JP2003113095A - Accelerating agent for dna synthesis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liver protecting agent for improvement, recovery of liver function failure or the like. SOLUTION: This invention provides the liver protecting agent for improvement and prevention of liver function failure or the like, or a food and a medicine or the like, suitable for reproduction of hepatic cell/liver/organ by including a sugar protein having 57 kDa of molecular weight and manifesting DNA synthesis promoting function close to a growth factor which promotes DNA synthesis functioning in S-period in the cell cycle such as epidermal growth factor and hepatocyte growth factor isolated and purified from a biomedical tissue, and having the DNA synthesis promoting effect on a serum-free medium in hepatocyte cultivation system, and having survival maintaining effect to cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hepatocyte DNA synthesis promoter, and further relates to one whose action mechanism is determined by the uptake of tritium thymidine by hepatocytes.

[0002]

2. Description of the Related Art In cell division, another set of the same DNA is produced by a mechanism called replication and is equally distributed to two cells. Cell cycle of eukaryotic cells
In CY), resting cells that have not undergone cell division are in G0 phase. Most human cells are in this state.
The cell cycle is roughly divided into four phases, G1 phase, S phase, G2 phase, and M phase, and is regulated by many proteins such as phosphorylase and cyclin. Accurate completion of chromosomal replication is essential for the accurate transmission of genetic information to the next generation. When DNA replication is delayed for some reason, there is a mechanism to stop the cell cycle and wait for the completion of replication, which is called a DNA checkpoint. The progression from the G1 phase to the S phase is called the G1 / S checkpoint, and the progression from the G2 phase to the M phase is called the G2 / M checkpoint, which is determined by the phosphorylation state of certain proteins.

On the other hand, a growth factor which acts on the S phase in the above cell cycle and promotes DNA synthesis has been isolated and purified from living tissues. Typical examples include epidermal growth factor (EGF) and hepatocyte growth factor (H
Growth factors such as GF (hepatocyte growth factor) are also DN
It is already known that it has an A synthesis promoting action. Other known growth factors are mainly isolated and purified from living tissues of mammals, for example, IGF-1 (insulin-
like growth factor-1), IGF-2 (insulin-likegrowth fa
ctor-2), TGF-α (transforming growth factor-α) and the like are known, and all have important biochemical actions in the process of promoting cell growth.

On the other hand, the inventors have found that the active substance has been isolated from royal jelly and is a glycoprotein having a molecular weight of 57 kilodaltons. As its biological activity, liver protection, blood glucose maintenance, hyperammonemia suppression, antistress, lactate accumulation suppression, aerobic exercise promotion, NK cell activity normalization,
The inventors have found a limit exercise amount enhancing action, a fatigue recovering action, a cell growth promoting action and the like. So far, the glycoprotein with a molecular weight of 57 kilodaltons in royal jelly has
It is not known that it has a DNA synthesis promoting action in the S phase of the cell cycle, and it has not been confirmed that it exhibits a DNA synthesis promoting action like growth factors such as HGF and EGF. Furthermore, it has not been known that it is useful for the regeneration of liver tissues and organs by removing insulin from the hepatocyte culture system and promoting DNA synthesis in a serum-free medium, and further having a cell survival maintenance effect. . Further, neither a drug nor a food containing a glycoprotein having a molecular weight of 57 kilodalton is known.

[0005]

The present invention has been made in view of such a situation, and promotes DNA synthesis in the S phase of the cell cycle to improve the survival effect of hepatocytes and liver tissues / organs. An object is to provide an excellent composition for recycling.

[0006]

[Means for Solving the Problems] In view of such a situation, the present inventors have conducted diligent research efforts, and as a result, a glycoprotein having a molecular weight of 57 kilodaltons in royal jelly was serum-free except insulin in the S phase of the cell cycle. DNA under culture conditions
It was found that an excellent composition for liver tissue / organ regeneration can be provided by promoting the synthesis and adding it to a drug or a food, and completed the invention. That is, the present invention relates to the following technology. <1> A DNA synthesis promoter comprising royal jelly. <2> The royal jelly contains 9 proteins as shown below.
<1 characterized by containing at least wt%
> The DNA synthesis promoter according to [1]. 1) Non-denaturing polyacrylamide gel of proteins in royal jelly forms a single band in electrophoretic migration. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodaltons. 3) Includes the amino acid sequences of amino acid numbers 1 to 8 in Sequence Formula 1. <3> The DNA synthesis promoter according to <1> or <2>, wherein the promotion of DNA synthesis is judged by the uptake of tritium-labeled thymidine in hepatocytes by 57 kilodalton protein. <4> The DNA synthesis promoter according to <1> to <3>, wherein the DNA synthesis promoter is a hepatocyte DNA synthesis promoter. <5> A composition for hepatocyte / liver tissue / liver protection and / or organ regeneration, which comprises the DNA synthesis promoter according to any one of <1> to <4>. <6> Pharmaceutical or food, <5>
The composition according to. Hereinafter, the present invention will be described focusing on the embodiments.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Qualitative Method of 57-kilodalton Protein in Royal Jelly by Electrophoresis The 57-kilodalton protein in royal jelly of the present invention was analyzed by electrophoresis for protein composition and Characterizing the molecular weight of a protein. The method of electrophoresis is not particularly limited as long as the effective protein can be specified, but a preferable method is to use a water-soluble royal jelly protein (3% royal jelly aqueous solution (W / V)) on a polyacrylamide gel (1).
It is a method of electrophoresing at a current of 20 mA at 0% homogeneity) and staining with Coomassie Brilliant Blue to identify the protein. The molecular weight of the protein of the present invention in such electrophoresis was determined to be 57 kilodaltons by SDS-polyacrylamide gel electrophoresis of the purified protein.

(2) Method for preparing 57-kilodalton protein in royal jelly 0.7% by weight of lyophilized royal jelly was added to 10 mM.
Dissolved in Tris-HCl buffer (pH 7.0) of UF10
10,000 times (Miniplate 100; ultrafiltration), concentrated 6 times, desalted 7 times, and the filtrate was further subjected to UF30,000 (Mi
The product was concentrated 8 times by niplate 30; ultrafiltration) 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 separated by anion exchange chromatography and gel filtration chromatography to separate a protein having a molecular weight of 57 kilodaltons. The anion exchange chromatography may be performed according to a commonly known method, for example, using DEAE-Toyopearl 650M manufactured by Tosoh Corporation as a column and using 20 mM Tris-HCl buffer (pH
7.0) as the developing solution A, 20 mM Tris-HCl buffer (pH
7.0) and 1 M NaCl as a developing solution B, the flow rate was detected at an absorbance of 5 ml / min and 280 nm with a gradient, and the fraction No. fraction was 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 commonly known method. As such a preferable example, for example, HiLoa manufactured by Pharmacia Co., Ltd.
Using d16 / 60 Superdex 600 as a column, 0.15 M sodium chloride-containing 50 mM potassium phosphate buffer pH 7.0 was used as a developing solution, and the flow rate was 1.
Fraction N was set to 0 ml / min, detected by absorbance at 280 nm, and fractionated at 2.0 ml / tube
o. A protein having a molecular weight of 57 kilodaltons can be detected at 35 to 43. (The same protein was confirmed by electrophoresis.) From the result of gel filtration analysis of known molecular weight, it was confirmed that the above protein was a 57 kilodalton molecular weight protein. Also, this protein is
The present inventor has found that it is a glycoprotein because it is digested with N-glucosidase F and its molecular weight after digestion becomes 48 kilodaltons.

(3) Method for measuring DNA synthesis promoting activity by the 57-kilodalton protein of the present invention The liver of a Wistar male rat 7 weeks old (body weight: about 150 g) was perfused with Hanks buffer and then perfused with a collagenase solution. Then hepatocytes were obtained. It contained 100 mg / l of penicillin and streptomycin, and 5% FBS.
Was washed twice with L15 medium containing 100 mg / ml of penicillin and streptomycin,
Dexamethasone 10-6M, Aprotinin 0.7 μg /
37 ml in L15 medium containing 5 ml of 5% FBS.
Cultivated in a CO2 incubator for 3 hours at 2 ° C, and then cultured in a serum-free medium containing a DNA synthesis promoter under the same conditions.
It was cultured for 2 hours. Furthermore, tritium-labeled thymidine (2 μCi / ml) was added to the same medium, and after culturing at 37 ° C. for 2 hours, the medium was removed, washed twice with PBS, and washed with 10% TC.
A (trichloroacetic acid) 0.5N Na after fixing at 4 ° C
After solubilizing the cells with OH, the cells are neutralized with 0.5N HCl, and tritium-labeled thymidine incorporated into the cells is measured by a liquid scintillation counter (Aloka, LSC-700).

(4) Hepatocyte / liver tissue / liver protective agent of the present invention and composition for liver organ regeneration The hepatocyte / liver tissue / liver protective agent and composition of liver organ regeneration of the present invention are: It is characterized by containing a protein having a molecular weight of 57 kilodaltons isolated and purified from royal jelly. The above-mentioned protein having a molecular weight of 57 kilodaltons is effective for treating liver diseases, and when it is administered to humans, acute liver injury due to carbon tetrachloride and the like, liver injury due to alcohol intake, chronic liver injury due to viral infection, etc. Ameliorating effect on liver disease is obtained. In addition, it promotes DNA synthesis of hepatocytes at a site damaged or lost by a surgical operation due to hepatitis, cirrhosis, liver fibrosis, liver disease such as liver cancer, and exerts an effect in organ regeneration. Indications for the therapeutic drug for liver disease of the present invention include acute hepatitis, chronic hepatitis, alcoholic hepatitis, viral hepatitis, alcoholic fatty liver, alcoholic cirrhosis, liver fibrosis, liver cancer and the like. The protein of the present invention having a molecular weight of 57 kilodalton is as it is,
Alternatively, it can be administered to animals and humans together with a conventional pharmaceutical carrier. A preferred effective daily dose is a molecular weight of 57.
The amount of protein in kilodaltons is 0.01 to 100
mg / kg body weight / day, more preferably 0.5 to 5
0 mg / kg body weight / day. The dosage form is not particularly limited and may be appropriately selected and used as needed. Oral preparations such as tablets, capsules, granules, fine granules and powders, parenteral preparations such as injections and suppositories may be used. Can be mentioned. The above formulation is
Manufactured by conventional methods. Any conventionally known component can be added to the extent that the physiological activity of the protein of the present invention having a molecular weight of 57 kilodalton is not impaired. Such optional ingredients include excipients such as sucrose and lactose, binders such as starch and gelatin, disintegrating agents such as sodium carboxymethyl cellulose and carboxymethyl cellulose calcium, soybean lecithin, surfactants such as sucrose fatty acid ester, talc and wax. Lubricants such as type, light anhydrous silicic acid, glidants such as dried aluminum hydroxide gel, physiological saline,
Examples include diluents such as an aqueous glucose solution, flavoring agents, coloring agents, bactericides, preservatives, and fragrances.

[0011]

EXAMPLES The present invention will be described in more detail below with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

<Example 1> DN with a molecular weight of 57 kilodalton protein of the present invention
Method for Measuring A Synthesis Promoting Activity The liver of a Wistar male rat 7 weeks old (body weight: about 150 g) was perfused with Hanks buffer and then with a collagenase solution to obtain hepatocytes. It contained 100 mg / l of penicillin and streptomycin, and 5% FBS.
Was washed twice with L15 medium containing 100 mg / ml of penicillin and streptomycin,
Dexamethasone 10-6M, Aprotinin 0.7 μg /
37 ml in L15 medium containing 5 ml of 5% FBS.
Cultivated in a CO2 incubator for 3 hours at 2 ° C, and then cultured in a serum-free medium containing a DNA synthesis promoter under the same conditions.
It was cultured for 2 hours. Furthermore, tritium-labeled thymidine (2 μCi / ml) was added to the same medium, and after culturing at 37 ° C. for 2 hours, the medium was removed, washed twice with PBS, and washed with 10% TC.
A (trichloroacetic acid) 0.5N Na after fixing at 4 ° C
After solubilizing the cells with OH, the cells were neutralized with 0.5N HCl, and the tritium-labeled thymidine incorporated into the cells was measured with a liquid scintillation counter (Aloka, LSC-700). As shown in Table 1, the samples added as growth factor-like DNA synthesis promoters were royal jelly of the present invention, molecular weight 57 kilodalton protein, bovine serum albumin, HGF, and insulin. It can be seen that the protein of the present invention having a molecular weight of 57 kilodalton promotes hepatocyte DNA synthesis as seen in the HGF-added group. This result indicates that the protein having a molecular weight of 57 kilodalton is a mitogen that promotes DNA synthesis of primary cultured hepatocytes, which means that it has an excellent liver protecting action and organ regeneration action.

[0013]

[Table 1]

<Example 2> 10-week-old Wistar rats (male, 200 to 230 g), each group consisting of 10 rats were laparotomized, the liver was ligated, and the half lobe of the liver was excised and treated with penicillin. And then sutured. For one group, solid feed was crushed, and 10 wt% of 57-kilodalton protein was mixed therein to form a pressure, which was used as a feed, which was administered for 2 weeks. The other group was administered a normal solid diet for 2 weeks. 1
Five days later, the laparotomy was performed, and the liver weight was measured. As a result, it was found that in the 57 kg dalton protein-containing solid diet administration group, it was 8.64 ± 0.
It was 98 g, whereas the normal solid feed administration group was
It is 6.98 ± 0.56 g, and it is clear that the 57-kilodalton protein has a liver regeneration promoting function.

Example 3 As a pathological condition model corresponding to each hepatitis type, an effective substance for treating liver diseases was evaluated using a model of toxic liver damage caused by carbon tetrachloride. That is, 4% of carbon tetrachloride / olive oil was added to the rats after preliminary breeding.
After oral administration of ml / kg and fasting for 20 hours, the test substances shown in Table 1 were orally administered at 50 mg / kg. 200 mg of the test substance was added to 1 wt% Triton.
It was emulsified in 10 ml of -X100 physiological saline and administered by a sonde. There were four rats in each group. Water and food were taken ad libitum until the administration of carbon tetrachloride, fasted after the administration of carbon tetrachloride, and only water was given as it was. Blood was collected 24 hours after the administration of the test substance, the blood was left at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 10 minutes to obtain serum.

About this serum, total pyruvin (T-Bi
l) (Jendraski method), total cholesterol (T-Cho) (cholesterol oxidase method), triglyceride (TG) (GPO-p-chlorophenol method), transaminase (GOT, GPT) (POP)
-TOOS method), alkaline phosphatase (Alpas
e) (phenyl phosphate substrate method) was quantified using a kit manufactured by Wako Pure Chemical Industries, Ltd.

T-Bil and T-Ch of each test substance administration group
Table 6 shows the effect on the values of o, TG, GOT, GPT and Alpase as average values. In addition, the standard in Table 6 shows a group not administered with carbon tetrachloride, and the control shows a group administered with carbon tetrachloride but not a test substance. As can be seen from the results in Table 1, the values of total pyruvin, total cholesterol, triglyceride, and transaminase (GOT, GPT) were significantly decreased relative to the control, and the efficiency was improved by administering the molecular weight of 57 kilodalton protein of the present invention. The effect of improving liver dysfunction was recognized.

[0018]

[Table 2]

<Examples 4 to 6> According to the formulations shown in Table 2,
Created a healthy food. That is, the prescription ingredients were tumbling-phase granulated with 10 parts by weight of water and compressed into tablets to obtain a health food in the form of tablets. All of these showed an excellent liver function improving effect and had a fatigue recovery effect. The unit of numerical values in the table is parts by weight.

[0020]

[Table 3]

<Examples 7 to 9> Health foods were prepared according to the formulations shown in Table 4. That is, the prescription ingredients were stirred and solubilized to obtain a health food as a drink preparation. All of these showed an excellent liver function improving effect and had a fatigue recovery effect. The unit of numerical values in the table is parts by weight.

[0022]

[Table 4]

<Examples 10 to 12> According to the formulation of Table 5, a drug for injection preparation was prepared by dissolving with stirring at room temperature. The unit of numerical values in the table is parts by weight. In a model of toxic liver injury due to carbon tetrachloride, transaminases GOT and GPT had the effect of lowering the value.

[0024]

[Table 5]

<Example 13> According to the formulation shown below,
A liver protection solution for living liver transplantation was prepared. That is, the formulation components were stirred and solubilized at room temperature, and filter sterilized to obtain a liver protection solution. When the liver excised from bullfrog was stored for 1 hour in this preservation solution and frozen sections were prepared and observed under a microscope, the cell death was 25% less than that of Ringer's. From this, it is understood that the hepatoprotective liquid of the present invention has an excellent hepatoprotective action.

[0026]

[Table 6]

[0027]

INDUSTRIAL APPLICABILITY According to the present invention, epidermal growth factor (EGF: epidermal growth factor), which has been isolated and purified from living tissue, is a growth factor that acts at S phase in the cell cycle and promotes DNA synthesis.
owthfactor) and hepatocyte growt (HGF)
57 kilodalton which has a DNA synthesis promoting action similar to that of growth factors such as h factor) and has a DNA synthesis promoting effect even in a serum-free medium in a hepatocyte culture system and further has a cell survival maintaining effect. It is possible to provide a hepatoprotective agent for the improvement and prevention of liver dysfunction and the like by including the glycoprotein of No. 1 in foods and medicines, and foods and medicines suitable for the regeneration of hepatocytes, liver tissues and organs. I can.

[0028]

[Sequence list]                       SEQUENCE LISTING &#60; 110 &#62; Pola Chemical Industry Co., Ltd. &#60; 120 &#62; DNA synthesis promoter &#60; 130 &#62; P2001085 &#60; 140 &#62; &#60; 141 &#62; 2001-10-04 &#60; 160 &#62; 1 &#60; 170 &#62; PatentIn Ver.2.0

[0029] &#60; 210 &#62; 1 &#60; 211 &#62; 25 &#60; 212 &#62; PRT &#60; 213 &#62; Apis mellifera &#60; 220 &#62; &#60; 221 &#62; UNSURE &#60; 222 &#62; (24) &#60; 223 &#62; Xaa = unknown &#60; 400 &#62; 1 Asn Ile Leu Arg Gly Glu Ser Leu Leu Lys Lys Leu Pro Ile Leu His  1 2 10 10 Glu Met Lys Phe Phe Asp Tyr Xaa Asp              20 25

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 14/435 A61K 37/02 F term (reference) 4B018 LB10 LE01 MD20 MD76 ME02 ME14 MF01 4C084 AA02 BA01 BA07 BA22 CA49 ZA75 ZB22 4C087 BB22 CA16 CA44 ZA75 ZB22 4H045 AA10 AA30 BA18 CA51 EA01 EA27 FA71 GA10 GA22 GA23 HA06

Claims (6)

[Claims] 1. A DNA synthesis promoter comprising royal jelly. 2. The DNA synthesis promoter according to claim 1, wherein the royal jelly contains 9% by weight or more of a protein shown below. 1) Non-denaturing polyacrylamide gel of proteins in royal jelly forms a single band in electrophoretic migration. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodaltons. 3) Includes the amino acid sequences of amino acid numbers 1 to 8 in Sequence Formula 1. 3. The DNA synthesis promoter according to claim 1 or 2, wherein the promotion of DNA synthesis is judged by the incorporation of tritium-labeled thymidine into hepatocytes by the 57-kilodalton protein. 4. The D according to claim 1, wherein the promotion of DNA synthesis is the promotion of DNA synthesis in hepatocytes.
NA synthesis promoter. 5. The DN according to any one of claims 1 to 4.
A composition for hepatocyte / liver tissue / liver protection and / or liver organ regeneration, which comprises an A synthesis promoter. 6. The composition according to claim 5, which is a medicine or food.