JP2007297324A - Peptide, method for producing the same and angiotensin-converting enzyme inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new peptide and provide a method for producing the same, an angiotensin-converting enzyme inhibitor (ACE inhibitor) containing the new peptide as an effective component, a pharmaceutical composition containing the ACE inhibitor and health food or food. <P>SOLUTION: The peptide comprises a specific L-amino acid sequence produced by decomposing a protein extracted from spirulina with a protease and isolating from the decomposed product. The ACE inhibitor contains the peptide as an effective component. The pharmaceutical composition, the health food and the food containing the ACE inhibitor are provided. The method for producing the peptide has processes to treat microalgae with a protease in an aqueous solution to extract peptides into the aqueous solution, subsequently remove water-insoluble materials in the aqueous solution and fractionate the obtained peptides. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel peptide having a specific amino acid sequence, a method for producing the same, an angiotensin converting enzyme inhibitor containing the peptide, and a pharmaceutical composition, health food, and food containing the enzyme.

Angiotensin is a polypeptide renin secreted in the renal vein is made to act on the renin substrate contained in alpha ₂ globulin fraction of the plasma, firstly it angiotensin I is made, which is biologically active Does not have. Angiotensin I loses two amino acids and is converted into angiotensin II by the action of angiotensin converting enzyme (hereinafter abbreviated as ACE) mainly in the pulmonary circulation in the circulating blood.
Renin and angiotensin constitute an important pressor system in the body and are called the renin-angiotensin system, but the main active substance is angiotensin II. The most important action of angiotensin II clinically is a blood pressure raising action, and is the most powerful pressor substance in the body that contracts vascular smooth muscles to increase blood pressure. Its vasoconstrictive action is strong in arterioles but weak in venous systems, and strong in kidney and visceral blood vessels, but weak in limbs, brain, heart and lung. Angiotensin II plays an important role in the body's sodium balance and blood pressure maintenance. When sodium disappears and blood pressure decreases, renin and angiotensin II increase, and directly through aldosterone secretion, sodium retention and blood pressure increase. Work. In hypertensive diseases, it plays an important role in the origin or maintenance mechanism of hypertension (Non-patent Document 1).

As described above, the ACE inhibitor specifically inhibits the production of angiotensin II by inhibiting the action of the converting enzyme that produces angiotensin II from angiotensin I, thereby reducing the plasma concentration of angiotensin II and lowering the blood pressure. Indicates.
So far, ACE inhibitors include natural products or substances derived from natural products, snake venom-derived bradykinin-enhancing factor (Non-Patent Document 2), gelatin collagenase digestion-derived 6 peptides (Non-Patent Document 3), cattle Peptides derived from casein tryptic digest (Non-patent Document 4), five hexapeptides derived from sardine muscle (Patent Document 1), tetrapeptide derived from seaweed (Patent Document 2), and pentapeptide (Patent Document 3), Examples include ginseng-derived pentapeptide (Patent Document 4) and chlorella-derived pentapeptide (Patent Document 5), both of which can be ACE inhibitors.
Furthermore, although proposals have been made for di- and tripeptides (Patent Document 6 and Patent Document 7) having a short chain length obtained by the synthesis method, although they have been passed for a long time since they were discovered, they are still There is no report that development is progressing.

In recent years, health foods containing peptides having the ACE inhibitory action have been developed in order to improve health awareness. For example, as peptides extracted from wakame, jelly foods containing Phe-Tyr, Val-Tyr, or Ile-Tyr, lactic acid beverages containing Val-Pro, or Ile-Pro-Pro, etc. have been developed and sold. ing.
However, the peptide consisting of the L-amino acid sequence represented by SEQ ID NO: 1 of the present invention has not been known so far, and neither the peptide has an excellent ACE inhibitory action nor its production method is known at all. It was.

Next, regarding the method of treating microalgae with a proteolytic enzyme, Patent Document 8 discloses that nutrient components are abundant, there are few tastes and smells peculiar to microalgae, and there are precipitates and insolubles even when added to food. There is a description regarding a method for producing a microalgae water extract that does not precipitate.
However, in these conventional techniques, the peptide consisting of the L-amino acid sequence represented by SEQ ID NO: 1 disclosed in the present invention is obtained, the peptide has an excellent ACE inhibitory action, and is extremely useful as a blood pressure lowering agent. Up to now, it has not been described or suggested for superiority.
Medical Science Dictionary, 94 pages, 1982, Kodansha S. H. Ferrea et al: Biochemistry, 9, 3583 (1970). G. Oshima et al: Biochim. Biophs. Acta, 566, 128 (1979) S. Maruyama et al. : Agric. Biol. Chem. 46, 1393 (1983) Japanese Patent No. 2046483 Japanese Patent No. 2678180 Japanese Patent Laid-Open No. 10-36391 Japanese Patent No. 2920829 Japanese Patent No. 2990354 Japanese Patent No. 948071 JP-A-6-16568 JP 2004-204034 A

  The subject of this invention is providing the novel peptide, its manufacturing method, the ACE inhibitor which uses it as an active ingredient, and the pharmaceutical composition, health food, or foodstuff containing the said ACE inhibitor.

As a result of intensive studies to solve the above problems, the present inventors have obtained the following knowledge. That is,
(1) Peptides can be obtained by treating microalgae with proteolytic enzymes. (2) As a result of isolation and identification from peptides, a peptide comprising the L-amino acid sequence represented by SEQ ID NO: 1 is obtained. (3) The novel peptide has been found to have an excellent ACE inhibitory action, and the present invention has been completed.

That is, the present invention provides a peptide consisting of the L-amino acid sequence represented by SEQ ID NO: 1.
The present invention also provides a peptide comprising the L-amino acid sequence represented by any one of SEQ ID NOs: 2 to 5 and having ACE inhibitory activity.
Moreover, this invention provides the ACE inhibitor which uses the peptide which consists of L-amino acid sequence represented by sequence number 1 as an active ingredient.
Moreover, this invention provides the ACE inhibitor which uses the peptide which consists of L-amino acid sequence represented by any one of sequence number 2-5 as an active ingredient.
The present invention also provides a pharmaceutical composition, health food or food containing such an ACE inhibitor.
In addition, the present invention treats microalgae with a proteolytic enzyme in an aqueous solution to extract peptides into the aqueous solution, then removes insolubles in the aqueous solution, and fractionates the obtained peptides. It has a process, and provides a method for producing the peptide represented by SEQ ID NO: 1.

  When the peptide or ACE inhibitor of the present invention is used, the action of the converting enzyme that produces angiotensin II from angiotensin I is inhibited, the production of angiotensin II is specifically suppressed, the plasma concentration of angiotensin II is decreased, and the blood pressure is decreased. Shows the effect. Therefore, the ACE inhibitor of the present invention is effective as a blood pressure lowering agent.

Hereinafter, the present invention will be described in detail.
The peptide represented by SEQ ID NO: 1 of the present invention has an excellent ACE inhibitory action, and exhibits a blood pressure lowering action and a bradykinin inactivation inhibiting action. Therefore, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension, therapeutic agent, normalization of organ circulation and improvement of long-term prognosis (life extension effect) for these congestive heart failure, treatment of heart failure Useful as an agent.
Moreover, since the peptide of the present invention has a sequence structure of only L-amino acids and is gradually degraded by protease in vivo after administration, the toxicity is extremely low and the safety is extremely high (LD ₅₀ > 2000 mg / kg). Rat oral administration).
Moreover, the peptide of this invention includes what has an amino acid sequence represented by either of sequence number 2-5, and has ACE inhibitory activity. In SEQ ID NOs: 2 to 5, Xaa represents any natural amino acid.

Next, a method for producing the peptide represented by SEQ ID NO: 1 of the present invention will be described.
The microalgae used in the present invention is a microalgae that grows in water and is a phytoplankton having a size of several microns to several hundred microns among photosynthetic plants, such as Spirulina genus, Chlorella. Genus (Chlorella), genus Aphanizomenon, genus Fischerella, genus Anabaena, genus Nostoc, genus Aphanothem, genus Haematocoun (cc) (Scenedesmus) genus, etc., which are produced on an industrial scale and whose safety has been confirmed, Spirulina, Chlorella, Haematococcus, Donariella , It is preferable that belong to the genus Scenedesmus.

  Spirulina is a fine unicellular microorganism belonging to the genus Arthrospira and Spirulina that has been included in the cyanobacteria and has been collectively referred to as Spirulina. Arthrospira platensis, Arthrospira maxima, Arthrospira geitleri, Arthrospira sir sul, Sul, Sul But, But artificially it can be cultured, because the easy availability, Arthrospira platensis, Arthrospira maxima, Arthrospira-Geitoreri, the Arthrospira-Saiamize preferred.

Chlorella is a microalgae of the genus Chlorella, which is easily available and excellent in safety. For example, Chlorella vulgaris, Chlorella regularis, Chlorella pyrenoidosa (Chlorella pyrenoidosa) ), Chlorella ellipsidea, etc. are preferred.
Haematococcus is an algae belonging to the genus Hematococcus belonging to the Chlamydomonas family, which is useful as an astaxanthin-producing alga that is a carotenoid pigment with high added value as food and feed.
Donariella is a microalgae found in the Dead Sea of Israel, and is a microalgae rich in carotenoids that have antioxidant activity, and is particularly useful as an algae producing β-carotene.
Senedesmus is a green algae belonging to the genus Senedesmus, and is called the Japanese name “Ikadamo”. Algae such as (Scenedesmus chlorellodes), Senedesmus costuratus, Senedesmus nanus, Senedesmus obliquus, etc. are known.

  Examples of the microalgae used in the present invention include raw microalgae, microalgae subjected to a drying treatment, and components derived from microalgae. Raw microalgae can be obtained, for example, by harvesting microalgae cultured in water by a method such as centrifugation or filtration. Although raw microalgae can be used as it is after harvesting from the culture pond, it is preferably washed with water or physiological saline. Examples of the microalgae subjected to the drying treatment include those obtained by subjecting the raw microalgae obtained by the above method to freeze-drying treatment or spray-drying treatment. Examples of components derived from microalgae include those obtained by subjecting microalgae to mechanical treatment such as ultrasonic irradiation and homogenization, and those obtained by subjecting microalgae to chemical treatment such as enzyme treatment. It is done.

Furthermore, in the present invention, residual microalgae can be used as a raw material for producing the peptide represented by SEQ ID NO: 1. The term “residual microalgae” as used herein refers to microalgae obtained after extracting useful components other than the peptide of the present invention, the polypeptide from which the peptide is derived, and the protein contained in the microalgae in the extraction medium.
Extraction can be performed with water, hot water, an organic solvent, a supercritical extraction gas, or the like, and these can be used alone or in combination. Examples of the organic solvent used include alcohol solvents such as ethanol, hydrocarbon solvents such as hexane, and ketone solvents such as acetone.
For example, it is possible to extract with water-containing ethanol, ethanol, acetone, or a mixture of two or more of these at room temperature.
The extraction operation can also be performed with carbon dioxide gas or the like used for supercritical extraction.
The extraction temperature may be any temperature at which the intended useful substance can be extracted most effectively, but in general, a temperature from room temperature to the boiling point of the medium can be mentioned.
Even when extraction is performed using residual microalgae obtained by performing these extraction operations as raw materials, the ACE inhibitory action of the obtained peptides can be suitably used without being reduced. Effective use is possible, which is industrially preferable.
For example, when the microalga is Spirulina, the contained phycocyanin is useful as a blue pigment, but conventionally, the residue after extraction of phycocyanin with water has been discarded. However, according to the present invention, the ACE inhibitory action of the peptides obtained does not decrease even when the enzyme treatment is performed using the residual spirulina after extraction of phycocyanin, and can be suitably used.
For example, when the microalgae is chlorella, the contained chlorophyll is useful as a green pigment or health food, but even if the residual chlorella after extraction of chlorophyll with water is used, peptides having an ACE inhibitory action can be obtained. Obtainable.
For example, when the microalgae are hematococcus, peptides having an ACE inhibitory action can be obtained even by using the residual hematococcus after extracting the contained astaxanthin with an organic solvent. As the organic solvent, for example, extraction with water-containing ethanol at room temperature, ethanol, acetone, or a mixture of two or more thereof can be performed.
For example, when the microalga is Donaliella, peptides having an ACE inhibitory action can be obtained even by using the residual Donariella after extracting the contained β-carotene with an organic solvent. As the organic solvent, for example, extraction with water-containing ethanol at room temperature, ethanol, acetone, or a mixture of two or more thereof can be performed.

  The proteolytic enzyme used in the present invention can be used without particular limitation as long as it is an enzyme that hydrolyzes protein at pH 2.0 to 9.0, and may or may not be purified. Examples of proteolytic enzymes include proteolytic enzymes derived from animal digestive systems (digestive enzymes) such as pepsin, trypsin, chymotrypsin, and elastase, proteolytic enzymes derived from the genus Aspergillus, and proteins derived from the genus Bacillus. Degrading enzymes, proteolytic enzymes derived from the genus Rhizopus, proteolytic enzymes derived from the genus Penicillium, proteolytic enzymes derived from microorganisms such as proteolytic enzymes derived from the genus Mucor, papain, bromelain, ficin, etc. Plant-derived proteolytic enzymes and the like. As the proteolytic enzyme, a proteolytic enzyme derived from a microorganism or an animal-derived proteolytic enzyme is preferable, and a proteolytic enzyme derived from Aspergillus genus, a proteolytic enzyme derived from Bacillus genus, or pepsin is more preferable.

The peptides referred to in the present invention are proteolytic enzyme-treated products containing peptides, amino acids, and proteins obtained by treating microalgae with proteolytic enzymes. An included peptide may be included.
The peptides may be in the form of a solution, a suspension, or a dried product obtained by distilling off the medium.
In the present invention, the treatment of microalgae with proteolytic enzymes is, for example,
(1) Add powdered or liquid proteolytic enzyme to water suspension of microalgae (2) Add water to dried microalgae and powdered proteolytic enzyme (3) To dried microalgae It can be performed by a method such as adding water and a proteolytic enzyme.

The concentration of the microalgae in the treatment step is preferably a concentration at which the action efficiency of the proteolytic enzyme is good and the post-treatment is easy, and the microalgae solid content concentration is preferably 1 to 30% by mass, and 5 to 20% by mass. More preferred.
As the usage-amount of a proteolytic enzyme, 1-100000 units (U) are preferable with respect to 1 g of microalgae solid content, and 10-50000U is more preferable. Here, 1 unit was measured by the method described on pages 146 to 147 of “Biochemical Experimental Method 31 Proteolytic Enzyme II Tsuru Daisuke, Funatsu Katsuku 1993” published by Academic Publishing Center. Specifically, 1U uses heat-denatured casein as a substrate, prepares 1 ml of a 1% by weight substrate aqueous solution to which a proteolytic enzyme is added, and measures the absorbance at 280 nm of this aqueous substrate solution. Is the amount of enzyme that raises by 0.001.

The pH of the treatment is preferably adjusted to 2.0 to 9.0. When the pH exceeds 9.0, the water extract obtained has a taste and odor peculiar to microalgae and not only the flavor of the food deteriorates, but also precipitates and floats in acidic foods such as soft drinks. This is not preferable because water-insoluble components are easily generated. In order to adjust the pH, a compound used in normal food production such as sodium hydroxide or hydrochloric acid may be used.
The treatment may be carried out by standing or stirring, but stirring is preferred. The treatment temperature is preferably 30 to 75 ° C., more preferably 35 to 60 ° C., because the working efficiency of the proteolytic enzyme is good.
The treatment time is preferably 1 to 36 hours, and more preferably 2 to 24 hours.

After extracting the water-soluble peptides into the aqueous solution, the insoluble matter removing step described later is performed, but before that, the proteolytic enzyme may be deactivated as necessary. In order to deactivate, for example, it may be allowed to stand for 5 to 20 minutes in an environment of 70 to 95 ° C.
After the water-soluble peptides are extracted into an aqueous solution, insoluble matters in the aqueous solution are removed. The method for removing the insoluble matter is not limited as long as it is a means capable of solid-liquid separation. For example, the filtration method using a filter medium such as filter paper or filter cloth, the decantation method for collecting the supernatant, the filter press method, the centrifugal separation Examples include a separation method. Among them, a method of collecting the supernatant such as a centrifugal separation method and a filter press method capable of industrially processing in large quantities is preferable, and a centrifugal separation method is particularly preferable.
Centrifugation is not particularly limited as long as it can remove insolubles from the processing solution, but gravitational acceleration is preferably 1,000 to 30,000 G and preferably 10 seconds to 2 hours, and gravitational acceleration is 3,000 to 15,000 G. The condition of 1 to 30 minutes is more preferable. There are disperse type centrifuges, alpha type centrifuges, and shear press type centrifuges as centrifuges. However, since the workability is improved, the disperse type centrifuges and alpha centrifuges are improved. Combination continuous centrifugation is preferred.

  The peptides obtained above can be used for beverages and the like as they are, but may be purified by filtration or the like. Moreover, if necessary, after further sterilization, it may be dried and used for food. Further, it can be pulverized. As the drying method, there are a freeze-drying method, a spray-drying method and the like, but the spray-drying method is preferable because it is economical. In addition, when drying, it is possible to add dextrin or the like to the microalgae extract and dry it to adjust the powder physical properties.

  The peptide of the present invention can be obtained by performing fractionation by a known method from the obtained peptides, for example, various chromatographic methods such as ion chromatography, molecular sieve chromatography, reverse phase chromatography, The methods usually used as peptide separation means such as membrane separation treatment are carried out alone or in combination in any order.

  The analysis of the amino acid sequence of the obtained peptide can be performed by a generally known Edman method. The outline of this method will be described. First, a chemical reagent that is covalently bonded only to the N-terminal free amino group is first added to the peptide, and then a weak acid is added to activate the reagent. Only the cleaved and the cleaved amino acids are identified by chromatography. Subsequently, the same operation is sequentially repeated on the peptide shortened by one amino acid to determine the amino acid sequence in the peptide. Also in the present invention, the amino acid sequence of the novel peptide was determined by this method.

  The novel peptide of the present invention can be obtained by the above-mentioned microalgae proteolytic enzyme, but can also be obtained by a usual synthesis method such as a liquid phase method or a solid phase method. In order to carry out the synthesis, preferably, the L-form amino acid corresponding to the amino acid residue from the C-terminal side (carboxyl terminal side) of the novel peptide is sequentially bound to the polymeric solid-phase support by the solid-phase method. What is necessary is just to combine. The resulting synthetic peptide is cleaved from the polymeric solid support using trifluoromethanesulfonic acid, hydrogen fluoride, etc., then the amino acid side chain protecting group is removed, and a reversed-phase column is used. The desired peptide can be obtained by purification by a conventional method using high performance liquid chromatography or the like.

Moreover, the novel peptide of the present invention can be mass-produced by recombinant DNA technology. For example, an arbitrary oligonucleotide capable of encoding a novel peptide can be incorporated into a vector, and an appropriate host can be transformed with this and cultured.
In the same manner as described above, the peptides of the present invention represented by SEQ ID NOs: 2 to 5 are prepared by incorporating any oligonucleotide encoding them into a vector, transforming an appropriate host using this, and culturing the same. Can be obtained. Alternatively, it can also be obtained by a synthesis method as described above.

For measuring the ACE inhibitory action of the obtained peptide, a commonly known method, for example, developed by Cushman and Cheung et al. Is simple and widely used. In the measurement, for example, Hip-His-Leu (Hip is hippuric acid residue) is used as a substrate on which ACE acts, and the released hippuric acid after ACE treatment is measured to evaluate the enzyme inhibitory action. For example, a sample solution and a substrate are placed in a test tube, held in a thermostat, and an ACE solution is added, stirred, and reacted. Hydrochloric acid is added to stop the reaction, and then ethyl acetate is added to recover the liberated hippuric acid. After distilling off ethyl acetate, water is added to dissolve hippuric acid, and then the absorbance is measured with a spectrophotometer.
ACE inhibitory activity is calculated according to the following formula: Inhibition rate (%) = {(E _C -E _S ) / (E _C -E _B )} × 100. Here, E _S is the absorbance upon addition of the sample solution, absorbance when E _C is distilled water was added instead of the sample solution, E _B represents the absorbance when reacted by adding a pre-1N hydrochloric acid . The sample concentration in the reaction solution when the inhibition rate is 50% is defined as the IC ₅₀ value.

The ACE inhibitor of the present invention comprises the peptide represented by SEQ ID NO: 1 or the peptide represented by any of SEQ ID NOs: 2 to 5 and having a ACE inhibitory activity as an active ingredient. In addition, as long as this ACE inhibitor contains an effective amount of such a peptide, peptides obtained in an intermediate step of the above production method may be used.
The ACE inhibitor of the present invention exhibits a blood pressure lowering action and a bradykinin inactivation inhibiting action. Therefore, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension, therapeutic agent, normalization of organ circulation and improvement of long-term prognosis (life extension effect) for these congestive heart failure, treatment of heart failure Useful as an agent.
Moreover, since the peptide which is an active ingredient of the ACE inhibitor of the present invention has a sequence structure of only L-amino acids and is gradually degraded by in vivo protease after administration, the toxicity is extremely low and the safety is extremely high. High (LD ₅₀ > 2000 mg / kg; rat oral administration).

The ACE inhibitor of the present invention can be used as a pharmaceutical composition, health food or food.
The pharmaceutical composition can be in the form of a tablet, powder, granule, injection, capsule or the like. Examples of additives used for tablets, capsules, granules, and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate, etc., starches as binders, hydroxypropyl methylcellulose, etc., carboxymethylcellulose and its potassium salts as disintegrants, Examples of the lubricant include stearic acid and its salts, talc, and waxes. In preparation of the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. These can be used to prepare each form. In a sterile composition for injection, the novel heptapeptide of the present invention is dissolved or suspended in water for injection, physiological saline and a sugar alcohol injection solution such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol by a conventional method. It can be made cloudy to make an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as necessary. The preparation containing the novel heptapeptide of the present invention is in the form of a lyophilized product or a dry powder, and can be used by dissolving in a normal solubilizing agent such as water or physiological saline at the time of use.
In the case of health food or food, such a peptide can be blended with the raw material of the food and can be produced according to a normal production method of the food.

  The daily intake of the peptide of the present invention varies depending on age, sex, body weight, symptoms, etc., but is generally about 0.01 to 10 mg / day, particularly preferably 0.1 to 5 mg / day. This amount can be taken in 1 to several times.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

(Extraction and purification of peptide represented by SEQ ID NO: 1)
750 ml of distilled water was added to 25 g of Spirulina powder to obtain a Spirulina suspension. This suspension was applied to an ultrafiltration membrane (fractionated molecular weight 5000) to obtain a non-passing liquid (Spirulina protein) having a molecular weight of 5000 or more. This solution was adjusted to pH 2.0 with 1 M hydrochloric acid, 0.17 g of pepsin (manufactured by Merck & Co., Enzyme No. EC3.24.23.1) was added, and hydrolysis was performed while stirring at 37 ° C. for 20 hours. . The decomposition reaction solution was adjusted to pH 7.0 with 1M sodium hydroxide and boiled at 95 ° C. for 20 minutes. After cooling, the precipitate was removed by centrifugation (12,500 G, 1 hour), and the supernatant was passed through a membrane filter (cellulose acetate, 0.45 μ). This flow-through was applied to an ultrafiltration membrane (fractionated molecular weight 5000), and the obtained flow-through was lyophilized to obtain 7.7 g of spirulina protein-derived peptide powder. After dissolving 20 mg of this peptide powder in 2 ml of ultrapure water, high performance liquid chromatography (HPLC) was performed. Nomura Chemical Co., Ltd. trade name: Develosil ODS-5 (4.5 mm ID × 250 mm) is used as the column, and mobile phase (solution A); 0.1 vol% trifluoroacetic acid (hereinafter abbreviated as TFA) Aqueous solution / acetonitrile = 97/3 (v / v), (liquid B); 0.1 volume% TFA aqueous solution / acetonitrile = 20/80 (v / v), and the concentration of liquid B in liquid A is 0 to 36 Chromatography was performed at a flow rate of 0.8 ml / min with a detection wavelength of 215 nm while applying a concentration gradient to volume%. As a result, a peptide fragment peak having a high ACE inhibitory activity was obtained at an elution time of 102.7 minutes. The results of this HPLC analysis are shown in FIG. In FIG. 1, the horizontal axis represents the retention time by HPLC, and the vertical axis represents the peak area at 215 nm.

The amino acid analysis of the peptide fragment thus obtained was Gly; 2.01, Lys; 1.00, Thr; 0.95, Ile; 1.17, and Met; 0.66.
The amino acid sequence of the peptide fragment was determined using a protein sequencer GI000A and PTH analyzer type I090 manufactured by Hewlett-Packard Company. As a result, it was confirmed that the novel peptide according to the present invention was a novel peptide represented by the L-form amino acid sequence represented by SEQ ID NO: 1.
The peptide at room temperature was white powder.

(Residue treatment 1 blue pigment extraction)
300 g of a 1% calcium chloride dihydrate solution was added to 10 g of Spirulina powder, and the mixture was allowed to stand at 25 ° C. for 16 hours to extract Spirulina blue pigment. To the obtained extract, 5 g of disodium hydrogenphosphate 12 hydrate was added, stirred well, and centrifuged (10,000 G, 1 hour). As a result, 11.7 g (including inorganic salts) of Spirulina blue pigment extraction residue was obtained. Distilled water (10 mL) was added to 1 g of the residue thus obtained to obtain a Spirulina suspension. The suspension was adjusted to pH 2.0 with 1M hydrochloric acid, 0.01 g of pepsin (Merck, enzyme number EC 3.4.23.1) was added, and hydrolysis was performed while stirring at 37 ° C. for 20 hours. went. Thereafter, the same operation as in (Example 1) was performed to obtain the target peptide.

(Residual treatment 2 Extraction of spirulina extract)
Distilled water 100mL was added to Spirulina powder 10g, and Spirulina extract was extracted at 121 degreeC for 1 hour. After allowing to cool, the pH of the extract was adjusted to 4 with citric acid and centrifuged (10,000 G, 1 hour). As a result, 8.7 g of Spirulina extract extraction residue was obtained. Distilled water (10 mL) was added to 1 g of the residue thus obtained to obtain a Spirulina suspension. The suspension was adjusted to pH 2.0 with 1M hydrochloric acid, 0.01 g of pepsin (Merck, enzyme number EC 3.4.23.1) was added, and hydrolysis was performed while stirring at 37 ° C. for 20 hours. went. Thereafter, the same operation as in (Example 1) was performed to obtain the target peptide.

(Synthesis of peptide represented by SEQ ID NO: 1)
The C-terminal of the C-terminal was prepared in a conventional manner by a 9-fluorenylmethoxycarbonyl (hereinafter abbreviated as F-moc) method using a multi-item simultaneous solid phase method automatic peptide synthesizer PSSM-8 manufactured by Shimadzu Corporation. A peptide having the amino acid sequence shown in SEQ ID NO: 1 was synthesized from the side Met.
The obtained unpurified synthetic peptide was dissolved in distilled water and purified by HPLC using a trade name: Inertsil PREP ODS (4.5 mm ID × 250 mm) manufactured by GL Sciences. (A) 0.1 volume% TFA-containing distilled water and (B) 0.1 volume% TFA-containing acetonitrile solution were used as the mobile phase, and (B) the liquid was subjected to a concentration gradient method of 5 to 65 volume% in 30 minutes. Chromatography was performed at a flow rate of 1.5 ml / min. The elution fraction that was detected at an ultraviolet wavelength of 220 nm and showed the maximum absorption was collected and lyophilized to obtain the target peptide.
The synthetic peptide obtained in this example had the same retention time as a result of analysis under the same HPLC conditions as the peptide isolated in Example 1.

(Measurement of angiotensin converting enzyme inhibitory activity)
ACE (manufactured by Sigma, enzyme number EC 3.4.15.1) 1.5 mU and synthetic substrate Hip-His-Leu (manufactured by Sigma) 6.5 mM were used for measurement according to a generally known method. That is, the produced hippuric acid was extracted with ethyl acetate and measured by absorbance at 228 nm. The inhibition rate was calculated from the following equation, where Es is the absorbance in the test solution, Ec is the value when the buffer solution is added instead of the test solution, and Eb is the value when the reaction stop solution is added and reacted in advance. .
Inhibition rate (%) = {(Ec−Es) / (Ec−Eb)} × 100
The inhibitory activity IC ₅₀ value of the ACE inhibitor was expressed as the concentration (M) of the sample required to inhibit the enzyme activity of ACE by 50%.
The inhibitory activity (IC ₅₀ value) of the novel peptide of the present invention against angiotensin converting enzyme was 0.29 μM.

(Production of preparation)
Tablets were produced with the following formulation.
3 g of the peptide obtained in Example 1, 31 g of reduced maltose starch syrup, 8 g of sucrose fatty acid ester, 20 g of crystalline cellulose, 68.5 g of lactose, 68.5 g of dextrin, and 1 g of sweetener (stevia) were mixed, and this mixture was compressed into a compressed tablet machine. To produce tablets (200 mg × 1000 tablets).

(Production of food)
A drink was prepared according to the following formulation.
3 mg of the peptide obtained in Example 1, 4.0 g of sugar, 2.5 g of fructose-glucose liquid sugar, 0.25 g of acidulant, 0.8 g of fragrance, and 100 ml of the total volume with purified water, and a drink (100 mL) Manufactured.

  The present invention can be used as an antihypertensive agent for hypertensive patients.

The result of having analyzed the peptide obtained in Example 1 by HPLC is shown.

Claims (21)

  A peptide comprising the L-amino acid sequence represented by SEQ ID NO: 1.   A peptide comprising the L-amino acid sequence represented by any one of SEQ ID NOs: 2 to 5 and having angiotensin converting enzyme inhibitory activity.   An angiotensin converting enzyme inhibitor comprising a peptide having the L-amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.   An angiotensin converting enzyme inhibitor comprising as an active ingredient a peptide comprising the L-amino acid sequence represented by any one of SEQ ID NOs: 2 to 5.   The angiotensin converting enzyme inhibitor according to claim 3 or 4, which is a blood pressure lowering agent.   A pharmaceutical composition comprising the angiotensin converting enzyme inhibitor according to claim 3 or 4.   A health food containing the angiotensin converting enzyme inhibitor according to claim 3 or 4.   A food containing the angiotensin converting enzyme inhibitor according to claim 3 or 4.   It comprises a step of treating microalgae with a proteolytic enzyme in an aqueous solution to extract peptides into the aqueous solution, then removing insolubles in the aqueous solution, and fractionating the obtained peptides. A method for producing the peptide represented by SEQ ID NO: 1.   The method for producing a peptide according to claim 9, wherein the microalgae is Spirulina.   The method for producing a peptide according to claim 10, wherein the Spirulina is a residual Spirulina after passing through an extraction step with water, an organic solvent, or a supercritical extraction gas.   The method for producing a peptide according to claim 9, wherein the microalga is Chlorella.   The method for producing a peptide according to claim 12, wherein the chlorella is a residual chlorella after an extraction step with water, an organic solvent, or a supercritical extraction gas.   The method for producing a peptide according to claim 9, wherein the microalga is Haematococcus.   The method for producing a peptide according to claim 14, wherein the hematococcus is a residual hematococcus after an extraction step using water, an organic solvent, or a supercritical extraction gas.   The method for producing a peptide according to claim 9, wherein the microalgae is Dunaliella.   The method for producing a peptide according to claim 16, wherein the Donariella is a residual Donariella after an extraction step with water, an organic solvent, or a supercritical extraction gas.   The method for producing a peptide according to claim 9 to 17, wherein the proteolytic enzyme is a microorganism-derived proteolytic enzyme.   The method for producing a peptide according to claim 18, wherein the proteolytic enzyme derived from a microorganism is one or more selected from the group consisting of a proteolytic enzyme derived from the genus Aspergillus and a proteolytic enzyme derived from the genus Bacillus. .   The method for producing a peptide according to claim 9 to 17, wherein the proteolytic enzyme is an animal digestive enzyme. The method for producing a peptide according to claim 20, wherein the animal digestive enzyme is pepsin.

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