KR102147868B1 - Vasodilator and antiphlogistics containing extract of luffa cylindrica - Google Patents

Abstract

The present invention relates to a vasodilator and an anti-inflammatory agent containing a loofah extract as an active ingredient, and more particularly, to a vasodilator that can exhibit vascular relaxation efficacy by inducing NO (nitric oxide) production in vascular endothelial cells and NO in immune cells. It relates to anti-inflammatory drugs showing anti-inflammatory effects by inhibiting the production of (nitric oxide). The vasodilator according to the present invention can be used for the prevention, treatment or amelioration of blood vessel constriction such as hypertension, arteriosclerosis, blood circulation disorder, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure.

Description

Vasodilator and anti-inflammatory agent containing loofah extract as an active ingredient {VASODILATOR AND ANTIPHLOGISTICS CONTAINING EXTRACT OF LUFFA CYLINDRICA}

The present invention relates to a vasodilator and an anti-inflammatory agent containing a loofah extract as an active ingredient, and more particularly, to a vasodilator that can exhibit vascular relaxation efficacy by inducing NO (nitric oxide) production in vascular endothelial cells and NO in immune cells. It relates to anti-inflammatory drugs that can exhibit anti-inflammatory effects by inhibiting the production of (nitric oxide).

Nitric Oxide (NO) is a free radical in the body that regulates various biological functions such as vasodilation, nerve signal transmission, and immune regulation, and is involved in various inflammatory reactions.If excessively produced, it causes inflammatory reactions and damages tissues. And, as a result, chronic inflammation.

In addition to cytotoxicity, nitric oxide is also known as a cell differentiation or intracellular signal transducer. It can activate macrophages by treating inflammation-inducing Lipopolysaccharide (LPS) to confirm NO production. iNOS and cyclooxygenase- 2 (COX-2) is a representative inflammatory factor of immune cells. When an inflammatory reaction occurs, various inflammatory factors (pro-inflamatory mediatos) are produced and produced by NO and COX-2 by iNOS, an inducible nitric oxide synthase. It is divided into prostagrandin E2 (PGE2) and others. In the case of iNOS, it is expressed when there is stimulation of various inflammatory cytokines such as LPS.

On the other hand, the tonicity of vascular smooth muscle is neurotransmitter (norepinephrine, adenosine triphophate, NO), endothelium-derived relaxing factor (EDRF, endothelium-derived hyperpolarrizing factor: EDHF, endothelins), metabolic effects (PO ₂ , PCO ₂ . pH), humoral substances (norepinephrine, epinephrine, vasopressin), and muscular effects (determined by several important factors such as blood vessel elasticity, internal pressure)). In particular, vascular endothelial cells are known to play an important role in blood pressure control by releasing endothelium-derived relaxing factor and endothelium-derived constricting factor. The main body of this EDRF (endothelium-derived relaxing factor) is nitric oxide (NO). NO can be produced from smooth muscle, blood cells, and neurons of the central nervous system in addition to vascular endothelial cells, and at least three kinds of nitric oxide synthase isotopes It is synthesized from the amino acid L-arginine by action. In addition, vascular relaxation factors such as NO are synthesized and secreted in endothelial cells even under normal conditions, and are used in agonists such as acetylcholine, histamine, P substance P, and isoproterenol. Synthesis and secretion are increased. The action of organic nitrate, which has been used in the treatment of hypertension, is achieved by increasing cGMP by activating soluble guanylate cyclase. Also, since this drug generates NO by non-enzymatic action, the blood vessels of EDRF The relaxation action was also found to be due to cGMP by the activity of soluble guanylate cyclase. Recently, several herbal or 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 of Jogu-deung (釣鉤藤) decoction (extract) increases blood vessel relaxation effects in a nitric oxide-dependent manner, and Goto et al. reported that in a series of research results, 釣鉤藤 decoction solution lowered blood pressure in spontaneous hypertensive white papers. It was reported that hot water extract relaxes blood vessels in the thoracic aorta in a nitric oxide-dependent manner. In addition, lithospermic acid B of Dansam and protein components of Cordyceps sinensis are known to increase vascular relaxation in a nitric oxide-dependent manner.

The loofah is a fruit of the Cucurbitaceae family Luffa cylindrica Roemer. It is an edible food ingredient notified by the Ministry of Food and Drug Safety and is widely used as a beverage and other food ingredients. In addition, in oriental medicine, it is commonly used as a medicinal name, apple-lac (絲瓜絡), and it has a sweet taste, mild nature, and non-toxicity, so it has been used for medicinal purposes by drying the reticulum from which the skin was removed. In oriental medicine, scrubbers relieve pain in the chest and sides, abdominal pain, and back pain, clear blood stagnation, reduce liver fever, reduce heat in the lungs, stop phlegm and cough, and are inflammatory in the lungs, stomach, and liver. It has been mainly used for diseases. As a pharmacological ingredient, the mesh tissue contains saponin, lutein, and citrulline, and the seeds contain 20 to 40% of fatty oil, and ingredients such as linoleic acid, palmitic acid, stearic acid, and oleic acid are included. Research on loofah is mainly focused on whitening and reducing inflammation as a cosmetic ingredient, and it is only used for coughing, phlegm, and asthma in oriental medicine, so the efficacy of loofah on the cardiovascular system has not been properly reported.

Republic of Korea Patent Publication No. 10-2009-0064823

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

In addition, the present invention provides a health functional food for preventing or improving inflammation containing a scrubber extract as an active ingredient.

In addition, the present invention provides a health functional food for vascular relaxation containing a scrubber extract as an active ingredient.

The present invention provides a vasodilator containing a scrubber extract as an active ingredient.

The scrubber extract may be extracted with a solvent selected from the group consisting of water, ethanol, methanol, flophyllene glycol, ether, chlorform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, isopropanol, and mixtures thereof. I can. Most preferably, it may be an extract extracted using water as a solvent.

The scrubber extract may have an endothelial nitric oxide synthase (eNOS) enhancing activity.

The scrubber extract may increase the production of nitric oxide (NO) in vascular endothelial cells.

The scrubber extract may exhibit vascular relaxation effects by regulating the NO-cGMP pathway (nitric oxide-cyclic guanosine monophosphate) in vascular endothelial cells.

The vasodilator may be used for treatment, prevention or improvement of diseases caused by vasoconstriction.

The disease 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 health functional food for relaxation of blood vessels including a scrubber extract.

In addition, the present invention provides an anti-inflammatory agent containing the scrubber extract as an active ingredient.

The scrubber extract is extracted with a solvent selected from the group consisting of water, ethanol, methanol, flophyllene glycol, ether, chlorform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, isopropanol, and mixtures thereof. It may be an extract, more preferably an extract extracted with ethanol or methanol.

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

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

In addition, the present invention can provide a health functional food for preventing or improving inflammation comprising a scrubber extract.

In addition, the present invention can provide a food composition for relaxation of blood vessels or a food composition for preventing or improving inflammation containing a scrubber extract as an active ingredient.

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

The food composition may contain food additives acceptable food.

Specifically, the food composition includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline as a carrier, excipient, and diluent. Cellulose, polyvinyl pyrrolidone, water, methylhydroxy, hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The food composition extract may be used in various ways such as food and beverage for relaxation of blood vessels. Specifically, there are gum, tea, vitamin complexes or health supplements, and may be used in the form of powders, granules, tablets, capsules, or beverages.

In the case of manufacturing a health drink using the food composition, there is no particular limitation on the liquid component other than containing the scrubber extract, and various flavoring agents or natural carbohydrates, etc. may be included as an additional component, as in a conventional beverage.

In addition to the above, the food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, ilginic acid and salts thereof, organic acids, protection It may contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, carbonates used in alcohol or carbonated beverages, and the like.

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

In addition, the present invention can provide a quasi-drug composition or pharmaceutical composition for vascular relaxation using a scrubber extract as an active ingredient. In addition, it is possible to provide a quasi-drug composition for preventing or improving inflammation using a scrubber extract as an active ingredient, and a pharmaceutical composition for preventing or treating inflammation.

A pharmaceutically acceptable additive may be added to the composition.

Each of the above compositions may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

The composition includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, poly Vinyl pyrrolidone, water, methylhydr, oxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, and the like may be added.

In the case of formulation, it can be prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants or surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and such solid preparations include at least one or more excipients, such as all, calcium carbonate, sucrose, and lactose. It can be prepared by mixing (lactose) or gelatin.

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

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions lyophilized formulations, and suppositories.

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

As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper or glycerogelatin may be used.

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

In addition, the composition of the present invention may be administered to mammals such as mice, mice, livestock, or humans by various routes, and by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection. Can be administered.

In the above 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 scrubber extract, but is not limited thereto.

According to the present invention, it is possible to provide a vasodilator or anti-inflammatory agent containing the scrubber extract as an active ingredient. The vasodilator according to the present invention can realize vascular relaxation effect by increasing the production of nitric oxide in vascular endothelial cells. In addition, the vasodilator according to the present invention can be used for the prevention, treatment or amelioration of blood vessel constriction such as hypertension, arteriosclerosis, blood circulation disorders, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure. In addition, the scrubber extract according to the present invention can implement anti-inflammatory effects by increasing the production of nitric oxide in immune cells.

FIG. 1 is a graph showing the results of analyzing the vascular smooth muscle relaxation effect of hot water scrubber extract in a thoracic aortic section of a rat contracted by pretreatment with phenylephrine (1 x 10 ^-6 M) (e(-): blood vessels Endothelial cells are removed, e(+): vascular endothelial cells are not removed), WLC: water extract of Luffa cylindrica. Each value: mean ± SE of 4 experiments, *P<0.05, **P<0.01, vs. control.
FIG. 2 is a graph showing the results of analyzing changes in the blood vessel relaxation effect of hot water extracts of loofah by L-NAME and MB pretreatment. Each value: mean ± SE of 4 experiments, *P<0.05, **P<0.01, vs. control.
3 is a graph showing the results of analyzing changes in the blood vessel relaxation effect of hot water extracts of scrubbers by pretreatment with verapamil. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control.
4A is a graph showing the results of analyzing changes in the blood vessel relaxation effect of hot water extracts of scrubbers by pretreatment with glibenclamide. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control.
Figure 4b is a graph showing the results of analyzing the change in the blood vessel relaxation effect of the hot water scrubber extract by TEA pretreatment. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control.
Figure 4c is a graph showing the results of analyzing changes in the blood vessel relaxation effect of the hot water scrubber extract by 4AP (4-Aminopyridine) pretreatment. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control
FIG. 5A is a graph showing the results of analyzing changes in the blood vessel relaxation effect of hot water extract of loofah by pretreatment of propranolol. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control.
5B is a graph showing the results of analyzing changes in the blood vessel relaxation effect of hot water extracts of scrubbers by atropine pretreatment. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control.
6 is a graph showing the results of analyzing changes in the blood vessel relaxation effect of hot water extracts of scrubbers by pretreatment of indomethacin. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control.
7 is a graph showing the results of analyzing the change in the blood vessel relaxation effect of the hot water extract of the scrubber by pretreatment with ODQ. Each value: mean ± SEM of 4 experiments, *P<0.05, **P<0.01, vs. control.
8 is a graph showing the results of analyzing the effect of the hot water extract of the scrubber 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, vs. control.
9 and 10 are graphs showing the results of checking the cytotoxicity of each extraction solvent of the scrubber extract.
11 is a graph showing the results of analyzing the effect of the scrubber extract on NO generation by extraction solvent.
12 is a diagram showing the results of confirming the expression levels of iNOS and COX-2 by extraction solvent of the scrubber extract. (A) methanol extract, (B) ethanol extract, (c) hot water extract.

Hereinafter, the present invention will be described in more detail through examples. Objects, features, and advantages of the present invention will be easily 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 introduced herein are provided in order to sufficiently convey the spirit of the present invention to those of ordinary skill in the art. Therefore, the present invention should not be limited by the following examples.

Example 1. Preparation of scrubber extract

The scrubber used in the experiment was purchased domestically (Gyeongsan, Gyeongbuk) and used for the experiment. Wash the scrubber well to remove impurities, dry, and dip 100 g of dry weight in 1 liter of first distilled water (or methanol or ethanol) for 30 hours, and then extract hot water exports, methanol extracts, and ethanol extracts with an electric kettle for 2 hours without pressure. Respectively obtained. After the scrubber is extracted, it is filtered with Advantec Filter paper NO.2 (Toyo Roshi Kaisha Ltd, Japan), and the filtrate is concentrated with a rotary evaporator (Buchl, Swiss), and the concentrate is converted into an ultra-low temperature freezer (Operon, Korea). After freezing in, a freeze-dried product was obtained using a Freeze Dryer (Operon, Korea). (61.4 g, yield 6.14%). This was dissolved in an appropriate amount of tertiary distilled water in each experiment and used.

Experimental Example 1. Analysis of Vascular Relaxation Effect of Hot Water Extract of Scrubber

Preparation of physiological nutrient solution

Krebs solution (Krebs-Henseleit Solution) was used to maintain stable physiological activity of thoracic aortic vessels, and the composition is 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 , 10 mM glucose, pH 7.4)

Fabrication of vascular tissue section

The experimental animals used healthy Sprague-Dawley male white papers of about 250 to 330 g. Before the start of the experiment, anesthesia was performed under carbon dioxide supersaturation, and the abdominal cavity was immediately opened and the thoracic aorta was separated. The separated thoracic aorta was put in Kreb's solution at 4℃ supplied with oxygen, and the connective tissue and fat were removed, and then a ring-shaped section of about 3-4 mm in length was made.

Measurement of blood vessel tension

The thoracic aortic section of the white paper was fixed at the bottom of an organ bath containing Krebs solution at 37℃ saturated with 95% O ₂ and 5% CO ₂ gas, and the other end was a force-displacement transducer (Grass FT). 03, GRASS Instrument, MA, USA), and the isometric tension of blood vessels was measured using a physiological recorder (Grass Model 7E, Grass Instrument, MA, USA) connected to a force-displacement transducer. Thereafter, for stabilization of blood vessels, fresh Kreb's solution was supplied at 10 minute intervals for 60 minutes, and a basal tone of 1 g was applied to the vascular section. When the basal tone of 1g is kept constant, it is contracted with 1x10 ^-6 M of phenylephrine, and after 5 minutes, it is relaxed with 1 x 10 ^-6 M of ACh (acetylcholine). If the relaxation rate is more than 80%, the vascular endothelium It was determined that the cells were not damaged and the drug was tested. When using a pre-treatment drug to confirm the mechanism of the hot water scrubber extract, the drug is pretreated in an organ bath for 10 minutes and contracted with phenylephrine, and then the vascular relaxation reaction caused by the hot water scrubber extract is observed in a concentration-dependent manner. Measured. When conducting a vascular endothelial cell-independent experiment, the vascular endothelial cells are scraped off with a thin stainless steel and removed, contracted with phenylephrine, and then relaxed with ACh, where the relaxation rate is less than 5%. Proceeded. The vascular relaxation rate of the drug was calculated as a percentage (%) based on the contraction of blood vessels by phenylephrine (1 uM).

reagent

ACh, phenylephrine, L-NAME (NG-nitroargininemethyl ester), indomethacin, glibenclamide, TEA (tetraethylammonium), verapamil, 4AP, methylene blue, atropine, propranolol, etc. used in this experiment are Sigma Chemical Co. (St. Louis, MO, USA).

Statistical processing

The experimental results were tested for statistical significance using Sigma Plot, and statistical significance was determined when p was 0.05 or less through the Students t-test or one-way ANOVA test.

1-1. Concentration-dependent Vascular Smooth Muscle Relaxation Effect of Loofah Hot Water Extract

In the thoracic aortic section of rats contracted by pretreatment with phenylephrine (1 x 10 ^-6 M), the hot water extract of a loofah relaxed the blood vessels in a concentration-dependent manner (Fig. 1). In addition, the vascular relaxation effect of the hot water scrubber extract did not appear when the vascular endothelial cells were completely removed (FIG. 1).

1-2. Vascular relaxation effect upon inhibition of nitric oxide synthase (NOS)

In order to confirm whether the thoracic aortic relaxation effect of the hot water scrubber extract is related to the nitric oxide system, L-NAME (1 x 10 ^-5 M) and MB (2.5x 10 ^-5 M), which are non-selective nitric oxide synthesis inhibitors, were used to organ As a result of pretreatment in a bath for 10 minutes and measurement of vascular relaxation effect, the vascular relaxation effect of the scrubber was statistically significantly suppressed by the pretreatment of L-NAME and MB (FIG. 2).

1-3. Ca ²⁺ Vascular relaxation effect when the passage is blocked

To measure the effect of Ca ²⁺ channel blocking on the vascular relaxation effect by the scourer hot water extract, Ca ²⁺ channel blocker verapamil (1 x 10 ^-5 M) was pretreated and then the degree of relaxation by the scourer hot water extract was measured. As a result of the measurement, the vascular relaxation effect of the scrubber was significantly suppressed by the pretreatment of verapamil (FIG. 3).

1-4. K ⁺ Vascular relaxation effect when the passage is blocked

To measure whether K ⁺ channel blockade affects the vasodilating effect of the scourer hot water extract, the non-selective K ⁺ channel inhibitor, TEA (1 x 10 ^-5 M) and the ATP-sensitive K ⁺ channel inhibitor, glibenclamide ( After pretreatment with glibenclamide, 1 x 10 ^-5 M) and Ca ^{2 +} -associated K ⁺ pathway inhibitor 4AP (1 x 10 ^-5 M), we measured the vasodilating effect of the hot-water scrubber extract, in the case of TEA and 4AP. There was no significant difference from the group without pretreatment, and when pretreatment with glibenclamide, the vascular relaxation effect of the scrubber was significantly suppressed (FIG. 4).

1-5. Vascular relaxation effect upon blocking autonomic nervous system receptors

To investigate the relationship between the vascular relaxation effect of the hot water extract of a loofah with the nervous system, propranolol, a non-selective β-adrenergic antagonist, and atropine, an anti-muscarinic drug, were pretreated with 1 x 10 ^-6 M, respectively. After that, as a result of measuring the vascular relaxation effect of the hot water extract of the scrubber, the vascular relaxation effect of the scrubber was significantly suppressed by pretreatment with propranolol and atropine (FIG. 5).

1-6. Vascular relaxation effect upon blocking COX(Cyclooxygenase)

Vascular relaxation effect of hot-water scrubber extract after pretreatment of indomethacin (1 x 10 ^-5 M), a non-selective cyclooxygenase (COX) inhibitor, to observe whether the vasodilator effect by hot water scrubber extract is related to prostacyclin. As a result of measuring, it was confirmed that the vascular relaxation effect of the scrubber was significantly suppressed by pretreatment of indomethacin (FIG. 6).

1-7. Inhibitory effect of vasoconstriction by ODQ pretreatment

After pretreatment with ODQ, a soluble guanylate cyclase inhibitor, vascular relaxation effect was measured, it was confirmed that the vascular relaxation effect by hot water extract on a scrubber was suppressed (Fig.7).

1-7. Vasoconstriction inhibitory effect

To find out whether the hot-water scrubber extract affects blood vessel constriction by phenylephrine, the hot-water scrubber extract was treated with 1 x 10 ^-4 g/ml and 1 x 10 ^-3 g/ml, respectively, and then phenylephrine. (phenylephrine, 1 x 10 ^-8 M-1 x 10 ^-5 M) was administered. As a result, blood vessel constriction due to phenylephrine was significantly reduced compared to the control (vehicle) (FIG. 8).

Various types of vasoactive factors secreted from vascular endothelial cells can regulate vascular smooth muscle relaxation.These vasoactive factors include nitric oxide (NO), prostacyclin, and endothelial cell-derived hyperpolarization factors ( endothelium-dependent hyperpolarizing factor (EDHF) and the like are known. In the above experimental results, it was confirmed that the hot water extract of the scrubber was found to relax the blood vessel constriction induced by phenylephrine in the thoracic aorta extracted from white paper in a concentration-dependent manner. However, after removing vascular endothelial cells, the vascular relaxation effect of the scrubber hot water extract was completely blocked, which means that the vascular relaxation effect of the scrubber is closely related to these vascular endothelial cells-derived vascular activating factors. In addition, according to the experimental results, the vasodilating effect of the hot water extract of the scrubber was statistically significantly suppressed by pretreatment of L-NAME and MB, which are nitric oxide synthesis inhibitors. This result may be evidence indicating that NO among these endothelial cell-derived vascular activating factors is crucially related to the vasodilating effect of the hot water scrubber extract.

In addition, various drugs such as autonomic nervous system, calcium ion channel, potassium ion channel, COX (cyclooxygenase), and nitric oxide production blockers were tested to find out the relationship between endothelial cell-derived vascular relaxation factors and the vascular relaxation effect of the scrubber hot water extract. Was used in To determine whether nitric oxide (NO), the body of endothelial cell-derived vascular relaxation factor, is involved in the vascular relaxation effect of the loofah hot water extract, first pretreatment with L-NAME, a nitric oxide synthesis inhibitor, was measured. Completely blocked. Nitric oxide (NO), a small molecule produced in vivo, was found to have a large blood pressure lowering effect as an endothelial cell-dependent vascular relaxation factor. Nitric oxide is synthesized from L-arginine by nitric oxide synthase (NOS), and there are three isotopes of nitric oxide synthase in blood vessels. The first type is brain-type nitric oxide synthase (bNOS, nNOS, NOS Ⅰ), which was first discovered in brain tissue and plays a role in synthesizing nitric oxide as a neurotransmitter. The second isotope is an inducible nitric oxide synthase (iNOS, NOS Ⅱ), which synthesizes nitric oxide, which mainly acts on the immune system. The third isotope is a vascular endothelial nitric oxide synthase (ecNOS, eNOS, NOS III), which is mainly distributed in vascular endothelial cells and acts to relax blood vessels, and is activated by acetylcholine to relax blood vessels. These nitric oxides activate cytoplasmic guanylate cyclase to increase the production of cGMP and relax vascular smooth muscle through a signaling system. In this experiment, the relaxation effect of the scrubber was suppressed by pretreatment of L-NAME, so it is believed that the vascular relaxation effect by the hot water extract of the scrubber occurs via the nitric oxide system. ACh, a generally well-known vascular relaxation agonist, causes NO-mediated endothelial-dependent relaxation effects, and it is known that the release of NO by ACh is via the endothelial M3 receptor. The increase of Ca ^{2+ in} endothelial cells by ACh is the result of Ca ²⁺ outflow from intracellular storage and Ca ²⁺ influx from extracellular by IP3. Nitric oxide synthase (NOS) is activated by increased Ca ²⁺ , and nitric oxide (NO) is released using L-arginine as a substrate. Nitric oxide produced at this time is diffused to the vascular smooth muscle, and again activates cytoplasmic guanylate cyclase. Cytoplasmic guanylate cyclase converts GTP (guanosinetriphosphate) to cGMP, and cGMP activates cGMP-dependent protein-activating enzymes again. This enzyme relaxes the vascular smooth muscle by inhibiting the influx of Ca ²⁺ into the vascular smooth muscle and reducing the sensitivity to the contractile factors of intracellular Ca ²⁺ . If the NO/cGMP system is involved in the vascular relaxation effect by the hot water scrubber extract, soluble guanylate cyclase activated by nitric oxide is also thought to be involved in the relaxation effect of blood vessels by the hot water scrubber extract. After pretreatment, the vascular relaxation effect was measured, and it was confirmed that the vascular relaxation effect by the hot water extract on the scrubber was suppressed. These results may mean that the vascular relaxation effect by the hot water scrubber extract occurs via the NO/cGMP system. From these results, it is believed that the hot-water scrubber extract relaxes blood vessels by increasing the synthesis of nitric oxide in vascular endothelial cells and activating soluble guanylate cyclase to increase cGMP production.

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 a vasodilating action. In order to determine whether the vascular relaxation effect of hot water extract of loofah is related to the production of prostacyclin, indomethacin, a cyclooxygenase inhibitor, was pretreated and the relaxation effect of loofah was measured. It was confirmed that the vasodilating effect was suppressed by pretreatment of indomethacin, and it was determined that the prostacyclin system was related to the vasodilating action of the hot water extract of the loofah.

Since the vascular smooth muscle tone is due to the difference in the vascular smooth muscle cell membrane potential, the change of the ion channel between the cell membranes allows a direct explanation of the relaxation action. 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 , Cell membrane potential of vascular smooth muscle is mainly regulated by the outflow and inflow of Ca ²⁺ and K ⁺ ions. Vascular tonicity is regulated by the membrane potential, and when the membrane potential increases, Ca ²⁺ increases through VOC (Voltage Operated Channel), which is a voltage-dependent Ca ²⁺ channel. This increase in Ca ²⁺ activates the contractile protein, resulting in contraction, which shows electro-mechanical coupling. On the other hand, the K ⁺ channel induces hyperpolarization of the cell membrane upon activation and relaxes the blood vessel by inhibiting the inflow of Ca ²⁺ through VOC. This is also known as an important endogenous vascular relaxation mechanism in regulating vascular tone. The K ⁺ pathway in vascular smooth muscle includes the Ca ²⁺ -activated K pathway (KCa), ATP-sensitive K (KATP), and voltage-gated K (Kv) pathways. The endothelium-dependent hyperpolarizing factor , EDHF) activates the K ⁺ pathway and causes membrane hyperpolarization. The outflow of K ^{+ into} the cell by the activation of the K ⁺ channel causes hyperpolarization of the cell membrane, leading to inactivation of the voltage-sensitive Ca ²⁺ channel, leading to a decrease in the intracellular Ca ²⁺ concentration, which eventually leads to vascular relaxation. do. Inhibitors for the K+ pathway include 4aminopyridine that blocks Kv, TEA that blocks KCa, and glibenclamide, a sulfonylurea-based drug, and glibenclamide It blocks only KATP without affecting the KCa pathway in smooth muscle. To find out whether the vascular relaxation effect of the hot water scrubber extract occurs via Kv, KCa or KATP, the results of measuring the vascular relaxation effect after pretreatment with Kv blocker 4AP, KCa blocker TEA and KATP blocker glibenclamide were measured. , In the case of 4AP, there was no significant difference from the non-pretreatment group, and the pretreatment of glibenclamide had a statistically significant effect on the vasodilation effect of the scrubber. These results suggest that the vascular relaxation effect of the scrubber is also partially related to the K ⁺ pathway.

The concentration of Ca ²⁺ in blood vessels is the most important factor in the contraction and relaxation of blood vessels. When the intracellular Ca ²⁺ concentration increases, it binds to calmodulin and promotes phosphorylation of the myosine light chain (MLC), causing smooth muscle contraction. In order to find out whether the vascular relaxation effect of the scrubber hot water is related to the Ca ²⁺ pathway, pretreatment with verapamil, an L-type Ca ²⁺ blocker, and then measuring the vascular relaxation effect, pretreatment with verapamil It was confirmed that the vascular relaxation effect of the scrubber hot water extract was significantly reduced, and thus the vasculature relaxation effect of the scrubber was judged to be related to the Ca ²⁺ pathway.

Vascular smooth muscle tone is also regulated by the regulation of the autonomic nervous system. In the above experiment, in order to find out whether the hot water extract of loofah relaxes blood vessels via receptors in the autonomic nervous system, an anti-cholinergic receptor that selectively blocks atropine and β receptors that selectively blocks muscarinic receptors. Treatment with the adrenaline drugs propranolol and atropine was found to significantly inhibit the vasodilating effect of the hot water extract of the loofah.The vascular relaxation effect of the loofah is related to the muscarinic receptor among cholinergic receptors. It is expected to be.

In addition, in this experiment, it was found that after the pretreatment of the hot water extract of the loofah, blood vessel constriction by phenylephrine was significantly suppressed. Since contraction by phenylephrine, a selective α1 agonist in the thoracic aorta, is maintained by increasing the intracellular Ca ²⁺ concentration through the Ca ²⁺ pathway, the scourer hot water extract partially directly antagonizes the adrenergic α1-receptor. It is thought to have an effect or inhibit the influx of Ca ²⁺ into cells through the α1 receptor.

The NO/cGMP system not only relaxes vascular smooth muscle, but also inhibits proliferation of vascular smooth muscle cells, platelets, and leukocyte adhesion, lowers osmoticity of endothelial cells, and inhibits the synthesis of extracellular interstitial proteins. The decrease in NO production in vascular endothelial cells is due to dysfunction or damage of vascular endothelial cells, which is a major pathological factor of arteriosclerosis and hypertension. In addition, chronic inhibition of NO synthase in animal models causes vasculitis such as hypertension. Therefore, it can be seen that the increase of the vasodilator activity through the recovery of the NO/cGMP system has a very important meaning in the treatment of cardiovascular diseases.

Taken together, the experimental results show that the hot water scrubber extract exhibits a vascular endothelial cell-dependent relaxation effect via NO/cGMP system, and these results suggest that it can be a useful therapeutic agent for cardiovascular diseases.

In conclusion, the scourer hot water extract relaxed vascular smooth muscle in an endothelial cell-dependent manner, and this effect appeared in a concentration-dependent manner, and blocking the NO/cGMP signaling system completely inhibited it. Furthermore, when the vascular tissue was reacted with the hot water extract of the scrubber, the production of cGMP increased, and this increase did not appear when the vascular endothelial cells were removed or when nitric oxide synthase or cytoplasmic guanylate cyclase was blocked. In addition, the vascular relaxation effect of the hot water scrubber extract was significantly influenced by blocking prostacyclin production, blocking K ⁺ pathway, blocking Ca ²⁺ pathway, blocking muscarinic receptors and β-adrenergic receptor blockers. From these results, it is thought that the vascular smooth muscle relaxation mechanism of the scourer hot water extract activates the NO/cGMP pathway in endothelial cells, and appears through the Ca ²⁺ pathway and K ⁺ pathway associated therewith.

Experimental Example 2. Confirmation of cytotoxicity by extraction solvent of scrubber extract (1)

In order to confirm the cytotoxicity of the scrubber (apple) extract to mouse macrophages (Raw264.7 cells), MTT assay was performed for each extraction solvent.

As an experimental method, Raw264.7 cells were dispensed into a 48 well plate at 2×10 ⁵ cells/well and incubated in a 37°C, 5% CO ₂ incubator for 24 hours. After treating the cultured Raw264.7 cells with a scrubber extract extracted with methanol, ethanol and hot water at a concentration of 10, 30, 50, 100, and 300 ug/ml, respectively, the MTT reagent was added to a final concentration of 0.5 mg/ml. Put in a well and incubated for 4 hours in a 37°C, 5% CO ₂ incubator. After incubation, the culture medium was discarded, treated with DMSO to dissolve formazan, and 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 cells, except that the cell viability was significantly reduced at a concentration of 300 ug/ml of the methanol extract of the scrubber (Fig. 9).

Experimental Example 3: Confirmation of Cytotoxicity by Extraction Solvent of Scrubber Extract (2)

In order to confirm the cytotoxicity of LPS, an inflammation-inducing cytokine on mouse macrophages (Raw264.7 cells) of the loofah (apple) extract, MTT assay was performed for each extraction solvent.

As an experimental method, Raw264.7 cells were dispensed into a 48 well plate at 2×10 ⁵ cells/well and incubated in a 37°C, 5% CO ₂ incubator for 24 hours. Methanol, ethanol, and hot water-extracted scrubber extracts were treated in the cultured Raw264.7 cells at concentrations of 10, 30, 50, and 100 μg/ml, respectively, and 1 hour later, 1 μg/ml of LPS was treated and then cultured. After incubation, the MTT reagent was added to each well so that the final concentration was 0.5 mg/ml, incubated in a 37°C, 5% CO ₂ incubator for 4 hours, discarded, and treated with DMSO to dissolve formazan, followed by ELISA The absorbance was measured at 570 nm with a reader. As a result, in the case of methanol, ethanol and hot water extracts of the scrubber, the cytotoxicity by LPS was suppressed in a concentration-dependent manner (10, 30 ug/ml). As a result, it was shown that the scrubber extract significantly inhibited cytotoxicity in a concentration-dependent manner in methanol, ethanol and hot water extracts.

Experimental Example 4: Confirmation of NO generation by extraction solvent of scrubber extract

Inflammatory response is an in vivo defense mechanism against stimuli, and can cause various inflammatory diseases when overreacted. LPS is an inflammatory substance, and various inflammatory mediators such as NO, PGs, and inflammation-inducing cytokines IL-6 and IL-β are induced in Raw264.7 cells.

As an experimental method, Raw264.7 cells were dispensed into a 48 well plate at 2×10 ⁵ cells/well, incubated in a 37°C, 5% CO ₂ incubator for 24 hours, and then methanol, ethanol and hot water extracted into Raw264.7 cells The extracts were treated at concentrations of 10, 30, 50, and 100 μg/ml, respectively, and 1 hour later, 1 μg/ml of LPS was treated and cultured. After adding the same amount of Griess reagent as 50 μl of the culture solution and reacting at room temperature for 10 minutes, the absorbance was measured at 540 nm with an ELISA reader. As a result, it was confirmed that, in the case of methanol and ethanol extracts, the production of NO by LPS was significantly reduced in a concentration-dependent manner (10-300 ug/ml) (FIG. 11).

Experimental Example 5: Confirmation of iNOS and COX-2 expression levels by extraction solvent of scrubber extract

In order to check the expression level of iNOS and COX-2 protein for inhibition of NO production, methanol, ethanol and hot water extracts of apple rock extracts extracted with methanol, ethanol and hot water were added to Raw264.7 cells cultured in 6 well plates at concentrations of 10, 30, and 100 μg/ml, respectively. After the treatment, 1 µg/ml of LPS was treated and cultured after 1 hour, and the quantified sample was checked for expression using the Immunoblot assay. As a result, the iNOS expression level increased by LPS was significantly decreased in a concentration-dependent manner (10-300 ug/ml), but in the case of COX-2, it was confirmed that there was no difference in the expression level change by extraction solvent (Fig. 12). )

When the extraction method for each solvent was different, the methanol extract showed cellularity at a concentration of 300 ug/ml, and the ethanol and hot water extract did not show cytotoxicity. When inducing NO production by LPS treatment, methanol, ethanol and hot water extracts significantly inhibited cytotoxicity at low concentrations, and NO production in methanol and ethanol extracts significantly decreased in a concentration-dependent manner. In addition, when checking the expression levels of iNOS and COX-2, which are representative inflammatory factors, in the case of iNOS, it was confirmed that methanol and ethanol extracts decreased the expression level of the iNOS protein increased by LPS in a concentration-dependent manner. In the case, it was confirmed that there was no difference regardless of the extraction solvent or concentration. Based on the above results, the scrubber extract showed that methanol and ethanol extracts inhibited LPS-induced NO production, and later confirmed before inflammatory reactions and various inflammatory mediators such as PGs, inflammation-inducing cytokines IL-6 and IL-β. The substance is induced, but it seems that confirmation is necessary.

Claims (14)

As a vasodilator containing hot water extract of loofah fruit as an active ingredient,
The vasodilator is a vasodilator for preventing or treating diseases caused by vasoconstriction,
The disease caused by constriction of blood vessels is a vasodilator, characterized in that it is selected from the group consisting of hypertension, arteriosclerosis, blood circulation disorder, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure.
delete delete The method according to claim 1,
The loofah fruit hot water extract is a vasodilator, characterized in that it has an endothelial nitric oxide synthase (eNOS) enhancing activity.
The method according to claim 1,
The loofah fruit hot water extract increases the production of nitric oxide in vascular endothelial cells.
delete delete As a health functional food containing hot water extract of loofah fruit,
The health functional food is a health functional food for preventing or improving diseases caused by vasoconstriction,
The disease caused by constriction of blood vessels is a health functional food, characterized in that it is selected from the group consisting of hypertension, arteriosclerosis, blood circulation disorder, headache, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure.
delete delete delete delete delete delete

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