KR101145237B1 - Alkaloid compounds as activators of DDAH promoter from Evodia rutaecarpa and compositions for prevention and treatment effects of islet cellular apoptosis and diabetic nephropathy containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a composition extracted from Evodia rutaecarpa containing an alkaloid component having activity for preventing or treating cellular dysfunction and diabetic complications. More specifically, the present invention relates to a composition comprising a sewage oil extract comprising compounds 1 to 5 separated from an ethyl acetate fraction which increases the activity of DDAH (dimethylarginine dimethylaminohydrolase) after solvent fractionation of the crude alcoholic extract of sewage oil. . The composition comprising any one of the compounds according to the present invention as an active ingredient can be usefully used for the prevention or treatment of pancreatic beta cell death and diabetic nephropathy through an increase in DDAH activity.

Description

Alkaloid compounds derived from filthy pears that activate DC, and compositions for preventing or treating pancreatic beta cell death and diabetic nephropathy using the active ingredient {Alkaloid compounds as activators of DDAH promoter from Evodia rutaecarpa and compositions for prevention and treatment effects of islet cellular apoptosis and diabetic nephropathy containing the same as an active ingredient}

The present invention relates to a composition containing an alkaloid component extracted from Evodia rutaecarpa having the activity for preventing and treating diabetic complications. More specifically, after the solvent fractionation of the crude alcoholic extract of sessile oil, the prevention of beta cell death and diabetic nephropathy comprising at least one of compounds 1 to 5 separated from the ethyl acetate fraction that increases the activity of dimethylarginine dimethylaminohydrolase (DDAH) and A therapeutic composition.

Diabetes mellitus is a chronic disease that is characterized by chronic hyperglycemia. If the metabolic control is not properly performed, it is a chronic disease that causes microvascular complications such as retinopathy, nephropathy and neuropathy, and chronic complications such as arteriosclerosis and diabetic nephropathy. The number of diabetic patients in Korea is expected to increase to 92,000 in type 1 and 1.2 million in type 2 in 1994, and to 130,000 in type 1 and 1.7 million in type 2 in 2010. However, this figure is estimated only for overt patients with obvious hyperglycemia, and the oral glucose tolerance test is expected to more than double the number, and even complications such as chronic complications, amputations and limbs may occur. Diabetes is already a big social problem. Diabetes is the leading cause of adult blindness (24,000 patients / year), and more than one-third of dialysis patients are diagnosed with diabetic renal failure, which is the most important cause of adult renal failure. As described above, the patient does not die due to the nature of the disease, but suffers from chronic complications such as eye disease-related complications and kidney complications. As a result of the astronomical cost of managing the complications, more than US $ 100 billion is spent annually in the US. Billions of dollars have been spent on diabetes, making it the largest single chronic disease.

As is well known, the epidemiological characteristics of type 2 diabetes in Korea differ from those in the West, only about 20% of patients with body mass index of 27kg / m ² or more and only 35% of patients with body mass index of 25kg / m ² . It is reported as an obese type. Type 2 diabetes mellitus in Korea is non-obesity type, and more patients are treated with insulin. Type 2 diabetes is originally known to be a metabolic disease caused by insulin resistance in peripheral tissues and the insufficiency of beta cells. In the case of impaired glucose tolerance with increased insulin resistance, insulin secretion is increased to compensate for insufficient insulin of beta cells, and at the same time, beta cells proliferate and suppress the rise of blood glucose, but when the failure to compensate occurs beta cell failure, insulin secretion These factors cause increased apoptosis, resulting in a decrease in the amount of beta cells, leading to an increase in blood sugar and accelerating diabetes.

NO (Nitric oxide) is an important substance involved in essential body reactions such as oxidative stress response, various defense mechanisms, neurotransmission, function regulation. NO is produced through NOS (Nitrous Oxide Systems), which is an endogenous NOS inhibitor known in the human body. In addition, it has been confirmed that it is associated with various diseases caused by diabetes. Most ADMA is broken down into citrulline and dimethylarginine by an enzyme called dimethylarginine dimethylaminohydrolase (DDAH). Therefore, since ADMA concentration is determined by the enzymatic activity of DDAH, DDAH regulates NOS activity and NO production through ADMA, and decreasing ADMA concentration through improvement of DDAH activation or dysfunction is a target that regulates NO production. Can be. To date, in an effort to induce the activity of DDAH for reducing ADMA concentration, some studies have been conducted to prevent oxidative stress or to reduce oxidized DDAH, but studies to fundamentally regulate it as a compound are insufficient. Interesting facts have been reported to involve ER stress in beta cell dysfunction and development of insulin resistance (Science, 306, 425-426, 2004), chondrocytes and microglial cells. Beta cell function based on the results of endoplasmic reticulum stress (J. Cell Physiol., 204 (1), 45-50, 2005). It was confirmed that it is closely related to the decrease and the development of insulin resistance. This suggests that increased NOS activity in beta cells induces endoplasmic reticulum stress and is involved in beta cell deterioration and insulin resistance development. In addition, it has been reported that cytokines such as IL-1β are involved in beta cell destruction and apoptosis, and NO is mainly involved in insulin secretion disorder caused by cytokines. Therefore, the regulation of NO can prevent the destruction of beta cells and improve the secretion of insulin secretion, so the regulation of the activity of DDAH involved in NO production can be a new target of diabetes treatment.

Diabetic nephropathy is the most important causative disease of patients with end-stage renal failure, and according to the 2002 statistics of the Nephrology Society, it accounts for 40% of all causative diseases of dialysis patients. It is expected that the frequency is similar worldwide, especially in patients with end-stage renal failure due to type 2 diabetes, which will double the current level after 10 years. The reason why the diabetic nephropathy is remarkably increased is that the nephropathy as a diabetic complication increases as the onset age of diabetes increases and the life of the diabetic patient is extended. Another clinical significance of diabetic nephropathy is the significant increase in mortality. In other words, type 1 diabetes patients with overt proteinuria have a 20 to 40 times higher mortality rate than normal patients, whereas type 1 diabetes patients with normal albuminuria have only two times higher mortality rates than normal patients. The domestic market for diabetes treatment reached about 300 billion won in 2005, and the domestic market of aniotensin converting enzyme (ACE) inhibitors and aniontensin receptor blocker (ARB), known to inhibit the progression of diabetic nephropathy, in 2005 was 32.85 billion won. To reach According to the Korean Nephrological Society's '2004 Current Status of Renal Replacement Therapy in Korea', diabetic nephropathy accounted for 43.4% of the causes of chronic renal failure, followed by hypertension glomerulosclerosis 16.2% and chronic glomerulonephritis respectively. In Korea, the increasing trend of kidney disease caused by chronic diseases such as diabetes due to population aging is being fixed.

Recently, the concentration of ADMA has been reported to be a strong predictor of renal impairment in patients with chronic kidney disease (J. Am. Soc. Nephrol., 16 (8), 2456-2461, 2005). Increasing ADMA levels are involved in the development and progression of tubulointerstitial ischemia, and it is reported that increased DDAH activity may improve renal function in diabetic nephropathy by significantly reducing ADMA and decreasing NO (Nephrol). Dial.Transplant., 24 (4), 1162-1169, 2009). Thus activating DDAH may enable the treatment of diabetic nephropathy.

In general, it is highly likely that new active ingredients can be found from natural medicines used in traditional medicine, rather than efforts by experimental modification of existing drugs. Since it has been used has the advantage of less toxicity concerns by drugs. Therefore, the present inventors have discovered that licorice exhibits strong activity as a result of searching for over 1,500 natural materials using a method of measuring the increase in DDAH activity in order to develop new low-toxic diabetic complications and nephropathy from natural products. After confirming the purification and structure of the compound was completed the present invention.

Osuyu ( Evodia rutaecarpa ) is a deciduous small shrub that grows in southern Korea. Immature fruit is used as a herbal medicine, and it is used as dry, dry, detoxification, and diuretic for sewage. The sewage extract has been reported to have analgesic effects and increase blood flow in experimental animals. When the sewage oil extract is used as a patent and literature related to such sewage oil, "whitening cosmetic composition containing sewage oil extract (Korea Patent No. 345225)", "cholesterol esterase inhibitor composition containing sewage oil extract (Korea Registered Patent No. 399529) And the like, but this is not related to the present patent which identifies a compound showing activity as an extract.

In the case of a patent using a compound known from filthy oil, a triglyceride lowering agent containing a quinolone alkaloid compound that is an acylcoa: diacylglycerol acyltransferase activity inhibitor (Korean Patent No. 577320, 2006) is claimed. Quinolone alkaloid is different from the compound of the present patent, and the indole-based compound claimed in the "composition for preventing and treating metabolic diseases containing an indole derivative as an active ingredient (Korean Patent No. 807718)" is also an indole-based indole system claimed in this patent. It is different from the compound. In addition, there is a therapeutic agent for dementia caused by Alzheimer's disease using dihydroebodiamine which is a compound of the present patent (Korean Patent No. 203456).

The compounds related to this patent are used to describe the effects on the body weight, visceral fat, and lipolysis of rats, and thus to detect lipid metabolism improving agents or anti-obesity agents in food, feed, or human or animal having lipid metabolism improving or anti obesity. The claimed patent (European Patent EP 0 852 117 A1, US Patent US 6,214,831 B1, PCT Publication 1999-0036207) is a patent claiming to improve lipid metabolism and obesity, inducing suicide of renal cells and pancreatic cells by regulating DDAH, which is the content of this patent. The mechanism is different from the improvement of type 2 diabetes symptoms by inhibiting apoptosis and preventing or treating pancreatic cell death and diabetic nephropathy, and the claimed activity is also different.

Therefore, an object of the present invention is to provide a compound containing a compound having a DDAH activating action by purely purified from sewage oil, and has a beta-cell killing and diabetic nephropathy prevention or treatment effect as an active ingredient. Another object of the present invention is to provide a composition containing as an active ingredient a compound exhibiting the effect of preventing and treating beta cell death and diabetic nephropathy obtained by purely purified from sewage.

The present inventors selected sewage milk as a candidate plant through a process of investigating DDAH activation by collecting various native plants and herbal medicines in view of the above description.

The DDAH active compound according to the present invention is used for separation and extraction of plant components from licorice by extraction with organic solvent alcohol (methanol, ethanol, propanol, etc.) or water-soluble alcohol, distribution of organic solvent and water, method by column chromatography, etc. A well-known method can be obtained easily or in combination suitably. The crude extract can be further purified according to a commercial method as needed.

Thereafter, the effluent oil was extracted with ethanol, purified purely by chromatography, and the evodiamine (compound 1 ) represented by the following Chemical Formula 1 among a plurality of alkaloid compounds whose structure was confirmed through chemical structure and physicochemical characterization. the bases represented by the 2-carboxylic gateum the pin (compound 2), carboxylic pin (compound 3) and Nord-keto yaw fishy (compound 4), Kane Toyo fishy (compound 5) a high activating effect on DDAH 7-hydroxy bases The present invention was completed by finding out.

The compound is characterized in that it is used for at least one of the drug substance for activating the DDAH enzyme for the beta cell death and diabetic nephropathy prevention or therapeutic effect.

(Formula 1)

The compound may be derived from sewage oil. The compound may be derived from ethanol extract. The pharmaceutical composition for activating the DDAH enzyme according to the present invention is characterized by comprising at least one compound of the above compounds as an active ingredient.

The composition for preventing or treating beta cell death and diabetic nephropathy according to the present invention is characterized by comprising at least one compound of the above compounds as an active ingredient.

The compound comprises the steps of extracting the sewage oil with ethanol and analyzing and separating and purifying the alkaloid compound for activating the DDAH enzyme using high pressure liquid chromatography (HPLC) using chromatography; Examining the chemical structure and physicochemical properties of the alkaloid compound obtained in the step; Investigating the activation of the DDAH enzyme of the compound of the step; And it can be obtained through the manufacturing process comprising the animal experimental step by oral administration and measurement of the beta-cell death prevention effect of the fraction containing the compound obtained in the above step.

From the interpretation of the physicochemical properties and nuclear magnetic resonance spectra, the inventors have confirmed that the compounds according to the invention are a series of compounds analyzed by the above formulas ( 1) to (3) . The beta-cell killing effect of the most compound, evodiamine, and the effect of animal by oral administration were investigated.

The activating compound of the DDAH enzyme according to the present invention can be used alone or suitably as a known method used for the separate extraction of plant components, such as organic solvent (alcohol, ether, acetone, etc.) extraction, hexane and water distribution, column chromatography, etc. It can be easily obtained by combining. The crude extract can be further purified according to a commercial method as needed.

The chromatography used in the present invention includes silica gel column chromatography, L-20 column chromatography, thin layer chromatography and high performance liquid chromatography (TLC). high performance liquid chromatography) and the like.

The alkaloid compound according to the present invention acts on the activation of the DDAH enzyme and thus can be advantageously used as it is effective in preventing or treating beta cell death and diabetic nephropathy.

The alkaloid compounds according to the present invention can be easily separated from sewage oil and have high stability, and thus can be used as additives for food and medicine.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, or the like. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated, the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.001 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 1 mg / kg / day to 200 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The extract of the present invention can be administered to mammals such as mice, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

The present invention also provides a health functional food for the prevention of atherosclerosis comprising the sewage oil extract and a food supplement acceptable food supplement. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the compound of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, etc. And health functional foods. Specifically, the present invention provides a dietary supplement for the prevention and treatment of beta cell death and diabetic nephropathy, including an extract containing licorice extract as an active ingredient and a food acceptable additive.

As described above, the extract derived from sewage oil of the present invention and the compound represented by Chemical Formulas 1 to 3 separated therefrom have a strong activating action of the DDAH enzyme, and inhibit suicide induction of cells caused by abnormal overexpression of NO. The effects of prevention and treatment for diseases such as diabetic nephropathy and diabetes complications caused by the destruction of pancreatic cells can be expected.

In addition, licorice is traditionally prescribed and used in many herbal medicines such as licorice of oriental medicine in oriental medicine, and it is a natural product that has been proven to be stable and extracted from it.It is very useful for medicine, cosmetics, and food industry. And application.

Hereinafter, the present invention will be described in more detail with reference to Examples and Formulation Examples, but the scope of the present invention is not limited by the following Examples and Formulation Examples.

Example 1 Isolation and Purification of DDAH or ANT Activating Material

3 kg of sewage fruit were extracted with 10 L of ethanol for 1 week at room temperature. The ethanol extract was concentrated under reduced pressure, and the extract (345 g) was suspended in water, and then solvent fractions were sequentially fractionated with n-hexane, ethyl acetate and butanol, and then the DDAH activity of each solvent fraction was measured. Strong activity was shown. 48 g of ethyl acetate fractions were subjected to silica gel column chromatography under a solvent gradient of hexane-acetone 1: 0 to 0: 1 to obtain 6 fractions (fr. 1 to 6). Of these fractions, fractions 2, 3 and 5 showed strong activity, 200 mg of compound 2 (rutaecarpine) was subjected to crystallization from fraction 2, and 1 g of compound 1 (evodiamine) was subjected to the same process from fraction 3 ) Reverse phase (RP-18) column chromatography was carried out using fraction 5 as a mixed solvent of MeOH-H ₂ O and sequentially increasing the ratio from 2: 1 to 5: 1 to obtain five subfractions (fr 5.1-5.5). Subfraction F5.3 was purified by HPLC using 43% MeCN-H ₂ O [RS Tech Optima Pak C ₁₈ column 20 × 150 mm; 10 μm particle size 2 ml / min; UV detection: 254 nm] to give compound 3 (7-hydroxyrutacarpine, t _R = 28.5 min), compound 5 (ketoyovirin, t _R = 51.2 min), compound 4 (norketovirin, t _R = 65.0 min). (See Figure 1).

Example 2: Structural Analysis of Isolated Compound

In Example 1, the structure of five compounds isolated from sewage was analyzed. The chemical structure of the compound was analyzed based on ¹ H and ¹³ C-NMR analysis of molecular weight and nuclear magnetic resonance analysis obtained using an ESI mass spectrometer.

As a result, the structure-determined compound had the structure shown in the formula ( 1) and summarized the ¹ H, ¹³ C-NMR results of the compound is shown below.

(Chemical properties)

Evo-diamine (Evodiamine, compound 1): Yellowish powder; ¹ H NMR (500 MHz , Acetone- d ₆ ) δ (ppm): 10.32 (1H, br, s, 1-NH), 7.93 (1H, dd, J = 1.5, 7.5, H-19), 7.54 (1H , br, d, J = 7.5, H-9), 7.47 (1H, ddd, J = 1.5, 7.5, 8.0, H-17), 7.41 (1H, br, d, J = 8.0, H-16), 7.41 (1H, br, d, J = 8.0, H-12), 7.14 (1H, br, t, J = 8.0, H-18), 7.08 (1H, br, t, J = 7.5, H-10) , 7.04 (1H, ddd, J = 0.5, 7.5, 8.0, H-11), 6.06 (1H, s, H-3), 4.77, 3.22 (each 1H, m, H-5), 1.96 (2H, m , H-6), 2.80 (3H, s, N-CH ₃ ); ¹³ C NMR (125 MHz , Acetone- d ₆ ) δ (ppm): 165.2 (C-21), 150.8 (C-15), 138.1 (C-13), 135.0 (C-2), 134.0 (C-17 ), 130.8 (C-19), 129.3 (C-10), 125.1 (C-8), 123.1 (C-11), 122.4 (C-18), 120.2 (C-9), 119.3 (C-16) , 113.4 (C-20), 112.5 (C-12), 110.8 (C-7), 70.8 (C-3), 41.3 (C-5), 37.3 (N-CH ₃ ), 20.7 (C-6) .

Carboxylic pin (Rutaecarpine, Compound 2) on a rutile: Light yellow amorphous powder; ¹ H NMR (600 MHz , CDCL ₃ ) δ (ppm): 9.67 (1H, br, s, 1-NH), 8.34 (1H, dd, J = 1.2, 7.8, H-19), 7.70 (1H, ddd) , J = 1.8, 1.2, 7.2, H-9), 7.65 (1H, d, J = 7.5, 8.0, H-17), 7.41 (1H, br, d, J = 8.0, H-16), 7.41 ( 1H, br, d, J = 8.0, H-12), 7.14 (1H, br, t, J = 8.0, H-18), 7.08 (1H, br, t, J = 7.5, H-10), 7.04 (1H, ddd, J = 0.5, 7.5, 8.0, H-11), 6.06 (1H, s, H-3), 4.77, 3.22 (each 1H, m, H-5), 1.96 (2H, m, H -6), 2.80 (3H, s, N-CH ₃ ); ¹³ C NMR (150 MHz , CDCL ₃ ) δ (ppm): 165.2 (C-21), 150.8 (C-15), 138.1 (C-13), 135.0 (C-2), 134.0 (C-17), 130.8 (C-19), 129.3 (C-10), 125.1 (C-8), 123.1 (C-11), 122.4 (C-18), 120.2 (C-9), 119.3 (C-16), 113.4 (C-20), 112.5 (C-12), 110.8 (C-7), 70.8 (C-3), 41.3 (C-5), 37.3 (N-CH ₃ ), 20.7 (C-6).

Carboxylic pin (7-Hydroxyrutaecarpine, compound 3) in the 7-dihydro-oxy rutile: Pale yellow powder; ¹ H NMR (500 MHz , Acetone- d ₆ ) δ (ppm): 10.32 (1H, br, s, 1-NH), 8.23 (1H, dd, J = 1.5, 8.5, H-19), 7.78 (1H) , ddd, J = 1.5, 8.5, 9.0, H-17), 7.71 (1H, br, d, J = 8.0, H-9), 7.62 (1H, br, d, J = 9.0, H-12), 7.46 (1H, br, t, J = 8.0, H-18), 7.31 (1H, br, t, J = 7.5, H-10), 7.26 (1H, br, d, J = 9.0, H-16) , 7.14 (1H, br, t, J = 7.5, H-11), 6.91 (1H, dd, J = 1.5, 5.0, H-5), 3.55 (1H, dd, J = 1.5, 17.0, H-6eq ), 3.43 (1H, doublet of doublets, J = 5.0, 17.0, H-6ax); ¹³ C NMR (125 MHz , Acetone- d ₆ ) δ (ppm): 161.7 (C-21), 148.9 (C-3), 145.2 (C-15), 139.9 (C-13), 135.4 (C-17 ), 127.9 (C-19), 127.8 (C-18), 127.5 (C-2), 127.3 (C-8), 126.8 (C-16), 125.8 (C-10), 122.4 (C-20) , 120.9 (C-11), 120.8 (C-9), 115.7 (C-7), 74.8 (C-5), 28.3 (C-6).

Nord Kane Toyo fishy (Norketoyobyrine, Compound 4): Pale yellow amorphous powder; ¹ H NMR (500 MHz , Acetone- d ₆ ) δ (ppm): 10.04 (1H, br, s, 1-NH), 8.16 (1H, dd, J = 1.5, 7.5, H-19), 7.86 (1H , br, d, J = 8.0, H-12), 7.77 (1H, ddd, J = 1.5, 7.5, 8.0, H-17), 7.44 (1H, br, d, J = 8.5, H-16), 7.39 (1H, br, d, J = 8.0, H-18), 7.30 (1H, ddd, J = 0.5, 7.5, 8.0, H-18), 7.24 (1H, s, H-14), 7.11 (1H , ddd, J = 0.5, 7.5, 8.0, H-10), 7.06 (1H, ddd, J = 0.5, 7.5, 8.0, H-11), 4.32, (2H, m, H-5), 3.10 (2H , m, H-6); ¹³ C NMR (125 MHz , Acetone- d ₆ ) δ (ppm): 162.2 (C-21), 151.7 (C-3), 141.9 (C-2), 137.8 (C-13), 135.8 (C-17 ), 128.9 (C-19), 128.6 (C-8), 123.5 (C-15), 123.4 (C-18), 123.3 (C-12), 122.2 (C-10), 119.7 (C-11) , 119.6 (C-9), 116.6 (C-7), 115.0 (C-16), 112.8 (C-20), 112.2 (C-14), 43.2 (C-5), 24.7 (C-6).

Kane Toyo fishy (Ketoyobyrine, compound 5): Light yellow amorphous powder; ¹ H NMR (500 MHz , Acetone- d ₆ ) δ (ppm): 9.88 (1H, br, s, 1-NH), 8.17 (1H, br, d, J = 7.5, H-19), 7.77 (1H , br, t, J = 8.0, H-11), 7.45 (1H, br, d, J = 8.0, H-10), 7.39 (1H, dd, J = 2.0, 7.5, H-12), 7.31 ( 1H, br, t, J = 7.5, 8.0, H-18), 7.27 (1H, d, 8.5, H-9), 7.24 (1H, s, H-14), 6.76 (1H, dd, J = 2.0 , 8.5, H-17), 4.32, (2H, m, H-5), 3.84 (3H, s, -OCH ₃ ), 3.10 (2H, m, H-6); ¹³ C NMR (125 MHz , Acetone- d ₆ ) δ (ppm): 162.3 (C-21), 154.9 (C-16), 151.7 (C-3), 141.9 (C-2), 135.9 (C-11 ), 133.1 (C-13), 129.3 (C-8), 129.1 (C-19), 124.1 (C-7), 124.0 (C-9), 123.5 (C-18), 116.6 (C-20) , 115.1 (C-10), 112.9 (C-12), 112.8 (C-15), 112.5 (C-14), 101.6 (C-17), 55.9 (-OCH ₃ ), 43.0 (C-5), 24.7 (C-6).

Example 3: Activity Measurement Using DDAH Promoter

It was confirmed using the DDAH promoter-luciferase system to measure the activity of DDAH using compounds 1-5 analyzed in Examples 1 and 2. The promoter is located at the front of the DNA sequence that contains the genetic information of the gene to regulate transcription, and transcription transcription factor is coupled to the promoter to bring RNA polymerase for RNA synthesis. Therefore, the luciferase gene located behind the nucleotide sequence of the promoter also increases the amount of expression as the promoter is more active can confirm the activity of the promoter. In addition, since DDAH is a mitochondia protein, mouse endothelial cell (endothelioma) bEnd. 3 cells from which mitochondria are relatively distributed were selected to measure promoter activity.

First, bEnd.3 cells were collected by treatment with 0.25% trypsin, and then the supernatant was removed and suspended in DMEM medium free of bovine serum. 3 μl of lipoectamine (lipopectAMINE, life Technologies, Inc) was added to 100 μl of DMEM without bovine serum in a cell suspension, mixed slowly, and left at room temperature for 15 minutes, and 50 ng of pDDAH II-Luc or mpDDAH- Β-galactosidase as a standard for consistently balancing the efficiency of Luc (plasmid with luciferase gene), 0.1 ug of pCMV-b-galactosidase (transfection, the way DNA enters animal cells) (plasmid with [beta] -galactosidase) gene], 0.1 µg of DDAH expression vector, or a microneedle tube containing a vector to be used as a control, slowly dropping for 30 seconds, mixed and left for another 15 minutes at room temperature. The mixture of lipofectamine and DNA-plasmid and b-End.3 cells were slowly mixed and incubated at room temperature for 20 minutes in suspension, then aliquoted in a 60 mm culture vessel and incubated for 24 hours in a CO ₂ incubator. After addition of 1 ㎍ / ㎖ each, it was incubated for another 24 hours. bEnd.3 cells were washed twice with PBS, 500 μl of extract solution was added to each culture vessel, and the cells were disrupted and centrifuged at 14,000 rpm for 5 minutes. The obtained supernatant was luciferase and β-galactosidase. Used to measure the activity of. Luciferase activity was obtained from 100 μl of supernatant, 100 μl of 20 nM D-luciferin (substrate developed using luciferase) and 300 μl of reaction solution (20 mM glycylglycin, 12.5 mM MgSO4, 3 mM EGTA, 15 mM potassium). phosphate, 1 mM DTT, 1 mM ATP) was measured by a luminometer (luminometor, Berthold Clinolumat). Luciferase activity was corrected by measuring the activity of β-galactosidase in order to correct the gene injection efficiency. The activity of β-galactosidase was determined using 30 μl of cell extract, 66 μl of ONPT (4 mg / ml), and 204 μl of reaction solution (0.1 M sodium phosphate, 1 mM MgCl ₂ , 45 mM β-mercaptoethanol, pH 7.5). After reacting in a thermostat, the absorbance was measured using a spectrophotometer.

Figure 2 is a result confirming the activity of the DDAH of alkaloid compounds 1 to 5 isolated from the sewage extract. As shown in Figure 2 as a result of measuring the DDAH promoter activating action of alkaloid compounds 1-5 obtained from sewage oil was confirmed that the compound increases the activity of the promoter of DDAH more than two times on average concentration of 5㎍ / ㎖ It was.

Example 4 Measurement of Preventing Beta Cell Death of Evodiamine Isolated from Sewage Milk

Next, an MTT assay was conducted to determine whether the sewage-derived compound has an effect of preventing beta cell death. The compound was carried out using evodiamine, which is typically present in many of the compounds derived from sewage oil through the results of Examples 1 and 2.

First, the insulin-secreting cell line, HIT-T15 cells were cultured for 48 hours, and then treated with 10 mM 2-deoxyglucose (2-DG), which induces cell death, and the evodiamine (10 ㎍ / ml) was 2- 1 hour prior to deoxyglucose treatment, the reaction was carried out. MTT (5 mg / mL) solution dissolved in PBS buffer was added to the cells for 4 hours, after which the supernatant was removed, and 250 μl of DMSO was added and the absorbance was confirmed with an ELISA reader.

3 is a result of inducing beta cell death using 2-deoxyglucose. As shown in FIG. 3, beta cells were killed about 50% in the group treated with 2-deoxyglucose, but beta cells were not nearly killed in the group treated with 2-deoxyglucose with evodiamine. Can be.

Example 5 Measurement of Proteinuria Reduction Effect of Evodiamine in Diabetic Nephropathy Animal Model

One week after 200-250 g of male SD rats at 8 weeks of age, one kidney was excised and injected once intraperitoneally with 60 mg / kg of streptozotocin, a substance known to induce diabetes. Diabetes was induced when the blood glucose from the tail was 300 mg / dl or more within 1 week. Diabetic rats were orally administered with evodiamine (25 mg / kg / day) for 8 weeks. The untreated group used as a control group was administered a normal food without addition of drugs. Weekly blood glucose and urine ketone tests were performed to subcutaneously inject insulin at 2 to 3 U / kg if necessary, and urine was collected for 24 hours to measure albuminuria levels.

4 shows the albuminuria levels of urine collected for 24 hours. As shown in FIG. 4, the increased albuminuria in diabetic rats showed an effect that the group administered with evodiamine was significantly reduced to a level similar to that of the control group compared to the group treated with streptozotocin alone.

Example 6: Toxicity Test

6-1. Acute Toxicity

This experiment was conducted to investigate the toxicity of the evodiamine extracted from sewage milk to the animal body acutely (within 24 hours) and to determine the lethality. 20 ICR mouse strains, which are general mice, were assigned to 10 control groups and 10 experimental groups. In the control group, only the solvent containing no compound (PEG-400 / tween-80 / EtOH, 8/1/1, v / v / v) was administered, and the experimental group used evodiamine extracted from sewage oil in Example 5. Orally at a 40-fold (1.0 g / kg) concentration of 25 mg / kg. Each mortality rate was 24 hours after administration. Both control group and experimental group treated with 1.0 g / kg lycokalcon A survived and showed no acute toxic effects.

6-2. Organ and Histotoxicity

Evodiamine extracted from sewage milk was administered at a concentration of 25 mg / kg for 8 weeks to test OLETF rats (Rat). In order to investigate the effects on the organs (tissues) of the animals, blood was collected after 4 weeks from animals of the experimental group administered with evodiamine and the control group administered with solvent only, and then GOT (glutamate-oxalate-transferase) and GPT (glutamate). Blood concentrations of -pyruvate-transferase, creatin kinase and creatinine were measured. As a result, in the case of GOT and GPT known to be related to hepatotoxicity, creatinine, which is an indicator of kidney toxicity, and creatine kinase, which is an indicator of muscle toxicity, there was no difference in the experimental group of ricokalcon A compared with the control group. In addition, the heart, lung, pancreas, adrenal gland, liver, kidneys and muscles were cut from each animal and histological observations were performed by optical microscopy through conventional tissue fabrication procedures, but specific abnormalities or toxic effects were induced. Could not be confirmed.

Formulation Example 1: Pharmaceutical Formulation

1-1. Manufacture of tablets

200 g of sewage extract or a compound isolated therefrom was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

1-2. Preparation of Injection

1 g, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to prepare 100 ml of a sewage oil extract according to the present invention or a compound isolated therefrom. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

Preparation Example 2 Food Preparation Example

2-1. Preparation of Cooking Seasonings

The sewage oil extract according to the present invention or a compound separated therefrom was prepared in a health promotion cooking seasoning at 0.2 to 10 parts by weight, respectively.

2-2. Manufacture of flour food products

Sewage oil extract according to the present invention or a compound separated therefrom is added 0.1 to 5.0 parts by weight to each flour, and using the mixture to prepare bread, cakes, cookies, crackers and noodles to prepare a health-promoting food.

2-3. Preparation of soups and gravy

The sewage oil extract according to the present invention or the compounds separated therefrom were added 0.1 to 1.0 parts by weight to soups and broth to prepare meat products for health promotion, soups and noodles for noodles.

2-4. Manufacture of dairy products

Sewage oil extract according to the present invention or a compound separated therefrom is added 0.1 to 1.0 parts by weight to each milk, using the milk to prepare a variety of dairy products such as butter and ice cream.

Preparation Example 3 Beverage Preparation Example

3-1. Vegetable Juice Manufacturing

0.5 g of sewage extract or a compound isolated therefrom was added to 1,000 ml of tomato or carrot juice to prepare a vegetable juice for health promotion.

3-2. Fruit juice manufacturing

The fruit juice for health promotion was prepared by adding 0.1 g of sewage extract or a compound isolated therefrom to 1,000 ml of apple or grape juice.

Figure 1 shows the spectrum of HPLC analysis from the extract of sewage oil with ethanol.

Figure 2 measures the activity of the DDAH promoter of the compound isolated from sewage.

Figure 3 is treated with 2-deoxyglucose and evodiamine in HIT-T15 cells.

4 shows the results of albuminuria after 24 hours measured after administration of evodiamine to a diabetic nephropathy animal model at a concentration of 25 mg / kg for 8 weeks.

Claims (7)

1 selected from the group consisting of evodiamine, rutaecarpine, 7-hydroxyrutaecarpine, norketoyobyrine, and ketoyobyrine A pharmaceutical composition for the prevention or treatment of diabetic nephropathy induced from pancreatic beta cell death containing sewage ethanol extract containing at least one compound. 1 selected from the group consisting of evodiamine, rutaecarpine, 7-hydroxyrutaecarpine, norketoyobyrine, and ketoyobyrine Health functional food for the prevention or improvement of diabetic nephropathy induced from pancreatic beta cell death containing sewage ethanol extract containing more than one compound. The method of claim 2, The health functional food is selected from the group consisting of nutritional products, including drinks, meat, sausages, bread, candy, snacks, noodles, dairy products including ice cream, soups, beverages including ionic drinks, alcoholic beverages and vitamin complexes Health functional foods for the prevention or improvement of diabetic nephropathy induced by pancreatic beta cell death, characterized in that. delete The method of claim 1, The pharmaceutical composition is a pharmaceutical composition for preventing or treating diabetic nephropathy induced from pancreatic beta cell death, characterized in that to increase the activity of dimethylarginine dimethylaminohydrolase (DDAH) enzyme. 1 selected from the group consisting of evodiamine, rutaecarpine, 7-hydroxyrutaecarpine, norketoyobyrine, and ketoyobyrine Pharmaceutical composition for the prevention or treatment of diabetic nephropathy induced from pancreatic beta cell death, characterized in that it contains more than one compound. The method of claim 6, The pharmaceutical composition is a pharmaceutical composition for preventing or treating diabetic nephropathy induced from pancreatic beta cell death, characterized in that to increase the activity of dimethylarginine dimethylaminohydrolase (DDAH) enzyme.

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