JP2001213791A - Prophylactic for hyperammonemia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide prophylactic for inhibiting the generation of blood ammonia thereby preventing the rise of blood ammoniac level that becomes a problem in the case of congenital urea cycle enzyme deficiency or impairment of hepatic function. SOLUTION: The prophylactic for hyperammonemia consists of a royal jelly which is characterized in containing protein of molecular weight of 57 kilo dalton.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a preventive agent for hyperammonemia, which is suitable for recovering and improving urea cycle reduction due to abnormal liver function.

[0002]

2. Description of the Related Art Blood ammonia, which increases after exercise and the like, is produced by deamination from amino acids in the course of protein metabolism and is used for urea synthesis in the liver. Bacteria in the small intestinal mucosa and large intestine). Ammonia detoxification depends on the urea cycle in hepatocytes, and urea is excreted in urine from the kidney. Therefore, in the case of decreased activity of the urea cycle due to decreased liver function, increased production of ammonia in the intestine, and influx of portal venous blood into the large circulatory system by the portal vein, the blood ammonia concentration may be reduced. It is said to be high. Therefore, many people with congenital urea cycle enzyme deficiency and liver dysfunction have hyperammonemia. The presence of ammonia excreted into the blood without conversion to urea in this way may damage the living body due to its toxicity.Therefore, there is a method for lowering the ammonia concentration in the blood and an increase in the ammonia concentration in the blood. Preventive measures are needed to avoid this. However, there is still no specific medicine for reducing the activity of the urea cycle due to the decrease in liver function and for suppressing the increase in blood ammonia concentration in people with congenital urea cycle enzyme deficiency, and such a drug is desired. Was. This is because when a person with such a disease exercises suddenly or excessively, the protein is digested, ammonia is produced, and this is excreted into the blood, and symptoms may appear. Because it is very dangerous.

[0003] On the other hand, royal jelly is known as a material having a nourishing and tonic effect, and health foods and the like containing it as a main component are widely sold, but all of its physiological activities have not yet been determined. Therefore, there is a lot difference in the biological activity between the lots, and it is not controlled as an activity value at present.

[0004] Further, it is not known at all that royal jelly activates the urea cycle in the liver and suppresses the production of blood ammonia.

In addition, the active substance is a protein having a molecular weight of 57 kDa, and the royal jelly containing the protein is
It was not known to have a hyperammonemia preventive effect. Further, it has not been known that the protein is an active substance having such an effect, and royal jelly can be used as a preventive agent for hyperammonemia without lot variation by controlling this content. The action of the hyperammonemia preventive agent referred to herein means an action suitable for suppressing the amount of ammonia produced in the blood which increases at the time of fatigue such as after exercise and for recovering and improving liver function. .

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and has been developed to reduce the amount of ammonia present in blood due to congenital urea cycle enzyme deficiency and decreased liver function. An object of the present invention is to provide a means for controlling and preventing the manifestation of hyperammonemia.

[0007]

In view of such circumstances, the present inventors have made intensive research efforts, and as a result, royal jelly has an inhibitory effect on blood ammonia production, and its active substance has a molecular weight of 5
They found that it was a 7 kilodalton protein, and completed the invention. Hereinafter, the present invention will be described focusing on embodiments.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Royal jelly which is a preventive agent for hyperammonemia of the present invention The preventive agent for hyperammonemia of the present invention is characterized by comprising royal jelly. The chemical composition of royal jelly varies slightly depending on the analyst, but the water content is 65 to 7%.
It is said to contain 0%, 15-20% protein, 10-15% carbohydrates, 1.7-6% fat, and 0.7-2% ash.
Regarding the biological and pharmacological effects of royal jelly,
Anti-aging, enzymatic, antibacterial, antitumor, and blood / circulatory effects are known. Hyperammonemia preventive agent of the present invention, fulminant hepatitis, cirrhosis, congenital urea cycle enzyme deficiency, congenital amino acid metabolism disorder,
It is used for diseases showing abnormally high levels of blood ammonia, such as portal vein integrated shunt, Reye's disease, and shock symptoms. The royal jelly, which is a hyperammonemia preventive agent of the present invention, comprises 1) a non-denaturing polyacrylamide gel of proteins in the royal jelly, which forms a single band upon electrophoresis, and 2) SDS-polyacrylamide gel under reducing conditions. It has a molecular weight of about 57 kilodaltons as measured by electrophoresis. 3) It is characterized by containing a protein containing an amino acid sequence of amino acids 1 to 8 (N-terminus) of Sequence Formula 1. Further, the present inventors have found that this protein is a glycoprotein because it is digested by N-glucosidase F and the molecular weight after digestion becomes 48 kDa.
Here, the content of the protein is preferably at least 9% by weight or more. This protein is GP
It can be quantified by high-performance liquid chromatography using a C column. In the liquid chromatography of the royal jelly, the peak area of the protein is 9% by weight or more of the total peak area, which indicates that the production of ammonia according to the present invention is suppressed. It is preferable to use as an agent. If the peak area of the protein is less than 9% by weight of the total peak area, the effect of the hyperammonemia preventive agent of the present invention is too small, which is not preferable.

(2) Method for qualifying 57-kilodalton protein in royal jelly
Dissolved in Tris-HCl buffer (pH 7.0), and UF10
6-fold concentration and desalting 7 times with Miniplate 100 (Ultrafiltration), and the filtrate was further filtered with UF 30,000 (Mi
(Niplate 30; ultrafiltration), concentrated 8 times, and desalted once to obtain a fraction having a molecular weight of 100,000 to 30,000. The sample having a molecular weight of 100,000 to 30,000 can be fractionated by anion exchange chromatography or gel filtration chromatography to separate a protein having a molecular weight of 57 kDa. The anion exchange chromatography may be performed according to a commonly known method. For example, using a DEAE-Toyopearl 650M manufactured by Tosoh Corporation as a column, a 20 mM Tris-HCl buffer (pH 7.0) is used.
0) as a developing solution A, 20 mM Tris-HCl buffer (pH 7.0);
0) and 1 M NaCl as the developing solution B, the gradient N was detected at a flow rate of 5 ml / min and absorbance at 280 nm, and fractionated at 2.5 ml / tube.
o. From 119 to 127, a protein fraction having a molecular weight of 57 kDa can be detected. The fraction may be further subjected to gel filtration chromatography according to a generally known method. Preferred examples of such a fraction include, for example, HiLoad 16 manufactured by Pharmacia.
Using / 60 Superdex600 as a column,
Using 50 mM potassium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride as a developing solution, the flow rate was set to 1.0 ml.
/ Min, the column temperature was set at 35 ° C., the absorbance was detected at 280 nm, and fraction No. 2 was fractionated at 2.0 ml / tube. A protein having a molecular weight of 57 kDa can be detected in 35 to 43. (The same protein was confirmed by electrophoresis.) From the results of gel filtration analysis of known molecular weight, the protein was determined to be a 57-kilodalton monomer protein.

(3) Method for measuring the preventive action of royal jelly on hyperammonemia in mice (anti-fatigue test) As experimental animals, ddY mice, 5 weeks old, female, 10 animals per group were used. The effect of royal jelly on ammonia production in mice was measured by using a Kyoto University Matsumoto-style mouse momentum measuring water tank. A group of 10 mice were allowed to swim for 15 minutes, then rested for 10 minutes, orally administered royal jelly at a dry weight of 0.7 g / kg, rested for another 20 minutes, allowed to swim for 15 minutes, and collected blood and collected blood. Was measured by using ammonia test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, as a control experiment, an experiment without royal jelly was similarly performed. The anti-fatigue effect can be tested by measuring the effect of royal jelly on the effect of inhibiting blood ammonia production.

(4) Composition of the Present Invention The composition of the present invention is characterized by containing royal jelly containing a protein having a molecular weight of 57 kDa. Here, the composition can be applied without any particular limitation as long as it is involved in the preventive agent for hyperammonemia, and examples thereof include compositions such as cosmetics, medicines, and foods. Particularly preferred among these are food and medicine. This is because it has a track record as a royal jelly preparation. The royal jelly containing the protein having a molecular weight of 57 kilodaltons, preferably containing 9% by weight or more based on the total amount of the proteins in the royal jelly, causes the above-mentioned congenital urea cycle enzyme deficiency in terms of enhancing the suppression of ammonia production in a living body. Shows significant effect. The indication of the hyperammonemia preventive agent of the present invention includes fulminant hepatitis, cirrhosis, congenital amino acid metabolism disorder, portal vein shunt, Reye's syndrome, shock symptoms and the like. The royal jelly of the present invention containing a protein having a molecular weight of 57 kilodaltons can be administered to animals and humans as it is or together with conventional pharmaceutical carriers. A preferable effective daily dose is 0.01 to 100 mg / k as a protein having a molecular weight of 57 kDa.
g body weight / day, more preferably 0.5 to 50 mg / day.
kg body weight / day. The dosage form is not particularly limited and may be appropriately selected and used as needed. Tablets, capsules, granules, fine granules, oral preparations such as powders, parenteral preparations such as injections, suppositories and the like may be used. No. The above preparations are manufactured by a conventional method. Commonly known optional components can be added to such an extent that the physiological activity of royal jelly containing the protein of the present invention having a molecular weight of 57 kDa 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 calcium carboxymethylcellulose, surfactants such as soy lecithin and sucrose fatty acid esters, talc and wax. Lubricants, such as light anhydrous silicic acid, glidants such as dried aluminum hydroxide gel, diluents such as physiological saline and aqueous glucose solution, flavoring agents, coloring agents, disinfectants, preservatives, fragrances, etc. No.

[0012]

EXAMPLES 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 only these Examples.

<Example 1> Experiment of royal jelly preventive action on hyperammonemia in mice (anti-fatigue test) (Experimental method) ddY mouse, 5 weeks old,
Females, 10 animals per group were used. By using the Kyoto University Matsumoto-type mouse momentum measurement water tank, the mouse ammonia production inhibition effect by the excessive exercise was measured using a mouse ammonia production model. A group of 10 mice was allowed to swim for 15 minutes, and after resting for 10 minutes, royal jelly (molecular weight 5
Oral administration of a dry weight of 0.7 g / kg with a protein content of 7 kilodalton (10.2% by weight), a rest for 20 minutes, a swim for 15 minutes, blood collection and blood ammonia test for ammonia level It was measured by using Wako (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, the same experiment was carried out for the deteriorated royal jelly administration group having a protein content of 3.4% by weight and a protein content of 57 kilodalton and having no royal jelly as a control experiment. By measuring the effect of royal jelly on the suppression of blood ammonia level, the effect of preventing hyperammonemia was measured. Table 1 shows the results. It turns out that the blood jelly level was suppressed in the royal jelly administration group. This indicates that royal jelly has an effect of controlling the amount of ammonia in blood.

[0014]

[Table 1]

Examples 2 to 4 Pharmaceuticals were prepared according to the following prescription. That is, the prescription components were subjected to tumbling phase granulation together with 10 parts by weight of water, and tabletted to obtain a tablet-like drug. All of these had an excellent hyperammonemia preventive action. The units of the numerical values in the table represent parts by weight.

[0016]

[Table 2]

<Examples 5 to 7> Health foods were prepared according to the following formulations. That is, the ingredients were stirred and solubilized to obtain a health food of a drink preparation. All of these had an excellent effect of suppressing an increase in the amount of ammonia in blood. The units of the numerical values in the table represent parts by weight.

[0018]

[Table 3]

[0019]

According to the present invention, it is possible to provide a preventive agent for hyperammonemia which is suitable for improving and preventing congenital urea cycle enzyme deficiency and the like.

[0020]

[Sequence List] SEQUENCE LISTING <110> POLA KASEI KOGYO CO., LTD. <120> Hyperammonemia preventive agent <130> P2000134 <140> <141> 2000-10-25 <160> 1 <170> PatentIn Ver.2.0

<210> 1 <211> 25 <212> PRT <213> Apis mellifera <220> <221> UNSURE <222> (24) <223> Xaa = unknown and # 60; 400and # 62; 1 Asn Ile Leu Arg Gly Glu Ser Leu Leu Lys Lys Leu Pro Ile Leu His 1 2 10 15 Glu Met Lys Phe Phe Asp Tyr Xaa Asp 20 25

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 7/08 C07K 14/435 C07K 14/435 A61K 37/02 (72) Inventor Hirotaka Miyazaki Yokohama, Kanagawa Prefecture 560 Kashio-cho, Totsuka-ku Pola Totsuka Research Institute

Claims (5)

[Claims] 1. A preventive for hyperammonemia comprising royal jelly. 2. The royal jelly contains at least 9% by weight of a protein described below,
The agent for preventing hyperammonemia according to claim 1. 1) Formation of a single band in non-denaturing polyacrylamide gel electrophoresis of proteins in royal jelly. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodalton. 3) Includes the amino acid sequence of amino acid numbers 1 to 8 of SEQ ID NO: 1. 3. A composition comprising the hyperammonemia preventive agent according to claim 1 or 2. 4. It is a food or a medicine,
A composition according to claim 3. 5. The composition according to claim 3, wherein the composition recovers or improves liver function abnormality or congenital urea cycle enzyme deficiency.