JP3815726B2 - Royal jelly freshness-keeping agent consisting of buffer solution - Google Patents

Description

【００１５】
【表２】

【００１６】
＜実施例６〜８＞
以下に示す処方に従って、健康食品を作成した。即ち、処方成分を撹拌可溶化しドリンク製剤の健康食品を得た。これらのものは何れも分子量５７キロダルトンタンパク質の安定性に優れていた。表中の数値の単位は重量部を表す。
【００１７】
【表３】

【００１８】
＜実施例９〜１１＞
以下に示す処方に従って、健康食品を作成した。即ち、処方成分を撹拌可溶化しドリンク製剤の健康食品を得た。これらのものは何れも分子量５７キロダルトンタンパク質の安定性に優れていた。表中の数値の単位は重量部を表す。
【００１９】
【表４】

【００２０】
＜実施例１２〜１４＞
以下に示す処方に従って、健康食品を作成した。即ち、処方成分を撹拌可溶化しドリンク製剤の健康食品を得た。これらのものは何れも分子量５７キロダルトンタンパク質の安定性に優れていた。表中の数値の単位は重量部を表す。
【００２１】
【表５】

【００２２】
＜実施例１５〜１７＞
以下に示す処方に従って、クリームを作成した。イ）、ロ）の成分を８０℃に加熱し、ロ）をイ）に徐々に加えることにより乳化し、冷却撹拌しクリームを得た。
これらのものは何れも分子量５７キロダルトンタンパク質の安定性に優れていた。尚、表中の数値の単位は重量部を表す。
【００２３】
【表６】

【００２４】
【発明の効果】
本発明によれば、ローヤルゼリー中の分子量５７キロダルトンタンパク質の分解を緩衝液を加えることにより内因性プロテアーゼによる分解を防ぎ、以てローヤルゼリーを製剤中に安定に含有させる手段を提供することができる。
【００２５】
【配列表】

【００２６】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a composition such as food, cosmetics or pharmaceuticals containing a royal jelly and a buffer and / or a salt thereof as a freshness-preserving agent.
[0002]
[Prior art]
Royal jelly is known as a material with nourishing tonic, and health foods based on this are widely sold, but the body of its physiological activity has not yet been determined, so that There is a large lot difference in physiological activity, and the current situation is that it is not controlled as an activity value. This is because the bioactive main body has not been determined yet. For this reason, development of means for maintaining physiological activity has been desired. Further, in the royal jelly, 1) a single band is formed in non-denaturing polyacrylamide gel electrophoresis of the protein in the royal jelly, and 2) the molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57. 3) it is also not known to contain a protein comprising the amino acid sequence of amino acid numbers 1 to 8 of SEQ ID NO: 1, and the protein is a protein having a molecular weight of 57 kilodalton in royal jelly, and It is not known at all that it is a physiologically active substance. In addition, it was not known that the molecular weight 57 kilodalton protein is weakly decomposed by heat and the physiological activity is reduced accordingly. Therefore, a means for stabilizing the molecular weight 57 kilodalton protein has not been known.
[0003]
Buffer, on the other hand, refers to a mixture of substances that resists a significant change in the pH of the solution when a small amount of H + or OH − ions are added. In general, a buffer solution is a conjugate acid. And a conjugated base. Buffers can be used to stabilize pH and to measure enzyme activity for biochemical research, etc., or to dilute media used for culturing microorganisms, gel filtration and gel chromatography It is used for purposes such as swelling agents and gel stabilizers during electrophoretic analysis.
[0004]
In addition, among proteases that are proteolytic enzymes, there are proteases that require metal ions in the process of their activity expression. To suppress the activity, chelating agents such as ethylenediaminetetraacetic acid and / or salts It is also known to suppress the activity by adjusting the pH. However, since it is not known at all that the active body of royal jelly is a protein, no attempt has been made to suppress the degradation of the royal jelly protein during storage and to stabilize the royal jelly.
[0005]
[Problems to be solved by the invention]
This invention is made | formed in view of such a situation, and makes it a subject to provide the means to stabilize the bioactive component in a royal jelly and maintain the bioactivity.
[0006]
[Means for solving problems]
In view of such a situation, as a result of intensive research efforts, the present inventors have found that the 57 kilodalton protein in royal jelly is the active body of royal jelly, and this can be stabilized by stabilizing it. I found out. As a result of pursuing such stabilization means, royal jelly is selected from malic acid and / or salt thereof, citric acid and / or salt thereof, succinic acid and / or salt thereof, phosphoric acid and / or salt thereof, etc. Through the addition of two or more kinds of buffer solutions, it was found that the 57 kilodalton protein, which is the active body, is stabilized without being degraded, and has been completed. Hereinafter, the protein may be simply expressed as “a protein of 57 kilodalton” or “molecular weight of 57 kilodalton protein”. That is, the present invention relates to the following technique.
(1) A composition comprising a royal jelly and a buffer solution.
(2) The composition according to (1), wherein the royal jelly contains 9% by weight or more of the protein shown below per royal jelly total protein.
1) Proteins in royal jelly form a single band in non-denaturing polyacrylamide gel electrophoresis.
2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodaltons.
3) The amino acid sequence of the amino acid numbers 1-8 of sequence formula 1 is included.
(3) The buffer contains at least one selected from malic acid and / or salt thereof, citric acid and / or salt thereof, succinic acid and / or salt thereof, phosphoric acid and / or salt thereof The composition according to any one of (1) to (2), wherein
(4) The composition according to any one of (1) to (3), wherein the concentration of the buffer solution is 0.1 to 2 mol.
(5) The composition according to any one of (1) to (4), wherein the pH of the buffer solution is 3.5 to 4.5.
(6) The composition according to any one of (1) to (5), wherein the buffer solution inhibits protease activity in the royal jelly.
(7) The composition according to any one of (1) to (6), which is a food, cosmetic or pharmaceutical product.
Hereinafter, the present invention will be described with a focus on embodiments.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
(1) Royal jelly which is an essential component of the composition of the present invention The composition of the present invention is characterized by having a royal jelly as an essential component. The chemical composition of royal jelly is somewhat different depending on the production area, but moisture 65-70%, protein 15-20%, carbohydrate 10-15%, fat 1.7-6%, ash 0.7-2 % Is included. As for the biological and pharmacological actions of royal jelly, anti-aging action, enzyme action, antibacterial action, antitumor action, blood / circulatory action, etc. are known. The royal jelly of the present invention is characterized by containing a protein having a molecular weight of 57 kilodaltons. This protein has been confirmed by the present inventors for the first time as an active body of royal jelly, such as a limiting momentum enhancing action, a hepatocyte proliferation promoting action, a blood ammonia concentration inhibiting action, a blood lactate accumulation inhibiting action, etc. Has been found to express the activity of. Here, although it is content of this protein, in order to fully exhibit the said effect, it is preferable that at least 9 weight% or more is included. This protein can be quantified by high performance liquid chromatography using a GPC column. In the royal jelly liquid chromatography, the peak area of the protein is 9% by weight or more of the total peak area. This is because it sufficiently acts as a physiologically active ingredient in foods, cosmetics and pharmaceuticals. If the peak area of the protein is less than 9% by weight of the total peak area, it is not preferable in terms of quality as a royal jelly which is an essential component of the present invention. The preferable content of royal jelly in the composition of the present invention is 1 to 50% by weight, more preferably 5 to 30% by weight, based on the total amount of the composition. This is because of the combination of the expression of effects and the degree of freedom of prescription.
[0008]
(2) Qualitative method of molecular weight 57 kilodalton protein in royal jelly by anion exchange chromatography and gel filtration chromatography Lyophilized royal jelly was dissolved in 10 mM Tris-HCl buffer (pH 7.0) at 0.7% by weight. , UF 100,000 (Miniplate 100; ultrafiltration), concentrated 6 times and desalted 7 times, and the filtrate was further concentrated 8 times with UF30,000 (Miniplate 30; ultrafiltration), once desalted and molecular weight 100,000 ~ 30,000 fractions were obtained. The sample having a molecular weight of 100,000 to 30,000 can be separated by anion exchange chromatography and gel filtration chromatography to separate a protein having a molecular weight of 57 kilodaltons. Anion exchange chromatography may be carried out according to a generally known method. For example, DEAE-Toyopearl650M manufactured by Tosoh Corporation is used as a column, 20 mM Tris-HCl buffer (pH 7.0) is used as a developing solution A, 20 mM. Fraction No. obtained by detecting a flow rate of 5 ml / min at an absorbance of 280 nm by a gradient using Tris-HCl buffer (pH 7.0) and 1M NaCl as a developing solution B, and fractionating with 2.5 ml / tube. A protein fraction having a molecular weight of 57 kilodaltons can be detected at 119 to 127. Further, this fraction may be subjected to gel filtration chromatography according to a generally known method. As such a preferable example, for example, HiLoad16 / 60Superdex200 manufactured by Pharmacia Co., Ltd. is used as a column. 15 mM sodium phosphate-containing 50 mM potassium phosphate buffer pH 7.0 was used as a developing solution, the flow rate was set to 1.0 ml / min, the column temperature was set to 35 ° C., the absorbance was detected at 280 nm, and fractionation was performed with 2.0 ml / tube. Fraction No. A protein with a molecular weight of 57 kilodaltons can be detected at 35-43. (The same protein was confirmed by electrophoresis) From the result of gel filtration analysis with a known molecular weight, the protein was determined to be a 57 kilodalton monomer protein with a molecular weight. In addition, the present inventors have found that this protein is a glycoprotein because it is digested with N-glucosidase F and has a molecular weight of 48 kilodalton after digestion.
[0009]
(3) Qualitative method of protein having a molecular weight of 57 kilodaltons in royal jelly by electrophoresis The protein having a molecular weight of 57 kilodaltons in the royal jelly of the present invention is analyzed for the structure of the protein by electrophoresis, and the molecular weight of the protein as an active ingredient is determined. It is characterized by specifying. The method of electrophoresis is not particularly limited as long as the effective protein can be identified, but a preferable method is to convert a water-soluble royal jelly protein (10% royal jelly aqueous solution (W / V)) into a polyacrylamide gel (10% uniform). In this method, electrophoresis is performed at a current of 20 mA, and the protein is identified by staining with Coomassie Brilliant Blue. The molecular weight of the protein of the present invention in such electrophoresis was determined to be 57 kilodalton by SDS-polyacrylamide gel electrophoresis of the purified protein.
[0010]
(4) Buffer solution which is an essential component of the composition of the present invention The composition of the present invention is characterized by containing a buffer solution. Any buffer known in the art can be used without particular limitation. Specific examples of the buffer used in the composition of the present invention include boric acid and / or a salt thereof, cacodylic acid. And / or salt thereof, acetic acid and / or salt thereof, veronal acetic acid and / or salt thereof, collidine and / or salt thereof, hydrochloric acid and / or salt thereof, acetic acid and / or salt thereof, glutaric acid and / or salt thereof, Carbonic acid and / or salt thereof, malic acid and / or salt thereof, citric acid and / or salt thereof, succinic acid and / or salt thereof, phosphoric acid and / or salt thereof, HEPES-KOH buffer solution, Kellenberger buffer solution, It is preferable to contain one or more selected from PBS buffer, Tris-HCl buffer, and lithium citrate buffer. Among them, it is most preferable to contain at least one selected from malic acid and / or a salt thereof, citric acid and / or a salt thereof, succinic acid and / or a salt thereof, phosphoric acid and / or a salt thereof. . Moreover, it is preferable that the density | concentration of a buffer solution is a 0.1-2 mol density | concentration range as the property, and it is preferable that the pH of a buffer solution is 3.5-4.5. Special products include Good's buffer, which uses amphoteric electrolyte amino acids, especially N-substituted taurines or N-substituted glycines. This property is 1) easily soluble in water and non- It is relatively insoluble in polar solvents, 2) difficult to pass through biological membranes, and 3) has low ionic strength. These buffers are dozens of zwitter-ionic buffers developed by NEGood et al. (1966), many of which are buffer agents (reagents used to make buffers). However, some of them are not aliphatic amines or zwitterionic buffers. This buffer solution can be prepared by neutralizing these buffer solution to a desired pH with sodium hydroxide or hydrochloric acid. In addition, as commercially available products, MESA (MOPS-EDTA-sodium acetate buffer solution, 10-fold concentrated) TAE (Tris-acetic acid-EDTA buffer solution, 10-fold concentrated), TBE (Tris-boric acid) manufactured by Kishida Chemical Co., Ltd. -EDTA buffer, TPE (Tris-phosphate-EDTA buffer, 10 times concentrated), TE (Tris-EDTA buffer, 10 times concentrated), TNE (Tris-sodium chloride-EDTA buffer, 10 times concentrated) SSC (citric acid-saline solution, 20-fold concentration), SSPE (sodium chloride-EDTA-phosphate buffer solution, 20-fold concentration), 1M Tris HCl (pH 8.0), and the like can also be used.
[0011]
(5) Composition of the present invention The composition of the present invention comprises a royal jelly and a buffer (liquid), preferably malic acid and / or a salt thereof, citric acid and / or a salt thereof, succinic acid and / or a salt thereof, 1 type or 2 types or more chosen from phosphoric acid and / or its salt are characterized by the above-mentioned. The composition of the present invention has a molecular weight of 57 kilodalton protein which is a physiologically active ingredient of royal jelly by adding malic acid and / or salt thereof, citric acid and / or salt thereof, phosphoric acid and / or salt thereof to royal jelly. However, a suitable content can be maintained without being decomposed. Here, the composition can be applied without any particular limitation, and for example, compositions such as foods, cosmetics, and pharmaceuticals can be preferably exemplified. This is because there is a track record as a royal jelly preparation. Moreover, royal jelly is effective in improving normal fatigue accumulation, and a fatigue recovery effect can be obtained by administering it to a person. The indications of the composition of the present invention include rough skin, weak constitution, recovery of physical strength during and after illness, before and after childbirth, and improvement of chronic fatigue common symptoms. The royal jelly of the present invention can be administered to animals and humans as it is or together with a conventional pharmaceutical carrier. A preferred effective daily dose is 0.1 to 1000 mg / kg body weight / day, and a more preferred dose is 5 to 500 mg / kg body weight / day. In foods and pharmaceuticals, the dosage form is not particularly limited, and is appropriately selected and used as necessary. Oral preparations such as drinks, tablets, capsules, granules, fine granules, powders, Examples include parenterals such as injections and suppositories. The above preparation is manufactured by a conventional method. Usually known optional components can be blended to such an extent that the physiological activity of the royal jelly of the present invention is not impaired. Such optional ingredients include excipients such as sucrose and lactose, binders such as starch and gelatin, disintegrants such as sodium carboxymethylcellulose and carboxymethylcellulose calcium, surfactants such as soybean lecithin and sucrose fatty acid ester, talc, wax Lubricants such as light silicic acid anhydride, glidants such as dry aluminum hydroxide gel, diluents such as physiological saline and glucose aqueous solution, flavoring agents, coloring agents, bactericides, preservatives, flavors, etc. Can be mentioned. In cosmetics, in addition to the above essential components, optional components that are usually used in external preparations for skin can be contained. Such optional components include, for example, hydrocarbons such as petrolatum and microcrystalline wax, esters such as jojoba oil and gay wax, triglycerides such as beef tallow and olive oil, higher alcohols such as cetanol and oleyl alcohol, stearic acid, Increase in fatty acids such as oleic acid, polyhydric alcohols such as glycerin and 1,3-butylene glycol, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, ethanol, carbopol, etc. Preferred examples include a sticky agent, an ultraviolet absorber, an antiseptic, an antioxidant, a pigment, and powders. The composition of the present invention can be obtained by treating these essential components and optional components according to a conventional method.
[0012]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.
[0013]
<Examples 1-3>
Each buffer solution was added to 50 g of fresh royal jelly so that each buffer was 1 M, and left at 40 ° C. for 1, 2, 3, and 4 days. As a control experiment, a control group with no buffer solution was treated in the same manner. This was centrifuged at 5000 rpm for 10 min at 4 ° C., and the 57 kilodalton protein content in the supernatant was measured with a gel filtration column. For the measurement, using a TSK-GEL G3000SW column manufactured by Tosoh Corporation, 0.1M potassium phosphate buffer pH 7.0 containing 0.3M sodium chloride was used as a developing solution, the flow rate was 0.3 ml / min, and the column temperature was adjusted. A method was used in which the temperature was set to 35 ° C. and the absorbance was detected at 280 nm. When a buffer solution was added as shown in Table 1, the molecular weight of 57 kilodalton protein was less degraded over time, whereas the control group without the buffer solution was degraded over time ( Table 1: Examples 1-3). In addition, citric acid-sodium citrate buffer (pH 4.0) and malic acid-sodium malate buffer (pH 4.0) inhibited endogenous protease activity (Table 2: Examples 4 and 5). . In this way, each buffer can stabilize a protein having a molecular weight of 57 kilodaltons by inhibiting the protease in the royal jelly.
[0014]
[Table 1]

[0015]
[Table 2]

[0016]
<Examples 6 to 8>
A health food was prepared according to the prescription shown below. That is, the prescription ingredients were solubilized with stirring to obtain a health food as a drink preparation. All of these were excellent in stability of a molecular weight 57 kilodalton protein. The unit of numerical values in the table represents parts by weight.
[0017]
[Table 3]

[0018]
<Examples 9 to 11>
A health food was prepared according to the prescription shown below. That is, the prescription ingredients were solubilized with stirring to obtain a health food as a drink preparation. All of these were excellent in stability of a molecular weight 57 kilodalton protein. The unit of numerical values in the table represents parts by weight.
[0019]
[Table 4]

[0020]
<Examples 12 to 14>
A health food was prepared according to the prescription shown below. That is, the prescription ingredients were solubilized with stirring to obtain a health food as a drink preparation. All of these were excellent in stability of a molecular weight 57 kilodalton protein. The unit of numerical values in the table represents parts by weight.
[0021]
[Table 5]

[0022]
<Examples 15 to 17>
A cream was prepared according to the formulation shown below. The ingredients of a) and b) were heated to 80 ° C. and emulsified by gradually adding b) to b) and cooled and stirred to obtain a cream.
All of these were excellent in stability of a molecular weight 57 kilodalton protein. In addition, the unit of the numerical value in a table | surface represents a weight part.
[0023]
[Table 6]

[0024]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the decomposition | disassembly by endogenous protease can be prevented by adding a buffer solution to the decomposition | disassembly of the molecular weight 57 kilodalton protein in a royal jelly, Therefore The means to make a royal jelly stably contain in a formulation can be provided.
[0025]
[Sequence Listing]

[0026]

Claims (1)

A royal jelly containing at least 9% by weight of the protein shown below per royal jelly, and malic acid and / or salt thereof, citric acid and / or salt thereof, succinic acid and / or salt thereof, phosphoric acid and the like And a buffer solution having a concentration of 0.1 to 2 mol and a pH of 3.5 to 4.5, which is selected from one or two or more selected from salt or salt thereof. composition.
1) Proteins in royal jelly form a single band in non-denaturing polyacrylamide gel electrophoresis.
2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodaltons.
3) The amino acid sequence of the amino acid numbers 1-8 of sequence formula 1 is included.