KR20090030831A - Antihypertensive effect of extract from styela clava, in vitro and in vivo - Google Patents

Abstract

An antihypertensive composition is provided to secure an effect of hypertension suppression by blocking a process of an Angiotensin I-Converting Enzyme becoming an Angiotensin II. An antihypertensive composition comprises styela clava extract. The styela clava extract is extracted by water or an organic solvent such as a 70% ethanol. The styela clava extract is extracted at a temperature of 20°C or 70°C. A method for preparing the styela clava extract is composed of the following steps of: washing the styela clava; freeze-drying the styela clava; and pulverizing the styela clava to obtain powder type extract.

Description

Antihypertensive composition using midder extract {Antihypertensive effect of extract from Styela clava, in vitro and in vivo}

Figure 1 is a flow chart of the production separation process of crude extract liquid of antihypertensive substances

Figure 2 shows the angiotensin I-converting enzyme inhibitory activity results from the water-soluble extract extracted at 20 ℃

Figure 3 shows the results of angiotensin I-converting enzyme inhibitory activity from the water-soluble extract extracted at 70 ℃

4 shows the results of angiotensin I-converting enzyme inhibitory activity from 70% ethanol extract extracted at 20 ° C.

5 shows the results of angiotensin I-converting enzyme inhibitory activity from 70% ethanol extract extracted at 70 ℃

Figure 6 Figure 4 is angiotensin I-converting enzyme inhibitory results from 100% ethanol extract extracted at 20 ℃ midderok

FIG. 7 shows the results of angiotensin I-converting enzyme inhibitory activity from 100% ethanol extracts extracted from medders at 70 ° C.

Figure 8 shows the results of blood pressure drop after oral administration of hypertension-induced rats from the aqueous extracts

The present invention relates to anti-hypertensive effect with an aqueous extract of Styela clava, which when More specifically, the one of the marine life in Styela clava (Styela clava) in blood pressure from the water-soluble and organic solvent extract is extracted at 20 ℃ and 70 ℃ for chemical experiments showing a specific antihypertensive action angiotensin I- converting in (in vitro) enzyme (angiotensin I-converting enzyme) an aqueous extract of animal experiment shows a mechanism of inhibition (in vivo) hypertensive rats (Spontaneously hypertensive rat in After oral administration to), antihypertensive composition using Styela clava extract having antihypertensive effect using water-soluble and organic solvent extracts will be.

Hypertension is a prevalent disease worldwide, affecting 15-20% of adults and causing serious chronic diseases such as atherosclerosis, stroke, myocardial infarction and diabetes. It is a disease that causes normal blood pressure to slowly decay, which causes circulatory disease and, when combined with the aforementioned disease, develops into a chronic degenerative disease with a high mortality rate (Frohlich ED. 1982. Hemodynamic factors). in the pathogenesis and maintenance of hypertension Fed A SOC Exp Biol 41:. 2400-2408).

 Angiotensin I converting enzyme (ACE) belongs to the zinc metal protease group and surrounds the pulmonary vascular endothelium. Angiotensin I is a peptide with 10 peptides that is degraded by renin, which is cleaved by angiotensin I converting enzyme (ACE) to dipeptide (His-Leu) at the C end. Angiotensin II, a potent vasoconstrictor with two peptides, is produced (Cuttiss C, Chon JN, Vrobel T, Francious JA, 1978. Role of the renin-angiotensin system in the systemic vasoconstriction of chronic congestive heart failure. 58: 763-770). The angiotensin II thus produced constricts arterial blood vessels and raises blood pressure, thereby promoting the release of aldosterone from adrenaline to increase resorption of sodium and water in the kidney. In addition, ACE decomposes and inactivates bradykinin, which acts to expand blood vessels and increase intestinal motility, resulting in elevated blood pressure (Cushman DW, Cheung HS. 1971. Spectorophotometric assay and properties of the angiotensin) I-converting enzyme of rabbit lung.Biochem pharmacol 20: 1637-1648). As a result of the study of ACE inhibitors to increase blood pressure, angiotensin I converting enzyme (ACE) inhibitors Alacepril, Benazepril, Captopril, and Silas for the treatment of hypertension or heart disease. Cilazapril, Enalapril, Fosinopril, Lisinopril, Perindopril, Moexipril, Ramipril, Tandora Many studies have been done, including Tandolapril and Zofenopril. However, these chemotherapy drugs have a lot of interest in finding ACE inhibitors derived from natural products in foods because of side effects such as flatulence, taste disorders and skin redness.

 Among the food ingredients, ACE inhibitory effect is mainly researched mainly on the peptides (peptides) obtained from the enzyme hydrolyzate of various food proteins. Based on this, the peptides are chemically synthesized to affect the inhibitory effect. Gum is being examined for C and N terminal amino acids. In Japan, casein DP, a soft drink containing casein-derived dodecapeptide, is commercially available as one of the health foods.

Marine organisms have a search function for a variety of new bioactive substances due to the unique metabolic processes and unique environment that terrestrial organisms lack. In addition, many studies on terrestrial organisms have been conducted, but marine organisms are still highly expected because of limited research (Park JC. 1996. Screening of marine natural products on inhibitory effect of the formation) of lipid peroxidation.J Kor Soc Food Sci Nutr 35 (3), 278-283 ).

 As described above, most of the chemical synthetic hypertension treatments have a problem of side effects such as flatulence, taste disorders and skin redness.

Therefore, an object of the present invention is to devise to improve the various problems as described above, in order to obtain an antihypertensive effect from the marine organisms, such as to extract the water at 20 ℃ and 70 ℃ to prepare a water-soluble and organic solvent extract Since the water-soluble and organic solvent extracts have antihypertensive activity as such, the water-soluble and organic solvent extracts of the anti-hypertensive composition using the water-soluble and organic solvent extracts of the natural antihypertensive agent Midderok and containing the same Its purpose is to provide a mesotheli extract.

The present invention relates to an antihypertensive composition using an extract of Styela clava having an antihypertensive effect, and more specifically, the water-soluble extract of Styela clava , which is one of marine organisms, at 20 ° C. and 70 ° C. Anti-hypertensive activity in vitro from the organic and organic solvent extracts and showed an angiotensin I-Converting Enzyme inhibitory mechanism in vitro . After taking oral administration in hypertensive rats (Spontaneously Hypertensive Rat), the medicinal herb (Styela clava) extract having the antihypertensive effect using water-soluble and organic solvent extracts extracted from 20 ℃ and 70 ℃ It relates to the antihypertensive composition used.

 Referring to the configuration of the present invention through examples and experimental examples are as follows.

Example 1 Preparation of Materials

The material used for the test was purchased from marine organisms, Styela clava , to remove foreign substances, washed several times with water to remove water, and then pulverized. The material was stored for 24 hours in a deep freezer (-70 ° C) and frozen. Dry (2) to dry completely. The dried sample was pulverized (3) to a size of 27 mesh or less using a grinder.

Example 2 Preparation of Extract Solution of Midderb-derived Antihypertensive Substance

 Figure 1 shows a flow chart of the preparation and separation process of crude extract of the antihypertensive substance of the mesothelioma, which will be described in detail, in the preparation of water-soluble and organic solvent extracts of medeok, the selection and washing process of 1), lyophilization process (2) freeze-drying the selected meadak and powdering process (3) to crush the dried meaduk to 27 mesh Aha and 100L of water and organic solvent in 100g of the powdered meaduk The extraction process (4) carried out at 20 ℃ and 70 ℃ with a manufacturing reaction time of 24 hours and the filtrate was centrifuged by centrifugation at 2000 rpm for 10 minutes (5) and clarified after centrifugation The filtrate was prepared using an Whatman No. 1 filter paper (6) and then stored at 4 ° C. under reduced pressure filtration to prepare an extract of the antihypertensive substance derived from midderm.

Experimental Example 1 Measurement of Yield of Extract from Water-Soluble and Organic Solvent Extract

 The yield of the extract from the water-soluble and organic solvent extract was obtained according to the preparation method according to the manufacturing method from the midderk, the amount was calculated as a percentage (%), the yield is shown in Table 1.

Experimental Example 2 Measurement of General Components of Midderb Extract

 According to the AOAC method (1990), the general components of the extract were analyzed by 105 ℃ atmospheric pressure drying method, crude fat as Soxhlet extraction method, crude protein as Kjeldahl method, and crude ash as 550 ℃ dry ash method. Carbohydrate content is expressed as the total amount of solids minus moisture, ash, crude protein, and crude fat (Table 2).

Experimental Example 3 Angiotensin I-Converting Enzyme Inhibitory Activity of Extract

 As an angiotensin I-converting enzyme (ACE) inhibitory activity measurement method, a slight application of Cushman and Cheung method was used to measure the hippuric acid produced by the action of ACE on the substrate Hip-His-Leu. Substrate solution was prepared by mixing 5mM Hip-His-Leu in 100mM sodium borate buffer containing 300mM NaCl (pH 8.3), and adding 80µl of crude extract to the incubator at 37 ° C for 3 minutes before ACE. 20 μl of (25mU / mL) was added and subjected to enzymatic reaction for 30 minutes. After the reaction was completed, 250 µl of 1M hydrochloric acid (HCl) was added with stirring to stop the reaction. After the reaction was stopped, 1.7 mL of ethyl acetate was added to the hips to extract hippuric acid, and the mixture was stirred well. After the reaction was centrifuged (800xg, 15 minutes), 1 mL of the supernatant was transferred to a microtube and centrifuged for about 40 minutes. After all the ethyl acetate was volatilized in the concentrator, 1 mL of distilled water was added and mixed well, and then the absorbance value was measured at 228 nm.

 Their results are as follows.

 First, Figure 2 shows the results of angiotensin I-converting enzyme inhibitory activity from the water-soluble extract extracted with water at 20 ℃, angiotensin I-converting enzyme inhibitory activity from the water-soluble extract (water) extracted at 20 ℃, At concentrations of 1.62 ㎍ / mL, the inhibitory activity was 50%, and at 200 ㎍ / mL, the angiotensin I-converting enzyme showed 80% or more inhibitory activity, and as the concentration of the extract increased, it was angiotensin I-converting enzyme. Inhibitory activity was increased.

 Figure 3 shows the results of angiotensin I-converting enzyme inhibitory activity from the water-soluble extract extracted with water at 70 ℃, angiotensin I-converting enzyme inhibitory activity from the water-soluble extract (water) extracted from the extract at 70 ℃, the concentration of the extract Inhibitory activity was 50% at 0.96 ㎍ / mL, and angiotensin I-converting enzyme inhibitory activity was at least 80% at 200 ㎍ / mL, and angiotensin I-converting enzyme inhibitory activity was observed as the concentration of the extract increased. This increased.

 Figure 4 shows the angiotensin I-converting enzyme inhibitory activity results from the organic solvent extract extracted from the organic solvent with 70% ethanol at 20 ℃, angiotensin I-converting enzyme inhibition from the organic solvent extract extracted with 70% ethanol at 20 ℃ In the activity of the extract concentration of 1.74 ㎍ / ㎖ showed 50% inhibitory activity, 200 ㎍ / ㎖ angiotensin I-converting enzyme inhibitory activity was shown to have about 80% inhibition, the extract concentration Increasing angiotensin I-converting enzyme inhibitory activity was increased.

 5 shows angiotensin I-converting enzyme inhibitory activity from the organic solvent extracts extracted with 70% ethanol at 70 ° C., and angiotensin I-converting enzyme inhibitory activity from the organic solvent extracts extracted with 70% ethanol at 70 ° C. Silver, the extract concentration of 0.93 ㎍ / ㎖ showed 50% inhibitory activity, 200 ㎍ / ㎖ angiotensin I-converting enzyme inhibitory activity showed more than 80% inhibitory activity, and as the concentration of the extract increased angiotensin I-converting enzyme inhibitory activity was shown to increase.

 FIG. 6 shows angiotensin I-converting enzyme inhibitory activity results from organic solvent extracts extracted with 100% ethanol at 20 ° C., and angiotensin I-converting from organic solvent extracts extracted with 100% ethanol at 20 ° C. Enzyme inhibitory activity showed 50% inhibitory activity at the extract concentration of 1.2 ㎍ / ml, and angiotensin I-converting enzyme inhibitory activity at 80 µg / ml showed over 80% inhibition. Increasing angiotensin I-converting enzyme inhibitory activity was increased.

 7 shows angiotensin I-converting enzyme inhibitory activity results from organic solvent extracts extracted with 100% ethanol at 70 ° C., and angiotensin I-converting enzyme from organic solvent extracts extracted with 100% ethanol at 70 ° C. Inhibitory activity of the extract was 0.91 ㎍ / ㎖ showed 50% inhibitory activity, 200 ㎍ / ㎖ angiotensin I-converting enzyme inhibitory activity was about 80%, the extract concentration Angiotensin I-converting enzyme inhibitory activity increased with increasing.

Experimental Example 4 Effect of Crude Extract Solution on Blood Pressure Drop in Hypertensive Rats

 After insertion of a “ㅏ” type catheter into the carotid artery of the hypertensive rat, the rats were stabilized for more than a week and the crude extract was administered to the catheters, and blood pressure was observed in real time.

 Fig. 8 shows the result of blood pressure drop after oral administration of hypertensive rats from the aqueous extracts of the mesothelidae extract. After administration, the real-time blood pressure was measured by using a blood pressure meter through the opposite forelimb, and the degree of blood pressure drop was expressed as a percentage (%), which showed the effect of the overall blood pressure drop, especially between 250 and 450 minutes. In addition, blood pressure lowered by up to 40 percent (%).

 Putting the above results together,

 As shown in Figure 2, Figure 3, the result of angiotensin I-converting enzyme inhibitory activity from the water-soluble extract extracted from the water at 20 ℃ or 70 ℃, the concentration of the extract extracted at 20 ℃ is 1.62 ㎍ / ㎖, extracted at 70 ℃ The extract concentration showed 50% inhibitory activity at 0.96 ㎍ / ml, and the inhibitory activity was about 80% at 200 ㎍ / ml, and the angiotensin I-converting enzyme inhibitory activity as the concentration of the extract increased. This increased.

 As shown in FIGS. 4 and 5, the angiotensin I-converting enzyme inhibitory activity result from the organic solvent extract obtained by the extraction of the middera with 70% ethanol at 20 ° C. or 70 ° C., the concentration of the extract extracted at 20 ° C. was 1.74 μg /. The concentration of the extract extracted at ㎖, 70 ℃ showed 50% inhibitory activity at 0.93 ㎍ / ㎖, the inhibitory activity at about 200 ㎍ / ㎖ all showed an inhibitory activity of about 80%, and as the concentration of the extract increased angiotensin I-converting enzyme inhibitory activity was shown to increase.

 6 and 7 also show that the angiotensin I-converting enzyme inhibitory activity from the organic solvent extract extracted from 100% ethanol at 20 ° C. or 70 ° C., the concentration of the extract extracted at 20 ° C. was 1.2 μg / ml at 70 ° C. The concentration of the extracted extract showed inhibitory activity of 50% at 0.91 ㎍ / mL, and inhibitory activity of 80% at all 200 ㎍ / mL, and angiotensin I-converting enzyme inhibition as the concentration of the extract increased. The activity was shown to increase.

As described above, it was confirmed that the antihypertensive active substance was extracted from the water-soluble and organic solvent extracts of the medodeok according to the present invention at 20 ° C and 70 ° C. Water-soluble extract has the effect that can be utilized as an active ingredient of natural antihypertensive without side effects.

Claims (4)

In the antihypertensive composition, the antihypertensive composition using the mesotheli extract, characterized in that it contains an extract of Styela clava The method according to claim 1, wherein the extract is a water-soluble extracted with water and an organic solvent extract extracted with 70% ethanol, the organic solvent extract extracted with water and ethanol extracted with each water extracted at 20 ℃ or 70 ℃ using Antihypertensive composition The method of claim 2, wherein the water-soluble extract and the organic solvent extract extracted with 70% ethanol from each of the water extracted at 20 ° C or 70 ° C inhibits the angiotensin I-converting enzyme to block the process of angiotensin II to prevent hypertension Antihypertensive Composition Using Extracts The antihypertensive composition according to claim 2, wherein the water-soluble extract extracted from the meadak has water-lowering effect of the rat that induced the hypertension.

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