JPH0521092B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to an oral food useful as a health food for preventing or treating hypertension. [Prior Art] Today, hypertension is one of the diseases that accounts for the highest mortality rate in Japan, and its prevention and treatment have become urgent and important issues. Hypertension is divided into secondary hypertension and essential hypertension. It is well known that the renin-angiotensin system in particular is deeply involved. In other words, the renin-angiotensin system contains angiotensin converting enzyme (hereinafter referred to as "Angiotensin Converting Enzyme"), which is involved in blood pressure regulation.
This enzyme produces an active peptide (angiotensin) that has a smooth muscle contraction effect on blood vessel walls, resulting in a strong increase in blood pressure. Synthetic antihypertensive drugs have already been developed that suppress increases in blood pressure by inhibiting the enzymatic activity of ACE, and the proline derivative captopril (manufactured by Squip) has been put into practical use as a new drug that can be administered orally. The present inventors have already discovered several types of peptides, such as Phe
−Phe−Val−Ala−Pro−Phe−Pro−Glu−Val
−Phe−Gly−Lys, Ala−Val−Pro−Tyr−Pro
-Gln-Arg, Phe-Phe-Val-Ala-Pro, etc. were isolated and purified, and these peptides were converted into ACE.
discovered that it has an inhibitory effect (Japanese Patent Application Laid-open No. 1983-
109425, JP 61-36226, and JP 59-44323). Generally, bioactive peptides are said to be unable to exhibit effective bioactivity when administered orally, for reasons such as being hydrolyzed and inactivated by gastrointestinal enzymes or being poorly absorbed from the gastrointestinal tract. , many reports have been made. For example, most oligopeptides are degraded into amino acids in the brush border or epithelial cells of the small intestine [DM
Physiological Reviews, 55 (4),
537 (1975)], when a high concentration of glycine oligopeptide was administered to the rabbit duodenal intestine using rabbits, the absorption into the plasma of the portal vein and femoral artery, and furthermore the absorption into the lymph of the mesenteric membrane, was investigated. The results of the investigation showed that only low concentrations of glycine dipeptide appeared in plasma and lymph, and that tripeptides and tetrapeptides were mostly hydrolyzed at the brush border and could not be detected [Peters & Mac Mahon; Clinical Science, 39 , 811 (1970)]. On the other hand, there is an example where it is stated that even if the immunostimulating substance is a peptide, it can be administered orally in some cases (Japanese Patent Application Laid-open No. 17522/1983). Although they are called physiologically active substances, immunostimulatory substances are not absorbed from the gastrointestinal tract and exhibit their physiological activity through blood circulation, but rather they are activated by a local immune response mediated by Beyer's patches in the intestinal tract. In order to express the
Vol. Spring Special Issue (1984) 175-186]. However, the above peptides isolated and purified by the present inventors are found in tissues through blood circulation.
Since it is carried to ACE and exerts a blood pressure lowering effect by blocking it, it is thought that oral administration cannot exhibit physiological activity, and the effectiveness of intravenous administration has only been investigated. Administration was not investigated. Since hypertension is a chronic disease, it is preferable that drugs for preventing or treating hypertension can be taken orally, without relying on a doctor, such as by intravenous injection. [Problems to be solved by the invention] Under these circumstances, as a result of extensive searches for compounds for the prevention or treatment of hypertension that can exert an ACE inhibitory effect even when taken orally, we found that the conventional Contrary to common sense, although it is a peptide, Phe−Phe−Val−Ala−Pro−Phe−
Pro−Glu−Val−Phe−Gly−Lys, Ala−Val−
Pro−Tyr−Pro−Gln−Arg and Phe−Phe−Val
It was discovered that the ACE inhibitory effect of -Ala-Pro is not inactivated even when taken orally, and based on this finding, the present invention was completed. An object of the present invention is to provide an oral food with low toxicity and ACE inhibitory action that can be used routinely and continuously for the prevention or treatment of hypertension. [Means for solving the problem] The present invention provides Phe-Phe-Val-Ala-Pro-Phe
−Pro−Glu−Val−Phe−Gly−Lys (hereinafter,
(abbreviated as CEI ₁₂ ), Ala−Val−Pro−Tyr−Pro
-Gln-Arg (hereinafter abbreviated as CEIβ ₇ ) and Phe
−Phe−Val−Ala−Pro (hereinafter abbreviated as CEI ₅ )
Provided is an oral food product characterized by containing one or more peptides selected from the following. The above CEI ₁₂ and CEIβ ₇ are, for example,
As described in Japanese Patent Publication No. 58-109425 and Japanese Patent Application Laid-Open No. 61-36226, casein from cows has a pH of 5.0~
Each product can be obtained as a single product by separating and purifying the reaction solution obtained by hydrolyzing with trypsin at 9.0 and removing trypsin and undecomposed casein. Alternatively, both may be contained in the oral food of the present invention as a mixture without being isolated. Furthermore, CEI ₅ can be produced by hydrolyzing CEI ₁₂ as a raw material with a proline-specific endopeptidase in a phosphate buffer (PH 7.0) (Japanese Patent Laid-Open Publication No. 599-4432). In the production of these peptides, commonly used purification methods such as mass fractionation by high performance liquid chromatography and purification using ion exchange material sheets, and common biochemical means can be used in appropriate combinations. Furthermore, the above peptides can be produced by conventional peptide synthesis methods. The above-mentioned peptides are usually separated and obtained as a powder, and then used as is, or more preferably, mixed with a suitable non-toxic carrier for oral administration (ingestion) to form an oral ingestion composition having an appropriate shape and form. It is shaped into a shape and used for oral intake for the prevention or treatment of hypertension. Non-toxic carriers for oral administration include pharmaceutically acceptable carriers that do not chemically change with the above peptides, such as excipients, lubricants, binders, colorants,
Examples include flavoring agents and flavoring agents. The shape and form are not particularly limited, and may be taken directly as a powder or as an aqueous solution, but may be in the form of pharmaceutical preparations for oral administration, such as sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, etc. Tablets, capsules, troches, powders,
It may also be formed into fine granules, granules, etc. It may also be in the form of solid or liquid foods or luxury goods, such as confectionery, powdered tea, alcoholic beverages, sports drinks, and the like. As a method for producing an oral food having a specific shape and form, conventional methods for manufacturing formulations or foods are used, except for avoiding harsh conditions that would reduce the ACE inhibitory activity of the above peptides. be able to. The content of the above-mentioned peptides in the oral food of the present invention is generally preferably 0.1% to 40% by weight. In addition, the intake amount of the oral food of the present invention is CEI ₁₂
{ACE inhibitory activity (ID ₅₀ ) is 77 μM (sample concentration in the enzyme reaction solution when measuring ACE inhibitory activity: hereinafter abbreviated as final concentration)}, convert it to the weight of the peptide. 0.5 per day for adult men
A range of ~30 mg/Kg is appropriate, and for the other peptides mentioned above, an appropriate range is selected based on CEI ₁₂ , depending on their inhibitory activity (ID ₅₀ ), purity, intestinal absorption, etc. The oral food of the present invention having the above-mentioned structure exhibits a considerable blood pressure lowering effect even when taken orally, as shown in the Examples, and has extremely low toxicity as shown in the Reference Examples. Therefore, the oral food of the present invention can be continuously orally administered (ingested) for the purpose of alleviating hypertension tendency or regulating blood pressure. As described above, the oral food of the present invention can be expected to be effective not only for treating hypertension, but also for preventing hypertension when taken as a health food. [Example] Next, as reference examples, preparation of ACE solution, method for measuring ACE inhibitory activity, mixture of CEI ₁₂ and CEIβ ₇ ,
Manufacturing and toxicity testing of CEI ₁₂ , CEIβ ₇ , and CEI ₅ ; Example 1, in which aqueous solutions of these peptides were orally administered to rats and blood pressure was measured; various oral foods were prepared; The present invention will be explained in more detail by showing Example 2 in which blood pressure was measured after orally administering the drug to patients. Reference example (Preparation of ACE solution) Dissolve 5g of Rabbit Lang Acetone Powder (Sigma) in 50ml of 0.1M borate buffer (PH8.3), centrifuge at 40000xg for 40 minutes, The supernatant was further diluted 5 times with the above buffer,
It was made into an ACE (angiotensin converting enzyme) solution. (Measurement of ACE inhibitory activity) A 0.03 ml sample was placed in a test tube, and 250μ of Hipryl-L-Histidyl-L-
Leucine (Aldrich Chemical Company)
Chem.Co.), final concentration 5mM, NaCl 300mM
including. ) and incubate at 37℃ for 10 minutes, then add the above
0.1 ml of ACE solution was added and reacted at 37°C for 30 minutes.
Thereafter, 0.25 ml of 1N hydrochloric acid was added to stop the reaction, and then 1.5 ml of ethyl acetate was added and the mixture was vigorously stirred for 15 seconds. Thereafter, the mixture was centrifuged at 3500 rpm for 15 minutes, and 1 ml of the ethyl acetate layer was collected. The ethyl acetate layer was heated at 120° C. for 30 minutes to remove the solvent.
After removing the solvent, 1 ml of distilled water was added, and the absorption of the extracted hyperlic acid (absorbance at 228 nm) was measured.
This was defined as enzyme activity. Note that under these conditions, the absorbance at 228 nm is 0.256 when the sample does not contain an inhibitor. The inhibition rate was calculated using the following formula. Inhibition rate = (A-B)/A x 100% A: Absorbance at 228 nm without inhibitor (0.256) B: Absorbance at 228 nm with addition of inhibitor Inhibitory concentration when inhibition rate is 50% Set ID to ₅₀ . (Production of a mixture of CEI ₁₂ and CEIβ ₇ ) 2 g of bovine casein (manufactured by Wako Pure Chemical Industries) was added to 50 ml of 40
Suspend in mM phosphate buffer (PH7.4) and add trypsin (150 units/mg, manufactured by PL Biochemicals)
5 mg was added and allowed to react at 37°C overnight. After the reaction,
2.4 ml of concentrated hydrochloric acid was added to the product to denature and precipitate trypsin and undegraded casein. the precipitate
After centrifugation at 9000xg for 30 minutes, the supernatant was concentrated 2-3 times under reduced pressure. Next, the obtained concentrate was added to a Sephadex G-25 column and purified by elution with distilled water. The column conditions during purification are as follows. Column: Inner diameter 2.5 cm x height 95 cm Sample addition amount: 10 ml Flow rate: 1.0 ml/min Elution: Distilled water The fraction with the highest ACE inhibition activity at this time was collected and ultrafiltrated (membrane membrane YC-05,
After desalting and concentration using a molecular weight cut-off of 500, lyophilization was performed to obtain 40 mg of white powder. This powder has a CEI ₁₂ and
ID ₅₀ = 2.1mg/ml for crude composition mainly composed of CEIβ ₇
(Sample concentration added to the test tube when measuring ACE inhibitory activity: hereinafter abbreviated as sample concentration). (Production of CEI ₁₂ ) In the same manner as in Production Example 1, bovine casein was digested with trypsin and treated with a Cephadex G-25 column. The obtained active fraction was desalted and added to an SP-Sephadex C-25 column. 0～
Elution was performed with a linear gradient of 0.5M ammonium formate (PH7.0). Column conditions are as follows. Column: Inner diameter 2.5 cm x height 50 cm Sample addition amount: 5 ml Flow rate: 0.4 ml/min Elution: 0 to 0.5 M ammonium formate (PH7.0) linear concentration gradient The fraction with maximum ACE inhibition activity at this time is collected and collected under pressure. Concentrated. Next, add the above concentrate again to Cephadex G-
25 column and eluted with distilled water. Column conditions are as follows. Column: inner diameter 2.5 cm x height 65 cm Flow rate: 0.4 ml/min Elution: distilled water The maximum active fraction at this time was collected and concentrated under reduced pressure. Next, the obtained concentrate was subjected to thin layer chromatography using a preparative silica gel plate (developing solution:
Ethanol/25% ammonia water = 77/23 (v/
The active spots (Rf=0.44) were scraped off, extracted with methanol, and dried under reduced pressure to obtain 9 mg of white powder. This powder has a CEI _{of 12} and an ID ₅₀ of 77μM
(final concentration). (Manufacture of CEIβ ₇ ) 1 g of bovine casein (manufactured by Wako Pure Chemical Industries) was added to 25 ml of 40
Suspend in mM phosphate buffer (PH7.4) and add trypsin (150 units/mg, manufactured by PL Biochemicals)
After adding 2.5 mg and reacting at 37°C for 18 hours, concentrated hydrochloric acid was added to the reaction solution to a concentration of 0.5M to stop the reaction. After removing the precipitate by filtration, the filtrate was adjusted to pH 7.0 with an aqueous caustic soda solution, added to a Sephadex LH-20 column, and eluted with distilled water. The column conditions are as follows. Column: 3.0 cm inner diameter x 112 cm height Flow rate: 1.0 ml/min Elution: distilled water The fraction with the highest ACE inhibition activity at this time was collected and concentrated under reduced pressure. Next, this concentrate was added to SP-Sephadex C-
25 column and eluted with a linear gradient of 0-0.5M ammonium formate (PH7.0). Column conditions are as follows. Column: 2.0 cm inner diameter x 47 cm height Flow rate: 0.4 ml/min Elution: 0 to 0.5 M ammonium formate (PH7.0) linear concentration gradient At this time, two active fractions were obtained. The first one was CEI ₁₂ . Second
Concentrate the second fraction under reduced pressure and use it as a sepadex.
It was applied to an LH-20 column and eluted with distilled water. The column conditions are as follows. Column: inner diameter 2.0 cm x height 62 cm Flow rate: 0.4 ml/min Elution: distilled water The fraction with the highest ACE inhibition activity at this time was collected, concentrated under reduced pressure, and dried to obtain 3 mg of white powder. This powder had a CEIβ ₇ and an ID ₅₀ of 15 μM (final concentration). (Production of CEI ₅ ) 4 mg of CEI ₁₂ white powder produced in Production Example 2 was added to 40
Dissolve in 1 ml of mM phosphate buffer (PH7.0) and add Flavobacterium meningosepticum purine-specific endopeptidase (Seikagaku Corporation).
After adding 2 units and reacting at 37℃ for 3 hours,
The enzyme was inactivated by boiling for 10 minutes, and the inactivated enzyme was removed by ultrafiltration using a CF25 membrane (manufactured by Amicon). The filtrate was treated with Radial-Pak C-
18 column (manufactured by Waters) and eluted with a phosphate buffer solvent. The preparative conditions for this high performance liquid chromatography are as follows. Column: Inner diameter 0.8cm x height 10cm Flow rate: 1ml/min Elution: Phosphate buffer (10mM KH ₂ PO ₄ +50mM
_Na2SO4 ; _PH3.0 )/ _CH3CN =60/40(v/
v) The active fraction (monitored by absorbance at 210 nm) was collected, desalted using a Setsupack C ₁₈ cartridge (manufactured by Waters), and then concentrated under reduced pressure.
After drying, 1 mg of white powder was obtained. This powder had a CE I ₅ and an ID ₅₀ of 6 μM (final concentration). (Acute toxicity test) (1) Sample The mixture of CEI ₁₂ and CEIβ ₇ produced above was used as a sample, and a 10% aqueous solution thereof was used. (2) Experimental animals Animals: ICR mice (CLEA Japan) Number of samples: 20 males and 20 females Weight at start of test: Males 24-26g Females 22-24g Rearing conditions during the period: Temperature 22±2℃ Humidity 50± 5% solid sample (CE-2, CLEA Japan) Drinking tap water ad libitum. (3) Test method After preliminarily rearing the animals for one week, they were subjected to experiments in groups of 10 animals. Fasted for 16 hours before oral administration, the experimental group received 3 g of sample (10
% aqueous solution 30 ml)/Kg, and tap water 30 ml/Kg for the control group.
Kg, each was forcibly administered orally using a gastric tube. For 7 days after administration, the animals were observed daily for survival and general symptoms. (4) Test results There were no animal deaths. We also observed general symptoms such as weakness, wasting, collapse, crouching, crawling on the stomach, lying on the side, changes in body hair color, changes in skin temperature, sweating, piloerection, hair loss, fur contamination, increase or decrease in breathing rate, irregular breathing, and asthma. There was no abnormality at all. From the above results, it was found that the product is extremely safe as an oral food. Example 1 13-week-old male spontaneously hypertensive (SHR) rats (Charles River Japan, 10 rats per group) were
Test animals were used as test animals that were acclimatized for 4 weeks in an animal room at 23 ± 2°C and humidity of 55 ± 5%, with free access to water and feed (manufactured by Oriental Yeast, MF). CEI ₁₂ manufactured in
A solution prepared by dissolving a mixture of CEIβ ₇ in distilled water (prepared immediately before administration) was orally administered at a dose of 2.8 g/Kg body weight in terms of solid content.
Blood pressure was measured 3 hours and 5 hours after administration. Blood pressure was measured using a non-heated, non-invasive rat blood pressure monitor (DSR801A manufactured by Toiden).
Systolic blood pressure values were measured 10 times in a row using the cuff method, and the average value was calculated. The results are shown in Table 1.

[Table] From this result, the mixture of CEI ₁₂ and CEIβ ₇ showed a remarkable antihypertensive effect even when administered orally;
It was found that the effect continued even after hours. Manufactured in the same manner as above and according to the reference example.
When aqueous solutions of CEI ₁₂ , CEIβ ₇ and CEI ₅ were orally administered to rats as test animals, a hypotensive effect was observed as described above. Example 2 (Preparation of tablets) 8.0 parts of a mixture of CEI ₁₂ and CEIβ ₇ produced as in Reference Example, 8.3 parts of lactose, 0.01 part of sucrose, and 1.5 parts of carboxymethylcellulose were added to a 70% aqueous ethanol solution containing 1.0 part of hydroxypropylmethylcellulose. The mixture was suspended in 25 parts, kneaded, and dried under vacuum to obtain a dried product. To this dried product, add 0.2 parts of magnesium stearate and 1.0 part of sodium starch glycolate.
tablets (400 mg per tablet) were prepared according to a conventional method. (Preparation of hard capsules) 8.0 parts of a mixture of CEI ₁₂ and CEIβ ₇ prepared as in the reference example, 10.0 parts of lactose, and 1.0 part of cross-linked polyvinylpyrrolidone were added to 25 parts of a 70% ethanol aqueous solution containing 1.0 part of hydroxypropyl methylcellulose. After suspending and kneading the mixture, the mixture was dried under vacuum to obtain a dried product. This dried material is sized into 20 mesh pieces,
Fill into No. 5 hard capsules and make hard capsules (100
mg/capsule). (Preparation of blowing agent) Mix 8.0 parts of the mixture of CEI ₁₂ and CEIβ ₇ produced as in the reference example, 8.5 parts of sodium bicarbonate, and 1.5 parts of sucrose, and mix this with hydroxypropyl cellulose.
Add 1.0 part of a 70% ethanol aqueous solution containing 0.15 parts, mix, and vacuum dry to obtain a dried product (Dry product 1)
I got it. Separately, 1.25 parts of tartaric acid, 0.25 parts of sucrose, and 0.25 parts of bailish starch were mixed, and 0.5 parts of a 70% ethanol aqueous solution containing 0.1 part of hydroxypropyl cellulose was added to the mixture. After kneading, the mixture was vacuum-dried to form a dried product (dried product). Product 2) was obtained. The above dried products 1 and 2 and 0.25 part of peppermint were mixed and compressed into 400 mg tablets according to a conventional method to obtain a foaming agent. (Preparation of lozenge) Mix 6.0 parts of lactose, 5.0 parts of sucrose, 1.0 part of powdered tragacanth, and 0.05 part of peppermint, and add 8.0 parts of a mixture of CEI ₁₂ and CEIβ ₇ prepared according to the reference example to 3.5 parts of distilled water. The dissolved solution was added and kneaded well. Next, the above-mentioned mixture was spread with a rolling pin on a glass plate sprinkled with starch to form a sheet with a thickness of about 5 mm, which was punched out with a mold and dried to form lozenges (1.5 g/piece). did. (Oral administration experiment) In the same manner as in Example 1, 13-week-old male spontaneously hypertensive (SHR) rats (Japan Charles River Co., Ltd., 1
Group of 10 animals) were placed in an animal room at a temperature of 23 ± 2°C and a humidity of 55 ± 5%, with water and feed (Oriental Yeast, MF).
The test animals were given ad libitum and acclimatized for 4 weeks, and the test animals were given oral food prepared as described above, i.e., tablets, hard capsules,
Foaming agents and troches were orally administered, and blood pressure was measured immediately before and 3 hours after administration to examine the antihypertensive effect. The average weight of the rats was 0.3 kg. The dosage is 6 g (720 mg/kg of body weight) of tablets, alarm agents, and lozenges as the amount of each food product.
) and, if necessary, crushed them into appropriate sizes and administered them. In addition, hard capsules are available in 5 capsules (500
mg)/mouse was administered orally by gavage. The results are shown in Table 2. The blood pressure reduction value in the table is the value obtained by subtracting the blood pressure 3 hours after administration from the blood pressure immediately before administration.

[Table] From the results, it was revealed that all the orally ingested foods had a clear blood pressure lowering effect. [Effects of the Invention] The present invention shows that the oral food contains an ACE inhibitory peptide as an active ingredient, which was conventionally believed to be inactivated by oral intake. Since it exhibits a lowering effect and has very low toxicity, it has the effect of being able to be ingested continuously on a daily basis for the prevention and treatment of hypertension without bothering a doctor.

Claims (1)

[Claims] 1 Phe－Phe－Val－Ala－Pro－Phe－Pro－
Glu−Val−Phe−Gly−Lys, Ala−Val−Pro−
Tyr−Pro−Gln−Arg and Phe−Phe−Val−Ala
An oral food product characterized by containing one or more peptides selected from -Pro.