KR20110005483A - Pharmaceutical composition for improving anti-inflammatory effect and vascular diseases, and preventing and treating hypertension comprising lophatherum gracile brongn extract - Google Patents

Abstract

PURPOSE: A composition containing Lophatherum gracile Brongn extract for preventing or treating inflammatory diseases or vascular diseases is provided to suppress NO generation and induce blood vessel relaxation. CONSTITUTION: A pharmaceutical composition for preventing and treating inflammatory diseases contains Lophatherum gracile Brongn extract as an active ingredient. A pharmaceutical composition for preventing and treating vascular diseases contains the Lophatherum gracile Brongn extract as an active ingredient. The Lophatherum gracile Brongn extract is isolated using water, low alcohol of 1-4 carbon atoms or mixture thereof. The content of Lophatherum gracile Brongn extract is 0.0001-30 weight%. The pharmaceutical composition additionally contains pharmaceutically acceptable carrier. A health food for preventing or treating inflammatory diseases or vascular diseases contains the Lophatherum gracile Brongn extract.

Description

Pharmaceutical composition for improving anti-inflammatory effect and vascular diseases, and preventing and treating hypertension comprising Lophatherum gracile Brongn extract}

The present invention relates to a composition for preventing or treating inflammatory diseases and vascular diseases including bile duct extract, and more particularly, the anti-inflammatory effect by inhibiting iNOS by inhibiting iNOS in immune cells, including bile duct extract, The present invention relates to a pharmaceutical composition for improving anti-inflammatory and vascular diseases, and for preventing and treating hypertension, which induces vascular relaxation by promoting eNOS and inducing NO production in vascular endothelial cells.

Nitric oxide (NO) is known not only as a pharmacological messenger of the nervous system, but also as an important regulator such as cellular differentiation or intracellular signal transduction, in addition to inflammatory responses, immune system and cytotoxicity.

Nitric oxide synthase (NOS) is classified into two types, constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS). Isomers of constitutive nitric oxide synthase include endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). Endothelial nitric oxide synthase is present in vascular endothelial cells and platelets to relax blood vessels and regulate vascular homeostasis. Neuronal nitric oxide synthase is a nitric oxide that acts like a neurotransmitter in the central and peripheral nervous systems. And inducible nitric oxide synthase is known to be present in immune cells such as macrophages, hepatocytes, neutrophils and the like. In addition, nNOS and eNOS are always present in cells, but iNOS is known to be expressed only when exposed to interferon, lipopolysaccaride (LPS) and various inflammatory-induced cytokines.

An inflammatory response is a mechanism for repairing and repairing the damaged area when an invasion that causes any organic change, such as physical action, chemicals, or bacterial infection, is applied to a living body or tissue. Vascularly active substances are released to increase vascular permeability, causing inflammation. However, persistent inflammatory reactions promote mucosal damage, and as a result, some cause diseases such as cancer.

Inflammatory reactions produce a variety of pro-inflammatory mediatos, which include NO produced by inducible nitric oxide synthase (iNOS) and prostagradnin produced by cyclooxygenase-2 (COX-2). E ₂ (PGE ₂ ), and the like. These inflammatory factors activate the nuclear factor-kB (NF-kB), a transcription factor of the inflammatory response, resulting in excess NO and PGE ₂ , resulting in inflammation. iNOS is expressed during the stimulation of interferon-gamma, lipopolysaccharide (LPS), and various inflammatory cytokines. PGE ₂ is produced from Arachidonic acid by cyclooxygenase (COX). COX has two similar forms. COX-1 is expressed in almost all tissues and produces prostagrandin, which regulates physiological functions such as regulating blood flow in the kidneys or protecting the cells of the stomach. In contrast, COX-2 is expressed in macrophage in response to microbial infection or damage or stress of various factors. INOS and COX-2 expression, NO and PEG ₂ production are representative inflammatory factors of immune cells. Inflammatory factors include tumor necrosis factor-alpha (TNF-) and interleukin-1beta (IL-1), which are proinflammatory cytokines.

Innate immunity is a classic idiom defense that activates rapidly through a variety of mechanisms to protect against bacterial, viral infections and stress. However, persistently excessive immune responses promote tissue damage, resulting in sepsis and chronic inflammation in some [Brown et al., 2007]. During inflammatory processes, pro-inflammatory cytokines, nitric oxide (NO) and prostaglandin E2 (PEG2) are produced in large quantities by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) [Posadas et al. , 2000].

On the other hand, cardiovascular diseases are often caused by the usual indifference due to the weak initial symptoms. This is why hypertension, a representative disease of cardiovascular diseases, is called a silent killer. Hypertension is more than 30% (34.4% of males and 26.5% of females) among adults over 30 years of age, which is one of the most prevalent diseases, and increases rapidly with age (2.3% in their 30s, 8.7% in their 40s). , 23.2% in the 50s, 35.6% in the 60s, and 42.4% over the age of 70). Organ damage associated with hypertension itself includes cardiac hypertrophy, congestive heart failure, retinopathy, cerebrovascular injury, and / or kidney failure. Blood pressure, which is the pressure exerted by the dynamic contraction of the heart and applied to the walls of blood vessels as it flows through the blood vessels, significantly reduces blood pressure and ischemic heart disease and stroke, especially when there are other risk factors such as hyperlipidemia and diabetes. Urgently relieve high blood pressure.

The mechanisms that control contraction and relaxation in vascular smooth muscles vary widely. One of the most important mechanisms among these mechanisms is endothelium-derived relaxing factor, the body is nitric oxide (NO). Nitric oxide in blood vessels is produced in vascular endothelial cells by nitric oxide synthase (NOS) using L-arginine as a substrate.

Vascular endothelial cell-releasing factor is synthesized and secreted in endothelial cells even under normal conditions and synthesized and secreted by agonists such as acetylcholine, histamine, substance P and isoproterenol. Is increased. Herbal medicines and herbal medicines that relax blood vessels by increasing the production of nitric oxide in vascular endothelial cells have also recently been reported, such as Kuramochi et al. (Kuramochi T, et al., Life Sci. , 54 (26)). , pp2061-2069, 1994) reported that lyotropic liquids increased nitric oxide-dependent vasorelaxant effects, and Goto et al. (Goto H, et al., Am. J. Chinese Med ., 27). (3-4), pp339-345, 1999) reported that, in a series of studies, precoating fluids decreased the blood pressure of spontaneous hypertension and methanol extracts relaxed nitric oxide-dependent blood vessels of the thoracic aorta. In addition, lithospermic acid B (lithospermic acid B) and protein components of Cordyceps sinensis are known to increase vascular relaxation depending on nitric oxide. In particular, Chen et al. (Chen ZY, et al., Life Sci ., 63 (22), pp1983-1991, 1998) reported that the ethanol extract of the hawthorn relaxes the mesenteric artery nitric oxide-dependently. (Kim, SH., Et al., Life Sci ., 67, pp121-131, 2000) reported that procyanidins isolated from hawthorn nitric oxide-dependently relax blood vessels and increase the production of vascular cGMP. Kang et al. (Kang, DG., Et al., Vascul.Pharmacol. , 39 (6), pp281-286, 2002). Also, an alkaloid compound, berberine, which is isolated from rhubarb, is NO / cGMP-based. Medicinal extracts such as mistletoe, ginkgo biloba, windproof, Angelica and motherwort have been shown to relax the blood vessels in a dependent manner. Procyanidine, flavonoid Sesquiterpene lactone compounds have been found (Nishida S, et al., Life Sci., 72 (23), pp2659-67, 2003; Lee TH, et al., Planta. Med., 68 (6) ), pp 492-6, 2002; Yuzurihara M, et al., Eur. J. Pharmacol ., 444 (3), pp183-9, 2002).

On the other hand, the nature of bile leaves is cold, the taste is sweet, and there is no poison. The wall is cut down, the fever is lowered, and the stroke is sore that you can't speak, the fever gets stronger and your head hurts. Nausea, mania and radiating pain, coughing, coughing, dizziness and falling of a pregnant woman, span of a child, and zen wind To heal.

Some people boil tea with the leaves of oleander, but not many people know that the oleander is surprisingly effective in all kinds of incurable diseases. Bile is an herb with amazing medicinal properties that are far superior to ginseng. Among the bamboos, the weakest because of the one of the biliary leaves, diabetes, high blood pressure, gastritis, gastric ulcers, chronic hepatitis, cancer, such as incurable diseases are not often cured. Bile leaves have effects such as lowering heat, releasing poison, removing phlegm and urine, and treating inflammation and suppressing cancer cells. Bile leaves suppress cancer cells and do no damage to normal cells. According to experiments in Japan, bile leaf extract has 100% inhibitory effect on liver ascites and cancer cells, and 70-90% of tumor cells were reduced after 30 days when bile leaf extract was fed to rats that carried cancer cells. do.

The major components of bile leaves include the triterpenoid compounds arundoin, cylindrin, taraxerol and friedelin. In addition, the ground part is said to contain phenolrP, amono acids, organic acids and sugars.

The inventors of the present invention, while studying the anti-inflammatory and vascular relaxation effects associated with the production of NO in a variety of natural products, in particular, herbal medicines, the bile duct extract has an excellent anti-inflammatory and vascular relaxation effect by controlling the degree of NO production according to the type of cells By discovering that, the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising the extract of biliary leaves as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition for preventing and treating vascular diseases comprising the bile leaf extract as an active ingredient.

Still another object of the present invention is to provide a method of treating inflammatory diseases or vascular diseases using a pharmaceutical composition comprising bile leaf extract as an active ingredient.

Still another object of the present invention is to provide a health functional food for preventing or improving an inflammatory disease comprising a bile leaf extract.

Still another object of the present invention is to provide a health functional food for preventing or improving vascular diseases including bile leaf extract.

In one embodiment, the present invention relates to a pharmaceutical composition for preventing and treating inflammatory diseases comprising a bile leaf extract as an active ingredient.

The bile leaves extract used in the present invention can be used without limitation, such as cultivated or commercially available, the method for producing the bile leaves extract may use a conventional extraction method in the art. Specifically, the dried, frozen or naturally dried perilla leaf is appropriately cut to about 1 to 15 times the dry weight of purified water (water), preferably a lower alcohol having 1 to 4 carbon atoms or their volume. It can be extracted using a mixed solvent of. More preferably ethanol is used, most preferably 99% ethanol. The extraction method may use hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction, and preferably cold and ultrasonic extraction. In addition, the extract may be filtered and lyophilized to be used for purification and concentration.

Bile leaf extract, which is the main active ingredient included in the composition of the present invention, has iNOS (inducible nitric oxide synthase) inhibitory activity. In addition, the bile leaf extract may inhibit the inflammatory response by inhibiting NO (nitric oxide) production in immune cells.

In the present invention, "iNOS" is a calmodulin-independent enzyme, and IL-1β (interlukin-1β), IFN-γ (interferon-γ), TNF-α (immunocells) in immune cells such as macrophages, hepatocytes, and neutrophils. Induced by cytokine (cytokines) such as tumor necrosis factor-α) and inflammatory factors such as bacterial endogenous bacterial LPS (lipopolysaccharide), to produce a variety of inflammatory reactions by generating NO. Since the composition comprising the bile duct extract of the present invention exhibits such iNOS inhibitory activity, it inhibits NO production in immune cells and inhibits the inflammatory response caused by the inflammatory factor, thereby being useful for the prevention or treatment of inflammatory diseases. have.

In the present invention, "inflammatory disease" means any inflammatory disease that can be caused by an inflammatory response, in particular NO mediated inflammatory disease, for example, rheumatoid arthritis, autoimmune disease, Parkinson's disease, artery Sclerosis, multiple sclerosis, edema, sepsis, septic shock, psoriasis, cancer, encephalomyelitis, meningitis, pancreatitis, peritonitis, vasculitis, lymphocytic choriomeningitis, glomerulonephritis, uveitis, ileitis, hepatitis, nephritis and inflammatory bowel disease However, it is not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for preventing and treating vascular diseases comprising the bile duct extract as an active ingredient.

The main active ingredient included in the composition of the present invention, the bile leaf extract has eNOS (endothelial nitric oxide synthase, endothelial nitric oxide synthase) enhancing activity. In addition, the bile duct extract by inducing NO (nitric oxide) production in vascular endothelial cells, can induce a relaxing action of vascular smooth muscle. In addition, blood pressure can be lowered through this vasodilation effect. Therefore, the composition of the present invention can be used, in particular, as a composition for the prevention and treatment of hypertension.

In the present invention, "eNOS" is calcium or calmodulin dependent, is present in vascular endothelial cells, and regulates vasodilation and blood flow by producing NO. Since the composition comprising the bile duct extract of the present invention exhibits such eNOS enhancing activity, by promoting NO production in vascular endothelial cells, thereby relaxing vascular smooth muscle, thereby expanding blood vessels to lower blood pressure, various vascular diseases It can be useful for the prevention or treatment of.

In addition, the composition of the present invention is characterized by relaxing the blood vessels via the NO / cGMP signaling system. NO derived from vascular endothelial cells diffuses into vascular smooth muscle and activates soluble guanylate cyclase and plays an important role in relaxing vascular smooth muscle by increasing cGMP.

In the present invention, "vascular disease" includes all vascular diseases that may be caused by abnormal pressure of blood vessels, for example, hypertension, coronary artery disease, cerebrovascular disease, peripheral vascular disease, aortic disease, renal dysfunction , Erectile dysfunction, hypertensive heart disease, diabetes, heart failure and visual impairment, and preferably hypertension, but is not limited thereto.

In the present invention, 'prevention' refers to any action that inhibits or delays the onset of inflammatory diseases and vascular diseases by administering a composition comprising bile leaf extract, and 'treatment' refers to inflammatory diseases and vascular diseases by administering the composition. Means any action that improves or changes beneficially.

The pharmaceutical composition comprising the bile duct extract of the present invention as an active ingredient may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered component.

The composition of the present invention may be prepared by filling the capsule containing the bile duct extract of the present invention without excipients or uniformly and sufficiently in contact with the atomized solid carrier, liquid carrier or both, and molding the product into the desired formulation, if necessary. Can be used. Examples of suitable carrier excipients include starch, water, saline, ethanol, glycerol, Ringer's solution and dextrose solution, and the like (Remington's Pharmaceutical Science, 19 ^th Ed., 1995, Mack Publishing Company, Easton PA) and the like. As disclosed, it may be formulated into a suitable formulation known in the art.

At this time, the bile duct extract of the present invention may be formulated to be contained in an amount of 0.0001 to 30% by weight, preferably 0.1 to 10% by weight based on the dry weight relative to the total weight of the composition.

In addition, the composition of the present invention can be applied to any formulation containing it as an active ingredient, it can be prepared in oral or parenteral formulations. Pharmaceutical formulations of the invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.

Oral dosage forms containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate It may include a lubricating oil, such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient, for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method or aerosol for spraying by inhalation through the respiratory system It can be formulated as. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. For infusion into suppositories, it may be formulated as a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

In another aspect, the present invention relates to a method for treating an inflammatory disease or a vascular disease comprising administering a composition comprising the bile duct extract.

As used herein, the term "administration" refers to introducing a pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be various oral or parenteral routes as long as the target tissue can be reached. It may be administered through, or specifically, can be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal route. . Preferably oral administration.

The treatment method of the present invention comprises administering the composition in a pharmaceutically effective amount. Appropriate total daily usage may be determined by the practitioner within the scope of good medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, It is desirable to apply differently depending on the route of administration and the rate of release of the composition, the duration of treatment, and the various factors and similar factors well known in the medical arts, including drugs used with or concurrent with the specific composition. Therefore, the preferred dosage of the composition of the present invention can be determined in view of the above, and can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg divided once to several times per day based on the dry weight of the bile duct extract.

The composition of the present invention may be used alone for the prevention and treatment of inflammatory diseases or vascular diseases, or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers.

The methods of preventing or treating inflammatory diseases or vascular diseases of the present invention are applicable to any animal in which such diseases may occur, and animals are not only humans and primates but also cows, pigs, sheep, horses, dogs and cats, and the like. Contains livestock.

In still another aspect, the present invention relates to a functional food for preventing or improving inflammatory diseases comprising bile leaf extract.

In still another aspect, the present invention relates to a health functional food for preventing or improving vascular diseases including bile leaf extract.

The bile duct extract of the present invention may be added to a health functional food for the purpose of preventing or improving inflammatory diseases or vascular diseases.

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

The health functional food may be prepared by adding other food materials according to the commerciality and consumer preference of the final product. The effective amount of the extract may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment), preferably 1 to 20% by weight, more preferably based on the dry weight of the bile leaves relative to the total raw materials. Preferably 1 to 5% by weight.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages, vitamin complexes, and the like, and includes all of the functional foods in a conventional sense. In particular, the bile berry extract of the present invention can be prepared as a health functional food in the form of a formulation by further comprising a food additive acceptable food formulation, the form of the formulation is a tablet, capsules, pills, liquids, etc. It is possible.

In addition, the health functional food of the present invention is a variety of flavors, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonating agents And the like may be further contained. In particular, the natural carbohydrate may be a sugar such as glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, polysaccharides such as dextrin, cyclodextrin, xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like can be used. The proportion of such additives is not critical but is preferably 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.

Composition comprising bile leaf extract according to the present invention as an active ingredient exhibits an anti-inflammatory effect by inhibiting NO production by inhibiting iNOS in immune cells, and inducing vascular relaxation by inducing NO production by enhancing eNOS in vascular endothelial cells. The present invention can be usefully applied to the development of drugs and foods for the prevention and treatment of various inflammatory diseases or vascular diseases.

Hereinafter, the configuration and operation of the present invention through the preferred embodiment of the present invention will be described in more detail. However, the following examples are provided to help the understanding of the present invention, and the scope of the present invention is not limited to the following examples. Details that are not described herein will be omitted since those skilled in the art can sufficiently infer technically.

Example 1 Preparation of Bile Leaf Extract

After purchasing the bile leaves, first crushed roughly with a crusher, and then finely crushed again using a crusher to powder. 1.2 kg of the powdered bile leaves were immersed in 10 L of 99% ethanol to sufficiently extract the samples, and then soaked at room temperature for 10 days. At this time, the sample was sufficiently extracted by using an ultrasonic wave analyzer (100 W, 40 kHz).

The dried bile leaf extract extracted for 10 days was first filtered through a cotton cloth to remove miscellaneous matter and debris. Secondly, only the clear decoction liquid was separated and filtered under a vacuum using a Watman NO.3 filter paper. Finally, the filtered bile ethanol extract was concentrated using a reduced pressure concentrator, and then completely frozen at -21 ° C and lyophilized in vacuum over about 2 to 3 days to remove alcohol. The composition powder of the present invention made by freeze drying was finely pulverized, sieved to a powder state, and then stored in a -70 ° C. cryogenic freezer.

38.21 g of lyophilized bile leaf extract was obtained by the above method. In addition, powdered bile leaf extract was dissolved in purified water and used for the experiment.

Example 2. Confirmation of anti-inflammatory effect

2-1. Cytotoxicity of Bile Extracts

MTT assay was performed to determine the cytotoxicity of the bile duct extract prepared in Example 1 on mouse macrophage (Raw 264.7). Raw 264.7 cells were treated with various concentrations of bile leaf extract, and then Raw 264.7 cell viability was examined (FIG. 1).

Specifically, to measure the toxicity of the cells of the bile duct extract was dispensed in 96 well plate of raw 264.7 cells 3 × 10 ⁴ cells / mL and incubated in 37 ℃, 5% CO ₂ incubator for 24 hours. After culturing for 18 hours with various concentrations of bile leaf extract in cultured cells, 50 μl of 5 mg / mL MTT solution dissolved in PBS (phosphate buffered saline) was added to each well, and then incubated at 37 ° C. in 5% CO ₂ incubator. Incubated for hours. After incubation, the culture medium was discarded, and 100 μl of DMSO was dissolved to dissolve formazin, and the absorbance was measured at 570 nm with a microplate reader.

As a result, the bile leaf extract did not reduce the concentration-dependent survival rate in Raw 264.7 cells. These results show that the bile leaf extract is not toxic to the cells.

2-2. Confirmation of NO Production of Immune Cells Treated with Bile Extracts

Inflammatory response is a complex process mediated by various immune cells activated by external antigens, and macrophages are caused by various inflammations such as NO, PGs, and inflammatory cytokines TNF- and IL-1 by inflammatory agents such as LPS. Intermediate substances are derived.

In this experiment, exposure of macrophage RAW 264.7 cells to LPS for 18 hours could confirm NO production. In order to examine the effect of bile leaf extract on the production of NO, the concentration of bile leaf extract in RAW 264.7 cells Treatment and simultaneous LPS 1 μg / mL were incubated for 18 hours. Griess Reagent (1% sulfanilamide in 5% phosphoric acid + 1% -naphthylamide in H ₂ O) in the same amount as 100 μl of the culture was added and reacted at room temperature for 10 minutes, and the absorbance was measured at 540 nm with an ELISA reader. NO concentration in the culture was determined using a standard curve of concentration of sodium nitrite (NO ₂ ⁻ ).

As a result, NO production was significantly increased in Raw 264.7 cells to 10.64 ± 0.53 uM compared with the control group 5.33 ± 0.21 uM when LPS alone treatment, and in the experimental group treated with bile leaf extract compared with LPS alone treatment group It was shown that NO production decreased depending on the concentration.

2-3. Measurement of iNOS Expression Levels of Immune Cells Treated with Bile Extracts

To investigate the relevance of iNOS protein to NO production inhibition, the expression level of iNOS protein in the cytoplasm of Raw 264.7 cells was examined using immunoblot analysis (FIG. 3). In the experimental group treated with LPS (1ug / ml) alone and LPS and bile leaf extract, the expression level of iNOS was markedly decreased, and the iNOS protein amount was decreased when LPS and bile leaf extract were treated simultaneously.

Example 3. Confirmation of Vascular Relaxation Effect

3-1. Isolation of the thoracic aorta

In order to measure the vasorelaxant effect of the bile leaf extract prepared in Example 1, a healthy SD (Sprague-Dawley) -based white paper weighing about 250 to 300 g was subjected to cervical distal bone, and the thoracic aorta was separated. The isolated thoracic aorta was transferred to a cold crab solution (pH) containing 118 mM NaCl, 4.7 mM KCl, 1.1 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ , 1.5 mM CaCl ₂ , 25 mM NaHCO ₃ , and 10 mM glucose. 7.4) were made into sections of about 3 mm length, with the connective tissue and fat removed.

3-2. Measurement of Vascular Relaxation Effect of Bile Extracts

It was examined whether the bile duct extract prepared in Example 1 exhibited vascular relaxation effect in the thoracic aorta of SD rats. In particular, to determine whether such vascular relaxation effect is blood and endothelial cell-dependent, comparative experiments were performed after the endothelial cells were completely removed (FIG. 4).

A physiological recorder equipped with a force-displacement transducer (Grass FT 03, GRASS Instrument, Mass., USA) after immobilization of the chest aortic section in a Krebs solution at 37 ° C saturated with 95% O ₂ -5% CO ₂ gas. (Grass Model 7E, Grass Instrument, Mass., USA). First, 1x 10 ^-6 M phenylephrine was contracted, and after 10 minutes, 1 x 10 ^-6 M acetylcholine (ACh) was relaxed to measure the safety of vascular endothelial cells, and then washed three times with Krebs solution.

The measurement of the vasorelaxant effect of bile leaf extracts by various drugs was first pretreated for 20 minutes, contracted with phenylephrine, and then the concentration-dependent relaxation of the bile leaf extracts was observed. When vascular endothelial cell-independent experiments were performed, the vascular endothelial cells were removed with a small cotton swab, and then phenylephrine contraction and ACh relaxation reactions were performed to confirm the removal of vascular endothelial cells.

As a result, it was confirmed that bile leaf extract showed relaxation effect in concentration-dependent manner in the thoracic aorta. In addition, after the endothelial cells were removed by physical method ((-) endothelium), the relaxation effect of bile leaf extract showed that the relaxation was not achieved. It can be seen that bile leaf extract is relaxed in endothelial cells.

The mechanisms that regulate contraction and relaxation in vascular smooth muscle vary widely, the most important of which is endothelium-derived relaxing factor (EDRF), the most representative of which is nitric oxide (NO). Nitric oxide in blood vessels is produced in vascular endothelial cells by nitric oxide synthase (NOS) using L-arginine as a substrate. Related to this isoform of NOS is vascular endothelial cell type nitric oxide synthase (eNOS), which mainly distributes vascular endothelial cells and acts to relax blood vessels. Nitric oxide (NO) produced by eNOS activates soluble guanylate cyclase (sGC) in vascular smooth muscle cells to increase the production of cyclic guanosin-3 and 5-mono-phosphate (cGMP), which leads to vascular smooth muscle Relaxes. Increased cGMP activates cGMP dependent prtein kinase, which inactivates myosin light chain kinase (MLCK) and activates myosin light chain (MLC) phosphatase. Increased cGMP also inhibits several pathways that increase smooth muscle intracellular Ca ²⁺ , lowering intracellular Ca 2+ concentrations, thereby relaxing vascular smooth muscle.

After treatment with non-specific nitric oxide inhibitor L-NAME (1 × 10 ^-4 M) in order to determine whether the vasorelaxant effect of bile leaf extract is related to nitric oxide in the thoracic aorta of the SD rat, Vascular relaxation effect of the bile duct extract was measured (FIG. 5). In addition, in order to determine whether the vasorelaxant effect caused by these bile duct extracts was related to cGMP, ODQ (1 × 10 ^-3 M), a cytoplasmic sGC inhibitor, was pretreated and the vascular relaxation effect of the bile foliate extract was measured (FIG. 5). .

As a result, the vasorelaxant effect of bile leaf extract was significantly suppressed in the vessels pretreated with L-NAME. These results demonstrate that bile leaf extract increased NO production through eNOS, and the increased NO diffused into vascular smooth muscle to relax vascular smooth muscle. When the blood vessels were pretreated with ODQ, the vasorelaxant effect of the oleander leaf extract was significantly suppressed. From these results, it can be seen that the vasorelaxant effect by the bile leaf extract is via the NO / cGMP signaling system.

3-3. Measurement of cGMP (cyclic guanosin-3,5, -monophosphate)

The amount of cGMP produced according to the concentration of bile leaf extract in the thoracic aorta of the SD rats according to the present invention was examined (FIG. 6). The concentration of bile leaf extract was used 1 × 10 ^-4 g / ml, 3 × 10 ^-4 g / ml, 1 × 10 ^-3 g / ml.

In addition, L-NAME (1 × 10 ^-4 M) and ODQ (1 × 10 ^-5 M) in order to determine the amount of cGMP produced through the NO / cGMP pathway of bile leaf extract in the thoracic aorta of SD rats according to the present invention. After the pretreatment was determined cGMP production (Fig. 7).

To measure cGMP in vascular tissues, chest aortic sections were equilibrated in Krebs solution for 30 minutes with 95% O ₂ -5% CO ₂ gas and then 1 x 10 ^-4 M 3-isobutyl-1- methylxanthine (IBMX) was added. Add 1 x 10 ^-6 M phenylephrine to equilibrate for 10 minutes, and then react with Habaekcho ethanol extract (3 x 10 ^-5 , 1 x 10 ^-4 , 3 x 10 ^-4 g / ml) for 3 minutes by concentration. Let. Vascular tissues were immediately placed in liquid nitrogen to stop the reaction, and then stored at -76 ° C and used for cGMP concentration measurement. The supernatant obtained by centrifugation at 13,000 g for 15 minutes was extracted four times using homogenized in the presence of 6% trichloroacetic acid. Extracts were concentrated using a Speed-vac concentrator and cGMP measurements were analyzed using radioimmunoassay. Briefly, 100 μl of diluted cGMP antibody (Calbiochem-Novabiochem, Co., San Diego, CA, USA) was added to a 50 mM sodium acetate buffer (pH 4.8) with a final volume of 100 μl. USA) and ¹²⁵ I coupled cGMP (10,000 cpm per 100 μl, specific activity 2200 ci / mM, Dupont-New England Nuclear, Wilmington, DE, USA) were added together and reacted at 4 ℃ for 24 hours. Antibody-bound ¹²⁵ I-cGMP and non-binding ¹²⁵ I-cGMP were separated using charcoal, and radioactivity was measured using a counter.

As a result, the increase in cGMP production through bile leaf extract was concentration-dependent, and cGMP production of bile leaf extract was significantly suppressed in blood vessels pretreated with L-NAME. These results demonstrate that bile leaf extract increases NO production through eNOS, and the increased NO diffuses into vascular smooth muscle and is involved in cGMP production. In addition, when the blood vessels were pretreated with ODQ, cGMP production by bile leaf extract was significantly inhibited. These results show that bile leaf extract has a vasodilation effect through NO / cGMP signaling system.

1 is a graph showing the results of experiments to determine whether the bile duct extract in the mouse macrophages according to the present invention having cytotoxicity.

Figure 2 is a graph showing the results of experiments to determine the inhibitory effect of the bile leaf extract on NO induced by LPS in mouse macrophages according to the present invention.

Figure 3 is a graph showing the results of experiments to determine the inhibitory effect of the bile leaf extract on LPS-induced iNOS in mouse macrophages according to the present invention.

Figure 4 is a graph showing the results of experiments to determine whether the relaxation effect and vascular relaxation effect of biliary leaf extract in the thoracic aorta of the SD-based white paper according to the present invention is endothelial cell-dependent.

Figure 5 is a graph showing the results of experiments to determine the relationship between the nitric oxide system and cGMP relaxation effect of bile leaf extract in the thoracic aorta of the SD rats according to the present invention.

Figure 6 is a graph showing the results of experiments to determine the cGMP production amount of bile leaf extract in the thoracic aorta of the SD rats according to the present invention.

Figure 7 is a graph showing the results of experiments to determine the relationship between nitric oxide and sGC in cGMP production of bile leaf extract in the thoracic aorta of the SD-based white paper according to the present invention.

Claims (15)

Pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising the extract of biliary leaves as an active ingredient. The pharmaceutical composition for preventing and treating inflammatory diseases according to claim 1, wherein the bile leaf extract has an inducible nitric oxide synthase (iNOS) inhibitory activity. The pharmaceutical composition for preventing and treating inflammatory diseases according to claim 1, wherein the bile leaf extract inhibits NO (nitric oxide) production in immune cells. The method of claim 1, wherein the inflammatory disease is rheumatoid arthritis, autoimmune diseases, Parkinson's disease, arteriosclerosis, multiple sclerosis, edema, sepsis, septic shock, psoriasis, cancer, encephalomyelitis, meningitis, pancreatitis, peritonitis, A pharmaceutical composition for preventing and treating inflammatory diseases, characterized in that the disease is selected from the group consisting of vasculitis, lymphocytic choroiditis, glomerulonephritis, uveitis, ileitis, hepatitis, nephritis and inflammatory bowel disease. A pharmaceutical composition for preventing and treating vascular diseases comprising the bile leaf extract as an active ingredient. The pharmaceutical composition for preventing and treating vascular disease according to claim 5, wherein the bile duct extract has eNOS (endothelial nitric oxide synthase) enhancing activity. The pharmaceutical composition for preventing and treating vascular diseases according to claim 5, wherein the bile duct extract promotes nitric oxide production in vascular endothelial cells. The pharmaceutical composition for preventing and treating vascular diseases according to claim 5, wherein the bile duct extract induces vascular smooth muscle relaxation. According to claim 5, The vascular disease is selected from the group consisting of hypertension, coronary artery disease, cerebrovascular disease, peripheral vascular disease, aortic disease, renal dysfunction, erectile dysfunction, hypertensive heart disease, diabetes mellitus, heart failure and vision disorders Characterized in that the disease, the pharmaceutical composition for preventing and treating vascular diseases. The pharmaceutical composition according to claim 1 or 5, wherein the bile duct extract is extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof. The pharmaceutical composition according to claim 1 or 5, wherein the content of the bile duct extract is 0.0001 to 30% by weight on a dry weight basis with respect to the total weight of the composition. The pharmaceutical composition according to claim 1 or 5, wherein the composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 1 or 5, wherein the dosage of the bile leaf extract is 0.0001 to 100 mg / kg per day on a dry weight basis. Functional food for preventing or improving inflammatory diseases comprising bile leaf extract. Health functional foods for preventing or improving vascular diseases, including bile leaf extract.

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