JP2005255670A - Hypotensive peptide derived from royal jelly - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a safe ACE(angiotensin-converting enzyme) inhibitor having high hypotensive effect. <P>SOLUTION: A hypotensive peptide as the ACE inhibitor, i.e. having angiotensin-converting enzyme inhibitory activity, is obtained by treating a royal jelly material with trypsin. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a royal jelly-derived protein degradation product, a method for producing the same, a food containing the protein degradation product, and a preparation for oral consumption. The proteolysate of the present invention has an angiotensin converting enzyme inhibitory action and a bradykinin-induced intestinal contractile response enhancing action, and is effective in preventing or treating hypertension.

  Hypertension is a disease that has no subjective symptoms and, if left untreated, causes secondary diseases such as heart disease and cerebrovascular disorder, and the treatment and prevention of hypertension is a major issue. It is known that angiotensin converting enzyme (hereinafter referred to as ACE) is deeply involved in hypertension, and hypertension can be prevented or treated by inhibiting ACE.

  On the other hand, since prevention or treatment of hypertension requires long-term ingestion, the extremely low side effects are an important factor, and the development of foods that prevent hypertension is required.

  Royal jelly is a milky-white jelly-like substance secreted from the hypopharyngeal gland and large chin gland by the bees to grow the queen bee, and has a long intake history as a human food, and has a wide range of nutrition, tonic, constitution improvement, etc. It is said that there is an effect. Studies have been made on enzymatic degradation products of the proteins, and royal jelly-derived peptides having ACE inhibitory activity are also known (Patent Documents 1 to 3, Non-Patent Documents 1 to 6).

However, these peptides are for industrialization from the viewpoint of the balance of the amount obtained by hydrolysis of royal jelly material and the strength of ACE inhibitory activity, immediate effect, side effect, duration of action, ingested form, and storage stability. There was room for improvement.
Japanese Patent No. 30686656 JP2002-145899 JP 2002-338594 A Journal of Nutritional Biochemistry 13 (2002) pp.80-86 Diagnosis and New Drug, Vol. 39, No. 2, 2002, pp. 85-90 Journal of Japanese Society for Food Science and Technology, Vol. 50, No. 10, pages 457-462 (2003). Journal of Japan Society for Food Science and Technology, Volume 50, No. 6, pp. 286-288 (2003). Journal of Japanese Society for Food Science and Technology, Vol. 50, No. 7, pp. 310-315 (2003) Journal of Japan Society for Food Science and Technology, Vol. 51, No. 1, pp. 34-37 (2004).

  An object of the present invention is to provide a proteolytic product having no side effects and having an effect of preventing hypertension, a method for producing the same, a food containing the proteolytic product, and a preparation for oral consumption.

  Another object of the present invention is to provide an ACE inhibitor having immediate effect, sustained effect, and storage stability, and a prophylactic or therapeutic agent for hypertension containing the inhibitor.

As a result of repeated examinations in view of the above problems, the present inventor can prevent hypertension by hydrolyzing a royal jelly material such as raw royal jelly or a denatured protein obtained by ethanol precipitation of dried royal jelly with a proteolytic enzyme such as trypsin. It has been found that possible proteolytic products can be obtained.
In addition, the proteolysate of the present invention has a strong ACE inhibitory action, but has a stronger preventive or therapeutic action on hypertension than expected from the ACE inhibitory action, and part of the action has a hypotensive action. It is presumed to be based on the degradation inhibitory action of bradykinin.

  The proteolytic product has low side effects such as allergic action, is a safe food derived from royal jelly, and has an antihypertensive action or antihypertensive action particularly useful for humans with high blood pressure such as normal high blood pressure or mild hypertension. It is particularly useful as a food product such as food for specified health use.

  When the proteolytic product of the present invention was administered to a human with normal high value and mild hypertension, a significant blood pressure lowering effect was confirmed, and blood pressure increased slowly after the end of intake, there was no rebound exceeding the pre-intake value, No abnormalities in pulse rate, body weight / BMI, blood test, urinalysis, no side effects that may have causal relationship with royal jelly degradation products such as dry cough, skin symptoms, digestive symptoms, etc., confirmed to be highly safe It was.

The present invention provides the following inventions.
1. A protein degradation product having an angiotensin converting enzyme inhibitory effect obtained by treating a royal jelly material with trypsin.
2. Item 2. The protein degradation product according to Item 1, wherein the royal jelly material is an alcohol-denatured protein of royal jelly.
3. Proteolysate derived from royal jelly material

A proteolysate having a residual ratio (molar ratio) after enzymatic degradation of Lys of 70% or less of the royal jelly material with respect to Gly represented by
4). Proteolysate derived from royal jelly material

A proteolytic product in which the content ratio (molar ratio) of Leu is 1.3 times or more that of the royal jelly material, based on Lys represented by
5). It is derived from royal jelly material and substantially contains at least one selected from the group consisting of (1) inorganic salts and (2) acidic free amino acids, basic free amino acids, free amino acids having alcoholic hydroxyl groups, free Ala and free Gly Item 5. A proteolytic product according to item 3 or 4.
6). Item 6. The proteolysate according to Item 5, which is derived from a royal jelly material and contains substantially no (1) inorganic salts and (2) free acidic amino acids and free basic amino acids.
7). Derived from royal jelly material, (1) inorganic salts and (2) acidic free amino acids (Asp, Glu), basic free amino acids (Lys, His, Arg), free amino acids with alcoholic hydroxyl groups (Thr, Ser), free Item 7. The protein degradation product according to Item 6, which is substantially free of Ala and free Gly.
8). Item 6. The protein degradation product according to any one of Items 3 to 5, which has a sugar content of 35% by weight or less and does not substantially discolor under accelerated test conditions at 40 ° C. for 2 months.
9. Item 3-6 having a maximum peak in the molecular weight range of about 200-1500 and a sharp peak of about 200-300 molecular weight, with substantially no component having a molecular weight of 10,000 or more by analysis with a gel filtration column. The protein degradation product in any one of.
10. Item 7. The protein degradation product according to any one of Items 3 to 6, wherein the average molecular weight is in the range of about 700 to 6000.
11. When the proteolysate is passed through a column (4.6 mmφ × 150 mm) of a styrene / divinylbenzene copolymer having the following characteristics, the content (%) of the component concerned represented by the following formula is 35% or more, preferably 40 % Or more of the protein degradation product / styrene / divinylbenzene copolymer according to any one of Items 1 to 10 Particle size distribution: 30 μm
Pore diameter: 250mm
Functional group; none Applicable pH range: Full range.

And involvement ingredient content _{(%) = A 1 / A} T × 100
T ₁ : Tryptophan retention time
T ₂ : 10-hydroxy-δ-2-decenoic acid retention time
A ₁ : Total peak area from the peak detected at the T ₁ elution position to the peak detected at the T ₂ elution position
A _T : Sum of all peak areas.
12 Item 12. The protein degradation product according to Item 11, wherein the content of the concerned component is 58 ± 5%.
13. Item 13. The proteolysate according to any one of Items 1 to 12, comprising at least one of the following nine peptides.
Glu-Trp-Lys;
His-Pro-Ala-Leu;
Thr-Phe;
Ser-Pro-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Thr-Pro-Phe;
Ser-His-Ser-Gly-Leu-Tyr;
Tyr-Ser-Pro-Leu.
14 The protein degradation product is passed through a column of a styrene / divinylbenzene copolymer having the following characteristics, and water (fractions 1, 2), 20% methanol (fraction 3), 40% methanol (fraction 4), 60% methanol ( Item 11. The protein degradation product according to any one of Items 1 to 11, comprising fractions 3 to 6 among fractions eluted with 80% ethanol (fraction 6) and 100% ethanol (fraction 7).
15. In fraction 3
Glu-Trp-Lys,
His-Pro-Ala-Leu and
Thr-Phe
At least one peptide selected from the group consisting of: and in fraction 4, Thr-Phe;
Ser-Pro-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Thr-Pro-Phe;
Ser-His-Ser-Gly-Leu-Tyr;
Tyr-Ser-Pro-Leu
Item 15. The proteolysate according to Item 14, comprising at least one peptide selected from the group consisting of:
16. Item 16. A prophylactic or therapeutic agent for hypertension, comprising the protein degradation product according to any one of items 1 to 15 as an active ingredient.
17. Item 15. A food containing the protein degradation product according to any one of Items 1 to 15.
18. Item 18. The food according to Item 17, which is a food for preventing hypertension.
19. Item 16. A preparation for oral consumption containing the protein degradation product according to any one of Items 1 to 15.
20. A prophylactic or therapeutic agent for hypertension containing at least one peptide selected from the following seven peptides as an active ingredient:
Glu-Trp-Lys;
His-Pro-Ala-Leu;
Thr-Phe;
Ser-Pro-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Thr-Pro-Phe;
Ser-His-Ser-Gly-Leu-Tyr;
Tyr-Ser-Pro-Leu.
21. Item 21. A food containing the protein degradation product according to Item 20.
22. Item 22. The food according to Item 21, which is a food for preventing hypertension.
23. Item 21. A preparation for oral consumption containing at least one peptide according to Item 20.
24. Item 20. The preparation according to Item 19, which is a food form comprising an apparent food and / or supplement.
25. Item 16. An angiotensin converting enzyme inhibitor comprising the protein degradation product according to any one of Items 1 to 15 as an active ingredient.
26. Item 16. The method for producing a protein degradation product according to any one of Items 1 to 15, wherein the royal jelly material is treated with trypsin.
27. Proteolytic product obtained by trypsin treatment is treated with a synthetic adsorbent, and consists of (1) inorganic salts and (2) acidic free amino acids, basic free amino acids, free amino acids having alcoholic hydroxyl groups, free Ala and free Gly. Item 27. The method according to Item 26, wherein at least one member selected from the group is removed.
28. Item 26. The protein product obtained by trypsin treatment is adsorbed on a styrene-divinylbenzene copolymer, washed with water to remove inorganic salts and water-soluble amino acids, and then eluted with hydrous alcohol. Method:
29. Item 29. The method according to Item 28, wherein the styrene-divinylbenzene copolymer has the following characteristics:
Particle size distribution:> 250μm (including more than 90% particles with a particle size larger than 250μm)
Pore diameter: 400mm
Functional group: None Applicable pH range: Full range.
30. A novel peptide consisting of any of the following five peptides:
His-Pro-Ala-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Tyr-Ser-Pro-Leu;
Ser-His-Ser-Gly-Leu-Tyr.

The proteolysate derived from the royal jelly material of the present invention exhibits a strong ACE inhibitory action when taken orally and is effective in preventing or treating hypertension. Moreover, by using trypsin as an enzyme, the ACE inhibitory activity per unit weight and the yield of proteolysate can be improved as compared with the case of using pepsin and chymotrypsin, and the effective use of royal jelly material can be achieved. Can do.
The protein degradation product of the present invention has a reduced protein content of a molecular weight of 20,000 or more that can be an antigen, and a preferred protein degradation product has little or no protein having a molecular weight of 10,000 or more that can be an antigen. Therefore, the protein degradation product of the present invention can suppress allergic reaction more strongly, and almost no allergic reaction is observed.
Furthermore, the present invention and the proteolysate are safe with no side-effects such as dry cough, skin symptoms, and digestive symptoms, with no abnormality in pulse, body weight / BMI, blood test, and surface test.

  The proteolysate of the present invention that has been desalted has a very low content of salts, particularly sodium chloride, which can serve as pressurizing substances. In addition, the desalting operation reduces the content of other components such as free amino acids and sugars, suppresses chemical reactions such as hygroscopicity and Maillard reaction, and a more stable proteolysate can be obtained.

  By taking the proteolysate of the present invention for a long period of time, blood pressure can be lowered slowly and continuously, and normal blood pressure subjects can prevent high blood pressure and various diseases caused by high blood pressure. .

  Since the proteolytic product of the present invention has a long duration of action (8 to 12 hours) and blood pressure gradually decreases, when taking the proteolytic product, fluctuations in blood pressure per day are small, and blood pressure control is possible. Easy. For example, if it is taken in the morning, the blood pressure is lowered during the activity time, and the blood pressure is not lowered while sleeping, so that it has an ideal action pattern when taken by a person with higher blood pressure. In addition, the blood pressure rises slowly after discontinuation of administration, and no rebound that exceeds the blood pressure before administration is observed.

  The proteolysate of the present invention has a negative result for mutagenicity and antigenicity tests, is a highly safe material with little blood pressure fluctuation and little effect on heart rate and body weight. is there.

  The proteolysate of the present invention is suitable for ingestion by normal high- or mildly hypertensive persons or hypertensive patients who have risk factors for hypertension (genetic, environmental predisposition, etc.).

In the present invention, the royal jelly material means a material containing a protein of royal jelly, and is not particularly limited as long as this requirement is satisfied. For example, raw royal jelly, dried royal jelly, royal jelly is ethanol precipitated, ammonium sulfate treated, heated Protein precipitates (including ethanol precipitates) obtained by protein denaturation such as treatment, or materials containing proteins after fractionation by various means such as extraction, filtration, centrifugation, and chromatography of royal jelly Means.
The royal jelly may be anything, and widely used by bees secreted to grow queen bees. Specifically, the royal jelly is a milky white jelly-like substance produced by mixing secreted products of the bees from the hypopharyngeal gland and the greater vagina.

  As the protein fraction derived from royal jelly, a protein fraction collected by alcohol precipitation of royal jelly can be suitably used. In particular, it is preferable to use an ethanol-denatured protein of royal jelly that is by-produced as a water-insoluble protein during the production of a royal jelly beverage. The ethanol-denatured protein can be obtained as a protein having low solubility in water of a residue obtained by extracting a physiologically active substance for beverages such as decenoic acid from royal jelly using about 50 to 100% by volume of ethanol.

The enzymatic lysate of the royal jelly material of the present invention can be obtained by hydrolysis with trypsin. Trypsin includes active α-trypsin and β-trypsin produced by limited hydrolysis of trypsinogen, and either of them may be used. In addition, trypsin-related enzymes having similar substrate specificity and catalytic mechanism such as thrombin, plasmin, kallikrein, and urokinase can also be used as trypsin. Furthermore, a mutant enzyme obtained by modifying one or more amino acids of trypsin (including trypsin-related enzymes) by substitution, addition, deletion, insertion, or the like may be used. Such mutant trypsin and trypsin-related enzymes are also included in the trypsin used for enzymatic degradation of the royal jelly material of the present invention. Trypsin is derived from mammals such as cows, pigs, sheep, and humans. For the hydrolysis of the royal jelly material, trypsin extracted from the pancreas of a mammal such as cow or pig may be used, or trypsin obtained by gene recombination may be used.
The conditions for treating the royal jelly material with trypsin include, for example, about 15 ° C. to about 60 ° C., preferably about 20 ° C. to about 50 ° C., particularly around 37 ° C. in an aqueous solvent, about 1 to 72 hours, preferably 3 to It can be preferably carried out by treating for about 48 hours. The amount of trypsin used is, for example, about 0.01 to 3 g, preferably about 0.05 to 1 g, per 100 g of protein. Trypsin may be a free enzyme or an enzyme immobilized on a carrier. As trypsin, specifically, bovine trypsin made by Roche Diagnostics and porcine trypsin made by Novozyme can be preferably used.
The pH of the enzyme reaction solution is about 6 to 10, preferably about 7 to 9. When royal jelly material is hydrolyzed with trypsin, the pH gradually decreases, so alkali metal hydroxides (eg, sodium hydroxide, potassium hydroxide), alkali metal carbonates (eg, sodium carbonate, potassium carbonate), alkali metal hydrogen carbonate It is preferred to use a base such as a salt (eg, sodium bicarbonate, potassium bicarbonate) to keep the pH within a certain range.
Water can be suitably used for the reaction solution, but it may be carried out in an aqueous solvent such as hydrous ethanol containing a small amount of a water-miscible organic solvent such as ethanol.
The reaction solution of the enzyme degradation product is subjected to heat treatment, and if necessary, further post-treatment such as centrifugation, filtration, extraction, desalting, freeze-drying and spray-drying (or spray-drying), and the protein degradation product of the present invention Can be obtained. For example, when raw royal jelly or dried royal jelly is used as the royal jelly material, components such as saccharides derived from royal jelly can be removed. In one preferred embodiment of the present invention, the royal jelly material proteolysate of the present invention has a molecular weight measured by a gel filtration column of about 200 to 7000, particularly about 250 to 6000, and a molecular weight of about 200 to 300. And a maximum peak in the molecular weight range of about 200-1500. The estimated molecular weight of the protein degradation product estimated from the measurement result of the gel filtration column is about 700 to 6000, preferably about 800 to 5000.

  In another preferred embodiment of the present invention, the proteolysate of the royal jelly material of the present invention comprises sugars, salts, acidic free amino acids, basic free amino acids, free amino acids having an alcoholic hydroxyl group, free Ala and free Gly. And at least one selected from the group consisting of: As described above, when the royal jelly material is trypsinized, the pH of the reaction solution decreases as the hydrolysis of the protein proceeds. Therefore, a base such as sodium hydroxide is added to adjust the pH of the reaction solution to that of trypsin. It is preferable to maintain the pH at about 6 to 10, particularly about 7 to 9. The thus added base (for example, sodium hydroxide) is adjusted to a pH of about 3.5 to 7, preferably about 4 to 5.5 by adding an acid (for example, hydrochloric acid) to the reaction solution after the enzyme reaction. , Leading a base (eg sodium hydroxide) to the corresponding salt (eg sodium chloride). It is desirable to remove a large amount of salt generated at this time, particularly in the case of a salt containing sodium such as sodium chloride, because sodium is a pressurizing substance. This desalting can be carried out, for example, by using a desalting column. Specifically, a hydrophobic synthetic adsorbent containing styrene / divinylbenzene copolymer (for example, Diaion HP-20 (Mitsubishi Chemical). ), Sepabeads SP-70 (Mitsubishi Chemical Co., Ltd.) is used to adsorb the proteolysate, and salts such as sodium chloride are washed away with water, and then an appropriate organic solvent such as methanol, ethanol, acetonitrile is removed. By elution, a protein degradation product that is desalted and substantially free of inorganic salts can be obtained. When a synthetic adsorbent is used for desalting, for example, acidic or basic free amino acids which are difficult to be adsorbed by a hydrophobic synthetic adsorbent such as Asp, Glu, Lys, and Arg are removed together with inorganic salts, while Phe, Leu , Ile, Trp, Val, etc., which are hydrophobic and easily adsorbed on the synthetic adsorbent, are not easily removed by a desalting operation. It should be noted that free amino acids having alcoholic hydroxyl groups such as Ser and Thr, and also free Ala and free Gly are not easily adsorbed by hydrophobic synthetic adsorbents, so they are removed together with inorganic salts like acidic or basic free amino acids. Easy to be.

  In one embodiment of the present invention, a relatively large amount of free Lys is produced in the proteolysate of royal jelly, but most of it is washed away with water by desalting using a synthetic adsorbent column. The proteolysate of the fraction eluted and recovered in step 3 is substantially free of free Lys.

  In another embodiment of the present invention, a large amount of free Ala is also present in the proteolysate of royal jelly, and most of the free Ala is also washed and removed by desalting using a synthetic adsorbent column. The proteolysate of the fraction eluted and collected by the above method does not substantially contain free Ala.

  Here, “substantially free of free amino acids” means that the content of free amino acids is about 0.01 μmol / 10 mg or less, preferably about 0.001 μmol / 10 mg or less, more preferably 0.0001 μmol / 10 mg or less. It means that.

In the present specification, “acidic amino acid” means Glu and Asp, basic amino acid means Lys, His, Arg, and amino acid having an alcoholic hydroxyl group means Thr, Ser.
In one preferred embodiment, the content of salts (particularly sodium chloride) in the proteolysate of the present invention is 1% by weight or less, more preferably 0.2% by weight or less, still more preferably 0.1% by weight or less, Especially below the detection limit.

  As described above, the desalting treatment reduces the concentration of basic free amino acid, acidic free amino acid, alcoholic free amino acid, free Ala, free Gly, etc., but there is little or no decrease in ACE inhibitory activity. This is presumably because the contribution rate to the ACE inhibitory activity such as basic free amino acids, acidic free amino acids, alcoholic free amino acids, free Ala, and free Gly is low.

In another preferred embodiment of the present invention, the proteolysate of the present invention has a Lys residual ratio (compared by the number of moles) based on Gly of 25 to 70%, 30 to 60% of the royal jelly material. %, 35-55%, 40-50% or less, 42-48%, or 44-46%. For example, regarding the protein degradation product obtained in the examples of the present invention,
Royal jelly material (ethanol precipitate) before enzymatic degradation
Lys (1.09 mol) per Gly (1 mol)
Enzymatic degradation product (sample collected after desalting by column treatment packed with synthetic adsorbent)
Lys (0.49 mol) per Gly (1 mol)
So, when calculating the residual rate of Lys according to Equation 1,
100 × (0.49 / 1.09) = 44.95%.

  Although there are some fluctuations depending on the royal jelly material and degradation conditions, it is clear that when the protein degradation product is adsorbed with a synthetic adsorbent and desalted, the concentration of free Lys is lower than that of the original degradation product. It is.

In another preferred embodiment of the present invention, the proteolysate of the present invention has a Leu content (molar ratio) of about 1.3 to 3.5 times that of the royal jelly material, based on Lys. For example, it is about 1.5 to 3 times, about 1.7 to 2.7 times, or about 2 to 2.5 times. For example, regarding the protein degradation product obtained in the examples of the present invention,
Royal jelly material (ethanol precipitate) before enzymatic degradation
Leu (1.08 mol) with respect to Lys (1 mol)
Enzymatic degradation product (sample collected after desalting by column treatment packed with synthetic adsorbent)
Leu (2.49 mol) relative to Lys (1 mol)
Therefore, when calculating the Lys content ratio (molar basis) according to Equation 1,
2.49 / 1.08 = about 2.3 times.

  In the proteolysate of the present invention, when a higher molecular weight protein, trypsin or the like that has not been decomposed remains, it can be removed by ultrafiltration or the like, if necessary.

  In another preferred embodiment of the present invention, the proteolysate of the present invention preferably has a low saccharide content derived from royal jelly. The saccharide (sugar content) is not particularly limited as long as the quality of the protein degradation product is not impaired, but is usually 35% by weight or less, preferably 30% by weight or less, more preferably 25% by weight or less. Examples of the sugar include monosaccharides or disaccharides such as glucose, fructose, galactose, maltose, and sucrose. These carbohydrates are preferably removed as much as possible because they cause a gradual reaction (for example, Maillard reaction) with the proteolytic product during storage and cause the proteolytic product to brown or become highly hygroscopic.

  The proteolysate of the present invention may be produced by desalting treatment after the enzyme treatment, and it is also possible to collect fractions having stronger ACE inhibitory activity by treatment with a column or the like. Then, if necessary, the same purification may be repeated to separate a peptide having an ACE inhibitory action, and the physiologically active peptide may be isolated or concentrated and used as a proteolysate having an ACE inhibitory action.

  For example, the ACE-inhibiting peptide is subjected to a synthetic adsorbent, a gel filter, a molecular sieve, a reverse phase chromatograph, etc. with respect to the proteolytic product of the present invention, and an appropriate eluent such as water, methanol, ethanol or a mixed solution thereof. The peptide having strong ACE inhibitory activity can be isolated by eluting and dividing into various fractions, and isolating the peptide by a conventional method such as preparative HPLC for the fraction having strong ACE inhibitory activity, and determining the structure. .

  In one preferred embodiment of the present invention, a proteolytic product obtained by trypsinizing a royal jelly material is adsorbed on an HP-20 column, which is one of the synthetic adsorbents, and fractionated into seven fractions according to the following scheme 1. Can do.

In the present specification, the fraction (fraction) may be abbreviated as “Fr” or “P”. For example, fraction 4 may be described as “Fr4” or “P4”.
Scheme 1
855 mL of royal jelly protein hydrolyzate (equivalent to 50 g as lyophilized product)
↓
Loaded on Diaion HP20 (column size: φ11cm × 20cm, capacity: about 1.9L)
↓ Cleaning solution: Use 3260 mL of water ↓ Eluent: 20%, 40%, 60%, 80%, 100% MeOH
↓ ・ Use 20% methanol 2460mL ↓ ・ Use 40% methanol 2340mL ↓ ・ Use 60% methanol 2720mL ↓ ・ Use 80% methanol 2300mL ↓ ・ Use 100% methanol 3200mL wash solution and each eluate ↓ Concentrate using an evaporator (40 ℃) Each concentrate ↓
Lyophilized water-washed fraction: fractions 1 and 2
20% methanol elution fraction: fraction 3
40% methanol elution fraction: fraction 4
60% methanol elution fraction: fraction 5
80% methanol elution fraction: fraction 6
100% methanol elution fraction: fraction 7

Fraction No. fractionated according to Scheme 1 above. The yield of 1 to 7 and the ACE inhibition rate are shown in Table 1 below, and the graph of fractions 1 to 7 is shown in FIG.

*: The concentration of the sample solution in each fraction is 0.5 mg / mL. The inhibition rate was measured according to the method described in Example 1 below.
Of the above fractions, products containing fractions 3-6 are preferred. Although fraction 7 may be contained, since fraction 7 has a low yield, a proteolysate containing fractions 3 to 6 exhibits a sufficient antihypertensive action. Further, although fractions 1 and 2 can exhibit an ACE inhibitory action, they contain an amino acid and an inorganic salt (for example, NaCl), and therefore need not be included in the proteolysate of the present invention.
When the protein degradation product of the present invention is passed through a column (4.6 mmφ × 150 mm) of a styrene / divinylbenzene copolymer (trade name: MCI-GEL, CHP55Y, manufactured by Mitsubishi Chemical Corporation) having the following characteristics, The component content (%) expressed is 35% or more, preferably 40% or more, more preferably 50% or more, still more preferably 50 to 70%, particularly preferably 58 ± 5%, most preferably 58 ± 3%. Good to be:
And involvement ingredient content _{(%) = A 1 / A} T × 100
T ₁ : Tryptophan retention time
T ₂ : 10-hydroxy-δ-2-decenoic acid retention time
A ₁ : Total peak area from the peak detected at the T ₁ elution position to the peak detected at the T ₂ elution position
A _T : Sum of all peak areas.
・ Characteristics of styrene / divinylbenzene copolymer Particle size distribution: 30μm
Pore diameter: 250mm
Functional group; none Applicable pH range: Full range.

Column: 4.6mmφ × 150mm
A ₁ mainly includes fraction 4 and fraction 5.
The measurement of the content of the components involved in the royal jelly protein hydrolyzate (specimen) can be carried out, for example, under the following conditions (test method).
Test method Weigh 0.50 g of this product, add 10 mL of water / methanol mixture (1: 1) and dissolve to make 50 mL. Filter this solution through a membrane filter (0.45 μL), and use the filtrate as the test solution.
Alternatively, weigh 0.50 g of this product, dissolve with 10 mL of water / methanol mixture (1: 1) and phosphate buffer (pH 8.0) ¹⁾ 10 mL, and then add 15 mL of water and 5 mL of dilute acetic acid ²⁾ Shake and add water to make 50 mL. Filter this solution through a membrane filter (0.45 μL), and use the filtrate as the test solution.
Separately, tryptophan ³⁾ 1 mg and glycyl-L-leucyl-L-tyrosine ⁴⁾ 5 mg are weighed, acetic acid / sodium acetate buffer solution (pH 4.0) ⁵⁾ Add ⁵ mL to dissolve, and add water to make 10 mL. Use liquid.
10-Hydroxy-δ-2-decenoic acid standard ⁶⁾ Weigh 5 mg and add 0.5 mL of water / methanol mixture (1: 1) to dissolve. Add 0.5 mL of solution A to this solution and mix well.
Perform liquid chromatography with 50 µL each of the test solution and comparative solution under the following operating conditions. The retention times of the tryptophan and 10-hydroxy-δ-2-decenoic acid in the comparison solution are defined as T ₁ and T ₂ and detected at the T ₂ elution position after the peak detected at the T ₁ elution position in the test solution. Measure the total peak area A ₁ and the total peak area _AT of all the peak areas up to the peak before the calculated peak, and calculate the content of the components involved by the following formula.

Involved component content (%) = A ₁ / A _T × 100

Operation condition detector: UV absorption photometer (measurement wavelength: 275nm)
Column: A stainless steel tube with an inner diameter of 4.6 mm and a length of 150 mm is packed with 30 μm styrene / divinylbenzene copolymer reverse phase polymer ⁷⁾ .
Column temperature: Constant temperature around 50 ° C Mobile phase A: Water / methanol mixture (199: 1)
B: Methanol / water mixture (19: 1)
Flow rate ^* : 1mL / min
Gradient condition ^*
0 min → 10 min B.CONC 8% hold
10 minutes → 12 minutes B.CONC 8% → 50% straight line
12 minutes → 22 minutes B.CONC 50% hold
22 minutes → 30 minutes B.CONC 50% → 90% straight line
30 minutes → 45 minutes B.CONC 90% hold
45.01 minutes → 55 minutes B.CONC 8% Retention Area measurement range: Approximately 1.5 times the retention time of 10-hydroxy-δ-2-decenoic acid *: The following system suitability is satisfied for flow rate and gradient conditions Adjust accordingly.
System suitability System performance: When liquid chromatography is performed with 50 µL of the reference solution under the above operating conditions, tryptophan, glycyl-L-leucyl-L-tyrosine, and 10-hydroxy-δ-2-decenoic acid are eluted in this order. , The resolution of tryptophan and glycyl-L-leucyl-L-tyrosine is 3 or more, and the resolution of glycyl-L-leucyl-L-tyrosine and 10-hydroxy-δ-2-decenoic acid is 5 or more.
note:
・ Phosphate buffer solution (pH 8.0): Preparation is in accordance with the official food additives.
・ Dilute acetic acid: Preparation is in accordance with the official food additives.
・ L-tryptophan: C ₁₁ H ₁₂ N ₂ O ₂ Wako Pure Chemical Industries, Ltd. Use reagent grade or equivalent quality.
・ Glycyl-L-Leucyl-L-tyrosine: C ₁₇ H ₂₅ N ₃ O ₅ Fruca (Riedel de Haan) Co., Ltd. Use 98% or more or equivalent quality.
・ Acetic acid / sodium acetate buffer (pH 4.0): Dissolve 5.44 g of sodium acetate trihydrate in 900 mL of water, add acetic acid (100) dropwise, adjust the pH to 4.0, add water to make 1000 mL. Do it.
・ 10-Hydroxy-δ-2-decenoic acid standard product: (E) -10-hydroxy-2-decenoic acid standard product (C ₁₀ H ₁₈ O ₃ ) Wako Pure Chemical Industries, Ltd. or equivalent Use.
・ Styrene / divinylbenzene copolymer reverse phase polymer: Use MCI-GEL CHP55Y manufactured by Mitsubishi Chemical Corporation or equivalent.
FIG. 8 shows an example of a chromatogram relating to the content of participating components.

Next, the components involved in fractions 3 to 6 (peptides) are isolated and identified as follows: Fractionation with large-diameter column
HPLC condition column: TSK-gel ODS 120T 55mmφ × 300mm Tosoh Guard column: TSK-gel ODS 120T 45mmφ × 55mm Tosoh Mobile phase: Water / acetonitrile mixture ^* / 0.05% TFA
Flow rate: 42mL / min
Column temperature: Room temperature Detection wavelength: 220nm
・: Acetonitrile concentration 7-25%, 50% (wash out)
・ Fractionation with medium-diameter column
HPLC condition column: COSMOSIL-5C18-AR II 20 mmφ × 250 mm Nacalai Tesque and / or COSMOSIL-5C18-AR II 10 mmφ × 250 mm Nacalai Tesque Mobile phase: Water / methanol mixture ^* / 0.05% TFA
Flow rate: 8mL / min or 2mL / min
Column temperature: 40 ° C
Detection wavelength: 220nm
・: Methanol concentration Isocratic mode 2 to 35%, 50% (wash out)
Gradient mode A solution 0% (0.05% TFA), B solution 50%

・ Confirmation of purity of each fraction <HPLC conditions>
Detector: UV spectrophotometer (measurement wavelength: 220 nm)
Column: COSMOSIL-5C18-AR II 4.6mmφ × 250mm Nacalai Tesque Mobile phase: Water / methanol mixture ^* / 0.05% TFA
Flow rate: 0.4mL / min
Column temperature: 40 ° C
・: Methanol concentration 2-40%
・ Preparative sample (number of fractions)

Measurement of ACE inhibition rate The ACE inhibition rate was measured for 295 fractions with a purity of 85% or more.
A part of the sample having a purity of 85% or less was also measured.

ACE inhibition rate measurement method Sample solution (concentration in reaction solution: 0.1 mg / mL) 25 μL
↓ ACE solution (rabbit lung derived angiotensin I converting enzyme: 25mU / mL) 50μL
Preincubation 37 ° C-5 min
↓ Substrate solution (Hippuryl-L-Histidyl-L-Leucine: 12.5 mM) 50 μL
Incubation 37 ° C-60 min
↓ Hydrochloric acid (9 → 200) 125μL
↓ Internal standard solution (m-hippuric acid: 0.625 mM) 100 μL
↓ Ethyl acetate 750μL
↓ Vortex mixer 10sec × 2
Centrifugation (2000rpm, 5min)
↓
Organic layer 500μL
↓ Dry at 60 ℃ under nitrogen gas flow ↓ Mobile phase 500μL
↓ Dissolve with vortex mixer
Inject 20 μL into HPLC

<HPLC conditions>
Detector: UV spectrophotometer (measurement wavelength: 228nm)
Column: L-column ODS 4.6mmφ × 150mm Chemical Substance Evaluation and Research Organization Mobile phase: 0.01M phosphate buffer (pH3.0) / acetonitrile mixture (17: 3)
Flow rate: 1.0mL / min
Column temperature: 40 ° C
Injection volume: 20μL
Measurement of constituent amino acids The fractions of 166 specimens having an isolated amount of 2 mg or more or an ACE inhibition rate of 20% or more were measured for the amino acid concentration after acid hydrolysis to determine the constituent amino acids.

Method for measuring constituent amino acids <Acid hydrolysis method-1: isolated peptide not containing tryptophan>
Sample solution (1mg / mL 50% methanol solution) 50μL
↓ Vacuum dry using Pico-Tag (Waters) (room temperature)
Residue ↓ + 6mol / L hydrochloric acid solution (containing 1% phenol) 0.2mL (put in reaction vial)
↓ Hydrolysis by Pico-Tag method (110 ° C, 22 hours)
↓ After cooling ↓ Use Pico-Tag to dry under reduced pressure (room temperature)
Residue ↓ + 0.02mol / L Hydrochloric acid test solution 0.25mL (5-fold dilution)
20 μL / Amino acid analysis <Acid hydrolysis method-2: isolated peptide not containing tryptophan>
Sample solution (1mg / mL 50% methanol solution) 50μL
↓ Vacuum dry using Pico-Tag (Waters) (room temperature)
Residue ↓ + 4mol / L methanesulfonic acid solution (containing 0.2% tryptamine) 20μL
↓ + water 0.2mL (put in reaction vial)
↓ Hydrolysis by Pico-Tag method (110 ° C, 22 hours)
↓ After cooling ↓ + 4mol / L potassium hydroxide solution 22μL
↓ Use Pico-Tag to dry under reduced pressure (room temperature)
Residue ↓ + 0.05mol / L Hydrochloric acid test solution 0.25mL (5-fold dilution)
20μL / Amino acid analysis

<Amino acid analysis conditions>
(Post-label method using ninhydrin reaction)
Equipment: Hitachi L-8500 Hitachi High-Speed Amino Acid Analyzer Column: 4.6 mm ID x 80 mm Hitachi Custom Ion Exchange Resin (# 2622SC / LotNo. K99272)
(Na type, high separation analysis, gradient method buffer: buffer L-8500PH-KIT for Hitachi high-speed amino acid analyzer (Mitsubishi Chemical or Wako Pure Chemical Industries, Ltd.)
V1: PH-1 (PH-1 500mL with ethanol (99.5) 65mL and water to 1000mL)
V2: PH-2
V3: PH-3 (unused)
V4: PH-4
V5: PH-RG
Reaction solution: Ninhydrin sample solution-L8500 set (Wako Pure Chemical Industries, Ltd.)
Flow rate: Buffer 0.26mL / min
Reaction solution 0.30mL / min
Injection volume: 20μL
Measurement wavelength: 570nm (Pro: 440nm)

・ Determination of amino acid sequence
Based on the measurement results of the ACE inhibition rate and the constituent amino acids, 18 isolates having relatively high ACE inhibitory activity, a clear amino acid composition ratio, and 2 to 6 constituent amino acids were analyzed using LC-MS / We tried to analyze the amino acid sequence by MS method.
Operating conditions for structural analysis by LC-MS / MS Method of preparing sample solution Each was dissolved in 400 μL of ultrapure acetonitrile (1: 1) and about 100 μg / 0.01% 0.01% formic acid solution / acetonitrile (1: 1). Dilute to a concentration of approximately 25 μg / mL.
HPLC conditions Equipment used: 1100Series (Agilent Technologies)
Column: Cadenza CD-C18, 3μm, 2mmφ × 50mm
Mobile phase: A solution 0.01% formic acid solution
: B solution 0.01% formic acid solution / acetonitrile (10:90)
0.01M phosphate buffer (pH3.0) / acetonitrile mixture (17: 3)
Elution condition: 0-8min B solution 8%
8-12min B solution 90%
12 ~ 13min B liquid 8%
Flow rate: 0.1mL / min
Column temperature: 40 ° C
Injection volume: 5μL
Operating conditions for MS and MS / MS Equipment used: API 4000 (Applied Biosystems / MDS Sciex)
Column: Turbo ion spray (positive)
Nebulizer gas: 18 psi
Curtain gas: 10 psi
Ion spray voltage: 5500V
Orifice voltage: Set as appropriate Measurement mass range: Set as appropriate Collision gas: Level 4 (Nitrogen)
Collision gas energy: Set as appropriate Product ion measurement range: Set as appropriate Measurement result In the 1548 sample shown in (d) above, ACE inhibitory activity was measured on 295 samples based on the HPLC purity measured simply, and the results were Based on the results, amino acid analysis was performed on 166 samples, and the composition was measured. Furthermore, regarding the amino acid sequence, 18 specimens were subjected to measurement, and the structures of nine types of peptides contained in the RJ protein hydrolyzate could be identified.

Specifically, the analysis of amino acid sequences by LC / MS / MS was attempted for 18 specimens having a relatively high ACE inhibitory activity rate, a clear amino acid composition ratio, and 2 to 6 constituent amino acids.
As a result, 9 specimens (I to IX) were able to identify the structures, and the structures of 3 peptides from the Fr 3 portion and 6 peptides from the Fr 4 portion of the crude fraction of HP-20. Could be identified.
Table 3 below shows the ACE inhibitory activity IC50 values determined from the structural formulas and the measurement results of the ACE inhibition rate.

The proteolysate of the present invention (including the seven peptides identified as active or involved components, hereinafter the same) is used as a free (free) proteolysate after adjusting to the isoelectric point pH. Or after adjusting the pH to the acidic or basic side from the isoelectric point, the solvent may be removed by freeze drying, spray drying, or the like, or precipitated by adding a solvent such as ethanol. It can be obtained in the form of an addition salt (an inorganic acid salt such as hydrochloride or an organic acid salt such as fumarate) or a base salt (for example, an alkali metal salt such as Na or K). The base salt preferably suppresses the content of sodium as a pressurizing substance as much as possible. Such a proteolysate may be used in the form of a solution, but it is usually desirable to isolate and use it in the form of a solid, for example, a crystal, amorphous or powder.

  The proteolytic product of the present invention can be used as a food by itself, or alone or together with a suitable non-toxic carrier for ingestion, diluent or excipient (tablet, uncoated tablet, sugar-coated tablet, effervescent tablet, Film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, solutions, suspensions, emulsions, pastes, creams, injections (amino acid infusions, electrolyte infusions, etc.) Or a preparation for food or medicine such as sustained release preparations such as enteric tablets, capsules and granules. The content of the proteolysate in the preparation can be appropriately selected, but is generally in the range of 0.01 to 100% by weight.

  Specific examples of foods that can be prepared by adding and blending the protein degradation product (royal jelly enzyme degradation product) according to the present invention include, for example, beverages (soft drinks (coffee, cocoa, juice, mineral beverages, tea Beverages, green tea, black tea, oolong tea, etc.), milk drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, alcoholic beverages (Japanese liquor, western liquor, fruit liquor, honey liquor, etc.), spreads (custard cream, butter cream, peanut cream, chocolate) Cream, cheese cream, etc.), paste (fruit paste, vegetable paste, sesame paste, seaweed paste, etc.), pastry (chocolate, donut, pie, cream puff, eclair, muffin, waffle, gum, gummy, jelly, candy, cookies, crackers , Biscuits, snacks, cakes, puddings, etc.) Japanese confectionery (rice cakes, rice crackers, karinto, arabe, dumplings, rice cakes, daifuku, bean mochi, rice cakes, rice cakes, buns, castella, anmitsu, sheep kan, etc.), ice confectionery (ice cream, ice candy, sorbet, shaved ice, etc.), retort food ( Curry, beef bowl, Chinese bowl, miso soup, rice cracker, miso soup, soup, meat sauce, demiglace sauce, meatball, hamburger, oden seeds, red rice, yakitori, chawanmushi, etc.), instant food (instant ramen, instant udon, instant soba, instant yakisoba) , Instant spaghetti, instant wonton noodles, instant shirako, miso soup, powdered soup, powdered juice, hot cake mix, etc.), bottled / canned, jelly-like foods (jelly, agar, terrine, jelly-like beverages, etc.) , Seasoning (salt, natural salt, soy sauce, mirin, vinegar, sugar, honey, miso, dressing, umami seasoning , Complex seasoning, sauce, mayonnaise, ketchup, sprinkle, tentsuyu, noodle soup, dashi soup, Chinese soup base, Chinese soup base (meat tofu base, chinjaoulose base), bouillon, grilled meat sauce, cold shabu Soy sauce, curry roux, stew roux, etc.), beekeeping products (honey, royal jelly, propolis, pollen, pollen, dairy products), dairy products (milk, cheese, yogurt, fresh cream, etc.), processed fruits ( Jam, marmalade, syrup pickled, dried fruit, etc.), processed vegetables (vegetable jam, etc.), processed cereals (noodles, pasta, bread, rice noodles, etc.), pickles (takuan, Nara pickled, kimchi pickled, Fukujin pickled, rakkyo pickled, Chinese cabbage) Pickles, mustard pickles, shiba pickles, shallow pickles, pickles, etc.), pickles (immediate pickles, kimchi pickles, etc.), fish products (kamaboko, chikuwa, hanpen, etc.) ), Livestock meat products (ham, sausage, salami, bacon, etc.), delicacies (sakisume, saki cod, sea urchin salt, squid salty, octopus salty, scallops, puffed squid, smoked squid ), Dried food (seasoned seaweed, etc.), side dishes (seafood, fried food, fried food, grilled food, boiled food, vinegared food, etc.), frozen food (fried shrimp, croquette, spring roll, tonkatsu, shumai, dumplings, hamburger steak) , Takoyaki, Imagawa-yaki (rotary roasting), meat buns, buns, etc.), fat and oil foods (salad oil, margarine, butter) and the like. Foods that can be prepared by adding and blending the royal jelly proteolysate of the present invention include so-called health foods, functional foods, dietary supplements, supplements, foods for specified health use, combined foods for the sick and the sick. (Ministry of Health, Labor and Welfare, a kind of special-purpose food) or food for the elderly (Ministry of Health, Labor and Welfare, a kind of special-purpose food). Capsule (including both hard capsules and soft capsules), chewable type, syrup type, drink type and the like can also be used. Preparation of the food which added and mix | blended the royal jelly enzyme degradation product based on this invention can be performed by a well-known method in itself.

  Foods such as foods for specified health use containing the proteolytic product of the present invention have normal high blood pressure (systolic blood pressure 130-139 mmHg, diastolic blood pressure 85-89 mmHg) and mild hypertension (systolic blood pressure 140-159 mmHg or dilated). It has the effect of preventing hypertension for both humans with a blood pressure of 90-99 mmHg.

  The proteolysate of the present invention exhibits an effective antihypertensive effect particularly in subjects with mild hypertension and normal high levels, but has little or no effect on the blood pressure of normal to low blood pressure subjects. It has the ideal characteristics.

  The proteolytic product of the present invention is absorbed directly from the oral mucosa or gastrointestinal tract by oral ingestion, or exhibits a strong and sustained ACE inhibitory action after being hydrolyzed and absorbed in the gastrointestinal tract. It can be ingested continuously for the purpose of prevention, alleviation of the tendency to hypertension or blood pressure regulation, and is useful as a functional food for preventing hypertension, particularly a food for specified health use. When the proteolysate or preparation of the present invention is used for this purpose, it is generally 10 mg to 10 g per day, preferably 0.1 to 5 g, more preferably 0.5 to 3 g, especially 0 for an adult weighing 60 kg. Ingested in the range of 5 to 1 g.

  In addition, the proteolysate of the present invention can be used in combination with or mixed with royal jelly, propolis, honey, pollen, honey, etc.

  The proteolysate of the present invention has little or no side effects such as allergic reactions, has a long duration of action, and can be expected to have a sufficient antihypertensive action when taken (administered) once a day. Furthermore, although it has an immediate effect, it does not cause a rapid decrease in blood pressure or a large decrease in blood pressure when swallowed in large quantities, and has a highly safe antihypertensive action. In addition, the antihypertensive action in humans is excellent.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
(1) Preparation of proteolytic product of royal jelly material After dissolving 1 g of dried royal jelly (protein content 0.4 g) in 10 ml of distilled water and adjusting the pH to 8, 0.25% by weight of trypsin (bovine) Pancreatic origin (Roche) was added and incubated for 24 hours. The ACE inhibition rate was measured for samples at 3 hours, 6 hours, and 24 hours after the start of incubation. The pH of the reaction solution was maintained at about pH 8 by adding sodium hydroxide.

Incubation was carried out in the same manner using chymotrypsin (pH 7) or pepsin (pH 2) instead of trypsin, and the ACE inhibition rate was measured according to the following (2) for the samples 3 hours, 6 hours and 24 hours after the start of incubation.

(2) Measurement of ACE inhibitory activity Take 25 μl of the proteolysate 4 mg / mL (0.8 mg / mL as the reaction solution concentration) obtained as described above in a test tube, and add 25 mU / mL ACE solution from rabbit lung to this. ^{* 1.} 100 μl of borate buffer (pH 8.3) was added and incubated at 37 ° C. for 5 minutes. As a substrate, 12.5 mM hippuryl-L-histidyl-L-leucine (HHL, manufactured by Peptide Research) ^{* 2} solution / borate buffer (pH 8.3) 50 μl was added and incubated at 37 ° C. for 60 minutes. Next, 125 μL of 0.5 M hydrochloric acid solution was added to stop the reaction, 100 μL of 0.625 mM m-methyl hippuric acid acetonitrile solution ^{* 3} was added as an internal standard solution, and 750 μL of ethyl acetate was added to extract the reaction product hippuric acid. Thereafter, the mixture was stirred and centrifuged with a vortex mixer (2000 rpm, 5 min). 0.5 mL of the organic layer was taken and evaporated to dryness under a nitrogen gas stream at 60 ° C., and the residue was dissolved in 0.5 mL of HPLC mobile phase to obtain a sample solution.

Separately, add 25 μl of borate buffer (pH 8.3) ^{* 4} to a test tube, add 25 mU / mL ACE solution ^{* 1} from bovine lung ^{* 1} and borate buffer (pH 8.3) ^{* 4} 50 μl to this at 37 ° C. After incubating for 5 minutes, the same procedure as for the sample solution was followed to obtain a standard solution. In addition, 75 μl of borate buffer (pH 8.3) was placed in a test tube, incubated at 37 ° C. for 5 minutes, and thereafter operated in the same manner as the sample solution to obtain a blank test. The sample solution, the standard solution and 20 μl of the blank test solution were measured by HPLC under the following conditions, and the ratio of the peak area of hippuric acid to the peak area of each m-methylhippuric acid was determined.
<HPLC conditions>
Detector: UV (measurement wavelength: 228nm)
Column: L-column ODS 4.6 × 150mm (Chemicals Evaluation and Research Institute)
Column temperature: 40 ° C
Mobile phase: 10 mM KH2PO4 (pH 3.0) / acetonitrile = 17/3
Flow rate: 1.0mL / min
Inflow volume: 20μL

The inhibition rate (IC (%)) was calculated according to the following formula based on the ratio of the peak area of hippuric acid to the peak area of each m-methylhippuric acid obtained by the HPLC method.

Inhibition rate (IC (%)) = {(Rs−Rt) / (Rt−Rb)} × 100
Rs; ratio of peak area of hippuric acid to peak area of m-methylhippuric acid in sample solution
Rt; ratio of peak area of hippuric acid to peak area of m-methylhippuric acid in standard solution
Rb; ratio of peak area of hippuric acid to peak area of m-methylhippuric acid in the blank test solution

* 1: 25 mU / mL ACE solution Add 1 mL of pH8.3 borate buffer solution to 0.25 U or 1 U / vial of angiotensin I converting enzyme (Sigma) from rabbit lung (0.25 U or 1 U / mL, Frozen).

This solution is appropriately diluted with a pH 8.3 borate buffer solution to prepare a 25 mU / mL solution (prepared at the time of use).
* 2: 12.5 mM Hip-His-Leu (substrate solution)
Add pH 8.3 borate buffer to Hippuryl-L-Histidyl-L-Leucine (HHL · H ₂ O, molecular weight 447.8, manufactured by Peptide Laboratories), and dissolve by heating in a boiling water bath for 3 minutes to 12.5 mM. Prepare the solution.
{HHL · H ₂ O 5.5935 g + pH 8.3 borate buffer 1 mL: 12.5 mM (prepared when used)}
* 3: Internal standard solution (3 → 25000)
To 12 mg of m-methylhippuric acid (molecular weight: 193.20), a water / acetonitrile mixture (9: 1) is added and dissolved to make 100 mL, which is used as an internal standard solution (stored in a refrigerator).
* 4: pH 8.3 borate buffer
A solution: 0.2M boric acid solution (including 0.3M sodium chloride)
Boric acid 12.4g + Sodium chloride 17.5g + Water → 1000mL
Liquid B: 0.05M sodium tetraborate solution (including 0.3M sodium chloride)
Adjust the pH to 8.3 by adding solution B to solution A (store in refrigerator)

The results are shown in Table 4 and FIG.

  From the above results, it was found that the trypsin hydrolyzate had the strongest ACE inhibitory activity.

  In the enzyme degradation product after 24 hours of incubation, proteins with a molecular weight of 20000 or more were hardly observed in the protein degradation product of the present invention by SDS-PAGE, but were confirmed as bands in the pepsin degradation product and chymotrypsin degradation product. .

  Furthermore, according to chromatogram analysis by HPLC using a wide pore column for COSMOSIL-5C18-AR-300 (4.6 x 150 mm) protein separation as the column, it was confirmed that the amount of peptide produced in the proteolysate was the largest in trypsin It was done.

From these results, it was confirmed that trypsin was most preferable.
Example 2
Instead of dried royal jelly 1 g (protein content 0.4 g), ethanol was added to raw royal jelly and precipitated, and then the ethanol-denatured protein of royal jelly was treated for 24 hours in the same manner as in Example 1. When the ACE inhibition rate of this trypsin-treated product was determined, an ACE inhibition rate almost equivalent to that of the royal jelly of Example 1 was obtained.

  The reaction solution treated with trypsin for 24 hours is adjusted to pH 4.5, applied to a synthetic adsorbent (Separbeads SP-70 (Mitsubishi Chemical)), washed with water to remove sodium chloride, acidic or basic free amino acids, sugars, etc. After removal, elution with ethanol to obtain a desalted proteolysate of the present invention.

  The dried royal jelly used in Example 1 (dried RJ), the trypsin degradation product obtained before desalting obtained in Example 1 (before desalting; dried RJ-enzyme), the ethanol-denatured protein of royal jelly used in Example 2 (RJ-denatured), its proteolysate (before desalting (RJ-denaturing-enzyme); and after desalting, washing fraction after column adsorption (1W), ethanol elution fraction of column adsorbed product (1E) amino acids Regarding the composition ratio, the amino acid composition of each sample is a decomposed product decomposed by methanesulfonic acid, and the free amino acid is a membrane filter filtered (pore size diameter: 0.45 μm), and the amino acid concentration is measured (Hitachi L-8500 type high-speed amino acid). The results are shown in Table 5, Table 6, Table 7, Fig. 2 and Fig. 3. In Tables 5 and 2, Gly is 1 mol. Table 6 and Fig. 3 are relative values when Lys is 1 mol, and Table 7 shows the measured values of free amino acids, and from these results, acidic or basic release by column treatment is shown. Amino acids, free Ala, and free Gly were removed by washing with water, and it was confirmed that a large amount of free aromatic and aliphatic amino acids were distributed in the column adsorption fraction.

  Furthermore, 25 mg of trypsin degradation product treated for 24 hours is taken, 20 mL of mobile phase is added and sonicated to dissolve to make exactly 25 mL, and the gel filtration chromatography (GFC) analysis is conducted on 10 μL of the following to investigate the molecular weight distribution. did.

<GFC analysis>
Column: TSKgel G2000SW _XL 7.8mmI.D. × 30cm
Mobile phase: 0.1% TFA / acetonitrile flow rate: 1.0 mL / min
Column temperature: 25 ° C
Detection: UV (220nm)
The GFC analysis results are shown in FIG.

Example 3 Structural analysis of components involved in royal jelly (RJ) protein hydrolyzate 100 g of ethanol-denatured protein of royal jelly was collected and enzyme was incubated with trypsin at 37 ° C. for 24 hours at pH 8.0 according to the method described in Example 1. Disassembled. The reaction solution was heated in a boiling water bath for 30 minutes to inactivate the enzyme, adjusted to pH 4.5 with hydrochloric acid, and centrifuged (10000 rpm, 20 minutes) to obtain a supernatant obtained from a membrane filter (0.8 μm). ) To obtain 1160 mL of RJ protein hydrolyzate.

The obtained RJ protein hydrolyzate was eluted with water, 20%, 40%, 60%, 80% methanol and methanol (100%) using Diaion HP20 according to Scheme 1 above, and fraction 1 (Fr1 ) To Fraction 7 (Fr7).
Next, details of the isolation and identification of the nine participating components (peptides) identified from fractions 3-6 are given below:
Isolation and identification of components involved in royal jelly protein hydrolyzate
1. Isolated identification peptide
HP20 Fr 3 fraction isolated peptide I: Glu-Trp-Lys (P30615-1)
Isolated peptide II: His-Pro-Ala-Leu (P30615-3-7-3)
Isolated peptide III: Thr-Phe (P30618-3-3)
HP20 Fr 4 fraction isolated peptide IV: Ser-Pro-Leu (P40408-3-2)
Isolated peptide V: Asn-Leu-Tyr (P41219-2-1)
Isolated peptide VI: Leu-Ser-Tyr (P41210-4-1-2)
Isolated peptide VII: Thr-Pro-Phe (P41211-3)
Isolated peptide VIII: Ser-His-Ser-Gly-Leu-Tyr (P40323-3-4)
Isolated peptide IX: Tyr-Ser-Pro-Leu (P40528-3-2)

2. Preparation of specimens for identification of participating components (1) Preparation of royal jelly protein hydrolyzate Add 1200 mL of water to 100 g of royal jelly ethanol-denatured protein, add 20% sodium hydroxide solution with stirring in a 37 ° C water bath, and adjust the pH to 8. The solution was adjusted to 0, 250 mg of trypsin was added, and enzymatic degradation was performed at 37 ° C. for 24 hours while maintaining the pH at 8.0 with a 1 mol / L sodium hydroxide test solution, followed by heating in a boiling water bath for 30 minutes. Diluted hydrochloric acid (1 → 2) was added to this, adjusted to pH 4.5, centrifuged (10000 rpm, 20 minutes), the resulting supernatant was filtered through a membrane filter, and 1160 mL of royal jelly protein hydrolyzate Got.

(2) Coarse fractionation with Diaion HP20 Load 855 mL of royal jelly protein hydrolyzate onto a glass column (11 cmφ x 20 cm, capacity: approx. 1.9 L) filled with Diaion HP20, a synthetic adsorbent, and add 20% water, Elution is performed in the order of 40%, 60%, 80% and 100% methanol, and the fraction eluted with water (hereinafter referred to as Fr) 1 and 2 is eluted with 20%, 40%, 60%, 80% and 100% methanol. Minutes were Fr3, 4, 5, 6 and 7, respectively. Concentrate each fraction with an evaporator, and then freeze-dry. Obtain the Fr3 fraction (5.38 g), Fr4 fraction (8.34 g), Fr5 fraction (11.58 g) and Fr6 fraction (4.84 g). It was set as the sample for identification of a participating component.

3. Isolation and identification of peptides involved in royal jelly protein hydrolyzate (1) Isolation and identification of peptides involved from Fr3 fraction Isolated peptide I: Glu-Trp-Lys (P30615-1)
High-performance liquid chromatographic method using a large-diameter preparative ODS column for the Fr3 fraction roughly fractionated by HP20 [Column: TSK-gel ODS 120T (55mmφ × 300mm), mobile phase: 7%, 15%, 25% and 50% acetonitrile (containing 0.05% TFA), flow rate: 42 mL / mL, column temperature: room temperature, detection wavelength: UV 220 nm. The following fractionation was performed by HPLC].

  About 4 g of Fr3 fraction, the mobile phase was switched in stages [7% acetonitrile (containing 0.05% TFA): 0 to 94 minutes, 15% acetonitrile (containing 0.05% TFA): 94 to 121 minutes, 25% acetonitrile ( 0.05% TFA): 121-135 minutes, 50% acetonitrile (0.05% TFA): 135 minutes-] Re-fractionation, retention times (hereinafter Rt) were obtained from 61.5-83.4 minutes and 98.0-112.4 minutes The procedure of adding ethanol to the fraction and concentrating was repeated until the TFA odor disappeared, and then the residue was dissolved in a small amount of water, freeze-dried, and P3 (6) (6th peak of fraction 3) (0.471). g), and P3 (8) (the eighth peak of fraction 3) (0.107 g, used for isolation of peptide III).

  For P3 (6) (0.471g), a medium-diameter semi-preparative ODS column (COSMOSIL 5C18 AR-II 20mmφ × 250mm) was used, flow rate: 8mL / mL, column temperature: 40 ° C, detection wavelength: UV220nm Under the conditions described above, Rt was determined by a concentration gradient method (0 min: Bconc 0% → 360 min: Bconc 75%) using 0.05% TFA for mobile phase A and 50% methanol (containing 0.05% TFA) for mobile phase B. The peak portions of 57.6-77.1 minutes and 84.2-100.9 minutes were collected, and the obtained fractions were concentrated and freeze-dried, respectively, and purified fractions P30615-1 (35.7 mg) and P30615-3 (107.0 mg, Which was subjected to isolation of peptide II).

The obtained P30615-1 was analyzed by LC / MS / MS based on the purity by HPLC and the ratio of the constituent amino acids obtained by amino acid analysis after acid hydrolysis. -Trp-Lys was determined.

Isolated peptide II: His-Pro-Ala-Leu (P30615-3-7-3)
For the previous P30615-3 (107.0 mg) with low purity, again using a medium-diameter semi-preparative ODS column, the mobile phase was changed from 8% methanol (containing 0.05% TFA) to 50% methanol (containing 0.05% TFA). ) (61.3 minutes), the peak portion of Rt of 68.9-70.2 minutes was collected, concentrated and freeze-dried to obtain P30615-3-7 (9.2 mg).

  For P30615-3-7 (9.2 mg), using an ODS column with a smaller aperture and higher resolution [column: COSMOSIL 5C18 AR-II 10 mmφ × 250 mm, mobile phase: 8% methanol (containing 0.05% TFA) , Flow rate: 2 mL / mL, column temperature: 40 ° C., detection wavelength: UV 220 nm], Rt was subjected to fractionation and purification for the peak portion of 57.1 to 59.8 minutes, and fractionated purified product P30615-3-7-3 (0.7 mg) was obtained.

The obtained P30615-3-7-3 was subjected to structural analysis by LC / MS / MS based on the purity by HPLC and the ratio of the constituent amino acids obtained by amino acid analysis after acid hydrolysis. The structure of -7-3 was determined to be His-Pro-Ala-Leu.

Isolated peptide III: Thr-Phe (P30618-3-3)
For P3 (8) (0.107 g) obtained by re-fractionation using a large-diameter ODS column, Rt was determined by using a medium-diameter semi-preparative ODS column and a concentration gradient method under the same conditions as for isolated peptide I. The peak part of 82.6-92.0 minutes was fractionated and purified, and P30618-3 (12.2 mg) was obtained.

  For P30618-3 (12.2 mg), using an ODS column with a smaller bore (inner diameter 10 mm), using 10% methanol (containing 0.05% TFA) as the mobile phase, fractionation of the peak portion of Rt 39.7 to 45.2 min -Purification operation was performed to obtain P30618-3-3 (3.7 mg).

The obtained P30618-3-3 was subjected to structural analysis by LC / MS / MS based on the purity by HPLC and the ratio of the constituent amino acids obtained by amino acid analysis after acid hydrolysis, and P30618-3-3 The structure of was determined to be Thr-Phe.

(2) Isolation and identification of participating peptides from Fr4 fraction Isolated peptide IV: Ser-Pro-Leu (P40408-3-2)
About 5 g of Fr4 fraction (8.34 g) roughly fractionated by HP20, high-performance liquid chromatography using a large-diameter preparative ODS column [column: TSK-gel ODS 120T (55 mmφ x 300 mm), mobile phase: 10% and 50% acetonitrile (containing 0.05% TFA), flow rate: 42 mL / mL, column temperature: room temperature, detection wavelength: UV 220 nm].

  When the mobile phase was 10% acetonitrile (containing 0.05% TFA), the fractions with Rt of 52.1-83.5 minutes and 118.9-179.0 minutes were concentrated and lyophilized to obtain P4 (2) (second peak of fraction 4) (0.536 g) and P4 (4) (the second peak of fraction 4) (without lyophilization, after concentration to dryness, were used for isolation of peptides V to VIII). The mobile phase was switched to 50% acetonitrile (containing 0.05% TFA) after 179 minutes, and the fraction with Rt of 179.0 to 205.0 minutes was concentrated and freeze-dried to isolate P4 (5) (2.162 g, peptide IX Were provided).

  For P4 (2) (0.536 g), again using a large-diameter preparative ODS column, changing the mobile phase to 8% acetonitrile (containing 0.05% TFA) and concentrating the fraction with Rt of 76.2 to 90.2 min. Freeze-dried to obtain P40121-13 (91.4 mg).

  For the obtained P40121-13 (91.4 mg), using a semi-preparative ODS column with medium caliber, 12.5% methanol (containing 0.05% TFA) as the mobile phase, and the peak portion of Rt 42.6-51.6 min Preparative and purification operations were performed to obtain P40408-3 (12.1 mg).

  For the obtained P40408-3 (12.1 mg), again using a medium-diameter semi-preparative ODS column, using 0.05% TFA for mobile phase A and 50% methanol (containing 0.05% TFA) for mobile phase B Using the concentration gradient method (0 min: Bconc 0% → 360 min: Bconc 100%), the peak portion of Rt was 95.1-99.1 min. Was subjected to fractionation and purification, and the fractionated purified product P40408-3-2 (1.2 mg) was obtained. Obtained.

The obtained P40408-3-2 was subjected to structural analysis by LC / MS / MS based on the purity by HPLC and the ratio of the constituent amino acids obtained by amino acid analysis after acid hydrolysis, and P40408-3-2 The structure of was determined to be Ser-Pro-Leu.

Isolated peptide V: Asn-Leu-Tyr (P41219-2-1)
For P4 (4) obtained by re-fractionation using a large-diameter preparative ODS column, again using a large-diameter preparative ODS column and 10% methanol (containing 0.05% TFA) as the mobile phase. Re-fractionation was performed, and the fractions obtained from Rt of 63.0-82.7 min, 82.7-108.0 min, 108.0-126.5 min, and 141.0-163.4 min were concentrated and lyophilized to obtain P41206-3 (133.1 mg, peptide P41206-4 (177.8 mg), P41206-5 (74.2 mg, used to isolate peptide VI), and P41206-7 (41.7 mg, used to isolate peptide VII) )

  About 100 mg of the obtained P41206-4 (177.8 mg), using a semi-prepared ODS column with a medium caliber, using 12.5% methanol (containing 0.05% TFA) as the mobile phase, the peak portion of Rt 121.9-153.5 min The product was subjected to preparative and purification operations to obtain P41219-2 (23.38 mg).

  The obtained P41219-2 (23.38 mg) was again subjected to fractionation and purification under the same conditions, and P41219-2-1 (12.3 mg) was obtained from the peak portion of Rt 118.3-142.9 min.

The obtained P41219-2-1 was subjected to structural analysis by LC / MS / MS based on the purity by HPLC and the ratio of the constituent amino acids obtained by amino acid analysis after acid hydrolysis, and P41219-2-1 The structure was determined as Asn-Leu-Tyr.

Isolated peptide VI: Leu-Ser-Tyr (P41210-4-1-2)
For P41206-5 (74.2 mg) obtained by performing the refractionation operation twice with a large-diameter preparative ODS column, a medium-diameter semi-preparative ODS column was used, and 17% methanol ( Using 0.05% TFA), the peak portion of Rt of 65.5-80.7 min was collected and concentrated to dryness to obtain P41210-4 (not lyophilized). The obtained P41210-4 was again subjected to fractionation and purification under the same conditions, and P41210-4-1 (12.7 mg) was obtained from the peak portion of Rt 63.5 to 85.3 minutes.

  Furthermore, for P41210-4-1 (12.7 mg), a medium-diameter semi-preparative ODS column was used, the mobile phase was 15% methanol (containing 0.05% TFA), and the peak portion of Rt was 92.0 to 102.7 min. -Purification operation was performed to obtain a preparative purified product P41210-4-1-2 (5.18 mg).

The obtained P41210-4-1-2 was subjected to structural analysis by LC / MS / MS based on the purity by HPLC and the ratio of constituent amino acids obtained by amino acid analysis after acid hydrolysis. The structure of -1-2 was determined as Leu-Ser-Tyr.

Isolated peptide VII: Thr-Pro-Phe (P41211-3)
For P41206-7 (41.7 mg) obtained by performing re-fractionation with a large-diameter preparative ODS column twice, a medium-diameter semi-preparative ODS column was used, and 15% methanol ( 0.05% TFA was used, and the peak portion of Rt was 70.4-88.6 min., Fractionation and purification were performed to obtain P41211-3 (5.8 mg).

The obtained P41211-3 was analyzed by LC / MS / MS based on the purity by HPLC and the ratio of the constituent amino acids obtained by amino acid analysis after acid hydrolysis. -Pro-Phe was determined.

Isolated peptide VIII: Ser-His-Ser-Gly-Leu-Tyr (P40323-3-4)
For P41206-3 (133.1 mg) obtained by re-fractionation using a large-diameter preparative ODS column twice, a medium-diameter semi-preparative ODS column was used, and 13% methanol ( 0.05% TFA) was used for fractionation and purification of the peak portion of Rt from 73.1 to 84.8 minutes to obtain P40323-3 (10.5 mg).

  Furthermore, for the obtained P40323-3 (10.5 mg), using an ODS column (inner diameter 10 mm) and using 12.5% methanol (containing 0.05% TFA) as the mobile phase, the peak portion of Rt was 73.9-77.1 min. Preparative purification was performed to obtain a preparative purified product P40323-3-4 (1.0 mg).

The obtained P40323-3-4 was subjected to structural analysis by LC / MS / MS based on the purity by HPLC and the ratio of the constituent amino acids obtained by amino acid analysis after acid hydrolysis, and P40323-3-4 Was determined to be Ser-His-Ser-Gly-Leu-Tyr.

Isolated peptide IX: Tyr-Ser-Pro-Leu (P40528-3-2)
For P4 (5) (2.162g) obtained by re-fractionation using a large-diameter preparative ODS column, again using a large-diameter preparative ODS column and 15% acetonitrile (containing 0.05% TFA) as the mobile phase The fraction solution with Rt of 60.0-70.0 min was concentrated and freeze-dried to obtain P40413-5 (75.8 mg).

  The obtained P40413-5 (75.8 mg) was concentrated on a semi-preparative ODS column with medium diameter using 0.05% TFA for mobile phase A and 50% methanol (containing 0.05% TFA) for mobile phase B. According to the method (0 min: Bconc 30% → 360 min: Bconc 100%), the peak portion of Rt was 75.4-86.0 min, and fractionation and purification were performed to obtain P40528-3 (5.9 mg).

  Furthermore, with respect to the obtained P40528-3 (5.9 mg), Rt was 43.4 ~ using a medium-diameter semi-preparative ODS column again using a single solvent of 22.5% methanol (containing 0.05% TFA) as the mobile phase. The peak of 46.4 minutes was subjected to fractionation / purification operation to obtain a fractionated purified product P40528-3-2 (1.6 mg).

The obtained P40528-3-2 was subjected to structural analysis by LC / MS / MS based on the purity by HPLC and the ratio of constituent amino acids obtained by amino acid analysis after acid hydrolysis, and P40528-3-2 Was determined to be Tyr-Ser-Pro-Leu.

Example 4: In vivo test of protein degradation product (rat)
I. Blood pressure lowering action at the time of single oral administration 1) Test substance Using ethanol-denatured protein of royal jelly (RJ) as a raw material, in the same manner as in Example 2, a RJ ethanol-denatured protein was treated with trypsin and then desalted, It used for the following experiment.

2) Animals used
Crj: SHR (male, 8 weeks old, Japan Charles River) was purchased and used for experiments at 9 weeks of age (body weight 235-260 g) after quarantine acclimation breeding. Rats are individually housed in one section of a wire mesh 5-unit cage, temperature: 23 ± 2 ° C, humidity: 30-70%, ventilation: 12 times / hour, lighting time: 12 hours (light period: 6: 30- 18:30) was kept in an animal breeding room adjusted to the environment, and solid feed (CRF-1, Oriental Yeast Co., Ltd.) and tap water were freely consumed.

3) Experimental method A group of 6 SHRs was used in the experiment. The RJ-denatured protein hydrolyzate was dissolved in distilled water for injection and was orally administered once at a dose of 10 mL / kg. The control group was similarly administered distilled water for injection. Rats were placed in a heat-retaining box set at 40 ° C. and warmed for 10 minutes, and then blood pressure (systolic blood pressure, mean blood pressure and diastolic phase) under awakening using the tail cuff method with a sphygmomanometer (TK-370C, Neuroscience) Blood pressure) and heart rate were measured. The blood pressure was measured before administration of RJ-denatured protein hydrolyzate and 4, 8, 12, 16, 20, and 24 hours after administration.

4) Statistical processing Data were expressed as mean ± standard error. The significant difference test with the control group was performed by one-way analysis of variance. When a significant difference was observed, Dunnett's multiple comparison test was performed between the RJ-denatured protein hydrolyzate and the control group. The significance level was 5% (analysis software: StatView, manufactured by SAS Institute Inc.)
5) Results FIG. 5 shows changes in systolic blood pressure measured before RJ-denatured protein hydrolyzate administration, 4, 8, 12, 16, 20 and 24 hours after administration. The systolic blood pressure before administration of the control group and the RJ-denatured protein hydrolyzate 3 g / kg group was 226 ± 6 and 225 ± 7 mmHg, respectively. The systolic blood pressure of the control group showed a substantially constant value until 24 hours after administration, and changed within a range of 217 to 229 mmHg. The systolic blood pressure of the RJ-denatured protein hydrolyzate 3 g / kg group remained at a value 10 to 20 mmHg lower than that of the control group 4 to 16 hours after administration, and the systolic blood pressure at 8, 12 and 16 hours after administration. Was significantly lower compared to the control group.

2. Mean blood pressure The transition of mean blood pressure measured before administration of RJ-denatured protein hydrolyzate, 4, 8, 12, 16, 20, and 24 hours after administration was measured. The mean blood pressure before administration of the control group and the RJ-denatured protein hydrolyzate 3 g / kg group was 173 ± 4 and 166 ± 4 mmHg, respectively. The average blood pressure of the control group showed a substantially constant value until 24 hours after administration, and changed within the range of 164 to 176 mmHg. The average blood pressure of the RJ-denatured protein hydrolyzate 3 g / kg group was 10 to 17 mmHg lower than that of the control group at 8 to 20 hours after administration, and the average blood pressure at 16 hours after administration was compared with that of the control group. Was significantly lower.

3. Diastolic blood pressure Changes in diastolic blood pressure measured before administration of RJ-denatured protein hydrolyzate, 4, 8, 12, 16, 20, and 24 hours after administration were measured. The diastolic blood pressure before administration of the control group and the RJ-denatured protein hydrolyzate 3 g / kg group was 150 ± 4 and 139 ± 4 mmHg, respectively. The diastolic blood pressure of the control group showed a substantially constant value until 24 hours after the administration, and changed within the range of 140 to 151 mmHg. The diastolic blood pressure of the RJ-denatured protein hydrolyzate 3 g / kg group was 10 to 13 mmHg lower than that of the control group at 8 and 12 hours after the administration, and the diastolic blood pressure 8 hours after the administration was compared with that of the control group. It was significantly lower compared.

4). Heart rate Changes in heart rate measured before administration of RJ-denatured protein hydrolyzate, 4, 8, 12, 16, 20 and 24 hours after administration were measured. The heart rates before administration of the control group and the RJ denatured protein hydrolyzate 3 g / kg group were 468 ± 15 and 445 ± 6 times / min, respectively. The control group and the RJ denatured protein hydrolyzate 3 g / kg group had no apparent effect on heart rate.
II. Blood pressure lowering effect when administered orally for 8 weeks 1) Test substance Using ethanol-denatured protein of Royal Jelly (RJ) as a raw material, a trypsin hydrolyzate of RJ-ethanol-denatured protein was prepared in the same manner as in Example 2, and the following experiment was conducted. It was used for.

2) Animals used
Crj: SHR (male, 8 weeks old, Japan Charles River) was purchased and used for experiments at 9 weeks of age (body weight 235-260 g) after quarantine acclimation breeding. Rats are individually housed in one section of a wire mesh 5-unit cage, temperature: 23 ± 2 ° C, humidity: 30-70%, ventilation: 12 times / hour, lighting time: 12 hours (light period: 6: 30- 18:30) was kept in an animal breeding room adjusted to the environment, and solid feed (CRF-1, Oriental Yeast Co., Ltd.) and tap water were freely consumed.

3) Experimental method A group of 11 SHRs was used in the experiment. The test substance was dissolved in distilled water for injection and orally administered once a day for 8 weeks at a dose of 20 ml / kg. The control group was similarly administered distilled water for injection. Rats were placed in a heat-retaining box set at 40 ° C. and warmed for 10 minutes, and then blood pressure (systolic blood pressure, mean blood pressure and diastolic phase) under awakening using the tail cuff method with a sphygmomanometer (TK-370C, Neuroscience) Blood pressure) and heart rate were measured. The measurement was carried out once a week before the start of test substance administration and after the start of administration, and 8 hours after the administration. Furthermore, blood pressure and heart rate were measured in the same manner 1 and 2 weeks after the end of the administration period. Body weight was measured once a week.

  The composition of the experimental group is as follows.

Experimental group Control group (distilled water administration)
2. RJ ethanol-denatured protein hydrolyzate (3g / kg)
3. RJ ethanol-denatured protein (3g / kg)
4) Statistical processing The results were expressed as mean ± standard error. Comparison between groups was analyzed using Dunnett's multiple comparison test. A risk rate of less than 5% was judged as significant.

5) Results Systolic blood pressure FIG. 6 shows changes in systolic blood pressure measured once weekly 8 hours after administration of the test substance. The systolic blood pressure of the control group administered with distilled water increased with the progress of the test period, and the blood pressure increased at a rate of about 10 mmHg / week from before administration to 4 weeks after administration, and thereafter until the end of the experiment period. The rate of increase was about 2 to 5 mmHg / week. The systolic blood pressure of the RJ-denatured protein hydrolyzate 3 g / kg group showed an increase in blood pressure in almost the same manner as that of the control group until 3 weeks after administration, but after 4 weeks of administration, the blood pressure increased compared to the control group. The suppression pattern was shown. The systolic blood pressure at 5 weeks after the administration was 207 ± 3 mmHg, and then remained at a value about 10 mmHg lower than the systolic blood pressure of the control group until the end of the administration period (8 weeks after the administration). The systolic blood pressures at 7 and 8 weeks after administration were 214 ± 3 mmHg and 210 ± 3 mmHg, respectively, which were significantly lower than those of the control group. After the end of the administration, it was 217 ± 3 mmHg after 1 week of withdrawal, and 221 ± 2 mmHg after 2 weeks of withdrawal, and gradually recovered to the same systolic blood pressure as the control group.

  On the other hand, the systolic blood pressure of the RJ-denatured protein 3 g / kg group fluctuated with the same transition as the control group during the administration period. The systolic blood pressure during the drug holiday also showed the same level of blood pressure as the control group.

2. Mean blood pressure Transition of mean blood pressure was measured once a week 8 hours after administration of the test substance. The average blood pressure of the control group increased with the progress of the test period, and increased at a rate of about 10 mmHg / week until 3 weeks after administration. The average blood pressure 4 weeks after administration was 158 ± 4 mmHg, and thereafter changed at a value of 163-170 mmHg until the end of the experiment period. The average blood pressure of the RJ-denatured protein hydrolyzate 3 g / kg group showed a blood pressure increase similar to that of the control group until 2 weeks after administration, but after 3 weeks of administration, the blood pressure increase pattern was suppressed compared to the control group. Indicated. The average blood pressure after 3 weeks of administration was 153 ± 2 mmHg, and then remained at a value about 10 mmHg lower than the average blood pressure of the control group until the end of the administration period. The mean blood pressure at 7 and 8 weeks after administration was 161 ± 2 and 158 ± 1 mmHg, respectively, which was significantly lower than that of the control group. During the drug holiday after the end of the administration, 165 ± 2 mmHg was obtained after 1 week of drug withdrawal, and 164 ± 3 mmHg after 2 weeks of drug withdrawal, and the blood pressure gradually recovered to the same level as the control group. The mean blood pressure of the RJ-denatured protein 3 g / kg group was 167 ± 2 mmHg, which was similar to that of the control group. The mean blood pressure during the drug holiday was the same level as the control group.

3. Diastolic blood pressure Transition of diastolic blood pressure was measured once weekly 8 hours after administration of the test substance. The diastolic blood pressure of the control group increased with the progress of the test period. The blood pressure increased at a rate of about 10 mmHg / week up to 3 weeks after administration, and the diastolic blood pressure after 3 weeks of administration was 137 ± 5 mmHg, and thereafter remained at a value of 140 to 144 mmHg until the end of the experimental period. The diastolic blood pressure of the RJ-denatured protein hydrolyzate 3 g / kg group showed an increase in blood pressure similar to that of the control group until 3 weeks after administration, but after 3 weeks of administration, the pattern of suppressing the increase in blood pressure compared to the control group showed that. The diastolic blood pressure at 3 weeks after administration was 132 ± 3 mmHg, and thereafter remained at a value about 10 mmHg lower than the diastolic blood pressure of the control group until the end of the administration period. Was significantly lower than. During the drug holiday after the end of the administration, it became 140 ± 3 mmHg after 1 week of drug withdrawal and 135 ± 3 mmHg after 2 weeks of drug withdrawal, and gradually recovered to the same level of diastolic blood pressure as in the control group. The diastolic blood pressure of the RJ-denatured protein 3 g / kg group had almost the same blood pressure fluctuation pattern as that of the control group. The blood pressure was the same as that in the control group even after the administration was completed.

4). Heart rate and body weight Heart rate and body weight were measured once weekly 8 hours after administration of the test substance. There was no significant variation in heart rate and weight gain of each group throughout the experimental period, and RJ-denatured protein hydrolyzate and RJ-denatured protein had no obvious effect on heart rate and weight gain.
Example 5: In vivo test of protein degradation product (human)
1) Test substance A trypsin degradation product of RJ ethanol-denatured protein was obtained in the same manner as in Example 2. This was adsorbed on Diaion HP20 according to the above-mentioned scheme 1, washed with water to remove fractions 1 and 2, and then 80% A proteolysate of the present invention containing fractions 3 to 6 eluted with ethanol was prepared and subjected to the following experiment.

2) Subjects Normal to high blood pressure to mild hypertension (blood pressure level; systolic blood pressure 130 to 159 mmHg, diastolic blood pressure 85 to 99 mmHg) 67 patients who are equal in terms of systolic blood pressure, diastolic blood pressure, age, and gender Divided into 4 groups. There were no significant differences between groups in age, blood pressure, pulse rate, height, weight, or body mass index (BMI).

3) Test food and intake method Tablets designed to contain 166.7 mg of the above-mentioned royal jelly proteolysate containing fractions 3 to 6 per tablet (hereinafter referred to as test meal) and tablets not containing the royal jelly proteolysate ( Hereinafter, placebo) was used.

  Table 8 shows a nutrient composition table of each test meal.

  Ingestion was conducted for 4 weeks, and the test was conducted 2 weeks and 1 week before ingestion, the start date of ingestion, 1 week after ingestion, 2 weeks, 3 weeks and 4 weeks, and 1 week after the end of ingestion. I went twice.

  As shown in Table 9, the test meal is combined with a placebo, and the royal jelly proteolysate intake is set to 0 mg / day, 500 mg / day, 1000 mg / day, and 5000 mg / day. After the meal, 15 tablets were taken in total, 30 tablets a day.

4) Examination method and statistical processing All subjects performed blood pressure, pulse rate, weight and height measurements, blood tests, urinalysis, examinations and interviews all at the same time. Data were expressed as mean ± standard error. For comparison of blood pressure, pulse rate, body weight, and BMI, Bonferroni multiple comparison test was performed. The results for systolic blood pressure are shown in FIG.

5) Results and discussion Systolic blood pressure As a result of a 4-week continuous ingestion test between 4 groups, a significant decrease in systolic blood pressure was observed at 1 and 4 weeks after ingestion in the 1000 mg / day ingestion group compared to the ingestion day, 5000 mg / day In the intake group, the systolic blood pressure significantly decreased 3 weeks and 4 weeks after the intake.

  From these results, it was revealed that the protein degradation product of the present invention has a dose-dependent antihypertensive action.

  In any group, the systolic blood pressure of the test food group recovered slowly after the end of intake, and no rebound phenomenon was observed that excessively exceeded the pre-intake value.

2. Other test items In the urine test, there was no change in the estimated daily excretion of Na and K, and the antihypertensive effect of the proteolytic product of the present invention was not due to the effect of salt intake, but was due to the proteolytic product. It has been suggested.

  There were no medically significant changes in various blood tests, other urine tests (protein, occult blood, urobilinogen), body weight, and BMI.

In particular, no findings considered to be adverse events (side effects) were observed at any dose. This revealed the high safety of the proteolysate of the present invention.

Example 6: Effect of proteolysate on bradykinin contraction (guinea pig isolated ileum)
1) Test substance A trypsin degradation product of RJ ethanol-denatured protein was obtained in the same manner as in Example 2. This was adsorbed on Diaion HP20 according to the above-mentioned scheme 1, washed with water to remove fractions 1 and 2, and then 80% A proteolysate of the present invention containing fractions 3 to 6 eluted with ethanol was prepared and subjected to the following experiment.

  The control drug enalapril (ACE inhibitor) was purchased from SIGMA. Fractions 3, 4, 5 and enalapril were dissolved in water for injection (Otsuka Pharmaceutical Co., Ltd.). Since the royal jelly proteolysate (RJ-H) consisting of fractions 3 to 6 and fraction 6 were insoluble in distilled water, they were used by dissolving in borate buffer (pH 8.3). For each test substance, a 30 mg / ml solution was prepared as a stock solution, diluted with distilled water for injection, and tested for a solution having a predetermined concentration (final concentrations 3, 10, 30, 100, 300 μg / ml).

2) Test material
Harley guinea pig (male, 6 weeks old, SLC Japan) was purchased and temperature (23 ± 2 ℃), humidity (55 ± 15%), ventilation (number of times: 15 times / hour or more) in a barrier system environment The animals were preliminarily reared (acclimated) under free feeding in an animal room that was adjusted to an environment of lighting (12-hour lighting: 7 am to 7 pm). After the acclimatization, guinea pigs were exsanguinated after bruising the head under diethyl ether anesthesia, and the intestinal tract (ileum) was removed, and a cylindrical specimen having a length of about 2 cm was prepared according to a conventional method. The specimen was immediately placed in Tyrode's solution heated to 27 ° C. The composition of Tyrode solution used as a nutrient solution is as follows.

  137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.1 mM MgCl2, 0.4 mM NaH2PO4, 11.9 mM NaHCO3, 5.5 mM glucose.

3) Test method The specimen is suspended in a Magnus tube filled with Tyrode's solution (15 ml), a static tension of about 1 g is applied, and an isotonic tension transducer (MODEL ME-4013, M.E. Commercial, Intestinal movements were recorded on a recorder (MULTICORDER MC6625, GRAPHTEC, Yokohama) via Tokyo. Bradykinin (5 ng / ml) is used as a contraction agent, and the sample is contracted by repeatedly adding a contraction agent at a single concentration. After the contraction response is almost stable, a test substance is added. After a minute, a contractile agent was added and the tension was measured. The test was performed at 27 ° C., and 4 samples per group were used. 0.15 ml each of the test substance and the contractile agent was added to 15 ml of Tyrode's solution (100-fold dilution). The shrinkage height is calculated by measuring the shrinkage height due to the shrinkage agent alone and the shrinkage height due to the shrinkage agent when the test substance is added on the recording paper, and the ratio of the latter to the former (control) (enhancement rate:% of control). Calculated.

4) Statistical processing The measurement results were expressed as mean ± standard error. Concentration (AC30; 30%) that increases bradykinin contraction by 30% using 4 doses (10 to 300 μg / ml or 0.3 to 10 μM) that show linearity from the dose-response curve of each test substance (EXSAS: linear regression line) augmentation). The results are shown in Table 10. The following results revealed that the proteolysate of the present invention has a bradykinin contraction enhancing action.

Below, the formulation example containing the protein degradation product of this invention is shown.
Formulation Example 1 (grain)
The following raw materials were mixed by a conventional method and molded with a tableting machine to produce an 8 mm diameter uncoated tablet. This uncoated tablet was film-coated for food by a conventional method to complete a granular health food.
(Composition per grain)

Formulation Example 2 (Drink)
After thoroughly stirring the following materials in water, the total amount was 100 ml.
(Composition for one bottle)

Formulation Example 3 (Capsule)
The following ingredients were uniformly mixed, passed through a 60 mesh sieve, and the whole powder was filled into a No. 2 gelatin capsule.
(Composition of one capsule)

Formulation Example 4 (Granule)
The following materials were granulated according to a conventional method and packed into stick-shaped aluminum.
(Composition for one bottle)

Formulation example 5 (source)
The following ingredients were mixed thoroughly and stirred well at the time of use, and used for salads and fries.
(Composition for one person)

The measurement result of the ACE inhibitory activity of Example 1 is shown. The relative amino acid composition when Gly is 1 mol is shown. The relative amino acid composition when Lys is 1 mol is shown. The result of GFC analysis is shown. The transition of the systolic blood pressure at the time of single administration of a test substance is shown. The change in systolic blood pressure after administration of the test substance for 8 weeks and withdrawal for 2 weeks is shown. Fraction No. fractionated according to Scheme 1. The graph of 1-7 is shown. FIG. 8 shows an example of a chromatogram relating to the content of participating components. In FIG. 8, the “comparative solution” includes tryptophan, glycyl-L-leucyl-L-tyrosine and 10-hydroxy-δ-2-decenoic acid standard. Moreover, a test solution is the protein degradation product of this invention containing the fractions 1-7. The result of the systolic blood pressure (all test subjects) of Example 5 is shown.

Claims (30)

A proteolysate having an angiotensin converting enzyme inhibitory effect obtained by treating a royal jelly material with trypsin. The protein degradation product according to claim 1, wherein the royal jelly material is an alcohol-denatured protein of royal jelly. Proteolysate derived from royal jelly material

A proteolysate having a residual ratio (molar ratio) after enzymatic degradation of Lys of 70% or less of the royal jelly material with respect to Gly represented by Proteolysate derived from royal jelly material

A proteolytic product in which the content ratio (molar ratio) of Leu is 1.3 times or more that of the royal jelly material, based on Lys represented by It is derived from royal jelly material and substantially contains at least one selected from the group consisting of (1) inorganic salts and (2) acidic free amino acids, basic free amino acids, free amino acids having alcoholic hydroxyl groups, free Ala and free Gly The protein degradation product according to claim 3 or 4, which is not present. The proteolysate according to claim 5, which is derived from a royal jelly material and is substantially free from (1) inorganic salts and (2) free acidic amino acids and free basic amino acids. Derived from royal jelly material, (1) inorganic salts and (2) acidic free amino acids (Asp, Glu), basic free amino acids (Lys, His, Arg), free amino acids with alcoholic hydroxyl groups (Thr, Ser), free The proteolysate according to claim 6, substantially free of Ala and free Gly. The proteolysate according to any one of claims 3 to 5, which has a sugar content of 35% by weight or less and does not substantially discolor under accelerated test conditions at 40 ° C for 2 months. A component having a molecular weight of 10,000 or more is not substantially recognized by analysis with a gel filtration column, and has a maximum peak in a molecular weight range of about 200 to 1500 and a sharp peak with a molecular weight of about 200 to 300. 7. The protein degradation product according to any one of 6. The protein degradation product according to any one of claims 3 to 6, wherein the average molecular weight is in the range of about 700 to 6000. When the proteolysate is passed through a column (4.6 mmφ × 150 mm) of a styrene / divinylbenzene copolymer having the following characteristics, the content (%) of the component concerned represented by the following formula is 40% or more: Properties of protein degradation product / styrene / divinylbenzene copolymer according to any one of 1 to 10 Particle size distribution; 30 μm
Pore diameter: 250mm
Functional group; none Applicable pH range: Full range.
And involvement ingredient content _{(%) = A 1 / A} T × 100
T ₁ : Tryptophan retention time
T ₂ : 10-hydroxy-δ-2-decenoic acid retention time
A ₁ : Total peak area from the peak detected at the T ₁ elution position to the peak detected at the T ₂ elution position
A _T : Sum of all peak areas. The protein degradation product according to claim 11, wherein the content of the concerned component is 58 ± 5%. The proteolysate according to any one of claims 1 to 12, comprising at least one of the following nine peptides.
Glu-Trp-Lys;
His-Pro-Ala-Leu;
Thr-Phe;
Ser-Pro-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Thr-Pro-Phe;
Ser-His-Ser-Gly-Leu-Tyr;
Tyr-Ser-Pro-Leu. The protein degradation product is passed through a column of a styrene / divinylbenzene copolymer having the following characteristics, and water (fractions 1, 2), 20% methanol (fraction 3), 40% methanol (fraction 4), 60% methanol ( The proteolysate according to any one of claims 1 to 11, comprising fractions 3 to 6 among fractions eluted with fraction 5), 80% ethanol (fraction 6), and 100% ethanol (fraction 7):
Particle size distribution:> 250μm (including more than 90% particles with a particle size larger than 250μm)
Pore diameter: 400mm
Functional group: None Application pH range: Full range. In fraction 3
Glu-Trp-Lys,
His-Pro-Ala-Leu and
Thr-Phe
At least one peptide selected from the group consisting of:
Thr-Phe;
Ser-Pro-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Thr-Pro-Phe;
Ser-His-Ser-Gly-Leu-Tyr;
Tyr-Ser-Pro-Leu
The proteolysate according to claim 14, comprising at least one peptide selected from the group consisting of: A prophylactic or therapeutic agent for hypertension comprising the protein degradation product according to any one of claims 1 to 15 as an active ingredient. The foodstuff containing the protein degradation product in any one of Claims 1-15. The food according to claim 17, which is a food for preventing hypertension. The formulation for oral ingestion containing the protein degradation product in any one of Claims 1-15. A prophylactic or therapeutic agent for hypertension containing at least one peptide selected from the following nine peptides as an active ingredient:
Glu-Trp-Lys;
His-Pro-Ala-Leu;
Thr-Phe;
Ser-Pro-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Thr-Pro-Phe;
Ser-His-Ser-Gly-Leu-Tyr;
Tyr-Ser-Pro-Leu. The foodstuff containing the protein degradation product of Claim 20. The food according to claim 21, which is a food for preventing hypertension. A preparation for oral consumption comprising at least one peptide according to claim 20. The preparation according to claim 19, which is in the form of a food comprising an apparent food and / or supplement. An angiotensin converting enzyme inhibitor comprising the proteolysate according to any one of claims 1 to 15 as an active ingredient. The method for producing a protein degradation product according to any one of claims 1 to 15, wherein the royal jelly material is treated with trypsin. Proteolytic product obtained by trypsin treatment is treated with a synthetic adsorbent, and consists of (1) inorganic salts and (2) acidic free amino acids, basic free amino acids, free amino acids having alcoholic hydroxyl groups, free Ala and free Gly. 27. The method according to claim 26, wherein at least one selected from the group is removed. 27. The protein degradation product obtained by trypsin treatment is adsorbed on a styrene / divinylbenzene copolymer, washed with water to remove inorganic salts and water-soluble amino acids, and then eluted with hydrous alcohol. the method of: The method of claim 28, wherein the styrene-divinylbenzene copolymer has the following properties:
Particle size distribution:> 250μm (including more than 90% particles with a particle size larger than 250μm)
Pore diameter: 400mm
Functional group: None Applicable pH range: Full range. A novel peptide consisting of any of the following five peptides:
His-Pro-Ala-Leu;
Asn-Leu-Tyr;
Leu-Ser-Tyr;
Tyr-Ser-Pro-Leu;
Ser-His-Ser-Gly-Leu-Tyr.

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