JP2732056B2 - Antihypertensive and vasodilator - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an agent for lowering blood pressure and a vasodilator, and more particularly, to a highly safe and novel blood pressure lowering agent using a natural product derived from fish meat. And vasodilators. (Related Art) Conventionally, the renin-angiotensin system has been considered to be one of the important factors of essential hypertension, and angiotensin plays a central role in the renin-angiotensin system as a vasopressor. Several drugs having an action of suppressing the activity of I-converting enzyme (ACE) are known. However, most of the practically used blood pressure lowering agents and vasodilators are synthetic drugs, and very few of them have natural products as active ingredients and are practically used. On the other hand, regarding fish components, the anti-atherosclerotic effect of eicosapentaenoic acid has been focused on fish oil components, which are often contained in sardines and horse mackerel, etc., and in fish meat, fish protein derived from Bacillus bacteria and plant-derived proteases An antihypertensive comprising a hydrolyzate of soybean protein or soybean protein as an active ingredient has already been proposed (Japanese Patent Application Laid-Open No. 62-169732). However, as in the present invention, no examples have been reported focusing on the bioactive effect of a peptide mixture obtained by autolyzing fish meat and then treating it with a protease.
Nothing is known about the combined use of the peptide mixture and egg yolk. (Problems to be Solved by the Invention) As described above, known antihypertensive agents and vasodilators are
Side effects such as excessive sedation, weakness, thirst and the like are inevitable, few are satisfactory in terms of efficacy, and most of them can only be administered parenterally, and there are very few oral preparations at present. (Means for Solving the Problems) The present invention has been made for the purpose of developing an antihypertensive and vasodilator which does not have the above-mentioned disadvantages, has high safety, and has no side effects. In particular, in view of safety and low toxicity, the present inventors have considered that a substance derived from a natural product rather than a synthetic artificial compound is preferable. Since antihypertensive drugs are usually taken for a long period of time, they can be administered orally.Particularly, it is necessary to screen for substances that do not pose any problems in terms of safety. Attention was paid to the arteriosclerosis-preventing action of saturated fatty acids, and the idea that useful physiologically active substances may be present in other components of fish and shellfish was given. We have obtained new findings that the hydrolyzate produced by proteolytic enzymes after autolysis of seafood meat is not only hypotensive but also has a vasodilator effect and is extremely safe. As a result of further study and research, the present invention has been completed. According to the present invention, a hydrolyzate of a fish muscle after autolyzing digestion by a protease and, if necessary, egg yolk are further used as an active ingredient. In order to carry out the present invention, fish is used as a raw material, and firstly, it is processed by a meat extractor, a deboner or the like to separate fish meat quality. The raw material is preferably as fresh as possible. The separated fish meat may be divided into about 10 kg of surimi and used as it is for the next processing, but may be used at -20 to -50C, for example, -30C.
℃, freeze quickly by blowing cold air of about -20 to -25 ℃
And may be used as needed as needed. As fish, red fish such as sardine, horse mackerel, tuna, bonito, saury, mackerel; white fish such as flounder, Thailand, kiss, konoshiro, cod, herring, and yellowtail; cartilage fish such as shark, ray;
Freshwater fish such as smelt, carp, char, and yamame; deep sea fish such as shark and anglerfish, as well as shrimp, crab, octopus, and mysid can be appropriately used. After slicing in this way, the fish muscle is crushed by a crusher, etc.
保持 60 ° C., preferably 40-55 ° C., for 1-5 hours to allow self-digestion, followed by the addition of enzymes to decompose. As an enzyme, a proteolytic enzyme is used. For example, ground fish meat (after further addition of water) is heated to an appropriate temperature for the enzyme (depending on the enzyme used, but about 20 to 60 ° C.). ~ 9), add protease and add
Treat for 1 to 30 hours. As the proteolytic enzyme, any enzyme capable of decomposing a protein can be used alone or in combination. Its origin can be obtained from microorganisms in addition to animals and plants, and in addition to pepsin, renin, trypsin, chymotrypsin, papain, bromelain, bacterial proteases, filamentous fungal proteases, actinomycetes proteases and the like can be widely used.
As these enzymes, those which are commercially available are generally used, but unpurified enzymes, culture solutions containing the enzymes, solid or liquid enzyme contents such as noodles may also be used as necessary according to the purpose. Can be. When using 0.1% protease solution, when processing red meat such as sardine as fish meat, pH 4.
The enzyme treatment is completed after about 17 hours at 1, 45 ° C or about 4 hours at pN5.9, 48 ° C. Next, after neutralization, the enzyme is inactivated by maintaining the temperature at 80 ° C. or higher for 5 to 30 minutes to obtain a decomposition solution. The decomposition product thus obtained can be used as it is as the active ingredient of the present invention. In the present invention, instead of directly enzymatically hydrolyzing fish meat, it is preliminarily self-digested and then treated with proteolytic enzymes, so that the yield of the digestion solution in which the peptide mixture as an active ingredient is dissolved is greatly increased. (As a result, the amount of insoluble decomposed residues generated is greatly reduced), and an effect excellent from an industrial point of view is achieved. The enzymatic decomposition solution may be subjected to ultrafiltration according to a conventional method to obtain a peptide solution having a molecular weight cut off of about 500 to 5,000, which may be used as the active ingredient of the present invention. Furthermore, the enzyme-decomposed solution prepared as described above is filtered through a vibrating screen or the like, and if necessary, subjected to a Jector treatment, and then centrifuged at, for example, 10,000 to 30,000 rpm using a Sharpless centrifuge or the like. This is concentrated by a conventional method (to about 30 Bx) and then centrifuged again to obtain a peptide stock solution. Next, the peptide stock solution is added with activated carbon, stirred, decolorized, and filtered. Add alcohol to the filtrate and leave to stand. As the alcohol, methanol, ethanol, propanol, isopropanol, butanol and the like are suitable, but other alcohols may be used.
The alcohol is used in a cold state (0 to 10 ° C.), and is used in an amount equal to or more than 30 times that of the peptide stock solution. After the addition of the cold alcohol, the mixture is left in a cold room for 30 minutes to 3 hours or more to form a precipitate. The resulting precipitate is hydrolyzed to form a solution, which is then chromatographed.
The precipitate may be used as it is for the various uses described above. The above solution is treated with an ion exchange resin. As the resin, a cation exchange resin, in particular, a strongly acidic cation exchange resin is used. For example, the above solution is injected into a column filled with these resins, washed with water, and then eluted with an alkaline aqueous solution such as dilute alkaline water to obtain an eluate. The obtained eluate is subjected to a method such as concentration under reduced pressure to remove ammonia, then concentrated if necessary, and powdered by a method such as freeze-drying according to a conventional method to obtain a desired basic peptide. As described above, the eluate is
It may be concentrated or diluted and used for various purposes. The peptide thus obtained was a novel one which was unknown in the literature and was extremely useful, and was named DAN-100. This DAN-100 can also be used as an active ingredient of the present invention, and all the components produced in the process of preparing DAN-100 can be used in the present invention. The physicochemical properties of peptide DAN-100 are as follows. 1. Elemental analysis value C: 41.5%, H: 6.7%, N: 16.9%, O: 34.9% 2. Molecular weight The molecular weight is estimated to be 10,000 or less and 500 or more by Sephadex G-25 column chromatography. 3. Melting point 173 ° C 4. Specific light intensity ▲ [α] ²⁰ _D ▼ 23 ゜ 5. UV spectrum No specific absorption is observed. 6. IR spectrum 7. As shown in Fig. 7. Solubility in solvent Soluble in polar solvents such as water, methanol and DMSO, but insoluble in non-polar solvents such as chloroform and hexane 8. Color reaction Ninhydrin reaction + Biuret reaction + Copper-Folin reaction + phenol-sulfuric acid reaction-9. Basic, acidic, neutral distinction Weak basic, pH 9.60 (10% solution) 10. Color and shape of substance White powder 11. Water content 4 4.01% (normal 12. Drying method) 12. Salt content 8.19% (measured by potentiometric titration as Cl) 13. Total nitrogen and amino acid nitrogen TN: 16.81% (micro locale d'al method) Amino acid form -N: 2.46% (formol method) The thus obtained fish meat decomposition solution or a processed product thereof is used as an active ingredient as it is, or after being concentrated or diluted, or as a dry powder such as a freeze-dried treatment. Since the peptide substance according to the present invention exhibits excellent hypotensive and vasodilatory effects as apparent from the test examples described below, the oral composition of a medicament for prevention and treatment of these diseases, infusion or nutritional food, health food, etc. Can be used extensively as objects. When used as a medicament, it can be administered orally or parenterally. In the case of oral administration, for example, tablets, granules, powders, capsules, and powders can be prepared in the usual manner. In the case of parenteral administration, for example, injection preparations, drops, suppositories It can be used as an agent or the like. As described above, antihypertensive and vasodilators which have been practically used in the past were very few orally administrable ones, but quite surprisingly, as apparent from the test examples described later, the aforementioned When the fish digestion solution according to the present invention and its related substance are orally administered in combination with egg yolk, the absorption of the peptide is promoted, and a remarkable in vivo drug effect is exhibited. It is one of the features. As egg yolk, besides chicken eggs, quail, duck and other bird eggs are mainly used, but eggs of other animals can also be used as appropriate. As the yolk, the yolk itself may be used as it is, or may be diluted with a solvent such as water or physiological saline and used as an egg yolk solution, and the dilution ratio may be freely determined as appropriate. Approximately 10-50% to achieve both economic efficiency and effectiveness
It is convenient to use egg yolk fluid. For administration, add 5 to 50 parts of fish meat decomposed solution to the yolk (liquid).
% May be used by dissolving them, or both may be simply mixed and used, or both may be put in a capsule and mixed in the body. As described above, the use of egg yolk can provide the desired drug effect even when administered orally. Therefore, not only the safety is improved and the side effects are reduced, but also various formulations such as capsules, tablets, drinks and the like can be used as oral compositions such as preventive foods, nutritional health foods and the like by controlling the active ingredient content at the same time. , Very practical. The medicinal component according to the present invention has no or very low toxicity due to its natural origin and is a food, and is extremely safe (LD ₅₀ > 3,000 mg / kg subcutaneous,> 5,000 mg / kg orally; Rat). The active ingredient according to the present invention varies depending on its type, administration method, patient condition, age, etc., but is about 0.1 to 6000 mg / kg /.
Days, and is preferably administered 1 to 4 times a day. In addition, when taken by a healthy person for the purpose of prevention, there is no particular limitation on the dose, the number of times of administration, and the like. If necessary, it can be used in combination with other drugs. Hereinafter, the present invention will be described in more detail with reference to Test Examples, Production Examples, and Preparation Examples. Test Example 1 Sardine peptide (enzyme digestion solution of sardine muscle prepared by the production example described below was subjected to ultrafiltration and the molecular weight cut off was 500 to 5).
000 peptide solution obtained by freeze-drying and powdering), rats were divided into the following three groups to test the antihypertensive effect. 1) intravenous injection group 2) physiological saline solution oral administration group 3) 30% yolk aqueous solution oral administration group. Rats used were 10-week-old Hos: SHR (spontaneously hypertensive rats) purchased from Hoshino Experimental Animal Co., Ltd., and preliminarily bred for 1 week. Administration was performed as follows, and after administration, blood pressure was measured over time using a pessimistic tail arterial blood pressure device (PS-100 manufactured by Riken Co., Ltd.), and the results in Table 1 were obtained. . Intravenous administration: 1 ml / animal obtained by dissolving sardine peptide in physiological saline to give a dose of 117 mg / kg was administered to rats. Oral administration: (a) Sardine peptide dose of 4000mg / kg
6 ml / animal was prepared by dissolving sardine peptide in physiological saline. (B) 6 ml / anim of sardine peptide dissolved in 30% egg yolk so that the dose of sardine peptide is 4000 mg / kg
al was administered. (The measured values were measured five times repeatedly, the highest and lowest values were rejected, and the average value of the three points was shown.) From the above results, the following can be found. 1) The intravenous administration clearly showed a hypotensive effect. 2) When the sardine peptide was dissolved in egg yolk and administered orally, the blood pressure significantly decreased. The details of its mechanism of action must be addressed in future studies, but this is because egg yolk
It was speculated that it was to promote cytosis absorption. Test Example 2 In this test example, a sardine peptide was dissolved in egg yolk,
The relationship between the dose and the decrease in blood pressure was tested using Nifedipine as a positive control test using SHR rats as in Test Example 1. A predetermined amount of sardine peptide was dissolved in yolk water to give a 30% yolk solution and orally administered. After administration, the blood pressure in the tail artery blood pressure device (manufactured by Riken Co., Ltd.)
Blood pressure was measured over time using PS-100). Measurement is 5
The maximum and minimum values were rejected, and the average of the three points was used as the measured value to obtain the progress shown in FIG. That is, 1) The blood pressure lowering amount certainly increased with the increase of the dose. 2) After administration, the amount of decrease increased up to about 3 hours, and the antihypertensive effect was continued even after 6 hours from administration. In the case of Nifedipine, which was used as a control sample, the blood pressure decreased rapidly to 46.3 mmHg 30 minutes after administration, but decreased to 14.9 mmHg 3 hours after administration, and 4 of 6 animals 6 hours later.
The head had risen to the pre-dose blood pressure value. The hydrolysis products according to the present invention, such as DAN-2, its celite filtration product, and DNA-100, all showed similar test results. From the above results, the effect of the yolk was clarified as follows. 1) When dissolved in egg yolk water and orally administered, it showed a positive antihypertensive effect according to the dose. 2) The anti-hypertensive effect reached a maximum in a few hours and persisted after 6 hours. Test Example 3 Each of sardine peptides 2, 4, 8 and 16 g / animal was dissolved in 20 ml of purified water, and orally administered to rabbits individually using a cannula. Rabbit ears were shaved to give a visual observation of the blood vessels before administration, and changes in the ear blood vessels were observed to perform a vasodilatation test. Rabbit is Std: NZW (New Zealand White), 1
Males of 4 to 15 weeks old and having a volume of 4.0 ± 0.5 kg were purchased from Shizuoka Prefectural Experimental Animal Agriculture Cooperative and bred for 1 week before healthy animals were subjected to the test. The time of observation is 20, 30, 60 and 120 minutes before medication,
Each time a photograph was taken at the same magnification, the results shown in FIGS. 3 and 4 were obtained. As a result, for 2, 4, 8, and 16 g / animal, vasodilation was observed at 20 minutes after sample administration, and 120 minutes after administration.
Even after a lapse of minutes, it was observed that vasodilation continued without recovery. The hydrolyzed peptides of the present invention, such as DNA-2, its celite filtrate, and DAN-100, all showed the same test results. Production Example 1 Fresh sardines were treated with a deboner and minced. The minced fish meat was divided into 10 kg surimi. After pulverizing the mixture with a pulverizer, an equal amount of water was added, the mixture was heated to 45 to 48 ° C., transferred to a stirring tank and kept at the same temperature for 2 to 3 hours for autolysis digestion. The pH was then adjusted to 4.1. In addition to this, Denateam (commercially available protease product)
A 0.1% solution was added, and the mixture was kept at 45 ° C. for 17 hours to perform enzymatic degradation. Neutralized and then boiled for 15 minutes to inactivate the enzyme. This is filtered through a vibe screen (150 mesh)
The filtrate was subjected to a Jector treatment at 5000 rpm, followed by treatment with a Sharpless centrifugal separator (15,000 rpm), concentration by heating under normal pressure until 20Bx, and centrifugation again with Sharpless (15,0).
(00 rpm) to obtain a peptide stock solution (DNA-2). DAN-2 was subjected to liquid chromatography as follows,
The results in the figure were obtained. Apparatus and measurement conditions Separation was performed by HPLC. Apparatus: Toyo Soda CCPM Column: Asahipak GS-320 7.6mmID × 50cmL Eluent: 6M Guanidine hydrochloride Flow rate: 0.5ml / min. Detector: UV (230nm) As is clear from the drawing, DAN-2 is 1 It has a broad molecular weight distribution of about 10,000 to 100, and a wide part of 10,000 to several hundreds (peak top 22
Min) and several peaks of several hundred or less. Next, the Brix of DAN-2 was adjusted to 35, this was filtered through celite, and the resulting peptide solution was similarly subjected to liquid chromatography to obtain the results shown in FIG. As is clear from the drawing, this substance has a molecular weight of 400
It is recognized that it is composed of a wide range of components of about 5000 to several hundreds (peak top 27 minutes) centering on those of about 500 and several component peaks of several hundreds or less. Production Example 2 In the same manner as in Production Example 1, sardines were self-digested, and pH was adjusted.
After the adjustment, enzymatic degradation was performed using denazyme, and neutralization and deactivation treatments were performed. Then, this was subjected to ultrafiltration to obtain a sardine peptide. The liquid chromatogram of the sardine peptide is as shown in FIG. 7 and consists of 10,000 to several hundreds of broad components with a molecular weight of about 2,000, and component peaks of several hundreds or less are recognized. I couldn't. Production Example 3 Take 1840 ml of the peptide stock solution (pH 6.22) obtained above,
100 g of activated carbon was added thereto, followed by stirring for 60 minutes, followed by filtration to obtain 1500 ml of a filtrate (pH 6.12). 100 ml of the filtrate was taken, 2,000 ml of cold ethanol was added thereto, and the mixture was allowed to stand in a cold room for 60 minutes to cause precipitation. Water was added to the resulting precipitate to obtain a solution (E). Production Example 4 Take 100 ml of the above solution (E), and use it in Dowex 50W
It was injected into a column (φ5 × 15 cm) packed with [H +].
Then, using water 2000ml were washed with water resin, 2N-NH ₄ OH250
The resulting eluate was subjected to ammonia removal by a conventional method, and lyophilized to obtain a white powder of peptide DAN-100. Formulation Example 1 (1) Sardine peptide (lyophilized powder) 10 g (2) Sodium chloride 9 g (3) Chlorobutanol 5 g (4) Sodium bicarbonate 1 g All components are dissolved in distilled water (1000 ml), and 1 ml per 1000 brown ampules The mixture was dispensed, replaced with nitrogen gas, and sealed to produce an injection. Formulation Example 2 (1) Sardine peptide (lyophilized powder) 10 g (2) Egg yolk powder 3 g (3) Lactose 87 g (4) Corn starch 29 g (5) Magnesium stearate 1 g (1), (2), (3) and 17 g Of cornstarch, granulated together with a paste made from 7 g of cornstarch, 5 g of cornstarch and (5) were added to the granules, and the obtained mixture was compressed with a compression tablet machine to give tablets.
00 pieces were produced. Preparation Example 3 20 kg of sardine peptide solution, 30 kg of egg yolk, 10 kg of granulated sugar
Was added to make the total amount 100 kg, and this was mixed well. Next, sterilize at 90-95 ° C for 15-30 seconds, and raise the product temperature to 70 ° C.
The mixture was quenched to the extent, filled into brown bottles previously sterilized in an amount of 100 g each, immediately sealed, and 1,000 drinks were produced. (Effects of the Invention) According to the present invention, an excellent antihypertensive agent and a vasodilator can be obtained by using a hydrolyzate of fish meat muscle obtained by autolyzing fish meat and then treating it with a protease. Can be. These drugs are not only excellent in direct drug efficacy, but also excellent in side effects, safety, and yield. In addition, by using it in combination with egg yolk, it becomes possible to prepare an oral preparation with enhanced peptide absorption, which simplifies administration and can significantly reduce phytotoxicity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the IR spectrum of peptide DAN-100. FIG. 2 is a graph illustrating the hypotensive effect of the sardine peptide. FIG. 3 and FIG. 4 are photographs showing the morphology of the organism, and are photographs showing the vasodilatory effect when the sardine peptide was administered at 2 g and 4 g per animal, respectively. FIGS. 5, 6, and 7 are liquid chromatograms of DAN-2, its celite filtrate, and sardine peptide, respectively.

Claims (1)

(57) [Claims] A blood pressure lowering and vasodilator composition comprising sardine-derived peptide DAN-100 having the following physicochemical properties. 1. Elemental analysis value C; 41.5%, H; 6.7%, N; 16.9%, O; 34.9% 2. Molecular weight It is estimated that the molecular weight is 10,000 or less and 500 or more by Sephadex G-25 column chromatography. 3. Melting point 173 ° C 4. Specific light intensity ▲ [α] ²⁰ _D ▼ = 23 ゜ 5. UV spectrum No specific absorption is observed. 6. IR spectrum As shown in Figure 1. 7. Soluble in solvents Soluble in polar solvents such as water, methanol and DMSO, but insoluble in non-polar solvents such as chloroform and hexane 8. Color reaction Ninhydrin reaction + Biuret reaction + Copper-Folin reaction + Phenol-sulfuric acid reaction-9. Basic, acidic, neutral Weak basic, pH 9.60 (10% solution) 10. Substance color, white powder 11. Moisture content 4.01% (normal pressure) (Drying method) 12. Salt content: 8.19% (measured by potentiometric titration as Cl) 13. Total nitrogen and amino acid nitrogen T-N: 16.81% (micro locale d'al method) Amino acid form -N: 2.46% (formol method). 2. A hypotensive and vasodilator composition comprising sardine-derived peptide DAN-100 having the following physicochemical properties and egg yolk. 1. Elemental analysis value C; 41.5%, H; 6.7%, N; 16.9%, O; 34.9% 2. Molecular weight It is estimated that the molecular weight is 10,000 or less and 500 or more by Sephadex G-25 column chromatography. 3. Melting point 173 ° C 4. Specific light intensity ▲ [α] ²⁰ _D ▼ = 23 ゜ 5. UV spectrum No specific absorption is observed. 6. IR spectrum As shown in Figure 1. 7. Soluble in solvents Soluble in polar solvents such as water, methanol and DMSO, but insoluble in non-polar solvents such as chloroform and hexane 8. Color reaction Ninhydrin reaction + Biuret reaction + Copper-Folin reaction + Phenol-sulfuric acid reaction-9. Basic, acidic, neutral Weak basic, pH 9.60 (10% solution) 10. Substance color, white powder 11. Moisture content 4.01% (normal pressure) (Drying method) 12. Salt content: 8.19% (measured by potentiometric titration as Cl) 13. Total nitrogen and amino acid nitrogen T-N: 16.81% (micro locale d'al method) Amino acid form -N: 2.46% (formol method).