KR102147058B1 - Composition for prevention, improvement or treatment of diabetes with extracts from Scrophularia Buergeriana, Siberian ginseng, Lycii fructus root, Momordica charantia Linnaeus, Rosa rugosa - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating, or treating diabetes containing a Scrophularia Buergeriana extract, a Siberian ginseng extract, a Lycii fructus root extract, a Momordica charantia linnaeus extract, and a Rosa rugosa root extract. The composition containing a Scrophularia Buergeriana extract, a Siberian ginseng extract, a Lycii fructus root extract, a Momordica charantia linnaeus extract, and a Rosa rugosa root extract of the present invention is a natural material that has few side effects and is effective, and exhibits an alleviation, prevention, and treatment effect of diabetes.

Description

Composition for prevention, improvement or treatment of diabetes with extracts from Scrophularia Buergeriana, Siberian ginseng, Lycii fructus root , Momordica charantia Linnaeus, Rosa rugosa}

The present invention prevents, improves or treats diabetes containing scrophularia buergeriana extract, Siberian ginseng extract, Lycii fructus root extract, bitter gourd ( Momordica charantia Linnaeus ) extract and glycosyllium ( Rosa rugosa ) root extract It relates to a dragon composition.

In modern people, adult diseases such as diabetes and obesity are increasing rapidly due to lack of exercise and dietary habits, and social security medical expenses are increasing exponentially. Among them, diabetes is a type of metabolic disease such as insufficient secretion of insulin or inability to perform normal functions, and is characterized by high blood sugar in which blood glucose concentration is increased.

Diabetes is divided into insulin dependent diabetes mellitus and non-insulin dependent diabetes mellitus. Among them, insulin-independent diabetes refers to a case in which the function of insulin decreases and blood sugar rises. Although the medical treatment method for insulin-independent diabetes is developing in various ways, the cure rate is very low as the diabetes treatment developed so far, and serious side effects are accompanied by long-term use.

Existing various diabetes treatments promote insulin secretion, inhibit the decomposition of accumulated fat, inhibit glucose neoplasm, increase intracellular glucose inflow through activation of GLUT2 or GLUT4 protein, and promote intracellular glucose degradation metabolism through activation of glucokinase. Although it tries to lower blood sugar based on the principle of digestion and absorption inhibition function, all these reactions are a series of linked metabolic processes, so if integrated control is not performed, blood sugar reduction is very limited and inefficient. Therefore, it is necessary to discover a natural material capable of reducing and improving diabetes symptoms without showing side effects.

In Korean Patent Registration No. 10-1321203 (Registration Date: October 16, 2013), a composition for the prevention and improvement of diabetes containing the extract of fresh herbaceous leaves from which the water-soluble component was removed from the residue, after the juice of the fresh herbaceous green juice, Is described. Republic of Korea Patent Laid-Open Patent No. 10-2014-0034620 (published date: Mar 20, 2014), diabetes treatment comprising an extract of a mixture of arrow tree, mistletoe, cuchipon, Joritdae, Duchung tree, gasiocarpus and licorice as an active ingredient, It is described for a composition for improvement or prevention.

The present invention aims to provide a composition for improving, preventing or treating diabetes of a natural material having fewer side effects and having good effects by developing a composition containing a hyeon ginseng extract, a gasoline extract, a wolfberry root extract, a bitter gourd extract and a glycosyllium root extract.

The present invention provides a food composition for improving diabetes, characterized in that it includes all of the hyeonsam extract, gasoline extract, goji japonica root extract, bitter gourd extract, and glycosyllium root extract.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes, characterized in that it includes all of the hyeon ginseng extract, gasoline extract, goji japonica root extract, bitter gourd extract, and glycosyllium root extract.

The composition containing the present invention of the present invention is a natural material material having few side effects and a good effect, and exhibits an improvement, prevention and treatment effect of diabetes.

(A) of Figure 1 is a schematic diagram depicting the metabolic process of liver cells using elevated glucose in blood, (B) is nicotinamide-N-methyltransferase (Nicotinamide-N-methyltransferase,) remaining acetyl coenzyme A used as energy NNMT) is a schematic diagram depicting a biochemical reaction that is excreted in urine through inhibition of activity.
2 is a graph showing the cytotoxicity measurement results of natural material extracts.
3 is a graph showing the results of glucose uptake when natural material extracts are added under different conditions (0.1%, 0.45%, 0.7%) of glucose concentration in the medium.
Figure 4 is a graph showing the results of glucose absorption (glucose uptake) when different concentrations of the hyeonsam extract and the mulberry bark extract added.
5 is a schematic diagram showing the relationship between HNF-1α protein and GLUT2 mRNA expression known as a mechanism of action for glucose uptake.
Figure 6 shows the results of HNF-1αmRNA and protein expression (A) and GLUT 2 mRNA and protein expression (B) when adding hyeonsam extract and mulberry bark extract to hepatocytes (HepG2 cells).
7 is a graph showing the results of measuring glucokinase (GK) activity when natural extracts are added under different conditions (0.1%, 0.45%, 0.7%) of glucose concentration in the medium.
8 is a graph showing the results of measuring glucokinase (GK) activity when the concentrations of the extract and the extract of the mulberry bark are varied.
9 is a glucokinase (GK) mRNA and protein expression test results when adding the extract and Morus bark extract to hepatocytes (HepG2 cells).
10 is a schematic diagram illustrating the mechanism of action of regulatory proteins known as mechanisms of action related to glucokinase (GK) activation in hepatocytes (HepG2 cells).
Figure 11 shows the results of experiments on PIK3 protein expression (A) and SREBP-1C protein expression (B) when adding the extract and Morus bark extract to hepatocytes (HepG2 cells).
12 is a graph showing the result of measuring pyruvate dehydrogenase (PDH) activity when natural extracts are added under different conditions (0.1%, 0.45%, 0.7%) of glucose concentration in the medium.
13 is a graph showing the results of measuring pyruvate dehydrogenase (PDH) activity when the concentration of the extract of Goji japonica root extract was varied.
14 is a result of pyruvate dehydrogenase (PDH) mRNA expression when adding Goji japonica root extract to hepatocytes (HepG2 cells).
15 is a schematic diagram illustrating an enzymatic reaction related to activation of pyruvate dehydrogenase (PDH) in hepatocytes (HepG2 cells).
FIG. 16 is a result of an experiment on expression of phosphorylated pyruvate dehydrogenase (pPDH, inactive state) and pyruvate dehydrogenase (PDH, activated state) proteins when adding goji root extract in hepatocytes (HepG2 cells).
FIG. 17 shows ATP-citrate lyase (ACL) protein and phosphorylated ATP-sheet when natural extracts were added under different conditions (0.1%, 0.45%, 0.7%) of glucose concentration in the medium. This is a graph showing the rate of expression of a phosphorylated ATP-citrate lyase (pACL) protein.
FIG. 18 is a graph showing the expression ratio of ATP-citrate lyase (ACL) protein and phosphorylated ATP-citrate lyase (pACL) protein at different concentrations of bitter gourd extract to be.
19 is a graph showing the result of measuring the activity of nicotinamide-N-methyltransferase (NNMT) when natural extracts are added.
20 is a graph showing the result of measuring the activity of alpha glucosidase (α-glucosidase) when natural extracts are added.
Figure 21 is a table showing the complex blending ratio of each natural material extracts having an activating effect of the metabolic process of sugar degradation.
22 is a graph showing the cytotoxicity measurement results of natural material composite extracts with different combinations.
23A) is a graph showing the result of expression of acetyl coenzyme A carboxylase (ACC) protein in hepatocytes (HepG2 cells) when processing complex extracts of natural materials with different combinations, and FIG. 23 B) of is an active form (phosphorylated acetyl coenzyme A carboxylase, phosphorylated acetyl CoA carboxylase, pACC) and inactive form (acetyl coenzyme A carboxylase, pACC) in liver cells (HepG2 cells) when treating complex extracts with different combinations This is a graph showing the results of expression of a proteinase, Acetyl CoA carboxylase, ACC).
24A) is a graph showing the result of expression of acetyl coenzyme A carboxylase (ACC) protein in hepatocytes (HepG2 cells) when processing complex extracts of natural materials with different combinations, and FIG. 24 B) of is the active form (phosphorylated acetyl coenzyme A carboxylase, phosphorylated acetyl CoA carboxylase, pACC) and inactive form (acetyl coenzyme A carboxylase) in hepatocytes (HepG2 cells) when treating complex extracts of natural materials with different combinations. This is a graph showing the results of expression of a proteinase, Acetyl CoA carboxylase, ACC).
25A) and B) are graphs showing the result of expression of a fatty acid synthetase (FAS) protein in hepatocytes (HepG2 cells) when processing complex extracts of natural materials with different combinations.
26A) and 26B) are graphs showing changes in the amount of malonyl coenzyme A (Malonyl-Co A) produced in hepatocytes (HepG2 cells) when processing complex extracts of natural materials with different combinations.

In general, when carbohydrates ingested through a meal become glucose through digestion, they are absorbed by the intestine and transferred to the blood. As a result, when the blood sugar concentration rises, in the case of normal people, insulin is secreted to move sugar to the brain and muscles and use it for metabolic reactions that make necessary substances such as energy, while also moving to liver cells to activate various metabolisms using blood sugar. To bring blood sugar back to normal. Various metabolisms using blood sugar in hepatocytes are 1) stored as glycogen and then stored for use when blood sugar is low, 2) metabolized to substances or energy required by hepatocytes, 3) acetyl coenzyme A ( Acetyl Coenzyme A and Acetyl Co A) are decomposed and then converted into fatty acids through a series of reactions, and then stored.

However, in the case of a diabetic patient, the above metabolic process is not well performed, and thus the state of high glucose in the blood continues to be maintained, causing serious metabolic abnormalities. Although research and development on medical preparations continues to treat such diabetes, it is still impossible to cure it, and because the response of blood sugar control is a series of linked metabolic processes, there is a problem that it is very limited and inefficient if it cannot be controlled in an integrated manner. Therefore, in the present invention, it was attempted to discover natural materials capable of reducing and improving diabetes symptoms without causing side effects.

Meanwhile, the core principle of the present invention is a metabolic process in which hepatocytes use elevated glucose in the blood, as shown in FIG. 1. Specifically, 1) GLUT2 protein, which is involved in the movement of glucose in blood into hepatocytes (HepG2), must be activated, and 2) glucose introduced into cells is decomposed to acetyl coenzyme A (Acetyl Co A). The activities of glucokinase (GK) and pyruvate dehydrogenase (PDH), the main enzymes involved in the reaction, should be increased.

In addition, 3) activity of ATP-citrate lyase (ACL), which decomposes citric acid produced to treat excess acetyl coenzyme A in mitochondria, is increased. In connection with the process of 3), when the metabolic reactions of 1) and 2) are increased, the amount of acetyl coenzyme A produced in the mitochondria increases. In general, when acetyl coenzyme A is produced, it must be converted to energy through a complete oxidation reaction, but it is produced in excess and cannot be oxidized to energy, and it is combined with oxalic acid to become citric acid and then comes out of the mitochondria. Thereafter, citric acid is decomposed into acetyl coenzyme A and oxalacetic acid again by ATP-citrate lyase (ACL).

At this time, the produced acetyl coenzyme A is converted to malonyl coenzyme A by acetyl coenzyme A carboxylase (ADD), and then transferred to the fatty acid synthetase complex. It is the normal metabolism that is converted into and stored, which may lead to obesity. Therefore, 4) acetyl coenzyme A must be excreted in the urine to prevent obesity from being converted and stored into fatty acids. This inhibits the activity of nicotinamide-N-methyltransferase (NNMT), an enzyme related to this. Is done. Through this, the over-produced acetyl coenzyme A is combined with polyamines (Acetyl-polyamines) to be discharged into the urine.

Therefore, the present invention finds natural materials that can control the metabolic reactions of 1)-4) above, scientifically verifies the efficacy and mechanism of action, and uses a composition formulated with them to intrahepatocytes without the help of insulin or diabetes drugs. The ability to promote the metabolism of glucose degradation was confirmed. In addition, in order to reduce digestion and absorption by decomposing carbohydrates transferred to the small intestine through meals into glucose, a natural substance capable of inhibiting the activity of α-glucosidase, a digestive enzyme, was discovered to create the final complex. It was completed, and it is intended to provide an excellent effect to lower blood sugar, which has been elevated after a meal.

The present invention provides a food composition for improving diabetes, characterized in that it includes all of the hyeonsam extract, gasoline extract, goji japonica root extract, bitter gourd extract and glycosyllium root extract. In addition, it provides a pharmaceutical composition for preventing or treating diabetes, characterized in that it comprises all of the hyeon ginseng extract, ginseng extract, goji japonica root extract, bitter gourd extract, and glycosyllium root extract.

Meanwhile, in the present invention, the composition may preferably increase intracellular glucose utilization metabolism.

Hyeonsam ( Scrophularia Buergeriana ) is a perennial herbaceous plant belonging to the Hyeonsam family, and is also known as the middle (重臺), Hyundai (玄臺), Gwijang (鬼藏), and Chukma (逐馬). Hyeonsam is grown in mountainous areas, and is used as an antipyretic agent for sore throat, boils, and lymphadenitis, and there are island hyeonsam ( S. takesimensis ) growing in Ulleungdo, large gaehyeonsam ( S. kakudensis ) and Tohyeonsam ( S. koraiensis ) growing in the mountains. Hyeonsam extract of the present invention may be involved in the influx of glucose into cells.

Siberian ginseng is a deciduous shrub of the plant umbel, Araliaceae, and is also known as Siberian ginseng . Gashiogalpi grows in deep mountain valleys all over the country, and is a shrub with leaves. It is known as a superior medicinal material than ginseng because of its abundant useful medicinal substances. Gasiogalpi extract of the present invention may be involved in the activation of glucokinase (Glucokinase).

Lycii fructus is a fruit of the Lyciiaceae tree belonging to the Gajitsu family, and contains betaine, zeaxanthin, carotene, thiamine, vitamins A, B1, B2, and C. When taken for chronic hepatitis, cirrhosis, etc., inflammation is removed and the function is activated, and in the private sector, it is used as Gugicha or Gugiju. In the present invention, Goji japonica root was used, and the Goji japonica root extract of the present invention may be involved in the activation of pyruvate dehydrogenase.

Yeoju ( Momordica charantia Linnaeus ) is a vegetable that has an elongated and uneven shape like a goblin bat and is widely used in Southeast Asian foods such as Thailand. It is also called'bitter cucumber' because of its characteristic bitter taste, and is mainly processed into medicinal or health foods. The bitter gourd extract of the present invention may be involved in the expression of ATP-citrate lyase.

Rosa rugosa is a deciduous shrub belonging to the dicotyledonous Rosaceae Rosaceae, and is also known as the narcissus tree, the lysaceae, and the head flower. The flowers are beautiful, have a unique scent, and the fruit is beautiful, so they are used as a perfume ingredient and medicinal material. Gaehedanghwa (var. kamtschatica ), Mancheophaedanhwa (for. plena ), Minhaedanghwa ( var.chamissoniana ), and white halibut There are types such as. In the present invention, glycosylation root was used, and the glycosylation root extract of the present invention may be involved in the inhibition of nicotinamide-N-methyl-transferase (NNMT) activity.

On the other hand, in the present invention, it is preferable to pulverize and use natural materials for extraction, such as hyeonsam, ginseng, goji root, bitter gourd, and glycolysis root to increase extraction efficiency, and a water extraction method was used.

The food composition for improving diabetes according to the present invention includes, for example, meat, grains, caffeine beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, gum, dairy products, ice cream, alcoholic beverages, It may be any one selected from alcohol, vitamin complexes, and other health supplements, but is not limited thereto.

On the other hand, the food composition according to the present invention may include conventional adjuvants or additives, or sweeteners, such as licorice, vitamin C, citric acid, nicotinic acid, sodium benzoate, aspartame, saccharin, pectin, malitol, sorbitol, depending on the form of the desired composition. By adding one or more components selected from the group consisting of xylitol, guar gum, skim milk powder, and oligosaccharides, preference or taste may be increased.

The pharmaceutical composition for preventing and treating diabetes of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of carriers, excipients or diluents that can be used include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, There are microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and one or more of these may be used. In addition, when the prophylactic and therapeutic agent is a drug, a filler, an anti-aggregating agent, a lubricant, a wetting agent, a fragrance, an emulsifying agent or a preservative may be additionally included.

The formulation of the pharmaceutical composition for preventing and treating diabetes of the present invention may be in a preferred form depending on the method of use, and in particular, a method known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is good to adopt and formulate. Examples of specific formulations include PLASTERS, GRANULES, LINIMENTS, LEMONADES, POWDERS, SYRUPS, LIQUIDS AND SOLUTIONS, and EX ( EXTRACTS), ELIXIRS, FLUID EXTRACTS, emulsions (EMULSIONS), suspensions (SUSPESIONS), DECOCTIONS, INFUSIONS, tablets, SUPPOSITIORIES, injections ( INJECTIONS), SPIRITS, CATAPLASMA, Capsules, Troches, Tinctures, Pastes, PILLS, Soft or Hard Gelatin Capsules It can be any one selected.

The dosage of the pharmaceutical composition for preventing and treating diabetes of the present invention is preferably determined in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. For example, the pharmaceutical composition for preventing and treating diabetes of the present invention may be administered at least once at 0.1 to 100 mg/kg (body weight) per day based on the active ingredient. However, the above dosage is only an example for illustration, and may be changed by a doctor's prescription according to the state of the user.

Hereinafter, the content of the present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

[Production Example: Preparation of natural material extract]

In this preparation example, natural materials purchased from native villages (gasiogalpi, hyeonsam, goji berry root bark, goji berry root, mulberry bark, bitter gourd, glycolysis root, etc.) were washed with running water, dried, and then pulverized. 10 g of each pulverized natural material was added to 10.7 times of distilled water and extracted with stirring in a 60°C shaking incubator for 24 hours. After extracting for 24 hours, centrifuged at 3,000 rpm for 30 minutes, filtered under reduced pressure with filter paper with 0.45 um, and lyophilized each sample was used in the experiment.

In the following, an experiment was conducted to confirm the metabolic ability of the natural material extracts of the present preparation example to use intracellular glucose.

[Experimental Example 1: Hepatocyte (HepG2 cell) culture and cytotoxicity measurement and experimental method]

In this experimental example, the following experiments were conducted to confirm the cultivation of hepatocytes (HepG2 cells) and measurement of hepatocyte (HepG2) toxicity to the natural material extracts of the preparation example.

1) Hepatocyte (HepG2 cell) culture

HepG2 cells stored in cryopreservation were thawed in a water bath set at 37°C, and then cultured in a cell incubator at 37°C and 5% CO ₂ using MEM containing 10% FBS and 1% PEST. When the cells were cultured more than 80% of the culture dish, the cells were removed using trypsin-EDTA and subcultured in a new culture dish to secure sufficient cells. Cells were cultured using 0.1% glucose low glucose media, 0.45% glucose normal glucose media, and 0.7% glucose high glucose media. . In the selection material experiment, cells were cultured under the conditions of low sugar medium, normal sugar medium, and high sugar medium, and cells were cultured under high sugar medium conditions in the mechanism verification experiment and combination addition experiment. In the following, HepG2 cells cultured by the above method were used.

2) Cytotoxicity measurement

Toxicity evaluation of natural extracts on hepatocytes (HepG2 cells) was measured using 3-[4,5-dimethyl-thiazole-2-yl]-2,5-di-phenyl-tetrazolium bromide (MTT) reduction method. . MTT tetrazolium (yellow color) is reduced to MTT formazan (purple color) by a reducing agent present in the mitochondria of normal cells, and the reduction action by reductase does not occur in abnormal cells.

HepG2 cells were dispensed into a 96 well plate at 1 × 10 ⁵ cells/mL at 100 uL each, cultured for 24 hours, and then the material was diluted in a medium to which FBS was not added, treated with cells, and cultured for 24 hours. The culture medium containing the material was removed, MTT (0.5 mg/mL) was dissolved in the culture medium, dispensed at 100 uL, and cultured at 37°C for 4 hours, and the culture medium was removed. After adding 100 uL of DMSO to the formazan formed in each well, it is dissolved using a shaker, and after 30 minutes, using a UV/vis spectrophotometer (Opiwen 2120 UV plus, Mecasys Co. Ltd., Korea) Absorbance was measured at 570 nm. Cytotoxicity was confirmed based on the absorbance value of the control group.

Figure 2 shows when the natural material extracts were individually treated in hepatocytes (HepG2 cells) at various concentrations, by concentration (0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.5mg/mL, 5.0 mg/mL) is the result of measuring cytotoxicity. Cytotoxicity was found in some of the natural extracts (Hyunsam 5.0mg/mL, Goji berry root bark 2.5mg/mL, 5.0mg/mL, Goji berry root 5.0mg/mL, bitter gourd 5.0mg/mL, Glycoris root 5.0mg/mL). The experiment was conducted only in a safe range.

[Experimental Example 2: Measurement of glucose uptake by natural material extract]

In this experimental example, the following experiment was conducted to measure glucose uptake of the natural material extracts of the preparation example.

The effect of each natural material extract on glucose uptake in hepatocytes (HepG2 cells) was tested using a glucose uptake colorimetric assay kit. 2-Deoxyglucose (2-DG) has a structure similar to glucose and is absorbed together when glucose is absorbed in the cell metabolism process, and 2-DG-6-phosphate (2-DG- 6-phosphate, 2-DG6P). 2-DG6P is no longer metabolized and accumulates in cells, and 2-DG6P accumulates in proportion to the amount of 2-DG absorbed. By measuring the accumulated 2-DG6P, the degree of sugar influx into the cell can be measured.

Hepatocytes (HepG2 cells) were dispensed with 100 uL at 1 × 10 ⁵ cells/mL in 96 well plates, cultured for 24 hours, and cultured for 12 hours in a medium containing no FBS for serum starvation. After starvation, 2% BSA was dissolved in KRPH (Krebs ringer phosphate HEPES) buffer and incubated for 40 minutes. 100 nM of insulin and a natural material, 10 mM 2-Deoxyglucose (2-DG) were treated for 20 minutes. After washing the cells 3 times with PBS, 58 uL of assay buffer and 2 uL of enzyme mix A were treated, and then reacted at 37°C for 1 hour. After treatment with 90 uL of the extraction buffer, it was heated at 90°C for 40 minutes, cooled with ice, and then 12 uL of neutralization buffer was added. Using 20 uL of glutathione reductase and 16 uL of DTNB [5-5'(2-nitrobenzoic acid)], 2 uL of recycling mix was used to react at 37°C for 40 minutes, and absorbance was measured at 412 nm. I did.

First, when natural material extracts were added under different conditions (0.1%, 0.45%, 0.7%) with different glucose concentrations in the medium to create conditions similar to normal or hyperglycemic conditions (diabetes-like conditions), hepatocytes (HepG2 cells) As a result of experimenting how the amount of glucose introduced into the cells changes, as shown in FIG. 3, when adding natural material extracts compared to the untreated control group, the amount of glucose introduced into the cells was increased regardless of the glucose concentration in the medium. It was confirmed that it can be. In particular, it was observed that the ginseng extract and the mulberry bark extract significantly increased the amount of glucose introduced into the hepatocytes (HepG2 cells), and statistically similarly increased the amount of glucose introduced into the cells even when treated with insulin. It was selected as the best candidate material.

In addition, as a result of testing how the amount of glucose introduced into hepatocytes (HepG2 cells) changes when the added concentrations of the selected materials, hyeon ginseng extract and mulberry bark extract were varied, hepatocytes (HepG2) were tested as the concentration of these extracts increased. Cells) was observed to increase the amount of glucose introduced into the cells, and in particular, it was observed that the increase was the highest when 1.5mg/ml of Hyeonsam extract was added.

On the other hand, various transcription factors that regulate the expression of GLUT protein and GLUT 2 mRNA when glucose is introduced into hepatocytes, hepatocyte nuclear factors 1 alpha, HNF-1α, and HNF. -4α, forkhead box A2 (FOXA2), sterol regulatory element binding protein 1C (SREBP-1C), CCAAT/enhancer binding protein-beta (CCAAT enhancer binding protein-beta, C/EBP-β) and the like are known to be involved, and in particular, HNF-1α, FOXA2, and C/EBP-β are known as major regulators in the human GLUT2 gene promotor (Ban N et al. , 2002. Hepatocyte nuclear factor-1alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter.Diabetes 51(5): 1409-1418.28, 29). The relationship between HNF-1α protein and GLUT2 mRNA expression when glucose is introduced into hepatocytes is shown in FIG. 5. Thus, when the selected materials, hyeonsam extract and mulberry bark extract, were added, the relationship between HNF-1α protein and GLUT2 mRNA expression when glucose was introduced into hepatocytes was examined.

Dispense HepG2 cells into 6-well plates at 1x10 ⁶ cells/mL, incubate for 24 hours, treat the material, and incubate for 24 hours, then RIPA lysis buffer (10 mM Tris-HCl, 0.1 M EDTA). , 10 mM NaCl, 0.5% Triton X-100, pH 7.4,) mixed with a protease inhibitor cocktail, 1 mM PMSF, dissolved at 4℃, and centrifuged (8,000 rpm, 10 min, 4℃ ) The obtained protein was quantified using the Brandford method and used in the experiment.

The same amount of protein (40 ug) and sample buffer were mixed at 4:1 and heated at 100° C. for 10 minutes. The prepared protein sample was SDS-PAGE using a Bio-Rad mini-gel system, and then the protein was transferred to a polyvinylidene fluoride membrane (0.45 um). Membrane was blocked in tris-buffered saline (TBS) containing 0.1% tween 20 and 5% skim milk for 1 hour. Then, it was reacted for 1 hour in 5% skim milk to which the primary antibody was added, and washed 3 times for 5 minutes with TBS-T (TBS containing 0.1% tween-20). The washed membrane was added with 5% skim milk to which a secondary anti-rabbit IgG conjugate horseradish peroxidase antibody was added, and 1 hour at room temperature. After reacting for 5 minutes, it was washed 3 times. After washing, it was sensitized to the x-ray film using enhanced chemiluminescence. The protein band developed on the film was quantified using Image J (National institutes of health, Bethesda, MD, USA) software, and β-actin was used as the internal standard protein. When measuring the protein expression level in the following other experimental examples, the same method was used.

As a result of the experiment, it was observed that both HNF-1α mRNA and protein expression were increased as shown in FIG. 6A), and both HNF-1α increased as shown in FIG. 6B). It was observed to increase GLUT2 mRNA and protein expression through (see Fig. 6A). Therefore, the mechanism of action could be verified.

[Experimental Example 3: Measurement of glucokinase (GK) activity by natural material extract]

In this experimental example, the following experiment was conducted to measure the glucokinase (GK) activity of the natural material extracts of the preparation example. In this experimental example, hepatocytes (HepG2 cells) cultured in Experimental Example 1 were used.

Hexokinase colorimetric assay kit was used to determine the effect of each natural material extract on GK activity in hepatocytes (HepG2 cells). Glucose by GK is phosphorylated to glucose-6-phosphate (G-6-P), and G-6-P dehydrogenase (G-6) -PD) was used to measure GK activity by using the reduction of Nicotinamide adenine dinucleotide (NAD) to NADH. After culturing natural materials in hepatocytes (HepG2 cells) for 24 hours, the cells were recovered, and the protein was quantified and adjusted to 40 ug/50 uL per well, followed by 34 uL of assay buffer and 2 uL of enzyme mix. , After treatment with 2 uL of developer, 2 uL of coenzyme, and 10 uL of hexokinase, absorbance was measured at 450 nm every 3 minutes.

After a meal, the digested and absorbed glucose into the cells must be converted to glucose-6 phosphate by glucokinase (GK) to start decomposition and metabolism. As a result of the experiment on the effect of natural material extracts on the increase in glucokinase (GK) activity in hepatocytes (HepG2 cells), as shown in FIG. 7, the gasoline extract is irrespective of the glucose concentration in the medium (0.1%, 0.45%, 0.7%) was found to increase the glucokinase activity most significantly.

When the concentrations of the selected material, the extract of the extract of the bark bark and the extract of the mulberry bark were varied, the effect of the experiment on the activity of glucokinase (GK) in the hepatocytes (HepG2 cells) was tested. As a result, the addition of these extracts as shown in FIG. It was observed that as the concentration increased, the activity of glucokinase (GK) in hepatocytes (HepG2 cells) increased. In particular, it was observed that the glucokinase (GK) activity increased significantly when added with 1.5 and 2 mg/ml of the gasoline extract, compared to the addition of insulin, indicating a very excellent material. In addition, as a result of confirming how the selected materials, gasiogapi extract and mulberry bark extract affect the expression of glucokinase (GK) mRNA and protein in hepatocytes (HepG2 cells), as shown in FIG. 9, glucokinase ( It was confirmed that glucokinase, GK) mRNA and protein expression were increased, and this was a result supporting the result of FIG. 9.

Meanwhile, glucokinase (GK) mRNA expression is regulated by SREBP-1C and phosphatidylinositol 3-kinase (PI3K), and SREBP-1C acts on the promoter of GK (Desvergne B). , Michalik L, Wahli W. 2006. Transcriptional regulation of metabolism. Physiol Rev 86(2): 465-514.), this mechanism of action is shown in FIG. Therefore, it was tested how the addition of the selected materials, the extract of the extract and the extract of the mulberry bark, affects the expression of PI3K protein and SREBP-1C protein in hepatocytes (HepG2 cells).

As a result, it was observed to increase the expression of PI3K protein in both the gasiocarpone extract and the mulberry bark extract as shown in FIG. 11A), and both the gasiocarpone extract and the mulberry bark extract increase the SREBP-1C protein expression as shown in FIG. 11B). Was observed. Through the results of FIGS. 9 and 11, it was confirmed that the activation of glucokinase (GK) in hepatocytes (HepG2 cells) was mechanically reacted when adding the extract and the mulberry bark extract.

[Experimental Example 4: Dehydrogenase (Pyruvate dehydrogenase, PDH) activity measurement by natural material extract]

In this experimental example, the following experiment was performed to measure the activity of the natural material extracts of the preparation example for measuring Pyruvate dehydrogenase (PDH) activity.

The effect of each natural material extract on the PDH activity in HepG2 cells was performed using a PDH activity assay kit. It was measured using the principle that NAD ⁺ is reduced to NADH by PDH activity. After culturing the material in HepG2 cells for 24 hours, the cells were recovered, and the protein was quantified and adjusted to 100 ug/200 uL per well, incubated at room temperature for 3 hours, washed twice with 1X stabilizer, and then 20X reagent Mix 11 uL , 0.2 mL of 1X buffer, 2.25 uL of 100X coupler, 2.25 uL of 100X reagent Dye were treated, and absorbance was measured at 450 nm every 3 minutes.

Glucose introduced into cells is converted to glucose-6 phosphate by glucokinase (GK), and then converted to pyruvic acid through glycolysis, and then pyruvic acid dehydrogenase in mitochondria (Pyruvate dehydrogenase, PDH) is produced as acetyl coenzme A (Acetyl co A). Experimental results for the effect of natural material extracts on the increase of pyruvate dehydrogenase (PDH) activity in hepatocytes (HepG2 cells), as shown in FIG. 12, the concentration of glucose concentration in the medium of hyeon ginseng extract, goji root bark extract, goji berry root extract Regardless of (0.1%, 0.45%, 0.7%), the activity of pyruvate dehydrogenase (PDH) was most significantly increased, and it was found that the increase effect was similar to that of insulin addition.

When the concentration of the extract of Goji japonica root extract, which is the material selected above, was tested, how it affected the activity of pyruvate dehydrogenase (PDH) in hepatocytes (HepG2 cells), as shown in FIG. As the concentration of addition increases, the activity of pyruvate dehydrogenase (PDH) in hepatocytes (HepG2 cells) increases.In particular, when 1.5 and 2 mg/ml of Goji root extract are added, pyruvate dehydrogenase (Pyruvate dehydrogenase) increases. PDH) activity was observed to increase similarly to when insulin was added, indicating a very good material.

In addition, as a result of confirming how the selected material, goji root extract, affects the expression of pyruvate dehydrogenase (PDH) mRNA in hepatocytes (HepG2 cells), as shown in FIG. 14, when compared with the control group, pyruvate dehydrogenase , PDH) mRNA expression was confirmed to increase, which was a result supporting the result of FIG. 13.

On the other hand, in order to activate pyruvate dehydrogenase (PDH) in liver cells, an enzyme in an inactivated state (phosphorylated pyruvate dehydrogenase, pPDH) is activated (pyruvate dehydrogenase, pyruvate dehydrogenase) by increased pyruvate. , PDH). A schematic diagram related to this is shown in FIG. 15. Therefore, in order to prove whether the result of FIG. 14 is due to the activation of pyruvate dehydrogenase (PDH), it is necessary to verify whether the enzyme of pPDH is increased or the enzyme of PDH is increased. Therefore, it was tested how the addition of the selected material, Goji root extract, on the expression of pPDH and PDH proteins in hepatocytes (HepG2 cells).

As a result, it was observed that the expression of the pPDH protein decreased and the expression of the PDH protein increased when the Goji root extract was added as shown in FIG. 16, and it was confirmed that the expression of the PDH protein was significantly increased even when compared with the addition of insulin. there was. From these results, it was confirmed that when the extract of Goji japonica root extract was added, the activation of pyruvate dehydrogenase (PDH) was mechanically reacted.

[Experimental Example 5: Measurement of ATP-citrate lyase (ACL) activity by natural material extract]

In this experimental example, the following experiment was conducted to measure the activity of ATP-citrate lyase (ACL) of the natural material extracts of the preparation example.

Acetyl coenzyme A, which cannot be oxidatively decomposed into energy through the citric acid cycle in the process of glucose decomposition and metabolism, is combined with oxaloacetic acid in the mitochondria, then becomes citric acid, and then comes out of the mitochondria. It is decomposed into acetyl coenzyme A and oxalacetic acid again by ATP-citrate lyase (ACL). The acetyl coenzyme A produced at this time is converted to malonyl CoA by acetyl coenzyme A carboxylase (ACC), and then converted to fatty acid by the action of the fatty acid synthetase complex. It is converted and saved.

If the decomposition reaction of acetyl coenzyme A and oxalic acid by ATP-citrate lyase (ACL) is not activated, acetyl coenzyme A accumulates in the cell and interferes with the metabolism of glucose, resulting in elevated blood sugar levels after eating. Diabetes is induced by inhibiting return and maintaining high blood sugar for a long time. Therefore, activation of ATP-citrate lyase (ACL) plays an important role in the induction of diabetes.

ATP-citrate lyase In an inactive state (ATP-citrate lyase, ACL) and activated (phosphorylated ATP-citrate lyase. pACL), citric acid is decomposed into acetyl coenzyme A and oxalic acid. )and The change in the percentage of active form (pACL) is measured. Experimental results of the effect of natural extracts on the expression of ATP-citrate lyase (ACL) protein and phosphorylated ATP-citrate lyase (pACL) protein in hepatocytes (HepG2 cells) , As shown in FIG. 17, it was verified that the bitter gourd extract was a very excellent material by increasing the pACL/ACL ratio most significantly.

When the concentration of bitter gourd extract, the material selected above, was varied, ATP-citrate lyase (ACL) protein in hepatocytes (HepG2 cells) and phosphorylated ATP-citrate lyase , pACL) As a result of testing how it affects protein expression, as shown in FIG. 18, when the added concentration of the extract was increased to 2 mg/ml, the ratio of intracellular inactive form (ACL) and active form (pACH) increased. Could be observed.

[Experimental Example 6: Measurement of nicotinamide-N-methyltransferase (NNMT) activity by natural material extract]

In this experimental example, the following experiment was performed to measure the activity of nicotinamide-N-methyltransferase (NNMT) of the natural material extracts of Preparation Example.

In liver cells, 1) GLUT2 protein, which is involved in the movement of glucose in blood into hepatocytes (HepG2), is activated, and 2) the reaction to decompose glucose introduced into cells to acetyl coenzyme A (Acetyl Co A). The activities of glucokinase (GK) and pyruvate dehydrogenase (PDH), which are the major enzymes involved, are increased, and 3) ATP- decomposes citric acid generated for treatment of excess acetyl coenzyme A. Even if the three reactions that increase the activity of citrate lyase (ATP-citrate lyase, ACL) are activated to improve diabetes, if acetyl coenzyme A, which is increased through the process of 1)-3) above, cannot be oxidatively decomposed into energy, eventually It is converted into fatty acids and stored, resulting in obesity, which can lead to another metabolic abnormality. Therefore, increased acetyl coenzyme A must be excreted into the urine, and the only enzyme that plays this role is nicotinamide-N-methyltransferase (NNMT), if the activity of this enzyme can be inhibited. , Over-produced acetyl coenzyme A is combined with polyamines (Acetyl-polyamines) to be excreted in urine.

As a result of the experiment on the effect of natural material extracts on the inhibition of nicotinamide-N-methyltransferase (NNMT) activity, as shown in Fig. 19, the extracts of fresh wood and glycosyllium root have significant NNMT enzyme activity. Was inhibited, and it was confirmed that the result was significantly more inhibited than N-methyl nicotinamide, a drug generally used to inhibit the activity of NNMT enzyme.

[Experimental Example 7: Measurement of α-glucosidase activity by natural material extract]

In this experimental example, the following experiment was performed to measure the α-glucosidase activity of the natural material extracts of Preparation Example.

Apart from metabolism using glucose decomposition in liver cells, alpha-glucosidase, an enzyme that digests and decomposes ingested carbohydrates into glucose in order to reduce digestion and absorption by decomposing carbohydrates transferred to the small intestine through meals into glucose. ) To find a natural substance that can inhibit the activity. As a result of the experiment on the effect of natural material extracts on the inhibition of α-glucosidase activity, as shown in FIG. 20, it was confirmed that the Morus bark extract had the most excellent inhibitory effect.

[Example 1: Glucose decomposition and metabolism activity by natural material complex extract (Hyunsam extract, Ginseng extract, Gugija root extract, bitter gourd extract, Glycoris root extract)]

In this example, as confirmed from the results of the above experimental examples, the materials selected by showing partial activation effects of each of the glucose degradation utilization metabolism (Hyunsam extract, Gasiocarp extract, Goji berry root extract, bitter gourd extract, Glycoris root extract ) Was used in a complex combination to verify whether there was a substantial change in the metabolism of glucose degradation in hepatocytes (HepG2 cells). When the natural material extracts are mixed and used, since glycolysis and metabolism may be activated due to the interaction between the materials, various verifications were made by preparing a natural material complex extract by combining all or part of the mixture by varying the mixing ratio as shown in FIG. I wanted to.

1) Cytotoxicity measurement

It was attempted to evaluate the cytotoxicity of the natural material composite extract mixed with all or part of the mixture by varying the mixing ratio, and the experimental method was the same as described in Experimental Example 1. As a result of evaluating the toxicity to hepatocytes (HepG2 cells) by treating the natural material composite extracts as shown in FIG. 22, cytotoxicity was not observed in all formulations.

2) Expression patterns related to Acetyl Co A carboxylase (ACC) protein by natural material complex extracts (Horse ginseng extract, Ginseng extract, Goji root extract, and bitter gourd extract)

Among the acetyl coenzyme A carboxylase (ACC) enzymes, ACC1 is expressed in the cell substrate and is known to activate adipogenesis in hepatocytes and adipocytes (Tong L, Harwood HJ. 2006. Acetyl-coenzyme A carboxylases: versatile targets for drug discovery.Journal of Cellular Biochemistry. 99(6):1476-88), ACC2 is expressed in the outer mitochondrial membrane and is known to regulate mitochondrial β-oxidation (Nguyen P, Leray V, Diez M, Serisier S, Le Bloc'J, Siliart B, Dumon H. 2008. Liver lipid metabolism. Journal of Animal Physiology and Animal Nutrition. 92: 272-283).

Acetyl CoA carboxylase (ACC) has a function of converting acetyl coenzyme A, which has been increased through activation, to malonyl coenzyme A (Malonyl CoA) by activating total sugar utilization. Thus, some of the material that activates the three reactions of glucose degradation utilization metabolism (activating GLUT2 protein, increasing glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increasing the activity of ATP-citrate lyase (ACL)) is partially (Fig. 23). Mixtures 1 to 3) or all (mixture 4 in Fig. 23) were added to investigate the change in the activity to verify whether the selected materials and the combination of the materials were effective.

As a result of the experiment, as shown in FIG. 23A), when all the materials (mixture 4 in FIG. 23) were added and some (mixtures 1 to 3 in FIG. 23) were added to activate the three reactions Acetyl CoA carboxylase (ACC) 1 was significantly increased compared to the insulin-added group. In addition, all of the materials that activate the three reactions of glucose degradation utilization metabolism (activation of GLUT2 protein, increase in glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increase in activity of ATP-citrate lyase (ACL)) (Fig. The addition of mixture 4) was found to increase both acetyl coenzyme A carboxylase (ACC) 1 and 2 than when some (mixtures 1 to 3 in FIG. 23) were added.

In addition, natural material complex extracts are active (phosphorylated acetyl CoA carboxylase, pACC) and inactive form (Acetyl CoA carboxylase, ACC) proteins in hepatocytes (HepG2 cells). As a result of experimentation to see how it affects the expression, three reactions of glucose degradation utilization metabolism (GLUT2 protein activation, glucokinase (GK) and pyruvate dehydrogenase (PDH) activity increase, ATP-citrate lyase) as shown in Fig. 23B. When all (mixture 4 in FIG. 23) or some (mixtures 1 to 3 in FIG. 23) were added to activate (increased activity of (ACL)), protein expression of the active form (pACC) was significantly compared to the control group. Increased, and there was no significant change in the protein expression of the inactive form (ACC).

3) Measurement of the activity of nicotinamide-N-methyltransferase (NNMT) by natural material complex extracts (Horse ginseng extract, Ginseng extract, Gugija root extract, bitter gourd extract, Glycoris root extract)

Among the natural extracts, a material that activates three reactions of glucose degradation and utilization metabolism (activating GLUT2 protein, increasing glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increasing activity of ATP-citrate lyase (ACL)), and The purpose of this study was to confirm the effect on the expression of acetyl coenzyme A carboxylase (ACC) protein in hepatocytes (HepG2 cells) by adding materials that inhibit NNMT activity.

As a result of the experiment, three reactions of glucose degradation utilization metabolism (GLUT2 protein activation, glucokinase (GK) and pyruvate dehydrogenase (PDH) activity increase, and ATP-citrate lyase (ACL) activity increase) as shown in FIG. 24A). Inhibit the activity of nicotinamide-N-methyl-transferase (NNMT) to the experimental group in which all or part of the activated material (mixture 4 in FIG. 24) or part (mixtures 1 to 3 in FIG. 24) was added. It was confirmed that the addition of the glycolysis root extract, which is the material), did not increase both acetyl coenzyme A carboxylase (ACC) 1 and 2 compared to the control group and the insulin-added group.

These results show that when only extracts that increase the metabolism of glucose decomposition and utilization in hepatocytes (Hyunsam extract, Ginseng extract, Gugija root extract, and bitter gourd extract) were added, increased acetyl coenzyme A was expressed by activation of ACC 1 and ACC 2. The reaction to convert to neyl coenzyme A (Malonyl CoA) is activated, but when an extract (glycosylated root extract) that inhibits NNMT activity is added, increased acetyl coenzyme A is combined with polyamines (Acetyl-polyamines) and urine It is believed that the effect of reducing the amount of acetyl coenzyme A synthesized as fatty acids is exhibited because the activation of ACC 1 and ACC 2 cannot occur.

In addition, among the natural material extracts, three reactions of glucose degradation and utilization metabolism (activation of GLUT2 protein, increase in glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increase in activity of ATP-citrate lyase (ACL)) are activated. In the active form (phosphorylated acetyl CoA carboxylase, pACC) and inactive form (acetyl coenzyme A carboxylase, pACC) in hepatocytes (HepG2 cells) by adding a combination of materials and materials that inhibit NNMT activity. CoA carboxylase (ACC) protein expression was tested to see what effect it had.

As a result of the experiment, three reactions of glucose degradation utilization metabolism (activation of GLUT2 protein, increase in glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increase in activity of ATP-citrate lyase (ACL)) as shown in FIG. 24B) When glycotinamide-N-methyl-transferase (NNMT) activity inhibitory material is added to the experimental group in which all or part of the activating material is added, the active form (pACC ) And inactive (ACC) protein expression was significantly reduced.

This result is similar to the result of FIG. 23, since increased acetyl coenzyme A is released into urine after being combined with polyamines (Acetyl-polyamines), the activation of ACC is remarkably reduced, so acetyl coenzyme synthesized into fatty acids. It is believed that the effect of decreasing the amount of A appears.

3) Confirmation of the expression pattern of fatty acid synthetase (FAS) protein by natural material complex extracts (Hyunsam extract, Ginseng extract, Gugija root extract, bitter gourd extract, Glycoris root extract)

Among the natural material extracts, the three reactions of glucose degradation and utilization metabolism (activating GLUT2 protein, increasing glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increasing the activity of ATP-citrate lyase (ACL)) The purpose of this study was to find out how the combination additives affect the expression of fatty acid synthetase (FAS) proteins in hepatocytes (HepG2 cells).

As a result of the experiment, as shown in Fig.25A), three reactions of glucose degradation utilization metabolism (GLUT2 protein activation, glucokinase (GK) and pyruvate dehydrogenase (PDH) activity increase, ATP-citrate lyase ( The expression of fatty acid synthetase (FAS) protein in all experimental groups in which all (mixture 4 in FIG. 25) or part (mixtures 1 to 3 in FIG. 25) was added to activate the (ACL) activity). In particular, when all the materials (mixture 4 in Fig. 25) were added, a significant increase was observed.

In addition, among the natural material extracts, three reactions of glucose degradation and utilization metabolism (activation of GLUT2 protein, increase in glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increase in activity of ATP-citrate lyase (ACL)) are activated. The purpose of this study was to confirm the effect on the expression of the fatty acid synthetase (FAS) protein in hepatocytes (HepG2 cells) by adding materials and materials that inhibit NNMT activity.

As a result of the experiment, three reactions of glucose degradation utilization metabolism (activation of GLUT2 protein, increase in glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increase in activity of ATP-citrate lyase (ACL)) as shown in FIG. When nicotinamide-N-methyl-transferase (NNMT) activity inhibiting material is added to the experimental group in which all or part of the activating material is added, fatty acids in all experimental groups It was confirmed that the protein expression of the synthetase complex (fatty acid synthetase (FAS)) did not show a significant increase, and showed similar values to the control group and the insulin-added group.

These results show that when a material that inhibits nicotinamide-N-methyl-transferase (NNMT) activity is additionally added, the increased acetyl coenzyme A is bound to polyamines (Acetyl-polyamines). ), the amount of fatty acid synthetase (FAS) protein is not increased because the amount of fatty acid synthesized as fatty acid decreases after being excreted in urine.

4) Changes in malonyl coenzyme A (Malonyl-Co A) production by natural material complex extracts (Horse ginseng extract, Ginseng extract, Goji root extract, bitter gourd extract, Glycoris root extract)

Among the natural material extracts, the three reactions of glucose degradation and utilization metabolism (activating GLUT2 protein, increasing glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increasing the activity of ATP-citrate lyase (ACL)) The purpose of this study was to determine how the combination addition had an effect on the change of malonyl-Co A production in hepatocytes (HepG2 cells).

The human malonyl coenzyme A ELISA Kit was used to determine the effect of natural extracts on malonyl-CoA content in HepG2 cells. Malonyl coenzyme A (Malonyl-Co A) content was measured using a quantitative sandwich (quantitative sandwich) ELISA method. After culturing the material in HepG2 cells for 24 hours, the cells were recovered, homogenized with PBS, frozen twice to destroy the cell membrane, and then centrifuged at 5,000 x g for 5 minutes. After collecting only the supernatant, quantify the protein, and dispense 100 uL per well. After reacting for 2 hours at 37° C., 100 uL biotin-antibody (1x) was dispensed without washing, and then reacted at 37° C. for 1 hour. After washing twice with 200 uL wash buffer, 100 uL HRP-avidin (1x) was dispensed and reacted at 37° C. for 1 hour. After washing twice with 200 uL wash buffer, dispense 90 uL TMB substrate, block the light at 37°C for 15-30 minutes, react, and dispense 50 uL stop solution. . Absorbance was measured at 450 nm every 5 minutes.

As a result of the experiment, three reactions of glucose degradation utilization metabolism (GLUT2 protein activation, glucokinase (GK) and pyruvate dehydrogenase (PDH) activity increase, and ATP-citrate lyase (ACL) activity increase) as shown in FIG. The amount of malonyl coenzyme A (Malonyl-Co A) produced was significantly increased in all experimental groups to which all or part of the activated material (mixture 4 in FIG. 26) or part (mixtures 1 to 3 in FIG. 26) was added.

In addition, among the natural material extracts, three reactions of glucose degradation and utilization metabolism (activation of GLUT2 protein, increase in glucokinase (GK) and pyruvate dehydrogenase (PDH) activity, increase in activity of ATP-citrate lyase (ACL)) are activated. The purpose of this study was to confirm the effect on the change in the amount of malonyl-Co A produced in hepatocytes (HepG2 cells) by adding materials and materials that inhibit NNMT activity.

As a result of the experiment, the three reactions of glucose degradation utilization metabolism (GLUT2 protein activation, glucokinase (GK) and pyruvate dehydrogenase (PDH) activity increase, and ATP-citrate lyase (ACL) activity increase) as shown in FIG. When glycotinamide-N-methyl-transferase (NNMT) activity is suppressed in the experimental group in which all or part of the activating material is added, the extract of glycosyllium root extract is added. It was confirmed that the production amount of Neil Coenzyme A (Malonyl-Co A) did not increase significantly, but rather showed a lower value than the control group.

These results show that when a material that inhibits nicotinamide-N-methyl-transferase (NNMT) activity is additionally added, the increased acetyl coenzyme A is bound to polyamines (Acetyl-polyamines). ), the amount excreted into urine increases, and the amount synthesized into Coenzyme A (Malonyl-Co A) decreases.

Claims (2)

Including all of the Hyeonsam extract, Ginseng ginseng extract, Goji berry root extract, bitter gourd extract and Glycoris root extract,
The Hyeonsam extract, Ginseng extract, Goji japonica root extract, bitter gourd extract, and Glycoris root extract are mixed in a weight ratio of 0.3: 0.5: 1: 1.5: 1,
The Hyeonsam extract increases the amount of glucose introduced into hepatocytes,
The gasoline extract increases glucokinase activity,
The wolfberry root extract increases pyruvate dehydrogenase activity,
The bitter gourd extract increases ATP-citrate lyase activity,
The glycosylation root extract inhibits nicotinamide-N-methyltransferase (NNMT) enzyme activity, thereby activating the metabolism of glucose utilization in hepatocytes, lowering blood sugar, and expressing a fatty acid synthase complex (FAS) protein. Food composition for improving diabetes, characterized in that to reduce the.
Including all of the Hyeonsam extract, Ginseng ginseng extract, Goji berry root extract, bitter gourd extract and Glycoris root extract,
The Hyeonsam extract, Ginseng extract, Goji japonica root extract, bitter gourd extract, and Glycoris root extract are mixed in a weight ratio of 0.3: 0.5: 1: 1.5: 1,
The Hyeonsam extract increases the amount of glucose introduced into hepatocytes,
The gasoline extract increases glucokinase activity,
The wolfberry root extract increases pyruvate dehydrogenase activity,
The bitter gourd extract increases ATP-citrate lyase activity,
The glycosylation root extract inhibits nicotinamide-N-methyltransferase (NNMT) enzyme activity, thereby activating the metabolism of glucose utilization in hepatocytes, lowering blood sugar, and expressing a fatty acid synthase complex (FAS) protein. A pharmaceutical composition for preventing or treating diabetes, characterized in that reducing the.

Images