KR101497505B1 - Pharmaceutical or food composition comprising Disporum smilacinum - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a food composition which is safe and has excellent long-term endocrine cell stimulating activity due to its low side effects, and comprises Aspergillus oryzae L. extract as an active ingredient. The composition of the present invention may be useful as a pharmaceutical composition or food composition for the prevention and improvement of diabetes, the improvement of heart disease, the prevention of appetite or the prevention and improvement of obesity, the improvement of arteriosclerosis, the neuroprotective action, have. In addition, the composition of the present invention may affect secretory stimulation of GLP-1, CCK or 5-HT or hTGR5 activation.

Description

[0001] The present invention relates to a pharmaceutical composition or a food composition containing a sweet potato,

The present invention relates to a pharmaceutical composition or a food composition. More particularly, the present invention relates to a pharmaceutical composition useful for a specific disease or disease containing, as an active ingredient, an extract that activates 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. For example, specific endocrine hormones or neuropeptides secreted from intestinal endocrine cells have been reported to protect cardiomyocytes from ischemia and to be effective in improving cardiac function in patients suffering from heart disease.

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.

The GLP-1 may be used for the treatment of diabetes, obesity, heart disease, cerebrovascular disease and neurogenic inflammation (Salcedo I et al., Neuroprotective and neurotrophic actions of glucagon-like peptide-1 Glucagon-like peptide-1 (GLP-1) and its split products (Burgmanier M et al., Proc. Natl Journal of Pharmacology (2012) 166, 1586-1599) GLP-1 (9-37) and GLP-1 (28-37) stabilize atherosclerotic lesions in apoe ^{- / -} mice. Atherosclerosis (2013) 231, 427-435].

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 cardiomyocyte protection from ischemia and cardiopulmonary effects in patients with heart failure concerns (Sokos, GG et al. 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 induce antidiabetic effect by inhibiting glucose production, and it is known to have an anti-obesity effect.

The CCK secretion is known to involve G protein-coupled receptors (GPCRs) and signaling molecules (G proteins, PLCβ2) and transient receptor potential (TRP) channels.

And 5-HT is a hormone secreted from Enterochromaffin cells (EC cells) in the whole gastrointestinal tract. It is known to cause an anti-obesity effect through appetite suppression.

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 the balance between the energy absorbed and the energy consumed. If this balance is broken, it will lead to obesity, 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 provides a pharmaceutical composition or food composition exhibiting an effect of treating diabetes, improving heart disease, inhibiting appetite or anti-obesity, improving atherosclerosis, neuroprotective action, .

Another object of the present invention is to provide a method for treating or preventing diabetes in an individual or patient, which comprises administering an effective amount of a pharmaceutical or food composition containing a safe extract as an active ingredient to a subject or a patient in need thereof, A method for achieving effects such as improvement of heart disease, suppression of appetite or anti-obesity, improvement of arteriosclerosis, neuroprotective action and treatment or improvement of liver disease.

Another technical problem to be solved by the present invention is to provide a composition which affects secretion stimulation of GLP-1, CCK or 5-HT or hTGR5 activation.

In order to accomplish the above object, the present invention provides a pharmaceutical composition comprising Disporum smilacinum extract as an active ingredient, having intestinal endocrine cell activity, and consequently, a therapeutic effect of diabetes mellitus; Appetite suppressant effect; Anti-obesity effect; Improvement of heart disease; Improvement of arteriosclerosis; Neuroprotection; Or a liver disease treating or improving effect.

The present inventors have completed the present invention by confirming that the extract of Aphanomyceans 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 is demonstrated through experiments that the Aphanomycephalus extract can exhibit effects such as increased insulin gene expression, glucagon secretion inhibition effect, and blood glucose lowering, and thus provides a use for preventing and improving diabetes mellitus.

In another embodiment of the present invention, it is demonstrated through experimentation that Aphanomyceae extract has an appetite suppressing effect and promotes satiety feeling, and provides an anti-obesity effect through an appetite suppression effect of Aphanomyceae extract.

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

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

In another embodiment of the present invention, the present invention provides a use for the neuroprotective effect of Acanthopanax senticosus extracts by revealing that it activates the brain Arcuate nucleus in hypothalamus (ARC) and activates vagus nerves do. More specifically, the Acanthopanax senticosus extract of the present invention can treat neurodegenerative diseases and cerebrovascular diseases, treat neuroinflammation, and improve neurogenesis.

Another embodiment of the present invention provides the use of Aphanomyceae extract for the treatment or amelioration of liver diseases. More specifically, the Aphanomycephalus extract of the present invention is effective for reducing hepatic inflammation, enhancing enterohepatic blood flow, improving liver function, treating cholestatic liver diseases, pruritus, Painless jaundice treatment effect, intestinal function improvement effect, neurotransmitter promotion effect can be expected.

In order to achieve still another technical object, the present invention provides a composition which affects secretion stimulation of GLP-1, CCK or 5-HT or hTGR5 activation.

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

In another embodiment of the present invention, it is demonstrated through experimentation that the Aphanomycephalus extract stimulates CCK secretion, activates GPCRs by intracellular calcium concentration and cAMP increase, and provides a use for the CCK secretion stimulating effect of Aphanomyceae extract.

In another embodiment of the present invention, it has been experimentally demonstrated that Aphanomyceae extract stimulates 5-HT secretion and induces a change in intracellular calcium concentration, and provides a use for the 5-HT secretion stimulating effect of Aphanomyceae extract.

The A. thaliana extract of the present invention refers to an extract obtained by a conventional method in the field of the present invention and can be further purified by extraction, fractionation, separation, filtration and the like.

For example, the Acacia var. Umbraculifera extract according to the present invention may be a solvent extract or a dried product thereof. The extraction solvent at the time of extraction of Agarius can be used without restriction as long as it is a solvent used for extracting natural products, 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 extraction solvent may be 10 to 100 times, preferably 20 to 80 times, more preferably 30 to 50 times the weight of the tablet, but is not limited thereto.

Examples of the method for extracting Aphanomycephalus 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, 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, it can be prepared by shredding and crushing Aescularius japonica, extracting the extracting solvent with a volume of 1 to 20 times the volume of the crushed Pygmina crassifolia and then selectively (depressurizing) the concentrate, drying or refining. 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.

Also, according to another embodiment of the present invention, the Acanthops grandiflorum extract for the intestinal endocrine cell activation may be a fraction of Acanthopanax senticosus extract.

The Acanthaceae fraction of the present invention can be obtained by fractionating said Acanthopanax senticosus extract (preferably a lower alcohol extract having 1 to 4 carbon atoms) with a solvent having a lower polarity than water, such as hexane, chloroform, butanol and ethyl acetate, Or chloroform, but is not limited thereto.

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

The fractions may be prepared by suspending or dissolving the dried Aphanopodiaceae extract in a suitable solvent such as water, ethanol, or a mixed solvent thereof, using a liquid Aphaerai extract before drying, and then dissolving the dried Aphaeroid extract in a suitable solvent such as hexane, chloroform, butanol or ethyl acetate This low solvent 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 extract according to the present invention can be used in combination with glucagon-like peptide-1 (GLP-1, GLP-1- (7-36) amide), cholecystokinin (CCK), serotonin , Or neuropeptides by activating intestinal endocrine cells that secrete endocrine hormones and / or neuropeptides such as 5-hydroxytryptamine, 5-HT, and the like.

Accordingly, the present invention provides a method for preventing or treating an appetite-suppressing or anti-obesity effect, diabetes prevention and improvement, heart disease-improving effect, arteriosclerosis treatment Or an improvement effect, a neuroprotective effect, or a liver disease treatment or amelioration effect.

The present invention also provides a food composition for treating or ameliorating an appetite suppression or anti-obesity, diabetes prevention and improvement, heart disease improvement, arteriosclerosis improvement, neuroprotection, and / or liver disease, comprising Aspergillus oryzae as an active ingredient can do.

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 pharmaceutical composition of the present invention can be administered orally or parenterally depending on the disease to be treated and the condition of the subject, an injection (for example, intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant) Rectal, rectal, transdermal, topical, and the like, but the present invention 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, which is an appetite suppressant or anti-obesity effect, prevention and improvement of diabetes, improvement of heart disease, neuroprotective action or treatment of liver disease, the Acanthamo extract according to the present invention is administered at a daily dose of about 0.001 mg / kg to about 10 g / kg can be administered, and a daily dosage of about 0.01 mg / kg to about 1 g / kg is preferred. However, the above dosage may vary depending on the degree of purification of Aegis extract, the patient's condition (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.

In the case of using the A. thaliana extract of the present invention as a food composition, there is no particular limitation on the type of food. Examples of the food to which the Aphaenaea extract can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, , Beverages, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The Pineapple extract of the present invention can be added directly to the food or used together with other food or food ingredients, and can be suitably used according to a conventional method. 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 ingredients other than the above-mentioned AQUARIUM extract as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates, ≪ / RTI > 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 pineapple extract of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, , Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

The present invention also relates to a method of preventing or treating an appetite suppression or an anti-obesity effect, comprising administering to a patient or an individual in need thereof a therapeutically effective amount or an amount effective for improving health, Improvement of atherosclerosis, neuroprotection and / or treatment or improvement of liver disease.

That is, the present invention provides a pharmaceutical or health functional food application having an appetite suppressant or anti-obesity effect, diabetes prevention and improvement or heart disease improvement, arteriosclerosis improvement, neuroprotective action and / do.

The present invention provides an anti-diabetic, preventive or ameliorative treatment of heart disease, an appetite suppressant and an anti-obesity treatment, an improvement of arteriosclerosis, a neuroprotective action and / or treatment or improvement of liver disease. The pharmaceutical composition or the food composition according to the present invention can be used safely and safely with the use of natural extracts, and can be used for the prevention of diabetes, prevention or improvement of heart disease, an effect of appetite suppression and anti-obesity, improvement of arteriosclerosis, The treatment or improvement effect is excellent.

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

Outposts except for the root of Disporum smilacinum A. Gray used in this experiment were purchased from Hwasun Gun, Jeollanamdo. First, the sample was lyophilized and then ground. 40 ml of 50% ethanol was added to 1 g of the sample, and the mixture was extracted at room temperature for 3 days. The extract was filtered twice using a filter paper (Whatman No. 1, England), concentrated using a rotary vacuum evaporator (B-480, Buchi, Switzerland), lyophilized and powdered.

Hereinafter, experiments showing the results of the experiment and the results showing that the Aphanomycephalus extract and its fractions obtained by the above method are effective for the secretion of endocrine hormone or neuropeptide.

Example 2 GLP-1 secretion stimulation using NCI-H716 cells, GPR119 activation experiment

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. In the KRB buffer at 37 ° C in 5% CO ₂ The cells were washed three times with KRB buffer, and then 0.5 mL of 0.1 N HCl was added and the cells were reacted at 150 rpm for 20 minutes at room temperature. 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 50% ethanol extract of A. japonicus in NCI-H716 cells

sample density GLP-1 secretion
(fold of control) Control group 1.000 + 0.088 Agaricus (50% ethanol extract) 100 μg / mL 1.502 0.0802 * 250 μg / mL 1.956 + 0.0495 * 500 μg / mL 3.145 + 0.338 ** glucose 100 mM 2.694 + 0.024 *

As shown in the above Table 1, it was found that Glucagon (50% ethanol extract) had a GLP-1 secretion effect in a concentration-dependent manner in NCI-H716 cells.

2-2. NCI - H716  50% Ethanol Extract of Acanthopanax senticosus Intracellular Ca ^{2 +}  Concentration change

sample density Ca ^{2 +} reaction
(? RFU) Control group 9.181 ± 0.632 Agaricus (50% ethanol extract) 100 μg / mL 23.132 + 0.724 *** 250 μg / mL 26.959 ± 0.161 *** 500 μg / mL 31.062 ± 4.084 ** glucose 100 mM 10.240 + - 0.352 **

According to the results shown in Table 2, 100, 250 and 500 μg / mL of 50% ethanol extract of A. japonica significantly increased intracellular calcium concentration in the cells compared to the control. The increase in intracellular calcium concentration is believed to be due to the activation of GPCRs, thus activating calcium ions. Thus, the extract of A. japonicus activates GPCRs to increase GLP-1 secretion, to prevent and improve diabetes, to improve heart disease, Suppression, and anti-obesity effect.

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.

2-3. NCI - H716  50% Ethanol Extract of Acanthopanax senticosus Intracellular cAMP  Increase effect

density cAMP
(fold of control) Control group 1.000 ± 0.100 Agaricus (50% ethanol extract) 500 μg / mL 10.203 + - 1.612 * OEA (oleoylethanolamide) 20 μM 6.813 + - 1.634 *

As shown in the above Table 3, it was found that the Aegari (50% ethanol extract) was more effective in increasing intracellular cAMP than the control group. In other words, cAMP concentration, which is highly related to calcium concentration, was measured, and it was judged that the Acanthopanax senticosus extract had an effect on the increase of GLP-1 secretion. Therefore, it is thought that it will have the effect of treating obesity such as appetite suppression and satiety, and it is believed that it will show a cardiac disease improving effect through cardiac cell cooperating action, and it will be effective for diabetes treatment 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  50% Ethanol Extract of Acanthopanax senticosus TGR5  Activity enhancement effect

CHO-K1 cells transfected with the TGR5 plasmid were used, and 50% ethanolic extract of Agaricus 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)

density TGR5 activity measurement
(fold of control) Control group 1.000 ± 0.120 Agaricus (50% ethanol extract) 10 μg / mL 1.900 + - 0.275 * 50 μg / mL 2.389 + - 0.540 * 100 μg / mL 3.741 + - 0.214 ** Lithocholic acid (LCA) 5 [mu] M 3.483 ± 0.119 ***

As shown in Table 4 above, TGR5 activity was increased by 50% ethanol extract of A. japonica in the cells transfected with hTGR5. Therefore, the activation of TGR5 promotes the secretion of GLP-1, thereby exhibiting the therapeutic effect of diabetes, the therapeutic effect of heart disease, the suppression of appetite, and the anti-obesity effect. Thus, TGR5 activated by the Aphanomycephala extract increases the secretion of GLP- , And fullness. It is believed that it will be effective for the treatment of diabetes by enhancing insulin secretion.

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

4-1. STC -1 cells by 50% ethanol extract CCK Secretory effect

sample density Fold of control
(mean ± SEM) t-test P-Value Control group 1% dmso 1.00 + - 0.01 50% Ethanol Extract 100 μg / ml 1.61 + - 0.23 * 0.043 250 μg / ml 1.92 ± 0.22 * 0.036 500 μg / ml 3.91 + - 0.52 ** 0.006 AITC 0.1 mM 21.57 ± 0.59 *** <0.001

Fold of control = sample value / untreated control value

+/- S.E.M. (n = 9) *** P < Control group

According to the results shown in Table 5, it was found that the Acanthopanax senticosus extract had an effect on CCK secretion activation.

4-2. STC -1 cells, 50% ethanol extract of Agaricus Ca ^{2 +}  Effect on reaction

sample density ΔRFU
(mean ± SEM) t-test P-Value Control group 0.5% dmso 75.93 + - 4.26 Aegari
50% ethanol extract 100 μg / ml 87.68 + - 2.84 * 0.05 250 μg / ml 93.58 + - 3.02 * 0.05 500 μg / ml 114.97 + - 9.20 * 0.018 AITC 0.1 mM 105.41 ± 2.12 ** 0.003

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

According to the results shown in Table 6, the 80% ethanol extract of A. japonica significantly increased the intracellular calcium concentration in the cells compared to the control. The increase in intracellular calcium concentration is believed to be due to the activation of GPCRs and activation of calcium ions. Therefore, it is believed that the extract of A. japonicus activates GPCRs, which may be effective in the treatment of obesity such as appetite suppression and satiety, And it is thought to be effective in the treatment of diabetes by increasing insulin secretion.

4-3. STC Effect of 50% Ethanol Extract on the Cell Membrane Potential Changes

sample density ΔRFU
(mean ± SEM) t-test P-Value Control group 0.5% dmso 59.12 ± 2.09 50% Ethanol Extract 500 μg / ml 66.23 + - 0.77 *** <0.001 KCl 0.1 mM 17.01 + - 0.93 *** <0.001

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

The results are shown in Table 7, and incubation was carried out in an incubator while keeping the temperature 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

The results showed that the 50% ethanol extract of A. japonicus affects the cell membrane potential, suggesting that the activation of GPCRs has activated the intracellular ion transport Therefore, it is believed that the Aphanomycephalus extract activates GPCRs to increase GLP-1 secretion, and may have a therapeutic effect on diabetes, an improvement in heart disease, an appetite suppression effect, and an anti-obesity effect.

Example 5 Measurement of 5-HT using RIN cells

1) Cell culture

RIN cells were inoculated at a concentration of 5.0 × 10 ⁴ cells / well in RPMI medium 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin) and incubated at 37 ° C. in 5% CO ₂ Respectively. 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 the cells, 5- HT  Secretory effect

sample density Fold of control
(mean ± SEM) t-test P-Value Control group 1% dmso 1.00 + - 0.26 50% Ethanol Extract 500 μg / ml 8.25 ± 0.17 ** 0.01 AITC 300 μM 8.11 ± 2.70 ** 0.01

Fold of control = sample value / untreated control value

Values are presented as mean ± SEM (n = 9) ** P <0.01, *** P <0.001 vs. Control group

According to the results shown in the above Table 8, the Aphanomycephalus extract was found to affect 5-HT secretion, which is known to have an anti-obesity effect through appetite suppression.

5-2. RIN14B  50% Ethanol Extract of Agaricus from Cells Ca ^{2 +}  Effect on reaction

sample density ΔRFU
(mean ± SEM) t-test P-Value Control group 0.5% dmso 3.34 ± 0.44 Aegari
50% ethanol extract 500 μg / ml 18.02 + - 0.72 *** <0.001 AITC 50 uM 13.44 ± 1.78 ** 0.005

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 9 above, the ΔRFU value of RIN14B cells treated with Ahnsung extract was higher than the ΔRFU value of AITC, which is known to promote 5-HT secretion. From these results, it is suggested that Ahnryeuri extract affects channel activation in TRP involved in CCK secretion.

&Lt; Example 6 > Animal experiment using Ahnsaburi extract

2) Glucose tolerance tests, 3) Gastric emptying rate, 4) Food intake measurement, 5) Glucose tolerance test, 5) Gastric emptying rate, ) Chloride secretion effect experiment.

(1) Experimental method

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

Cryptosporidium extract was injected into the stomach and c-Fos immunostaining was performed to analyze the activation of the brain area. Experimental animals were fasted for 12 hours, and then orally administered to a 500 mg / kg BW extract dissolved in distilled water and allowed to stand 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 (500 mg / kg BW) 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 by Izbeki and Doihara (1, 2). The rats used in this experiment were aspirated for 1, 2, 3, 4, 5, 6, and 7 days after the start of the experiment. 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 food extracts to the stomach and to measure food intake, rats were fasted one day before the start of the experiment. The food intake was measured 120 minutes after oral administration of Phellinus linteus extract (500 mg / kg) in distilled water. 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 rats, Ornithine extract (500 mg / kg BW) was orally administered to 5 rats per group after oral administration. 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

And the activation of the brain region was analyzed by c-Fos immunostaining. When GLP-1, CCK, and 5-HT are secreted from the intestine by stimulation of Ahnryeuri extract, they secrete into the blood and act 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, A. tuberculosis 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, the blood glucose level increase after 30 minutes by the glucose load of the present invention was lowered by 29.4% to 72.5 mg / dl compared to the control group of 102.7 mg / dl, and the increase of blood glucose level after 60 minutes was 110.7 mg / dl in the control group And the area under the curve (AUC) was lowered to 13960 mg / dl min by 20% compared to 17465 mg / dl min in the control group, which is shown in Table 10 below.

Effect of Acanthopanax senticosus extract 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  Acorn extract 75.5 ± 2.9 148 ±
15.6 160 ±
21.0 135 ±
8.5 120 ±
9.9 13960 ±
1111

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)

The rate of emptying the stomach was lowered by the administration of 100, 250, 500 mg / kg of Agaricus extract (see Table 11 below). This may prolong the time the food stays on, thereby reducing the food intake and thus producing an anti-obesity effect.

The effect of Aegari on the slowing of stomach emptying Experimental group Oral dose (mg / kg BW) Gastric emptying rate (%) Control group - 90 ± 7.4 Acanthopanax senticosus extract group 100 75 ± 3.9 250 47 ± 2.8 500 29 ± 1.9

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. Samples were orally administered 30 min before dyeing.

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

As shown in Table 11, it was found that the rate of emptying the gastrointestinal tract in a dose-dependent manner of the extract was lower in the group administered with the Aphanomycephalus extract. This can prolong the time the food stays on and thus produce an anti-obesity effect.

4) Food intake  Measure ( Anti-obesity  Effect check)

The effects of Ahnigiri extract on stomach uptake after 120 min were measured. As a result, the Acanthopanax extract decreased the dietary intake by 68% (see Table 12).

Effect of Acanthopanax senticosus extract 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 Acanthopanax senticosus extract group 500 0.61 + 0.09

According to the results shown in Table 12 above, the dietary intake for 2 hours was much lower in the Acanthopanax extract group than in the control group. These results suggest that the anti - obesity effect in the group administered with Ahnarii extract is superior.

5) Chloride  Effect on secretion (confirmation of digestion promoting effect)

In order to indirectly evaluate the effect of Aegari on the gastric acid secretion, the concentration of chloride in the serum of rats was measured. As a result of measuring the chloride concentration in the blood after 30 minutes from oral administration of Ahnryeuri extract (500 mg / kg BW), the administration of Ahnrynuri extract increased the chloride concentration in the serum by 52.8% compared to the control group (see Table 13 below) ). This suggests that hydrochloric acid due to gastric acid stimulation is secreted into the blood, which can be expected to promote digestion.

Effect of Ahnarii Extract on the Serum Chloride Concentration in Rats Experimental group Oral dose (mg / kg BW) Serum chloride concentration (mg / dL) Control group - 265 ± 19.1 Acanthopanax senticosus extract group 500 405 ± 30.7

According to the results shown in Table 13, it was found that the serum chloride concentration in the rats fed with the Aphanomyceae extract was higher. From the above results, it was thought that Ahnimari extract promotes gastric acid secretion and promotes digestion.

Claims (15)

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