JP2010195720A - Tripeptide having osteocyte proliferating action and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tripeptide which has weak side effects and osteocyte proliferating actions and a method for producing the same. <P>SOLUTION: The tripeptide having osteocyte proliferating actions is a tripeptide having a structure of Lys-Glu-Ile and osteocyte proliferating actions and all amino acids are L-amino acids and are bonded by a peptide bond. The method for producing the tripeptide comprises a step of adding a protease to an egg membrane obtained from a fish belonging to genus Gadus, Gadidae family, and Clupeidae family and heating the resulting mixture. The amount of the protease added is preferably 0.03-0.5 times the weight of the egg membrane, and the treating temperature is preferably 20-40°C. Furthermore, the obtained tripeptide Lys-Glu-Ile having osteocyte proliferating actions is utilized as an active ingredient of a food preparation, a cosmetic preparation, and a pharmaceutical preparation. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a tripeptide having a bone cell proliferation action and a method for producing the same.

In recent years, the number of patients with osteoporosis and arthritis has been increasing in Japan, and it has become a social problem related to a decrease in the working population and an increase in medical expenses. Possible causes of osteoporosis include destruction of bone tissue due to lack of sex hormones, muscle atrophy, and weakening of joints.

One of the causes of arthritis is destruction of bone tissue, which is caused by inhibition of bone cell proliferation and degeneration.

Various treatment methods have been developed for the purpose of treating osteoporosis. As pharmacotherapy, hormonal treatment such as estrogen preparations, intake of calcium preparations, and surgical treatment are performed. However, a complete treatment method has not yet been established.

For example, in the case of hormonal treatment such as estrogen preparations, estrogen has a risk of developing breast cancer because of its proliferative effect on the mammary gland.

Furthermore, in the case of calcium preparations, the burden on the kidney is large, and it has not been used for a long time.

There are few examples of inventions derived from natural products having a bone cell proliferation action, and there are examples of collagen production promoters, cosmetics, foods and pharmaceuticals containing the same, and external preparations for preventing or ameliorating skin diseases (for example, see Patent Document 1). .)

In addition, there are inventions of collagen production promoters, functional foods containing them, and pharmaceuticals, but their effects are limited (see, for example, Patent Document 2).

Therefore, this time, a tripeptide derived from a natural product and a method for producing the same will be described for the purpose of improving osteoporosis.
JP 2003-137807 A JP 2002-255847

As described above, the estrogen preparation for osteoporosis has a risk of side effects, and the calcium preparation may have a problem of side effects on the kidneys.

The present invention has been made paying attention to the problems existing in the prior art as described above. The object is to provide a tripeptide with low side effects and showing an excellent bone cell proliferation action.

Moreover, it is providing the efficient manufacturing method of the tripeptide which shows a bone cell proliferation effect | action.

In order to achieve the above object, the invention described in claim 1 relates to a tripeptide having a Lys-Glu-Ile structure and a bone cell proliferating action.

The invention according to claim 2 is the efficiency of a tripeptide having a bone cell proliferating action comprising a step of adding a protease to an egg membrane obtained from any of the fishes of the cod family, cod family, and herring family, and heating it. It relates to a typical manufacturing method.

Hereinafter, the best mode for carrying out the present invention will be described in detail.

First, the tripeptide having the bone cell proliferation action of the present embodiment has a structure of Lys-Glu-Ile.

Lys is L-type lysine. Glu is L-type glutamic acid. Ile is L-type isoleucine. These amino acids are linked by peptide bonds.

The bone cell proliferation action here is an action that activates the proliferation of bone cells, and has a function of repairing bone tissue in the case of osteoporosis or arthritis.

Since this tripeptide consisting of Lys-Glu-Ile acts on both the beta-type estrogen receptor and bFGF (basic growth factor) receptor of bone cells, it is effective against various types of osteoporosis and arthritis. It is.

That is, in menopause-type osteoporosis, sex hormones such as estrogen are decreased, and this tripeptide activates a beta-type estrogen receptor to proliferate bone cells.

In senile osteoporosis and inflammatory arthritis, bFGF decreases and bone cells decrease, so this tripeptide activates the bFGF receptor to proliferate bone cells.

Since this tripeptide acts on the beta-type receptor of estrogen, it is highly safe. That is, since breast cancer and uterine cancer develop by acting on alpha-type estrogen receptor, this tripeptide does not act on alpha-type estrogen receptor, and there is little risk of developing breast cancer and uterine cancer.

This tripeptide can be produced by chemical or biochemical synthesis. Further, it can be produced by decomposing with a protease from peptides such as soybean, milk and egg.

This tripeptide is used therapeutically for bone abnormalities such as osteoporosis, arthritis, and hypogrowth.

It is used as a pharmaceutical orally or parenterally, and as a quasi-drug, it is used by blending with tablets, capsules, drinks, soaps, toothpastes and the like.

Examples of oral preparations include tablets, capsules, powders, syrups, and drinks. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, excipient, swelling agent, lubricant, sweetener, flavoring agent and the like.

The tablets can also be coated with shellac or sugar. Moreover, in the case of the said capsule, liquid carriers, such as fats and oils, can be further contained in said material. In the case of the above syrup and drink, sweeteners, preservatives, pigment flavoring agents and the like can be contained.

Next, a method for producing a tripeptide having a bone cell proliferating action, comprising a step of adding a protease to an egg membrane obtained from any one of the fishes of the cod family, cod family, and herring family and heating it, will be described.

The tripeptide having a bone cell proliferation action has a structure of Lys-Glu-Ile.

The raw material is egg membrane obtained from any fish of the cod family, cod family, or herring family. The country of origin of raw materials is good in any country. Since it is fresh, it is preferably collected in the suburbs of Japan.

As the fish of the family Madaraceae, the Madara scientific name Godus macrocephalus is preferable because it has abundant catch, collects madaraco as an egg and ingests it as food, and the safety of the hen's egg membrane is also confirmed.

As the fish of the cod family, walleye pollack, komai, and cod are used. Of these, the walleye pollack scientific name Theragra charcogram is preferable because it has abundant catch, collects octopus as an egg, and ingests it as food, and the safety of the octopus egg membrane is also confirmed.

Herring family name is used in herring family name, Cupea pallasii, and the catch is abundant, casks are collected as eggs and consumed as food, and the safety of casserole egg membrane is confirmed. ,preferable.

The trapped fish can be frozen or cryopreserved and then thawed and broken into egg membranes.

The egg membrane is washed with clean water or seawater after removing blood and deposits. If washing is insufficient, the efficiency of the subsequent protease degradation process may be reduced.

The egg membrane is preferably collected and then stored frozen or at a low temperature. When harvested in large quantities, cryopreservation is preferred.

Since the efficiency of the manufacturing process is good, the egg membrane is preferably pulverized with a pulverizer or the like, and the size of the pulverized product is preferably 10 to 10,000 μm.

As the protease, any of neutral, acidic, and basic proteases can be used. Neutral protease is preferable because it eliminates the need for a neutralization step as compared with acidic and basic proteases.

As the neutral protease, protease A, protease N, protease M, Sumiteam FP, Sumiteam LP, and Denateam AP are preferable from the viewpoint of stability to heat, and in particular, protease N manufactured by Amano Enzyme from the viewpoint of high processing ability. Sumiteam LP is more preferable. These proteases can also be used in combination.

The amount of the protease added is preferably 0.03 to 0.5 times, more preferably 0.05 to 0.4 times, and 0.08 to 0.3 times the weight of egg membrane. Is more preferable.

When the amount of the protease added is less than 0.03 times the amount, the protease treatment may not be sufficiently performed. When the amount exceeds 0.5 times the amount, the protease is expensive, so the cost is high. There is a risk.

The protease is added and warmed. The heating temperature is preferably 21 to 48 ° C. When this processing temperature is lower than 21 ° C., the treatment with protease may not proceed.

Moreover, when this heating temperature exceeds 48 degreeC, there exists a possibility that the peptide and protein derived from an egg membrane may change and the function as a peptide may fall.

The protease treatment is performed in a stirring state for efficient execution of the treatment. The stirring speed is preferably 10 to 100 times / minute, more preferably 30 to 80 times / minute, and further preferably 30 to 70 times / minute.

After heating with the protease, it is filtered. Filtration with filter paper is used for filtration, and suction filtration is preferable from the viewpoint that time can be shortened.

The filtered liquid is preferably cooled after being boiled at 80 to 95 ° C. for 5 to 20 minutes. If the boiling temperature is below 80 ° C., protease inactivation may not be performed. Moreover, when it exceeds 95 degreeC, there exists a possibility that the activity of the obtained tripeptide may fall.

If the boiling time is less than 5 minutes, protease inactivation may not be performed. Moreover, when it exceeds 20 minutes, there exists a possibility that the activity of the obtained tripeptide may fall.

The heated liquid is stored at 1 to 15 ° C. This process has the purpose of inactivating, disinfecting proteases and eliminating unstable products. That is, when a substance that cannot be decomposed or an unstable substance is left at a low temperature, it may precipitate. These insolubles are excluded.

The standing time at the above temperature is preferably 1 to 18 hours, more preferably 2 to 10 hours, and further preferably 3 to 8 hours.

As described above, after being left to stand, it is filtered. Filtration with filter paper is used for filtration, and suction filtration is preferable from the viewpoint that time can be shortened.

The filtrate obtained by the filtration described above is a liquid, and a tripeptide is obtained in a concentrated solution and lyophilized state. The resulting tripeptide is a mixture of various peptides. From the viewpoint of reducing the capacity and maintaining stability, lyophilization is preferred.

Furthermore, it is preferable to store it frozen or at low temperature from the viewpoint of maintaining stability. In this way, an extract containing the tripeptide Lys-Glu-Ile is obtained.

Furthermore, it is preferable because the tripeptide Lys-Glu-Ile is obtained by separating and purifying the tripeptide Lys-Glu-Ile. The above tripeptide is mixed with an extraction solvent, and the extract extracted with the extraction solvent is applied to a separation carrier or resin and eluted with the separation solvent.

Hereinafter, the embodiment will be specifically described with reference to examples and test examples.

After harvesting madara harvested in the Sea of Japan, it was dismantled and tarako was collected with the egg membrane attached. The egg membrane was extracted from this octopus and washed with tap water. The egg membrane washed with water was pulverized with a mixer (high-speed pulverizer, manufactured by Nihon Leeing).

5 kg of tap water was added to 1 kg of the pulverized product, and 45 g of protease N (manufactured by Amano Enzyme) was further added. The mixture was warmed at 38 ° C. for 2 hours and stirred at an average speed of 50 times / minute. The obtained solution was filtered with a filter paper (No. 2 manufactured by Toyo Roshi Kaisha, Ltd.) to obtain the obtained filtrate.

The filtrate was boiled at 92 ° C. for 10 minutes and cooled. This cooled liquid was filtered again with the above filter paper, and the filtrate was freeze-dried with a freeze dryer (manufactured by Toyo Riko) to obtain 34 g of powder.

A tripeptide having bone cell proliferation action was separated from the powder and purified. That is, the extract was subjected to a device equipped with a DEAE cellulose (Wako Pure Chemical) packed column (3.5 cm diameter, 50 cm length), washed with water, and separated by flowing a 1M sodium chloride aqueous solution. Furthermore, this fraction was subjected to Sepharose, flushed with water, and lyophilized to obtain 1.3 g of the desired fraction. This was designated as Sample 1.

The obtained fraction was subjected to high performance liquid chromatography (HPLC) to analyze the target tripeptide. In addition, for the detection of a fraction having a bone cell proliferation action, the proliferation property of mouse bone cells described below was used as an index.

Below, the analysis of the tripeptide which has a bone cell proliferation effect | action by a high performance liquid chromatography (HPLC) and a nuclear magnetic resonance apparatus (NMR) is demonstrated.
(Test Example 1)

In this test, a tripeptide having a bone cell proliferation action was analyzed. That is, the specimen 1 obtained in Example 1 and the tripeptide Lys-Glu-Ile chemically synthesized by Takara Bio were subjected to analysis by HPLC equipped with a photodiode array (manufactured by Shimadzu Corporation).

As a result, it was found that the tripeptide Lys-Glu-Ile having a bone cell proliferation action was present in the fraction having a molecular weight of 200 to 500 in the specimen 1. It was identical to the analysis result of the chemically synthesized tripeptide Lys-Glu-Ile.

Sample 1 was subjected to HPLC using a CAPCELLPACK C18 column. Furthermore, as a result of analysis by NMR (manufactured by Varian), Lys-Glu-Ile was identified.

Hereinafter, detection of bone cell proliferation action using mouse-derived bone cells will be described.
(Test Example 2)

A pregnant mouse on the 17th day of gestation was euthanized and the uterus was removed. Fetuses were collected from this, and bone tissue was separated by collagenase treatment.

10,000 bone cells were seeded in a 35 mm diameter petri dish and cultured at 37 ° C. under 5% carbon dioxide using a culture solution (MEM medium containing 10% fetal bovine serum). After culturing for 2 days, the sample 1 aqueous solution obtained in Example 1 above was added at a final concentration of 0.1 mg / mL under conditions not containing estrogen, cultured, and the number of cells 48 hours after addition was measured. Counted.

As a result, when Sample 1 was added, the growth rate was 192% compared to the control group to which the solvent was added, and the bone cells showed growth.

The tripeptide exhibiting the bone cell proliferation action and the method for producing the same of the present invention improves the QOL of patients suffering from bone diseases such as osteoporosis, arthritis, and bone disorders. And contribute to the medical field, food industry and pharmaceutical industry.

Egg membranes of fish such as cod are discarded as industrial waste, and there is a risk of polluting the environment. By effectively using this waste, it contributes to the improvement of the environment.

Claims (2)

Tripeptide having a bone cell proliferating action having a structure of Lys-Glu-Ile 2. The method for producing a tripeptide having bone cell proliferation activity according to claim 1, comprising a step of adding a protease to an egg membrane obtained from a fish of the family Spotted family, cod family, and herring family and heating