells were placed on a microscope and fluorescence absorbance was measured at 340 nm and 380 nm. The images were checked every 3 seconds in chronological order and the relative proportions of F340 and F380 (380 nm) were analyzed for changes in intracellular calcium levels using Metafluor software (Molecular devices, USA).

Intracellular Ca using cell-based assays ²⁺  Measure

Intracellular Ca ²⁺ measurements were performed using Flex Station ™ III (Molecular Devices, Sunnyvale, Calif., USA). Cells were plated 24 hours prior to measurement and treated with 5 μM Fluo-4-AM (Molecular Probes, Eugene, OR, USA) as a marker for measuring changes in intracellular calcium content Respectively. The cells were then washed with assay buffer (140 mM NaCl, 10 mM HEPES, 2 mM CaCl ₂ 2H ₂ O, 2 mM EGTA, 1 mM MgCl ₂ 6H ₂ O, 10 mM glucose and 5 mM KCl) Was added and the mixture was allowed to stand at room temperature for 15 minutes. Each of the extracts and the compositions were treated to measure fluorescence absorbance at 486 nm (F486) and 516 nm (F516). The change in intracellular calcium amount was expressed as [Delta] RFU (Relative Fluorescence Unit) and analyzed using Softmax software (Molecular devices, USA).

Analysis of effects on GLP-1 secretion (Anti-diabetic and anti-obesity efficacy analysis)

Glucagon-like peptide-1 (GLP-1), a hormone secreted from the gut, is involved in antidiabetic actions by amplifying glucose-stimulated insulin secretion and inhibiting beta cell death. In addition, GLP-1 is also involved in anti-obesity action by reducing gastric emptying rate and inhibiting appetite. Therefore, GLP-1 secretion material can antidiabetic and anti-obesity effect.

1. Cell culture

NCI-H716 cells, which are human-derived cell lines, were obtained from ATCC (the American Type Culture Collection (Manassas, VA). This cell line is a suspension cell cell isolated from the colon adenocasinoma of a Caucasian adult male (33-year-old), which mainly secretes GLP-1 It is known as a cell line. To maintain cell line proliferation and culture, cells were maintained in 10% FBS bovine serum), 2 mM L-glutamine, 100 IU / ml penicillin and 100 / ml streptomycin. For cell attachment and differentiation, cells were cultured in a Becton Dickinson &apos; Co., Bedford, MA) was coated on a plate to obtain a 10% Glucose DMEM containing FBS, 2 mM L-glutamine, 100 IU / ml penicillin and 100 / ml streptomycin and used for the experiments.

2. Secretion effect of GLP-1 in NCI-H716 cells

The NCI-H716 cell line was cultured on a 96-well culture plate coated with Matrigel at a concentration of 2 x 10 ⁵ cells / mL for 3 days. On the day of the experiment, the cells were washed with 1X PBS (phosphated buffered saline), and KRB (Krebs-Ringer bicarbonate buffer, 128.8 mmol / l NaCl, 4.8 mmol / l KCl, 1.2 mmol / l KH ₂ PO _{4 , 1.2 mmol / l MgSO 4,} 2.5 mmol / l CaCl 2, 5 mmol / l and NaHCO _3, and 10 mmol / l HEPES, 0.2% BSA, was used to pH 7.4) was added to the sample for analysis herein by concentration And cultured at 37 ^° C for 1 hour. After incubation, the supernatant was collected (100 μL) for GLP-1 analysis and analyzed according to the manufacturer's manual using GLP-1 active ELISA kit (EGLP-35K, Millipore). The concentration of GLP-1 was calculated (pmole) based on the supplied GLP-1 standard.

Results and Discussion

KPs  Active ingredient of extract HPLC  analysis

Saponin-based standards and phenolic compounds such as Kalopanaxsaponin A and Carlopanaxsaponin B (hedrageni bis desmoside) used in this study, such as syringes, methyl syringes, chlorogenic acids, methyl syringes and proto Catecuic acid and the like are reported to be distributed in the skin and leaves, but the ratio of saponin-based compounds and phenolic compounds is known to vary depending on the site. Recent studies have shown that the shells contain less saponins than leaves and are present in high contents of lyrodendrine and syringine, and these two compounds are evaluated as important active substances for representing the sea tangle. On the other hand, (Open field) showed a high content of chlorogenic acid, and this compound was evaluated to exist as a functional compound. On the basis of this, component analysis of six KPs fractions showed the highest content of chlorogenic acid (cf. FIGS. 1 - 7). However, according to hTRPA1 action Evaluation of Ca ^{2 +} imaging is chlorogenic acid did not show any effect, it was confirmed that the methyl ache gate confirmed by HPLC resulting in a small amount in the specific action hTRPA1.

KPs Fraction hTRPA1  Activity evaluation

HTRPA1 activity was measured for KPsEtx, KPsEtx-DW and KPsEtx-EA. In hTRPA1, KPsEtx and KPsEtx-DW showed a time-dependent effect and were completely inhibited by RR treatment. As shown in FIG. 8, it seems that the intracellular Ca ^{2 +} influx appeared when AITC (Allyl isothiocyanate), which is a positive control of hTRPA1, is treated, and it is considered that the effect is about 30 μM AITC. Furthermore, it was judged that the intracellular calcium concentration by KPs extract in the hTRPA1-expressing stable cell line was further increased as shown by the RR completely suppressed and time-dependent pattern (FIG. 8).

Ca ^{2 +} Imaging  Used methyl Of syringe gate hTRPA1  Activity evaluation

as a result of the Ca ^{2 +} imaging in cells stably expressing the hTRPA1, hTRPA1 the hTRPA1 methyl ache gate in stable cells expressing the time-can be seen that the Sikkim dependently increase intracellular Ca ^{2 +} influx, which is methyl The syringegates show time-dependent activation of hTRPA1 (FIG. 9). In addition, it was confirmed that the action of methyl syringe gate is completely blocked when RU (Ruthenium Red) (Sigma-aldrich, USA), a general blocker of the TRP channel. Thus, methyl ache gate may increase the intracellular Ca ^{2 +} influx activates the hTRPA1 was seen through it indicates a variety of physiological responses by hTRPA1.

Using cell-based analysis hTRPA1  Activity evaluation

As the hTRPA1-specific activity of methyl syringe was identified in the previous experiment, HC-030031 [2- (1,3-) was identified as a change in the concentration of methyl syringe and the specific blocker of TRPA1 using the cell cytometry. Dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl) -N- (4-isopropylphenyl) acetamide] (Sigma-aldrich, USA) The reaction by was confirmed. As a result, EC ₅₀ of AITC and CALD, known as agonists of TRPA1, was 8.475 μM and 20.66 μM, respectively, and methyl syringe gate was identified as 178.5 μM (FIG. 10). Thus, it can be seen that the methyl syringe of the present invention is an hTRPA1 agonist with excellent ability to activate hTRPA1.

In addition, decreased significantly the Ca ^{2 +} responses by gate ache methyl ever the addition of 100 μM HC-030031 antagonists of hTRPA1 (Fig. 11).

GLP -1 on the secretion of Anti-diabetic  And Anti-obesity  efficacy)

The NCI-H716 cell line was used to confirm the GLP-1 secretion response to methyl syringe gate. As a result, GLP- was 4.2-fold at 100 μM of methyl syringe gate, 3.6-fold at 30 μM, 3.2-fold at 10 μM, and 2.5-fold at 3 μM compared to untreated control samples. 1 secretion was increased (FIG. 12). The 500 μM AITC used as a positive control showed a 3.9-fold secretion of GLP-1. Therefore, antidiabetic and anti-obesity effects can be expected for the methyl syringe which promotes GLP-1 secretion.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

delete Food composition for inducing irritant flavor selected from the group consisting of pungent taste and stinging containing methyl syringe gate as an active ingredient.
Food composition for the prevention or improvement of obesity containing methyl syringe as an active ingredient.
Pharmaceutical composition for the prevention or treatment of obesity, including methyl syringe as an active ingredient.
delete A method of activating a human Transient Receptor Potential A1 (hTRPA1) of a cell comprising treating the isolated cell with a methyl syringe as a hTRPA1 agonist.

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