KR20150077393A - Pharmaceutical or food composition comprising Geum aleppicum - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or food composition which reduces the side effects so as to be safe and which is highly active for stimulating enteroendocrine cells, and comprises Geum aleppicum as an effective ingredient. The composition of the present invention can be effectively used as a pharmaceutical composition or food composition for prevention and alleviation of diabetes, alleviation of heart diseases, alleviation or treatment of digestive disorders and malabsorption, anti-obesity or appetite suppression, alleviation of arteriosclerosis, neuroprotective effects, treatment or alleviation of liver diseases or the like. In addition, the composition of the present invention can promote the secretion of GLP-1, CCK or5-HT, or the activation of hTGR5, and can be used as a TRP, TRPA1, G protein and PLCβ2.

Description

[0001] The present invention relates to a pharmaceutical composition or a food composition comprising Geum aleppicum,

The present invention relates to a pharmaceutical composition or a food composition, and more particularly, to a pharmaceutical composition useful for a specific disease or disease containing, as an active ingredient, an extract that promotes the secretion of endocrine hormones or neuropeptides from intestinal endocrine cells or ≪ / RTI >

Enteroendocrine cells, which are endocrine cells present in the gastrointestinal tract, are stimulated by components that enter the intestinal lumen of the intestine, leading to the formation of gut hormones or nerves Secrete peptides (neuropeptides). Even if they are the same, they move to the target organs through the blood and are called hormones. When they act as vagal activation signaling substances, they are called neuropeptides.

Glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), serotonin (5-hydroxytryptamine, 5-HT ) And the like.

First, GLP-1 is secreted in L-cells, a kind of intestinal endocrine cells present in the ileum and colon.

(GLP-1): an emerging opportunity to treat neurodegenerative and cerebrovascular disorders. British Journal of Clinical Pathology & Glucagon-like peptide-1 (GLP-1) and its split products GLP-1 (9-37) and GLP-1 (28-37) stabilize atherosclerotic lesions in apoe ^{- / -} mice. Atherosclerosis (2013) 231, 427-435], and the like.

In the pancreas, it is involved in demonstrating the therapeutic effect of diabetes through glucose-dependent insulin secretion stimulation, insulin gene expression enhancement, pancreatic beta cell proliferation promotion effect, pancreatic beta cell survival promotion effect, glucagon secretion inhibition effect and blood glucose lowering effect, Slows the appetite, promotes satiety, and inhibits food intake, thus contributing to the effect of obesity treatment.

And it protects cardiocyte from cardiac myocyte through cardiomyocyte protection effect and cardiac function improvement in patients with heart attack. It improves digestive enzyme secretion in pancreas and small intestine, (Sokos, GG et < RTI ID = 0.0 > al . , Glucagon-like peptide-1 infusion improves left ventricular ejection fraction and functional status in patients with chronic heart failure. J. Card. Fail. (2006) 12: 694-699., Ban, K., et al . , Cardioprotective and vasodilatory actions of glucagon-like peptide-1 receptors are mediated through both glucagon-like peptide-1 receptor-dependent and -independent pathways. Circulation (2008) 117: 2340-2350. Reference).

The GLP-1 activates TGR5 and GPR119, one of the G protein-coupled receptors (GPCRs) (Reimann, F., et al . , Glucose sensing in L cells: a primary cell study. Cell Metab. (2008) 8: 532-539; Lauffer, LM, et al . , GPR119 is essential for oleoylethanolamide-induced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L-cell. Diabetes (2009) 58: 1058-1066. ) Or activation of α-gustducin (Jang, HJ, et al . , 2007. Gut expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1. Proceeding of the National Academy of Science 104, 1506915074.). In particular, the activation of G protein-coupled receptor (GPCR) TGR5 (GPR131) expressed in brown adipose tissue and muscle increases energy expenditure and results in the treatment of obesity, (See, for example, Lieu T et al., GPBA: AG protein-coupled receptor for bile acids and an emerging therapeutic target for disorders of digestion and sensation. British Journal of Pharmacology (2013) in press) (Pols TWH et al., TGR activation inhibition atherosclerosis by reducing macrophage inflammation. Cell Metabolism (2011) 14, 747).

Next, CCK is synthesized and secreted in I-cells, a kind of intestinal endocrine cells present in the duodenum and jejunum, and CCK secreted from the duodenal mucosa activates CCK-A receptors of vagus nerve fibers Is known to inhibit glucose production, induce an antidiabetic effect, inhibit food intake, bring about an anti-obesity effect, and participate in the regulation of digestion and absorption.

It is known that CCK secretion involves G protein-coupled receptors (GPCRs) and signaling molecules (G proteins, PLCβ2) and transient receptor potential (TRP) channels (FEBS Letters (2008) 582: 229-232).

5-HT is a hormone secreted from enterochromaffin cells (EC cells) in the gastrointestinal tract. It is known that 5-HT is involved in the regulation of digestion and absorption and has an anti-obesity effect through an appetite suppression (Gershon, MD, Tack, J. (2007) The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 132, 397-414).

Gastrointestinal hormone or neuropeptide secretion is known to play a role in maintaining the homeostasis (intestinal motility, secretion of digestive enzymes in the intestines, appetite, etc.) related to food intake and digestion by monitoring ingested food at the stage of digestion , Body homeostasis represents a balance between the energy absorbed and the energy consumed, and if this balance is broken, it can lead to diabetes, cardiovascular disease and the like.

There has been a continuing effort worldwide to develop drugs that are effective in serious diseases such as diabetes and cardiovascular disease. However, it is not easy to develop drugs with very low effects, such as obesity, hepatotoxicity, and edema, which are very effective.

On the other hand, medicines having anti-obesity effect are used for prevention or treatment of obesity, but these drugs are known to cause side effects such as oil stomach, abdominal bloating, dizziness, dryness, constipation, Development of strong natural functional materials is urgently required.

Accordingly, the technical problem to be solved by the present invention is to provide a pharmaceutical or food composition which is safe and has excellent endocrine endothelial cell activity and can exhibit various useful effects because of its low side effect.

In particular, the present invention is directed to a method of treating diabetes, comprising administering an effective amount of at least one of a diabetic therapeutic effect, an appetite suppressive effect, an anti-obesity effect, an improvement of a heart disease, an improvement of digestion and absorption disorder, an improvement of atherosclerosis, Or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for treating diabetes, an effect of treating diabetes, an appetite suppressing effect The present invention provides a method for achieving at least one of an inhibitory effect, an anti-obesity effect, an improvement of a heart disease, an improvement of atherosclerosis, an improvement of a digestive and absorption disorder, a neuroprotective action and a treatment or improvement of a liver disease.

Another object of the present invention is to provide a composition which acts on secretion stimulation of GLP-1, CCK or 5-HT or hTGR5 activation and consequently acts as TRP, TRPA1, G protein or PLCβ2 activator .

In order to accomplish the above object, the present invention provides a method for treating diabetes mellitus comprising: extracting Geum aleppicum Jacq. As an active ingredient and having intestinal endocrine cell activity; Appetite suppressant effect; Anti-obesity effect; Improvement of heart disease; Improving digestive and absorption disorders; Improvement of arteriosclerosis; Neuroprotection; Or a liver disease treating or improving effect.

The inventors of the present invention have completed the present invention by confirming that the extract of a big snake mug is effective for the secretion of endocrine hormone and / or neuropeptide secreted by the activation of intestinal endocrine cells among natural products which have been used for a long time and proved to be safe.

In one embodiment of the present invention, it has been revealed through experimentation that a large snake extract has an effect of increasing insulin gene expression, inhibiting the secretion of glucagon, and lowering blood glucose level, and thus provides a use for prevention and improvement of diabetes mellitus do.

In another embodiment of the present invention, it is demonstrated through experiments that a large snake extract has an appetite suppressing effect and promotes satiety, and provides an anti-obesity effect through inhibition of appetite of a large snake extract.

In another embodiment of the present invention, it was experimentally found that a large snake extract promotes digestive enzyme secretion in the pancreas and small intestine, and controls the intestinal motility and motility, It provides usages.

According to another embodiment of the present invention, it is revealed that a large snake extract has an excellent effect of protecting cardiomyocytes by regulating blood flow to the heart muscle, thus providing a use for the improvement of heart disease of a large snake extract do. More specifically, the large snake extract of the present invention protects cardiac cells from ischemia and can exhibit cardiac function-improving effects in patients suffering from heart attack.

In another embodiment of the present invention, it is confirmed that a large snake extract can prevent atherosclerosis, and the use of the extract for improving arteriosclerosis is provided.

In another embodiment of the present invention, it is revealed that the large snake extract activates the arcuate nucleus in hypothalamus (ARC) of the brain and activates the vagus nerves, Lt; / RTI > More specifically, the large snake extract of the present invention can be expected to treat neurodegenerative diseases and cerebrovascular diseases, treat neuroinflammation, and enhance neurogenesis .

In another embodiment of the present invention, the use of large snake extracts for the treatment or amelioration of liver diseases is provided. More specifically, the large snake extract of the present invention is effective for reducing hepatic inflammation, enhancing enterohepatic blood flow, improving liver function, treating cholestatic liver diseases, pruritus, And painless jaundice treatment, improvement of intestinal functions, and promotion of neurotransmission response can be expected.

In order to accomplish still another technical object, the present invention provides a composition which acts as secretion stimulus or effect of GLP-1, CCK or 5-HT or acts as TRP, TRPA1, G protein, PLCβ2 activator.

Specifically, in one embodiment of the present invention, it is demonstrated through experiments that a large snake extract stimulates GLP-1 secretion, and provides a use for a GLP-1 secretion stimulating effect of a large snake extract.

In another embodiment of the present invention, the activity of GPCRs is stimulated by intracellular calcium concentration and cAMP increase stimulated by CCK secretion, and the use of large snake extracts for CCK secretion stimulating effect is provided .

In another embodiment of the present invention, a large snake extract stimulates 5-HT secretion and induces a change in intracellular calcium concentration through experimentation, and provides a use for 5-HT secretion stimulation of a large snake extract .

In order to achieve still another technical object, the present invention provides a composition that acts as TRP, TRPA1, lipoprotein (G protein) or PLCβ2 activator.

Specifically, in one embodiment of the present invention, it is experimentally demonstrated that a large snake extract activates TGR5, and provides a use for the TGR5 activity effect.

In another embodiment of the present invention, it has been found that a large snake extract increases calcium concentration, and a large snake extract activates TRP and TRPA1 channels through experiments to provide a use for TRP and TRPA1 channel activity . Also, it is revealed that the intracellular calcium ion concentration is increased by activation of the G protein by the large snake extract of the present invention, and the application for the effect of the G protein activity is provided.

In another embodiment, PLCβ2 is activated by a large snake extract to increase intracellular calcium ion concentration and cAMP concentration, thus providing a use for PLCβ2 activity.

The large snake extract of the present invention means an extract obtained by a conventional method in the field of the present invention and may be further purified by methods such as extraction, fractionation, separation and filtration.

For example, the large snake extract according to the present invention may be a solvent extract of a large snake or a dried product thereof. The extraction solvent for the extraction of the large snake may be any solvent which can be used for extracting natural products without limitation, for example, purified water; Lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propyl alcohol and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol and propylene glycol; Hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether and dichloromethane; Or a mixed solvent thereof may be used, but it is preferable to use purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, propyl alcohol, butyl alcohol, or a mixed solvent thereof, and it is preferable to use purified water, ethanol, , And it is more preferable for the purpose of the present invention to use a mixed solvent of purified water and ethanol.

The amount of the extracting solvent may be 10 to 100 times, preferably 20 to 80 times, more preferably 30 to 50 times the weight of the large snake, but is not limited thereto.

Examples of the method for extracting the large snake extract include a conventional extraction method such as stirring, cold extraction, hot water extraction, immersion extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, May be used, and it is preferable to heat and reflux or extract at room temperature. The extraction temperature is preferably 10 to 30 ° C, and the extraction time is preferably 1 to 20 days, more preferably 1 to 5 days, but is not limited thereto.

More specifically, after crushing the large snake mushroom, the extraction solvent is added by 1 to 20 times the volume of the large snake crush, extracted and then selectively (decompression) concentrated, dried or purified. More specifically, an extraction solvent is added to extract an active ingredient substance as in the above-mentioned method, (a) heating at a temperature of 50 to 100 ° C for 1 to 20 hours in a state where a cooling condenser is installed to prevent evaporation of the solvent, (B) immersing the mixture at a temperature of 5 to 37 DEG C for 1 to 15 days to extract the active ingredient.

According to another embodiment of the present invention, the large snake extract for the intestinal endocrine cell activation may be a fraction of a large snake extract.

The large snake fraction of the present invention can be obtained by fractionating said large snake extract (preferably a lower alcohol extract having 1-4 carbon atoms) with a solvent having a lower polarity than water such as hexane, chloroform, butanol and ethyl acetate, But is not limited to, hexane or chloroform.

Particularly, for the purpose of achieving the effect of the present invention, it is preferable that the large snake extract according to the present invention is a hexane or chloroform fraction of a lower alcohol extract having 1-4 carbon atoms.

The fraction may be prepared by suspending or dissolving the large snake extract in a liquid form before drying or in a suitable solvent such as water, ethanol, or a mixed solvent thereof, or by using water such as hexane, chloroform, butanol or ethyl acetate A solvent having a lower polarity may be sequentially added to separate each layer, and then each layer may be obtained by concentration under reduced pressure and / or drying.

At this time, the vacuum concentration is preferably performed using a vacuum rotary evaporator, but not limited thereto.

In addition, " drying " according to the present invention may be vacuum drying, vacuum drying, boiling drying, spray drying, room temperature drying or freeze-drying, However, the present invention is not limited to this drying method.

The extracts of the great snails according to the present invention are glucagon-like peptide-1, GLP-1, GLP-1- (7-36) amide, cholecystokinin (CCK), seretonin Serotonin, 5-hydroxytryptamine, 5-HT), and the like, thereby stimulating the secretion of the endocrine hormone and / or neuropeptide.

Accordingly, the present invention provides a method for treating diabetic nephropathy, comprising administering to a mammal an effective amount of a compound of formula (I), wherein the large snake extract promotes secretion of endocrine hormones and neuropeptides, Improvement of disorder, improvement of arteriosclerosis, neuroprotective effect, or treatment or amelioration of liver disease, and the like.

The present invention also relates to a method of treating diabetes mellitus, comprising administering to a patient in need of such treatment a diabetic therapeutic effect, an appetite suppressing effect, an anti-obesity effect, an improvement of heart disease, an improvement of digestion and absorption disorder, an improvement of arteriosclerosis, A food composition for improvement can be provided.

That is, the composition according to the present invention can be used for medicines, health supplements, food additives, feed additives, and the like, but is not limited thereto.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments. The pharmaceutical composition according to the present invention can be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository and sterilized injection solution according to a conventional method Can be used. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

More specifically, for example, powders can be prepared by simple mixing of an extract of the present invention with an appropriate pharmaceutically acceptable excipient such as lactose, starch, microcrystalline cellulose and the like. The granule is an extract of the present invention; Suitable excipients which are pharmaceutically acceptable; And a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropylcellulose, followed by wet granulation using a solvent such as water, ethanol or isopropanol or dry granulation using a compressive force . Further, the tablet may be prepared by mixing the above granule with a suitable pharmaceutically acceptable salt such as magnesium stearate and then tableting it using a tablet machine.

The large snake composition of the present invention can be administered orally or parenterally depending on the disease to be treated and the condition of the individual, and can be administered orally, injectable (for example, intramuscular, intraperitoneal, intravenous injection, infusion, subcutaneous injection, implant) , Rectal, rectal, sublingual, transdermal, topical, and the like, but is not limited thereto. May be formulated into suitable dosage unit formulations, including those conventionally used and non-toxic, pharmaceutically acceptable carriers, additives, vehicles according to the route of administration. Depot formulations that are capable of sustained release of the drug over a period of time are also within the scope of the present invention.

In order to achieve the object of the present invention, the large snake extract according to the present invention may be administered at a daily dose of about 0.001 mg / kg to about 10 g / kg, and a daily dose of about 0.01 mg / kg to about 1 g / . However, the dosage may vary depending on the degree of purification of the large snake extract, the condition of the patient (age, sex, weight, etc.), severity of the condition being treated, and the like. For convenience, the total daily dose may be divided as needed and divided into several doses throughout the day.

When the large snake extract of the present invention is used as a food composition, the kind of the food is not particularly limited. Examples of foods which can be added with the above snake extract include dairy products including drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen noodles, gums, ice cream, Soups, beverages, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The large snake extract of the present invention can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of the extract in the food may be added in an amount of 0.01 to 50% by weight of the whole food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the food composition according to the present invention is not particularly limited to the other components except for containing the above-mentioned large snake extract as an essential ingredient in the indicated ratios. It is also possible to add various flavors or natural carbohydrates, . Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The flavoring agent may be a natural flavoring agent (tau martin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (for example, saccharin and aspartame).

The large snake extract of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

The present invention also relates to a method for the treatment of diabetic retinopathy, comprising administering to said patient or individual a therapeutically effective amount or an amount effective for improving health of said large snake extract, Prevention or improvement, improvement of digestive and absorption disorders, improvement of arteriosclerosis, neuroprotective action and / or treatment or improvement of liver disease.

That is, the present invention relates to a method for the treatment and / or prophylaxis of a large snake extract having an appetite suppressing effect, an anti-obesity effect, an improvement of a heart disease, prevention or improvement of diabetes, improvement of digestion and absorption disorder, improvement of arteriosclerosis, Provide medicinal or health functional food uses.

The present invention provides a diabetic therapeutic effect, an appetite suppressing effect, an anti-obesity effect, an improvement of heart disease, an improvement of digestion and absorption disorder, an improvement of arteriosclerosis, a neuroprotective action and / or treatment or improvement of liver disease. The pharmaceutical composition or food composition according to the present invention can be used safely and safely with fewer adverse effects by using natural extracts, and has a therapeutic effect on diabetes, an appetite suppressant effect, anti-obesity effect, improvement of heart disease, improvement of digestive and absorption disorder, Protective effect and / or treatment or amelioration of liver disease are excellent.

FIG. 1 is a photograph showing c-Fos activation in Caudal DVC (Dorsal vagal comlpex) by administration of a large snake extract.
Fig. 2 is a photograph showing c-Fos activation in Arcuate nucleus in hypothalamus (ARC) by administration of a large snake extract.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1: Preparation of a large snake extract

The 80% ethanol extract of the big snake used in this experiment was purchased from Jeju Techno Park (prevalence No. JBRI-10611). Specifically, the large branworm outposts other than roots were freeze-dried and then ground, and 50 ml of 80% by weight ethanol was added per 1 g of the sample, followed by extraction at room temperature for 3 days. The extract was filtered twice using a filter paper (Whatman No. 1, England), concentrated with a rotary evaporator (B-480, Buchi, Switzerland), lyophilized again and powdered was used as a sample .

Hereinafter, an experiment showing that the large snake extract obtained by the above method is effective for the secretion of endocrine hormones or neuropeptides, and the results are shown.

Example 2: Results in NCI-H716 cells

1) Cell culture

Human-derived cell lines, NCI-H716 cells, were obtained from the American Type Culture Collection (Manassas, Va.). Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU / ml penicillin, 100 / ml streptomycin.

2) Determination of GLP-1 secretion effect of sample

NCI-H716 cells were cultured in 96-wells at a concentration of 2 x 10 < ⁵ > cells / plate for 3 days. On the day of the experiment, the cells were rinsed with phosphate-buffered saline (PBS), washed with Krebs-Ringer bicarbonate buffer (KRB, 128.8 mmol / l NaCl, 4.8 mmol / l KCl, 1.2 mmol / l KH ₂ PO ₄ , 1.2 mmol / 1 mM MgSO ₄ , 2.5 mmol / l CaCl ₂ , 5 mmol / l NaHCO ₃ , and 10 mmol / l HEPES, 0.2% BSA, pH 7.4) and incubated at 37 ° C for 1 hour. After 1 hour incubation, the supernatant was collected (100 μL) for GLP-1 analysis and analyzed by GLP-1 activity ELISA kit (EGLP-35K, Millipore). The concentration of GLP-1 was calculated according to the supplied GLP-1 standard (pmole / mL), and the result was compared with the concentration secreted by stimulation of GLP-1 secreted by the stimulus of the sample, of control.

3) Measurement of changes in intracellular calcium concentration

Fluorescence-based assay was used to examine changes in intracellular calcium. Cells were collected at a concentration of 4.0 × 10 ⁴ cells / well in a 96-well plate. Fluorometric imaging plate reader (FLIPR ^™ ) was prepared using a calcium-5 assay kit purchased from Molecular Devices, Intracellular Ca ²⁺ concentration was measured using Flexstation III (Molecular Devices Co., Sunnyvale, Calif., USA). Fluorescence intensity of calcium stained for 120 seconds after injection of sample was measured at 488 nm excitation and 525 emmision. The results were as follows: maximum fluorescence value - minimum fluorescence value = relative fluorescence value And was automatically calculated as a fluorescence unit (delta relative fluorescent units (? RFU)).

Activation of the GPCRs activates the G protein complex (Gα-gustducin, Gα-transtducin, Gα14, Gβ3, Gβ1, Gγ13), which activates the phospholipase to activate the type Ⅲ IP3-receptor, it is possible to measure the activation of GPCRs by measuring the change of intracellular Ca ²⁺ ion concentration because it causes increase of Ca ²⁺ concentration in cytoplasm.

In addition, when TRP channels are activated, the calcium ions outside the cell membrane enter the cells, so that the measurement of the intracellular Ca ^{2 +} ion concentration may be able to measure the activation of TRP channels.

4) cAMP measurement method

NCI-H716 cell line in 24-well 1 x 10 ⁶ cells / mL concentration of culture and then cultured for 3 days. Experiment were cultured in the same day a sample and with 5% CO ₂ incubator in ℃ 37 in KRB buffer, the reaction medium is removed and cells are subjected to back washing with water three times to process the KRB buffer into 0.1N HCl 0.5mL 150 rpm, at room temperature The reaction was carried out for 20 minutes. The reacted cells were centrifuged at 1000 g for 10 minutes, and the supernatant was obtained. The cyclic AMP kit using enzyme immunoassay (EIA) was used.

5) Results

The concentrations of GLP-1 and cAMP were determined according to the EIA method. Changes in intracellular calcium concentration were expressed in relative fluorescence units (ΔRFU). All values were expressed as mean ± SEM (n = 9) in triplicate experiments. * p &lt;0.05; ** p &lt;0.01; *** p <0.001 vs control (control group)

2-1. GLP-1 secretion effect of 80% ethanol extract of large snake mug in NCI-H716 cells

density GLP-1 secretion
(fold of control) Control group 1.000 + 0.088 Large bananas (80% ethanol extract) 500 μg / mL 2.396 + - 0.226 *

As shown in Table 1 above, the large snakebug (80% ethanol extract) showed GLP-1 secretion in NCI-H716 cells. The effect of GLP-1 secretion from intestinal endocrine cells in the large snake (80% ethanol extract) stimulates glucose-dependent insulin secretion, insulin gene expression, pancreatic beta cell proliferation, pancreatic beta cell survival, Glucagon secretion inhibition effect, blood glucose lowering effect, etc., it can be expected to be effective in the treatment of diabetes.

It also has the effect of treating obesity by slowing the rate at which the stomach is emptied, suppressing the appetite, promoting the satiety and suppressing the food intake, and protecting the cardiomyocytes from ischemia and heart attack It is thought that it is possible to control the digestion and absorption by improving digestive enzyme secretion in the pancreas and small intestine.

It is also believed that the extract can be used to treat neurodegenerative diseases and cerebrovascular diseases, to treat neuroinflammation, and to enhance neurogenesis.

In addition, it has been shown that treatment of hepatic inflammation, enhancement of enterohepatic blood flow, improvement of liver function, treatment of cholestatic liver diseases, treatment of pruritus and painless jaundice , Intestinal functions, and neurotransmitter - stimulating effects, which may be useful for the improvement of liver disease.

In addition, the extract is believed to be effective in improving arteriosclerosis.

2-2. NCI - H716  In a cell Big snake  Of 80% ethanol extract Intracellular Ca ^{2 +}  Concentration change

density Ca ^{2 +} reaction
(? RFU) Control group 9.181 ± 0.632 Large bananas (80% ethanol extract) 500 μg / mL 35.003 ± 1.984 ** glucose 100 mM 10.240 + - 0.352 **

According to the results shown in Table 2, 500 μg / mL of a large snake-berry extract of ethanol extracts significantly increased intracellular calcium concentration compared with the control group. The increase in intracellular calcium concentration is thought to be due to activation of GPCRs and activation of calcium ion.

Therefore, it is considered that the large snake extract of the present invention has an effect of increasing insulin gene expression, inhibiting the secretion of glucagon, and lowering blood glucose, thus being effective for the treatment of diabetes, and having an appetite suppressing effect, I think.

In addition, it is believed that the effect of large snake mugwort extract on cardiomyocyte can be controlled by regulating intestinal motility and mobility to improve digestion and absorption of large snake mugwort extract and to protect cardiac muscle cells.

2-3. NCI - H716  In a cell Big snake  Of 80% ethanol extract Intracellular cAMP  Increase effect

sample density cAMP
(fold of control) Control group 1.000 ± 0.100 Large bananas (80% ethanol extract) 500 μg / mL 13.365 ± 3.321 * OEA (oleoylethanolamide) 20 μM 6.813 + - 1.634 *

As shown in Table 3 above, the larger ethanol extract of Snwumu than the control group was found to be effective in increasing intracellular cAMP. In other words, by measuring the cAMP concentration which is highly related to calcium concentration, it was judged that the large snake extract affects the increase of GLP-1 secretion. Therefore, it seems to be effective for obesity treatment such as appetite suppression and satiety improvement. It is believed that it will show a cardioprotective effect through cardioprotective action, and it is thought to be effective for the treatment of diabetes by increasing insulin secretion.

<Example 3> Measurement of TGR5 activity

1) Cell culture

CHO-K1 cells were cultured in Ham's F-12K medium containing 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin, .

2) Measurement of TGR5 activity

The hTGR5 plasmid DNA was purchased from Origene, Inc. The transcript variant 3 was purchased from Origene Inc., and the vector was pCMV6-XL5 (4.5 kb), and the insert, GPBAR1 (SEQ ID NO: TGR5) was 1.4 Kb. In order to obtain the TGR5 plasmid, plasmids were obtained using Invitrogen Purelink (High pure filter maxiprep kit) after transfection into transformed E. coli containing 100 μg / ml ampicillin. 400 ng / well of TGR5 expression plasmid (pCMV6-XL5 / TGR5) was transfected with CHO-K1 in 100 ng / well CRE-driven luciferase reporter plasmid using Neon ^™ (Invitrogen, Cergy Pontoise, France) after reaction by treating the sample for a time to measure the luciferase activity using a ^{dual-glo TM luciferase assay system kit} (promega). Luminescence was obtained using Victor 3 ^TM (PerkinElmer).

3) Results

3-1. CHO - K1  To the cell hTGR5 To Transfection  In the cell Big snake  Of 80% ethanol extract TGR5  Activity enhancement effect

CHO-K1 cells transfected with the TGR5 plasmid were used, and a large snake-like 80% ethanol extract was treated for 5 hours. TGR5 activity was assessed by Luciferase assay as mentioned in the previous method and all values are expressed as means ± SEM. n = 9. * p <0.05, *** p <0.001 vs control (control group)

sample density TGR5 activity measurement
(fold of control) Control group 1.000 ± 0.120 Large bananus (80% ethanol extract) 100 μg / mL 2.012 ± 0.332 * Lithocholic acid (LCA) 5 [mu] M 3.483 ± 0.119 ***

As shown in Table 4 above, the activity of TGR5 was increased by the extract of 80% ethanol of the great snake mushroom in the cells transfected with hTGR5. Therefore, it is thought that the large snake extract will be effective for treating obesity such as appetite suppression and satiety improvement, and it is believed that it will show the effect of improving heart disease through cardiac cell cooperating action, and it will be effective for treating diabetes by increasing insulin secretion do.

In addition, it is thought that a large snake extract can improve the digestive and absorption disorders by controlling the intestinal motility and motility.

It is also believed that the extract can be used to treat neurodegenerative diseases and cerebrovascular diseases, to treat neuroinflammation, and to enhance neurogenesis.

In addition, it has been shown that treatment of hepatic inflammation, enhancement of enterohepatic blood flow, improvement of liver function, treatment of cholestatic liver diseases, treatment of pruritus and painless jaundice , Intestinal functions, and neurotransmitter - stimulating effects, which may be useful for the improvement of liver disease.

In addition, the extract is believed to be effective in improving arteriosclerosis.

Example 4 CCK secretion experiment using STC-1 cells

1) Cell culture

STC-1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and maintained at 37 ° C and 5% CO ₂ .

Fetal serum (FBS), penicillin-streptomycin, and Dulbecco's modified eagle medium (DMEM) used for culture were purchased from Gibco.

2) CCK secretion measurement method

Cells were inoculated into a 24-well plate at a concentration of 2 × 10 ⁵ cells / well per well and incubated at 37 ° C. in 5% CO ₂ Lt; / RTI &gt; After 72 hours, the cells were washed twice with HEPES buffer (4.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, 140 mM NaCl, and 20 mM Hepes-Tris, pH 7.4) The reaction was carried out for 60 minutes. After completion of the reaction, the culture medium was collected and centrifuged at 10000 rpm for 10 minutes. The obtained supernatant was stored at -70 ° C for analysis. The kit used in the experiment is the enzyme immunoassay (EIA) kit (Phoenix Pharmaceuticals Inc., Belmont CA).

3) Measurement of intracellular calcium response

Poly-D-Lysine cotead black 96 -well clear-bottom plates (Thermo) to 8.0 x 10 ⁴ cells / well, and inoculated at a concentration of 37 ℃, 5% CO ₂ Lt; / RTI &gt; After 72 hours and incubated 60 min at 37 ° C with 5-Calcium assay kit (Moelcular device) to measure the Ca ^{2 +} reaction using a Flexstation III. Fluorescence wavelengths were 488 nm (ex) and 525 (em) for about 120 seconds. Compound injection was performed for 20 seconds and reaction was observed for 2 seconds.

4) Results

CCK  Secretory effect

4-1. STC -1 cells Big snake  By 80% ethanol extract CCK Secretory effect

sample density Fold of control
(mean ± SEM) t-test P-Value Control group 1.00 + - 0.01 80% ethanol extract of large snake mug 100 μg / ml 20.37 ± 3.94 ** 0.008 250 μg / ml 33.12 ± 3.40 *** <0.001 500 μg / ml 60.49 + - 2.21 *** <0.001 AITC 0.1 mM 21.57 ± 0.59 *** <0.001 1 mM 21.99 + - 0.64 *** <0.001 10 mM 25.59 ± 0.59 *** <0.001

Fold of control = sample value / untreated control value

± S.E.M. (n = 9) ** P <0.01, *** P <0.001 vs. Control (1% DMSO)

As shown in Table 5 above, large snake-mucilage 80% ethanol extracts in STC-1 cells were found to have an effect on CCK secretion activation.

4-2. STC -1 cells Big snake  By 80% ethanol extract CCK  Effects of inhibitors on secretion (1)

STC-1 cells were pretreated with 100 [mu] M ruthenium red or 50 [mu] M HC-030031 for 30 min. After adding 500 μg / ml of the extract, the cells were incubated at 37 ° C for 60 minutes. ± S.E.M. (n = 9) ** P <0.05, *** P <0.001 vs. Control group

sample density Inhibitor Fold of control
(mean ± SEM) t-
test P-
Value Inhibition
(%) Control group 1% DMSO 1.00 + 0.04 Big snake
80% ethanol extract 500
μg / ml No treatment 34.22 + - 0.98 RR 12.22 + - 0.41 *** <0.001 64.29 HC-030031 29.44 + - 0.82 * 0.02 13.97  AITC 0.1
mM No treatment 22.56 ± 0.65 RR 6.04 ± 0.73 *** <0.001 73.21 HC-030031 10.70 ± 0.92 *** <0.001 52.58

When the TRP antagonist, TRP antagonist, HC-030031, was cultured with the extract of 80% ethanol of the big snake, the CCK secretion by the extract of 80% ethanol of the big snake was inhibited by 47.89%, 6% . This means that the large snake-mulberry 80% ethanol extract acts as TRP agonist and TRPA1 agonist to secrete CCK.

Therefore, it is considered that the large snake extract of the present invention has an effect of increasing insulin gene expression, inhibiting the secretion of glucagon, and lowering blood glucose, thus being effective for the treatment of diabetes, and having an appetite suppressing effect, I think.

In addition, it is believed that the effect of improving the digestive and absorption disorders of the large snake mug extract may be controlled by controlling the long-axis contractility and mobility.

4-3. STC -1 cells Big snake  By 80% ethanol extract CCK Effect of Inhibitor on Secretion (2)

STC-1 cells were pretreated with 400 μM TPPO or 20 μM U-73122 for 30 min.

After adding 500 μg / ml of the extract, the cells were incubated at 37 ° C for 60 minutes.

Fold of control = sample value / untreated control value

± SEM (n = 9) *** P <0.001 vs. No treatment

sample density Inhibitor Fold of control
(mean ± SEM) t-test P-Value Inhibition (%) Control group 1% DMSO 1.00 + 0.07 Big snake
80% ethanol
extract 500
μg / ml No treatment 30.49 + - 0.45 TPPO 21.19 ± 0.24 *** <0.001 30.50 U-73122 14.35 ± 0.27 *** <0.001 52.93 DB 10
mM No treatment 25.01 + - 0.77 TPPO 17.05 + 0.03 *** <0.001 31.83 U-73122 15.53 + - 0.01 *** <0.001 37.92

As shown in Table 7 above, CCK secretion by the extract of 80% ethanol from the large snake mushroom was 30.50% and 52.93%, respectively, when the TPPO and PLCβ2 inhibitor, U-73122, It was hindered. This suggests that the large snakebug 80% ethanol extract acts as a chaperone activator and / or a PLCβ2 activator to secrete CCK.

Therefore, it is considered that the large snake extract of the present invention has an effect of increasing insulin gene expression, inhibiting the secretion of glucagon, and lowering blood glucose, thus being effective for the treatment of diabetes, and having an appetite suppressing effect, I think.

In addition, it is thought that the large snake extract has an effect of improving the digestive and absorption disorders by controlling the intestinal motility and motility.

Intracellular  Calcium reaction

4-4. STC -1 cells Big snake  80% ethanol extract Ca ^{2 +}  Effect on reaction

sample density ΔRFU
(mean ± SEM) t-test P-Value Control group 0.5% DMSO 75.93 + - 4.26 Big snake
80% ethanol extract 100 μg / ml 148.82 + - 4.69 ** 0.006 250 μg / ml 170.79 ± 2.46 *** 0.001 500 μg / ml 206.37 ± 5.37 *** <0.001 AITC 0.1 mM 105.41 ± 2.12 ** 0.003 1 mM 151.04 + - 5.65 *** <0.001 10 mM 168.68 ± 5.79 *** <0.001

± S.E.M. (n = 9) ** P <0.01, *** P <0.001 vs. The control (0.5% DMSO)

As shown in Table 8 above, it was found that the extract of 80% ethanol of the great snake mug was affected on the calcium ion secretion in a concentration-dependent manner. From the above results, it was thought that the large snake extract had an effect on the activation of GPCRs and had an antidiabetic effect, an appetite suppression effect, an anti-obesity effect, and a digestive and absorption disorder control effect.

4-5. STC -1 cells Big snake  By 80% ethanol extract Ca ^{2 +}  Effect of Inhibitor on Reaction (1)

STC-1 cells were pre-treated with 100 [mu] M ruthenium red or 50 [mu] M HC-030031 for 60 minutes. Then, 1 mg / ml of the extract was treated. +/- S.E.M. (n = 9) *** P < No treatment

sample density Inhibitor ΔRFU
(mean ± SEM) t-test P-Value Inhibition (%) Control group 0.5% DMSO 34.34 + 1.63 Big snake
80% ethanol
extract One
mg / ml No treatment 137.18 ± 3.10 RR 17.25 + - 2.77 *** <0.001 87.43 HC-030031 39.31 + - 1.02 *** AITC 0.1
mM No treatment 400.52 + - 6.90 RR 27.47 ± 1.77 *** <0.001 93.14 HC-030031 66.07 ± 2.56 *** <0.001 83.50

According to the results shown in Table 9, TRP antagonist RR, TRPA1 antagonist HC-030031 a large baemmu 80% when incubated with the ethanol extract of 52.68% of the Ca ^{2 +} secretion by greater baemmu 80% ethanol extract, respectively, 10.19 %. This result suggests that the extract of 80% ethanol of the large snake bruit acts as a TRP agonist and a TRPA1 agonist, resulting in the activation of TRP channels and the influx of calcium ions outside the cell membrane into the cells.

4-6. STC -1 cells Big snake  By 80% ethanol extract Ca ^{2 +}  Effect of Inhibitor on Reaction (2)

STC-1 cells were pretreated with 400 μM TPPO or 20 μM U-73122 for 60 min.

Then, 1 mg / ml of the extract was treated. +/- S.E.M. (n = 9) *** P < No treatment

sample density Inhibitor ΔRFU
(mean ± SEM) t-
test P-
Value Inhibition
(%) Control group 0.5% DMSO 71.14 ± 7.10 Big snake
80% ethanol
extract One
mg / ml No treatment 219.36 + - 31.64 TPPO 126.38 ± 16.25 ** 0.853 U-73122 22.23 + - 2.35 ** 0.003 89.37 DB 10
mM No treatment 217.79 ± 15.99 TPPO 100.56 ± 2.16 ** 0.002 53.83 U-73122 130.60 ± 6.04 ** 0.007 40.03

According shown in Table 10, lipoprotein inhibitor of TPPO, PLCβ2 inhibitor U-73122 a large baemmu 80% when incubated with the ethanol extract each have Ca ^{2 +} secretion by greater baemmu 80% ethanol extract of 20.33%, 78.80%. This is thought to be caused a large baemmu increase in Ca ^{2 +} concentrations in the 80% ethanol extract of cytoplasm acts lipoprotein activator and / or zero PLCβ2 enable them the big baemmu extract over was found that affects the GPCRs activated .

The results shown in Tables 9 and 10 suggest that the large snake extract has an antidiabetic effect, an appetite suppressant effect, an anti-obesity effect, a heart disease improving effect, and a digestive and absorption disorder-modulating effect.

4-7. STC -1 cells Big snake  Effect of 80% Ethanol Extract on Cell Membrane Potential Changes

sample density ΔRFU
(mean ± SEM) t-test P-Value Control group 0.5% DMSO 59.12 ± 2.09 80% ethanol extract of large snake mug 500 μg / ml 248.80 ± 3.25 *** <0.001 KCl 0.1 mM 17.01 + - 0.93 *** <0.001 1 mM 35.08 ± 3.43 ** 0.004 10 mM 270.08 + - 9.57 *** <0.001

± S.E.M. (n = 3-4) ** P <0.01, *** P <0.001 vs. The control (0.5% DMSO)

According to the results shown in the above Table 11, the extract of 80% ethanol of the big snake had an effect on the cell membrane potential change. These results suggest that the activation of GPCRs may activate the intracellular ion migration. Therefore, it is thought that the large snake extract activates GPCRs to increase GLP-1 secretion, and may have a therapeutic effect on diabetes, a cardioprotective effect, an appetite suppressant effect, an anti-obesity effect, and a digestive and absorption control effect.

Example 5 Measurement of 5-HT using RIN14B cells

1) Cell culture

RIN 14B cells were cultured in RPMI medium 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and incubated at 37 ° C and 5% CO ₂ .

Fetal serum (FBS), penicillin-streptomycin, and Dulbecco's Modified Eagle Medium (DMEM) used for culture were purchased from Gibco.

2) 5-HT secretion measurement method

Cells were inoculated into a 24-well plate at a concentration of 2 × 10 ⁵ cells / well per well and incubated at 37 ° C. in 5% CO ₂ Lt; / RTI &gt; After 72 hours, the cells were washed twice with 5-HT assay buffer (HBSS, 0.1% BSA, 1.25 mM CaCl 2, 2 μM flocetin, pH 7.4) and the material was added to 5-HT assay buffer and reacted at 37 ° C. for 60 minutes. After the reaction, the medium was collected and centrifuged at 10,000 rpm for 10 minutes. The obtained supernatant was stored at -70 ° C for analysis. The kit used for the experiment was the Serotonin (EIA) kit (Enzo Life Sciences)

3) Measurement of intracellular calcium response

Cells were inoculated at 5.0 × 10 ⁴ cells / well in black 96-well clear-bottom plates (Corning) and cultured at 37 ° C and 5% CO ₂ . After 48 hours and incubated 60 min at 37 ° C with 5-Calcium assay kit (Moelcular device) to measure the Ca ^{2 +} reaction using a Flexstation III. Fluorescence wavelengths were 488 nm (ex) and 525 (em) for about 120 seconds. Compound injection was performed for 20 seconds and reaction was observed for 2 seconds.

4) Results

5-1. RIN14B  In a cell Big snake  The effect of 5- HT  Secretory effect

sample density Fold of control
(mean ± SEM) t-test P-Value Control group 1% DMSO 1.00 + - 0.26 80% ethanol extract of large snake mug 100 μg / ml 10.54 + - 0.36 *** <0.001 250 μg / ml 24.46 ± 2.14 *** <0.001 500 μg / ml 33.29 + - 0.38 *** <0.001 AITC 300 μM 8.11 ± 2.70

Fold of control = sample value / untreated control value

Values are presented as mean ± SEM (n = 9) *** P <0.001 vs. Control group

According to the results shown in Table 12 above, the large snake extract has an effect on the secretion of 5-HT known to have an effective digestion-absorption regulating action and an anti-obesity effect through appetite suppression.

5-2. RIN14B  In a cell Big snake  The effect of 5- HT  Influence of inhibitors on secretion

RIN14B cells were pretreated with 100 [mu] M ruthenium red or 50 [mu] M HC-030031 for 30 min. After adding 500 μg / ml of the extract, the cells were incubated at 37 ° C for 60 minutes

Fold of control = sample value / untreated control value

Values are presented as the mean ± SEM (n = 3-4)

sample density Inhibitor Fold of control
(mean ± SEM) t-test P-Value Inhibition (%) Control group 1% DMSO 1.00 + - 0.19 Big snake
80% ethanol
extract 500
μg / ml No treatment 35.34 ± 4.03 RR 12.20 ± 6.45 65.49 HC-030031 17.38 ± 9.21 50.82  AITC 300uM No treatment 8.11 ± 2.70 RR 3.60 ± 1.80 55.56 HC-030031 9.02 + - 6.37 -11.27

When the TRP antagonist RR and the TRPA1 antagonist HC-030031 were cultured together with the extract of 80% ethanol of the big snake mucilage, the 5-HT secretion by the extract of 80% ethanol of the big snake was 65.49%, 50.82% %. This result suggests that the extract of 80% ethanol of the large snake bruit acts as a TRP agonist and a TRPA1 agonist, resulting in the activation of TRP channels and the influx of calcium ions outside the cell membrane into the cells.

5-3. RIN14B  In a cell Big snake  80% ethanol extract Ca ^{2 +}  Effect on reaction

sample density ΔRFU
(mean ± SEM) t-test P-Value Control group 0.5% DMSO 3.34 ± 0.44 Big snake
80% ethanol extract 100 μg / ml 11.08 + - 0.74 *** <0.001 250 μg / ml 25.24 ± 0.23 *** <0.001 500 μg / ml 37.04 + - 1.28 *** <0.001

The reaction of each well was determined by ΔRFU.

P < 0.05, ** P < 0.01, *** P < 0.001, etc.). Control group

As shown in Table 14 above, the RFU value of RIN14B cells treated with the large snake extract showed a larger value than that of the untreated control group. From these results, it is suggested that large snake extracts affect channel activation in TRP involved in CCK secretion.

5-4. RIN14B  In a cell Big snake  By 80% ethanol extract Ca ^{2 +}  Effect of Inhibitor on Reaction

RIN14B was pretreated with 100 μM ruthenium red or 50 μM HC-030031 for 60 min followed by addition of 500 μg / ml of extract.

± S.E.M. (n = 9) ** P <0.01, *** P <0.001 vs. No treatment

sample density Inhibitor ΔRFU
(mean ± SEM) t-test P-Value Inhibition (%) Control group 0.5% DMSO 3.61 ± 0.15 Big snake
80% ethanol
extract 500
μg / ml No treatment 24.99 + - 0.88 RR 12.68 + - 2.41 ** 0.003 49.24 HC-030031 11.11 ± 0.92 ** 0.003 AITC 300uM No treatment 13.42 ± 1.08 RR 4.51 ± 1.40 ** 0.005 66.36 HC-030031 3.98 ± 0.80 *** <0.001 70.31

According to the results shown in Table 15, TRP antagonist RR, TRPA1 antagonist HC-030031 a large baemmu 80% when incubated with the ethanol extract of 52.68% of the Ca ^{2 +} secretion by greater baemmu 80% ethanol extract, respectively, 10.19 %. This result suggests that the extract of 80% ethanol of the large snake bruit acts as a TRP agonist and a TRPA1 agonist, resulting in the activation of TRP channels and the influx of calcium ions outside the cell membrane into the cells.

&Lt; Example 6 > Animal experiment using a large snake extract

2) Glucose tolerance tests; 3) Gastric emptying rate; and 4) Food intake measurement. The results are shown in Table 1 below. 5) Chloride secretion effect experiment was performed.

(1) Experimental method

1) Activation of the central nervous system by immunological staining of brain tissue

Large snake extracts were administered into the stomach and c-Fos immunostaining was performed to analyze brain area activation. After fasting for 12 hours, the animals were orally administered the large snake extract (500 mg / kg BW) dissolved in distilled water and left for 2 hours. Then induce deep anesthesia with urethan. 200 mL of cold PBS is perfused through the heart, followed by 300 mL perfusion of 4% paraformaldehyde, then the brain is harvested and stored in 4% paraformaldehyde for 4 hours at 4 ^° C. Then, it was transferred to a 30% sucrose solution and sufficiently submerged at 4 ^° C. After the fixation, brain tissue is frozen using OCT solution. The frozen tissue is cut from the target area at a thickness of 30 μm at -20 ^° C. The target areas are Arcuate nucleus in hypothalamus (ARC) and Dorsal vagal complex (DVC). The cut tissue is transferred to PBS and washed with PBS. The tissue is immersed in 0.3% H ₂ O ₂ for 15 minutes and then washed three times with PBS. The tissue is incubated in PBS containing 3% normal goat serum for 30 minutes. The tissue was diluted with PBST containing 0.3% normal goat serum at a ratio of 1: 1000 with polyclonal rabbit c-Fos antibody (sc-25, Santa Cruz Biotech, USA) without washing and reacted at room temperature for 24 hours. After the reaction, the tissue is washed with PBST three times for 10 minutes. Then, the second anti-rabbit antibody (BA-1000, Vector, USA) was reacted at room temperature for 30 minutes and washed three times for 5 minutes. This tissue was reacted with avidin-giotinyated horseradish peroxidase complex (ABC) (PK-6100, Vector, USA) for 30 minutes at room temperature, washed with PBST three times for 5 minutes each, and then with DAB kit (Zytomed Systems, Germany) After color development, the reaction is terminated with PBS. The dyed tissue is transferred to a slide, dried thoroughly, and dehydrated. After completion of the dehydration reaction, cover slides are covered with a microscope.

After staining, the target area was determined according to the coordinates of Paxinos and Watson using a microscope (AXio 4.8.1, Zeiss, Germany), and the number of nuclei stained with c-Fos antibody on slides 3-6 And the average value is obtained.

2) In rats Oral glucose tolerance  Assessing Impact on Testing ( Glucose tolerance tests )

Sprague-Dawley rats (male, about 400 g body weight) are fasted for 16 hours and then administered orally with large snake extract (500 mg / kg BW) simultaneously with glucose (2 g / kg body weight). The tail tip was cut at 0, 30, 60, 90, and 120 minutes after the administration, and the blood was collected and the blood glucose level was measured using an Aquacheck blood glucose meter (manufactured by Roche). Glucose (2 g / kg body weight) was administered as a control group.

3) Gastric emptying rate  (Slowing the rate at which the stomach is emptied)

Gastric emptying rate was measured using the method of Izbeki and Doihara (1,2). The rats used in this experiment were fasted for one day before the start of the experiment and the large snake extracts (100, 250, and 500 mg / kg BW) were dissolved in distilled water and allowed to stand for 30 minutes after oral administration. Twenty minutes after oral administration of 0.05% phenol red solution (1.5 mL) to rats, the stomach is removed after cervical dislocation, washed in saline and homogenized in 0.1 N NaOH (100 mL). After stabilization at room temperature for 1 hour, the supernatant (5 mL) is mixed with 20% trichloroacetic acid (0.5 mL) and centrifuged at 3000 rpm at 4 ^° C for 20 minutes. The supernatant was mixed with 0.5 N NaOH (4 mL) and absorbance was measured at 560 nm by UV-spectrophotometer. The control group was administered with distilled water orally.

references

(1) F. Izbeki, T. Wittmann, S. Csati, J. Lonovics. 2004. The mechani of the inhibitory effect of ethanol on gastric emptying involve type A CCK receptors. Regulatory Peptides. 117: 101-105

(2) H. Doihara, K. Nosawa, E. Kawabata-Shoda, R. Kojima, T. Yokoyama, H. Ito. 2009. TRPA1 agonists delay gastric emptying in rats through serotonergic pathways. Naunyn-Schmied Arch Pharmacol. 380: 353-357

Gastric emptying rate was calculated as follows.

Gastric emptying rate (%) = 100 (A / B) x 100,

A: The amount of phenol red remained in the gastrointestinal tract 20 minutes after the oral administration of phenol red solution

B: The amount of phenol red in the gastrointestinal tract immediately after oral administration of the phenol red solution of the control group

4) Food intake  Measure

In order to administer large snake extracts into the stomach and to measure food intake, rats were fasted one day before the start of the experiment. (500 mg / kg) was dissolved in distilled water and the food intake was measured 120 minutes after oral administration (3). The control group was administered with distilled water orally.

references:

(3) A. Serrano, FJ. Pav, S. Tovar, F. Casanueva, R. Se, C. Di, FR Fonseca. 2011. Oleoylethanolamide: Effects on hypothalamic transmitters and gut peptides regulating food intake. Neurohparmacology 60: 593-601

5) Chloride  Secretory effect experiment

To determine the chloride content in the serum of the rats, 5 rats per group were sacrificed The extract (500 mg / kg BW) was orally administered and orbital blood was collected 30 minutes later. Blood was left at room temperature for 30 minutes, centrifuged at 3,000 rpm for 15 minutes, and the serum was separated and measured using a Chloride Assay Kit (BioAssay Systems, USA).

(2) Experimental results

1) Experimental results of central activation by immunostaining of brain tissue

Large snake extracts were injected into the stomach and brain activation was analyzed by c-Fos immunostaining. When GLP-1, CCK, and 5-HT are secreted from the intestine by stimulation of the large snake extract, they are secreted into the blood and function as hormones and also as neuropeptides. GLP-1, CCK, and 5-HT act as hormones directly activating the brain's Arcuate nucleus in hypothalamus (ARC). In addition, GLP-1, CCK, and 5-HT activates the vagus nerves and activates the dorsal vagal complex (DVC), which in turn activates the arcuate nucleus in hypothalamus (ARC) And the central body acquires this information and controls the physical activity by sending commands to peripheral tissues related to diabetes and anti-obesity (Yi CX and Tschop MH, Brain-gut-adipose-tissue communication pathways at a glance. & Mechanisms, 5, 583, 2012).

As a result, the large snake extract had an effect of activating DVC and ARC (see FIGS. 1 and 2). This suggests that GLP-1, CCK, and 5-HT secrete various antitubercular activities related to antidiabetic and anti-obesity.

2) In rats Oral glucose tolerance  Impact Assessment on Testing Anti-diabetic  Effect check)

As a result, In the extract, the increase in blood glucose level after 30 minutes by glucose load was 40.8% lower than that of control group by 102.8 mg / dl compared with 60.8 mg / dl in the control group. The increase of blood glucose level after 60 minutes was 67.7 mg / The area under the curve (AUC) was lowered by 20.8% to 13841 mg / dl min compared to 17465 mg / dl min in the control group, which is shown in Table 16.

Effects of large snake extracts on blood glucose elevation induced by oral glucose tolerance in rats Experimental group ^{1 )} Blood glucose level per hour ( mg / dL ) AUC ^{2 )} 0 minutes 30 minutes 60 minutes 90 minutes 120 minutes Control group ^{3 )} 65.3 ±
5.9 168 ±
22.9 176 ±
14.7 130 ±
15.1 121 ±
11.2 17465 ±
1500  Large snake extract 64.2 ±
5.1 125 ±
9.6 132 ±
11.2 121 ±
7.1 98 ±
5.2 13841 ±
984

¹⁾ Values are mean ± SD (n = 10).

²⁾ AUC: Area under the curve (area under the blood glucose level rise curve for 120 minutes after glucose load, in units of minmg / dl)

³⁾ Glucose alone group

3) Gastric emptying rat  (The rate at which the stomach is emptied) Anti-obesity  Effect check)

100, 250, and 500 mg / kg of the large snake extract decreased the rate at which the stomach was emptied (see Table 17). This may prolong the time the food stays on, thereby reducing the food intake and thus producing an anti-obesity effect.

The effect of the large snake extract administered slows the rate at which the stomach is emptied Experimental group Oral dose (mg / kg BW) Gastric emptying rate (%) Control group - 90 ± 7.4 Large snake extract administered group 100 73 ± 4.9 250 36 ± 2.3 500 29 ± 0.8

Twenty minutes after administration of phenol red, the amount of phenol red remaining in the stomach was measured to determine the rate at which the stomach was emptied.

Control group was treated with distilled water. Values are means SEM ( N = 6).

As shown in Table 17, it was found that the rate of emptying the stomach is low in the dose-dependent manner of the extract group administered with the large snake extract. This can prolong the time the food stays on and thus produce an anti-obesity effect.

4) Food intake  Measure ( Anti-obesity  Effect-related experiments)

Big snake The effect of the extract on stomach intake after 120 minutes of ingestion was measured. As a result, the large snake extract reduced the dietary intake by 79.5% (see Table 18 below).

Effects of large snake extract administration on the dietary intake of rats Experimental group Oral dose (mg / kg BW) Dietary intake (g) for 2 hours Control group - 1.9 ± 0.18 Large snake extract administered group 500 0.39 + - 0.05

According to the results shown in the above Table 18, the dietary intake for 2 hours was much lower in the large snake extract administered group than in the control group. These results suggest that the anti - obesity effect in the group treated with the large snake extract was superior.

5) Chloride  Effect on secretion (related to digestion promoting effect)

In order to indirectly evaluate the effects of large snake extract administration on gastric acid secretion, the concentration of chloride in serum of rats was measured. After 30 minutes of oral administration of large snake extract (500 mg / kg BW), the concentration of chloride in the blood was measured. As a result, administration of large snake extract increased the chloride concentration in the serum by 33.6% Reference). This suggests that hydrochloric acid due to gastric acid stimulation is secreted into the blood, which can be expected to promote digestion.

Effect of large snake extract on serum chloride concentration in rats Experimental group Oral dose (mg / kg BW) Serum chloride concentration (mg / dL) Control group - 265 ± 19.1 Large snake extract administered group 500 354 ± 18.5

According to the results shown in Table 19, it was found that the serum chloride concentration in the rats treated with the large snake extract was higher. Therefore, from the above results, it was thought that a large snake extract promotes gastric acid secretion and promotes digestion.

Claims (18)

Geum aleppicum ) extract as an active ingredient for the prevention or amelioration of diabetes. A pharmaceutical composition for improving or treating a heart disease, comprising a large snake extract as an active ingredient. A pharmaceutical composition for improving or treating digestive disorders or absorption disorders comprising a large snake extract as an active ingredient. An anti-obesity or an appetite suppressing pharmaceutical composition comprising a large snake extract as an active ingredient. A pharmaceutical composition for neuroprotection comprising a large snake extract as an active ingredient. A pharmaceutical composition for the treatment or amelioration of liver diseases comprising a large snake extract as an active ingredient. A pharmaceutical composition for treating or improving arteriosclerosis comprising a large snake extract as an active ingredient. [Claim 7] The pharmaceutical composition according to any one of claims 1 to 7, wherein the large snake extract stimulates the secretion of GLP-1, CCK or 5-HT. A food composition for prevention or improvement of diabetes comprising a large snake extract as an active ingredient. A food composition for improving or treating heart disease, comprising a large snake extract as an active ingredient. A food composition for improving or treating digestive disorders or absorption disorders comprising a large snake extract as an active ingredient. An antiobesity or an appetite suppressing food composition comprising a large snake extract as an active ingredient. A food composition for nerve protection comprising a large snake extract as an active ingredient. A food composition for the treatment or improvement of liver diseases comprising a large snake extract as an active ingredient. A food composition for the treatment or improvement of arteriosclerosis comprising a large snake extract as an active ingredient. 16. The food composition according to any one of claims 9 to 15, wherein the large snake extract stimulates the secretion of GLP-1, CCK or 5-HT. For stimulating GLP-1 secretion characterized by containing a large snake extract as an active ingredient; For CCK secretion stimulation; For secretory stimulation of 5-HT; Or hTGR5 activation. For activating TRP, characterized by comprising a large snake extract as an active ingredient; For TRPA1 activation; For activation of G protein; Or PLCβ2.

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