KR20060118258A - Inhibitor of angiotensin converting enzyme containing sagarchromic acid derivatives and pharmaceutical composition thereof - Google Patents

Abstract

An inhibitor of angiotensin-converting enzyme containing derivatives of chromic acid having the valence four and a pharmaceutical composition thereof are provided to prevent and treat cardiovascular diseases including hypertension without harmful effects on human by relaxing the blood vessel and reducing blood pressure. The inhibitor of angiotensin-converting enzyme contains at least derivatives of chromic acid having the valence four selected from chromic acid having the valence four represented by the formula(1), and chromanol having the valence four represented by the formula(2), wherein the derivatives of chromic acid having the valence four are extracted from brown algae; the brown algae is Sargassum sagamianum, Sargassum siliquastrum, Sargassum confusum, Sargassum hemiphyllum, Sargassum thunbergii, Sargassum igoldianu, Sargassum horneri, Sargassum fulvescens or Sargassum patens.

Description

Inhibitor of Angiotensin Converting Enzyme Containing Sagarchromic Acid Derivatives and Pharmaceutical Composition Thereof}

The present invention relates to an angiotensin converting enzyme activity inhibitors and pharmaceutical compositions thereof, and more particularly, to an angiotensin converting enzyme activity inhibitors and pharmaceutical compositions thereof having a sagachromic acid derivative extracted from seaweed as an active ingredient.

Hypertension is a chronic degenerative disease of the circulatory system and is explained by the physicochemical mechanism of the Renin-Angiotensin System (RAS), which plays an important role in the regulation of blood pressure and electrolyte balance. In particular, angiotensin converting enzyme (hereinafter referred to as 'ACE') is an enzyme that serves to convert angiotensin II from angiotensin I to dipeptide (His-Leu), which has a vasoconstrictive action. Angiotensin II increases hypertension by promoting strong blood pressure synergism and the secretion of the antidiuretic hormone aldosterone and inhibiting the excretion of water and sodium to increase the amount of circulating blood. Therefore, by inhibiting the action of ACE to prevent blood vessels contraction can exhibit a blood pressure lowering effect, these ACE inhibitors can be said to be a drug that provided a breakthrough in improving hypertension.

ACE activity screening is performed using a hypertension test animal and an in vitro test using ACE and a special substrate. As a representative ACE inhibitor, captopril, a chemical synthesis agent, has been developed and used as an antihypertensive agent, but there are many side effects such as dry cough, headache, anorexia, taste disorders, rash, and white blood cell reduction. Has focused on the development of ACE inhibitors.

Substances exhibiting ACE inhibitory activity are broadly classified into peptide and polyphenolic substances. Since peptide inhibitors have been reported from snake venom for the first time, it has been reported that tuna, sardine, mackerel, horse mackerel, bonito, shrimp, scallops, etc. Enzyme hydrolysates, milk proteins casein, egg albumin (gebumin), gelatin, and peptides isolated from animal plasma have been reported (G. Oshima, et al., Biochimica et Biophysica Acta, 566, 128, 1979; S. Maruyama, et al., Agric. Biol. Chem., 46 (5), 1393, 1982; S. Maruyama, et al., Agric. Biol. Chem., 51 (6), 1581, 1987; M. Kohmura, et al., Agric. Biol. Chem., 54 (4), 1101, 1990; Yasuo Ariyoshi, Trends in Food Sci.Tech., 4, 139, 1993; Song, KB, et al., Biotech.Tech., 10 (7), 479, 1996; Song, KB, et al., Agric.Chem. Biotech., 40 (1), 39, 1997).

On the other hand, as polyphenolic substances, extracts such as persimmon leaves, mandarin, tannins of green tea, aloe acetylmannin, chitosan oligosaccharides, onion seasonings, and tannins obtained from mandarin skin have been found to have an ACE inhibitory effect (Matsubara, Y). , et al., Agric. Biol. Chem., 49 (4), 909, 1985; Cho, Y., et al., Korean J. Food Sci.Tech., 25 (3), 238, 1993; Ryu , IW, et al., Korean J. Food Sci.Tech., 29 (1), 1269, 1997; Hong, SP, et al., Korean J. Food Sci.Tech., 30 (6), 1476, 1998 Park, EJ, et al., Food Sci. Biotech., 9 (3), 163, 2000). In Korea, there have been studies on ACE inhibitory peptides derived from doenjang, rice, squid, slaughter blood plasma, seaweeds and mushrooms (Yang, HC, et al., Agric. Chem. Biotech., 37 (6), 441) , 1994; Shin, JI, et al., J. Food Sci.Tech., 27 (2), 230, 1995; Ko, YS, et al., J. Korean Fish.Soc., 32 (4), 427 , 1999; Song, KB, et al., Korean Patent No. 215090; Song, KB, et al., Korean Patent No. 215091; Suh, HJ, et al., Food Res. Int., 34, 177 , 2001).

However, until now, researches on extracting ACE inhibitors from natural products have mainly focused on preparing functional peptides having an ACE inhibitory effect from hydrolysates obtained by hydrolyzing food proteins with proteases such as pepsin and trypsin. In this case, there is a disadvantage that the industrial use is not easy due to economic problems such as high production cost as well as very low yield during separation and purification.

In order to solve this problem, the present inventors confirmed that sagachromic acid derivatives extracted from seaweeds have an effect of inhibiting the activity of angiotensin converting enzyme to induce blood pressure reduction in SHR (Spontaneously Hypertensive Rats). On the basis of this, the present invention was completed.

Accordingly, it is an object of the present invention to provide an angiotensin converting enzyme activity inhibitor comprising the sagachromic acid derivative as an active ingredient. It is still another object of the present invention to provide pharmaceutical compositions and health foods containing sagachromic acid derivatives.

According to the invention,

Sagachromic acid represented by Formula 1;

      [Formula 1]

 Following sagachromool of formula (2); And

       [Formula 2]

       Sagachromenal of Formula 3

      [Formula 3]

An angiotensin converting enzyme activity inhibitor containing at least one sagachromic acid derivative selected from the group consisting of is provided.

Furthermore, according to the present invention, there is provided a pharmaceutical composition and health food containing a sagachromic acid derivative.

Hereinafter will be described in detail with respect to the present invention.

As described above, in the present invention, it was found that the sagachromic acid-derivatives extracted from seaweeds, particularly brown algae, inhibit the activity of angiotensin converting enzyme and have an effect of inducing blood pressure reduction in SHR (Spontaneously Hypertensive Rats).

Sagachromic acid derivatives having angiotensin converting enzyme activity inhibitory activity of the present invention are represented by the following Chemical Formulas 1-3.

       [Formula 1]

Structure of Saga Chromic Acid

[Formula 2]

Structure of Saga Chromol

      [Formula 3]

Structure of saga chromatin

The sagachromic acid derivative, which is an active ingredient of the angiotensin converting enzyme activity inhibitor according to the present invention, is extracted from seaweed, and brown algae is particularly preferable. Such brown algae include, but are not limited to, for example, Sargassum sagamianum , Sargassum siliquastrum, Sargassum confusum, Sargassum hemiphyllum, Sargassum thunbergii , Sargassum igoldianu, Sargassum horneri, Sargassum fulvescens, or Sargassum patens are preferred.

The method of extracting the sagachromic acid derivative from the brown algae is performed by a conventional organic solvent extraction method. The organic solvent used in the solvent extraction method is preferably methanol, ethanol, ethyl acetate, acetonitrile, acetone, water, and mixtures thereof, and more preferably water / ethanol. Optionally, this extraction process may be repeated two or more times in order to increase the yield of the tetravalent chromic acid derivative. The residue and solvent contained in the extract are removed using conventional separation and concentration means such as centrifuge and rotary evaporator, and may be subjected to additional separation and purification if higher purity is required, but in the present invention There is an economically advantageous advantage to use the extract without a separate additional process.

According to the present invention, an angiotensin converting enzyme activity inhibitor having a sagachromic acid derivative as an active ingredient can be used as a pharmaceutical composition or health food.

The pharmaceutical composition containing the angiotensin converting enzyme activity inhibitor according to the present invention is characterized in that it is for the prevention and / or treatment of hypertension or other cardiovascular diseases. Diseases useful for such pharmaceutical compositions include, but are not limited to, coronary artery disease, cerebrovascular disease, peripheral vascular disease, aortic disease, renal dysfunction, heart failure and vision disorders. Examples of the coronary artery disease include angina pectoris and myocardial infarction. Cerebrovascular diseases include stroke, cerebral hemorrhage, cerebral infarction, and the like. In addition, it can be applied to the treatment or prevention of diabetes mellitus, hyperlipidemia, arteriosclerosis.

The pharmaceutical composition according to the present invention may include only the angiotensin converting enzyme activity inhibitor alone or may be combined with a pharmaceutical carrier, an excipient and a diluent. Preferably, the angiotensin converting enzyme activity inhibitor is 20 to 20 based on the total weight. It is contained in 70% by weight.

The pharmaceutical composition according to the invention may be administered to the mammal in any way, for example orally or parenterally. Parenteral administration methods include, but are not limited to, subcutaneous, intravenous, intramuscular or intraperitoneal administration.

The pharmaceutical compositions of the present invention are prepared in all pharmaceutically acceptable forms and may be formulated into preparations such as injections, liquids, pills, tablets, capsules, powders and suspensions according to conventional methods in the pharmaceutical art. And, preferably, tablets, capsules, or liquids.

Capsules may contain active ingredients such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like and powdered carriers. Diluents can also be used to make compressed tablets. Pharmaceutical compositions in liquid form for oral administration may contain pigments and flavors as desired by the patient.

Pharmaceutical compositions in liquid form for parenteral administration may contain water, suitable oils, saline, aqueous dextrose (glucose) and glycols such as propylene glycol or polyethylene glycol as suitable carriers. It may preferably comprise a water soluble active ingredient salt, suitable stabilizers and buffers. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. The pharmaceutical compositions in liquid form for parenteral administration may also contain preservatives such as benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol.

Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art, and preferably 1 to 500 mg / kg. It may be administered in an amount of / day and may be administered once or several times a day.

In addition, angiotensin converting enzyme activity inhibitor containing a sagachromic acid derivative according to the present invention may be added to health foods for the purpose of improving and lowering blood pressure. Foods to which the angiotensin converting enzyme activity inhibitor can be added for the development of such health foods include, but are not limited to, various foods, beverages, snacks, confectionery, noodles, gums, ice creams, alcoholic beverages, Tea bags, instant teas, and vitamin complexes.

As such, when the angiotensin converting enzyme activity inhibitor of the present invention is added to foods, the amount is added at a suitable ratio of 0.1 to 20% by weight based on the total weight, and it does not limit the intake of such foods, but it is 1 mg to daily It is effective to take 1 g / kg, preferably 1 to 100 mg / kg.

As described above, the angiotensin converting enzyme activity inhibitor provided in accordance with the present invention can be used as a pharmaceutical composition or a health food, which is effective in preventing and treating hypertension or other cardiovascular diseases because it exhibits a blood pressure lowering effect by relaxing blood vessels. Edible seaweed extract has no toxicity to the human body, so it can be consumed for a long time in the form of health foods as well as pharmaceuticals.

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

Example 1

Seaweeds collected from offshore, Sargassum sagamianum , were first washed with distilled water to remove debris and dried in the shade before being crushed. 500 g of the extract was refluxed for 2 hours to elute the active substance using 20 times the amount of 10% ethanol solution. This process was repeated twice. Then, the residue was filtered off and the solvent extract was concentrated under reduced pressure using a rotary evaporator. The concentrated solution was suspended in 20 times distilled water, extracted three times using the same amount of ethyl acetate solvent, and the ethyl acetate fractions were concentrated under reduced pressure. The concentrate was loaded on 15 times the amount of silica gel and extracted and concentrated using a mixed solvent of ethyl acetate / acetone (volume ratio 9/1) to prepare a crude extract.

Using the crude extract as a sample, angiotensin converting enzyme inhibitory activity was measured according to the method of Cushman and Cheung. (Cushman, DW and Cheung, HS. Biochem. Pharmacol., 20, 1637. 1971) Furyl histidyl leucine (hippuryl-L-histidyl-L-leucine, HHL) was used, and HHL was dissolved in 50 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl to make 5 mM HHL. The enzyme was an angiotensin converting enzyme (ACE) isolated from rabbit lungs. The sample was placed in a substrate and the reaction solution to which the enzyme was added was reacted at 37 ° C. for 30 minutes, and then the reaction was stopped by adding 1N hydrochloric acid. Ethyl acetate was added to the reaction mixture, mixed for 30 seconds, centrifuged at 3000 rpm for 15 minutes, and only the supernatant ethyl acetate layer was taken and dried at 100 ° C. for 1 hour. After drying, distilled water was completely added to the hyplic acid remaining in the test spheres, and then absorbance was measured at 228 nm. For the control, 50 μl of sodium borate buffer was added instead of the sample solution.

As a result of measuring the inhibitory activity of the extract on angiotensin converting enzyme in the same manner as described above, as shown in Table 1, the inhibition rate of the crude extract was IC50 = 70 μg / ml.

Example 2-4

The crude extract prepared in Example 1 was filtered through a 0.2 μm membrane filter and loaded on high performance liquid chromatography. In high-performance liquid chromatography, the column was HP ODS Hypersil column, distilled water and methanol as solvent, and the supply of solvent was linear gradient over 15 minutes from 15% to 70% methanol at a flow rate of 1.0 ml / min. ) To isolate the tetravalent chromic acid derivatives of the formulas (1), (2) and (3). IC50 was calculated by measuring the inhibitory activity against ACE-converting enzyme for each substance and determining the concentration showing 50% inhibition rate. The results are shown in Table 1.

Example 5

IC50 was calculated by measuring the inhibitory activity against ACE converting enzyme using the mixture of the samples of Examples 2 to 4, and determining the concentration showing the inhibition rate of 50%. The results are shown in Table 1.

Comparative Examples 1 to 6

Catechin, resveratrol, green tea extract, grape seed extract, quercetin, and isoflavone, known as polyphenolic substances, were used as a control to measure the inhibitory activity against ACE converting enzyme. The results are shown in Table 1.

Inhibitory Activity of Angiotensin Converting Enzyme of Each Sample Composition of the sample IC50 (μg / ml) Example 1  Crude extract 70 Example 2  Sagachromic acid 99% of formula 1 38 Example 3  Sagachromanol of Formula 2 98% 30 Example 4  Sagachromenal 99% of Formula 3 32 Example 5  Sagachromic acid of formula (1): 60% + Sagachromenol of formula (2): 25% + Sagachromenal of formula (3): 15% 35 Comparative Example 1  Catechin 84 Comparative Example 2  Resveratrol 122 Comparative Example 3  Quercetin 97 Comparative Example 4  Isoflavone 91 Comparative Example 5  Green tea extract 310 Comparative Example 6  Grape seed extract 230

Example 6

Blood Pressure Induction Effect of Spontaneous Hypertensive Rat (SHR)

Experimental Animal and Extract Administration: Male Spontaneous Hypertensive Rats (SHR) with systolic blood pressure of 240 mmHg or more were divided into 20 experimental groups and 20 control groups. The nursery was operated with 25 ± 1 ℃ ventilator and the contrast was adjusted every 12 hours. During the breeding, water and feed were freely consumed. The composition of Example 5 in Table 1 was dissolved in 10% Tween 80 as a control solution 10% Tween80 as a control solution, the test solution in the experimental group, the control solution was administered orally once daily for 5 days. Dosing was done daily from 6:00 to 7:00 pm.

Blood pressure measurement: Blood pressure measurement was performed at 9:00 every morning for 7 days including the start date of ingestion. A blood pressure measurement method was performed using a Pulse Amplifier (Flat-bed Recosders & Amplifier Operating Instructions, USA), and a small animal noninvasive automatic blood pressure gauge (JAPAN) and a tail cuff were attached to the tail, and the tail was connected to a sensor. Mean systolic blood pressure was measured through the artery. The results are shown in Table 2.

Data processing: Student T-test was used for statistical analysis of the experimental data. The significance level was P <0.05.

Blood pressure drop effect of SHR Systolic Blood Pressure (mmHg, Mean ± SD) Diastolic blood pressure (mmHg, mean ± SD) Work Experimental group Control P value Experimental group Control P value 0 221 ± 34 226 ± 27 179 ± 19 180 ± 22 One 188 ± 30 224 ± 15 0.028 153 ± 21 181 ± 24 0.031 2 190 ± 21 228 ± 23 0.047 154 ± 16 179 ± 18 0.042 3 191 ± 26 212 ± 10 0.038 152 ± 18 174 ± 15 0.023 4 189 ± 22 212 ± 10 0.021 154 ± 22 182 ± 19 0.031 5 192 ± 16 225 ± 13 0.036 151 ± 24 179 ± 25 0.034 6 189 ± 19 222 ± 15 0.025 155 ± 19 182 ± 19 0.043 7 191 ± 16 219 ± 24 0.042 152 ± 14 179 ± 21 0.041

N = 20: experimental group and control group

As a result, the control group showed no decrease in systolic and diastolic blood pressure after repeated administration for 7 days, but the systolic blood pressure was decreased by 13.5% (p = 0.042) and diastolic blood pressure by 15.1% (p = 0.041) in the experimental group. .

Example 7

4-week repeated dose toxicity test in rats

In order to investigate the toxicity by repeated oral administration of the test substance, the media control and the test substance (Example 1) were repeatedly administered orally to 10 SD male and female rats for 4 weeks at doses of 1000, 333, and 111 mg / kg, respectively. The test results shown by administration were as follows.

(1) No dead animals associated with administration of the test substance were observed.

(2) No change of general symptoms related to administration of test substance was observed.

(3) No change in body weight was found in both male and female.

(4) No abnormalities related to the administration of the test substance were recognized in all the male and female groups in the feed and water intake.

(5) In the ophthalmologic examination, no abnormalities were observed in all groups of male and female.

(6) In urinalysis, no toxicological abnormalities were observed in all male and female groups.

(7) No toxicological changes were observed in the hematological examination by the administration of test substance in all groups of male and female.

(8) No toxicological changes related to the administration of test substance were observed in blood biochemical tests.

(9) At autopsy findings, no abnormal findings were observed in all male and female treatment groups.

(10) No significant change in organ weight was observed in all groups of male and female.

(11) Histopathological examination showed no toxicological changes due to the administration of the test substance.

In view of the above results, no toxicological changes were observed by repeated oral administration of 4 weeks in rats. Therefore, the NOEL of this test substance was judged to be 1000 mg / kg / day or more.

As shown in the results of the above example, the sagachromic acid derivatives isolated from various brown algae which are marine edible plants according to the present invention are harmless to the human body and have the activity of inhibiting the activity of angiotensin converting enzyme, thereby economically preparing ACE inhibitors. It is expected to have the effect of developing the process and the development of functional foods and medicines for improving hypertension using these substances.

The composition for lowering blood pressure containing the sagachromic acid derivative provided according to the present invention relaxes blood vessels and thus lowers blood pressure, and thus can be used for the prevention and treatment of hypertension or other cardiovascular diseases. There is no advantage in that the drug can be consumed for a long time in the form of food, of course.

Claims (10)

Sagachromic acid represented by Formula 1; [Formula 1]

Following sagachromool of formula (2); And [Formula 2]

  Sagachromenal of Formula 3   [Formula 3]

         Angiotensin converting enzyme activity inhibitor containing at least one or more sagachromic acid derivatives selected from the group consisting of: The angiotensin converting enzyme activity inhibitor according to claim 1, wherein the sagachromic acid derivative is extracted from brown algae. The method of claim 2, wherein the brown algae is twisted for Sargassum (Sargassum sagamianum), pretzel Sargassum (Sargassum siliquastrum), alssong the Sargassum (Sargassum confusum), jjakip Sargassum (Sargassum hemiphyllum), Sargassum thunbergii is (Sargassum thunbergii), keunip Sargassum (Sargassum igoldianu), Sargassum horneri, Sargassum fulvescens, or Sargassum patens . Sagachromic acid represented by Formula 1; [Formula 1]

Following sagachromool of formula (2); And        [Formula 2]

      Sagachromenal of Formula 3       [Formula 3]

        A pharmaceutical composition containing an effective amount of at least one or more sagachromic acid derivatives selected from the group consisting of: The pharmaceutical composition of claim 4, wherein the composition is for the prevention and / or treatment of hypertension and cardiovascular disease. The pharmaceutical composition according to claim 4, wherein the daily dosage of the composition is 0.1 to 500 mg / kg / day. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is administered orally or parenterally in the form of a capsule, tablet, or liquid. Sagachromic acid represented by Formula 1; [Formula 1]

Following sagachromool of formula (2); And        [Formula 2]

       Sagachromenal of Formula 3        [Formula 3]

      A health food containing an effective amount of at least one or more sagachromic acid derivatives selected from the group consisting of: The health food according to claim 8, wherein the health food is a beverage, snack, snack, cotton, gum, ice cream, alcoholic beverage, tea bag tea, instant tea, and vitamin complex. The health food according to claim 8, wherein the intake amount of the health food is 1-100 mg / kg daily.