KR20190089425A - Vasodilator and antiphlogistics containing extract of luffa cylindrica - Google Patents

Abstract

The present invention relates to a vascular relaxant and an anti-inflammation agent containing a Luffa cylindrica extract as an active component. More specifically, the present invention relates to the vascular relaxant which can exhibit a vasorelaxant effect by inducing nitric oxide (NO) production in vascular endothelial cells and the anti-inflammation agent which exhibits an anti-inflammation effect by inhibiting NO production in immunocytes. The vascular relaxant according to the present invention can be used for prevention, treatment or alleviation of diseases caused by vascular contractions such as hypertension, arteriosclerosis, blood circulation disorder, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction, heart failure and the like.

Description

TECHNICAL FIELD [0001] The present invention relates to a vasodilator and an anti-inflammatory agent,

The present invention relates to a vasodilator and an anti-inflammatory agent containing scrubs extract as an active ingredient, and more particularly, to a vasodilator and an anti-inflammatory agent that can induce nitric oxide (NO) production in vascular endothelial cells, The present invention relates to an anti-inflammatory agent capable of exhibiting anti-inflammatory activity by inhibiting the production of nitric oxide.

Nitric Oxide (NO) is a free radical in vivo that regulates various biological functions such as vasodilation, neurotransmission, and immunomodulation. It is involved in various inflammatory reactions, And as a result causes chronic inflammation.

In addition, NO is known to be a cell differentiation or intracellular signal transducer in addition to cytotoxicity. Lipopolysaccharide (LPS), which induces inflammation, is activated to activate macrophages, thereby confirming NO production. INOS and cyclooxygenase- 2 (COX-2) is a typical inflammatory factor of immune cells. When inflammatory reaction occurs, various inflammatory factors (pro-inflammatory mediatos) are produced and NO and COX-2 produced by iNOS, an inducible nitric oxide synthase Prostagrandin E2 (PGE2), and so on. In the case of iNOS, it is expressed when there is stimulation of various inflammatory cytokines such as LPS.

On the other hand, the degree of vascular smooth muscle tone is affected by norepinephrine, adenosine triphophate (NO), endothelium-derived relaxing factor (EDRF), endothelium-derived hyperpolarization factor (EDHF), endothelins, metabolic effects (PO ₂ , PCO ₂ . pH), humoral substances (norepinephrine, epinephrine, vasopressin), and muscular effects (blood vessel elasticity, pressure resistance). In particular, vascular endothelial cells play an important role in regulating blood pressure by releasing an endothelium-derived relaxing factor and an endothelium-derived constricting factor. The main body of this EDRF (endothelium-derived relaxing factor) is NO (NO), which can be produced from vascular endothelial cells as well as smooth muscle and blood cells and neurons of the central nervous system. At least three nitric oxide synthase isoenzymes (L-arginine), which is an amino acid. In addition, vascular relaxation factors such as NO are synthesized and secreted in endothelial cells even in normal state, and are inhibited by agonists such as acetylcholine, histamine, substance P and isoproterenol Synthesis and secretion are increased. The action of nitrate, which has been used in the treatment of hypertension, is caused by activating soluble guanylate cyclase to raise cGMP. Moreover, since this drug generates NO by non-enzymatic action, Relaxation was also found to be due to cGMP due to the activity of soluble guanylate cyclase. Recently, several herbal medicines and herbal medicines have been reported that relax blood vessels by increasing the production of nitric oxide in vascular endothelial cells. Kuramochi et al. Reported that the extract solution of the 鉤) extract increased nitric oxide-dependent vasorelaxation effect. Goto et al. Reported that the fish extract of the fish caught 鉤 藤 in the spontaneously hypertensive rats lowered the blood pressure The hydrothermal extract relaxes the blood vessels of the thoracic aorta in a nitric oxide-dependent manner. It is also known that the protein components of licorice (B) and licorice (B) are also increased in a nitric oxide-dependent manner.

Spruce is a fruit of Luffa cylindrica Roemer (Cucurbitaceae), and it is widely used as beverage and other food materials as edible food raw materials. In addition, in one room, it is commonly used as a medicine name called apple ring (shiitake), and it has been used as a medicinal product because it has a sweet taste and is pure and has no toxicity. In one room, loofah has the efficacy to alleviate the aches and pains of the chest and flank, to relieve pain such as abdominal pain and back pain, to reduce ejaculation, to reduce fever, to reduce the heat of the lungs and to stop the sputum and cough. It has been used mainly for diseases. It contains phytonutrients such as saponin, lutein and citrulline. The seeds contain 20 ~ 40% of lipid oil and contain linoleic acid, palmitic acid, stearic acid, and oleic acid. Research on scrubs is mainly focused on the reduction of whitening and inflammation in raw materials for cosmetics, and it is only to the extent that it is used in cocoon, sputum and asthma in the oriental medicine.

Korea Patent Publication No. 10-2009-0064823

The present invention provides a vasodilator and an anti-inflammatory agent containing scrubs extract as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating inflammation containing scrubs extract as an active ingredient.

The present invention also provides a health functional food for vascular restenosis comprising a scrub brush extract as an active ingredient.

The present invention provides a vasodilator comprising a scrub brush extract as an active ingredient.

Wherein the scrubber extract is extracted with a solvent selected from the group consisting of water, ethanol, methanol, propylene glycol, ether, chloroform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, isopropanol, . Most preferably, it may be an extract extracted with water as a solvent.

The scrubs extract may have endothelial nitric oxide synthase (eNOS) promoting activity.

The scrubs extract may increase the production of nitrogen oxides (NO) in vascular endothelial cells.

The scrubs extract may exhibit vasorelaxant efficacy by controlling the NO-cGMP pathway (vascular endothelial nitric oxide-cyclic guanosine monophosphate).

The vascular relaxant may be used for the treatment, prevention or amelioration of diseases caused by vasoconstriction.

The diseases caused by the vasoconstriction may be selected from the group consisting of hypertension, arteriosclerosis, blood circulation disorder, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure.

In addition, it is possible to provide a vascular resting health functional food containing a scrub brush extract.

The present invention also provides an anti-inflammatory agent containing a scrub brush extract as an active ingredient.

The scrub brush extract may be extracted with a solvent selected from the group consisting of water, ethanol, methanol, propylene glycol, ether, chloroform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, isopropanol, Extract, and more preferably an extract extracted with ethanol or methanol.

The scrubs extract may have iNOS (inducible nitric oxide synthase) inhibitory activity.

The scrubs extract can inhibit the production of nitric oxide in immune cells.

In addition, the present invention can provide a health functional food for prevention or improvement of inflammation including scrub brush extract.

Further, the present invention can provide a composition for vascular restenosis or a composition for preventing or improving inflammation, which comprises a scrub brush extract as an active ingredient.

The health functional food may be prepared using the food composition.

The food composition may include foodstuff acceptable food additives.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline Cellulose, polyvinylpyrrolidone, water, methylhydroxy, hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, and the like.

The food composition of the present invention can be widely used for foods and beverages for relaxing blood vessels. Specifically, it may be a gum, a tea, a vitamin complex or a health supplement food, and may be in the form of powder, granule, tablet, capsule or beverage.

When a health drink is prepared using the above food composition, there are no particular limitations on the liquid component in addition to a scrub extract, and it may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages.

In addition to the above, the food composition may further contain various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, Colloidal thickeners, pH adjusting agents, stabilizers, preservatives, carbonates used in glycerin, alcohols or carbonated drinks, and the like.

In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination.

Further, the present invention can provide a quasi-drug composition or a pharmaceutical composition for use as a vascular relaxant comprising a scrub brush extract as an active ingredient. In addition, it is possible to provide a quasi-drug composition for preventing or improving inflammation comprising a scrub brush extract as an active ingredient, and a pharmaceutical composition for preventing or treating inflammation.

Pharmaceutically acceptable additives may be added to the composition.

The compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterilized injection solutions according to a conventional method.

Examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, poly Vinyl pyrrolidone, water, methylhydroxy, hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be added.

In the case of formulation, it may be prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents or surfactants usually used.

Solid formulations for oral administration include tablets, pills, powders, granules and capsules, which may contain at least one or more excipients such as, for example, calcium carbonate, sucrose, lactose lactose, gelatin, and the like.

Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances or preservatives may be included have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsion-freeze-dried preparations and suppositories.

As the suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin or glycerogelatin can be used.

The preferred dosage of the composition varies depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by a person skilled in the art (those skilled in the art).

The composition of the present invention may also be administered to a mammal such as a mouse, a mouse, a domestic animal, or a human in a variety of routes and may be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine or intracerebroventricular injection ≪ / RTI >

In the composition, the ratio of the additive is generally selected in the range of 0.1 to 20 parts by weight based on 100 parts by weight of the scrub brush extract, but is not limited thereto.

According to the present invention, it is possible to provide a vasodilator or an anti-inflammatory agent containing a scrub brush extract as an active ingredient. The vascular relaxant according to the present invention can increase the production of nitric oxide in vascular endothelial cells, thereby realizing vascular relaxation efficacy. In addition, the vascular relaxant according to the present invention can be used for preventing, treating or ameliorating inflammatory diseases due to vasoconstriction such as hypertension, arteriosclerosis, blood circulation disorder, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure. In addition, the scrubs extract according to the present invention can increase anti-inflammatory activity by increasing the production of nitric oxide in immune cells.

1 is a graph showing the results of analysis of vascular smooth muscle relaxation effect of scouring hot water extract on a thoracic aortic cross section of a rat retracted by pretreatment with phenylephrine (1 x 10 ^-6 M) (e (-): Endothelial cells were removed, e (+): vascular endothelial cells were not removed), WLC: water extract of Luffa cylindrica. Each value: mean ± SE of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 2 is a graph showing the results of analyzing changes in vascular relaxation effect of scouring hot water extract by L-NAME and MB pre-treatment. Each value: mean ± SE of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 3 is a graph showing the results of analyzing changes in vasorelaxant effect of a scouring hot water extract by Verapamil pretreatment. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 4A is a graph showing the results of analyzing changes in vasorelaxant effect of scouring hot water extract by pre-treatment with glibenclamide. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 4B is a graph showing the results of analysis of changes in vasorelaxant effect of scouring hot water extract by TEA pretreatment. FIG. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 4C is a graph showing the results of analysis of changes in vasorelaxant effect of scouring hot water extract by 4AP (4-Aminopyridine) pretreatment. FIG. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control
FIG. 5A is a graph showing the results of analysis of changes in vasorelaxation effect of scouring hot water extract by propranolol pretreatment. FIG. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 5B is a graph showing the results of analysis of changes in vasorelaxant effect of scouring hot water extract by atropine pretreatment. FIG. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 6 is a graph showing the results of analysis of changes in vasorelaxant effect of scouring hot water extract by indomethacin pretreatment. FIG. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 7 is a graph showing the results of analyzing changes in vascular relaxation effect of scouring hot water extract by ODQ pretreatment. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
FIG. 8 is a graph showing the results of analysis of the effect of the scouring hot water extract on the vasoconstriction by phenylephrine. vehicle: thoracic aortic rings, WLC: water extract of Luffa cylindrica. Each value: mean ± SEM of 4 experiments, * P <0.05, ** P <0.01, etc. control.
Figs. 9 and 10 are graphs showing the results of cytotoxicity of extracts of scrub brush extracts for each extraction solvent. Fig.
Fig. 11 is a graph showing the results of analyzing the effect of extracting the scrub brush on the production of NO by the extraction solvent.
FIG. 12 is a graph showing the results of confirming the expression levels of iNOS and COX-2 in the extraction solvent of scrubs extract. FIG. (A) a methanol extract, (B) an ethanol extract, and (c) a hot-water extract.

Hereinafter, the present invention will be described in more detail with reference to Examples. The objects, features and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments described herein are provided to enable those skilled in the art to fully understand the spirit of the present invention. Therefore, the present invention should not be limited by the following examples.

Example 1. Preparation of scrub brush extract

The scrubbers used in the experiment were purchased from domestic sources (Gyeongsan, Gyeongbuk) and used in the experiments. After washing with water and drying, 100 g of dry weight was immersed in 1 L of primary distilled water (or methanol or ethanol), and the hydrothermal extract, methanol extract, and ethanol extract Respectively. The extract was filtered through Advantec Filter paper No. 2 (Toyo Roshi Kaisha Ltd., Japan), and the filtrate was concentrated using a rotary evaporator (Buchl, Swiss). The concentrate was applied to an ultra-low temperature freezer (Operon, Korea) And then lyophilized product was obtained using Freeze Dryer (Operon, Korea). (61.4 g, yield 6.14%). This was dissolved in an appropriate amount of tertiary distilled water for each experiment.

Experimental Example 1. Vascular relaxation effect analysis of scorpion hot water extract

Production of physiological nutrients

Krebs-Henseleit solution was used to maintain the stable physiological activity of the thoracic aortic blood vessels. The composition was as follows. (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, pH 7.4)

Production of blood vessel tissue section

Male Sprague-Dawley rats weighing 250 ~ 330g were used in the experiment. Before the start of the experiment, anesthesia was sacrificed by supersaturation of carbon dioxide, and the abdominal aorta was opened and the thoracic aorta was separated. The isolated thoracic aorta was placed in an oxygenated Kreb's solution at 4 ° C, and the connective tissue and fat were removed, and then a ring-shaped section of about 3 to 4 mm in length was made.

Measurement of blood vessel tension

The thoracic aortic cross-section of the rat was fixed at one end to the bottom of an organ bath containing 37 ° C Krebs solution saturated with 95% O ₂ , 5% CO ₂ gas mixture, and the other end was immersed in a force-displacement transducer (Grass FT The isometric tension of the blood vessels was measured using a physiological recorder (Grass Model 7E, Grass Instrument, MA, USA) connected to a force-displacement transducer. Then, fresh blood Kreb's solution was supplied for 60 minutes at 10-minute intervals to stabilize the blood vessels, and the blood vessel slice was given a base tension of 1 g. When the basal tension of 1g is maintained constant contractible phenyl LES printer (phenylephrine) of 1x10 ^-6 M and when this was more than 80% relaxation rate to relax to ACh (acetylcholine) 5 minutes later 1 x 10 ^-6 M endothelial The cells were judged not to be injured and the drug experiment was carried out. When the pretreatment drug is used for the confirmation of the mechanism of hot water extract of sponge, the drug is pretreated with an organ bath for 10 minutes and shrinked with phenylephrine, and then the blood vessel relaxation reaction by the scouring hot water extract is observed in a concentration dependent manner Respectively. The endothelial cells were scraped off with a thin stainless steel, contracted with phenylephrine and relaxed with ACh to achieve relaxation of less than 5% . The vascular relaxation rate of the drug was calculated as a percentage (%) based on the contraction of the blood vessel by phenylephrine (1 uM).

reagent

In the present study, ACh, phenylephrine, NG-nitroarginine methyl ester, indomethacin, glibenclamide, tetraethylammonium, verapamil, 4AP, methylenblue, atropine and propranolol were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Statistical processing

The test results were statistically analyzed using Sigma Plot. Student's t-test or one-way ANOVA test was used to determine statistical significance when p was less than 0.05.

1-1. Concentration-dependent relaxation of smooth muscle vascular smooth muscle

The sclerotial hot-water extracts relaxed the blood vessels in a concentration-dependent manner on the thoracic aortic cross-section of the rat retracted by pretreatment with phenylephrine (1 x 10 ^-6 M) (Fig. In addition, the vascular relaxation effect of the scouring hot water extract did not appear when the vascular endothelial cells were completely removed (Fig. 1).

1-2. Nitric oxide synthase (NOS) inhibition of blood vessel relaxation effect

The ^{L-NAME (1 x 10 -5} M) and MB (2.5x 10 ^-5 M), non-selective nitric oxide synthesis inhibitors in order to ensure that the thoracic aorta relaxation effect of the hot-water extract acid scrubber quality subtotal associated with each organ bath for 10 minutes and the vascular relaxation effect was measured. As a result, the relaxing effect of the loofah was inhibited statistically by pretreatment of L-NAME and MB (FIG. 2).

1-3. Ca ²⁺  Vascular relaxation effect during passage block

In order to measure the effects of Ca ²⁺ channel blockage on vasodilatory effects by scouring hot water extract, verapamil (1 x 10 ^-5 M), a Ca ²⁺ channel blocker, was pretreated and then relaxed by scouring hot water extract As a result, verapamil pretreatment significantly inhibited the relaxation effect of scarlet wounds (FIG. 3).

1-4. K ⁺  Vascular relaxation effect during passage block

(1 × 10 ^-5 M), which is a non-selective K ⁺ channel inhibitor, and glibenclamide, which is an ATP-sensitive K ⁺ channel inhibitor, to measure the effect of K ⁺ channel blockage on vasodilatory effects of scouring hot- ^{glibenclamide, 1 x 10 -5 M)} , Ca 2+ association K ⁺ channel inhibitor, 4AP (1 x 10 ^-5 M) to each of the pre-treatment after the results of the measurement of the vasodilator effect of the sponges, the hot-water extract, in the case of TEA, 4AP There was no significant difference from the untreated group, and when the glibenclamide was pretreated, the relaxation effect of the loofah was significantly inhibited (FIG. 4).

1-5. Vascular relaxation effect on autonomic nervous system blockade

In order to investigate the relationship between the relaxation effect and the nervous system, the propranolol (non-selective β-adrenergic antagonist) and atropine (anti-muscarinic drug) were pretreated with 1 × 10 ^-6 M The vascular relaxation effect of scouter hot water extract was measured, and as a result, pretreatment of propranolol and atropine significantly inhibited the relaxation effect of scallops (Fig. 5).

1-6. Vascular relaxation effect when COX (Cyclooxygenase) is blocked

In order to observe whether vascular relaxation effect of scorchion hydrothermal extracts is related to prostacyclin, it is necessary to pre-treat indomethacin (1 x 10 ^-5 M), a non-selective COX (cyclooxygenase) inhibitor, , It was confirmed that the vascular relaxation effect of the scrubber was significantly inhibited by the indomethacin pretreatment (FIG. 6).

1-7. Prevention of vascular contraction by ODQ pretreatment

As a result of pre-treatment of the soluble guanylate cyclase inhibitor ODQ and measurement of vasodilation effect, it was confirmed that the vascular relaxation effect by the hot-water extract was inhibited in the scrubber (Fig. 7)

1-7. Vascular contraction inhibitory effect

In order to investigate the effect of scorching hot water extract on phenylephrine-induced vasoconstriction, sclerotial hot water extracts were treated at 1 × 10 ^-4 g / ml and 1 × 10 ^-3 g / ml, respectively, (phenylephrine, 1 x 10 ^-8 M - 1 x 10 ^-5 M). As a result, vascular contraction due to phenylephrine was significantly reduced as compared with the vehicle (Fig. 8).

Several vasoactive factors secreted from vascular endothelial cells can regulate vascular smooth muscle relaxation. These vasoactive factors include nitric oxide (NO), prostacyclin, endothelium-derived hyperpolarizing factor endothelium-dependent hyperpolarizing factor, EDHF). From the above results, it was confirmed that sclerotin hydrothermal extract relaxed the phenylephrine-induced vasoconstriction in the concentration-dependent manner in the thoracic aorta extracted from the rat. However, the complete relaxation of the vascular relaxation effect of sclerotin hydrothermal extract after removal of vascular endothelial cells implies that vascular relaxation effect of scallop is closely related to the vascular endothelial cell-derived vascular activation factors. In addition, according to the results of the above experiment, the vascular relaxation effect of the scouring hot water extract was statistically significantly inhibited by pretreatment of L-NAME and MB, which are nitric oxide synthesis inhibitors. These results suggest that NO among the endothelium - derived vasoactive factors is crucially related to vasodilatory effects of scouring hot - water extracts.

In addition, various drugs such as autonomic nervous system, calcium ion channel, potassium ion channel, COX (cyclooxygenase) and nitric oxide synthase blocker were investigated in order to examine the vascular relaxation effect of endothelium-derived vascular relaxants and sclerotial hot- . In order to investigate the effect of nitric oxide (NO), the body of endothelium-derived vascular relaxation factors, on the vasodilation effects of scouter hot-water extracts, L-NAME, an inhibitor of nitric oxide synthase, was pretreated and the vascular relaxation effect It was completely blocked. Nitric oxide (NO), a small molecule produced in vivo, was found to be a potent endothelium-dependent vasodilatory factor and has a strong blood pressure lowering effect. Nitric oxide is synthesized from L-arginine by nitric oxide synthase (NOS), and there are three isoenzymes, nitric oxide synthase, in the blood vessels. The first type has been found in brain tissue as brain - type nitric oxide synthase (bNOS, nNOS, NOS Ⅰ) and plays a role of synthesizing nitric oxide mainly as a neurotransmitter. The second isozyme is an inducible nitric oxide synthase (iNOS, NOS Ⅱ), which synthesizes nitric oxide mainly acting on the immune system. The third isoenzyme is vascular endothelial nitric oxide synthase (eNOS, eNOS, NOS Ⅲ), which is mainly distributed in vascular endothelial cells, and is activated by acetylcholine to relax blood vessels. These nitric oxide activates cytoplasmic guanylate cyclase to increase the production of cGMP and relax vascular smooth muscle through the signaling system. In this experiment, relaxation effect of wrung remedy was suppressed by pretreatment of L-NAME, and it seems that vascular relaxation effect by scouring hot water extract occurs via the oxinoid system. In general, ACh, a well - known vascular relaxant, induces NO mediated endothelium - dependent relaxation effects, and the release of NO by ACh is known to pass through the endothelial M3 receptor. The endothelial Ca ²⁺ uptake by ACh is the result of Ca ²⁺ flux from the intracellular reservoir by IP3 and extracellular Ca ²⁺ influx. Nitric oxide synthase (NOS) is activated by increased Ca ²⁺ and nitric oxide (NO) is liberated from L-arginine as a substrate. The nitric oxide produced at this time diffuses into the vascular smooth muscle and activates cytoplasmic guanylate cyclase again. Cytoplasmic guanylate cyclase converts GTP (guanosinetriphosphate) into cGMP, and cGMP activates cGMP-dependent protein activating enzyme again. This enzyme relaxes the vascular smooth muscle by inhibiting the Ca ²⁺ entry into the vascular smooth muscle and reducing the susceptibility to intracellular Ca ²⁺ contraction. If the NO / cGMP system is involved in vasodilatory effect by scouring hot water extract, the soluble guanylate cyclase activated by nitric oxide is considered to be involved in vasorelaxant effect by scouring hot water extract. Thus, the soluble guanylate cyclase inhibitor ODQ After the pretreatment, blood vessel relaxation effect was measured. As a result, it was confirmed that the relaxation effect of hydrothermal extract was inhibited in the scrubber. These results may indicate that the vascular relaxation effect by the scouring hot water extract occurs via the NO / cGMP system. These results suggest that the scorpion hydrothermal extract increases the synthesis of nitric oxide in vascular endothelial cells and activates the soluble guanylate cyclase to increase the production of cGMP and relax the blood vessels.

Another endogenous factor that regulates vascular smooth muscle tone is prostacyclin (PGI2). Prostacyclin is produced from arachidonic acid by cyclooxygenase, and the produced prostacyclin has vasodilatory action. In order to investigate whether the vasodilating effect of scorch fluid hydrothermal extracts is related to the production of prostacyclin, indomethacin, which is a cyclooxygenase inhibitor, was pretreated and the wringing relaxation effect was measured. Indomethacin pretreatment inhibited the relaxation of blood vessels, suggesting that the prostacyclin system is involved in the vasodilatory action of scouring hot - water extracts.

Because the vascular smooth muscle tone is due to the vascular smooth muscle cell membrane potential difference, changes in intercellular ion channels can explain the direct relaxation effect. According Van Breeman ion pump or the like (ion pump) roneun Na ⁺ / K ⁺ pump and Ca ²⁺ pump, as well as Na ⁺ / H ^+, HCO ₃ of vascular smooth muscle cell membrane ^- There ^{are, Na + / Ca 2+ exchanger -} / Cl , The vascular smooth muscle cell membrane potential is regulated mainly by the outflow and inflow of Ca ²⁺ and K ⁺ ions. Vascular tone is controlled by the membrane potential and to increase the Ca ²⁺ through a VOC (Voltage Operated Channel) of Ca ²⁺ channels (channel) which depends on the voltage by increasing membrane potential. This increase in Ca ²⁺ activates the contractile proteins, resulting in contraction, which is electro-mechanical coupling. On the other hand, K ⁺ channel induces hyperpolarization of cell membrane during activation and relaxes blood vessels by inhibiting Ca ²⁺ influx through VOC. This is also known to be an important mechanism of endogenous vascular relaxation in controlling vascular tone. In the vascular smooth muscle, the K ⁺ channel is Ca ²⁺ -activated K channel, ATP-sensitive K channel and voltage-gated K channel. Endothelium-dependent hyperpolarizing factor , EDHF) activate K ⁺ channel and cause hyperpolarization. The intracellular flux of K ⁺ due to the activation of K ⁺ channel causes hyperpolarization of the cell membrane, leading to inactivation of voltage-sensitive Ca ²⁺ pathway, resulting in a decrease in intracellular Ca ²⁺ concentration and eventually inducing vasorelaxation do. Inhibitors for K + channels include 4aminopyridine blocking Kv, TEA blocking KCa, and glibenclamide, a sulfonylurea drug, glibenclamide, In smooth muscle, KCa channel is not affected but KAp is blocked. To investigate the effect of Kv, KCa or KATP on the vasorelaxation effect of spore extracts, 4AP, KEA, KEA, TEA and GLB were pretreated. , 4AP was not significantly different from non-pretreated group, and pretreatment with glibenclamide significantly affected the loosening effect of loofah. These results suggest that the relaxation effect of the loofah is partially related to the K ⁺ channel.

Intracellular Ca ²⁺ concentration is the most important factor in vessel contraction and relaxation. When the intracellular Ca ²⁺ concentration increases, it binds calmodulin and promotes phosphorylation of MLC (myosine light chain) to contract smooth muscle. The vasodilator effect of the hot-water scrubber to investigate whether associated with a Ca ²⁺ passage were pretreated with the L-type Ca ²⁺ blocker verapamil (verapamil) results of the measurement of the vasodilator effect, in the case where the pre-processing verapamil (verapamil) The vascular relaxation effect of sponge hydrothermal extract was significantly reduced, suggesting that the loosening effect of scallop is related to the Ca ²⁺ channel.

Tension of vascular smooth muscle is also regulated by regulation of the autonomic nervous system. In order to investigate whether the sclera hydrothermal extract relaxes the blood vessels via the autonomic nervous system receptor, atropine (atropine) selectively blocks the muscarinic receptor and cholinesterase When the adrenergic drugs propranolol and atropine were treated, it was found that the vascular relaxation effect of the scouring hot water extract was significantly inhibited, and the vascular relaxation effect of the scouring was correlated with the muscarinic receptor in the cholinergic receptors .

In addition, in this experiment, it was found that the vasoconstriction by phenylephrine was remarkably suppressed after the pretreatment of the scouring hot water extract. In the thoracic aorta, the selective α1-agonist phenylephrine contraction is maintained by intracellular Ca ²⁺ concentration through the Ca ²⁺ channel, so that the scorpion hot-water extract partially antagonizes the adrenergic α 1 -receptor Or inhibit the intracellular Ca ²⁺ influx through α1 receptors.

 The NO / cGMP system inhibits vascular smooth muscle cell proliferation, platelet and leukocyte adhesion as well as vascular smooth muscle relaxation, and plays a role in lowering the permeability of endothelial cells and inhibiting the synthesis of extracellular stromal proteins. Decreased NO production in vascular endothelial cells is due to dysfunction or damage of vascular endothelial cells, which is a major pathologic factor of arteriosclerosis and hypertension. In addition, the chronic inhibition of NO synthase in experimental models of animals leads to vasculitis, such as hypertension. Therefore, the increase of vascular relaxant activity through restoration of NO / cGMP system is very important for the treatment of cardiovascular diseases.

Taken together, these results suggest that the sclerotial hot water extract has a vascular endothelial cell-dependent relaxation effect via the NO / cGMP system, and that these results may be useful therapeutic agents for cardiovascular diseases.

In conclusion, the sclerotial hot - water extract relaxed vascular smooth muscle in endothelium - dependent manner, and this effect was dose - dependent and completely inhibited by blocking the NO / cGMP signaling system. Furthermore, when vascular tissues were reacted with sclerotin hydrothermal extract, the production of cGMP was increased. This increase was not observed in vascular endothelial cell depletion, nitric oxide synthase or cytosolic guanylate cyclase inhibition. In addition, vasorelaxant effect of scorpion hydrothermal extract was significantly influenced by blocking of production of prostacyclin, blockage of K ⁺ channel, blocking of Ca ²⁺ channel, blocking of muscarinic receptor and β-adrenergic receptor blocker. These results suggest that the vascular smooth muscle relaxation mechanism of sclerotin hydrothermal extract activates the NO / cGMP pathway in the endothelial cells and is associated with Ca ²⁺ and K ⁺ pathways.

EXPERIMENTAL EXAMPLE 2. Identification of Cytotoxicity of Solvent Extracts from Extracted Solvents (1)

MTT assay was performed to determine the cytotoxicity of mouse rosette (apple rock) extracts on mouse macrophages (Raw264.7 cells).

As a test method, Raw 264.7 cells were seeded at 48 × 10 ⁵ cells / well in a 48-well plate and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours. The cultured Raw264.7 cells were treated with methanol, ethanol, and hot water extracted rind extracts at concentrations of 10, 30, 50, 100, and 300 ug / ml, respectively. The MTT reagent was then added to a final concentration of 0.5 mg / ml well, and cultured in a 5% CO ₂ incubator at 37 ° C for 4 hours. After the culture, the culture medium was discarded, the formazan was dissolved by treating with DMSO, and the absorbance was measured at 570 nm with an ELISA reader. As a result, ethanol and hot water extracts did not show cytotoxicity in Raw264.7 cell (Fig. 9), except that cell viability was significantly decreased at a concentration of 300 ug / ml of scalloped methanol extract.

EXPERIMENTAL EXAMPLE 3: Examination of cytotoxicity of extraction solvent of extracts of scrub brush (2)

To determine the cytotoxicity of LPS treatment of inflammation - inducing cytokines on mouse macrophages (Raw264.7 cells) of extracts from rosemary (apple rock), MTT assay was performed for each extractant.

As a test method, Raw 264.7 cells were seeded at 48 × 10 ⁵ cells / well in a 48-well plate and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours. The cultured Raw264.7 cells were treated with methanol, ethanol and hot water extracts at a concentration of 10, 30, 50 and 100 μg / ml, respectively. After 1 hour, 1 μg / ml of LPS was treated and cultured. After incubation, the MTT reagent was added to each well to a final concentration of 0.5 mg / ml. The cells were incubated in a 5% CO ₂ incubator at 37 ° C for 4 hours. The culture medium was discarded and DMSO was applied to dissolve the formazan. absorbance at 570 nm. As a result, LPS - induced cytotoxicity was suppressed by concentration - dependent (10, 30 ug / ml) of methanol, ethanol and hot - water extract of scum. As a result, scrub extract significantly inhibited cytotoxicity in methanol, ethanol and hot water extracts in a dose - dependent manner.

Experimental Example 4: Determination of NO production by extraction solvent of extracts of scrub brush

Inflammatory reactions can lead to a variety of inflammatory diseases in the in vivo stage of the in vivo defense against stimulation. LPS is an inflammation-inducing substance, and various inflammatory mediators such as NO, PGs, IL-6 and IL-β are induced in Raw264.7 cell.

As a test method, Raw 264.7 cells were seeded at 48 × 10 ⁵ cells / well in a 48-well plate and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Raw 264.7 cell was washed with methanol, ethanol, The extracts were treated at the concentrations of 10, 30, 50, and 100 μg / ml, respectively, and after 1 hour, 1 μg / ml of LPS was treated and cultured. The same amount of Griess reagent as that of the culture solution (50 μl) was added and reacted at room temperature for 10 minutes. Absorbance was measured at 540 nm with an ELISA reader. As a result, it was confirmed that methanol and ethanol extract significantly decreased NO production by LPS to a concentration-dependent (10-300 ug / ml) (FIG. 11).

Experimental Example 5: Detection of iNOS and COX-2 Expression Levels by Extraction Solvent of Scrubber Extract

In order to confirm the expression level of iNOS and COX-2 protein against NO production inhibition, methanol, ethanol and apple juice extract extracted with hot water were added to Raw264.7 cell cultured on a 6-well plate at concentrations of 10, 30 and 100 μg / ml After 1 hour, LPS was treated with 1 μg / ml and cultured, and the quantified sample was analyzed by immunoblot assay. As a result, it was confirmed that the amount of iNOS expression increased by LPS was significantly decreased in a concentration-dependent manner (10-300 ug / ml), but COX-2 showed no significant difference in the expression level of each extractant )

The methanol extracts showed cellulite at a concentration of 300 ug / ml and the ethanol and hot - water extracts showed no cytotoxicity. Methanol, ethanol and hot water extract significantly inhibited cytotoxicity at low concentrations when LPS was induced by NO production. NO production was significantly decreased in methanol and ethanol extracts in a concentration - dependent manner. In addition, when iNOS and COX-2 expression levels, which are typical inflammatory factors, were confirmed, it was confirmed that the methanol and ethanol extracts of iNOS decreased the expression level of iNOS protein increased by LPS in a concentration-dependent manner, It was confirmed that there was no difference in the extraction solvent or concentration. Based on the above results, the scrub extract showed inhibition of LPS-induced NO production in the methanol and ethanol extracts, and it was confirmed that inflammatory mediators such as PGs, IL-6, IL-β and inflammatory mediators such as inflammation-inducing cytokines It is likely that confirmation is needed that the substance is induced.

Claims (14)

A blood vessel relaxant containing an extract of scrubs as an active ingredient.
The method according to claim 1,
Wherein the scrubber extract is extracted with a solvent selected from the group consisting of water, ethanol, methanol, propylene glycol, ether, chloroform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, isopropanol, &Lt; / RTI &gt;
The method according to claim 1,
Wherein the extract is a hot-water extract.
The method according to claim 1,
Wherein the scrubber extract has an endothelial nitric oxide synthase (eNOS) promoting activity.
The method according to claim 1,
Wherein the scrubber extract increases the production of nitric oxide in vascular endothelial cells.
The method according to claim 1,
Wherein said blood vessel relaxant is used for prevention, treatment, or improvement of a disease caused by vasoconstriction.
The method of claim 6,
Wherein the disease caused by the vasoconstriction is selected from the group consisting of hypertension, arteriosclerosis, blood circulation disorder, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure.
Vascular relaxation health functional food including scrub extract.
An anti-inflammatory agent containing scrub brush extract as an active ingredient.
The method of claim 9,
Wherein the scrubber extract is extracted with a solvent selected from the group consisting of water, ethanol, methanol, propylene glycol, ether, chloroform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, isopropanol, Lt; / RTI &gt;
The method of claim 9,
Wherein the extract is ethanol or methanol extract.
The method of claim 9,
Wherein the scrub brush extract has iNOS (inducible nitric oxide synthase) inhibitory activity.
The method of claim 9,
Wherein said scrubber extract inhibits the production of nitric oxide in immune cells.
A health functional food for prevention or improvement of inflammation including scrub brush extract.

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