JP5154964B2 - Contains royal jelly-degrading enzyme - Google Patents

Description

  The present invention relates to a royal jelly-degrading enzyme-containing material prepared from a honey bee larvae of 2-3 days old of a western honey bee (Apis melifera), a royal jelly-degrading enzyme contained in the royal jelly-degrading enzyme-containing material, the royal jelly-degrading enzyme-containing material, or the The present invention relates to a royal jelly degradation product characterized by being prepared using a royal jelly degrading enzyme, and a royal jelly degradation method using the royal jelly degrading enzyme-containing material or the royal jelly degrading enzyme.

  Royal jelly is a milky white jelly-like nutrient secreted by the worker bees from the hypopharyngeal gland and the greater vagina to grow the queen bee larvae. Since queen bees are genetically homogenous with worker bees, royal jelly is thought to contain some regulatory factor that induces differentiation into queen bees, but the substance has not yet been revealed. The elucidation of the regulatory factor and the mechanism for inducing differentiation into queen bees is useful for improving the efficiency of artificial production of queen bees and the like, and research is being conducted by researchers all over the world.

  Royal jelly varies depending on the bee group, variety, and the type of flower from which the bees collect nectar, pollen, etc. Usually, about 2/3 of the raw weight is water, and it is mainly composed of proteins, carbohydrates, and fatty acids. And In addition, it contains a lot of vitamins, minerals, etc. and is extremely nutritious. Moreover, although not scientifically proven, it has long been considered to have many pharmacological actions such as fatigue recovery action, antiallergic action, anticancer action, and immune enhancement action. For this reason, it is widely used as a dietary supplement and a pharmaceutical raw material.

  In recent years, proteins that account for about 40% of the dry weight of royal jelly have attracted attention as the main physiologically active substance of royal jelly. In particular, many inventions using the physiological activity of royal jelly protein degradation products have been reported. For example, (1) an invention related to an infection defense function enhancing agent characterized by containing a peptide having a molecular weight of 3,000 or less obtained by degrading a protein in royal jelly with a protease as an active ingredient has been reported (for example, , See Patent Document 1). Since the royal jelly proteolysate by protease is superior to the undegraded royal jelly protein in infection prevention function and has low viscosity and water solubility and excellent stability, It is an invention based on the knowledge that oral intake is easy. Moreover, the invention which concerns on the protein degradation product which has an angiotensin converting enzyme inhibitory effect formed by processing (2) royal jelly raw material with trypsin is also reported (for example, refer patent document 2).

  In addition, as an invention relating to a royal jelly degradation product for use as a food, (3) a low allergenized royal jelly with reduced allergenicity, comprising 10-hydroxydecenoic acid and a royal jelly-derived peptide component, the peptide An allergenic royal jelly characterized in that the sex component is composed of a component having a molecular weight of 4.5 kDa or less has also been reported (see, for example, Patent Document 3). In the invention, by proteolytic enzyme treatment and glycolytic enzyme treatment using β-mannosidase, among components derived from royal jelly, components exceeding a molecular weight of 4.5 kDa that easily cause allergies are decomposed and removed. The invention is based on the finding that it is easy to reduce allergenicity.

As described above, there are many reports on the various physiological activities of royal jelly degradation products, particularly royal jelly protein degradation products, but specific physiologically active substances and their detailed action mechanisms have been elucidated. There are very few things. The identification of bioactive substances and the elucidation of the mechanism of action contribute to the elucidation of the differentiation mechanism of bees and the development of more effective pharmaceuticals and functional foods for animals such as humans. Further research and research are highly desired. In order to effectively investigate the royal jelly degradation product, it is important to obtain a royal jelly degradation product that contains abundant bioactive degradation products such as functional peptides.
JP-A-8-59499 JP 2005-255670 A JP 2005-287411 A

  However, many of the methods currently used, including the method of (1) above, are merely degrading the royal jelly protein using a widely used protease or the like. Since the general-purpose protease or the like does not use royal jelly as an original substrate, there is a possibility that the function of a peptide that is originally useful may be inactivated by decomposition at an inappropriate place. Although this fear can be solved by using a protease or the like using royal jelly as a substrate, such an enzyme has not yet been found.

It is an object of the present invention to provide an optimal royal jelly-degrading enzyme and a royal jelly-degrading enzyme-containing product that are optimal for efficiently obtaining a biologically active degradation product such as a functional peptide from royal jelly.
Another object of the present invention is to provide a royal jelly degradation product characterized by being prepared using the royal jelly degrading enzyme-containing material, and a royal jelly degradation method using the royal jelly degrading enzyme-containing material. .

  As a result of earnest research to solve the above problems, the present inventors have found that royal jelly is a staple food, and the queen bee larvae whose differentiation into queen bees is controlled by feeding royal jelly is congenitally optimal for the degradation of royal jelly. The present invention was completed by separating a fraction containing an enzyme having the activity of degrading royal jelly protein from a larva body fluid extract of a queen bee larva.

That is, the present invention provides a royal jelly-degrading enzyme-containing material prepared from a honey bee larva of 2 to 3 days old from a western bee (Apis mellifera) by a method having the following steps.
(A) A step of separating the body suspension of the queen bee larvae into three layers of a white solid upper layer, a solution middle layer, and a precipitation lower layer by centrifuging at 6 ° C. or lower.
(B) A step of recovering the middle layer as a royal jelly-degrading enzyme-containing material.
In addition, the present invention provides a royal jelly-degrading enzyme-containing material, wherein the centrifugation is 8,000 to 12,000 × g for 5 minutes or more.
The present invention also provides a royal jelly-degrading enzyme-containing material prepared from a honey bee larva of 2 to 3 days old of a western bee by a method having the following steps.
(A ′) A step of preparing a milky white body tissue suspension by washing the queen bee larvae with ice-cold physiological saline and then straining them.
(B ′) After diluting the body tissue suspension with a 50 mM phosphate buffer having a pH of 7.0, centrifugation is performed at 10,000 × g and 5 ° C. for 10 minutes to obtain an upper layer of a white solid. The process of separating the middle layer of the yellowish-white turbid solution from the white to the lower three layers due to a slightly brown precipitate.
(C ′) A step of recovering the intermediate layer as a royal jelly-degrading enzyme-containing material.
Moreover, this invention provides the royal jelly degradation product prepared using the royal jelly degradation enzyme containing material in any one of the said description.
Moreover, this invention provides the royal jelly degradation product which has the following characteristics obtained by carrying out an enzyme process for 4 hours or more at pH 9 using the royal jelly degradation enzyme containing material in any one of the said.
(1) Non-specific cytopathic effect on nerve cells is less than that of unenzyme-treated royal jelly water-soluble fraction,
(2) The inhibitory effect on neuronal cell death induction by amyloid is almost equivalent to the unenzyme-treated royal jelly water-soluble fraction,
(3) The cell proliferation action on glial cells is increased as compared with the unenzymatic treated royal jelly water-soluble fraction,
(4) The infection inhibitory action against human rotavirus infection is higher than that of the unenzyme-treated royal jelly water-soluble fraction.
The present invention also provides an antioxidant comprising a royal jelly degradation product obtained by subjecting royal jelly to pH 9 for at least 4 hours using the royal jelly degrading enzyme-containing product as described above. is there.
The present invention also provides a cell proliferating agent comprising as an active ingredient a royal jelly degradation product obtained by subjecting royal jelly to enzymatic treatment at pH 9 for 4 hours or more using any of the above-mentioned royal jelly degrading enzyme-containing materials. is there.
In addition, the present invention provides an infection inhibitor comprising, as an active ingredient, a royal jelly degradation product obtained by subjecting royal jelly to enzyme treatment at pH 9 for 4 hours or more using any of the royal jelly degrading enzyme-containing materials described above. is there.
The present invention also provides a neuronal cell death induction inhibitor comprising, as an active ingredient, a royal jelly degradation product obtained by subjecting royal jelly to pH 9 for at least 4 hours using the royal jelly degrading enzyme-containing product according to any one of the above. To do.
The present invention also provides a method for degrading royal jelly, characterized by using the royal jelly-degrading enzyme-containing material described above .

With the royal jelly-degrading enzyme-containing material and the royal jelly-degrading enzyme of the present invention, the royal jelly can be degraded without fear of impairing the physiological activity of the functional peptide or the like contained in the royal jelly.
In addition, since the royal jelly degradation product degraded by the royal jelly-degrading enzyme-containing product of the present invention contains abundant functional peptides with high physiological activity, it is very efficient in investigating and studying the biological activity of royal jelly. A sample may be provided. Furthermore, by using the royal jelly degradation product of the present invention, it is possible to provide foods and the like that are more excellent in functionality and digestibility and absorbability than conventionally eaten royal jelly-containing foods and the like.
In addition, the royal jelly can be efficiently decomposed by the royal jelly decomposition method of the present invention.

  The queen bee larva in the present invention is not particularly limited as long as it is a queen bee larva of 2 to 3 days of western bee. It may be a fresh larva or a larva after freezing. Since it is considered that the activity of royal jelly (hereinafter abbreviated as RJ) degrading enzyme or the like is considered high, it is preferable to use live fresh larvae. Larvae can be frozen and stored by conventional methods, but in order to prevent inactivation of the RJ-degrading enzyme, it is preferably stored at −80 ° C. after quenching with liquid nitrogen or the like.

  The body tissue suspension in the present invention means a suspension of larva body tissues, mainly viscera and body fluids. The internal organs and the like are not particularly limited, but are preferably those in which the whole body tissue of a larva individual is suspended.

  The method for preparing a body tissue suspension from larvae is not particularly limited as long as it is a method for preparing a tissue suspension from animals or the like. For example, the whole larva body tissue, internal organs, or body fluid may be lined using a mesh or the like, or prepared using a mortar, a mixer, a homogenizer, or the like. Since a body tissue suspension can be prepared under mild conditions, it is preferable to use a mesh or the like. The mesh preferably has a mesh size of 80 mesh to 120 mesh. When using a mixer or the like, it is preferable to add physiological saline or the like in order to prevent excessive heat generation. In order to prevent the contamination of impurities, the larvae are preferably washed in advance with physiological saline or the like before storage when the frozen tissue is prepared.

  In order to prepare the RJ-degrading enzyme-containing product of the present invention, first, as a step (a), the body tissue suspension of a queen bee larva is centrifuged at 6 ° C. or lower to obtain a white solid upper layer, yellow The white turbid solution is separated into a middle layer and white to a lower three layers with a slightly brown precipitate. Prior to centrifugation, it is preferable to appropriately adjust the viscosity and concentration of the body tissue suspension by diluting with a neutral and low concentration buffer or the like. This is because when the viscosity or concentration of the body tissue suspension is high, the extraction efficiency of the RJ-degrading enzyme may be lowered, and workability may be deteriorated. Examples of the buffer include phosphate buffer (50 mM, pH 7.0) and physiological saline. The upper layer is a lipid component, and the lower layer is presumed to be an insoluble component of body tissue.

  The conditions for the centrifugation are not particularly limited as long as the body tissue suspension can be centrifuged in three layers, but it is 8,000 to 12,000 × g, 5 ° C. or less. It is preferable that the centrifugation process be performed for at least minutes. It is particularly preferable to centrifuge at 10,000 × g and 5 ° C. for 10 minutes. When the centrifugal speed is low or when the centrifugation time is short, there is a possibility that the three layers are not separated cleanly.

  Next, as step (b), the RJ-decomposing enzyme-containing material of the present invention can be prepared by recovering the middle layer of the three layers as a royal jelly-decomposing enzyme-containing material. The recovery method can be performed by a conventional method. In order to increase the accuracy of extraction, it is preferable to centrifuge the middle layer again under the same conditions as in step (a).

  After the step (b), it is preferable to filter the middle layer in order to remove insoluble matters such as insoluble tissue fragments mixed in the middle layer. In order to prevent contamination by microorganisms or the like, it is preferable to use a 0.2 μm filter or the like for the filtration.

  In addition, since the enzyme contained in the larva body fluid extract may inactivate the preparation of the larva body fluid extract or the preparation of the royal jelly-degrading enzyme-containing material from the larva body fluid extract, the centrifugation is performed for 3-6. It is preferable to carry out at ° C. Further, other operations are preferably performed under ice cooling.

  The RJ decomposition product of the present invention can be prepared by the RJ decomposition method of the present invention, that is, by using the RJ decomposing enzyme-containing material of the present invention. The RJ may be raw RJ or may be obtained by reducing dry powder RJ. In addition, the RJ may be from any production area, and may be produced by any bee species. Further, the RJ may be a protein fractionated from RJ by a conventional method. In particular, a water-soluble protein of RJ is preferable. This is because it can be expected that many substances having physiological activity are contained.

  For the water-soluble protein fractionation of RJ, any method may be used as long as it is a method capable of separating a water-soluble protein and a hydrophobic protein. Examples of the method include a method in which a suspension obtained by mixing a water-soluble solvent and RJ is extracted and purified by partitioning with an organic solvent such as ethyl acetate or butanol, or the suspension is allowed to stand and then centrifuged. There is a method of obtaining a supernatant by carrying out the above.

  For example, the RJ degradation product can be prepared by a method in which the RJ degrading enzyme-containing product of the present invention is added to RJ or a water-soluble protein of RJ and incubated. RJ or the like may be diluted in advance using a phosphate buffer or the like. Moreover, you may use the RJ degrading enzyme containing material which adjusted protein concentration and viscosity using the phosphate buffer etc.

  The incubation temperature and time are not particularly limited as long as the RJ degradation activity of the RJ degrading enzyme-containing product of the present invention is not inhibited, but the reaction temperature condition is preferably 30 to 40 ° C., 34 to 38 ° C. is particularly preferred. Moreover, pH 6.5-10 is preferable and pH 7-9.5 is especially preferable. When performed under reaction conditions of pH 7 to 7.5, proteins appearing as bands at about 51 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are mainly decomposed, and reaction conditions of pH 8.5-9 In the case of carrying out in step (1), protein appearing as a band at about 46 kDa by SDS-PAGE is mainly decomposed.

Hereinafter, the RJ decomposition method of the present invention will be described in more detail.
First, the protein concentration of the RJ-degrading enzyme-containing material prepared by the method described in Example 1 below is measured using the Lowry method with BSA as a standard substance, and then diluted with a 50 mM phosphate buffer solution at pH 7. Thus, a 6 mg / mL RJ-degrading enzyme solution was prepared.
Next, 1 mL of the RJ protein solution (30 mg / mL) prepared by the method described in Reference Example 2 below and 1 mL of the RJ degrading enzyme solution (6 mg / mL) were added to and mixed with 8 mL of the buffer solution. An enzyme reaction solution was prepared. The enzyme reaction solution was reacted at 37 ° C., ice-cooled to stop the reaction, and then the change in RJ protein was confirmed by SDS-PAGE. The separated RJ protein was stained with CBB R-250. Buffers include 50 mM acetate buffer at pH 4, 5, and 5.5, 50 mM phosphate buffer at pH 6, 6.5, 7, and 7.5, 50 mM Tri-HCl buffer at pH 8 and 9. Were used respectively.

  FIG. 1 is a diagram showing the results of SDS-PAGE for each pH. (A) pH 4, (b) pH 5, (c) pH 6, (d) pH 6.5, (e) pH 7, (f) pH 7.5, (g) pH 8, and (h) Is pH 9. The numbers above the lanes in each figure represent the respective reaction times. M represents a molecular weight marker. When the RJ protein solution prepared by the method described in Reference Example 2 described later was subjected to SDS-PAGE, strong bands were detected at positions of about 51 kDa and about 46 kDa. Arrow a indicates a band of about 51 kDa, and arrow a indicates a band of about 46 kDa. As a result, it was found that the RJ protein was decreased by the treatment with the RJ degrading enzyme solution, and the degradation rate was increased with the increase in pH. In particular, it was confirmed that the protein of about 51 kDa was mainly degraded under the reaction conditions of pH 7 to 7.5, and the protein of about 46 kDa was mainly degraded under the reaction conditions of pH 8.5 to 9. That is, it is clear from the results in FIG. 1 that the RJ-degrading enzyme-containing material prepared by the method described in Example 1 below contains an enzyme that degrades the RJ protein. In addition, since the RJ proteolytic pattern of the RJ-degrading enzyme-containing product is different under neutral conditions and alkaline conditions, it is estimated that the RJ-degrading enzyme-containing product contains at least two types of degrading enzymes. Is done.

  RJ degradation products obtained by subjecting RJ to enzyme treatment at pH 9 for 4 hours or more using the RJ-degrading enzyme-containing product of the present invention have various physiological activities. The physiological activity has several characteristics compared to the physiological activity of the unenzyme-treated RJ water-soluble fraction. For example, the RJ degradation product has almost the same inhibitory action on the induction of neuronal cell death by amyloid as the unenzymatic treated RJ water-soluble fraction, but the non-specific cytopathic action on neuronal cells is not treated with unenzyme It is less than the royal jelly water soluble fraction. Moreover, the cell proliferation action with respect to a glial cell is increasing rather than the royal jelly water-soluble fraction of a non-enzyme process. In addition, the inhibitory effect on infection against human rotavirus infection is higher than that of the unenzyme-treated royal jelly water-soluble fraction.

  The RJ degradation product obtained by subjecting RJ to an enzyme treatment at pH 9 for 4 hours or more using the RJ degradation enzyme-containing material of the present invention also has an antioxidant activity. Therefore, the RJ degradation product can also be used as an antioxidant. For example, the RJ degradation product can suppress proteolysis by radicals such as chlorine monoxide (ClO.) Due to its antioxidant activity. Since RJ is a food that has been ingested for a long time, the RJ degradation products are also very safe, and as with ascorbic acid, etc., antioxidants added to foods and drinks and the body are protected from oxidative stress It can also be expected to be useful as an active ingredient of supplements eaten for the purpose of eating.

  Since the RJ degradation product obtained by subjecting RJ to enzyme treatment at pH 9 for 4 hours or more using the RJ-degrading enzyme-containing product of the present invention has the physiological activity as described above, an antioxidant, a cell It can be used as an active ingredient such as a proliferation agent, infection inhibitor, nerve cell death induction inhibitor and the like. These physiologically active agents may be consumed alone as foods and drinks such as supplements, and can be used as additives to foods and beverages and pharmaceuticals as well as other food and beverage compositions and pharmaceutical compositions. .

  The method for producing these bioactive agents using the RJ degradation product as an active ingredient is not particularly limited as long as it does not inhibit the physiological activity of the RJ degradation product, and usually contains RJ or RJ degradation product. It can be manufactured using the method used when manufacturing the composition for eating and drinking or the pharmaceutical composition. The dosage form of these bioactive agents is not particularly limited, but is preferably a dosage form suitable for oral preparations. For example, it may be a soft capsule, a hard capsule, a tablet, or a syrup. In addition, the amount of the RJ degradation product contained in these bioactive agents is not particularly limited as long as the physiological activity of the RJ degradation product can exert an action effect. The RJ degradation product can be appropriately determined in consideration of the physiological activity, dosage form and the like.

  The intake amount of these bioactive agents containing the RJ degradation product as an active ingredient is not particularly limited as long as the physiological activity of the RJ degradation product can exert an action effect. It can be determined as appropriate according to the body weight, age, sex, and the like. For example, it is preferable to take 300 mg to 3 g per day at a time or divided into several times.

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

[Reference Example 1] Preparation of Larva Suspension Approximately 15 g of a 3-day-old western honey bee larva was collected and washed with ice-cold physiological saline. Thereafter, the queen bee larvae were lined up using an average 100 mesh cloth made of polyester ("Tetron", manufactured by Toray Industries, Inc.) to break the epidermis of the larvae and squeeze out body fluids and internal organs. Thereby, a milky white larvae suspension was prepared.

[Reference Example 2] Preparation of RJ protein solution To 10 mL of RJ (made in China), 20 mL of 50 mM phosphate buffer having a pH of 7.0 was added and mixed to prepare a diluted RJ solution. The RJ diluted solution was centrifuged at 25,000 × g and 5 ° C. for 20 minutes, then insoluble components were removed, and the supernatant was collected. Thereafter, the supernatant was diluted with a 50 mM phosphate buffer having a pH of 7.0 to prepare a 30 mg / mL RJ protein solution.

[Example 1]
1. Preparation of RJ-degrading enzyme-containing product A larvae suspension prepared by the method described in Reference Example 1 was diluted 2-fold with a pH 7 50 mM phosphate buffer, and then at 10,000 × g, 5 ° C. for 10 minutes. Centrifuged. The centrifugal separation separated the white solid into an upper layer, a middle layer of a yellowish-white turbid solution, and a white to slightly lower brown precipitate. The upper layer is presumed to be lipid, and the lower layer is presumed to be insoluble body tissue. After recovering the middle layer, it was further centrifuged at 10,000 × g and 5 ° C. for 10 minutes to separate into three layers, and the middle layer was recovered as an RJ-degrading enzyme-containing material. The RJ-degrading enzyme-containing material was filtered using a 0.2 μm cellulose acetate filter (DISMIC-25CS, manufactured by ADVANTEC) in order to remove insoluble tissue fragments that could not be removed and sterilize. Thereby, a yellow transparent RJ-degrading enzyme-containing material was obtained.

2. Degradation of RJ protein The protein concentration of the RJ-degrading enzyme-containing product was measured using the Lowry method with BSA as a standard substance, and a 3 mg / mL RJ-degrading enzyme solution was prepared using a pH 7 50 mM phosphate buffer. .
In 8 mL of buffer solution, 1 mL of RJ protein solution (30 mg / mL) prepared by the method described in Reference Example 2 and 1 mL of RJ degrading enzyme solution (6 mg / mL) prepared by the method described in Example 1 above. ) Was added and mixed to prepare an enzyme reaction solution. As the buffer, a 50 mM phosphate buffer having a pH of 7 or a 50 mM Tri-HCl buffer having a pH of 9 was used. The enzyme reaction solution was reacted at 37 ° C., ice-cooled to stop the reaction, and then RJ protein was separated by SDS-PAGE. After the gel stained with CBB R-250 was dried, a stained image was obtained using a fluorescence scanner Typhoon 9400 (manufactured by Amersham Biosciences).

  FIG. 2 is a diagram showing the results of SDS-PAGE for each pH. (A) is a dyed image at pH 7, and (b) is a dyed image at pH 9. The numbers above the lanes in each figure represent the respective reaction times, and M represents the molecular weight marker. The arrow A is about 51 kDa, the arrow A is about 46 kDa, the arrow C is about 33 kDa, and the arrow D is about 25 kDa. As a result, it was confirmed that the proteins of about 51 kDa and about 46 kDa were degraded at both pH 7 and 9. In particular, it was found that a protein of about 46 kDa was decomposed so as not to be detected in a short time of 4 hours after the start of the reaction at pH 9. Further, at pH 7, about 33 kDa and about 25 kDa proteins came to be detected as the reaction time passed.

  Using image analysis software ImageQuant (manufactured by GE Healthcare Bioscience), the band intensity of each band of the stained image in FIG. 2 was quantified. FIG. 3 shows the change in the band intensity over the course of the reaction time, with the band intensity at 0 hours from the start of the reaction being 1. (A) shows the intensity ratio of the band of about 51 kDa, (b) shows the intensity ratio of the band of about 46 kDa, and (c) shows the intensity ratio of the bands of about 33 kDa and about 25 kDa. In the figures of (a) and (b), ● represents a pH 7 band, and ○ represents a pH 9 band. Further, in the figure of (c), Δ represents a band of about 33 kDa at pH 7, and ▲ represents a band of about 25 kDa at pH 7.

  As shown in (a) and (b) of FIG. 3, it was confirmed quantitatively that the proteins of about 51 kDa and about 46 kDa were degraded at both pH 7 and 9. . Further, from the result of FIG. 3 (c), it was confirmed that the protein of about 33 kDa and about 25 kDa increased at pH 7 until 16 hours after the start of the reaction. From this result, it is suggested that the protein of about 33 kDa and about 25 kDa may be a degradation product formed by decomposing the protein of about 51 kDa or about 46 kDa by the RJ degrading enzyme contained in the RJ degrading enzyme solution. Is done.

[Example 2]
Except for the reaction time of up to 60 minutes, the RJ protein was decomposed in the same manner as in the reaction under the condition of pH 9 in Example 1, and the RJ protein was separated by SDS-PAGE. Quantified. 4A shows a stained image of the gel, and FIG. 4B shows an intensity ratio of a band of about 46 kDa. The numbers above the lanes in the figure represent the respective reaction times, and M represents the molecular weight marker. Further, the arrow A indicates about 51 kDa, and the arrow A indicates about 46 kDa.

  As a result, it was confirmed that approximately 46% of the protein of about 46 kDa was degraded by RJ degrading enzyme contained in the RJ degrading enzyme solution in a short time of 1 hour after the start of the reaction. According to FIG. (B), almost no decomposition occurred at the time of 5 minutes after the start of the reaction. This is because the RJ protein solution was kept at 37 ° C. before the enzyme reaction with the RJ degrading enzyme was started. It is presumed that there was a time lag before the enzyme reaction started because the pre-incubation was not performed.

[Example 3]
The RJ protein was degraded in the same manner as in the reaction under the condition of pH 7 in Example 1 except that the reaction time was up to 24 hours. Thereafter, the enzyme reaction solution whose reaction was stopped by cooling with ice was subjected to gel filtration chromatography using Superdex 200 HR 10/30 (Pharmacia) to fractionate proteins contained in the enzyme reaction solution. did. Gel filtration chromatography was performed using a 50 mM phosphate buffer (pH 7) containing 0.15 M sodium chloride as an eluent at a flow rate of 0.5 mL / min and 5 ° C. In order to detect the fractionated protein, the absorbance at 280 nm was measured using a UV detector.

FIG. 5 is a diagram showing the results of absorbance at 280 nm measured in Example 3. (A) is the result before the start of the reaction, (b) is the result of 8 hours after the start of the reaction, (c) is the result of 16 hours after the start of the reaction, and (d) is the result of 24 hours after the start of the reaction. In addition, the arrow A in the figure indicates the protein fraction of about 350 kDa, the arrow A is about 60 kDa, the arrow C is about 5 kDa, the arrow D is about 3 kDa, and the range E is several hundred Da.
As the reaction time elapses, it is clear from the results of FIG. 5 that the proteins of about 350 kDa and about 60 kDa decrease while the proteins of about 5 kDa, about 3 kDa, and several hundreds of Da increase. It is inferred that the increased protein such as about 5 kDa is a degradation product of a protein such as about 350 kDa. That is, it was confirmed from the result of fractionation by gel filtration chromatography that the RJ macromolecular protein was degraded by the RJ-degrading enzyme-containing product of the present invention.

[Example 4]
In the same manner as in Reference Example 2, an RJ protein solution (30 mg / mL) was prepared. Further, an RJ degrading enzyme solution (3 mg / mL) was prepared in the same manner as in Example 1 except that the concentration after dilution was 3 mg / mL.
An enzyme reaction mixture was prepared by adding 1 mL of the RJ protein solution (30 mg / mL) and 1 mL of the RJ degrading enzyme solution (3 mg / mL) to 8 mL of buffer solution. As the buffer, a 50 mM phosphate buffer having a pH of 7 or a 50 mM Tri-HCl buffer having a pH of 9 was used. The enzyme reaction solution was reacted at 4, 20, 37, and 50 ° C., respectively, and ice-cooled to stop the reaction. The time when the enzyme reaction solution was mixed in a thermostatic bath at each temperature was set as the reaction start time, and the reaction time was set to 4, 16, and 24 hours. The enzyme reaction solution after ice cooling was separated from the RJ protein in the same manner as in Example 1 to observe the influence of the reaction temperature.

As a result, at 37 ° C., as in Example 2, degradation of proteins of about 51 kDa and about 46 kDa was observed at both pH 7 and 9. In particular, at pH 9, the protein of about 46 kDa was degraded to below the detection limit at 4 hours from the start of the reaction, and the protein at about 51 kDa was also degraded to below the detection limit at the time of 16 hours from the start of the reaction.
In contrast, at 4 ° C., almost no degradation of proteins of about 51 kDa and about 46 kDa was observed at both pH 7 and 9. At 20 ° C., the degradation reaction proceeded more slowly than at 37 ° C., but protein degradation of about 51 kDa and about 46 kDa was observed. On the other hand, at 50 ° C., the degradation reaction was faster than at 37 ° C., and it was confirmed that most of the protein was degraded at pH 7 and 9 at 4 hours after the start of the reaction.

  That is, in the temperature range of 20 to 50 ° C., the RJ-decomposing enzyme-containing material of the present invention can decompose RJ, and in this temperature range, the higher the temperature, the higher the RJ-degrading enzyme of the present invention. It is clear that the progress of the decomposition reaction of RJ by the inclusion is rapid. However, generally, at 50 ° C., browning of RJ, denaturation of RJ protein, and the like may occur. For this reason, when decomposing | disassembling RJ with the RJ decomposing enzyme containing material of this invention, it is preferable to carry out at the temperature of 37 degreeC vicinity.

[Example 5]
The physiological activity on brain neurons possessed by the RJ degradation product obtained using the RJ-degrading enzyme-containing product of the present invention was examined. Specifically, the influence of RJ degradation products on neuronal cell death induction by amyloid in primary cultured mouse fetal hippocampal neurons was examined. Mouse fetal hippocampal neurons were spread from a mouse fetus by a conventional method in a 96-well multiwell plate in which glial cells were cultured to 4.0 × 10 ⁴ cells / well, 37 Culturing was performed at 5 ° C. in a 5% CO ₂ atmosphere. As a cell culture solution, B27 supplement-containing neuroversal medium (manufactured by Gibco) was used. In the experiment, cells after one week had been cultured.

First, 15 mL of 50 mM phosphate buffer having a pH of 7.0 was added to 5 mL of RJ (made in China) and mixed to prepare a diluted RJ solution. The RJ diluted solution was centrifuged at 25,000 × g and 5 ° C. for 20 minutes, then insoluble components were removed, and the supernatant was collected. Thereafter, the supernatant was freeze-dried to obtain an RJ water-soluble crude fraction. The RJ water-soluble crude fraction was diluted with 50 mM phosphate buffer at pH 7.0 to prepare a 30 mg / mL RJ water-soluble crude fraction solution.
Next, in place of the RJ protein solution prepared by the method described in Reference Example 2, the RJ water-soluble crude fraction solution was used, and all the examples except that the reaction time was 4 hours. In the same manner as in No. 1, the RJ protein contained in the RJ water-soluble crude fraction solution was decomposed to obtain an RJ decomposition product.

An amyloid peptide (manufactured by Peptide Institute) and an RJ water-soluble crude fraction solution or an RJ degradation product were added to a 96-well multiwell plate in which mouse fetal hippocampal neurons were cultured, and then cultured for 48 hours. The amyloid peptide was incubated at 37 ° C. for 48 hours at a concentration of 2 mg / mL, and then added so that the final concentration was 20 μg / mL. In addition, the RJ water-soluble crude fraction solution, the RJ degradation product obtained by degradation at pH 7 or the RJ degradation product obtained by degradation at pH 9 have a final concentration of each protein in the cell culture solution of 0, 31 respectively. .25, 62.5, 125, 250, 500, or 1,000 μg / mL. Neither amyloid peptide nor RJ degradation product was added, but the same amount of cell culture medium was added as a control.
Thereafter, dead cells were identified by immunostaining with anti-Map2 antibody or Hoechst 33342 dye staining, and the number of viable mouse fetal hippocampal neurons and the number of glial cells in each dish were measured. Furthermore, the number of mouse fetal hippocampal neurons in which cell deformation was observed was measured by microscopic observation. In addition, cell deformation means that a nerve cell changes from the form peculiar to the nerve cell which consists of a cell body which has a dendrite, and an axon. Such changes include, for example, protrusion formation, axonal atrophy, multiaxonization, neuronal aggregation, and neuronal death.

  Table 1 shows measurement results such as the number of mouse embryonic hippocampal neurons. The number of surviving neurons and the number of degenerated neurons are expressed with the number of control neurons as 100%, and the number of glial cells with the number of control glial cells as 100%. When amyloid peptide was added to mouse embryonic hippocampal neurons cultured primarily, about 60% of the neurons were killed and the survival rate was about 40%. When the RJ water-soluble crude fraction solution was added together with the amyloid peptide, the RJ water-soluble crude fraction solution inhibited nerve cell death induction by amyloid in a dose-dependent manner. Maximum survival recovered to 60-70%. The effect of the RJ water-soluble crude fraction solution was observed even at a low concentration of 62.5 to 250 μg / mL. However, when an RJ water-soluble crude fraction solution of 1,000 μg / mL or more was added, non-specific nerve cell deformation action was observed.

  When an RJ degradation product obtained by decomposition at pH 7 was added, almost the same effect was observed as when an RJ water-soluble crude fraction solution was added. On the other hand, when an RJ degradation product obtained by degradation at pH 9 was added, an inhibitory effect on the induction of neuronal cell death by amyloid was observed as in the RJ water-soluble crude fraction solution. Furthermore, as compared with the RJ water-soluble crude fraction solution and the like, the non-specific nerve cell deformation action was reduced and the cell proliferation action on glial cells was increased. It is presumed that this is because the proliferation of glial cells and the like is promoted, and thus the neuroprotective effect is exerted more strongly, so that the nonspecific nerve cell deformation action of the RJ water-soluble crude fraction solution is reduced. Is done.

  From the results of Table 1, the RJ degradation product obtained by carrying out the enzyme treatment at pH 9 for 4 hours or more using the RJ degradation enzyme-containing product of the present invention has an inhibitory effect on the induction of neuronal cell death by amyloid. It is almost the same as the treated royal jelly water-soluble fraction, and its non-specific cytopathic effect on neurons is significantly reduced compared to the unenzyme-treated royal jelly water-soluble fraction, and cell proliferation on glial cells. However, it is apparent that it has the characteristic that it is increased over the unenzyme-treated royal jelly water-soluble fraction.

[Example 6]
In order to examine the infection inhibitory activity against human rotavirus infection possessed by the RJ degradation product obtained using the RJ-degrading enzyme-containing product of the present invention, a neutralization activity test was conducted. Specifically, the effect of RJ degradation products on human rotavirus infection was observed by adding RJ or RJ degradation products together with human rotavirus to the cell culture of MA104 cells derived from fetal kidneys of rhesus monkeys. As RJ, an RJ water-soluble crude fraction solution prepared in the same manner as in Example 4 was used. In addition, as an RJ decomposition product, an RJ decomposition product obtained by decomposing at pH 7, which was prepared in the same manner as in Example 4 except that the reaction time was 24 hours, or an RJ decomposition product obtained by decomposing at pH 9, Each was used. Note that MA104 cells were cultured at 37 ° C. in a 5% CO ₂ atmosphere. As a cell culture solution, a 10% FCS (fetal bovine serum) and 10% TPB (tryptophosphate phosphate broth) -containing Eagle's MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) with some antibiotics added thereto was used. .

First, a human rotavirus solution (manufactured by Miyagi Cancer Center) was added to a cell suspension prepared using MA104 cells that had been cultured for 5 days in a 24 cm ² TC flask at 5.0 × 10 ⁵ cells / flask. Then, RJ water-soluble crude fraction solution or RJ degradation product was added. The human rotavirus solution was added so that the final concentration was 6.5 × 10 ⁴ FCFU / mL. In addition, in the RJ water-soluble crude fraction solution, the RJ degradation product obtained by degradation at pH 7, or the RJ degradation product obtained by degradation at pH 9, the final concentration of each protein in the cell culture solution is the concentration described in Table 2. It added so that it might become. Instead of the RJ degradation product, etc., an addition of an equal amount of cell culture medium was used as a control. Then, after culturing for 22 hours, P3-1 that recognizes Hatrotavirus strain PO-13 VP6 as a primary antibody, FITC-labeled goat anti-mouse IgG as a secondary antibody, and human rota by indirect fluorescent antibody method, respectively. The number of MA104 cells infected with the virus was measured.

  Table 2 shows the results of the human rotavirus neutralization activity test. The number of infected cells is expressed with the number of infected cells of the control as 100%. When the RJ water-soluble crude fraction solution was added, virus infection was promoted at a concentration of about 300 μg / mL or more, and a tendency that virus infection was suppressed at a concentration of about 300 μg / mL or less was observed. In other words, it was suggested that there is a duality of action: infection is promoted at high concentrations and infection is inhibited at low concentrations.

  On the other hand, it was observed that the RJ degradation product obtained by degradation at pH 7 promoted virus infection at a concentration of 37 μg / mL or higher, and suppressed viral infection at a concentration of 18.5 μg / mL or lower. In other words, the RJ degradation product obtained by degradation at pH 7 promotes infection at a high concentration and suppresses infection at a low concentration, but converts from infection suppression to promotion, like the RJ water-soluble crude fraction solution. The concentration was shifted to the low concentration side. On the other hand, when an RJ degradation product obtained by degradation at pH 9 having a concentration of 40 μg / mL or less was added, the infection inhibition effect was greater than when the RJ water-soluble crude fraction solution was added. In particular, an RJ degradation product obtained by degradation at pH 9 exhibited an infection inhibitory effect depending on the protein concentration at a concentration of 16.5 μg / mL or less. Moreover, when the reaction time was 4 hours or more, the concentration dependence of the infection inhibitory effect in a low concentration was more remarkable as the RJ degradation product having a longer reaction time.

  From the results of Table 2, the RJ degradation product obtained by subjecting the RJ degrading enzyme-containing product of the present invention to an enzyme treatment at pH 9 for 4 hours or more exhibits an infection inhibitory action against human rotavirus infection. It is clear that it has the characteristic that it is higher than the water-soluble fraction of royal jelly.

[Example 7]
In order to examine the cell proliferation activity of the RJ degradation product obtained using the RJ-degrading enzyme-containing product of the present invention, the WST-1 method was performed using rat IEC-6 cells derived from the small intestine. Specifically, IEC-6 cells are seeded at 2.0 × 10 ⁵ cells / mL and cultured for 24 hours, and then the culture solution is replaced with an RJ degradation product-containing culture solution and further cultured for 24 hours. . Then, after adding tetrazolium salt WST-1 (manufactured by Wako Pure Chemical Industries, Ltd.) to the culture medium and culturing for 2 hours, the amount of formazan product produced is measured by measuring the absorbance of the cell culture liquid at 450 nm. did. As the RJ decomposition products, RJ decomposition products obtained by decomposing at pH 7 prepared in the same manner as in Example 4 except that the reaction time was 24 hours, or RJ decomposition products obtained by decomposing at pH 9 were used. It was. IEC-6 cells were cultured by a conventional method.
As a result, in the RJ degradation product obtained by degradation at pH 7, when the concentration of the RJ degradation product is 1 mg / mL or less, the cell proliferation promotion tendency is 1 mg / mL or more. Each trend was observed.

  From the results of Examples 6 and 7, one of the reasons that the two aspects of the promotion and suppression of viral infection in Example 6 depending on the concentration of RJ protein was observed. It is assumed that there is. That is, it was suggested that the RJ protein and its peptide fragment may have the effect of controlling human rotavirus infection, partly because of control of cell proliferation.

[Example 8]
In order to analyze the cell growth activity of the RJ degradation product obtained using the RJ degradation enzyme-containing product of the present invention in more detail, the cell proliferation activity after desalting the RJ degradation product was examined.
1. Desalting treatment First, an RJ degradation product (hereinafter referred to as RJ) obtained by enzymatic treatment at pH 7 using the RJ degrading enzyme-containing material of the present invention in the same manner as in Example 4 except that the reaction time was 24 hours. A degradation product (sometimes referred to as pH 7)) and an RJ degradation product obtained by enzymatic treatment at pH 9 (hereinafter, sometimes referred to as RJ degradation product (pH 9)) were prepared.
The obtained RJ degradation product (pH 7) and RJ degradation product (pH 9) were each subjected to size fractionation using a Hi Trap Desalting column (manufactured by GE Healthcare Bioscience). According to the elution pattern obtained by measuring the absorbance (280 nm) and the conductivity, two fractions were fractionated: a desalted fraction (hereinafter, fraction D1) and a salt-containing fraction (hereinafter, fraction D2).
FIG. 6 is a chart obtained as a result of measuring absorbance (280 nm) and conductivity in column chromatography using a Hi Trap Desalting column. The solid line indicates the absorbance of the RJ decomposition product (pH 9), the dotted line indicates the absorbance of the RJ decomposition product (pH 7), the two-dot chain line indicates the conductivity of the RJ decomposition product (pH 9), and the one-dot chain line indicates the conductivity of the RJ decomposition product (pH 7). Each is shown. In the figure, “D1” is a fraction sorted as fraction D1, and “D2” is a fraction sorted as fraction D2.

2. Measurement of cell growth activity As a cell culture solution, a culture solution containing a fraction D1 (fraction D1 (pH 9)) of an RJ degradation product (pH 9) in a culture solution (control culture solution) not containing a growth factor such as FCS or In the case of culturing using each culture solution in the same manner as in Example 7 except that a culture solution containing the fraction D1 (fraction D1 (pH 7)) of the RJ degradation product (pH 7) was used in the control culture solution Cell proliferation was measured. A 10% FCS-containing culture solution was used as a positive control, and a control culture solution was used as a negative control. The amount of cell proliferation when cultured using each culture solution was expressed as the ratio of the amount of formazan product produced when each culture solution was added to the amount of formazan product produced when the control culture solution was added. .
FIG. 7 is a diagram showing the amount of cell proliferation when cultured using each culture solution. FIG. 7 (a) shows the results of culturing using a culture solution containing fraction D1 (pH 9), and FIG. 7 (b) shows using the culture solution containing fraction D1 (pH 7). It is a result at the time of culture | cultivation. In the figure, “NC” represents a control culture solution, and “PC” represents a 10% FCS-containing culture solution. Moreover, each density | concentration has shown the protein density | concentration derived from the fraction D1 in each culture solution.
As shown in the results of FIG. 7 (a), it was found that the amount of cell proliferation increased depending on the concentration of fraction D1 (pH 9) in the culture solution. In particular, when culture was performed using a culture solution having a fraction D1 (pH 9) concentration of 50.5 μg / mL, cell growth was promoted to the same extent as when a culture solution containing 10% FCS was used. However, in the case of a culture solution having a fraction D1 (pH 9) concentration of 84.2 μg / mL or more, a tendency for the cell growth amount to decrease depending on the concentration was observed. It was suggested that the effect (cell proliferation activity) has an optimum concentration.
On the other hand, as shown in the results of FIG. 7 (b), when the culture solution contains fraction D1 (pH 7), the amount of cell proliferation hardly changes, and fraction D1 (pH 7) contributes to cell proliferation. It became clear that there was no significant impact.
That is, also from the result of Example 8, it is clear that the RJ degradation product obtained by enzymatic treatment at pH 9 using the RJ degradation enzyme-containing product of the present invention has cell proliferation activity.

[Example 9]
In order to analyze the cell growth activity of the RJ degradation product obtained using the RJ degradation enzyme-containing product of the present invention in more detail, the cell proliferation activity after roughly purifying the RJ degradation product was examined.
1. Crude Purification The fraction D2 (fraction D2 (pH 9)) of the RJ degradation product (pH 9) and the fraction D2 (fraction D2 (pH 7)) of the RJ degradation product (pH 7) prepared in Example 8 were converted into Cosmosil® 5C ₁₈ -Rough purification was performed using an AR column (Nacalai Tesque). A 0.05% TFA (trifluoroacetic acid) solution was used as an adsorption buffer, and a 90% acetonitrile solution containing 0.05% TFA was used as an elution buffer. According to the elution pattern obtained by measuring the absorbance (215 nm and 280 nm) and the conductivity, two fractions, a non-adsorbed fraction (hereinafter referred to as fraction C1) and an adsorbed fraction (hereinafter referred to as fraction C2), were fractionated.
FIG. 8 is a chart obtained as a result of measuring absorbance (215 nm) and conductivity in column chromatography using a Cosmosil (registered trademark) 5C ₁₈ -AR column. The solid line indicates the absorbance of the fraction D2 (pH 9), the dotted line indicates the absorbance of the fraction D2 (pH 7), the two-dot chain line indicates the conductivity of the fraction D2 (pH 9), and the one-dot chain line indicates the conductivity of the fraction D2 (pH 7). . In the figure, “C1 (pH 7)” is the fraction C1 (pH 7), “C1 (pH 9)” is the fraction C1 (pH 9), “C2 (pH 7)” is the fraction C2 (pH 7), “C2 (pH 9)” Are fractions fractionated as fraction C2 (pH 9).

2. Measurement of cell proliferation activity of fraction C1 As a cell culture solution, a culture solution obtained by diluting fraction C1 of fraction D2 (pH 9) (fraction C1 (pH 9)) or fraction C1 of fraction D2 (pH 7) (fraction C1 (pH 7)) The amount of cell proliferation in the case of culturing using each culture solution was measured in the same manner as in Example 7 except that the culture solution diluted was used. A control culture solution was used for dilution of fraction C1 (pH 9) or fraction C1 (pH 7).
FIG. 9 is a diagram showing the amount of cell proliferation when cultured using each culture solution. FIG. 9 (a) shows the results of culturing using a culture solution containing fraction C1 (pH 9), and FIG. 9 (b) shows using the culture solution containing fraction C1 (pH 7). It is a result at the time of culture | cultivation. In the figure, “NC” represents a control culture solution, and “PC” represents a 10% FCS-containing culture solution.
As shown in the results of FIGS. 9 (a) and 9 (b), cell growth was promoted in the culture solution containing either fraction C1 (pH 9) or fraction C1 (pH 7). When both were compared, the tendency for the cell growth promotion effect to be higher was observed when the fraction C1 (pH 9) was included than when the fraction C1 (pH 7) was included.

3. Measurement of Cell Proliferation Activity of Fraction C2 As a cell culture solution, a culture solution containing fraction C2 (fraction C2 (pH9)) of fraction D2 (pH9) in a control culture solution or a control culture solution containing fraction D2 (pH7) The amount of cell proliferation when cultured using each culture solution was measured in the same manner as in Example 7 except that the culture solution containing fraction C2 (fraction C2 (pH 7)) was used.
FIG. 10 is a diagram showing the amount of cell proliferation when cultured using each culture solution. FIG. 10 (a) shows the results of culturing using a culture solution containing fraction C2 (pH 9), and FIG. 10 (b) shows using the culture solution containing fraction C2 (pH 7). It is a result at the time of culture | cultivation. In the figure, “NC” indicates a control culture solution. Moreover, each density | concentration has shown the protein density | concentration derived from the fraction C2 in each culture solution.
As a result, when fraction C2 (pH 9) is contained in the culture solution, the amount of cell proliferation increases in a concentration-dependent manner when the concentration in the culture solution is 3.42 μg / mL or less, and exceeds 3.42 μg / mL. Then, the tendency that the amount of cell proliferation decreased depending on the concentration was observed. On the other hand, when the fraction C2 (pH 7) is contained in the culture solution, the amount of cell proliferation increases in a concentration-dependent manner when the concentration in the culture solution is 4.67 μg / mL or less, and when it exceeds 4.67 μg / mL. In addition, a tendency for the amount of cell proliferation to decrease in a concentration-dependent manner was observed. Moreover, when both were compared, the tendency for the cell growth promotion effect to be higher was observed when the fraction C2 (pH 9) was included than when the fraction C2 (pH 7) was included.

  Therefore, from the results of Example 9, the RJ degradation product obtained by enzymatic treatment using the RJ-degrading enzyme-containing product of the present invention has cell proliferation activity and was obtained by enzymatic treatment under the reaction conditions of pH 7. It is clear that the RJ degradation product obtained by the enzyme treatment under the reaction condition of pH 9 has higher cell proliferation activity than the RJ degradation product.

[Example 10]
The antioxidant activity of the RJ degradation product obtained using the RJ-degrading enzyme-containing product of the present invention was examined.
1. Preparation of RJ degradation product A suspension obtained by adding pure water to RJ (made in China) was dialyzed against pure water for 72 hours using a dialysis membrane having a pore diameter of 10 kD. The dialysis membrane solution was collected, lyophilized, and then suspended again in pure water to prepare a 30 mg / mL RJ protein solution.
To 1 mL of the RJ protein solution, 1 mL of RJ-degrading enzyme content (3 mg / mL) and 8 mL of Tris-HCl buffer (pH 9.0) were added and incubated at 37 ° C. for 24 hours. Then, it dialyzed for 5 days with the pure water using the dialysis membrane with a hole diameter of 500 Da. The dialysis membrane liquid was collected and lyophilized to obtain an RJ degradation product. The RJ-degrading enzyme-containing material prepared in the same manner as in Example 1 was used.

2. Antioxidant activity measurement The antioxidant activity of the RJ degradation product was determined according to the method of Yanaiuchi et al. (Japan Food Science and Technology Journal, Vol. 51, No. 5, pp. 238-246, 2004). It was measured by investigating the influence of the RJ degradation product on the BSA degradation due to the above.
Specifically, first, by adding an RJ degradation product diluted with PBS to BSA-containing PBS (phosphate buffered saline), BSA (final concentration 40 μg / mL) and RJ degradation product (final concentration) PBS sample solutions containing 0, 0.3, 0.5, 1.0, or 2.0 μg / mL) were prepared. Sodium hypochlorite was added to the PBS sample solution to a final concentration of 1.7 mM and incubated at 37 ° C. for 30 minutes. Thereafter, the protein in the PBS sample solution was separated by SDS-PAGE and detected by CBB staining. After capturing a CBB-stained image with a scanner, the density of the band corresponding to BSA was analyzed. Regarding the BSA concentration of each PBS sample solution, the PBS sample solution to which sodium hypochlorite was not added was used as a blank, and the relative concentration when the BSA concentration of the blank was set to 1 was examined. Band concentration analysis was performed using analysis software ImageJ provided by the National Institutes of Health. Further, in place of the RJ degradation product, ascorbic acid (final concentration 0, 3.0, 4.0, 5.0, or 7.5 mM) or carnosine (final concentration 0, 2.5), which is a known antioxidant, is used. , 5.0, 7.5, or 10.0 mM), the BSA concentration was similarly examined.
FIG. 11 is a diagram showing the results of the BSA concentration of each PBS sample solution. FIG. 11 (a) shows the results when the RJ degradation product is added, FIG. 11 (b) shows the results when the ascorbic acid is added, and FIG. 11 (c) shows the results when the carnosine is added. . As a result, when no RJ degradation product was added, no band was detected by CBB staining for BSA, and almost all BSA was degraded. On the other hand, when the RJ degradation product was added, the relative concentration of BSA increased depending on the amount added to the PBS sample solution, similar to ascorbic acid and carnosine. This is presumably because the decomposition of BSA by chlorine monoxide produced from sodium hypochlorite was suppressed by the RJ decomposition product.
Further, based on the results shown in FIG. 11, the concentration at which BSA decomposition by chlorine monoxide is suppressed to 50% was calculated. The RJ decomposition product was 0.92 ± 0.24 mg / mL, and ascorbic acid was 0.67. It was ± 0.01 mg / mL (3.83 ± 0.08 mM), and in carnosine, it was 0.84 ± 0.10 mg / mL (3.65 ± 0.32 mM). That is, in the antioxidant power against BSA degradation by chlorine monoxide, the RJ degradation product is a mixture, but has an antioxidant power comparable to known antioxidants ascorbic acid and carnosine. Admitted.
From the results of Example 10, it is clear that the RJ degradation product obtained by enzyme treatment at pH 9 using the RJ degradation enzyme-containing product of the present invention has antioxidant activity.

  The RJ-degrading enzyme-containing product of the present invention contains an RJ-degrading enzyme having RJ as an original substrate, which is possessed by a queen bee larva, and is optimal for degrading RJ. It can also be used in fields such as the development of pharmaceuticals and foods using RJ.

Obtained as a result of SDS-PAGE to separate the RJ protein in the enzyme reaction solution after reacting the enzyme reaction solution mixed with the RJ protein solution and the RJ degrading enzyme solution at 37 ° C. It is the figure which showed the dyeing | staining image for every pH. (A) pH 4, (b) pH 5, (c) pH 6, (d) pH 6.5, (e) pH 7, (f) pH 7.5, (g) pH 8, and (h) Is pH 9. The numbers above the lanes in each figure represent the respective reaction times. M represents a molecular weight marker. Arrow A indicates a band of about 51 kDa, and arrow A indicates a band of about 46 kDa. In Example 1, it is the figure which showed the result of SDS-PAGE for every pH. (A) is a dyed image at pH 7, and (b) is a dyed image at pH 9. The numbers above the lanes in each figure represent the respective reaction times, and M represents the molecular weight marker. The arrow A is about 51 kDa, the arrow A is about 46 kDa, the arrow C is about 33 kDa, and the arrow D is about 25 kDa. In Example 1, the intensity of the band in the stained image of FIG. 2 was measured, and the band intensity at 0 hours from the start of the reaction was taken as 1, and the change in the band intensity over the course of the reaction time was shown. (A) shows the intensity ratio of the band of about 51 kDa, (b) shows the intensity ratio of the band of about 46 kDa, and (c) shows the intensity ratio of the bands of about 33 kDa and about 25 kDa. In the figures of (a) and (b), ● represents a pH 7 band, and ○ represents a pH 9 band. Further, in the figure of (c), Δ represents a band of about 33 kDa at pH 7, and ▲ represents a band of about 25 kDa at pH 7. In Example 2, it is the figure showing the result of having decomposed | disassembled RJ protein on the conditions of pH9. (A) shows the stained image of the gel obtained as a result of SDS-PAGE, and (b) shows the intensity ratio of the band of about 46 kDa. The numbers above the lanes in the figure (a) represent the respective reaction times, and M represents the molecular weight marker. Further, the arrow A indicates about 51 kDa, and the arrow A indicates about 46 kDa. In Example 3, it is the figure showing the result of the light absorbency of 280 nm measured. (A) is the result before the start of the reaction, (b) is the result of 8 hours after the start of the reaction, (c) is the result of 16 hours after the start of the reaction, and (d) is the result of 24 hours after the start of the reaction. In addition, the arrow A in the figure indicates the protein fraction of about 350 kDa, the arrow A is about 60 kDa, the arrow C is about 5 kDa, the arrow D is about 3 kDa, and the range E is several hundred Da. It is the chart obtained as a result of measuring a light absorbency (280 nm) and conductivity in the column chromatography using the Hi Trap Desalting column of Example 8. It is a chart figure obtained as a result of measuring a light absorbency (280 nm) and conductivity. The solid line indicates the absorbance of the RJ decomposition product (pH 9), the dotted line indicates the absorbance of the RJ decomposition product (pH 7), the two-dot chain line indicates the conductivity of the RJ decomposition product (pH 9), and the one-dot chain line indicates the conductivity of the RJ decomposition product (pH 7). Each is shown. In the figure, “D1” is a fraction sorted as fraction D1, and “D2” is a fraction sorted as fraction D2. In Example 8, it is the figure which showed the cell proliferation amount at the time of culture | cultivating using each culture solution. (A) is a result at the time of adding the culture solution containing the fraction D1 (pH9), (b) is a result at the time of adding the culture solution containing the fraction D1 (pH7). In the figure, “NC” represents a control culture solution, and “PC” represents a 10% FCS-containing culture solution. Moreover, each density | concentration has shown the protein density | concentration derived from the fraction D1 in each culture solution. 6 is a chart obtained by measuring absorbance (215 nm) and conductivity in column chromatography using the Cosmosil (registered trademark) 5C ₁₈ -AR column of Example 9. FIG. The solid line indicates the absorbance of the fraction D2 (pH 9), the dotted line indicates the absorbance of the fraction D2 (pH 7), the two-dot chain line indicates the conductivity of the fraction D2 (pH 9), and the one-dot chain line indicates the conductivity of the fraction D2 (pH 7). . In the figure, “C1 (pH 7)” is the fraction C1 (pH 7), “C1 (pH 9)” is the fraction C1 (pH 9), “C2 (pH 7)” is the fraction C2 (pH 7), “C2 (pH 9)” Are fractions fractionated as fraction C2 (pH 9). In Example 9, it is the figure which showed the cell proliferation amount at the time of culture | cultivating using each culture solution. (A) is a result at the time of adding the culture solution containing the fraction C1 (pH 9), (b) is a result at the time of adding the culture solution containing the fraction C1 (pH 7). In the figure, “NC” represents a control culture solution, and “PC” represents a 10% FCS-containing culture solution. In Example 9, it is the figure which showed the cell proliferation amount at the time of culture | cultivating using each culture solution. (A) is a result at the time of adding the culture solution containing the fraction C2 (pH 9), (b) is a result at the time of adding the culture solution containing the fraction C2 (pH 7). In the figure, “NC” indicates a control culture solution. Moreover, each density | concentration has shown the protein density | concentration derived from the fraction C2 in each culture solution. In Example 10, it is the figure which showed the result of the BSA density | concentration of each PBS sample solution. (A) is a result at the time of adding RJ decomposition product, (b) is a result at the time of adding ascorbic acid, (c) is a result at the time of adding carnosine.

Claims (10)

A royal jelly-degrading enzyme-containing product prepared from a 2-3 day old queen bee larva of a western bee (Apis mellifera) by a method having the following steps.
(A) A step of separating the body suspension of the queen bee larvae into three layers of a white solid upper layer, a solution middle layer, and a precipitation lower layer by centrifuging at 6 ° C. or lower.
(B) A step of recovering the middle layer as a royal jelly-degrading enzyme-containing material.   2. The royal jelly-degrading enzyme-containing material according to claim 1, wherein the centrifugation is 8,000 to 12,000 × g for 5 minutes or more. A royal jelly-degrading enzyme-containing material prepared from a honey bee larva of 2 to 3 days old of a western bee by a method having the following steps.
(A ′) A step of preparing a milky white body tissue suspension by washing the queen bee larvae with ice-cold physiological saline and then straining them.
(B ′) After diluting the body tissue suspension with a 50 mM phosphate buffer having a pH of 7.0, centrifugation is performed at 10,000 × g and 5 ° C. for 10 minutes to obtain an upper layer of a white solid. The process of separating the middle layer of the yellowish-white turbid solution from the white to the lower three layers due to a slightly brown precipitate.
(C ′) A step of recovering the intermediate layer as a royal jelly-degrading enzyme-containing material.   The royal jelly degradation product prepared using the royal jelly degradation enzyme-containing material according to any one of claims 1 to 3. A royal jelly degradation product having the following characteristics, which is obtained by subjecting royal jelly to an enzyme treatment at pH 9 for 4 hours or more using the royal jelly degradation enzyme-containing material according to any one of claims 1 to 3.
(1) Non-specific cytopathic effect on nerve cells is less than that of unenzyme-treated royal jelly water-soluble fraction,
(2) The inhibitory effect on neuronal cell death induction by amyloid is almost equivalent to the unenzyme-treated royal jelly water-soluble fraction,
(3) The cell proliferation action on glial cells is increased as compared with the unenzymatic treated royal jelly water-soluble fraction,
(4) The infection inhibitory action against human rotavirus infection is higher than that of the unenzyme-treated royal jelly water-soluble fraction.   The antioxidant which uses as an active ingredient the royal jelly degradation product obtained by carrying out the enzyme treatment of royal jelly at pH9 for 4 hours or more using the royal jelly degradation enzyme containing material in any one of Claims 1-3.   The cell growth agent which uses as an active ingredient the royal jelly degradation product obtained by carrying out the enzyme treatment of royal jelly at pH9 for 4 hours or more using the royal jelly degradation enzyme containing material in any one of Claims 1-3.   The infection inhibitor which uses as an active ingredient the royal jelly degradation product obtained by carrying out the enzyme treatment of royal jelly at pH9 for 4 hours or more using the royal jelly degradation enzyme containing material in any one of Claims 1-3.   A neuronal cell death induction inhibitor comprising, as an active ingredient, a royal jelly degradation product obtained by subjecting royal jelly to enzyme treatment at pH 9 for 4 hours or more using the royal jelly degrading enzyme-containing material according to any one of claims 1 to 3.   A royal jelly-decomposing enzyme-containing material according to any one of claims 1 to 3, wherein the royal jelly-decomposing method is used.

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