JP2005073502A - Food or food material having apoptosis-inducing ability - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an excellently safe food or food material having an apoptosis-inducing ability, obtained from an extract from Sarcodon aspretus (Berk.) S. Ito which is one kind of edible mushrooms, having slight adverse effects and useful for suppression or prophylaxis of various cancer diseases including leukemia. <P>SOLUTION: The food or food material is composed of the extract from the Sarcodon aspretus (Berk.) S. Ito obtained by extracting the Sarcodon aspretus (Berk.) S. Ito and has the apoptosis-inducing ability. The food or food material is composed of ergosterol peroxide derived from the Sarcodon aspretus (Berk.) S. Ito and has the apoptosis-inducing ability. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a food or a food material having an apoptosis-inducing ability, and in particular, is obtained from a mushroom extract, which is a kind of edible mushroom, and has an apoptosis-inducing ability effective for the suppression or prevention of various cancers including leukemia. It relates to food or food materials.
[0002]
[Prior art]
In recent years, for the purpose of maintaining health and preventing illness through daily eating habits, many studies have been made on ingredients that exhibit “functions that contribute to maintaining physical condition (tertiary functions of foods)” of foods. Their research extends to various agricultural, forestry and fishery ingredients (lipids, proteins, carbohydrates, minerals, etc.).
[0003]
On the other hand, the proportion of cancer (malignant neoplasm) in human death is high and is the first cause of death in Japan. Among the tertiary functions of food, what can be expected to have cancer prevention / treatment effects is the induction of apoptosis of cancer cells (apoptosis).
Apoptosis is phagocytosed and removed by macrophages and the like through reduction, fragmentation of DNA in nucleosome units, formation of apoptotic bodies, etc., by planned signal transduction after cells receive apoptosis-inducing stimulation. For this reason, the cell contents do not leak out and the surrounding cells / tissues are not inflamed like necrosis (necrosis).
[0004]
It is known that some anticancer drugs and substances showing anticancer activity currently used in clinical practice induce apoptosis in cultured cells, and apoptosis induction is thought to lead to cancer suppression. Yes. In particular, for the treatment of hematopoietic tumors such as leukemia, eradication of hematopoietic tumor cells is the basic principle of treatment, and the treatment with chemotherapeutic agents and radiation is derived from inducing apoptosis in hematopoietic tumor cells. Yes.
[0005]
As described above, since apoptosis induction is recognized as a safe cancer suppression method, various apoptosis inducers including anticancer agents have been conventionally found (for example, patents). Reference 1).
However, many of them are chemically synthesized products or hydrolysates of proteins that do not exist in nature, so safety is not always sufficient, including side effects when used for a long time. There was a problem.
In addition, natural anticancer substances derived from natural products have been studied in the past, but they have been selected for the long term because they have been selected using cytotoxin as an indicator and the mechanism of action has not been clarified. In some cases, there were problems with safety, such as concerns about side effects.
[0006]
[Patent Document 1]
Japanese Patent Laid-Open No. 2002-275068
[Problems to be solved by the invention]
An object of the present invention is to provide a food or food material that eliminates such conventional drawbacks, is derived from food, has few side effects, and has excellent safety and apoptosis induction ability. It is what.
[0008]
[Means for Solving the Problems]
The inventors of the present invention have made extensive studies to solve the above-described conventional problems. As a result, it was surprisingly found that an extract from a mushroom, a kind of mushroom (edible mushroom) that has been traditionally used as food, can achieve its purpose, and the present invention has been completed based on this finding. It came to do.
[0009]
That is, the present invention according to claim 1 provides a food or a food material having an apoptosis-inducing ability comprising a mushroom extract obtained by extracting mushroom. The present invention according to claim 2 provides the food or food material having apoptosis-inducing ability according to claim 1, wherein the mushroom extract exhibits a cancer suppression or prevention effect.
This invention which concerns on Claim 3 provides the foodstuff or foodstuff material which has the apoptosis induction ability which consists of an ergosterol peroxide derived from a mushroom.
The present invention according to claim 4 is a food or food having apoptosis-inducing ability according to claim 3, wherein the ergosterol peroxide derived from the mushroom is obtained by purifying the mushroom extract from which mushroom is extracted. The material is provided.
The present invention according to claim 5 provides a food or a food material having apoptosis-inducing ability according to claim 3 or 4, wherein an ergosterol peroxide derived from agar mushroom exhibits a suppressive or preventive effect on cancer.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Next, embodiments of the present invention will be described.
The present invention according to claim 1 relates to a food or food material having apoptosis-inducing ability, and comprises a mushroom extract obtained by extracting mushroom.
Sarcodon aspretus (Berk.) S. Ito used in the present invention according to claim 1 is an edible mushroom belonging to the genus Kotake.
Kotake is an edible mushroom traditionally eaten in the mountains. Kotake is said to be effective for anti-inflammatory skin, and there is a legend in Fukushima that it is good for rough hands. However, no food function has been clarified so far.
[0011]
In addition, about mushrooms, the research about cancer suppression is progressing so far, For example, it has been reported that Agaricus, Maitake, Shiitake, etc. have pharmacological effects, such as cancer suppression. In these reports, most reports indicate that the medicinal components of mushrooms are polysaccharides.
The center of these activities is said to be a complex with 1,6-β-D-glucan having 1,3 branches or a protein thereof.
The mushroom extract (crude extract) used in the present invention according to claim 1 does not contain these polysaccharides and glycoproteins at all.
Therefore, the present invention according to claim 1 is completely different from the cancer suppression derived from the immune activation of the human body by the polysaccharide.
In addition, components such as ergosterol peroxide derived from the mushroom extract obtained by purifying mushroom extract are not polysaccharides, the cancer suppression mechanism is not immunostimulatory, is apoptosis induction, and has been reported for mushrooms in the past. It is different from what is being done.
[0012]
The present invention according to claim 1 relates to a food or food material having apoptosis-inducing ability, and comprises a mushroom extract obtained by extracting mushroom.
As the mushroom, a dried product (preferably having a water content of about 10% or less) is generally used, but is not limited thereto.
When extracting the mushrooms, it is preferable to pulverize them beforehand with a pulverizer or the like. In addition to this, a material obtained by cutting a mushroom into an appropriate size can be used.
[0013]
The mushroom extract is obtained by extracting such mushrooms with, for example, water and / or an organic solvent, or critical extraction, and preferably with water and / or an organic solvent.
When water is used here, there are cold water, hot water, and hot water, but hot water is preferable.
The organic solvent is not particularly limited as long as it is generally used for extraction of plant components. For example, hydrophilic organic solvents such as methanol, ethanol, and acetone, and lipophilic organic solvents such as chloroform. Can be used. If necessary, water and an organic solvent can be used in combination.
Extraction is obtained by immersing and stirring pulverized mushrooms at normal temperature and pressure. Moreover, it can extract by heating and pressurizing. After extraction, the eluate is collected by filtration and used as it is, or if necessary, by further concentrating using a rotary evaporator or the like, a mushroom extract can be obtained.
[0014]
The food or food material having apoptosis-inducing ability comprising the mushroom extract of the present invention according to claim 1 thus obtained can be applied to various cancer diseases based on apoptosis-inducing ability (apoptosis-inducing action). it can. Until now, it has not been known at all that Kotake extract has the ability to induce apoptosis.
Applicable diseases include malignant neoplasms, more specifically brain tumors, oral cavity and pharynx, digestive organs and peritoneum (gastric cancer, colon cancer, liver cancer, pancreatic cancer), lung cancer, breast cancer, prostate cancer, testicular cancer, renal cancer, bladder cancer Lymph tissue and hematopoietic tissue (malignant lymphoma, multiple myeloma), uterine cancer, ovarian cancer and the like.
[0015]
As described in claim 2, the food or food material having apoptosis-inducing ability according to claim 1 exhibits the above-described cancer suppression or prevention effect.
[0016]
The mushroom extract can be used as it is as a food or food material, or can be used as a food or food material in combination with other foods or materials.
Here, food is a concept including beverages in addition to solids such as confectionery and bread. In addition, processed products such as boiled Kotake mushroom bodies and salted foods, and so-called functional foods and health foods are also included.
When used as such a food or contained in a food, the mushroom extract is generally used in the form of a liquid, granule, powder, etc., but is not limited thereto.
In addition, the mushroom extract can be used as a food material as it is or in combination with other mushrooms, vegetables and other appropriate materials.
[0017]
The present invention according to claim 3 relates to a food or a food material having apoptosis-inducing ability, and is composed of ergosterol peroxide derived from Kotake mushroom.
Ergosterol peroxide (ergosterol peroxide) is a known substance, but it has not been known that ergosterol peroxide is present in the mushroom. Moreover, it has not been known so far that ergosterol peroxide derived from Kotake has an apoptosis-inducing ability.
Furthermore, as described above, this ergosterol peroxide derived from mushrooms is not a polysaccharide, and its tumor suppression mechanism is not immunostimulatory and induces apoptosis, which is different from what has been reported for mushrooms in the past. it is conceivable that.
[0018]
Here, the ergosterol peroxide derived from the mushroom is obtained by purifying the mushroom extract from which mushroom is extracted as described in claim 4. As for the mushroom extract, it is as described in claim 1, and it is obtained by extracting mushroom, for example, with water and / or organic solvent or critical extraction, preferably with water and / or organic solvent. is there.
The ergosterol peroxide derived from the mushroom of the present invention according to claim 4 can be obtained by further purifying the mushroom extract obtained by extracting such mushroom. Purification can be performed by a known method using chromatography or the like.
[0019]
The food or food material having an apoptosis-inducing ability according to claim 3 or 4 exhibits a cancer suppression or prevention effect as described in claim 5, and is based on the apoptosis-inducing ability, as described above. It can be applied to various cancer diseases.
As described above, it has never been known that ergosterol peroxide derived from mushrooms has an ability to induce apoptosis.
[0020]
Ergosterol peroxide derived from mushrooms can be used as it is as a food or food material, or can be used as a food or food material in combination with other foods or materials.
Here, food is a concept including beverages in addition to solids such as confectionery and bread. In addition, processed products such as boiled Kotake mushroom bodies and salted foods, and so-called functional foods and health foods are also included.
When used as such a food or contained in a food, ergosterol peroxide derived from mushroom is generally used in the form of a liquid, granule, powder, etc., but is not limited thereto.
Moreover, the ergosterol peroxide derived from agar mushroom can be used as a food material as it is or in combination with other mushrooms, vegetables and other appropriate materials.
[0021]
In addition, a Kotake extract obtained by extracting Kotake with water and / or an organic solvent or an ergosterol peroxide derived from Kotake can also be used as an apoptosis inducer having apoptosis-inducing ability. Such an apoptosis-inducing agent can be in various dosage forms depending on the purpose of use, including general pharmaceutical preparations. For example, dosage forms, such as a liquid agent, a tablet, a granule, a powder agent, a capsule, are mentioned.
Thus, the dosage when used as an apoptosis-inducing agent varies depending on the type and symptoms of the disease to be applied and is difficult to define uniquely, but generally 0.1 to 40 mg / kg body weight per day for an adult, Preferably it is 0.5-20 mg / kg body weight.
[0022]
【Example】
Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.
[0023]
Preparation Example 1 (Preparation of mushroom extract)
After pulverizing 103.3 g (water content 3.7%) of a dried bamboo shoot (Sarkon aspretus (Berk.) S. Ito) from Fukushima Prefecture, the mixture was extracted with stirring in acetone. Subsequently, the eluate was collected by filtration, and concentrated with a rotary evaporator (40 ° C.) to obtain 5.27 g of a mushroom extract.
[0024]
Example 1 (Results of test for inhibition of proliferation of cancer cells and confirmation test of apoptosis-inducing effect for mushroom extract)
(1) Cancer Cell Growth Inhibition Test The effect of the mushroom extract obtained in Preparation Example 1 on the growth of human promyelocytic leukemia cell line HL60 (hereinafter referred to as HL60 cells) is 1 × 10 ⁵ cells. The number of viable cells showing trypan blue dye-exclusion ability after adding the mushroom extract obtained in Preparation Example 1 to 0.1 mg / ml to HL60 cells seeded to cells / ml and culturing for 24 hours Were measured using a hemocytometer. The result is shown in FIG. 1 together with the result of the control in which no mushroom extract was added. As the medium, RPMI1640 medium containing 10% fetal calf serum was used and cultured at 37 ° C. and relative humidity 100%.
[0025]
According to FIG. 1, it was found that when the mushroom extract was added, the number of viable cells was significantly reduced compared to the control without any mushroom extract, which was effective in suppressing the growth of cancer cells.
[0026]
(2) Confirmation of apoptosis induction effect Apoptosis induction effect of the mushroom extract obtained in Preparation Example 1 was obtained by adding 0.1 mg per ml of the mushroom extract obtained in Preparation Example 1 to HL60 cells. After culturing for 20 hours, it was confirmed by detecting fragmentation of nucleosome units of DNA characteristic of apoptosis and observing nuclear condensation / fragmentation. As the medium, RPMI1640 medium containing 10% fetal calf serum was used and cultured at 37 ° C. and relative humidity 100%.
Fragmentation of DNA at the nucleosome unit is performed by collecting HL60 cells cultured by adding a sample, lysing the cells by adding Lysis buffer, treating with ribonuclease (RNase), proteinase K, and ethylene. Electrophoresis on a 2% agarose gel containing bromide was observed on a UV transilluminator. The results are shown in FIG. 2 together with the results of the control in which no mushroom extract was added.
Condensation / fragmentation of nuclei was carried out by collecting cells cultured after adding a sample, fixing with 10% glutaraldehyde / PBS, fluorescently staining nuclear DNA with Hoechst 33258, and observing with a fluorescence microscope. The results are shown in FIG. In addition, FIG. 4 shows the results of a control in which no mushroom extract was added.
[0027]
As a result, the DNA of the cells cultured with the addition of the mushroom extract showed fragmentation of the nucleosome unit peculiar to apoptosis (FIG. 2), fragmentation and condensation of the nucleus (FIG. 3), and it was clearly apoptotic. became.
[0028]
As described above, when the mushroom extract was added to the culture medium of human promyelocytic leukemia cells (HL60 cells), morphological changes peculiar to apoptosis such as apoptotic body formation were observed under a microscope. Further, as a result of subjecting the DNA of this cell to agarose gel electrophoresis, DNA fragmentation peculiar to apoptosis was observed, and it was confirmed that apoptosis was observed.
From these results, it was clarified that the mushroom extract has an apoptosis-inducing function that is an important suppression mechanism of cancer suppression and is effective in suppressing the growth of cancer cells.
[0029]
As described above, since the mushroom extract induced apoptosis of HL60 cells, in Preparation Example 2, a substance having apoptosis-inducing activity was isolated from the mushroom extract.
[0030]
Preparation example 2 (isolation of ergosterol peroxide)
5.20 g of the mushroom extract obtained in Preparation Example 1 was added to a column (OASIS HLB column manufactured by Waters), distilled water, distilled water: ethanol (8: 2, 6: 4, 4: 6, 2: 8). ) And ethanol in sequence, and the eluate was concentrated to dryness on a rotary evaporator (40 ° C.). From the eluate of distilled water: ethanol 2: 8, 2.34 g of a fraction having apoptosis-inducing activity was obtained. This was dissolved in hexane and divided into a soluble part (2.05 g) and an insoluble part (0.29 g). This hexane-soluble part was added to a silica gel column, and eluted with hexane: ethyl acetate (10: 1), hexane: ethyl acetate (3: 1), and methanol. Four fractions (1: 968 mg, 2: 546 mg, 3 : 63.3 mg, 4: 351 mg). Fraction 3 having apoptosis-inducing activity was collected from the fraction, separated by silica gel thin layer chromatography using 2.5% methanol / dichloromethane as a developing solvent, and 13.7 mg of substance A was isolated as an apoptosis-inducing compound. did.
The isolated substance A was subjected to instrumental analysis. Nuclear magnetic resonance spectra ( ¹³ C, ¹ H, DQFCOCY, HSQC, HMBC, DEPT) were measured by a Bruker DRX800 at 298 K in CDCl ₃ .
The mass (MS) spectrum was measured by ionization with an electrospray ionization method using a Shimadzu Fourier transform cyclotron resonance mass spectrometer (high resolution). The molecular weight was compared and identified with the calculated values of both sodium salt and potassium salt.
[0031]
Example 2 (Results of cancer cell growth inhibition test and confirmation of apoptosis-inducing effect of ergosterol peroxide)
(1) Cancer Cell Growth Inhibition Test Cell A seeded so that substance A obtained in Preparation Example 2, that is, ergosterol peroxide, on the proliferation of HL60 cells is 5 × 10 ⁴ cells / ml In addition, the substance A obtained in Preparation Example 2, that is, ergosterol peroxide was added so as to be 10, 25, and 50 μM, respectively, and after culturing for 0 to 48 hours, the number of viable cells exhibiting trypan blue dye exclusion ability was obtained by hemocytometer Measurements were made using a board. The results (results when substance A is added at 10 μM, 25 μM, and 50 μM, respectively) are shown in FIG. 5 together with the control results obtained without any substance A obtained in Preparation Example 2.
[0032]
According to FIG. 5, the substance A obtained in Preparation Example 2, that is, ergosterol peroxide, suppresses cell growth in a concentration-dependent manner, and the medium containing 25 μM of substance A after 12 hours in the medium containing 50 μM of substance A. It can be seen that the cells were almost completely killed after 48 hours.
[0033]
(2) Confirmation test of apoptosis-inducing effect In Example 1 (2), the substance A obtained in Preparation Example 2, that is, ergosterol peroxide was used in place of the mushroom extract obtained in Preparation Example 1. Except for this, the apoptosis-inducing effect of substance A obtained in Preparation Example 2, that is, ergosterol peroxide, was confirmed in the same manner as (2) of Example 1.
FIG. 6 shows the results of electrophoresis of cell DNA (results obtained when substance A was added at 25 μM and 50 μM, respectively), together with the control results obtained without addition of substance A obtained in Preparation Example 2. Further, FIG. 7 shows the result of fluorescent staining with addition of 50 μM of substance A, and FIG. 8 shows the result of fluorescent staining with addition of 25 μM of substance A. Further, FIG. 9 shows the result of the control in which the substance A obtained in Preparation Example 2 was not added at all.
[0034]
As a result, the substance A, that is, the DNA of ergosterol peroxide exhibits apoptosis-specific nucleosome fragmentation (FIG. 6), chromatin aggregation and nuclear fragmentation (FIGS. 7 and 8), and induces apoptosis. It became clear.
[0035]
As described above, when substance A obtained by purification from an extract of mushrooms, that is, ergosterol peroxide, was added to the culture solution of HL60 cells, morphological changes peculiar to apoptosis were observed under a microscope. Further, as a result of subjecting the DNA of this cell to agarose gel electrophoresis, DNA fragmentation peculiar to apoptosis was observed, and it was confirmed that apoptosis was observed.
From these results, it became clear that substance A, that is, ergosterol peroxide has an apoptosis-inducing function, which is an important suppression mechanism of cancer suppression, and is effective in suppressing the growth of cancer cells.
[0036]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the foodstuff or food material which has few side effects and has the safety | security which is excellent in apoptosis is provided.
In other words, the food or food material having apoptosis-inducing ability of the present invention is obtained from an extract of the mushroom, which is a kind of edible mushroom, and has excellent side effects with few side effects.
Therefore, the present invention can be effectively used for suppression or prevention of various cancer diseases including leukemia.
[Brief description of the drawings]
1 is a graph showing the results of a growth inhibition test for a mushroom extract in Example 1. FIG.
FIG. 2 is an electrophoretogram image showing the results of a confirmation test of the apoptosis-inducing effect of the mushroom extract in Example 1.
FIG. 3 is a fluorescence micrograph image showing the confirmation test result of apoptosis-inducing effect of the mushroom extract in Example 1.
4 is a fluorescence micrograph image showing the confirmation test result of apoptosis-inducing effect for the control in Example 1. FIG.
5 is a graph showing the results of a cancer cell growth inhibition test for ergosterol peroxide in Example 2. FIG.
6 is an electrophoretogram showing the results of a confirmation test of the apoptosis-inducing effect of ergosterol peroxide in Example 2. FIG.
7 is a fluorescence micrograph image showing the confirmation test result of the apoptosis-inducing effect when 50 μM ergosterol peroxide is added in Example 2. FIG.
8 is a fluorescence micrograph image showing the confirmation test result of the apoptosis-inducing effect when 25 μM ergosterol peroxide is added in Example 2. FIG.
FIG. 9 is a fluorescence micrograph image showing the confirmation test result of the apoptosis-inducing effect of a control in which ergosterol peroxide was not added at all in Example 2.

Claims (5)

A food or food material having an apoptosis-inducing ability, comprising a Kotake extract obtained by extracting Kotake. The food or food material having apoptosis-inducing ability according to claim 1, wherein the mushroom extract exhibits a suppressive or preventive effect on cancer. A food or food material having an apoptosis-inducing ability comprising ergosterol peroxide derived from Kotake. The food or food material having apoptosis-inducing ability according to claim 3, wherein the ergosterol peroxide derived from mushroom is obtained by purifying a mushroom extract from which mushroom is extracted. The food or food material having an apoptosis-inducing ability according to claim 3 or 4, wherein an ergosterol peroxide derived from agar mushroom exhibits a suppressive or preventive effect on cancer.

Images