JP5250302B2 - Antihyperglycemic agent - Google Patents

Description

The present invention, by the action of neutral protease protein from royal jelly, more particularly blood glucose increase inhibitor comprising as an active ingredient a royal jelly peptidyl de obtained by heating with pentoses.

  Generally, royal jelly is a jelly-like substance made by mixing secretions secreted from the hypopharyngeal gland and the greater vagina by female honeybees 3 to 15 days after emergence, and contains specific proteins, fatty acids, minerals, etc. It is known that Conventionally, royal jelly has blood pressure lowering action, antitumor action, wound healing promotion, serum cholesterol lowering action, blood flow increasing action, sexual central aging prevention action, antibacterial action, growth promoting action, radiation and chemotherapeutic side effect reducing action. It is known to have various physiological effects such as an action on chronic diseases. Accordingly, royal jelly has been used not only for health foods with high nutritional value but also for uses such as pharmaceuticals and cosmetics.

As a form using a royal jelly, a method disclosed in Patent Document 1 is known. Patent Document 1 discloses an enzyme-treated royal jelly obtained by subjecting royal jelly to hydrolysis using an endo-type neutral protease derived from Bacillus subtilis. Such an enzyme-treated royal jelly contains an angiotensin converting enzyme inhibitory peptide having a blood pressure lowering action.
JP 2007-78 A

  The present invention has found that a useful effect on the living body, such as an effect of improving the antioxidant effect, can be obtained by further causing a pentose to act on a peptide produced by allowing a neutral protease to act on a protein derived from royal jelly. It is based on.

It is an object of the present invention is to provide a hyperglycemic inhibitor comprising as an active ingredient a royal jelly peptidyl de that may be utilized in various applications.

In order to achieve the above object, the blood sugar elevation inhibitor of the invention described in claim 1 hydrolyzes a protein derived from royal jelly with thermolysin, and the hydrolyzed protein together with xylose at 1 to 5 at 70 to 85 ° C. It contains a royal jelly peptide obtained by heating for a period of time as an active ingredient.

According to the present invention, it is possible to provide a hyperglycemic inhibitor comprising as an active ingredient a royal jelly peptidyl de that may be utilized in various applications.

Hereinafter, the embodiment which actualized the royal jelly peptide of this invention is described. Hereinafter, royal jelly is abbreviated as RJ.
The RJ peptide of this embodiment is obtained by hydrolyzing a protein derived from RJ with a neutral protease and heating the hydrolyzed protein and pentose. RJ includes, as main physiologically active components, for example, proteins, saccharides, organic acids such as 10-hydroxydecenoic acid, which are specific to RJ, lipids, vitamin Bs, vitamins such as folic acid, nicotinic acid, pantothenic acid, and various minerals Contains As RJ used for the production | generation of the RJ peptide of this embodiment, you may employ | adopt either RJ powder which dried and pulverized raw RJ or this raw RJ. Moreover, the production area of RJ is not specifically limited, For example, any of Japan, China, Brazil, European countries, Oceania countries, the United States, etc. may be sufficient. In addition, you may use what extracted the protein in RJ so that it may mention later.

  Since RJ contains a high amount of protein, a protease may be directly acted on using raw RJ or dried RJ powder as an RJ-derived protein that is a raw material for treating a neutral protease. In addition, raw RJ or dry RJ powder may be pretreated, for example, degreased using activated carbon, acetone, ethanol, hexane, activated carbon, or activated clay. Moreover, you may use what extracted or refine | purified protein from raw RJ or dry RJ powder using the well-known method. Methods for separating and purifying proteins include, for example, heating / cooling, acid / alkali, pressurization, protein precipitation using denaturing agents, protein precipitation using salting out, and various chromatography methods. The method to use etc. are mentioned. In addition, an insoluble fraction that is extracted when a hydrophilic organic solvent or a hydrous hydrophilic organic solvent is used to obtain a soluble fraction (RJ extract) is used as an RJ-derived protein that is a neutral protease-treated raw material. May be. The fraction insoluble in the extraction solvent mainly contains a high amount of water-soluble protein.

  In order to obtain the RJ peptide of this embodiment, the above RJ-derived protein is first subjected to protease treatment (protein hydrolysis treatment). Protease treatment is a treatment that hydrolyzes peptide bonds of RJ-derived proteins using a neutral protease to lower the molecular weight. By this treatment, hydrolyzed protein is generated from the RJ-derived protein. Examples of the neutral protease include a protease having an optimum pH in the vicinity of neutrality (pH 5.0 to 8.5, preferably pH 6.5 to 7.5). Neutral proteases include exo-type proteases that hydrolyze from the end of the peptide and endo-type proteases that degrade from the middle of the peptide. Any protease can be used. Specific examples of the neutral protease include thermolysin, Aspergillus oryzae, which are neutral proteases derived from Bacillus stearothermophilus (Bacillus thermoproteolyticus Rokko). And neutral protease derived from Bacillus subtilis. Among these, neutral protease derived from thermolysin, which is highly effective in improving antioxidant activity by enzyme treatment, is preferable.

  Thermolysin is classified as a metalloprotease, and the optimum pH is about 6.5 to 8.5. Thermolysin is an endopeptidase that cleaves peptide bonds containing amino residues with large hydrophobic side chains such as isoleucine, leucine, valine, phenylalanine, methionine. As a commercial item, Samoaase PC10F (made by Yamato Kasei Co., Ltd.) can be mentioned, for example. In addition, other neutral proteases having an optimum pH near neutrality and capable of cleaving the same site as thermolysin alone or in combination can also be used. The proteolytic enzyme treatment using thermolysin is preferably performed under conditions of 10 to 80 ° C, more preferably 40 to 75 ° C, and even more preferably 50 to 70 ° C.

  Protease treatment using a neutral protease is carried out by incubating a reaction solution containing RJ-derived protein, neutral protease and water (or buffer solution) under predetermined conditions. The treatment time for the protease treatment is appropriately set depending on the reaction temperature, enzyme activity (titer), etc., and is, for example, 0.1 to 24 hours. In this protease treatment, the reaction solution after the incubation may be heated to inactivate the protease.

  The reaction solution contains water or a buffer solution as a solvent for suppressing the increase in viscosity caused by the RJ-derived protein and allowing the protease treatment to proceed rapidly. It is desirable that the solvent contains 2 to 15 times, preferably 2 to 14 times, more preferably 3 to 10 times the amount of water or buffer, with respect to the weight of the processing raw material (protein). When less than twice the amount of solvent is added relative to the weight of the raw material to be treated, the increase in the viscosity of the solvent due to the protein cannot be sufficiently suppressed, so that it is difficult to rapidly proceed with the protease treatment. On the other hand, when a solvent exceeding 15 times the amount of the treatment raw material (protein) is added, there is a disadvantage that it takes a lot of time to powder the obtained enzyme-treated RJ.

  Pentose (pentose sugar) is blended in order to promote the production of melanoidin by Maillard reaction with the hydrolyzed protein (enzyme-treated RJ). The Maillard reaction is a browning reaction in which a reducing sugar (pentose) and an amino compound (hydrolyzed protein) are polymerized to produce melanoidin, which is a brown substance. By this browning reaction, the RJ peptide of the present embodiment having an antioxidant effect and a blood glucose elevation inhibitory effect is produced from the hydrolyzed protein. Examples of the pentose used in the Maillard reaction include aldo pentose and keto pentose. Aldopentoses include ribose, arabinose, xylose and lyxose. Ketopentose includes ribulose and xylulose. Of these, xylose and arabinose are preferred.

  The pentose may be added at any time before or after the protease treatment. Preferably, the pentose is added after protease treatment. Although the blending ratio of the pentose and the hydrolyzed protein in the Maillard reaction is not particularly limited, the mass of the hydrolyzed protein (dry mass) is preferably 1 to 20 and more preferably 5 to 15 with respect to the mass 1 of the pentose. Water or a buffer solution is contained as a solvent for allowing the treatment to proceed rapidly. The pH of the solvent is not particularly limited, but is preferably adjusted in the acidic to neutral range, more preferably in the range of pH 3 to 6 in order to proceed the reaction. The Maillard reaction generally proceeds under normal temperature to heating conditions. In the present embodiment, heat treatment is performed to advance the Maillard reaction using pentose. The heating temperature is preferably 50 to 100 ° C, more preferably 70 to 90 ° C, and further preferably 75 to 85 ° C. When the heating temperature is less than 50 ° C., it takes a long time for the Maillard reaction to proceed, and thus the productivity may be significantly reduced. On the other hand, if the heating temperature exceeds 100 ° C., the solvent may be significantly volatilized. Moreover, palatability may fall. The processing time of the Maillard reaction is appropriately set depending on the blending amount of pentose, the processing temperature, and the like, and is, for example, 0.1 to 24 hours, preferably 1 to 5 hours. When the treatment temperature increases, the reaction can be completed in a relatively short time.

  The RJ peptide of this embodiment has a high antioxidant effect and an inhibitory effect on blood sugar elevation. Therefore, it can be applied as an anti-oxidant and an anti-glycemic inhibitor for the purpose of obtaining those effects. As a specific blending form, it can be applied as a food / beverage product, a cosmetic product, a pharmaceutical product and the like for the purpose of obtaining those functions and effects.

  When applying the RJ peptide of this embodiment to food-drinks, it can be used by adding to various food materials or beverage materials. The form of the food or drink is not particularly limited, and may be any of liquid, powder, gel, and solid, and the dosage form is any of tablets, capsules, granules, and drinks. May be. Among these, a capsule is preferable because hygroscopicity is suppressed. As said food-drinks, you may mix | blend gelatinizer containing foodstuffs, saccharides, a fragrance | flavor, a sweetener, fats and oils, a base material, an excipient | filler, a food additive, a subsidiary material, a bulking agent etc. suitably as another component.

  When applying the RJ peptide of this embodiment to cosmetics, it can manufacture by mix | blending with a cosmetic base material. The form of the cosmetic may be any of emulsion, cream, powder and the like. By applying such a whitening cosmetic product to the skin, an effect such as an antioxidant effect can be obtained. Cosmetic bases are generally blended in common with cosmetics. For example, oil, purified water, and alcohol as main components, surfactants, moisturizers, antioxidants, thickeners, anti-seborrheic agents. At least one selected from an agent, a blood circulation promoter, a whitening agent, a pH adjuster, a pigment, a preservative, and a fragrance is appropriately blended.

  When the RJ peptide of this embodiment is used as a pharmaceutical, it is possible to employ any administration method such as intravascular administration or transdermal administration, in addition to administration by taking (oral intake). The RJ peptide of this embodiment is desirably administered by oral ingestion. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, etc. are mentioned. Moreover, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc. as an additive.

The effects exhibited by this embodiment will be described below.
(1) In this embodiment, an RJ peptide obtained by hydrolyzing an RJ-derived protein with a neutral protease and heating the hydrolyzed protein together with pentose has a high antioxidant effect. Therefore, it can be preferably applied to cosmetics, foods and drinks and pharmaceuticals intended for antioxidant action as antioxidants.

  (2) In this embodiment, an RJ peptide obtained by hydrolyzing an RJ-derived protein with a neutral protease and heating the hydrolyzed protein together with pentose has a high blood glucose rise-inhibiting action. . Therefore, it can be preferably applied to foods and drinks for the purpose of suppressing blood sugar elevation and pharmaceuticals used as a blood sugar rise inhibitor.

  (3) In the present embodiment, as the pentose used for obtaining the RJ peptide, at least one selected from xylose and arabinose is preferably used. Therefore, the antioxidant effect and blood glucose elevation inhibitory action of the finally obtained RJ peptide can be further enhanced.

  (4) In this embodiment, thermolysin is preferably applied as the neutral protease used to obtain the RJ peptide. Therefore, the antioxidant treatment and the blood glucose elevation inhibiting effect can be further enhanced by the enzyme treatment.

In addition, you may change the said embodiment as follows.
· RJ peptides of the above embodiments, food products that are applied to humans, not 及 beauty medicines only, supplement for domestic animals such as domestic animals, dietary supplements, may be applied as 及 beauty pharmaceuticals.

  The RJ peptide of the above embodiment is preferably applied to the treatment of patients with hyperglycemia such as diabetes. However, not only for therapeutic use, but also for healthy people to take for the prevention of hyperglycemia.

Although the test example is given below and the embodiment is described more specifically, the present invention is not limited to these.
(Test Example 1, relationship between Maillard reaction and heat treatment temperature)
The relationship between the Maillard reaction and the heat treatment temperature was tested. 1 kg of Chinese-produced RJ (solid content 35% (w / w)) was degreased with ethanol. Next, 10 times the amount of distilled water was added as a solvent to the solid content in the degreasing RJ, and the temperature was raised to 65 ° C. Next, after adjusting the pH of the solvent to 7.5 using 5N NaOH, thermolysin (2% with respect to the solid content in the degreased RJ) was added as a neutral protease to perform a hydrolysis reaction. As the thermolysin, Samoa PC10F (manufactured by Daiwa Kasei Co., Ltd.) was used. Samoaase PC10F has a titer of 90000 PU / g or more (1 PU = casein (final concentration 0.5%) at pH 7.2, 35 ° C., acid soluble low equivalent to 1 μg of tyrosine for 1 minute at the beginning of the reaction. The amount of enzyme required to produce a molecular degradation product).

  After the hydrolysis reaction for 4 hours, 50% of diatomaceous earth 600S was added as a filter aid to the solid content in the degreasing treatment RJ, and suction filtered. The filtrate was freeze-dried, and the resulting powder was designated as enzyme-treated RJ. After adding 1 part by weight of xylose and 100 parts by weight of distilled water to 9 parts by weight of this enzyme-treated RJ, the pH was adjusted to pH 5.0 using 3N HCl. The adjusted mixed solution was heat-treated at each temperature shown in Table 1 below for 3 hours. After cooling, the absorption wavelength (abs 460 nm) of the browning substance (melanoidin) generated by the Maillard reaction was measured. The results are shown in Table 1. In addition, the blank used what was not heat-processed.

  For palatability, sensory evaluation by taste was performed for those not subjected to heat treatment. No decrease in palatability was observed: ◯, slight decrease in palatability was observed: Δ, a decrease in palatability was clearly observed: evaluated as x.

As shown in Table 1, it is confirmed that the absorbance increases as the heat treatment temperature increases. It seems that the Maillard reaction progressed and the production of melanoidin was promoted. On the other hand, it was confirmed that the preference decreased slightly when the heat treatment temperature increased. It was confirmed that the heat treatment temperature is most preferably around 80 ° C. from the viewpoint of reactivity and palatability.

(Test Example 2: Production of browning substance by adding various sugars)
1 kg of Chinese-produced RJ (solid content 35% (w / w)) was degreased with ethanol. Next, 10 times the amount of distilled water was added as a solvent to the solid content in the degreasing RJ, and the temperature was raised to 65 ° C. Next, after adjusting the pH of the solvent to 7.5 using 5N NaOH, thermolysin (2% with respect to the solid content in the degreased RJ) was added as a neutral protease to perform a hydrolysis reaction. As the thermolysin, Samoa PC10F (manufactured by Daiwa Kasei Co., Ltd.) was used. Samoaase PC10F has a titer of 90000 PU / g or more (1 PU = casein (final concentration 0.5%) at pH 7.2, 35 ° C., acid soluble low equivalent to 1 μg of tyrosine for 1 minute at the beginning of the reaction. The amount of enzyme required to produce a molecular degradation product).

After the hydrolysis reaction for 4 hours, 50% of diatomaceous earth 600S was added as a filter aid to the solid content in the degreasing treatment RJ, and suction filtered. The filtrate was freeze-dried, and the resulting powder was designated as enzyme-treated RJ. For this enzymatic treatment RJ9 parts, real施例that shown in Table 2 1, 1 part by weight of sugar in Reference Example 2 and Comparative Example, and after the distilled water added to 100 parts by weight of a 3N HCl using The pH was adjusted to 5.0. The adjusted mixed solution was heat-treated at 80 ° C. for 3 hours. After cooling, the absorption wavelength (abs 460 nm) of the browning substance (melanoidin) generated by the Maillard reaction was measured. The results are shown in Table 2. In Comparative Example 1, no sugar was added and no heat treatment was performed. In Comparative Example 2, the heat treatment was performed without adding sugar.

As shown in Table 2, it was confirmed that Example 1 and Reference Example 2 produced many brown materials. On the other hand, in Comparative Examples 3 to 5 using hexose, almost no brown substance was generated by heating. In Comparative Examples 6 and 8 using disaccharides, almost no brown material was generated by heating. In Comparative Example 7 using tetrose alcohol (4-carbon sugar), almost no brown material was produced by heating.

(Test Example 3, blood glucose level increase inhibitory effect test)
In order to confirm the inhibitory effect of the RJ peptide on the increase in blood glucose level, rats were orally loaded with sucrose and the effect on the time course of the blood glucose level was examined.

  In this test example, 6-week-old male Wistar rats were obtained from Japan SLC and used. The animals were raised under the conditions of room temperature 23 ± 1 ° C., relative humidity 55 ± 10%, illumination time 12 hours / day (8:00 to 20:00) and fed with chow CRF-1 from Charles River Japan, For drinking water, tap water was freely consumed. After arrival, animals were preliminarily raised for one week, and animals with no abnormal health condition were used for the test.

  As a test sample, an RJ peptide (freeze-dried) using xylose as the sugar of Example 1 was used as a sample of the example. As a sample of Comparative Example 9, degreased RJ (freeze-dried) before protease (samoyase) treatment was used. As Comparative Example 10, an enzyme treatment RJ (freeze drying) after a Samoaase treatment under the same conditions as in Example 1 was used. As Comparative Example 11, RJ (freeze-dried) was used in which D-xylose was added as a saccharide to the degreased RJ before the Samoaase treatment and heat-treated under the same conditions as in Example 1. As a positive control, alkaboose having α-glucosidase inhibitory activity was used. Each sample was prepared by suspending in distilled water to 2 g / kg / 10 mL. Alkabose was adjusted to 10 mg / kg / 10 mL.

  After fasting 7-week-old male Wistar rats overnight (5 animals per group), blood was collected from the tail vein under non-anaesthesia, and blood glucose was directly measured using a blood glucose meter (Extra, manufactured by Abbott Japan). The concentration was measured and taken as the blood glucose level at 0 hour. Distilled water was used as a control. Each sample group was orally administered at a rate of 1 g / kg (body weight), and the positive control group was alkaboose 10 mg / kg (body weight). After 30 minutes from the administration, 2 g / kg (body weight) of sucrose solution was orally administered to all groups. Blood glucose concentration 30 minutes after oral administration of sucrose was measured. The inhibition rate was calculated assuming that the blood glucose level of the control was 0% inhibition rate, and the blood glucose level at 0 hours was 100% inhibition rate. Student's t-test is used to test the statistical difference in blood glucose level between the control group and each administration group. The obtained values were expressed as mean ± standard deviation, and the significance level in the test was less than 1%.

As shown in Table 3, it was confirmed that all the test groups including the positive control tend to have an inhibitory effect on the increase in blood glucose level. Among these, it was confirmed that Example 1 has a significant blood glucose level inhibitory action. From the above results, it was confirmed that the blood glucose level increase inhibitory ability obtained by heating RJ was significantly increased by peptide-forming the protein in RJ with neutral protease and further heating with pentose.

(Test Example 4, a radical scavenging ability test as an antioxidant activity is shown as a reference example )
Antioxidant action, which is one of the physiologically active actions of RJ peptide, was compared by radical scavenging ability test. This test example utilizes the fact that DPPH (1,1-Diphenyl-2-picrylhydrazyl) having a maximum absorption of 517 nm in a radical state is reduced by an antioxidant and fades. As a test sample, the sample (lyophilized) obtained in Example 1 and Comparative Examples 9 to 11 used in Test Example 3 was used.

Sample solutions were prepared by dissolving the samples of each example in absolute ethanol to concentrations of 1%, 2%, and 3%, respectively. To 100 μL of each sample solution, 1.9 mL of a 170 μM DPPH ethanol solution was added and mixed to obtain a DPPH ethanol sample solution, and then reacted at room temperature (22 ° C.) for 15 minutes. And the light absorbency in wavelength 517nm of each DPPH ethanol sample solution was measured using the spectrophotometer (Shimadzu Corporation UV-1200). In addition, the same operation was performed using only a solvent as a control. The DPPH radical scavenging rate was determined from the following equation. Then, a calibration curve was prepared to calculate 50% inhibitory concentration IC ₅₀ (mg / ml) (the test was measured at n = 2). The results are shown in Table 4. In Table 4, the lower the IC ₅₀ value, the stronger the antioxidant effect.

DPPH radical scavenging rate (%) = {(Ac−As) / Ac} × 100
Ac: Absorbance of control As: Absorbance when DPPH solution is added-Absorbance when only ethanol is added

As shown in Table 4, Example 1 in which degreased RJ was treated with Samoaase and Maillard reaction was performed using pentose was conducted in Comparative Example 10 where only Samoaase treatment was performed and Comparative Example 11 where only Maillard reaction was performed. It is confirmed that the DPPH radical scavenging rate is higher than that.

  Free radicals are considered as causative factors for various diseases such as malignant tumors, heart diseases, and cerebrovascular diseases. RJ peptides having the action of removing free radicals can be effective components for the prevention and treatment of these diseases. Therefore, it can mix | blend suitably as active ingredients, such as food-drinks, cosmetics, and a pharmaceutical aiming at the exhibit of free radical removal ability.

(Test Example 5, relationship between Maillard reaction and antioxidant activity is shown as a reference example )
The relationship between Maillard reaction and antioxidant activity was tested. By the method shown in Test Example 1, powdery enzyme-treated RJ was finally obtained from Chinese-produced RJ (solid content 35% (w / w)) using thermolysin. After adding 2 parts by weight of xylose and 200 parts by weight of distilled water to 18 parts by weight of this enzyme-treated RJ, the pH was adjusted to pH 5.0 using 3N HCl. The adjusted mixed solution was heat-treated at a temperature of 90 ° C. The reaction solution was collected every 30 minutes (maximum 7 hours), and the production of browning substance (melanoidin) generated by Maillard reaction was measured at the absorption wavelength (abs460nm) and the antioxidant action (DPPH radical scavenging rate) was also measured. . Antioxidant activity was measured according to the method of Test Example 4. The results are shown in FIG.

  As shown in FIG. 1, it is confirmed that the absorbance (abs 460 nm) increases with the heat treatment time. It is also confirmed that the antioxidant activity increases with the heat treatment time. That is, it is confirmed that the antioxidant activity is also increased in proportion to the production of the browning substance. It was inferred that the increase in the antioxidant activity by the heat-treated RJ peptide subjected to the enzyme treatment and the Maillard reaction was an action derived from the browning substance (melanoidin).

The graph which shows the relationship between a Maillard reaction and antioxidant activity.

Claims (1)

  Increase in blood glucose, comprising a royal jelly peptide obtained by hydrolyzing a protein derived from royal jelly with thermolysin and heating the hydrolyzed protein with xylose at 70 to 85 ° C. for 1 to 5 hours. Inhibitor.