KR101833428B1 - Pharmaceutical composition or prevention or treatment of diabetes comprising the extract of Angelica dahurica, fractions thereof or compound Phellopterin isolated therefrom as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diabetes or diabetic complications containing an Angelica dahurica extract, a fraction thereof or a phellopterin compound isolated therefrom as an active ingredient. In is confirmed that the Angelica dahurica extract, an n-hexane fraction thereof and the phellopterin compound show GPR119 agonistic activity and reduce glucose in mouse blood in in-vitro and in-vivo experiments. Therefore, the Angelica dahurica extract, the n-hexane fraction thereof and the phellopterin compound can be used for the pharmaceutical composition for preventing or treating diabetes or diabetic complications and health functional food for alleviating diabetes or diabetic complications.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for prevention and treatment of diabetes comprising an extract of Porphyra, fractions thereof or a pelopterin compound isolated therefrom as an active ingredient, and a pharmaceutical composition for prevention and treatment of diabetes comprising fractions thereof or compound Phellopterin isolated therefrom as an active ingredient}

The present invention relates to a pharmaceutical composition for the prevention and treatment of diabetes, comprising an Angelica dahurica extract, a fraction thereof, or a Phellopterin compound isolated therefrom as an active ingredient.

Diabetes mellitus can be classified into Type 1 and Type 2 diabetes. Type 1 diabetes is caused by genetic susceptibility and viral infection resulting in an immunological response to the pancreas, and autoimmune diseases And type 2 diabetes is the predisposing type of insulin resistance. The main pathogens are obesity, decreased insulin receptor, decreased tyrosine kinase activity, decreased muscle / adipose tissue type transporter in muscle tissue and adipose tissue, insulin receptor substrate-1 (IRS-1) deficiency in the intracellular environment.

Type 1 diabetes is known as one of the typical autoimmune diseases and is a chronic disease in which pancreatic [beta] -cells that produce insulin by self-activating T cells that recognize pancreatic [beta] -cells are destroyed (American Diabetes Association, Diabetes Care 2014, 37 Suppl 1, S81-90). On the other hand, type 2 diabetes is a common metabolic disorder that is very common worldwide and is characterized by insulin resistance and hyperglycemia. It has various pathological features such as insulin secretion defects, inappropriate hepatic glucose production and peripheral insulin resistance from pancreatic beta cells, and many therapeutic approaches have been tried for the treatment of this metabolic syndrome.

Representative methods for treating type 2 diabetes include non-drug therapies such as diet and exercise therapy, and are largely classified into drug treatment methods other than the non-drug therapy. Drug therapy is usually initiated when drug control methods such as diet and exercise do not achieve the goal of glycemic control and widely prescribed oral drugs can be classified according to the mechanism of action and structural characteristics of the drug, (Current Medical Research and Opinion 2007, 23, 945-952).

Metformin is a biguanide-based drug that acts mainly through inhibition of glucose production in the liver and decreased insulin resistance, and generally has no side effects, but lactic acidosis can be induced as a potential side effect (International Journal of Obesity 2008, 32, 61-72). The thiazolidinedione-based drugs include rosiglitazone and pioglitazone as PPARgamma agonists, which act through the improvement of insulin sensitivity and have been reported to exhibit side effects such as edema, weight gain, hepatotoxicity and heart failure (Proceedings of the National Academy of Sciences, United States of America 2009, 106, 18745-18750). Sulfonylurea (sulphonylurea) drugs act in a sugar-independent manner to promote insulin secretion in the beta cells of the pancreas, thus causing hypoglycemia and possibly causing weight gain. The meglitinide-based drugs also have similar mechanism and side effects to sulfonylurea drugs. Alpha-glucosidase inhibitors are used for post-prandial suppression, with adverse effects in the intestine. Insulin and insulin analogs are commonly used in combination with oral medications, which have side effects such as hypoglycemia and weight gain induction and limitations as an injection (Current Opinion in Drug Discovery & Development 2009, 12, 519-532).

In recent years, efforts have been made to develop drugs that can increase insulin secretion in a sugar-dependent manner to address hypoglycemia and weight gain associated with drug therapy for advanced type 2 diabetes (Forecast Insight: Antidiabertics, Datamonitor DMHC2447, 08/2008).

GLP-1 receptor agonists such as exenatide are analogs (GLP-1 mimetics) mimicking GLP-1 rapidly degraded by dipeptidyl peptidase-IV (DPP-IV) It is a drug that promotes glucose-dependent insulin secretion without inhibiting rapid degradation, inhibits glucagon secretion, gastric emptying and appetite, and shows the effect of GLP-1, which has a beta-cell protective effect. Therefore, it does not induce hypoglycemia, shows weight reduction effect, and has HbA1c lowering effect, but it has side effects such as intestinal adverse effects and pancreatitis, and limitations as an injection. On the other hand, DPP-IV inhibitors such as sitagliptin and vildagliptin act by inhibiting the degradation of GLP-1 as an oral drug and show a HbA1c-lowering effect similar to GLP-1 receptor agonist, No reduction effect is observed (Current Opinion in DrugDiscovery & Development 2009, 12, 519-532).

In this context, GPR119 is a promising target for the development of type 2 diabetes therapies and is being targeted for intensive research and development by major pharmaceutical companies. In adults, GPR119 is predominantly expressed mainly in the pancreas and intestines and increases glucose-dependent insulin secretion upon activation in pancreatic beta cells, similar to the GLP-1 receptor, and increases GLP-1 and / or GIP (glucose- dependent insulinotropic peptide is known to increase secretion of the corresponding secretion when activated in secretory cells. Unlike the GLP-1 receptor, which is a peptide ligand, GPR119 is a strong and selective ligand, (Endocrinology 2007, 148, 2598-2600; Expert Opinion on Drug Discovery 2008, 3, 403-413; Annual Reports in Medicinal Chemistry 2009, 44, 149-170; Expert Opinion on Therapeutic Patents 2009, 19, 1339-1359; Current Opinion in Drug Discovery & Development 2009, 12, 519-532).

Thus, GPR119 is a potential point of implementation of the GLP-1 receptor agonist efficacy as an oral drug. In 2006, APD-668 compound (Ortho-McNeil, Arena) as GPR119 agonist first entered Clinical Phase I GPR119 agonists such as APD-597 compounds (Ortho-McNeil, Arena), PSN-821 compounds (OSI), MBX-2982 compounds (Sanofi-Aventis, Metabolex), and GSK-1292263A compounds (GSK) Currently, PSN-821 compound (OSI), MBX-2982 compound (Sanofi-Aventis, Metabolex) and GSK-1292263A compound (GSK) have entered clinical phase II (Thomson Reuters Integrity).

Herbal botanical extracts have received much attention as complementary or alternative drugs for the treatment of diabetes. Angelica dahurica Bentham et Hooker f. (Umbelliferae, AD) have been widely used in Korea, China and Japan as traditional drugs effective for colds, headaches and toothache. Povidone may be selected from coumarin (PubChem CID: 323), phellopterin (PubChem CID: 98608), isoimperatorin (PubChem CID: 68081), imperatorin (PubChem CID: 10212) Including but not limited to the following: dexamethasone, oxypeucedanin (PubChem CID: 33306), byakangelicin (PubChem CID: 10211), pimpinellin (PubChem CID: 4825) , Antimutagenic, antibiotic, anticancer, and antioxidant.

Accordingly, the present inventors have made efforts to develop a diabetic therapeutic agent that is guaranteed efficacy and safety, and have found that an extract of Angelica dahurica having GPR119 agonizing activity, a fraction thereof and a compound isolated therefrom have glucose load GLP-1 and insulin secretion, and GRP119 agonist effects according to the present invention.

An object of the present invention is to provide a pharmaceutical composition and a health functional food for preventing or treating diabetes or diabetic complications containing Angelica dahurica extract, a fraction thereof or a phellopterin compound isolated therefrom as an active ingredient .

In order to achieve the above object, the present invention provides a pharmaceutical composition and a health functional food for preventing or treating diabetes or diabetic complications containing Angelica dahurica extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition and a health functional food for preventing or treating diabetes or diabetic complications containing the phellopterin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating diabetes or diabetic complications containing Angelica dahurica extract, a fraction thereof or a phellopterin compound isolated therefrom as an active ingredient, The n-hexane fraction and the pelopherin compound showed GPR119 (G protein-coupled receptor 119) agonistic activity in in vitro and in vivo experiments and confirmed that glucose in mouse blood was reduced, A pharmaceutical composition for preventing or treating complications, and a health functional food for improving diabetes or diabetic complications.

Figure 1 shows the activity of GPR119 activator according to the treatment of Angelica dahurica extract in Invitro:
A: GPR119-CRE-bla CHO-K1 cells were assayed for beta-lactamase activity as a reporter following the treatment with the peptone extract;
B: Measurement of cAMP activity by GLUTag and INS-1 cell lines upon treatment with Piper extract;
C: Results of GLP-1 secretion from GLUTag cell line treated with Piper extract;
D: Insulin secretion due to glucose stimulation in the INS-1 cell line treated with glutathione peroxidase;
n = 3-4 / group; * p <0.05, ** p <0.01 (solvent control comparison), ## p <0.01 (comparison solvent control in the presence of glucose).
FIG. 2 is a graph showing oral glucose tolerance test and insulin secretion according to the treatment of a gill extract in a mouse:
A: Oral load testing in C57BL6 mice;
B: serum insulin measurement after C57BL6 mouse glucose administration;
C: Oral load test in db / db mouse;
n = 7-9 / group; * p < 0.05, ** p < 0.01 (comparison of control group).
Figure 3 shows the results of measuring the activity of GPR119 activator according to the treatment of Angelica dahurica fractions in Invitro:
A: GPR119-CRE-bla beta-lactamase activity as a reporter following treatment with grit total crude (MeOH), n-hexane, ethyl acetate (EA), n-butanol (BuOH), or water extract in CHO- β-lactamase activity);
B: Measurement of active GLP-1 secretion from GLUTag cells by treatment with grit total crude (MeOH) and n-hexane;
C: Insulin secretion by glucose-stimulated total or n-hexane extract treatment in INS-1 cells;
n = 3-4 / group; * p < 0.05, ** p < 0.01 (comparison of control group).
Figure 4 shows the oral challenge test and insulin secretion according to the treatment of glutathione n-hexane fraction in mice:
A: C57BL6 mice (n = 15 / group) Oral load test;
B: serum insulin measurement after C57BL6 mouse glucose administration;
C: db / db mice (n = 10-11 / group) Oral load test;
* p < 0.05, ** p < 0.01 (compare PBS-treated group as a control group).
Fig. 5 is a diagram showing the chemical structure of a compound separated from a pea extract; Fig.
6 is a graph showing the activity of GPR119 activator according to the compound treatment separated from the gluten in Invitro:
A: Measurement of beta-lactamase activity as a reporter following treatment with imperatorin, phellopterin or isoimperatorin in GPR119-CRE-bla CHO-K1 cells result;
B: Measurement of active GLP-1 secretion by treatment with imperatorin or phellopterin in GLUTag cell line;
C: Results of measurement of insulin secretion due to glucose stimulation by treatment with imperatorin or phellopterin in INS-1 cell line;
n = 3-4 / group; * p < 0.05, ** p < 0.01 (control comparison); #p <0.05, ## p <0.01 (comparison of solvent control in the presence of glucose).
7 is a diagram showing the results of an oral load test according to the treatment with imperatorin or phellopterin in a mouse:
A: Oral load test after administration of imperatrin to C57BL6 mice;
B: Oral load test after administration of C57BL6 mouse pelopherin;
C: Oral load test after administration of pelopherin to diabetic db / db mice;
n = 8-12 / group; * p < 0.05, ** p < 0.01 (compare PBS-treated group as a control group).
FIG. 8 is a chart for confirming the reduction effect of glucose after a long-term treatment with a grit extract or a n-nucleic acid fraction in a diabetic mouse:
A: Results showing blood glucose concentration 3 weeks after treatment;
B: Oral load test result;
C: Weight change after 3 weeks of treatment
D: change in food intake;
* p < 0.05, ** p < 0.01 (comparison of PBS-treated group as a control group); # p < 0.05 (compared with the PBS-treated group as a control group after 3 weeks of treatment).

As used herein, the term "prophylactic " means any action that inhibits or slows the onset of a disease by administration of the composition of the present invention.

As used herein, the terms " treatment "and" improvement "refer to all actions by which administration of the composition of the present invention improves or alleviates the symptoms of the disease.

The term "administering" as used herein is meant to provide any desired composition of the invention to a patient in any suitable manner.

The term "patient" as used herein refers to all animals such as humans, monkeys, dogs, goats, pigs or rats having a disease whose symptoms can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating diabetes or diabetic complications containing Angelica dahurica extract or a fraction thereof as an active ingredient.

The grit extract may be extracted with water, C ₁ to C ₂ lower alcohols or a mixture thereof, and the lower alcohol may be ethanol or methanol, but is not limited thereto.

The grizzly extract is preferably, but not exclusively, prepared by a process comprising the steps of:

1) adding an extraction solvent to the grit;

2) filtering the extract of step 1);

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

In the above method, the granules of step 1) may be used without limitation such as cultivated or commercially available. The buckwheat may be flowers, stems, leaves or fruits.

In the above method, any conventional method known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used as the extraction method of the glutinous extract. It is preferable that the extraction solvent is added by 2 to 20 times the amount of the dried glycyrrhiza. The extraction temperature is preferably 20 to 50 DEG C, but is not limited thereto. The extraction time is preferably 10 to 100 hours, more preferably 24 to 96 hours, and most preferably 72 hours, but not always limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In addition, the fraction according to the present invention

1) adding the grit to water, a C ₁ to C ₂ lower alcohol or a mixture thereof to obtain a pea pot extract;

2) fractionating the extract obtained in step 1) with n-hexane, ethyl acetate and n-butanol to obtain a fraction; And

3) separating and purifying the compound by performing silica gel column chromatography on the n-hexane fraction obtained in the step 2).

Specifically, the fractions were prepared by suspending the granular extract obtained in the above step 1) in methanol, sequentially adding n-hexane, ethyl acetate and n-butanol to separate the respective layers, and then concentrating the n-hexane layer under reduced pressure And drying to obtain the n-hexane fraction, but is not limited thereto.

At this time, the vacuum concentration is preferably performed using a vacuum rotary evaporator, but is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying, room temperature drying or freeze drying, But is not limited thereto.

The diabetes mellitus is preferably type 2 diabetes but the present invention is not limited thereto. The diabetic complication may be at least one of obesity, coronary artery disease, ischemic stroke, peripheral vascular disease, intermittent claudication, myocardial infarction, postprandial lipidemia, Diabetic nephropathy, chronic renal failure, diabetic neuropathy, angina pectoris, thrombosis, atherosclerosis, hypertension, diabetic retinopathy, macular degeneration, cataract, diabetic nephropathy, glomerulonephritis, glomerulonephritis, , Myocardial infarction, transient ischemic attack, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia, insulin resistance, foot ulcer, ulcerative colitis, and endocardial dysfunction But is not limited thereto.

The composition preferably has GPR119 (G protein-coupled receptor 119) agonistic activity.

The therapeutically effective amount of the composition of the present invention may vary depending on a variety of factors, such as the method of administration, the site of administration, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in consideration of safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman ' s Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington ' s Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity, Kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and can be administered 1 to 6 times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof, which are commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Sodium chloride, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, antioxidants, buffers, And other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. The composition of the present invention may contain 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the above compound, based on the total weight of the composition.

The composition of the present invention may be administered orally or non-orally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally administered in accordance with a desired method, and the dose may be appropriately determined depending on the patient's body weight, The range varies depending on diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, more preferably one to several times a day.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances or preservatives are included .

Formulations for non-oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations or suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

In a specific embodiment of the present invention, the present inventors used dry distilled roots of gherkin to extract gherkin extracts with methanol (500 mL x 3) at room temperature using an ultrasonic apparatus, and then 27.5 g of concentrated The crude extract was resuspended and the concentrated residue was resuspended in 1000 mL of water and treated with 1000 mL of n-hexane, ethyl acetate or n-butanol sequentially To obtain 4.7 g of n-hexane fraction, 1.4 g of ethyl acetate fraction, 4.8 g of n-butanol fraction and 16 g of water fraction, respectively.

The present inventors measured the GPR119 activator in the extract of GPR119-CRE-bla CHO-K1 reporter cells using GPR119-CRE-bla CHO-K1 reporter cell line. The reporter activity was increased according to the treatment capacity of the extract of Phellinus linteus. (See Fig. 1A).

The present inventors also found that cAMP accumulates directly in enteroendocrine cell lines (GLUTag cells) and pancreatic beta-cell lines (INS-1 cells), which are known to express GPR119 Was measured. GPR119 binds to Gαs protein and induces cAMP production. As a result, it was confirmed that the amount of cAMP significantly increased in both GLUTag and INS-1 cells after treatment with 100 μg / mL of Pallet's extract (see FIG. Similar to these results, the amount of active GLP-1 was significantly increased upon treatment of the pomegranate extract in GLUTag cells (see FIG. 1c).

In addition, the present inventors measured glucose-activated insulin secretion in INS-1 cells in order to examine the effect of Pallet Extract on the beta-cells of the inner ear, since the GPR119 agonists increase the insulin secretion of the inner beta cells. As a result, insulin secretion was slightly increased when 3 mM glucose was treated, and insulin secretion was further increased when 17.5 mM glucose was treated. When 100 μg / mL of the extract of Palladium and 17.5 mM glucose were simultaneously treated (See Fig. 1d). Therefore, it was found that the extract of Phellodendron griseus acts as an agonist of GPR119.

The present inventors conducted an oral glucose tolerance test using an 8-week-old male wild-type C57BL6 mouse and a diabetic db / db mouse (C57BL / KsJ background) in order to examine whether or not the grit extract was involved in glucose regulation. As a result, it was confirmed that the amount of glucose present in the blood of the normal C57BL6 mouse treated with the extract of Phellodendron japonica extract was significantly smaller than that of the PBS treated group at 30 min and 60 min, and compared with the control group treated with PBS, C57BL6 mice showed a decrease in the amount of glucose by about 11.5% (see Fig. 2a).

In addition, the present inventors measured the amount of serum insulin after oral glucose administration in order to determine whether the enhanced glucose tolerance was increased by insulin secretion in a normal mouse treated with grit extract. As a result, it was confirmed that the amount of serum insulin was increased more in the normal mice to which grit extract was administered than in the PBS-treated control group (see FIG. 2B). These results indicate that increased insulin secretion contributes to lowering the amount of glucose in the blood of mice treated with grit.

In addition, the present inventors conducted an oral glucose tolerance test in order to confirm the blood glucose-lowering effect of obese mice in the treatment of grit extract. As a result, compared to the control group treated with PBS, the obese model mice treated with the grit extract showed a significant decrease in the amount of glucose present in the blood at each time point after administration of the glucose solution, and compared with the control group administered with PBS, The mice treated with the extract showed a 24.6% decrease in the amount of glucose (see FIG. 2C).

In order to evaluate the efficacy of various fractions of Phellinus linteus extract on GPR119 activity, GPR119-CRE-bla CHO-K1 reporter cells were treated with various concentrations of glutaraldehyde extract and n-hexane, ethyl acetate, n-butanol, The fractions were processed. As a result, it was confirmed that the reporter activity was highest in the cells treated with n-hexane fraction of the extract of Phellodendron gigantis (see Fig. 3A). The amount of active GLP-1 (FIG. 3b) and insulin secretion in INS-1 cells (FIG. 3c) in GLUTag cells were significantly increased in the n-hexane fraction of the pomegranate extract. These results suggest that the active compound that activates GPR119 is present in the n-hexane fraction of the extract.

The present inventors performed an oral glucose tolerance test after administration of n-hexane fraction to normal C57BL6 mice in order to examine the effect of the n-hexane fraction fractioned from the extract on the glucose control. As a result, it was confirmed that the amount of glucose in the blood of the mice administered with n-hexane fraction was significantly reduced (see FIG. 4A), compared to the control group in which only PBS was administered 30 minutes and 60 minutes after the administration of glucose. In addition, it was confirmed that the secretion of insulin significantly increased when the n-hexane fraction of the grit extract was administered alone (see FIG. 4B).

In addition, the present inventors performed an oral glucose tolerance test on obese db / db mice. As a result, it was found that when db / db mouse administered with n-hexane fraction at 30 and 60 minutes after glucose administration The amount of glucose in the blood was significantly reduced compared to the PBS-treated db / db control rats, and the amount of glucose decreased by 12.7% in the mice treated with the n-hexane fraction of PBS compared to the PBS-treated control group (See FIG. 4C).

Thus, the extract of Angelica dahurica of the present invention, its fractions, showed that it showed agonistic activity of GPR119 (G protein-coupled receptor 119) in in vitro and in vivo tests and decreased glucose in mouse blood , Diabetes, or diabetic complications.

The present invention also provides a pharmaceutical composition for preventing or treating diabetes or diabetic complications comprising, as an active ingredient, a phelopterin compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

.

.

The felopterin compound may be isolated from an extract of Angelica dahurica or a fraction thereof, but may be a compound isolated from other animals, plants, or microorganisms, or artificially synthesized through any method known in the art do.

The diabetes mellitus is preferably type 2 diabetes but the present invention is not limited thereto. The diabetic complication may be at least one of obesity, coronary artery disease, ischemic stroke, peripheral vascular disease, intermittent claudication, myocardial infarction, postprandial lipidemia, Diabetic nephropathy, chronic renal failure, diabetic neuropathy, angina pectoris, thrombosis, atherosclerosis, hypertension, diabetic retinopathy, macular degeneration, cataract, diabetic nephropathy, glomerulonephritis, glomerulonephritis, , Myocardial infarction, transient ischemic attack, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia, insulin resistance, foot ulcer, ulcerative colitis, and endocardial dysfunction But is not limited thereto.

The composition preferably has GPR119 (G protein-coupled receptor 119) agonistic activity.

The present invention includes all the possible solvates and hydrates, which can be prepared therefrom, as well as the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of the present invention in an excess amount of an aqueous acid solution, adding the salt to a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile &Lt; / RTI &gt;

By heating the same amount of the compound represented by the formula (1) and the acid or alcohol in water, and then evaporating and drying the mixture, or by filtering the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In a specific example of the present invention, the present inventors analyzed the glycocalyx extract and the n-hexane fraction extracted from the above with Agilent 1200 HPLC, and separated the total of three compounds. Compound 1 thus separated was imperatorin and compound 2 Phellopterin, and compound 3 was identified as isoimperatorin (see FIG. 5).

The present inventors have confirmed that imperatorin, phellopterin, and isoimperatorin, which are compounds obtained from n-hexane fractions fractionated from the extract of Phellinus linteus, are GPR119 activators. As a result, GPR119-CRE-bla When CHO-K1 cells were treated with imperatorin and phellopterin, the reporter activity was increased with increasing concentration, but not with isoimperatorin (see Fig. 6a) ). In addition, the treatment of GLUTag cells with imperatorin and phellopterin significantly increased HLP-1 secretion (see FIG. 6b) and increased glucose-activated insulin secretion in INS-1 cells (See FIG. 6C).

The inventors of the present invention found that imperatorin and phellopterin were first treated with imperatorin and phellopterin in C57BL6 mice and then glucose tolerance test was performed to confirm whether imperatorin or phellopterin enhances glucose tolerance oral glucose tolerance test). As a result, when the imperatorin was administered, the amount of glucose in the blood was similar to that of the PBS-treated control, but when the phellopterin was administered, the amount of glucose in the blood was significantly reduced as compared with the control (See Figs. 7A and 7B). From these results, it was found that phellopterin obtained from the Glycyrrhizae extract can act as an activator of GPR119.

The present inventors also confirmed that phellopterin improves glucose tolerance in obese db / db mice. As a result, rheumatoid rheumatoid arthritis (rheumatoid arthritis) (See FIG. 7C).

The present inventors measured the amount of glucose in the blood after 3 weeks of administration of the grit extract or n-hexane fraction in obese db / db mice to see if the amount of glucose in the blood decreased when the gully extract or n-hexane fraction was administered for a long time Respectively. As a result, it was confirmed that the amount of glucose in the blood was significantly reduced in the mice treated with n-hexane fraction compared with the control group administered with PBS, but the effect of glucose reduction was not observed in the mice administered with the extract (see FIG. 8A) .

In addition, the present inventors performed oral glucose tolerance test three weeks after the treatment of grit extract or n-hexane fraction. As a result, the mice treated with n-hexane fraction had higher glucose , The amount of glucose in the blood was significantly reduced at 15 minutes and 30 minutes after administration. In comparison with the PBS-treated control group, the amount of glucose decreased by about 8% in the mouse treated with Pallet's extract, and the mice treated with n-hexane fraction Showed a decrease in the amount of glucose by about 13% (see Fig. 8B).

In addition, the inventors of the present invention compared the body weights of mice consuming the same amount of food per day (see FIG. 8D). As a result, mice treated with the Pallet Extract had a pellet extract dose of 43.7g ± 0.7 Weighed to 44.8 g ± 1.0 after week and mice treated with n-hexane fraction changed from 42.8 g ± 0.7 before n-hexane fraction to 43.3 g ± 0.6 after 3 weeks of n-hexane fractionation , And mice administered with PBS were observed to weigh from 43.7 g ± 0.7 before administration of PBS to 47.3 g ± 1.0 after 3 weeks of PBS administration (see FIG. 8C). It was confirmed that the mice administered with the Pallet extract or the n-hexane fraction showed a slightly increased body weight in comparison with the PBS-treated control rats.

Accordingly, the phellopterin compound isolated from the Angelica dahurica extract of the present invention exhibited GPR119 (G protein-coupled receptor 119) agonistic activity in the in vitro and in vivo experiments, By confirming that glucose is reduced, it can be used in a pharmaceutical composition for the prevention or treatment of diabetes or diabetic complications.

The present invention also provides a health functional food for improving diabetes or diabetic complications comprising Angelica dahurica extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a health functional food for improving diabetes or diabetic complication, which comprises the phellopterin compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The health functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings.

There is no particular limitation on the kind of the food. Examples of foods in which the extract of Guerbet, its fractions, and the Pelopherin compound can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The composition for health functional beverage of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of guarbet, fractions thereof, and the felopterin compound of the present invention can be used as various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, , Stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the extract of Guiza, its fractions, and the felopterin compound of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The health functional food composition of the present invention may be various oral or parenteral formulations. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used.

Accordingly, the Angelica dahurica extract, its fractions or the phellopterin compounds isolated therefrom have an agonistic activity of GPR119 (G protein-coupled receptor 119) in in vitro and in vivo experiments, And confirming that glucose in the blood of the mouse is reduced, it can be used for health functional food for diabetes or diabetic complication improvement.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

<1-1> Vet ( Angelica dahurica ; AD) Crude extract crude extract and Fraction  Produce

The present inventors purchased glutinous roots from Kyungdong market (Seoul, Korea) to prepare the extract of Guridani, and stored the voucher specimen in HYU-AD-001 (HYU-AD-001) The following experiment was carried out for the preparation of the extract.

Concretely, the dry root of the peel was extracted with methanol (500 mL x 3) at room temperature using an ultrasonic apparatus, and 27.5 g of concentrated crude extract was obtained under vacuum. The concentrated residue was resuspended in 1000 mL of water and treated sequentially with 1000 mL of n-hexane, ethyl acetate or n-butanol, 4.7 g of hexane fraction, 1.4 g of ethyl acetate fraction, 4.8 g of n-butanol fraction and 16 g of water fraction.

<1-2> Vet Crude extract  And n- Hexane Fraction  High performance liquid Chromatography  Graph (HPLC) analysis

The present inventors analyzed the gully extract and n-hexane fractions extracted from the above Example <1-1> with Agilent 1200 HPLC.

Specifically, the mobile phase was flowed at a rate of 1 mL / min at 40 ° C using INNO C18 column (250 mm × 4.6 mm, 5 μm, Youngjin Biochrome, Korea). The mobile phase was 20-50% A for 0-20 min, A (acetonitrile with 0.1% formic acid) and B (0.1% formic acid) 50-70% A for 35 minutes and 75-100% A for 35-40 minutes. Total crude extract and n-hexane fraction were injected at 10 μl, and the chromatogram was observed at 254 nm. The three major peaks appeared at 19.1 minutes, 22.3 minutes and 26.2 minutes.

<1-3> Vet  Purification of the compound from the n-hexane fraction of the extract

The present inventors further purified the n-hexane fraction of the grit extract prepared in Example <1-1> by preparative HPLC.

Specifically, Inno C18 column (20.times.250 mm, 5 .mu.m, YoungJin Biochrom, Korea) was used and purification was performed by injecting 500 .mu.l of n-hexane fraction. The mobile phases were eluted with water containing 0.1% formic acid and 0.1% formic acid at a rate of 10 mL / min for 40-50 minutes at 40-100% A for 0-40 minutes While the mobile phase was flowed to 100% A. The chromatogram was observed at 254 nm and the separated compounds were identified by ESI-MS, ¹ H and ¹³ C NMR. Compound 1 was fractionated at 30.45-32.46 min, Compound 2 was fractionated at 32.20-33.15 min, and Compound 3 was collected at 35.05-36.05 min.

As a result, as shown in Fig. 5, a total of three compounds were isolated. Compound 1 thus isolated was identified as imperatorin, compound 2 as phellopterin, and compound 3 as isoimperatorin (Fig. 5).

In vitro (Invitro) Vet  Depending on the treatment of the extract GPR119  Identification of agonistic activity

<2-1> Vet  The β- lactamase  Identification of the activity of luciferase reporter expression

We used GPR119-CRE-bla CHO-K1 reporter cell line to measure the GPR119 activator in the pomegranate extract.

Specifically, the extract of Ganoderma lucidum was dissolved in dimethylsulfoxide (DMSO). (Gibco BRL, NY, USA) and 100 U / mL penicillin (Gibco BRL, NY, USA) were added to DMEM high glucose medium penicillin, 100 μg / mL streptomycin, 0.1 mM non-essential amino acids, 25 mM HEPES (pH 7.3), 100 μg / mL of Zeocin ™ and 600 μg / mL of hygromycin . The INS-1 rat insulinoma cells were cultured in RPMI-1640 medium (Gibco BRL, NY, USA) supplemented with 11 mM glucose, 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin , 50 μM beta-mercaptoethanol and 1 mM sodium pyruvate. GLUTag mouse enteroendocrine L cells were cultured in DMEM (Gibco BRL, NY, USA) medium supplemented with 5.5 mM glucose, 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. The GPR119-CRE-bla CHO-K1 cells were treated with different concentrations (1, 10, and 100 μg / ml) of Pallet's extract and the β-lactamase luciferase activity was measured.

For the GPR119 reporter assay, GeneBLAzer® T-REx GPR119-CRE-bla CHO-K1 cells containing human G-protein receptor 119 (GPR119) were inserted safely into the CRE-bla CHO-K1 cell line respectively. CHO-K1 cells contain the β-lactamase reporter gene under the control of the cAMP response element. GeneBLAzer (R) T-REx (TM) GPR119-CRE-bla CHO-K1 cells were seeded on a 96 well culture plate to give 2 x 10 ⁴ cells per 80 μL. The next day, 20 μL of assay media (Cat No. 10569) containing various concentrations of AD extract or compound were treated and incubated for 5 hours. After 5 hours, LiveBLAzer ™ -FRET B / G Substrate (Invitrogen, Carlsbad, Calif., USA) was added and cultured in the dark for 2 hours at room temperature. Blue / green emission ratios were calculated for each well by measuring the fluorescence values emitted at 460 nm (blue) and 530 nm (green) using a VICTOR3 microplate reader (PerkinElmer Life Sciences, Waltham, MA, USA) .

As a result, as shown in FIG. 1, the reporter activity was increased according to the treatment capacity of the extract of Phellinus linteus, and it was confirmed that intracellular cAMP production was promoted by the treatment with the extract of Phellinus linteus (FIG. 1A).

<2-2> Vet  Depending on the treatment of the extract cAMP  Confirm generation induction

GPR119 binds to Gαs protein and induces cAMP production. Therefore, the present inventors have found that the degree of accumulation of cAMP directly in the enteroendocrine cell line (GLUTag cells) and the pancreatic beta-cell line (INS-1 cells), which are known to express GPR119 Were measured.

Specifically, GLUTag and INS-1 cells were plated on a 12-well culture plate at a concentration of 1 × 10 ⁵ cells per well and cultured for 24 hours. After 24 hours, the cells were washed with PBS, and then Krebs-Ringer bicarbonate (KRB) buffer (116 mM NaCl, 1.8 mM CaCl ₂ · 2 (H ₂ O), 0.8 mM MgSO ₄ (H ₂ O), 5.4 mM KCl, 1 mM NaH ₂ PO _4. 2 (H ₂ O), 26 mM NaCHO ₃ , and 0.5% BSA, pH 7.4) for 30 min. After 30 minutes, the cells were harvested and lysates were obtained using 0.1 N HCl. The concentration of cAMP was measured using a cAMP ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer's protocol.

As a result, it was confirmed that the amount of cAMP significantly increased in both GLUTag and INS-1 cells after treatment with 100 μg / mL of Pallet's extract as shown in FIG. 1 (FIG. 1B). Similar to these results, the amount of active GLP-1 was significantly increased upon treatment of the pomegranate extract in GLUTag cells (FIG. 1c).

<2-3> Vet  Insulin secretion by extract treatment

GPR119 agonists increase insulin secretion in the beta-cell of interest. The present inventors measured the glucose-activated insulin secretion in INS-1 cells in order to examine the efficacy of the extract of Porphyra to beta-cells.

Specifically, INS-1 cells were washed in KRB buffer and then cultured in KRB buffer (0.1 mM glucose) containing glutaraldehyde extract for 2 hours to measure the amount of insulin secretion activated by glucose. After 2 hours, 3 or 17.5 mM glucose was added to KRB buffer and incubated at 37 ° C and 5% CO ₂ for 90 minutes. The amount of insulin was measured using a rat ultrasensitive ELISA kit (Alpco Diagnostics, NH, USA) according to the manufacturer's test method. The amount of insulin normalized to the amount of protein in the cell lysate.

As a result, as shown in FIG. 1, when 3 mM glucose was treated, the insulin secretion was slightly increased. When 17.5 mM glucose was treated, the insulin secretion was further increased, and 100 μg / When glucose was treated in the same manner, insulin secretion was significantly increased (FIG. 1d).

Therefore, it was found that the extract of Phellodendron griseus acts as an agonist of GPR119.

In the mouse model Vet  Depending on the treatment of the extract Glucose  Identify tolerance and insulin secretion efficacy

<3-1> Vet  In the blood of normal mice according to the extract treatment Glucose  Confirm reduction effect

The present inventors conducted an oral glucose tolerance test in order to determine whether the extract of Ganoderma extract is involved in glucose regulation.

Specifically, 8-week-old male wild-type C57BL6 mice and diabetic db / db mice (C57BL / KsJ background) were obtained from Orient Biotech (Gyeonggi-do, Korea) and Korea Biotechnology Research Institute. The mice were housed in a no-pathogen-free environment with a 12-h light / dark cycle and a temperature of 22-24 ° C (23 ± 1 ° C) for 12 hours. And were housed in an experimental animal center. All animal experiments were approved by the Animal Experimental Ethics Committee of the Cancer Diabetes Research Institute at Gachon University. For oral glucose tolerance test, mice were fasted for 14 hours and then administered with oral gavage extract (300 mg / kg body weight in PBS) or PBS as a control via oral intubation. After 30 minutes, the glucose solution was administered to the mouth by dissolving 2 g / kg body weight of mouse in PBS. Blood was obtained from the tail vein at intervals of 0, 5, 10, 15, 30, 60, 90 and 120 minutes. The amount of glucose in the blood was measured using a glucometer (LifeScan, Inc. One Touch Ultra).

As a result, as shown in FIG. 2A, the amount of glucose present in the blood in the normal C57BL6 mouse treated with the Pallet's extract was significantly smaller than that of the PBS-treated control at 30 minutes and 60 minutes, The amount of glucose in C57BL6 mice treated with Phellinus linteus extract was reduced by about 11.5% (Fig. 2a), compared to the control group administered with PBS.

<3-2> Vet  The effect of extract treatment on serum insulin level in normal mice

As shown in the above Example <3-1>, the present inventors investigated whether the increased tolerance of glucose in normal mice treated with glutathione peroxidase was due to an increase in insulin secretion, at 5, 10 and 15 minutes after oral glucose administration And the amount of serum insulin was measured. Serum insulin levels were measured using a mouse insulin ultrasensitive ELISA kit (Alpco Diagnostics, NH, USA).

As a result, as shown in FIG. 2B, it was confirmed that the amount of serum insulin in normal mice to which grit extract was administered increased more than that of PBS-treated control (FIG. 2B). These results indicate that increased insulin secretion contributes to lowering the amount of glucose in the blood of mice treated with grit.

<3-3> Vet  In the blood of obese mice according to the treatment of the extract Glucose  Confirm reduction effect

In order to examine the effect of reducing glucose, the inventors of the present invention conducted a glucose tolerance test in a db / db mouse, which is an obesity model in which the latency receptor gene has been destroyed and has no activity of a latency receptor, as in the above Example <3-1> oral glucose tolerance test).

As a result, as shown in FIG. 2C, it was confirmed that the amount of glucose present in the blood was significantly reduced in the obese model mice treated with the grit extract, as compared with the PBS-treated control. As a result, Compared with one control group, the amount of glucose decreased by 24.6% in mice treated with Pallet's extract (FIG. 2C).

In vitro (Invitro) Vet  n-hexane Fraction  GLP-1 activity and insulin secretion efficacy by treatment

In order to evaluate the efficacy of various fractions of Phellinus linteus extract on GPR119 activity, GPR119-CRE-bla CHO-K1 reporter cells were treated with various concentrations of glutaraldehyde extract and n-hexane, ethyl acetate, n-butanol, The fractions were processed.

Specifically, in order to measure the activated GLP-1 secretion, GLUTag cells were plated on a 24-well culture plate at a concentration of 1 × 10 ⁵ cells per well and cultured for 24 hours. After 24 hours, Krebs-Ringer bicarbonate (KRB) buffer (116 mM NaCl, 1.8 mM CaCl ₂ .2 (H ₂ O), 0.8 mM MgSO ₄ .7 (H ₂ O), 5.4 mM KCl, 1 mM NaH ₂ PO ₄ The cells were washed twice with ₂ mM H ₂ O, 26 mM NaCHO ₃ , and 0.5% BSA, pH 7.4, and the cells were washed twice with 10 mM glucose, 0.2 μM dipeptidyl peptidase-4 inhibitor, Mu] g / ml) in KRB buffer for 30 minutes. After 30 minutes, the supernatant was collected and GLP-1 secretion was measured using a GLP-1 (Active 7-36) ELISA kit (Alpco Diagnostics, NH, USA).

As a result, as shown in FIG. 3, it was confirmed that the reporter activity was the highest in the n-hexane fraction-treated cells of the extract of Gurley (FIG. 3A). The amount of active GLP-1 (FIG. 3b) and insulin secretion in INS-1 cells (FIG. 3c) in GLUTag cells were significantly increased in the n-hexane fraction of the pomegranate extract. These results suggest that the active compound that activates GPR119 is present in the n-hexane fraction of the extract.

In the mouse model Vet  n-hexane Fraction Glucose  Confirm adjustment effect

<5-1> Vet  n-hexane Fraction  Treatment of normal mouse blood Glucose  Confirm reduction effect

In order to examine the effect of the n-hexane fraction obtained from the extract of Phellinus linteus on the glucose control, the fraction of n-hexane in normal C57BL6 mice was administered at a dose of 300 mg / kg body weight as in Example <3-1> An oral glucose tolerance test was carried out 30 minutes after the administration alone.

As a result, as shown in FIG. 4, it was confirmed that the amount of glucose in the blood of the mice to which n-hexane fraction was administered was significantly reduced compared to the control group in which only PBS was administered 30 minutes and 60 minutes after the administration of glucose 4a). In addition, it was confirmed that the secretion of insulin significantly increased when the n-hexane fraction of Glycyrrhizae extract was administered alone (Fig. 4B).

<5-2> Vet  n-hexane Fraction  Treatment of obese mice with blood Glucose  Confirm reduction effect

The inventors performed an oral glucose tolerance test on obese db / db mice as in Example 3 above.

As a result, as shown in FIG. 4C, the amount of glucose in the blood of db / db mice administered with n-hexane fraction at 30 and 60 minutes after administration of glucose was considerably higher than that of PBS-treated db / db control mice (Fig. 4C, left). In addition, comparing the lower area of the graph, it was confirmed that the amount of glucose decreased by 12.7% in the mouse treated with the n-hexane fraction of guar gum compared to the control group administered with PBS (Fig. 4C, right).

Compounds isolated from the n-hexane fraction GPR119  Check for activator effect

The present inventors have found that imperatorin, phellopterin, and isoimperatorin, which are compounds obtained from n-hexane fractions fractionated from the pea extract in Example <1-3>, are GPR119 activators Experiments were carried out as described in Example 2 above.

As shown in FIG. 6, when GPR119-CRE-bla CHO-K1 cells were treated with imperatorin and phellopterin, the reporter activity was increased with increasing concentration, but isomerase isoimperatorin) did not increase (Fig. 6A). GLP-1 secretion was significantly increased when imperatorin and phellopterin were treated with GLUTag cells (FIG. 6b), confirming that glucose-activated insulin secretion also increased in INS-1 cells (Fig. 6C).

In the mouse model Felopterin ( phellopterin ) Treatment Glucose  Confirm tolerance

<7-1> Felopterin  Treatment of normal mouse blood Glucose  Confirm reduction effect

The present inventors treated imperatorin and phellopterin first in C57BL6 mice to confirm whether imperatorin or phellopterin improves glucose tolerance, and then, after <3> (Oral glucose tolerance test). The mice were fasted for 14 hours and then orally administered with imperatorin (30 mg / kg, in PBS, n = 9) or phellopterin (30 mg / kg in PBS, n = 7-8).

As a result, as shown in FIG. 7, when the imperatorin was administered, the amount of glucose in the blood was similar to that of the PBS-treated control, but when the phellopterin was administered, (Figs. 7A and 7B). From these results, it was found that phellopterin obtained from the Glycyrrhizae extract can act as an activator of GPR119.

<7-2> Felopterin ( phellopterin ) In obese mouse blood Glucose  Confirm reduction effect

The inventors performed an oral glucose tolerance test as in Example 3 to ascertain whether phellopterin improves glucose tolerance in obese db / db mice.

As a result, as shown in Fig. 7C, the mice administered with phellopterin showed a significant decrease in the amount of glucose in the blood as compared with the PBS-administered control (Fig. 7C).

Vet  Extract or n-hexane Fraction  Long-term treatment Glucose  Confirm reduction effect

<8-1> Vet  Extract or n-hexane Fraction  Observation of blood glucose reduction effect in obese mouse model after long-term treatment

The present inventors measured the amount of glucose in the blood after 3 weeks of administration of the grit extract or n-hexane fraction in obese db / db mice to see if the amount of glucose in the blood decreased when the gully extract or n-hexane fraction was administered for a long time Respectively.

Specifically, a group in which PBS (phosphate buffered saline) was administered so that 10 to 11 mice per one group were randomly assigned to 9 to 10 weekly db / db mice, and the administration of the guaride methanol extract prepared in Example <1-1> (Methanol extract of AD (Total)) and n-hexane fraction of AD (n-hexane). Total extracts and n-hexane fractions were dissolved in PBS at a concentration of 300 mg per kg of body weight and administered via oral intubation every day for 3 weeks. The amount of glucose in the blood, the weight, and the degree of eating were measured weekly.

As a result, as shown in FIG. 8A, the amount of glucose in the blood was significantly reduced in the mice treated with n-hexane fraction compared to the control group in which PBS was administered. However, (Fig. 8A).

<8-2> Vet  Extract or n-hexane Fraction  In the obese mouse model following long-term treatment, Glucose  Confirm reduction effect

In addition, the inventors performed an oral glucose tolerance test as in Example 3 after 3 weeks from the treatment of grit extract or n-hexane fraction.

As a result, as shown in FIG. 8B, in the mice treated with n-hexane fraction as compared with the control group administered with PBS, the amount of glucose in the blood significantly decreased at 15 minutes and 30 minutes after the administration of glucose, As a result, compared to the control group administered with PBS, the amount of glucose decreased by 8% and that of n-hexane fraction was decreased by 13% (FIG. 8b) .

<8-3> Vet  Extract or n-hexane Fraction  Long-term treatment confirms the effect of weight gain in obese mouse models

We also compared the body weights in the condition that mice consume the same amount of food per day (Figure 8d).

As a result, as shown in FIG. 8C, in the mice treated with the grit extract, the body weight was changed from 43.7 g ± 0.7 before the administration of the gully extract to 44.8 g ± 1.0 after 3 weeks of administration of the gully extract, and the mice treated with the n-hexane fraction Was weighted 42.8 g ± 0.7 before the n-hexane fraction and 43.3 g ± 0.6 after 3 weeks of the n-hexane fraction. PBS administered PBS was administered at a dose of 43.7 g ± 0.7 After the week, it was confirmed that the body weight was changed to 47.3g ± 1.0 (FIG. 8C).

It was confirmed that weight gain was reduced in the rats administered with Pallet's extract or n-hexane fraction compared with PBS-treated control rats.

Statistical analysis

Data are expressed as means ± standard deviation (mean ± SE). Significant differences were found in SPSS ver. One-way ANOVA and Duncan's post-hoc Duncan procedure using SPSS Inc. 10.0. Also, p <0.05 means statistically significant value.

Claims (14)

A pharmaceutical composition for the prevention or treatment of type 2 diabetes mellitus containing an n-hexane fraction of an Angelica dahurica extract as an active ingredient.
The pharmaceutical composition for preventing or treating type 2 diabetes according to claim 1, wherein the extract is extracted with water, a C ₁ -C ₂ lower alcohol or a mixed solvent thereof.
The pharmaceutical composition according to claim 2, wherein the lower alcohol is ethanol or methanol.
delete delete delete The pharmaceutical composition for preventing or treating type 2 diabetes according to claim 1, wherein the composition has GPR119 (G protein-coupled receptor 119) agonistic activity.
delete delete delete delete delete A health food for type 2 diabetes mellitus containing an n-hexane fraction of Angelica dahurica extract as an active ingredient. delete

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