JP3813808B2 - Method for producing antidiabetic agent using kefir - Google Patents

Description

【００４５】
（３）結果
結果を表１に示す。
表１に示すように８グレイでの２倍希釈液投与群から９グレイ以上のすべての群でコントロールに比べて小腸再生腺管数がケフィア投与群で統計的に有意の差で増加しており、ケフィアに放射線障害（小腸腺管死）を防ぐ効果があることが判明した。
表１で、ＭＦは対照群を示す。Ｐは統計上の有意の差があるを０．０５で示し、このＰが小さい程有意の差が大である事を示す。つまり、Ｐが０．０１以下とは有意の差が非常に大きい事を示す。
（４）考察
小腸腺管細胞は活発に分裂し、絨毛を形成するが、放射線により腺管が死滅すると、小腸は絨毛のない状態になり食物中の栄養成分の吸収ができなくなり、出血し生体は死滅する。このような放射線による消化管死を有効に防御できる薬剤もしくは食品はこれまでほとんど知られておらず、放射線障害を防御するケフィアの存在は大である。
【００４６】
（実施例１２）
ここまで、放射線によるマウス小腸腺管死をケフィアが抑制する効果があることを明らかにした。
次にケフィアの放射線防御効果として放射線による骨髄死の防御について実験した。ここでは、骨髄死を抑制できるかどうかを見るために、放射線照射後約１ヶ月間のマウスの生存率を調べた。１ヶ月間放射線照射マウスが生存した場合は骨髄死を免れたと考えることができる。
（１）方法
動物；６週齢雄Ｃｒｊ（チャールスリバー社製）：Ｂ６Ｃ３Ｆ１マウスを使用
（２）ケフィア上清の調製
ケフィア原液を１０，０００×ｇで３時間遠心分離した上清を用いた。
ケフィア上清：１０倍希釈液（×１０）
飲料水として投与（２日ごとに交換）
コントロール（蒸留水）
実験群；
コントロール区 １０匹
８Ｇｙ（グレイ）Ｃｏγ照射線照射区 １０匹
（３）実験方法
ケフィアを１週間前より投与し、コバルトγ線照射後２８日間の生存率を観察する。
観察；朝、昼、夜の一日３回観察
いつ何匹死んだか確認する。もし新鮮な死亡例は剖検し、病理検索を行う。
２８日後に屠殺、解剖時に組織重量、病理検索。
【００４７】
（４）結果
図１５に示すようにコントロール群ではすべてのマウスが２３日後には死んだが、ケフィア１０倍希釈液投与群では３匹が生存し、体重も増加してきて２８日後にも生き残った。
（５）考察
このことから、８Ｇｙの照射線による骨髄死をケフィア投与区の３匹のマウスが免れ、放射線による消化管死だけでなく、骨髄死をもケフィアが防御できることが示唆された。
【００４８】
第２の発明において、上記実施例では、ケフィアは日本ケフィア株式会社供与のケフィア（ケフィア原液）を使用したが、日本で製造、販売されたケフィア菌を使用したケフィアでも、ロシア以外のその他国由来のものでも、あるいはケフィア粒を求めて自分で培養したものを使用しても良い。あるいは濃縮物又は乾燥物を原料として用いることも可能である。本実施例は放射線障害防護剤としての作用について実験したが、放射線障害防護食品についても上記の実施例は適用される。
【００４９】
【発明の効果】
上記実施例に示すように、分子量が１０００以下のケフィア抽出物がグルコース取り込み細胞の活性酸素を低減させ、細胞の酸化ストレスを緩和させ、グルコース取り込みを制御するＰＩ３キナーゼの活性を促進させ、細胞のグルコース取り込みを増強させることにより、生体の恒常性維持をはかり、糖尿病、主としてＩＩ型糖尿病治療に有効であることが判明した。また、ケフィアを用いた糖尿病治療剤において、ケフィアの抽出液（ケフィア抽出物）が水溶性を有し、酸性、あるいは中性で安定、高圧蒸気滅菌にも安定であることが分かった。そして、ケフィアから、遠心分離、デカンテーション又は濾過の公知の方法にて残渣を分離して、その上澄液をゲル濾過、透析の公知の方法によって分子量１０００以下のケフィア抽出液を得ることによって、糖尿病治療剤が製造できることが分かった。
【００５０】
【図面の簡単な説明】
【図１】ケフィア上清（ｐＨ４．０、クロロホルム／メタノール抽出物）のＬ６筋管細胞に対するグルコース取り込み増強活性を測定をした結果を示すグラフである。
【図２】ケフィア上清（ｐＨ４．０、ｐＨ７．０、加熱処理）のＬ６筋管細胞に対するグルコース取り込み増強活性を測定をした結果を示すグラフである。
【図３】ケフィア上清（ｐＨ７．０、透析処理）のＬ６筋管細胞に対するグルコース取り込み増強活性を測定をした結果を示すグラフである。
【図４】ケフィア上清（ｐＨ７．０）のＬ６筋管細胞に対するグルコース取り込み増強活性を測定をした結果を示すグラフである。
【図５】ケフィア上清（ｐＨ４．０、ｐＨ７．０）の未分化Ｌ６細胞に対する細胞内酸化還元状態に与える効果を蛍光試薬ＤＣＦＨ−ＤＡを用いて測定した結果を示すグラフである。
【図６】ケフィア上清（ｐＨ７．０）のＬ６筋管細胞に対するグルコース取り込み増強活性を測定をした結果を示すグラフである。
【図７】ケフィア上清（ｐＨ７．０）のＢＩＯ−ＲＡＤＰ２カラムでゲルろ過を行った結果を示すグラフである。
【図８】ケフィア上清（ｐＨ７．０）のＬ６筋管細胞に対するグルコース取り込み増強活性を測定をした結果を示すグラフである。
【図９】ケフィア上清（ｐＨ７．０）のＬ６筋管細胞に対するグルコース取り込み増強活性を測定をした結果を示すグラフである。
【図１０】ケフィア抽出物の未分化Ｌ６細胞に対する細胞増殖に与える効果を測定した結果を示すグラフである。
【図１１】空腸での放射線照射による細胞のアポトーシスに対するケフィアの効果を示すグラフである。
【図１２】胸腺での放射線照射による細胞のアポトーシスに対するケフィアの効果を示すグラフである。
【図１３】脾臓での放射線照射による細胞のアポトーシスに対するケフィアの効果を示すグラフである。
【図１４】結腸での放射線照射による細胞のアポトーシスに対するケフィアの効果を示すグラフである。
【図１５】ケフィア投与による２８日間生存数（骨髄死）に対する効果を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a blood glucose lowering action or a radiation damage protecting action by kefir, which is a health food.
[0002]
[Prior art]
Kefir is a fermented milk loved by people in the Caucasus region of the Georgian Republic. By accident, lactic acid bacteria and yeast are mixed and fermented with lactic acid and alcohol in cows, goats, and sheep milk stored in goat leather bags. This is what happened. The Caucasus fermented milk kefir is made from stable kefir grains obtained through years of research by the Central Research Institute for Microbiology of the Russian Academy of Sciences, an independent national community, as a bacterial species. It enhances digestive function and is used in medical institutions as a diet for various diseases such as kidney disease and cardiovascular disease.
Kefir grains are sponge cakes of about 1 to 3 cm, and have about 20 types of lactic acid bacteria, yeast and acetic acid bacteria, and are generally called yogurt mushrooms in Japan. Kefir grains are mixed with milk and sealed at room temperature for a predetermined time to obtain kefir mixed with kefir grains (called kefir yogurt), which can be obtained simply by filtering and removing kefir grains. It is also used regularly as a health food by ordinary households.
[0003]
[Problems to be solved by the invention]
On the other hand, in recent years, non-insulin-dependent type II diabetes has been steadily increasing from Western diets, afflicting many Japanese. In this type II diabetes, it is impossible to maintain cellular homeostasis due to the inability to take up glucose in cells, which is the core physiological function of maintaining health, resulting in various complications and serious symptoms. Is not uncommon. In addition, the development of effective medicines for many people with radiation damage caused by accidents at modern nuclear power plants and even radiation damage is still insufficient.
The present invention can enhance glucose uptake of core cells in the treatment of type II diabetes, which accounts for 90% of Japanese diabetes. Pharmaceutical manufacturing method To provide the purpose And
[0004]
[Means for Solving the Problems]
The inventors of the present invention have intensively studied to solve the above problems, and as a result, kefir extract having a molecular weight of 1000 or less has been obtained. As an active ingredient By using it, it has succeeded in relieving cellular oxidative stress by reducing active oxygen, promoting the activity of PI3 kinase that regulates glucose uptake, enhancing cellular glucose uptake, and treating diabetes, mainly type II diabetes Drugs used for Manufacturing method As a result, the first invention has been completed.
[0005]
Diabetes treatment according to the first invention Medicine manufacture The kefir extract used in the preparation has water solubility, is acidic or neutral and stable, and is stable to autoclaving.
Here, acidic or neutral and stable means that a neutralized solution obtained by adding an alkaline solution to the kefir supernatant has activity.
Kefir As an active ingredient The manufacturing method of the used diabetes therapeutic agent is kefir (kefir undiluted solution), a residue is isolate | separated by well-known methods, such as centrifugation, decantation, or filtration, The supernatant liquid (it is called a supernatant) is gel-filtered and dialyzed A kefir extract (an example of an extract) having a molecular weight of 1000 or less obtained by a known method such as As an active ingredient It is characterized by using.
[0006]
The radiation damage protective agent containing kefir as an active ingredient in the second invention is the radiation damage of kefir considered to have many beneficial biological defense functions in addition to the action of reducing oxidative stress by the action of reducing active oxygen according to the first invention. As a result of diligent research by the present inventors for the purpose of application to keratinocytes, kefir has a protective action against apoptotic death of radiation-sensitive organs (bone marrow, spleen, thymus, small intestine, large intestine) that are generated in animals by irradiation. And the present invention was completed.
Here, the protective action of radiation protection agents is to reduce the biological effects of ionizing radiation and reduce radiation damage, reducing the chemical variables that occur following radical formation due to the chemical indirect action of radiation. It is thought that there is an effect. Therefore, it is necessary to administer the protective agent in advance before receiving irradiation.
[0007]
Apoptosis is a physiological process or form of cell death, which is distinguished from necrosis of accidental cell death, and apoptosis is often observed in cell death by irradiation. Therefore, the action of the radiation damage protective agent can also be known by examining the effectiveness of apoptotic death of living cells produced in animals by irradiation. Radiation damage generally varies depending on material factors such as temperature, chemical factors such as oxygen partial pressure and water content, and many other biological factors such as species, cell types, gene structure, cell division cycle, age, and physiological conditions. However, organs with higher cell divisions are more sensitive to radiation, and the effects of radiation protection agents are examined by observing apoptosis in radiation-sensitive organs such as bone marrow, spleen, thymus, small intestine, and large intestine. The unit of radiation used is gray (Gy) of absorbed dose.
[0008]
A health food using kefir, which contains a kefir extract having a molecular weight of 1000 or less as an active ingredient, contains a kefir extract in the ingredient, and thus has an anti-diabetic action and a radiation damage protective action, and an anti-diabetic food It can be used as a radiation protection food.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Kefir is kefir yogurt from which kefir grains are removed (culture medium), and most of its components are water, and it contains protein, sugar, alcohol and fat-soluble lipid in water in a colloidal form. .
The kefir used here was the one provided by Nippon Kefir Co., Ltd. (referred to as kefir stock solution). The fungus species were stored under appropriate conditions from the licensed Intorg company of the Russian Republic, subcultured to activate as necessary, and kefir grains sufficiently activated. If necessary, the solid content of milk is 4-18%, pectin is used as a heat stabilizer, and if the solid content is sterilized and cooled to 30-25 ° C, the mother starter (activated inoculum) 3-5% was added, cultured at an appropriate temperature and time in a fermentation tank, and the lactic acid concentration determined at the end point was used. Depending on the method of experiment, kefir (kefir undiluted solution) was used by sterilizing and bottling the cultured liquid, or freeze-dried and pulverized and packaged.
[0010]
Cells used for measurement of glucose uptake were used as L6 myotubes differentiated into muscle tissue having a function of taking up glucose in response to insulin stimulation in the living body. Differentiation treatment was performed by treating the confluent L6 cells in which the cells were fully grown in a petri dish with a 2% low serum treatment for 1 week.
[0011]
Cellular glucose uptake was performed by incorporating tritium-labeled 2-deoxyglucose into cells that had been treated with the kefir extract, lysing the cells, and then measuring the count value with a scintillation counter.
[0012]
A kefir supernatant prepared by the following method was used. The kefir stock solution was centrifuged at 5,000 × g for 1 hour at 4 ° C. to collect the supernatant, and the supernatant further centrifuged at 10,000 × g for 20 minutes was collected to collect the kefir supernatant 1 (pH 4.0 / kefir). A water extract) was obtained. The kefir supernatant has a pH of about 4.0 at this stage.
[0013]
The freeze-dried powder of kefir stock solution (also called culture supernatant) was suspended in a 10-fold volume of chloroform / methanol (2: 1), stirred at 4 ° C. for 24 hours, and then at 5,000 × g for 1 hour. When centrifuged, water-soluble substances such as sugars and proteins that are not soluble in chloroform precipitate below. Here, the supernatant (kefir chloroform / methanol extract) was collected, chloroform and methanol were removed by a vacuum concentrator, the resulting residue was dissolved in ethanol, and kefir supernatant 2 (kefir chloroform / methanol extract) was obtained. Obtained.
[0014]
The kefir supernatant 1 (pH 4.0) was autoclaved (high-pressure steam sterilization) at 120 ° C. for 20 minutes. Since precipitation occurred by the treatment, the supernatant was collected by centrifugation at 10,000 × g for 20 minutes to obtain Kefir supernatant 3 (heat treatment). The kefir supernatant 1 (pH 4.0) was neutralized with an aqueous sodium hydroxide solution to pH 7.0. Since neutralization caused precipitation, the supernatant was collected by centrifugation at 10,000 × g for 20 minutes to obtain kefir supernatant 4 (pH 7.0).
[0015]
The kefir supernatant 4 (pH 7.0) was dialyzed against water using a dialysis membrane having an exclusion limit molecular weight of 1,000. Since precipitation was generated by dialysis, the supernatant was collected by centrifugation at 10,000 × g for 20 minutes to obtain kefir supernatant 5 (dialysis treatment).
[0016]
【Example】
Hereinafter, the present invention will be described with reference to examples. However, the present invention is not limited to these examples.
[0017]
Example 1
Using rat-derived L6 cells differentiated into myotube cells, add 1/4 volume of kefir supernatant 1 (pH 4.0) and kefir supernatant 2 (kefir chloroform / methanol extract) of the L6 cell culture solution. After time treatment (sterile conditions, 37 ° C., 5% carbon dioxide / 95% air), the effect of kefir extract on glucose uptake was examined. The glucose uptake of L6 cells was measured by incorporating tritium-labeled 2-deoxyglucose into the cells.
[0018]
The result is shown in FIG. The vertical axis shows the amount of 2-deoxyglucose uptake relative to control (no insulin stimulation) as 1. The white of the graph is the result without insulin stimulation, and the black is the result with insulin stimulation.
As is clear from FIG. 1, in the kefir supernatant 1 (pH 4.0), the glucose uptake ability of L6 cells was enhanced. This potentiating effect was observed with or without insulin stimulation. On the other hand, in the kefir supernatant 2 (kefir chloroform / methanol extract), the uptake ability was rather suppressed. This shows that the kefir active ingredient is not fat-soluble but water-soluble.
[0019]
(Example 2)
In the same manner as in Example 1, rat-derived L6 cells differentiated into myotube cells were mixed with 1/4 volume of kefir supernatant 1 (pH 4.0), kefir supernatant 3 (heat treatment), and kefir Sei 4 (pH 7.0) was added, and the effect of kefir extract on glucose uptake was examined by treatment for 4 hours (sterile conditions, 37 ° C., 5% carbon dioxide / 95% air).
[0020]
The result is shown in FIG. The vertical axis shows the amount of 2-deoxyglucose uptake relative to control (no insulin stimulation) as 1. The white of the graph is the result without insulin stimulation, and the black is the result with insulin stimulation.
As is apparent from FIG. 2, the glucose uptake enhancing activity of the kefir supernatant was retained in both the heat treatment at 120 ° C. for 20 minutes and the neutralization treatment to pH 7.0. These facts revealed that the glucose uptake enhancing activity was acidic (pH 4.0), neutral (pH 7.0), and stable in autoclaving at 120 ° C. for 20 minutes.
[0021]
Example 3
Under the same conditions as in Example 1, kefir supernatant 4 (pH 7.0) and kefir supernatant 5 (dialysis treatment) were added, and the effect of kefir extract on glucose uptake was examined.
[0022]
The result is shown in FIG. The vertical axis shows the amount of 2-deoxyglucose uptake relative to control (no insulin stimulation) as 1. The white of the graph is the result without insulin stimulation, and the black is the result with insulin stimulation.
As is clear from FIG. 3, the glucose uptake enhancing activity of the kefir supernatant disappeared by dialysis treatment. From this, it was considered that the glucose uptake enhancing activity was a size passing through a dialysis membrane having an exclusion limit molecular weight of 1,000.
[0023]
Example 4
Kefir supernatant 4 (pH 7.0) was added under the same conditions as in Example 1, except that the treatment time was changed to 72 hours, and the glucose uptake enhancing activity of the kefir extract was examined.
[0024]
The result is shown in FIG. The vertical axis shows the amount of 2-deoxyglucose uptake relative to control (no insulin stimulation) as 1. The white of the graph is the result without insulin stimulation, and the black is the result with insulin stimulation.
As is clear from FIG. 4, the glucose uptake enhancing activity of the kefir supernatant increased linearly until 48 hours and thereafter reached a plateau (plane). From this, it was revealed that the glucose uptake enhancing activity increased in a time-dependent manner up to 48 hours, and then the effect was stably maintained.
[0025]
(Example 5)
Kefir supernatant 1 (pH 4.0) and kefir supernatant 4 (pH 7.0) were added to undifferentiated L6 cells, and the activity of inducing the intracellular redox state of the kefir extract to the reduced state was examined.
That is, kefir extract is added to L6 cells in a semi-confluent state (cells are grown to about half in a petri dish) in a 35 mm culture dish for 5 to 30 minutes, and after treatment, the cells are washed with HBSS (Hanks buffered saline). 5 μM fluorescent reagent DCFH-DA (dichlorofluoresce Inn Treatment with HBSS containing diacetate) for 5 minutes, followed by washing twice with HBSS, followed by DCF (dichlorofluorescein) in the cells using a laser biological microscope Inn ) Was measured and digitized, and used as an index of the redox state of the cells.
[0026]
The result is shown in FIG. The vertical axis shows the fluorescence intensity of DCF, which reacts with intracellular hydrogen peroxide and emits fluorescence, as a relative value with control (kefir extract untreated) as 1. The horizontal axis indicates the processing time. The white line in the graph is the result of kefir supernatant 1 (pH 4.0), and the black color is the result of kefir supernatant 4 (pH 7.0).
As is clear from FIG. 5, regardless of the pH state, the fluorescence intensity of DCF in cells treated with kefir supernatant 1 (pH 4.0) and kefir supernatant 4 (pH 7.0) decreased rapidly. The kefir extract was found to have an activity to quickly shift the intracellular redox state to the reduced state.
This phenomenon is thought to be due to a decrease in intracellular active oxygen. On the other hand, since the glucose uptake ability of cells is inhibited by oxidative stress due to active oxygen, this decrease in active oxygen is added to the action of the glucose uptake enhancing activity of the kefir supernatant. It may be involved.
[0027]
(Example 6)
Under the same conditions as in Example 1, kefir supernatant 4 (pH 7.0) was added to cells that had been treated with wortmannin, a PI3 kinase inhibitor, in advance and suppressed PI3 kinase, and kefir extract increased glucose uptake activity. I investigated.
[0028]
The result is shown in FIG. The vertical axis shows the amount of 2-deoxyglucose uptake relative to control (no insulin stimulation) as 1. The white of the graph is the result without insulin stimulation, and the black is the result with insulin stimulation.
As apparent from FIG. 6, the glucose uptake enhancing activity of the kefir extract was suppressed depending on the wortmannin concentration.
From these results, it is considered that the kefir extract activates the upstream side including PI3 kinase of the insulin signal transduction pathway centering on PI3 kinase that regulates cellular glucose uptake, and enhances the glucose uptake enhancing activity of the cell. It was.
[0029]
(Example 7)
50 ml of kefir supernatant 4 (pH 7.0) was applied to a P2 gel filtration column (bed 800 ml, flow rate 240 ml / hour) manufactured by BIO-RAD, and 6 fractions including a flow-through fraction (molecular weight 1,800 or more) ( Kefir P2 fraction) was collected. The results are shown in FIG. The vertical axis indicates absorbance, and the horizontal axis indicates elution time. The solid line of the graph indicates the absorbance at 280 nm, and the absorbance increases with the protein component. From FIG. 7, it can be seen that fractions 4 and 5 contain a lot of proteins that hinder the activity, and fraction 3 has a low protein content.
[0030]
Under the same conditions as in Example 1, the prepared kefir P2 fraction was added, and the effect of the kefir extract on glucose uptake was examined. The result is shown in FIG. The vertical axis shows the amount of 2-deoxyglucose uptake relative to control (no insulin stimulation) as 1. The white of the graph is the result without insulin stimulation, and the black is the result with insulin stimulation.
As apparent from FIG. 8, the kefir P2 fraction 3 had the highest glucose uptake enhancing activity.
[0031]
(Example 8)
10 ml of the previously prepared kefir P2 fraction 3 was applied to a DEAE-Toyopearl anion exchange column (bed 10 ml) manufactured by TOHSO Corporation, and the passed fraction was fractionated (DEAE column passing fraction). Since the anion in kefir P2 fraction 3 is adsorbed on the anion exchange column, the passed fraction contains cations or uncharged substances in kefir P2 fraction 3.
Next, the anion exchange column adsorbing the anion of kefir P2 fraction 3 was washed with a sodium phosphate buffer, and then the fraction eluted with 150 mM NaCl was fractionated (DEAE column 150 mM NaCl elution fraction). Here, the anion adsorbed on the anion exchange column is substituted with NaCl chloride ion, and the DEAE column 150 mM NaCl elution fraction contains an anion.
[0032]
Under the same conditions as in Example 1, the effects of the kefir extract on glucose uptake were examined for the DEAE column flow-through fraction and the DEAE column 150 mM NaCl elution fraction.
The result is shown in FIG. The vertical axis shows the amount of 2-deoxyglucose uptake relative to control (no insulin stimulation) as 1. The white of the graph is the result without insulin stimulation, and the black is the result with insulin stimulation.
As apparent from FIG. 9, the glucose uptake enhancing activity was retained in the fraction eluted with 150 mM NaCl in the DEAE column. Therefore, it can be seen that the DEAE column NaCl elution fraction containing anions in kefir P2 fraction 3 contains a glucose uptake enhancing substance. Therefore, it can be seen that the anionic substance in kefir P2 fraction 3 has a glucose uptake enhancing action.
[0033]
(Example 9 / reference test)
The effect of kefir supernatant (kefir extract) treatment on cell proliferation when undifferentiated L6 cells were cultured was examined. As the culture solution, the culture solution in which the above L6 cells were cultured was used. Cells are seeded on the day before treatment, and 1/4 volume of supernatant 1 or supernatant 4 of L6 cell culture is added, treated for 4 hours, washed with phosphate buffered saline, and then put into normal culture medium The cells were exchanged and the cell number was measured.
[0034]
The results are shown in FIG. The vertical axis represents the number of cells, and the horizontal axis represents the culture time. The white of the graph is the control, and the black is the result of the kefir extract.
As shown in FIG. 10, treatment with kefir extract for 4 hours had no effect on cell growth.
Therefore, it can be understood that the glucose uptake enhancing action of the kefir extract is not the growth action of glucose uptake cells.
[0035]
As described above, the active substance contained in the kefir extract is water-soluble, stable to alkali treatment, stable by high-pressure steam sterilization treatment at 120 ° C. for 20 minutes, freezing treatment, and acid treatment, but not extracted by chloroform / methanol. In other words, it has a property that is lost by dialysis using a dialysis membrane having an exclusion limit molecular weight of 1,000. Furthermore, it can be seen that the cellular glucose uptake enhancing action of the kefir extract is due to the reduction of the amount of active oxygen in the cell, the reduction of oxidative stress in the cell, and the promotion of PI3 kinase activity.
In addition, the effect of kefir extract to enhance glucose uptake of cells is increased by adding glucose to the cell culture over a period of 4 hours regardless of the presence of insulin stimulation, and further enhanced by prolonged treatment for 72 hours. I understand that
[0036]
From the above results, the kefir extract is separated from the residue by known methods such as centrifugation, decantation (tilting the supernatant) and filtration of the kefir (kefir stock solution), and the supernatant (supernatant) is adjusted to pH. Neutralize with sodium hydroxide until is neutral. The supernatant (supernatant) obtained by separating the precipitate that emerged by neutralization again by a known method such as centrifugation, decantation, or filtration is subjected to gel filtration, and a fraction having a molecular weight of 1,000 or less. By obtaining a kefir extract, a therapeutic agent for diabetes with a strong active effect can be produced.
[0037]
In the above examples, kefir provided by Nippon Kefir Co., Ltd. was used as it was to produce kefir extract, but kefir produced and sold in Japan was also used, even from other countries other than Russia. Or you may use what was cultivated by oneself in search of kefir grains.
In this example, kefir was used after sterilizing and bottling the cultured liquid, or freeze-dried and pulverized and stored in a package. Is As long as the dried product or other shapes are kefir, any shape can be used as a raw material. For the concentration or drying, a known method such as freeze concentration, freeze drying, vacuum concentration, vacuum drying, foam drying, or the like is used.
[0038]
Although the above examples were tested for action as a therapeutic agent for diabetes, health foods containing kefir extract with a molecular weight of 1,000 or less also have antidiabetic action from the above examples, and antidiabetic foods When I understand that
In addition, health foods using a kefir extract having a molecular weight of 1,000 or less can be added with other nutrients and substances to increase the nutritional value and to expect further effects in order to make it tasty.
[0039]
Next, the radiation damage protection action of the kefir extract according to the second invention will be described below. To examine whether kefir is effective in suppressing radiation-induced apoptosis in vivo, the amount of apoptosis induced by X-ray irradiation in the small intestine (jejunum), large intestine (colon), spleen, thymus, and bone marrow was quantified. . That is, after administration of kefir to rats, the effect of kefir on radiation damage in vivo was examined by examining the appearance of apoptosis in a short time after irradiation with a low dose in each organ of the rat and the change in LD50 value. Since radiation-induced damage to the digestive tract appeared immediately after irradiation, X-ray irradiation was observed in the rat organs 2 hours later. Since the effect on the bone marrow appears after about 30 days and survived after about 30 days, bone marrow death usually does not appear. Therefore, the number of survival after about 30 days was examined.
[0040]
Examples are shown below.
As kefir, kefir (kefir stock solution) provided by Nippon Kefir Co., Ltd. was used, and the supernatant was prepared by centrifugation and administered to rats.
[0041]
(Example 10)
An experiment was conducted using a kefir supernatant prepared below using male Wistar rats, 7 weeks old.
(1) Preparation of kefir supernatant
The kefir stock solution (kefir culture solution) was centrifuged at 10,000 × g for 2 hours, and then the supernatant was collected, added to a filtration device with Celite on the filter paper, filtered by suction, and the supernatant was 0.22 μm. And then sterilized by filtration to obtain a kefir supernatant.
(2) Administration to rats
The prepared supernatant was diluted 2-fold, placed in a drinking bottle and allowed to drink freely for 14 days. The kefir-administered group and the control group consisted of 3 to 5 animals per dose.
(3) X-ray irradiation
Rats were subjected to X-ray whole body irradiation using Toshiba EXS-300.
(200 KV, 15 mA condition, 0.5 mm Cu + 0.5 mm aluminum filter, dose rate 0.443 Gy / min) was used.
The kefir administration group and the control group were irradiated with 0.25 Gy, 0.5 Gy and 1 Gy.
(4) Irradiation and apoptosis count
Two hours after X-ray irradiation, the jejunum, colon, spleen, and thymus were removed from rats sacrificed under anesthesia and fixed in formalin. The jejunum and colon were cut in the long axis direction, both spleen and thymus were embedded in paraffin, and sliced into 3 μm sections. H & E (hematoxylin and eosin) staining was performed, and apoptotic cells in which the cells were condensed and fragmented were counted. The jejunum and colon were counted for apoptosis per crypt under a 400 × microscope, and the average value was used as the apoptosis index. More than 50 crypts were counted per rat. For the spleen and thymus, the number of apoptosis per total number of lymphocytes in one visual field was counted at a magnification of 1000 times for the white spleen and splenic cortex regions of the spleen, and the apoptosis rate (%) was used as the apoptosis index.
(5) Statistical processing
The experimental results are expressed as mean ± standard deviation, and Mann-Whitney U-test was used for the significance test.
[0042]
(6) Results
As shown in FIG. 11, in the jejunum, no significant difference was observed between the control group and the kefir group in the number of apoptosis per crypt at any dose of 0.25 Gy, 0.5 Gy, and 1 Gy.
As shown in FIGS. 12, 13, and 14, in the thymus, spleen, and colon, the ratio of the apoptosis index to the number of lymph was significantly suppressed at 1 Gy.
Moreover, it became clear that apoptosis by radiation damage of thymus and spleen cells can be significantly controlled at low doses (0.25 and 0.5 Gy).
Therefore, it was found that the number of apoptosis after X-body irradiation of 1 Gy or less was significantly suppressed in the thymus, spleen, and colon of kefir-administered rats. At doses of 1 Gy or less, kefir is thought to suppress cell radiation damage in some way in the thymus, spleen, and colon.
(7) Consideration
The thymus is an organ that produces T cells that play an important role in the immune system, and the spleen is an organ that plays an important role in the proliferation and functional expression of lymphocytes. Thus, it was suggested that kefir can prevent the decline in immunity caused by radiation damage and protect against various acute and chronic plagues caused by radiation.
[0043]
(Example 11)
Next, as a radiation protection effect of kefier, an experiment was conducted to protect against death of the small intestine, which is a highly sensitive organ of radiation.
Its purpose is acute radiation injury, and bone marrow death can be effectively prevented by bone marrow transplantation, but there are few effective techniques to prevent gastrointestinal death. Therefore, kefir was administered to mice to examine whether kefir could protect against glandular death caused by radiation in the small intestine.
(1) Method
Animal; 6-week-old male Crj (manufactured by Charles River): B6C3F1 mice used
After the stock solution was diluted 2 times (× 2), 4 times diluted solution (× 4), and 10 times diluted solution (× 10) were each administered as drinking water from 1 week before X-ray irradiation until the end of the experiment.
Distilled water was used as a control group. Change every 2 to 3 days and measure water intake.
Bait; MF bait manufactured by Oriental Yeast Co., Ltd. (measurement of intake)
X-ray irradiation; no filter / 450 cGy (centimeter gray) / body dose at a dose rate of 1 minute
Irradiation amount: 0, 7, 8, 9, 10, 12 Gy
Number of mice: 5 mice in each irradiation group. At the time of irradiation, two animals of each group are put and irradiated.
Slaughter; sacrificed by cervical dislocation 3 days after irradiation. After weighing, take out the small intestine, wash the contents, and fix with Carnoy's solution. H & E staining is performed and the number of regenerated gland ducts (crypts) in the small intestine is measured.
[0044]
[Table 1]

[0045]
(3) Results
The results are shown in Table 1.
As shown in Table 1, the number of small intestinal regenerating gland ducts increased in the kefir-administered group with a statistically significant difference from the control group in all groups of 9-gray or more from the 2-fold diluted solution with 8 gray. It was found that kefir is effective in preventing radiation damage (small intestinal gland death).
In Table 1, MF indicates a control group. P indicates that there is a statistically significant difference, and 0.05 indicates that the smaller the P, the greater the significant difference. That is, a significant difference from P of 0.01 or less is very large.
(4) Consideration
Small intestinal gland duct cells actively divide and form villi, but when the gland ducts are killed by radiation, the small intestine becomes villus-free and cannot absorb nutrients in food, bleeds and the living body dies. Few drugs or foods that can effectively protect against radiation-induced gastrointestinal death have been known so far, and the presence of kefir that protects against radiation damage is significant.
[0046]
(Example 12)
So far, it has been clarified that kefir has the effect of suppressing radiation-induced death of the mouse small intestinal gland.
Next, as a radiation protection effect of kefir, an experiment was carried out to prevent bone marrow death by radiation. Here, in order to see whether bone marrow death can be suppressed, the survival rate of mice during about one month after irradiation was examined. It can be considered that bone marrow death was avoided when the irradiated mouse survived for one month.
(1) Method
Animal; 6-week-old male Crj (manufactured by Charles River): B6C3F1 mice used
(2) Preparation of kefir supernatant
The supernatant obtained by centrifuging the kefir stock solution at 10,000 × g for 3 hours was used.
Kefir supernatant: 10-fold diluted solution (× 10)
Administer as drinking water (change every 2 days)
Control (distilled water)
Experimental group;
10 controls
8Gy (gray) Coγ irradiation irradiation group 10
( 3 )experimental method
Kefir is administered from one week before, and the survival rate for 28 days after the cobalt γ irradiation is observed.
Observation: Observation three times a day in the morning, noon and night
Check how many animals died. If a fresh death occurs, autopsy is performed and pathological search is performed.
Slaughtered 28 days later, tissue weight, pathological search at dissection.
[0047]
( 4 )result
As shown in FIG. 15, in the control group, all mice died after 23 days, but in the kefir 10-fold diluted solution administration group, 3 mice survived and their body weight increased and survived after 28 days.
( 5 Consideration
This suggested that three mice in the kefir-administered group were spared from bone marrow death caused by 8 Gy irradiation, and kefir could protect against bone marrow death as well as gastrointestinal death due to radiation.
[0048]
In the second embodiment, in the above example, the kefir used is kefir (kefir stock solution) provided by Nippon Kefir Co., Ltd. However, even kefir using kefir bacteria manufactured and sold in Japan is derived from other countries other than Russia. Or kefir grains that have been cultured on their own may be used. Alternatively, a concentrate or a dried product can be used as a raw material. Although the present Example experimented about the effect | action as a radiation damage protection agent, said Example is applied also to a radiation damage protection food.
[0049]
【The invention's effect】
As shown in the above examples, a kefir extract having a molecular weight of 1000 or less reduces the active oxygen of glucose uptake cells, relieves cellular oxidative stress, promotes the activity of PI3 kinase that controls glucose uptake, By enhancing glucose uptake, it was found that the homeostasis of the living body was maintained, and that it was effective for treating diabetes, mainly type II diabetes. In addition, in a therapeutic agent for diabetes using kefir, kefir extract (kefir extract) But It was found to be water-soluble, acidic or neutral and stable, and stable to high-pressure steam sterilization. And from kefir, centrifuge, decantation or filtration of The residue is separated by a known method, and the supernatant is subjected to gel filtration and dialysis. of It was found that a therapeutic agent for diabetes can be produced by obtaining a kefir extract having a molecular weight of 1000 or less by a known method.
[0050]
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the results of measuring the glucose uptake-enhancing activity of Kefir supernatant (pH 4.0, chloroform / methanol extract) on L6 myotube cells.
FIG. 2 is a graph showing the results of measurement of the glucose uptake enhancing activity for L6 myotubes of kefir supernatant (pH 4.0, pH 7.0, heat treatment).
FIG. 3 is a graph showing the results of measuring the glucose uptake enhancing activity of Kefir supernatant (pH 7.0, dialysis treatment) on L6 myotube cells.
FIG. 4 is a graph showing the results of measuring the glucose uptake enhancing activity of Kefir supernatant (pH 7.0) on L6 myotube cells.
FIG. 5 is a graph showing the results of measuring the effect of kefir supernatant (pH 4.0, pH 7.0) on the intracellular redox state of undifferentiated L6 cells using a fluorescent reagent DCFH-DA.
FIG. 6 is a graph showing the results of measuring the glucose uptake enhancing activity of Kefir supernatant (pH 7.0) on L6 myotube cells.
FIG. 7 is a graph showing the results of gel filtration using a Kefir supernatant (pH 7.0) with a BIO-RADP2 column.
FIG. 8 is a graph showing the results of measuring the glucose uptake enhancing activity of Kefir supernatant (pH 7.0) on L6 myotube cells.
FIG. 9 is a graph showing the results of measuring the glucose uptake enhancing activity of Kefir supernatant (pH 7.0) on L6 myotube cells.
FIG. 10 is a graph showing the results of measuring the effect of kefir extract on cell proliferation of undifferentiated L6 cells.
FIG. 11 is a graph showing the effect of kefir on cell apoptosis by irradiation in the jejunum.
FIG. 12 is a graph showing the effect of kefir on cell apoptosis by irradiation in the thymus.
FIG. 13 is a graph showing the effect of kefir on cell apoptosis due to irradiation in the spleen.
FIG. 14 is a graph showing the effect of kefir on cell apoptosis upon irradiation in the colon.
FIG. 15 is a graph showing the effect on the number of 28-day survival (bone marrow death) by kefir administration.

Claims (1)

A residue is separated from kefir by a known method such as centrifugation, decantation or filtration, and the supernatant is obtained to obtain a kefir extract having a molecular weight of 1000 or less by a known method of gel filtration or dialysis. The manufacturing method of the diabetes therapeutic agent which contains the kefir extract whose molecular weight is 1000 or less as an active ingredient .

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