JP2004051529A - New physiologically active peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new physiologically active peptide having angiotensin converting enzyme inhibiting activities and exhibiting persistent antihypertensive actions. <P>SOLUTION: The new physiologically active peptide is represented by Val-Trp-Ile-Ser. More specifically, it is obtained by hydrolyzing protein originating from a rape with a protease. An oil cake obtained by removing oils and fats from rape seeds is preferably employed as the protein originating from the rape, and subtilisin is preferably employed as the protease. <P>COPYRIGHT: (C)2004,JPO

Description

＊Ｐ＜０．０１：ペプチドを含まない同量の生理食塩水を投与した場合の血圧の変化の平均値（ｎ＝４）に対して有意水準１％で有意性を示した。
【００２９】
【発明の効果】
本発明の新規ペプチドは、アンジオテンシン変換酵素阻害活性を有し、しかも持続性のある血圧降下作用を示す。[0001]
[Industrial applications]
The present invention relates to a novel peptide which has an angiotensin converting enzyme inhibitory effect and is useful as a persistent antihypertensive agent.
[0002]
[Prior art]
It has been known that proteins contained in foods have various physiological activities as well as nutritional effects.
For example, angiotensin-converting enzyme inhibitory peptides and peptides that relax arterial blood vessels are known to exhibit blood pressure lowering activity.
Angiotensin converting enzyme is mainly present in lung, vascular endothelial cells and renal proximal tubules, and acts on angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) to cause angiotensin-converting enzyme. It is an enzyme that cleaves and releases a dipeptide (His ⁹ -Leu ¹⁰ ) from the C-terminus of I to generate angiotensin II having a strong pressor action. This enzyme also has the action of destroying and inactivating bradykinin, which is a hypotensive substance in the living body, and is strongly involved in the pressor system. Therefore, if the activity of such angiotensin converting enzyme is inhibited, it is considered that the blood pressure decreases, and it is clinically effective for preventing and treating hypertension.
As such angiotensin converting enzyme inhibitor, captopril which is a proline derivative was synthesized, and since its antihypertensive activity was confirmed, various angiotensin converting enzyme inhibitors have been synthesized, and in recent years, acquisition from natural products has been actively attempted. By the way.
[0003]
For example, {circle around (1)} Japanese Patent Application Laid-Open No. 2-36127 discloses an angiotensin conversion containing a peptide having a C-terminal amino acid sequence of Leu-Pro-Pro having a degree of polymerization of 3 to 5 by hydrolyzing γ-zein with thermolysin. Enzyme inhibitors have been reported. Also, {2} J. Biol. Chem. , 255 (2) 401 (1980), report a dipeptide having angiotensin converting enzyme inhibitory activity, and describe Val-Trp (IC ₅₀ = 1.6 μM) as one of such peptides.
[0004]
[Problems to be solved by the invention]
However, although the peptide disclosed in the above (1) and the peptide represented by Val-Trp described in the above (2) have angiotensin converting enzyme inhibitory activity, when they are orally administered to spontaneously hypertensive rats (hereinafter referred to as SHR), their blood pressure is reduced. It was found that the blood pressure returned to the original blood pressure within 3 hours after administration without sustaining a lowering effect.
In recent years, there has been a demand for a peptide that exhibits a significant blood pressure lowering effect even by oral administration and has a longer blood pressure lowering duration than conventional peptides and has a sustained blood pressure lowering.
[0005]
[Means for Solving the Problems]
The present inventors have intensively searched for a substance that has a sustained blood pressure lowering effect as a natural substance in order to solve such a problem, and found that the presence of a substance having such an activity in a composition obtained by hydrolyzing a rapeseed-derived protein with a protease. The present inventors have found that the substance is a peptide represented by Val-Trp-Ile-Ser and completed the present invention.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
The peptide of the present invention is represented by the sequence of Val-Trp-Ile-Ser and is a novel peptide which has not been described in any literature. Here, Val means valine, Trp means tryptophan, Ile means isoleucine, Ser means serine, and all such amino acids are in L-form.
[0007]
In producing the peptide of the present invention, although it is not particularly limited, it can also be produced by hydrolyzing a rapeseed-derived protein with a protease or by synthesizing by applying a conventional means of peptide synthesis, These methods will be described. Note that the former is preferable in that inexpensive raw materials can be used.
[0008]
First, a method for hydrolyzing rapeseed protein with a protease will be described.
As such a protein, rapeseed seeds, other rapeseed constituents, rapeseed tissue cultures and the like may be directly used, but it is easy to hydrolyze and purify the oilseed obtained by removing oils and fats from rapeseed seeds, and moreover, foods It is preferable because oil lees, an unused resource, can be effectively used.
The method for removing fats and oils is not particularly limited, but the fats and oils are usually removed by extraction with a solvent, pressing and the like.
[0009]
Examples of the protease include subtilisin, trypsin, chymotrypsin, pancreatin, thermolysin, pepsin, papain, alkaline protease, and the like. Subtilisin and pepsin are preferable, and subtilisin is particularly preferable.
[0010]
In hydrolyzing the oil cake with a protease, the oil cake may be dispersed in water and hydrolyzed as it is. However, after extracting the protein with a sodium chloride aqueous solution of about 1 M, dialysis, membrane treatment, column chromatography It is preferable to perform a pretreatment for removing low molecular substances such as glyoxylate by chromatography or the like because the peptide can be easily purified from the hydrolyzate.
In carrying out the hydrolysis, a dispersion of oil cake, preferably, an aqueous solution of the extracted protein pretreated as described above is adjusted to a protein concentration of 0.1 to 20% by weight, and the protease is added to the protein in an amount of 0.1 to 20% by weight. It is preferable to add about 1% by weight. If the amount is less than 0.1% by weight, hydrolysis is not sufficiently performed, and if the amount exceeds 1% by weight, no improvement in yield can be expected. At this time, the protease may be added as a powder, or may be added as an aqueous solution. After the addition of the protease, hydrolysis is carried out at a temperature of about 10 to 85 ° C, preferably 30 to 60 ° C for 0.1 to 48 hours, preferably 1 to 10 hours. The pH at this time is not particularly limited, and the pH is about 1 to 9, but it is preferably about 6 to 8 for subtilisin and about 1 to 3 for pepsin. After the start of the hydrolysis, the reaction is carried out with the degradation rate of the protein as a standard, and then the hydrolysis is stopped by adding an aqueous acid solution such as hydrochloric acid to the decomposition solution or by boiling.
[0011]
The hydrolyzate obtained by the above hydrolysis is a mixture containing the novel peptide of the present invention and other peptides, and the desired product is obtained by purifying and isolating the peptide of the present invention therefrom. However, in practical use, the mixture can be used as it is.
In purifying the peptide of the present invention from the hydrolyzed solution, the hydrolyzed solution is used as it is, or the hydrolyzed solution is filtered by a known operation such as centrifugation, followed by extraction, concentration, drying and the like. Differences in adsorption affinity for various adsorbents, differences in solubility or solubility in various solvents, differences in partitioning between two immiscible liquid phases, differences in elution rate based on molecular size, It is preferable to purify the peptide of the present invention by appropriately selecting a purification means utilizing a difference in precipitation property or a precipitation rate, and these purification means may be used alone or in combination in an arbitrary order, and may be repeated, if necessary. Applied.
[0012]
Next, a method for producing the peptide of the present invention by a peptide synthesis method will be described. Such a method is carried out by a liquid phase method or a solid phase method. In each case, a raw material having a reactive carboxyl group corresponding to one of two types of fragments bisected at an arbitrary position of a peptide bond, A method using an active ester such as 2- (1H-Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or a method using carbodiimide is performed by using a raw material having a corresponding reactive amino group. And the like.
[0013]
Functional groups that should not participate in the reaction in this condensation are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (Bz), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl-protecting group include groups capable of forming an alkyl ester, a benzyl ester and the like. In the case of the solid phase method, the carboxyl group at the C-terminus is chlorotrityl resin, chloromethyl resin, oxymethyl resin, P-alkoxy. It is bound to a carrier such as benzyl alcohol resin.
[0014]
After the completion of the condensation reaction, the protecting group is removed. In the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved, and the peptide is purified according to a usual method. Examples of such purification include methods such as reverse-phase liquid chromatography, ion exchange chromatography, and affinity chromatography.
Analysis of the synthesized peptide is performed by a method such as a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, LC-MS, or the like.
[0015]
The novel peptide of the present invention thus obtained is useful as a pharmaceutical, a functional food, and the like. Hereinafter, a case where the peptide is used as a pharmaceutical will be described.
The administration route of the peptide of the present invention may be any of oral administration, intravascular administration, and rectal administration, but oral administration is preferred. The dose of the peptide of the present invention to humans cannot be unconditionally determined depending on the administration method, the patient's symptoms, age, etc., but is usually 0.1 to 1000 mg, preferably 1 to 100 mg once to 1 to 3 times per day. It is preferable that The peptide of the present invention is usually administered in the form of a preparation. As the carriers and auxiliaries used in the preparation, substances commonly used in the field of preparation and which do not react with the peptide of the present invention are used.
[0016]
Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resin, methylcellulose , Gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, Fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macro Lumpur, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and the like.
[0017]
Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another appropriate medium when used. Tablets and granules may be coated by a known method. In the case of injections, the peptide of the present invention is prepared by dissolving the peptide in water.If necessary, the peptide may be dissolved in physiological saline or a glucose solution, or a buffer or a preservative may be added. Good.
[0018]
These preparations can contain the peptide of the present invention in an amount of 0.01% by weight or more, preferably 0.5 to 70% by weight. These formulations may also contain other components of therapeutic value.
[0019]
【Example】
Next, the present invention will be described more specifically with reference to examples.
Example 1
(Production and purification of peptide)
An oil dispersion obtained by extracting rapeseed seeds with hexane to remove oils and fats (10 g) was added to 100 ml of a 1 M aqueous solution of sodium chloride, sufficiently stirred and centrifuged to precipitate solids, and then the supernatant was added to water. After sufficient dialysis, the mixture was freeze-dried to obtain a powdery protein. Next, 1 g of the obtained protein was dissolved in water so as to have a concentration of 2% by weight, and 0.01 g of subtilisin (manufactured by Sigma, derived from Bacillus licheniformis, type VIII) was added thereto. The hydrolysis was carried out at a temperature of 5 ° C. for 5 hours. After the hydrolysis, the mixture was boiled for 10 minutes to stop the hydrolysis.
After cooling the hydrolyzate obtained above, the water-insoluble matter was concentrated by centrifugation and purified by high performance liquid chromatography. In such high performance liquid chromatography, a fraction having an inhibitory activity is firstly fractionated on an ODS-column, the fraction is further purified on a CN and a column, and further, an inhibitory activity fraction is sequentially purified on a 5PE-MS and 5NPE column. Thus, 0.015 mg of a single peptide was obtained.
As a result of analyzing the amino acid sequence of the peptide obtained by applying an automatic Edman degradation method using a gas-phase protein sequencer (“Type 492” manufactured by Applied Biosystems), the following sequence was confirmed.
Val-Trp-Ile-Ser
[0020]
(Chemical synthesis of peptide)
0.6 g of a commercially available Fmoc-Ser (tBu) resin (substitution ratio: 0.5 meq / g) was fractionated into a reaction vessel of a PS3-type peptide synthesizer (manufactured by Protein Technologies), and a new peptide was synthesized as follows. went.
First, the above resin was placed in a reaction vessel, and 1 mmol of Fmoc-Ile and 1 mmol of HBTU as an activator dissolved in 10 ml of dimethylformamide containing 0.4 M N-methylmorpholine were placed in a reaction vessel. In addition, a stirring reaction was performed at room temperature for 20 minutes.
[0021]
The Fmoc group was removed from the obtained resin in 20 ml of dimethylformamide containing 20% by volume of piperidine, and then Fmoc-amino acids were sequentially coupled from the C-terminus in the same manner as in the above-described method of coupling Fmoc-Ile. And Val-Trp-Ile-Ser (tBu) resin. The resin was dissolved in 10 ml of a deprotection solution (a mixture of 90% by volume trifluoroacetic acid, 5% by volume thioanisole, 3% by volume ethanedithiol, 2% by volume ethyl methyl sulfide, and 1% by volume methyl indole) at room temperature. After stirring for an hour, the peptide was released from the resin.
[0022]
To this was added 40 ml of cold ether to precipitate the peptide, which was further washed three times with cold ether to obtain a crude peptide. This ODS column _{(Cosmosil5C 18 -ARII, 20 × 250mm} ) by reverse phase chromatography on developed by linear gradient of acetonitrile containing 0.1 wt% trifluoroacetic acid, and purified, Val-Trp-Ile-Ser Was obtained. The product was analyzed using a protein sequencer (“492”, manufactured by Applied Biosystems) to confirm that the sequence was as described above.
[0023]
(Measurement of angiotensin converting enzyme inhibitory activity)
The angiotensin converting enzyme inhibitory activity of the above peptide was measured in the following manner according to the method of Cheung Cushman, which was improved by Yamamoto et al. [Journal of the Japanese Society for Chest Disease, (18), 1297 (1980)].
Enzyme substrate; Bz (benzyl) -Gly-His-Leu
Enzyme: Rabbit Lung Acetone Powder (Sigma)
Final concentration: 100 mM boric acid, 400 mM sodium chloride buffer (pH 8.3), 5 mM enzyme substrate, 3 μU enzyme
[0024]
The buffer, enzyme substrate, and enzyme were mixed to the final concentration described above, and the whole was adjusted to 200 μl with water, and then reacted at 37 ° C. for 30 minutes. The reaction was terminated using 200 μl of 1N hydrochloric acid. 1.5 ml of ethyl acetate was added to the reaction-terminated liquid, and the mixture was stirred with Vortex for 15 seconds and centrifuged. 1.0 ml was taken out from the ethyl acetate layer, ethyl acetate was distilled off, 1 ml of distilled water was added thereto to dissolve the residue, and the value of the ultraviolet absorption ₂₂₈ nm (OD ₂₂₈ ) of the extracted hippuric acid was measured. .
[0025]
Percent inhibition by the _{OD 228} of when reacted without peptide as 100%, was determined _{OD 228} when the reaction time of 0 minutes 0%. The concentration IC ₅₀ of the peptide at an inhibition rate of 50% was 30 μM.
[0026]
(Measurement of blood pressure lowering effect)
The above peptide was orally administered to a 20-week-old SHR (n = 4) at a rate of 25 μmol / kg using a sonde needle, and the blood pressure in the tail artery was measured over time by the tail-cuff method, The maximum hypotensive value in systolic blood pressure was examined.
As a result, a significant blood pressure lowering effect at 2 and 4 hours after administration [2 hours later: 12.5 ± 2.87 mmHg (P <0.01), 4 hours later: 9.5 ± 2.5 mmHg (P <0 .01)], a blood pressure drop of 7 mmHg was observed even after 6 hours, and a sustained blood pressure lowering effect was observed (Table 1).
[0027]
Comparative Example 1
Evaluation was performed in the same manner as in Example 1 using a commercially available Val-Trp (manufactured by Bachem).
As a result, the IC ₅₀ of the peptide was 1.6 μM.
When such a peptide was administered orally, a significant blood pressure lowering effect [2 hours later: 10.75 ± 2.69 mmHg (P <0.01)] was shown 2 hours after administration, but 7 mmHg 4 hours later. The blood pressure decreased to about 3.5 mmHg after 6 hours, and no sustained blood pressure lowering action was observed (Table 1).
[0028]
[Table 1]

* P <0.01: Significance was shown at a significance level of 1% with respect to the mean change in blood pressure (n = 4) when the same amount of physiological saline containing no peptide was administered.
[0029]
【The invention's effect】
INDUSTRIAL APPLICABILITY The novel peptide of the present invention has an angiotensin converting enzyme inhibitory activity and exhibits a sustained blood pressure lowering effect.

Claims (4)

A novel bioactive peptide represented by Val-Trp-Ile-Ser. The novel bioactive peptide according to claim 1, wherein the peptide is obtained by hydrolyzing a rapeseed protein with a protease. 3. The novel bioactive peptide according to claim 2, wherein the rapeseed-derived protein is oil cake obtained by removing oil and fat from rapeseed seeds. 3. The novel bioactive peptide according to claim 2, wherein the protease is subtilisin.