JPH04279597A - New peptide, angiotensinase inhibitory peptide and per oral composition containing the same peptide - Google Patents

Abstract

PURPOSE:To obtain the subject new peptide capable of inhibiting angiotensinase having vasopressor activity and useful for a health food, a medical drug, etc., for prevention of hypertension by decomposing royal jelly using a protease. CONSTITUTION:Royal jelly protein is added to a phosphate buffer solution (pH2) and a protease such as pepsin is further added thereto. After decomposition of the protein by incubation at 37 deg.C for 24hr, the enzyme is inactivated by heat treatment in boiling water for 10min. The treated solution is then allowed to cool and subsequently centrifuged to remove the insoluble materials. The resultant supernatant is fractionated by a gel filtration and active fractions are passed through an adsorptive resin column. The adsorbed fraction is subjected to gradient elusion by using ammonium formate and its active fraction is then further purified by high performance liquid chromatography, etc., thus obtaining the objective new peptide having an amino acid sequence of the formula and having an angiotensinase inhibitory effect.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to novel peptides and angiotensin-converting enzyme inhibiting peptides useful as pharmaceuticals, health foods, etc. for the prevention of hypertension, etc., and oral compositions containing them. .

0002

BACKGROUND OF THE INVENTION In today's aging society, adult diseases such as heart disease, cerebrovascular disorders, and cancer pose a serious threat to life. Hypertension accounts for a large proportion of these exacerbating factors, and treatment and prevention of hypertension have become a major issue. Hypertension includes secondary hypertension and essential hypertension, of which essential hypertension is mainly caused by excessive salt intake, renin-angiotensin-aldosterone system, and kallikrein-kinin-prostaglandin system. It is thought that the disease develops due to interactions such as dysregulation and excessive secretion of catecholamines.

Angiotensin converting enzyme (Angiotensin E) is used to regulate the renin-angiotensin system and kallikrein-kinin system.
nzyme (hereinafter referred to as ACE) is an enzyme that produces angiotensin 2, a pressor peptide, in the blood, while hydrolyzing kinin, a blood pressure lowering peptide. Therefore, inhibiting ACE will suppress the increase in blood pressure as a whole.

[0004] Based on this idea, the search for ACE inhibitors in natural and synthetic products has been energetically carried out, and proline derivative compounds have already been put into practical use due to their usefulness. On the other hand, it has also been reported that certain foods and Chinese herbal medicines have inhibitory effects on this enzyme, although the effects may be more or less strong (Journal of the Japanese Society of Agricultural Chemistry, Vol. 57, No. 11).
, 1143, 1983). Among these, ACE inhibitory peptides have only been isolated and purified for tryptic digests of casein, and the amino acid structure has also been elucidated (Japanese Patent Application Laid-Open No. 1983-1989-1).
No. 109425).

[0005]

[Problems to be Solved by the Invention] Among hypertension, essential hypertension in particular has a wide variety of causes, so there is no fundamental treatment, and most treatments are symptomatic. Therefore, daily blood pressure control is carried out by ingesting so-called antihypertensive drugs or health foods containing components that have a blood pressure lowering effect. Normal control of blood pressure is something humans desperately desire in order to reduce the exacerbating factors of adult diseases and to delay the aging process of the body.

[0006] Royal jelly, which has been eaten around the world since ancient times, is used to treat high blood pressure, diabetes, cancer, menopausal disorders, neuralgia,
It is reported to be useful for cerebrovascular disorders, etc. It is also said to bring about normal mental and physical development in young people, and many people who have eaten royal jelly live long lives. However, there are few reports that have elucidated the physiologically active substance in royal jelly, and most of them have looked at the effects of 10-hydroxydecenoic acid, which is said to be the most physiologically active substance in royal jelly.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a peptide capable of inhibiting angiotensin converting enzyme, which has an effect of increasing blood pressure, and exert a hypotensive effect, and to provide an orally ingestible composition in which this peptide can be used as a health food or a medicine. Our goal is to provide the following.

[0008]

[Means for solving the problem] That is, the first invention is based on S
er-Leu-Pro-Lys-Leu-His-Gl
A peptide having the structure u-Trp (hereinafter referred to as RJP)
8) is its gist. The second invention is Ser-Leu-Pro-Ile-Le
Its gist is a peptide having the structure u-His-Glu-Trp-Lys (hereinafter abbreviated as RJP9).

[0009] The third invention is Tyr-Asn-Glu-
A peptide having the structure Val-Pro (hereinafter referred to as R
JP5) is its summary. The gist of the fourth invention is an angiotensin-converting enzyme-inhibiting peptide obtained by decomposing royal jelly with a protease. The gist of the fifth invention is an orally ingestible composition containing the orally ingestible peptides of the first to fourth inventions.

Next, each invention will be explained in detail. The peptides RJP8, RJP9 and RJP5 in the first to third inventions are Ser-Leu-Pro-L, respectively.
ys-Leu-His-Glu-Trp, Ser-L
eu-Pro-Ile-Leu-His-Glu-Tr
p-Lys and Tyr-Asn-Glu-Val-P
It has a structure called ro, is manufactured by the manufacturing method described below, and the structure is determined by structural analysis. These peptides are peptides that inhibit the aforementioned ACE. The amino acids constituting the above peptide have the following meanings. That is, Ser is serine, Leu is leucine, P
ro is proline, Lys is lysine, His is histidine, Glu is glutamic acid, Trp is tryptophan, Ile is isoleucine, Tyr is tyrosine, Asn is asparagine, and Val is valine.

[0011] Next, the royal jelly-derived ACE-inhibiting peptide referred to in the fourth invention is, for example, RJPn (n represents the number of amino acids; for example, a polypeptide where n = 4 to 20) having an amino acid sequence as shown below. These can be used alone or as a mixture. These ACE-inhibiting peptides can be obtained by treatment with proteolytic enzymes such as trypsin, chymotrypsin, pepsin, bromelain, papain, and proline endopeptidase, as well as proteases derived from bacteria (e.g., proteases (subtilisin, thermolysin, nagase, etc.)). For example, RJP8 is prepared as follows. Honey-derived royal jelly with a pH of 5.0 to 9.0
The protease and undegraded royal jelly protein are precipitated and removed by treating the decomposed product with acid or heat under the following conditions.

Next, the supernatant is neutralized with an alkali if necessary, concentrated under reduced pressure, added to a column packed with a carrier for gel filtration (trade name: Toyo Pearl 40S, manufactured by Toyo Soda Co., Ltd.), etc., and added to a column filled with distilled water. The ACE-inhibited fraction is collected. Then, if necessary, by repeating the same purification or by repeating purification by ion exchange, hydrophobic column chromatography, etc., a peptide having the characteristic of inhibiting ACE can be obtained.

[0013] Also, high performance liquid chromatography using a reverse phase column [eluent: 0.05% trifluoroacetic acid (
The ACE-inhibiting fraction can be separated by gradient elution of acetonitrile/water containing TFA). Moreover, it can be obtained by an organic chemical synthesis method. Below, using a solid phase method using polystyrene resin, peptide R
Synthesize JP8. All the amino acids used are in the L form, and the functional groups are blocked as shown below. That is, Se
r(Tos), Lys(2-chlorophenyl
oxycarbonyl), His(Tos), G
lu(O-Bzl), Trp(Tos). (
) indicates a protecting group. In addition, the abbreviations of amino acid protecting groups represent the following substituents.

Boc: tert-butyloxy-carbony
l group Tos: p-toluenesulfonyl group Bzl: benzyl group PAM: p-methoxy phenyl acet
amidomethyl resinAAn: AA1 (AA1-P
AM) was deprotected using TFA to form H-AA1.
-PAM was synthesized and Boc-AA2 -OH was condensed with DCC (dicyclohexylcarbodiimide) in dichloromethane in dimethylformamide,
Boc-AA2-AA1-PAM is synthesized, and unreacted AA1-PAM is inactivated using acetic anhydride.

The obtained Boc-AA2-AA1-P
AM was again deprotected with TFA and Boc
-AA3 -OH is condensed, and in the same manner, AA8
Condensation reaction is carried out until Note that AA1 = Ser, AA2
=Leu,AA3 =Pro,AA4 =Lys,A
A5 =Leu, AA6 =His, AA7 =Glu
, AA8 = Trp. After the condensation reaction, a deprotection reaction is performed using hydrogen fluoride (HF), and Boc, PAM
and the side chain protecting group is removed to form the desired peptide RJ.
Get P8.

Royal jelly-derived A obtained as above
CE-inhibiting peptides are usually isolated in the form of a powder, and may be used as a composition in an appropriate shape or form together with a suitable non-toxic carrier for oral administration as a pharmaceutical product, or as an oral or enteral nutritional supplement. . Examples of compositions include ACE-inhibiting peptides together with pharmaceutically acceptable carriers (excipients, lubricants, binders, colorants, flavoring agents, flavoring agents) in the form of pharmaceutical preparations for oral administration, e.g. It is available in the form of tablets (sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, and granules.

[0017] It may also be in the form of solid or liquid medicines, foods, or luxury goods, such as confectionery, powdered tea, sports drinks, alcoholic beverages, ice cream, and yogurt. The content of the ACE inhibitory peptide in the above-mentioned orally ingested food can be appropriately selected depending on the dosage form, but is generally in the range of 0.01 to 100% by weight.

As shown below, the oral food of the present invention exhibits a strong ACE inhibitory effect as shown in the test examples shown later, and it can be used continuously for the purpose of preventing hypertension, alleviating hypertension tendency, or regulating blood pressure. It can be ingested, and its effectiveness can be demonstrated by using it as a health food for preventing high blood pressure. When using the oral food of the present invention for this purpose, it is generally appropriate to orally feed the food in the range of 0.01 to 50 mg/kg body weight per day.

Furthermore, the effect can be further improved by appropriately combining or mixing the active peptide with bee products such as royal jelly, propolis, honey, pollen, and bee larva. That is, the above-mentioned bee products may also be processed products (for example, heat-treated products, or products in various shapes such as freeze-dried powder). Also,
Orally edible compositions include the active ingredient together with a pharmaceutically acceptable carrier in the form of a pharmaceutical formulation for oral administration, e.g.
It can be used in the form of tablets, etc.) or in the form of foods and luxury goods.

[0020]

[Action] Peptides RJP8 and RJP of the first to third inventions
9 and RJP5 both exhibit the effect of inhibiting ACE. In the fourth invention, a peptide obtained by using royal jelly as a raw material and decomposing it with a proteolytic enzyme exhibits an angiotensin-converting enzyme inhibitory effect.

[0021] In the fifth invention, the composition containing the peptides of the first to fourth inventions is an orally ingested composition as a health food, and by inhibiting ACE, exerts an effect such as preventing hypertension. be done.

[0022]

[Examples] Examples embodying the present invention will be described below. That is, a production example of an ACE-inhibiting peptide, a structural analysis, a method for measuring the activity of the ACE-inhibiting peptide, and the results of an acute toxicity test of the royal jelly-derived ACE-inhibiting peptide, which is the active ingredient of the oral food of the present invention, will be explained. [Activity measurement of ACE inhibitory peptide] 1 g of rabbit lung was precipitated in acetone, and 5 ml of the dried powder (manufactured by Sigma) was added.
Dissolved in phosphate buffer (pH 8.3) and heated at 10,000G
After centrifugation for 30 minutes, the supernatant was diluted 3 times with the above buffer to obtain an ACE enzyme solution, and enzyme inhibition was measured. The measurement method is Biochem. Pharm, Vol. 20,
pp1637-1648, 1971 and Ana
l. Biochem. , Vol. 84, pp361-3
69, 1978. That is, 1mM tripeptide (Hip-His-Leu, manufactured by Peptide Institute) was added as a substrate to 100mM potassium phosphate buffer (containing 300mM sodium chloride) pH 8.3.
100 μl of ACE enzyme solution and 100 μl of sample solution were added, and after reacting at 37°C for 30 minutes, the reaction was terminated by heating in boiling water for 5 minutes.
This is a method in which the reaction product hippuric acid is derivatized with a trichlortriazine reagent, and the absorbance at a measurement wavelength of 382 nm is measured colorimetrically.

The inhibition rate was calculated using the following formula. Inhibition rate = (E0 - Es)/E0 x 100%E0
: Absorbance at 382 nm without inhibitor Es : Absorbance at 382 nm when containing inhibitor Inhibition rate 50%
The sample concentration at that time is defined as IC50. [ACE inhibition test] The results of ACE inhibition effect (IC50) are shown in Table 1.

[0024]

[Table 1]

In Table 1, CEI12, CEIβ7
, CEI6 indicates a casein-derived peptide. (Quoted from Food Chemical, Nov. 39, 1988.)
[Measurement of protein concentration] The concentration of protein in the sample was measured by the burette method. Conversion was performed using bovine serum albumin as a standard protein. [Production Example 1, Production Example of ACE Inhibiting Peptide] Add 2 g of royal jelly protein to 50 ml of phosphate buffer (pH 7), add 5 mg of trypsin, and incubate at 37° C. for 24 hours. Thereafter, it is heated in boiling water for 10 minutes. After cooling, insoluble materials were removed by centrifugation, and the resulting supernatant was transferred to a column (φ30
mm x 30 cm).

The unadsorbed fraction was added to a column (φ20 mm×30 cm) packed with a cation exchange resin (trade name: SP Toyopearl, manufactured by Toyo Soda Co., Ltd.), and the adsorbed fraction was subjected to gradient elution of ammonium formate. Elution conditions are 0-0.5M ammonium formate aqueous solution, pH 6.8
, flow rate 1.0 ml/min. Furthermore, ACE inhibitory fractions with a molecular weight of less than 5000 were collected using an Amicon concentrator, and after freeze-drying, 283 mg of ACE inhibitory peptide (white powder) was obtained.
I got it. This is RJP8, RJP9 and RJP5
It is a crude composition containing. [Production Example 2, Production Example of ACE Inhibiting Peptide] Add 2 g of royal jelly protein to 50 ml of phosphate buffer (pH 2), add 10 mg of pepsin, and incubate at 37° C. for 24 hours. Thereafter, heat treatment is performed in boiling water for 10 minutes. After cooling, insoluble matter was removed by centrifugation, and the resulting supernatant was added to a column (φ30 mm x 100 cm) packed with Toyopearl 40S, and eluted with distilled water.

The active fraction was added to a column (φ20 mm×30 cm) packed with SP Toyopearl, and the adsorbed fraction was subjected to gradient elution with ammonium formate. Elution conditions were 0-0.5M ammonium formate aqueous solution, pH 6.
8. The flow rate is 1.0 ml/min. Furthermore, using an Amicon concentrator, AC with a molecular weight of 500 or more and less than 5000
E-inhibitory fractions were collected and lyophilized, followed by ACE-inhibiting peptide 2.
58 mg (white powder) was obtained. This is RJP8, RJ
This is a crude composition containing P9 and RJP5. [Production Example 3, Production Example of RJP8] 2 g of royal jelly protein was added to 50 ml of phosphate buffer (pH 7), 5 mg of trypsin was added, and the mixture was incubated at 37° C. for 24 hours. Thereafter, the pH was adjusted to 1 with hydrochloric acid, insoluble materials were removed by centrifugation, and the resulting supernatant was concentrated under reduced pressure. This was concentrated to 5 ml, subjected to Sephadex G-25 column chromatography (φ30 mm x 100 cm), and eluted with distilled water, and the active fraction was collected and concentrated.

Furthermore, high performance liquid chromatography (H
PLC): Shimadzu LC-8A type; Column Shim
pack PREP-ODS (L) (φ50mm x 2
5 cm), and the active fraction was collected by gradient elution of acetonitrile containing 0.05% TFA. The flow rate was 10 ml/min, the detector was SPD-M6A, and the detection wavelength was 210 nm. Among the purified ACE-inhibiting peptides, the yield of RJP8 after freeze-drying was 15.
．． 3 mg (white powder). [Production Example 4, Production Example of RJP9 and RJP5] The ACE-inhibiting peptide obtained in Production Example 1 was further separated and purified by HPLC using a reversed phase column, and RJP9 was obtained from several ACE-inhibiting peptides in the same manner as in Production Example 3. and RJP5
I got it. [Example of amino acid primary structure and amino acid analysis] Next, amino acid primary structure analysis and amino acid analysis were performed.

The peptides of Production Examples 3 and 4 were shown to be the following polypeptides using a fully automatic protein primary structure analyzer PSQ-1 system manufactured by Shimadzu Corporation. In addition, RJP8
Analytical results supporting the amino acid composition of , RJP9 and RJP5 were obtained.

(Primary structure of RJP8) Ser-Leu-Pro-Lys-Leu-His-G
lu-Trp (primary structure of RJP9) Ser-Leu-Pro-Ile-Leu-His-G
lu-Trp-Lys (primary structure of RJP5) Tyr-Asn-Glu-Val-Pro (RJP8
(Amino acid composition of RJP9) Glu 14.30% Ser 14.28% His 14.32% Pro 14.25% Leu 28.72% Lys 14.16% Trp - (Unmeasurable) (Amino acid composition of RJP9) Glu 12.56 % Ser 12.37% His 12.41% Pro 12.43% Leu 25.76% Lys 12.67% Ile 12.36% Trp - (Unmeasurable) (Amino acid composition of RJP5) Glu 20.60% Val 21 .32% Pro 19.56% Asn 19.82% Tyr 19.96% [Acute toxicity test of each ACE inhibitory peptide] (1) Test method A 5% aqueous solution of each ACE inhibitory peptide was used as a sample, and IC
1 group of R mice with an average weight of 21.9 ± 0.4 g
0 animals were used in the experiment. The animals were fasted overnight before the test, and, for example, 5 g/Kg of the peptide was orally administered using a probe. After administration, the animals were observed for life, death, general symptoms, etc. for one week. (2) Test results For example, the LD50 of RJP5 is 5g/Kg or more,
There were no deaths. In addition, observation of general symptoms revealed no abnormal conditions such as collapse, piloerection, abnormal breathing, trembling, crawling on the stomach, or sweating. Note that the results for other ACE inhibitory peptides were also similar.

Next, production example 1, RJP8, RJP9
The antihypertensive effects of ACE inhibitory peptides and RJP5 were tested. [Experimental example 1, antihypertensive test of each ACE inhibitory peptide] (1)
Sample and administration method Peptide 1 obtained in Production Example 1, Production Example 3, and Production Example 4
Dissolved in physiological saline to give g/Kg body weight5
ml was forcibly administered orally using a sonde. (2) Experimental Animals Eight-week-old male spontaneously hypertensive rats (SHR) were preliminarily bred for one week, and then five rats were used in each group. (3) Blood pressure measurement Before and after administration, the blood pressure of each rat was measured over time using a non-invasive tail artery sphygmomanometer (RIKEN). Then, the average value was calculated. (4) Test results The results are shown in Table 2. As is clear from Table 2, a dose-dependent hypotensive effect was observed. Moreover, the decrease continued even 6 hours after administration. [Experimental Example 2, Blood Pressure Lowering Test of Health Food of Formulation Example 7] 3 g/kg body weight of the health food of Formulation Example 7 (propolis liquid mixed with RJP8), which will be described later, was forcibly administered using a sonde. As a control, one containing no ACE inhibitory peptide was used.

The following procedure was carried out in the same manner as in Experimental Example 1. The results are shown in Table 2. Next, the peptide of Production Example 1 or RJP8
were blended to prepare oral feeding compositions shown in Formulation Examples 1 to 7 below. [Blend example 1] Skimmed milk powder 8.0% by weight, vegetable oil 11
．． 0% by weight, sugar 14.0% by weight, stabilizer 0.
3% by weight, emulsifier 0.3% by weight, fragrance 0.1% by weight
, 1.0% by weight of the peptide of Production Example 1, 7.0% by weight of egg yolk, and 55.0% by weight of water were mixed and stirred to obtain ice cream. [Formulation example 2] Purified honey 12000mg, vitamin C 1000mg, sodium L-glutamate 10
10 mg of nicotinic acid amide, 5 mg of the peptide of Production Example 1, and an appropriate amount of fragrance were added with water to make a total volume of 50 ml, and the mixture was mixed and stirred to obtain a drink. [Formulation Example 3] Table salt was obtained by mixing 5% by weight of the peptide of Production Example 1 with common salt. [Formulation Example 4] Miso was prepared by kneading 0.5% by weight of the peptide of Production Example 1 into miso produced by a normal production method. [Blend Example 5] 100ml of soy sauce made using normal manufacturing method
Soy sauce was prepared by mixing 0.5% by weight of the peptide of Production Example 1 per liter. [Formulation Example 6] Bread was produced by adding 0.1% by weight of the peptide of Production Example 1 to bread dough produced by a conventional production method. [Formulation Example 7] 1% by weight of RJ per 100g of health food made by normal manufacturing methods, such as propolis food
A health food was prepared by adding P8.

[0033]

[Table 2]

[0034] In Table 2, the value of blood pressure reduction shows the value of pre-administration blood pressure value - post-administration blood pressure value. In addition, the blood pressure value before administration in each example is 150 to 160 mmHg. (Note 1) Based on Test Example 1 of JP-A-62-270533. The initial value of blood pressure in this case is 174 mmHg. (Note 2) This is the value 5 hours after administration.

As mentioned above, it has been said since ancient times that royal jelly is an effective antihypertensive substance that can be obtained by orally administering a composition containing peptides obtained by decomposing royal jelly with proteolytic enzymes. This proves its physiological effects. FIG. 1 is a graph showing the relationship between the number of fractions of the peptide obtained in Production Example 1 and the absorbance at a wavelength of 280 nm, and the ACE inhibitory activity. In the figure,
Solid line 1 represents the peptides RJP8 and RJ obtained in Production Example 1.
This is a line showing the relationship between the number of fractions and the absorbance for a crude composition containing P9 and RJP5. Moreover, broken line 2 is a line showing the ACE inhibitory activity of this crude composition. As can be seen from the figure, as the absorbance of the solid line 1 increases, the dashed line 2 also increases correspondingly, and a correlation is recognized. Then, RJP8 is placed at each peak position.
, RJP9 or RJP5, and by isolating it, RJP8, RJP9 and R
JP5 is obtained.

[0036]

[Effect of the invention] The peptide R as used in the first to third inventions
JP8, RJP9, and RJP5 are all novel peptides, and have the excellent properties of being highly safe and useful as orally ingested compositions such as pharmaceuticals or foods that are highly effective in the prevention and treatment of hypertension. be effective.

[0037] In the fourth invention, the peptide obtained by decomposing royal jelly with a proteolytic enzyme is particularly effective in preventing and treating hypertension. Fifth
In the invention, the peptides of the first to fourth inventions are orally ingestible, and a composition containing the peptides as a main component is an orally ingestible composition suitable as a health food for preventing hypertension, etc. This has the effect of

[Brief explanation of the drawing]

FIG. 1 is a graph representing an example of the present invention, showing the relationship between the number of peptide fractions and absorbance, and the angiotensin-converting enzyme inhibitory activity.

[Sequence list]

Sequence number: 1 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu Pro Lys Leu His G
lu Trp1 5 SEQ ID NO: 2 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu Pro Ile Leu His G
lu Trp Lys1
5 Sequence number: 3 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Asn Glu Val Pro1
5

Claims (5)

[Claims] [Claim 1] Ser-Leu-Pro-Lys-L
A peptide having the structure eu-His-Glu-Trp. [Claim 2] Ser-Leu-Pro-Ile-L
A peptide having a structure of eu-His-Glu-Trp-Lys. [Claim 3] Tyr-Asn-Glu-Val-P
A peptide having the structure ro. 4. An angiotensin-converting enzyme-inhibiting peptide obtained by decomposing royal jelly with a protease. 5. An orally ingestible composition containing the peptide of claims 1 to 4, which is orally ingestible.