JP3378279B2 - Peptide and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a peptide having opioid activity and angiotensin converting enzyme inhibitory activity, a salt thereof, a method for producing the same, and an angiotensin converting enzyme inhibitor comprising the peptide and its salt as an active ingredient.
Agent .

[0002]

2. Description of the Related Art It is widely known that there are a large number of bioregulators involved in higher life activities in foods, and these bioregulators are used to promote health, prevent diseases, and so on. In recent years, various attempts have been made to positively utilize such treatments and prevent aging. As such bioregulatory factors, various factors acting on the nervous system, digestive absorption system, circulatory system, biological defense / immune system, endocrine system, cell differentiation / proliferation system, etc. are known. Among them, some food proteins act on the nervous system to cause morphine-like anesthesia,
It has been found that there is a peptide unit having an analgesic activity or the like (so-called opioid activity), and such a peptide is generally called an opioid peptide.

[0003] Milk proteins, especially milk β-casein, are one of the food proteins from which such opioid peptides are derived. An opioid peptide derived from this protein includes β-casomorphin 7 [Bra
ntl et al., Hoppe-Seyler's Z. Physiol. Chem., 360, 121
1 (1979)], β-casomorphin 5 represented by SEQ ID NO: 18
[Henshen et al., Hoppe-Seyler's Z. Physiol. Che
m., 360, 1217 (1979)].
casomorphin 4 [Brantl et al., Life Sic., 28, 1903 (1
981)].

Opioid peptides derived from these milk proteins are isolated from commercially available casein peptone (hydrolyzed milk with hydrochloric acid or hydrolyzed with a complex enzyme consisting of trypsin, pepsin and pancreatin). The structure was determined. However, the amount of these peptides contained in casein peptone is extremely small, and the above-mentioned Brantl et al., Hoppe-Seyle
r's Z. Physiol. Chem., 360, 1211 (1979) shows that the yield from casein peptone is about 0.005%, and the yield from β-casein (assuming a casein content of 30%) is about 0.01.
It is stated to be 8%. Therefore, it has not been practically possible to isolate those peptides from commercially available casein peptone and use them as agents having opioid peptide activity. Various attempts have been made to obtain opioid peptides in large amounts from milk β-casein, but no effective production method has been reported yet.

[0005] Also, a renin-angiotensin system is known as a typical in vivo factor that causes an increase in blood pressure, and a kallikrein / kinin system is known as a typical in vivo factor that works to lower blood pressure. "ACE") is greatly involved in both of these systems. To briefly explain the mechanism, first, Renin
In the angiotensin system, the renal enzyme renin secreted into the blood acts on angiotensinogen in the blood to produce angiotensin I, a decapeptide. This angiotensin I does not show a blood pressure increasing effect, but when ACE acts on it, angiotensin II, which is an octapeptide, is produced. This angiotensin II contracts peripheral blood vessels and acts on the adrenal cortex to promote the production of aldosterone.Aldosterone acts on the kidney to cause sodium reabsorption and increase in fluid volume, resulting in an increase in cardiac output, All of them greatly increase blood pressure.

On the other hand, in the kallikrein / kinin system, kallikrein, a blood enzyme, acts on kininogen, a precursor protein in the blood, to produce free kinin. This kinin expands peripheral blood vessels and activates phospholipase A ₂ . Promotes the synthesis of prostaglandins and lowers blood pressure. However, this kallikrein-kinin system has AC
When E acts, ACE is-decomposing the kinin with enhanced action and activating action of phospholipase A ₂ peripheral vascular
Due to the inactivation, no drop in blood pressure occurs.

[0007] Therefore, when a substance (ACE inhibitor) that inhibits the above-described action of ACE is present, the production of angiotensin II, which is a blood pressure increasing substance, is suppressed, and the degradation of kinin, which acts as a blood pressure lowering substance, is prevented. Thus, it is possible to suppress a rise in blood pressure and decrease blood pressure. From this point, in recent years, various studies have been made on peptides having ACE inhibitory activity obtained from natural proteins, and it is expected that such peptides can be used as agents having ACE inhibitory activity.

[0008]

Under the circumstances described above, the present inventors have conducted research to obtain a peptide having opioid activity from milk protein in high yield. As a result, it was found that a strong opioid activity was present in the hydrolyzate obtained by using a protease derived from a microorganism as a milk protein, and the peptide mixture was further fractionated by reversed-phase chromatography using high-performance liquid chromatography. It was confirmed that a specific fraction was a peptide having opioid activity.

In particular, milk β-casein, which is abundantly contained in milk among milk proteins, is hydrolyzed using a microbial neutral protease or a microbial alkaline protease to form a peptide mixture. The fraction having opioid activity obtained therefrom was isolated and purified, and its structure was determined. As a result, a novel peptide comprising 10 amino acids represented by SEQ ID NO: 3 and a novel peptide represented by SEQ ID NO: 4 were shown. It was found to be a novel peptide having a sequence of 10 amino acids.

When the two kinds of novel peptides obtained above or a mixture containing the peptides is further hydrolyzed using aminopeptidase, the novel peptide represented by SEQ ID NO: 5 is deduced from the peptide of SEQ ID NO: 3. The peptides, and also the peptides of SEQ ID NO: 4, yielded novel peptides of SEQ ID NO: 6, and those peptides of SEQ ID NO: 5 and SEQ ID NO: 6 were found to have more potent opioid activity. Was.

[0011] Further, the above-obtained peptide represented by SEQ ID NO: 3 or SEQ ID NO: 4 or a mixture containing the peptide is hydrolyzed simultaneously or sequentially in any order using aminopeptidase and carboxypeptidase. Then, it was found that the peptides represented by SEQ ID NO: 6 and SEQ ID NO: 17 were obtained.

The present inventors examined the ACE inhibitory activities of the novel peptides represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. It has been found that certain peptides have not only an opioid activity but also an activity of inhibiting ACE activity.

[0013] Milk proteins, especially milk casein, are easily and abundantly available proteins, and the opioid activity and ACE inhibition are observed when the above-mentioned method using milk protein as a raw material is found by the present inventors. An active peptide can be easily obtained in a large amount.

Further, the present inventors have found that, in the nonapeptide represented by SEQ ID NO: 5 or SEQ ID NO: 6 obtained from milk β-casein, histidine (His) or proline at the second residue from the C-terminal amino acid thereof (Pro) with alanine (Ala), arginine (Arg), glutamic acid (Gl
u), novel nonapeptides represented by SEQ ID NOS: 7 to 11 substituted with other amino acids such as glycine (Gly) and leucine (Leu) were chemically synthesized, and their opioid activity and ACE inhibitory activity were measured. However, the nonapeptides represented by SEQ ID NOs: 7 to 11 were found to have extremely high opioid activity and ACE inhibitory activity, similarly to the nonapeptides represented by SEQ ID NOs: 5 and 6. . Then, based on such a discovery by the present inventors, the above-mentioned SEQ ID NO: 5 to SEQ ID NO: 11
The novel nonapeptides represented by SEQ ID NO: 1 (wherein Xaa represents an amino acid) encompassing nonapeptides of formula (1) generally have extremely high opioid activity and also have ACE inhibitory activity, and are extremely useful. Was revealed.

Similarly, the present inventors have found that in the decapeptide represented by SEQ ID NO: 3 or SEQ ID NO: 4 obtained from milk β-casein, 2
SEQ ID NO: 12 in which the histidine (His) or proline (Pro) at the residue is replaced with another amino acid such as alanine (Ala), arginine (Arg), glutamic acid (Glu), glycine (Gly), leucine (Leu), etc. To a novel decapeptide represented by SEQ ID NO: 16 and its opioid activity and AC
When the E inhibitory activity was measured, it was found that these novel decapeptides also had opioid activity and ACE inhibitory activity, like the decapeptides represented by SEQ ID NO: 3 and SEQ ID NO: 4. From the discovery by the present inventors, it is represented by SEQ ID NO: 2 (where Xaa represents an amino acid) encompassing the decapeptides of SEQ ID NOs: 3 to 4 and 12 to 16 described above. The novel decapeptides generally have high opioid activity and also have ACE inhibitory activity, indicating that they are highly useful.

Accordingly, the present invention relates to a peptide represented by SEQ ID NO: 1 (where Xaa represents an amino acid) or a salt thereof. Further, the present invention relates to SEQ ID NO: 2 (Xaa
Represents a amino acid) or a salt thereof.

The present invention relates to SEQ ID NO: 1 (X
aa represents an amino acid) or a peptide represented by SEQ ID NO: 2 (where Xaa represents an amino acid) and a salt thereof.
Angiotensin containing one or more species as active ingredients
Converting enzyme inhibitor (hereinafter referred to as “ACE inhibitor”)
There is) .

In the peptide represented by SEQ ID NO: 1 or SEQ ID NO: 2, the amino acid at the second position from the C-terminal; the type of Xaa is not limited and may be any amino acid, such as histidine (His) and proline (Proline). ), Alanine (Al
a), arginine (Arg), glutamic acid (Glu), glycine (G
Amines such as ly) and leucine (Leu) are suitable. In that case, each peptide has the amino acid sequence represented by SEQ ID NO: 3 to SEQ ID NO: 16.

Further, the present invention provides (1)β-casein
A neutral protease of Bacillus origin, Aspergillus
Neutral protease of origin, in Streptomyces origin
Neutral protease, Rhizopus-derived neutral protease and
Selected from alkaline proteases of Bacillus origin
Neutral or alkaline protein of microbial origin
Hydrolysis using ase, from the resulting hydrolyzate
Recovers peptides with opioid activity
And a method for producing an opioid peptide, wherein (1)
The opioid peptide produced by the method of
Opioid pep with obvious and unclear structure
Both tides are included.

The opioid peptide obtained by the above method (1) contains the novel peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4, and particularly as a neutral protease, a neutral protease of Bacillus origin or Aspergillus When a neutral protease of origin is used and an alkaline protease of Bacillus origin is used as the alkaline protease, the peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4 can be efficiently produced.

Further, the present invention provides (2)β-casein
A neutral protease of Bacillus origin, Aspergillus
Neutral protease of origin, in Streptomyces origin
Neutral protease, Rhizopus-derived neutral protease and
Selected from alkaline proteases of Bacillus origin
Neutral or alkaline protein of microbial origin
After hydrolysis using ase, aminopeptidase
Is further hydrolyzed using
It is characterized by recovering a peptide having a poid activity
And a method for producing an opioid peptide.

The opioid peptide obtained by the method (2) contains the novel peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6, and particularly as a neutral protease, a neutral protease of Bacillus origin or Aspergillus When a neutral protease of origin is used, an alkaline protease of Bacillus origin is used as an alkaline protease, and leucine aminopeptidase is used as an aminopeptidase, the peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6 can be efficiently prepared. Can be manufactured.

Further, the present invention provides (3)β-casein
A neutral protease of Bacillus origin, Aspergillus
Neutral protease of origin, in Streptomyces origin
Neutral protease, Rhizopus-derived neutral protease and
Selected from alkaline proteases of Bacillus origin
Neutral or alkaline protein of microbial origin
After hydrolysis using ase, additional aminopeptida
Enzyme and carboxypeptidase simultaneously or in any order
Hydrolyze by using each and every time, and from the resulting hydrolyzate
Recovers peptides with opioid activity
And a method for producing an opioid peptide. here
In, "Aminopeptidase and carboxypeptidase
Hydrolyze simultaneously or sequentially, regardless of order. "
Is a neutral or alkaline protease
The hydrolyzate obtained by using the aminopeptidase and
Hydrolysis by reacting boxypeptidase simultaneously
Or, or aminopeptidase and carboxypepti
Hydrolyzed using one of the enzymes
After hydrolysis with the remaining peptidase
You.

Among the opioid peptides obtained by the method (3), a novel peptide represented by SEQ ID NO: 6 and a known peptide represented by SEQ ID NO: 17 are contained. When a neutral protease of Bacillus origin or Aspergillus origin was used, an alkaline protease of Bacillus origin was used as an alkaline protease, leucine aminopeptidase was used as an aminopeptidase, and carboxypeptidase A was used as a carboxypeptidase, SEQ ID NO: 6 And the peptide represented by SEQ ID NO: 17 can be efficiently produced.

The present invention relates to a peptide represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 produced by any one of the above methods (1) to (3) and a salt thereof. Include.

In the present invention, β-casein is hydrolyzed by any of the above-mentioned methods (1) to (3) to produce opioid peptides represented by SEQ ID NOS: 3 to 6 and 17 The method includes SEQ ID NO: 3
The method for producing the peptide represented by SEQ ID NO: 17 is not limited to the above-mentioned methods (1) to (3), but may be performed by chemical synthesis, a combination of chemical synthesis and enzymatic hydrolysis, or another enzymatic hydrolysis method. Can also be manufactured.

For example, the respective peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4 are prepared by chemical synthesis or other methods other than the above (1), and are hydrolyzed using an aminopeptidase such as leucine aminopeptidase. In this case, the respective peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6 can be produced.

In the same manner as described above, each peptide represented by SEQ ID NO: 3 and SEQ ID NO: 4 is prepared by chemical synthesis or other methods other than the above (1), and the peptide is synthesized with an aminopeptidase such as leucine aminopeptidase and the like. The respective peptides represented by SEQ ID NO: 6 and SEQ ID NO: 17 can also be produced by using carboxypeptidase such as carboxypeptidase A and hydrolyzing them simultaneously or sequentially in any order.

Further, each peptide included in the peptides represented by SEQ ID NO: 1 or SEQ ID NO: 2 may be produced by chemical synthesis alone.

The peptides of the present invention described above, milk protein
Among them, it is particularly preferable to use β-casein.
The β- casein, purified from milk casein β
-Using casein alone or β containing other caseins and proteins such as milk α, κ casein
-Casein may be used.

[0031] The peptide of the present invention is derived from the hydrolysis of β-casein, a natural milk protein , in which case the amino acids constituting the peptide are all L-amino acids. is there. However, the amino acids constituting the novel peptide of the present invention included in the peptides represented by SEQ ID NO: 1 or SEQ ID NO: 2 are as follows:
The peptide is not limited to L-amino acids, and may be any optical isomer as long as it has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. For example, even if all of the amino acids consist only of L-amino acids or only D-amino acids, one of the amino acids constituting each peptide is an L-amino acid and the rest is a D-amino acid.
-It may be an amino acid;

The term “agent having opioid activity” as used herein refers to analgesia, anesthesia, emotion, respiration, pulsation,
Body temperature, gastrointestinal function, food intake, immunity, regulation of secretion of hormones such as insulin and somatostatin, promotion of electrolyte absorption,
It refers to an agent having an activity recognized as a so-called opioid activity involved in the regulation of contraction of the heart muscle and the like. In addition, the present onset
Ming's “ACE inhibitor” refers to ACE involved in increasing blood pressure
Refers to an agent that inhibits the action of, and can cause an increase in blood pressure and a decrease in blood pressure.

Although not limited, an example in which the peptide of the present invention is prepared by hydrolysis of milk β-casein will be described below.

[Method for producing peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4] As described above, milk β-casein is hydrolyzed using a neutral protease or an alkaline protease of microbial origin. And a peptide mixture containing the peptide represented by SEQ ID NO: 4. In this case, milk β-casein is dispersed and dissolved in distilled water or tap water, and hydrolyzed with a neutral protease or an alkaline protease derived from a microorganism to prepare a peptide mixture.

As a neutral protease of microbial origin,
Examples of the protease include thermolysin, a heat-resistant protease of Bacillus origin, metal protease of Aspergillus origin, metal protease of Streptomyces origin, and metal protease of Rhizopus origin. Examples of the alkaline protease of microbial origin include alkaline protease such as serine protease of Bacillus origin. The above neutral protease or alkaline protease can be used alone or in combination of two or more as long as the proteases do not adversely affect each other. When a plurality of neutral proteases are used, the hydrolysis may be carried out by simultaneously acting them, or the hydrolysis may be carried out by successively acting them one by one. In this case, it is preferable to use thermolysin derived from Bacillus, since the desired product can be obtained in high yield.

The above protease may be used in a free state or may be used after being immobilized. The protease is preferably used in a ratio of about 5,000 to 1,000,000 units per 10 g of dried milk β-casein.

The protease treatment is preferably carried out by selecting conditions such as optimum pH, temperature, amount of protease, treatment speed, treatment time, etc., according to each situation (eg, type of protease, use form of protease, etc.). . As an example that can be carried out efficiently, when using the protease such as thermolysin, the temperature is generally about 30 to 50 ° C.
Hydrolysis may be carried out at about 6 to 8 so that the solubility in 0.75 M trichloroacetic acid is about 40 to 80%.

When the desired hydrolysis state is achieved, the protease is inactivated by heating, the purified insoluble solid is separated and removed by an appropriate means such as centrifugation, and the supernatant containing the peptide mixture is removed. to recover.

Next, the supernatant containing the peptide mixture is treated by high-performance liquid chromatography (for example, high-performance liquid chromatography using a reversed-phase column) according to the method described below to obtain SEQ ID NO: 3 and SEQ ID NO: The peptide represented by 4 is isolated.

That is, the above supernatant was subjected to high performance liquid chromatography (the column used was, for example, Cosmosil 5C ₁₈ -AR, which is an ODS column manufactured by Nacalai Tesque, Inc.) to give a 0.1% aqueous solution of trifluoroacetic acid (solution A). And 0.1%
Acetonitrile solution containing trifluoroacetic acid (B
Solution B), eluted with a concentration gradient in which the concentration of Solution B increases linearly from 0% to 40%, and the acetonitrile concentration is about 32%.
Peak fraction in the range of ~ 33% (fraction A) and about 34-35
The peak in the% range (fraction B) is collected and its opioid activity is confirmed.

The opioid active fraction obtained above (fraction A,
The solvent is removed from fraction B) by drying or the like. The amino acid sequence of these dried products was examined using, for example, a protein sequencer (type 477-A) manufactured by Applied Biosystems, and the like.
Is a peptide represented by SEQ ID NO: 4.

[Method for producing peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6] As described above, the peptide represented by SEQ ID NO: 5 and SEQ ID NO: 6 is represented by SEQ ID NO: 3 and SEQ ID NO: 4 It is obtained by hydrolyzing a peptide or a mixture containing the peptide using aminopeptidase.

As the aminopeptidase, the use of leucine aminopeptidase is preferable because the desired product can be obtained in high yield. At that time, the aminopeptidase may be used in a free state or may be used after being immobilized. The amount of aminopeptidase used is determined whether the peptide represented by SEQ ID NO: 3 or SEQ ID NO: 4 is purely isolated or in the form contained in the peptide mixture. 10 g of dried substrate
Preferably, it is used at a rate of about 10,000 to 100,000 units per unit.

The aminopeptidase treatment is preferably performed by selecting reaction conditions according to the type of aminopeptidase. For example, when leucine aminopeptidase is used, the pH of the solution is adjusted to 6 to 8, and the temperature is adjusted to about 30 to 50 ° C.
For about 5 to 15 hours. When the desired hydrolysis has been achieved, the aminopeptidase is inactivated by heating, and the peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6 contained in the reaction solution are recovered.

As a substrate, purely isolated SEQ ID NO: 3
And when using the peptide represented by SEQ ID NO: 4,
By this aminopeptidase treatment, each of the peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6 is obtained with a yield of almost 100%.

As substrates, SEQ ID NO: 3 and SEQ ID NO: 4
In the case of using an enzymatically decomposed product of milk β-casein containing a peptide represented by the following formula, the peptides of SEQ ID NO: 5 and SEQ ID NO: 6 are isolated by high performance liquid chromatography using the above-mentioned reversed phase column. . The conditions were the same as those used for the peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4, and the peak fraction (fraction C) in which the acetonitrile concentration was in the range of about 29 to 30% was about 31 to 32%. (Fraction D) was collected, its opioid activity was confirmed, and the amino acid sequence was examined in the same manner as described above. Fraction C was the peptide of SEQ ID NO: 5, and fraction D was SEQ ID NO: 6. Confirm that it is a peptide.

[Method for producing β-casomorphin 7, a known peptide represented by SEQ ID NO: 17]
Β-casom which is a known peptide represented by SEQ ID NO: 17
orphin7 is obtained by hydrolyzing the peptide represented by SEQ ID NO: 5 or a mixture containing the peptide using carboxypeptidase.

Carboxypeptidase A is preferable when carboxypeptidase A is used, since the desired product can be obtained in high yield. At that time, carboxypeptidase is
It may be used in a free state or may be used after immobilization. The amount of the carboxypeptidase used is determined whether the peptide represented by SEQ ID NO: 5 is purely isolated or contained in the peptide mixture. Also dried substrate 1
It is preferred to use about 10,000 to 500,000 units per 0 g.

[0049] Carboxypeptidase treatment is also preferably performed by selecting reaction conditions according to the type of carboxypeptidase. For example, when using carboxypeptidase A, the pH of the solution is adjusted to 6-8, and the temperature is adjusted to about 30-5.
The hydrolysis is preferably performed at 0 ° C. for about 5 to 15 hours. At the time when the desired hydrolysis was achieved, carboxypeptidase was inactivated by heating, and β-casomorphin, a known peptide represented by SEQ ID NO: 17 contained in the reaction solution, was used.
Collect 7.

As a substrate, purely isolated SEQ ID NO: 5
When using the peptide represented by the formula, β-casomorphin 7, which is a known peptide represented by SEQ ID NO: 17, is obtained by this carboxypeptidase treatment in a yield of almost 100%.

In the case where an enzymatically decomposed product of milk β-casein containing the peptide represented by SEQ ID NO: 5 was used as the substrate, the product of SEQ ID NO: 17 was obtained by high performance liquid chromatography using the above-mentioned reversed phase column. The known peptide, β-casomorphin 7, is isolated. The conditions were the same as those used for the peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4, and the acetonitrile concentration was about 34-35.
% Of the fraction (fraction E), the opioid activity of this fraction was confirmed, and the amino acid sequence was examined in the same manner as described above. Fraction E was β-peptide, a peptide of SEQ ID NO: 17. c
Confirm that it is asomorphin7.

A method for producing the novel peptide of the present invention represented by SEQ ID NO: 1 or SEQ ID NO: 2 by chemical synthesis will be described by taking the peptide represented by SEQ ID NO: 3 as an example. Can be adopted. Chemical Synthesis Method of the Peptide of the Present Invention The peptide is synthesized using a SAM TWO-type automatic peptide synthesizer manufactured by Biosearch, USA, according to the standard protocol of the same. That is, Boc (butoxycarbonyl)-
After treating the Asn-resin with a deblock solution containing 45% trifluoroacetic acid, Boc-His is coupled in the presence of diisopropylcarbodiimide to form Boc-His-Asn.
Obtain resin. Hereinafter, deblocking and coupling are repeated in the same manner as described above to obtain Boc-Val-Tyr (Cl ₂ -Bzl) -P
ro-Phe-Pro-Gly-Pro-Ile-His-Asn resin is obtained.
This peptide resin is placed in hydrogen fluoride containing 10% anisole and stirred at 0 ° C. for 2 hours. After distilling off the hydrogen fluoride, the residue is washed with ether and the peptide is extracted with 30% acetic acid. The peptide represented by SEQ ID NO: 3 is obtained by purifying the extracted crude peptide using high performance liquid chromatography with an ODS column.

Further, the other peptide included in SEQ ID NO: 1 or SEQ ID NO: 2 can be synthesized and purified by the same operation as described above to obtain the desired peptide.

The peptide represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof produced as described above, and the chemical structure thereof have not yet been elucidated, but milk proteins, especially milk An agent having an opioid activity containing, as an active ingredient, an opioid peptide or a salt thereof obtained by hydrolyzing β-casein with a neutral protease or an alkaline protease of microbial origin is used for analgesia, anesthesia,
Emotion, respiration, pulsation, body temperature, gastrointestinal function, eating, immunity, regulation of hormones such as insulin and somatostatin,
It may be involved in the promotion of electrolyte absorption, the regulation of contraction of the heart muscle, etc., for example, as an analgesic anesthetic, a hypnotic, a gastrointestinal hormone secretagogue, an electrolyte absorption promoter, a diarrhea ameliorating agent by suppressing intestinal peristalsis, etc. It can be administered to humans and various animals.

Further, the ACE inhibitor of the present invention containing the peptide represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof as an active ingredient inhibits the action of ACE involved in increasing blood pressure by ACE inhibitory activity And, involved in blood pressure rise suppression, blood pressure drop, etc., for example, blood pressure rise inhibitor,
It can be administered to humans and various animals as antihypertensive agents and the like.

In the above-mentioned agent having opioid peptide activity and the ACE inhibitor of the present invention , the above-mentioned peptide and its salt may be used alone or in the form of a mixture containing two or more peptides. May be used. Suitable doses of these agents vary depending on various conditions such as age, weight, sex, symptoms, animal type, etc. of the human or animal to be administered.

Any of the above-mentioned agents can be administered orally or parenterally, and may be administered alone or together with a solid carrier or liquid carrier commonly used in the pharmaceutical industry. Alternatively, it can be used in combination or combination with other agents. The dosage form can be in any form such as tablets, pills, granules, capsules, powders, aqueous solutions, injections and the like. In addition, it has opioid activity
And the ACE inhibitor of the present invention can be added to or administered together with food or feed,
In that case, a peptide obtained from milk protein is desirable because of its high safety. Hereinafter, the present invention will be described specifically by way of examples, but the present invention is not limited thereto.

[0058]

Example 1 [Preparation of peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4 by hydrolysis of milk β-casein] 100 mg of milk β-casein was dispersed and dissolved in 10 ml of distilled water, and thermolysin (peptide study) was used. 0.5 mg (approx.
00 units) and incubated at 37 ° C. for 5 hours. After completion of the reaction, the enzyme was inactivated by heating at 90 ° C. for 20 minutes, centrifuged at 6,000 G for 20 minutes to remove insolubles, and the obtained supernatant was lyophilized to obtain a hydrolyzate of about 90 m.
g was obtained.

10 mg of the hydrolyzate obtained above was applied to Cosmosil 5C ₁₈ -AR, an ODS column manufactured by Nacalai Tesque, Inc.
(Internal diameter 20 mm, length 250 mm) was subjected to reverse phase chromatography. Then, using a 0.1% trifluoroacetic acid aqueous solution (Solution A) and an acetonitrile solution (Solution B) containing 0.1% trifluoroacetic acid, the concentration of Solution B increases linearly from 0% to 40%. It was eluted at a flow rate of 10 ml / min with a concentration gradient. The wavelength 220 at that time
The flowchart in nm is shown in FIG. The fractions in the flow chart shown in FIG. 1 were collected and their opioid activities were measured. The fractions eluted in acetonitrile concentration range of about 32 to 33% (fraction A) and about 34 to Opioid activity was observed in the fraction eluted in the range of 35% (fraction B). Thus, the opioid-active fractions (fraction A, fraction B) were collected and concentrated and dried.
In FIG. 1, the left vertical axis represents Ab of the eluted liquid.
s. 220, the vertical axis on the right shows the acetonitrile concentration (%) in the eluent.

The amino acid sequence of the dried product obtained above was examined using a protein sequencer type 477-A manufactured by Applied Biosystems Co., Ltd. It was confirmed that the peptide was represented by SEQ ID NO: 3 consisting of the sequence of Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn from the N-terminal. In addition, the dried product of Fraction B has all of the constituent amino acids in the L-form, and Val-Tyr-Pro-Phe-Pro-Pro-
It was confirmed to be a peptide represented by SEQ ID NO: 4 consisting of the sequence of Gly-Pro-Ile-Pro-Asn.

The yield (%) of each peptide represented by SEQ ID NO: 3 and SEQ ID NO: 4 from raw milk β-casein
The molecular weight and the molecular extinction coefficient at Abs. ₂₈₀ were about 1% and about 1.5%, respectively, which were extremely high yields.

When the Rf value was determined by thin-layer chromatography using a silica gel plate, butanol:
When the developing solvent of acetic acid: pyridine: water = 15: 3: 10: 12 was used, the Rf value was 0.49 for the peptide represented by SEQ ID NO: 3 and 0 for the peptide represented by SEQ ID NO: 4. It was .57.

Example 2 [Preparation of peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6 from peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4] In the same manner as in Example 1, milk β-casein was prepared. About 80 μg of the peptide represented by SEQ ID NO: 3 and about 1200 μg of the peptide represented by SEQ ID NO: 4 were purely isolated from 80 mg of the thermolysin hydrolyzate (the amount of each peptide was determined from its molecular weight and the molecular extinction coefficient in Abs.280). Asked).

After the peptide isolated above was dissolved in 1 ml of distilled water and adjusted to pH 7 with 1N NaOH, leucine aminopeptidase (manufactured by Sigma) was added to each peptide based on the weight thereof. About 5% (about 20 units)
Was added and incubated at 36 ° C. for about 16 hours. After the reaction was completed, the enzyme was inactivated by heating at 90 ° C. for 20 minutes, and then subjected to the same high performance liquid chromatography as used in Example 1 to purify the peptide contained in the reaction solution. From the hydrolyzate of the peptide of SEQ ID NO: 3, the fraction eluted at an acetonitrile concentration in the range of about 29 to 30% showed opioid activity, and the hydrolysis of the peptide of SEQ ID NO: 4 showed the acetonitrile concentration Is about 31-
Opioid activity was observed in the fraction eluted in the range of 32%. Therefore, these opioid active fractions were collected, concentrated and dried.

The dried product obtained above was analyzed for its amino acid sequence using the protein sequencer used in Example 1. The dried product obtained from the hydrolyzate of the peptide represented by SEQ ID NO: 3 was All of its constituent amino acids are L
Tyr-Pro-Phe-Pro-Gly-Pro-Il
The peptide was confirmed to be the peptide represented by SEQ ID NO: 5 consisting of the sequence of e-His-Asn. Further, in the dried product obtained from the hydrolyzate of the peptide represented by SEQ ID NO: 4, all of the constituent amino acids were in the L form, and Tyr-Pro-
It was confirmed to be a peptide represented by SEQ ID NO: 6 consisting of the sequence of Phe-Pro-Gly-Pro-Ile-Pro-Asn.

[0066] Each peptide represented by SEQ ID NO: 5 and SEQ ID NO: 6, the yield (%) from the peptide of SEQ ID NO: 3 and SEQ ID NO: 4, the molecular extinction coefficient at its molecular weight and Abs. ₂₈₀ When asked, all were almost 1
00%.

When the Rf value was determined by thin-layer chromatography on a silica gel plate in the same manner as in Example 1, the Rf value was 0.46 for the peptide represented by SEQ ID NO: 5.
And the peptide represented by SEQ ID NO: 6 was 0.59.

Example 3 Preparation of β-casomorphin 7, a known peptide represented by SEQ ID NO: 17 from a peptide represented by SEQ ID NO: 5, a peptide represented by SEQ ID NO: 5 obtained in Example 2 500 μg is dissolved in 1 ml of distilled water, and 1N NaOH
After adjusting the pH to 7 with carboxypeptidase A
(Sigma) was added to the peptide at about 5% (about 12.5 units) based on the weight thereof,
Incubated for hours. After completion of the reaction, the enzyme was inactivated by heating at 90 ° C. for 20 minutes, and then the reaction solution was subjected to the same high performance liquid chromatography as used in Example 1 to purify the peptide contained in the reaction solution. . From the hydrolyzate of the peptide of SEQ ID NO: 5, an opioid activity was observed in the fraction eluted at an acetonitrile concentration of about 34 to 35%. Therefore, this opioid active fraction was collected, concentrated and dried.

When the amino acid sequence of the dried product obtained above was examined using the protein sequencer used in Example 1, all of the constituent amino acids were in the L form, and
Β-casomo which is a peptide represented by SEQ ID NO: 17 consisting of the sequence of Tyr-Pro-Phe-Pro-Gly-Pro-Ile in order from the terminal
It was confirmed to be rphin7. Of β-casomorphin7 a known peptide represented by SEQ ID NO: 17, where the yield (%) from the peptide of SEQ ID NO: 5 was determined from its molecular weight and Abs. Molecular extinction coefficient at _280, approximately 100%. Further, Rf was determined by thin-layer chromatography using a silica gel plate in the same manner as in Example 1.
When the value was determined, the Rf value was 0.72.

Example 4 [Preparation of peptide represented by SEQ ID NO: 5 and SEQ ID NO: 6 by hydrolysis of milk β-casein] 100 mg of milk β-casein was dispersed and dissolved in 10 ml of distilled water, and thermolysin (Peptide Research Institute) was used. 0.5 mg (about 4,0
00 units) and incubated at 37 ° C. for 5 hours. After completion of the reaction, 5 mg (about 1,000 units) of leucine aminopeptidase (manufactured by Sigma) was further added, and
Incubated at 6 ° C. for about 16 hours. After the reaction, 9
The enzyme was inactivated by heating at 0 ° C. for 20 minutes, centrifuged at 6000 G for 20 minutes to remove insolubles, and the obtained supernatant was lyophilized to obtain about 80 mg of a hydrolyzate.

The hydrolyzate 10 mg obtained above was used in Example 1
The sample was subjected to the same high performance liquid chromatography as used in Example 1 and eluted. FIG. 2 shows a flowchart at a wavelength of 220 nm at that time. Each fraction in the flow chart shown in FIG. 2 was collected and its opioid activity was measured. The fraction (fraction C) eluted in the range of acetonitrile concentration of about 29 to 30% (fraction C) and about 31 to 31 Opioid activity was observed in the fraction eluted in the range of 32% (fraction D). Therefore, this opioid-active fraction (fraction C,
Fraction D) was collected and concentrated to dryness. In FIG. 2, the left vertical axis represents Abs. Of the eluted liquid. 220, the vertical axis on the right shows the acetonitrile concentration (%) in the eluent.

When the amino acid sequence of the dried product obtained above was examined using the protein sequencer used in Example 1, the dried product obtained from Fraction C was found to have all of its constituent amino acids in the L-form. Yes, Tyr-Pro-Phe-Pro-Gly-Pro-
The peptide was confirmed to be the peptide represented by SEQ ID NO: 5 consisting of the sequence of Ile-His-Asn. In addition, the dried product obtained from Fraction D had all of the constituent amino acids in the L-form, and Tyr-Pr
It was confirmed to be a peptide represented by SEQ ID NO: 6 consisting of the sequence of o-Phe-Pro-Gly-Pro-Ile-Pro-Asn.

The yield (%) of each peptide represented by SEQ ID NO: 5 and SEQ ID NO: 6 from raw milk β-casein
Was determined from its molecular weight and the molecular extinction coefficient at Abs. _280, and found to be about 0.9% and about 1.4%, respectively.

When the Rf value was determined by thin-layer chromatography on a silica gel plate in the same manner as in Example 1, the Rf value was 0.46 for the peptide represented by SEQ ID NO: 5.
And the peptide represented by SEQ ID NO: 6 was 0.59.

Example 5 [Preparation of β-casomorphin 7, a known peptide represented by SEQ ID NO: 17 by hydrolysis of milk β-casein] 100 mg of milk β-casein was dispersed and dissolved in 10 ml of distilled water, and thermolysin was dissolved. (Manufactured by Peptide Research Institute)
Add 0.5 mg (about 4,000 units) and add 5 mg at 37 ° C.
Incubated for hours. After the reaction, 5 mg of leucine aminopeptidase (manufactured by Sigma) (about 1,000 u) was added.
nits) and 5 mg of carboxypeptidase A (about 2,50
0 units) and incubated at 36 ° C. for about 16 hours. After completion of the reaction, the enzyme was inactivated by heating at 90 ° C. for 20 minutes, centrifuged at 6000 G for 20 minutes to remove insolubles, and the obtained supernatant was lyophilized to obtain a hydrolyzate of about 80%.
mg was obtained.

The hydrolyzate 10 mg obtained above was used in Example 1
The sample was subjected to the same high performance liquid chromatography as used in Example 1 and eluted. FIG. 3 shows a flowchart at a wavelength of 220 nm at that time. Each fraction in the flowchart shown in FIG. 3 was collected and its opioid activity was measured. The fraction (fraction E) eluted in the range of acetonitrile concentration of about 31 to 32% (fraction E) and about 34 to Opioid activity was observed in the fraction eluted in the range of 35% (fraction F). Therefore, these opioid-active fractions (fraction E, fraction F) were collected, concentrated and dried. In FIG. 3, the left vertical axis represents Abs. Of the eluted liquid. 220
The vertical axis on the right shows the acetonitrile concentration (%) in the eluent.

The dried product obtained above was analyzed for its amino acid sequence using the protein sequencer used in Example 1. The dried product obtained from Fraction E showed that all of the constituent amino acids were in the L form. , Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pr
It was confirmed to be a peptide represented by SEQ ID NO: 6 consisting of the sequence of o-Asn. Further, the dried product obtained from Fraction F had all of its constituent amino acids in the L-form, and Tyr-Pr
It was confirmed to be a peptide represented by SEQ ID NO: 17 consisting of the sequence of o-Phe-Pro-Gly-Pro-Ile.

The peptide represented by SEQ ID NO: 6 and the known peptide represented by SEQ ID NO: 17, β-cas
The yield (%) of omoriphin 7 based on the raw milk β-casein was determined from its molecular weight and the molecular extinction coefficient at Abs. _280, and was about 1.4% and about 0.7%, respectively.
Was confirmed.

When the Rf value was determined by thin-layer chromatography on a silica gel plate in the same manner as in Example 1, the peptide represented by SEQ ID NO: 6 was 0.59 in the same manner as in Example 2; Similarly, β-casomoriphin 7, a known peptide represented by SEQ ID NO: 17, was 0.72
Met.

Example 6 [Chemical synthesis of peptide represented by SEQ ID NO: 3 to SEQ ID NO: 16] The peptide represented by SEQ ID NO: 3 was synthesized by the following method. 0.2 g of commercially available Boc (butoxycarbonyl) -Asn-resin (substitution rate 0.5 meq / g) and methylene chloride 2 containing 45% trifluoroacetic acid and 2.5% anisole
0 ml of biosearch peptide synthesizer SAM T
The reaction was carried out at room temperature for 25 minutes in a WO reactor to remove the Boc group. Next, the Asn resin from which the Boc group had been removed was washed with methylene chloride, then neutralized with methylene chloride containing 10% diisopropylethylamine, and further washed with methylene chloride. This resin was mixed with 5 ml of a 0.4 M solution of Boc-His in dimethylformamide and 5 ml of a 0.4 M solution of diisopropylcarbodiimide in methylene chloride, placed in a reaction vessel, and reacted at room temperature for 2 hours with stirring.

The resin obtained above was washed successively with dimethylformamide, methylene chloride, methylene chloride containing 10% of diisopropylethylamine, methylene chloride and a methylene chloride / dimethylformamide mixed solution, and
c-His-Asn-resin was obtained. Continue with B as above
The removal of the oc group and the coupling of the Boc-amino acid were repeated to obtain Boc-Val-Tyr (Cl ₂ -Bzl) -Pro-Phe-Pro-Gly-Pr.
A product consisting of o-Ile-His-Asn resin was obtained.

The resin was placed in 20 ml of hydrogen fluoride containing 10% anisole and stirred at 0 ° C. for 2 hours to release the peptide from the resin. Thereafter, hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 30% acetic acid, which was lyophilized to obtain 100 mg of a crude peptide. This is an ODS column of Nacalai Tesque, Inc. Cosmosil 5C ₁₈ -
Purification by reverse phase chromatography using AR gave 30 mg of the final product. Amino acid composition (molar ratio)
Is: Val: Tyr: Pro: Phe: Gly: Ile: His: Asn = 1:
1: 3: 1: 1: 1: 1: 1. Further analysis by the protein sequencer used in Example 1 confirmed that the peptide was represented by SEQ ID NO: 3. Further, the Rf value of the product by thin layer chromatography was 0.49 similarly to the product of Example 1.

Also, in the case of the peptides represented by SEQ ID NOs: 4 to 16, similarly to the peptide represented by SEQ ID NO: 3, commercially available Boc-Asn-resin (substitution rate: 0.5)
The desired final product (each peptide) was prepared from 0.2 g of meq / g). At that time, the yield of the final product (each peptide), the amino acid composition (molar ratio) and the Rf value of the final product by thin-layer chromatography were as shown in Table 1 below.

[0084]

[Table 1] Sequence Yield Amino acid composition (molar ratio) Rf value number (mg) 3 30 Val: Tyr: Pro: Phe: Gly: Ile: His: Asn = 1: 1: 3: 1: 1: 1: 1: 1 0.49 4 40 Val: Tyr: Pro: Phe: Gly: Ile: Asn = 1: 1: 4: 1: 1: 1: 1 0.57 5 28 Tyr: Pro: Phe: Gly: Ile: His: Asn = 1: 3: 1: 1: 1: 1: 1 0.46 635 Tyr: Pro: Phe: Gly: Ile: Asn = 1: 4: 1: 1: 1: 1 0.59 730 Tyr: Pro: Phe: Gly: Ile: Ala: Asn = 1: 3: 1: 1: 1: 1: 1 0.59 8 22 Tyr: Pro: Phe: Gly: Ile: Arg: Asn = 1: 3: 1: 1: 1: 1: 1 0.76 9 20 Tyr: Pro: Phe: Gly: Ile: Glu: Asn = 1: 3: 1: 1: 1: 1: 1 0.54 10 18 Tyr: Pro: Phe: Gly: Ile: Asn = 1: 3: 1: 2: 1: 1 0.62 11 20 Tyr: Pro: Phe: Gly: Ile: Leu: Asn = 1: 3: 1: 1: 1: 1: 1 0.77 12 35 Val: Tyr: Pro: Phe: Gly: Ile: Ala: Asn = 1: 1: 3: 1: 1: 1: 1: 1 0.56 13 25 Val: Tyr: Pro: Phe: Gly: Ile: Arg: Asn = 1: 1: 3: 1: 1: 1: 1: 1 0.65 14 25 Val: Tyr: Pro: Phe: Gly: Ile: Glu: Asn = 1: 1: 3: 1: 1: 1: 1: 1 0.46 15 27 Val: Tyr: Pro: Phe: Gly: Ile: Asn = 1: 1: 3: 1: 2: 1: 1 0.53 16 22 Val: Tyr: Pro: Phe: Gly: Ile: Leu: Asn = 1: 1: 3: 1: 1: 1: 1: 1 0.66

The opioid activity and the novel peptide represented by SEQ ID NO: 3 to SEQ ID NO: 16 of each fraction in the above Examples 1 to 6, and the known peptide represented by SEQ ID NO: 17, β-casomorphin 7, The opioid activity of pro-β-casomorphin 7, a known peptide represented by SEQ ID NO: 20, was measured as follows.

<< Method for Measuring Opioid Activity >> Weight 200-
An ileum longitudinal muscle isolated from a 250 g guinea pig was isolated with an isotonic transducer (Nihon Kohden Kogyo Co., Ltd .:
TB 612-T) and apply 0.5 g tension.
Place Krebs in a 2 ml Magnus tube kept at 36 ° C.
Isotonic solution (NaCl 118 mM, KCl 4.75 mM, CaCl ₂ 2.
54 mM, MgSO ₄ 1.2 mM, NaHCO ₃ 25 mM, KH ₂ PO
₄ Dip the ileum longitudinal muscle into a solution filled with 1.19 mM and glucose 11 mM, and aerate from the cylinder (0 ₂ 95
%: CO ₂ 5%). After stabilizing for about 2 hours in this way, an electrical stimulus of 0.5 msec at 30 V was given,
The ileum longitudinal muscle was electrically contracted. After the electric contraction becomes stable,
Inhibitor containing aminopeptidase inhibitor 50μ
1 and each of the fractions in Examples 1 to 6 and the peptides represented by SEQ ID NOS: 3 to 17 were added,
Changes in electrical contraction of the ileum longitudinal muscle were recorded with a recorder.
Opioid activity was determined by inhibition of electrocontraction after addition of each of the fraction and peptide, and release of inhibition by addition of 20 μl of 10 ⁻⁴ M naloxone. Then, the concentration (IC ₅₀ ) of inhibiting the electrical contraction of the ileum longitudinal muscle by 50% was measured.

As a result, a novel peptide represented by SEQ ID NO: 3 to SEQ ID NO: 16 obtained in the above example, and a known peptide represented by SEQ ID NO: 17, β-casomorphin7
The IC _{50 for} the opioid activity was as shown in Table 2 below. For reference, the IC ₅₀ obtained by measuring the opioid activity of pro-β-casomorphin 7, which is a known peptide represented by SEQ ID NO: 20, in the same manner as described above is also shown in Table 2 below.

Further, the ACE inhibitory activity of the novel peptides represented by SEQ ID NOS: 3 to 16 and SEQ ID NO: 1
The ACE inhibitory activity of β-casomorphin 7, which is a known peptide represented by SEQ ID NO: 7, and the ACE inhibitory activity of pro-β-casomorphin 7, which is a known peptide represented by SEQ ID NO: 20, are as follows. It was measured.

<< Method for Measuring ACE Measurement Activity >> Measurement of ACE inhibitory activity was carried out according to Biochemical Pharamacology 20; 1937 (197
The following method was used according to the Cheung and Cushman method described in 1). Enzyme substrate : A solution prepared by dissolving 86 mg of Bz (benzyl) -Gyl-His-Leu in 8 ml of distilled water and 8 ml of phosphate buffer Enzyme solution : acetone powder of rabbit lung (manufactured by Sigma)
After grinding 1 g in 10 ml of 50 nM phosphate buffer,
Centrifuged supernatant 100 μl of the above enzyme substrate, 12 μl of the enzyme solution and a predetermined amount of the peptide represented by SEQ ID NO: 3 to SEQ ID NO: 17 were mixed, and distilled water was added to make the total volume 250 μl. 1.5 ml of ethyl acetate was added, stirred for 15 seconds and centrifuged. 1 ml was collected from the ethyl acetate layer, ethyl acetate was distilled off, and the residue was dissolved by adding 1 ml of distilled water. The ultraviolet absorption value at 228 nm (O
D ₂₂₈ ) was measured.

The ACE activity inhibition rate was determined by the peptide (inhibitor)
OD ₂₂₈ when reacted as above without
OD ₂₂₈ before the start of the reaction (when the reaction time is 0 minutes)
Is 0%, the concentration of the inhibitor at which the ACE activity inhibition rate becomes 50% was determined as IC ₅₀ . As a result, the novel peptide represented by SEQ ID NO: 3 to SEQ ID NO: 16 obtained in the above example and the IC ₅₀ of the ACE inhibitory activity of β-casomorphin 7, a known peptide represented by SEQ ID NO: 17
Was as shown in Table 2 below. For reference, pro-β-casomor, a known peptide represented by SEQ ID NO: 20
Table 2 also shows the IC ₅₀ when the ACE inhibitory activity of phin7 was measured in the same manner as described above.

[0091]

[Table 2] Opioid activity ACE inhibitory activity Type of peptide IC ₅₀ IC ₅₀ Peptide of nonapeptide contained in SEQ ID NO: 1 14 55 Peptide of SEQ ID NO: 6 11.4 450 Peptide of SEQ ID NO: 7 14 100 Peptide of SEQ ID NO: 8 24 115 SEQ ID NO: 9 Peptide 20 34 Peptide of SEQ ID NO: 10 37 37 Peptide of SEQ ID NO: 11 14 40 Decapeptide contained in SEQ ID NO: 2 Peptide of SEQ ID NO: 3 141 170 Peptide of SEQ ID NO: 4 270 250 Peptide of SEQ ID NO: 12 150 200 SEQ ID NO: 13 Peptide 150 200 peptide of SEQ ID NO: 14 170 150 peptide of SEQ ID NO: 15 120 200 peptide of SEQ ID NO: 16 140 250 known peptide of SEQ ID NO: 17 500 (β-casomorphin 7) known peptide of SEQ ID NO: 20 240 330 (pro-β -casomorphin7)

From the results in Table 2, the novel peptides of the present invention represented by SEQ ID NOS: 3 to 16 have opioid activity, and among them, the SEQ ID NO: 1 is included in the peptide represented by SEQ ID NO: 1. It can be seen that the nonapeptides represented by SEQ ID NOs: 5 to 11 have high opioid activity. In addition, it can be seen that the novel peptides of the present invention represented by SEQ ID NOs: 3 to 16 can inhibit the activity of ACE in a smaller amount than the known peptide represented by SEQ ID NO: 17.

[0093]

Since the peptide of the present invention or a salt thereof has an opioid activity, an agent containing the peptide or a salt thereof as an active ingredient can be administered in a very small amount to provide analgesia, anesthesia,
Emotion, respiration, pulsation, body temperature, gastrointestinal function, eating, immunity, regulation of hormones such as insulin and somatostatin,
It may be used as an analgesic anesthetic, a hypnotic, a gastrointestinal hormonal secretion enhancer, an electrolyte absorption enhancer, or a diarrhea ameliorating agent due to gastrointestinal transport muscle contraction, because it may have a function of promoting electrolyte absorption and controlling myocardial contraction. Can be expected. The peptide included in the peptides represented by SEQ ID NO: 2 has lower opioid activity than the peptide included in the peptides represented by SEQ ID NO: 1, but is hydrolyzed in vivo to produce SEQ ID NO: 1
And may be highly activated, and it is highly possible to use it as a prodrug.

Further, the peptide of the present invention represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof has an ACE inhibitory activity, and exerts an ACE inhibitory effect on blood pressure increase in a very small dose. Inhibition and blood pressure lowering function can be exhibited, so that it can be expected to be used as a blood pressure increase inhibitor and a blood pressure lowering agent.

The above opioid activators and ACE inhibitors
When used as a harm agents, peptides and salts thereof of the present invention are the water-soluble white powder, also administered very easily by any of the methods oral and parenteral dissolved in as such or water or the like be able to.

The novel peptide of the present invention represented by SEQ ID NO: 1 or SEQ ID NO: 2 and a salt thereof are low molecular weight compounds having a very simple structure in which only 9 or 10 amino acids are arranged. , And can be easily manufactured by chemical synthesis. Furthermore, the peptides represented by SEQ ID NOs: 3 to 6 and 17 can be used to convert milk proteins such as milk β-casein into neutral proteins of Bacillus origin.
Aase, a neutral protease from Aspergillus
Neutral protease of Reptomyces origin, Rhizopus origin
Of neutral protease and Bacillus origin
Neutral protease of microbial origin selected from Rotase
Or hydrolyzed using an alkaline protease,
Optionally further hydrolyzed by aminopeptidase
Or aminopeptidase and carboxypeptidase
By the method of the present invention in which hydrolysis is carried out , a high yield can be assuredly obtained.

[0097]

[Sequence list]

SEQ ID NO: 1 Sequence length: 9 Sequence type: Amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 2 Sequence length: 10 Sequence type: amino acid topology: Linear sequence type: Peptide

SEQ ID NO: 3 Sequence length: 10 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 4 Sequence length: 10 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 5 Sequence length: 9 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 6 Sequence length: 9 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 7 Sequence length: 9 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 8 Sequence length: 9 Sequence type: amino acid topology: Type of linear sequence: peptide

SEQ ID NO: 9 Sequence length: 9 Sequence type: amino acid topology: Linear sequence type: Peptide

SEQ ID NO: 10 Sequence length: 9 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 11 Sequence length: 9 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 12 Sequence length: 10 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 13 Sequence length: 10 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 14 Sequence length: 10 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 15 Sequence length: 10 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 16 Sequence length: 10 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 17 Sequence length: 7 Sequence type: amino acid topology: Linear sequence type: Peptide

SEQ ID NO: 18 Sequence length: 5 Sequence type: Amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 19 Sequence length: 4 Sequence type: amino acid topology: Type of linear sequence: Peptide

SEQ ID NO: 20 Sequence length: 8 Sequence type: amino acid topology: Type of linear sequence: Peptide

[Brief description of the drawings]

FIG. 1 shows the absorbance of a peptide mixture obtained by hydrolyzing milk β-casein with thermolysin through high performance liquid chromatography, and the adsorbed component is eluted with a specific eluent having a linear concentration gradient. 5 is a flowchart in which is measured.

FIG. 2 shows a peptide mixture obtained by hydrolyzing milk β-casein with thermolysin and leucine aminopeptidase through high performance liquid chromatography, and the adsorbed components are eluted with a specific eluent having a linear concentration gradient. It is the flowchart which measured the light absorbency of the said component.

FIG. 3: A peptide mixture obtained by hydrolyzing milk β-casein with thermolysin, leucine aminopeptidase and carboxypeptidase A is passed through high performance liquid chromatography, and the adsorbed components are separated by a specific eluent having a linear concentration gradient. It is the flowchart which measured the absorbance of the said component at the time of elution.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C07K 7/06 A23J 3/10 A23J 3/34 C12N 9/99 A61K 38/08 BIOSIS (DIALOG) CA (STN ) REGISTRY (STN)

Claims (9)

(57) [Claims] 1. A peptide represented by SEQ ID NO: 1 (where Xaa represents an amino acid) or a salt thereof. 2. A peptide represented by SEQ ID NO: 2 (where Xaa represents an amino acid) or a salt thereof. 3. An active ingredient comprising one or more of the peptide represented by SEQ ID NO: 1 (where Xaa represents an amino acid) or SEQ ID NO: 2 (where Xaa represents an amino acid) and salts thereof. Angiotensin converting enzyme inhibitor. 4. A β-casein , wherein a neutral protease of Bacillus origin, a neutral protease of Aspergillus origin,
Hydrolysis using neutral protease or alkaline protease of microbial origin selected from neutral protease of Streptomyces origin, neutral protease of Rhizopus origin and alkaline protease of Bacillus origin, the opioid activity from the resulting hydrolyzate A method for producing an opioid peptide, comprising recovering a peptide having the same. 5. The method according to claim 4, wherein the opioid peptide is a peptide represented by SEQ ID NO: 3 or SEQ ID NO: 4. 6. A β-casein , wherein a neutral protease of Bacillus origin, a neutral protease of Aspergillus origin,
After hydrolysis using a neutral or alkaline protease of microbial origin selected from neutral protease of Streptomyces origin, neutral protease of Rhizopus origin and alkaline protease of Bacillus origin, further hydrolysis using aminopeptidase. A method for producing an opioid peptide, comprising decomposing and recovering a peptide having opioid activity from the obtained hydrolyzate. 7. The method according to claim 6, wherein the opioid peptide is a peptide represented by SEQ ID NO: 5 or SEQ ID NO: 6. 8. A β-casein , wherein a neutral protease of Bacillus origin, a neutral protease of Aspergillus origin,
After hydrolysis using neutral protease or alkaline protease of microbial origin selected from neutral protease of Streptomyces origin, neutral protease of Rhizopus origin and alkaline protease of Bacillus origin, aminopeptidase and carboxypeptidase are simultaneously or A method for producing an opioid peptide, wherein the method is used successively in any order to further hydrolyze and recover a peptide having opioid activity from the obtained hydrolyzate. 9. The method according to claim 8, wherein the opioid peptide is a peptide represented by SEQ ID NO: 6 and / or SEQ ID NO: 17.