JP2001058953A - Apoptosis inducer derived from sake-lees or rice bran fermented extract lees and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an apoptosis inducer consisting of an organic solvent extract from sake-lees or rice bran fermented extract lees, easily takable without any anxiety similarly to foods, and useful as a prophylactic/therapeutic agent for cancer. SOLUTION: This apoptosis inducer which consists of an organic solvent extract from sake-lees or rice bran fermented extract lees, is obtained, preferably by adding an organic solvent (pref. ethanol) to sake-lees or rice bran fermented extract lees, agitating the mixture while grinding and heating to effect extraction followed by centrifugal separation to obtain a supernatant, which, in turn, is concentrated e.g. under reduced pressures. The extract thus obtained can be daily taken similarly to sports drinks or tea after processed for drinks, or can also be taken as a health food or the like through addition to food; furthermore, the extract can be processed into a form suitable for intake through processing into capsules, tablets, syrups and the like as the case with vitamin preparations or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an apoptosis-inducing substance composed of sake lees or fermented rice bran extract and a method for producing the same, and particularly useful as a cancer preventive and therapeutic agent obtained from sake lees or fermented rice bran extract. About the substance.

[0002]

2. Description of the Related Art At present, cancer is the leading cause of death, and people are very interested in its prevention and treatment.
Also, a number of methods for cancer prevention and treatment that can be routinely adopted have been proposed.

In recent years, studies on apoptosis, that is, cell self-destruction, have been actively conducted in the field of cancer research. Apoptosis is the formation of tissues and organs during biological ontogeny,
It is being elucidated that not only plays an important role in maintaining and defending the homeostasis of living organisms but also has a deep relationship with the occurrence of many diseases.

[0004] Abnormal control of cells by apoptosis is considered to be one of the causes of cancer formation. It is said that if cells that should be killed survive without causing apoptosis, that is, cell self-destruction, the cells will receive various stimuli and repeat mutations on the chromosome, eventually becoming cancer cells. Cancer cells can only proliferate after acquiring a resistance mechanism to apoptosis. That is, cancer is a disease caused by the decline of the apoptotic mechanism. From this, the process of canceration of cells with various gene mutations is related to the process of acquiring resistance to apoptosis.

For apoptosis to function properly,
Factors that stimulate apoptosis induction are required. It is said that this factor ultimately activates the process of cell killing and causes apoptosis.

[0006] It is known that many anticancer agents are factors that stimulate apoptosis of cancer cells. However,
It is difficult to set an appropriate amount and period of use of the anticancer agent, and further, its side effects impose a heavy burden on cancer patients.

[0007]

It is very important for the prevention and treatment of cancer to determine factors that induce apoptosis.

Therefore, the present invention focuses on a substance that is a stimulus for inducing apoptosis, and seeks to find an apoptosis-inducing substance from components extracted from sake lees and fermented rice bran extract with an organic solvent.

[0009]

That is, the present invention relates to an apoptosis-inducing substance comprising sake lees or rice bran fermented extract lees, which is characterized in that it comprises an organic solvent extract of sake lees or fermented rice bran extract lees. .

[0010] The invention of claim 2 is characterized in that an organic solvent is added to sake lees or fermented rice bran extract, stirred and extracted, and then centrifuged to obtain a supernatant, and the supernatant is concentrated. The present invention relates to a method for producing an apoptosis-inducing substance composed of sake lees or fermented rice bran extract lees.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the accompanying drawings. FIG. 1 is a schematic process chart showing one embodiment of the present invention, FIG. 2 is a graph showing the inhibitory effect of the ethanol extract of sake lees of Experiment 1 on the growth of cancer cells, and FIG. FIG. 4 is a schematic diagram showing the distribution of the converted DNA, FIG. 4 is a graph showing the inhibitory effect of cancer extract on the ethanol extract of the rice bran fermented extract extract in Experiment 3, and FIG. It is a schematic diagram which shows the distribution state of the DNA fragmented by the thing.

The present invention relates to an apoptosis-inducing substance consisting of sake lees or rice bran fermented extract lees, which comprises an organic solvent extract of sake lees or fermented rice bran extract lees. It is intended to suppress proliferation.

In one embodiment of the present invention, sake lees, a by-product of sake production using white rice and the like as a raw material, and fermented rice bran extract lees, a by-product of a bath preparation obtained by fermenting rice bran, are used. Extraction is performed using an organic solvent in order to remove the components in the sake lees and the rice bran fermented extract lees. The organic solvent is preferably a fat-soluble one such as alcohol. Particularly, ethanol is a desirable solvent because it is highly safe for the human body and it is not necessary to completely remove the solvent.

The method for producing an apoptosis-inducing substance consisting of sake lees or fermented rice bran extract lees is performed as follows. An organic solvent is added to the sake lees or the rice bran extract extract lees, and the sake lees or the rice bran fermented extract lees are stirred for a certain period of time while being ground and heated. The components of sake lees or fermented rice bran extract lees are extracted by the organic solvent. Subsequently, centrifugation is performed to obtain a supernatant. Then, the supernatant is concentrated by vacuum concentration or the like. In addition,
Dilution with a solvent and filtration sterilization may be performed as appropriate.

By the above-mentioned production method, an organic solvent extract can be obtained from sake lees or rice bran fermented extract lees. For example, it can be processed into a beverage and ingested on a daily basis with the same feeling as a sports drink or tea. The extract can be ingested as a health food or the like by adding the extract. Further, the extract can be processed into capsules, tablets, syrups, and the like, such as vitamin preparations, so that the extract can be consciously processed.

[0016]

EXAMPLE An example for confirming the action of an apoptosis-inducing substance of an organic solvent extract obtained from sake lees or rice bran fermented extract lees as a raw material is shown below, and will be described with reference to the schematic process diagram of FIG.

Example 1 Example 1 was performed using sake lees as a raw material. To 10 g of sake lees, 90 ml of ethanol having an alcohol content of 50% was added and homogenized at room temperature for 5 minutes. 50
While maintaining the temperature at ６０60 ° C., stirring was performed for 5 hours. This was centrifuged at 9000 × g at 4 ° C. for 10 minutes to obtain a supernatant. The obtained supernatant was concentrated under reduced pressure at 50 ° C. for 3 hours using an evaporator. The obtained concentrate was dissolved in ethanol having an alcohol content of 50%, and sterilized by filtration through a Millipore filter (0.22 μm).

In this embodiment, ethanol having an alcohol content of 50% is used as the organic solvent. However, extraction of the components in the sake lees and the rice bran fermented extract lees is carried out within the range of alcohol content of 40 to 60%. I just need.

Using the ethanol extract of sake lees thus obtained, an experiment was carried out to examine the effect of the apoptosis-inducing substance contained therein to inhibit the growth of cancer cells.
And Experiment 2 were performed.

(Experiment 1) Human lymphocytic leukemia Molt
4B cells were treated with 10% bovine serum and penicillin G (50 IU / m
1) and streptomycin (50 μg / ml) in RPMI1640 medium. 4-5 × 10
⁵ cells / ml human lymphocytic leukemia Molt 4
50% ethanol for B cells (for control sample), sake lees 5
A 0% ethanol extract (for an experimental sample) was added to each, and the mixture was cultured at 37 ° C., 95% air and 5% CO ₂ to obtain a control sample and an experimental sample. Three days later, the number of human lymphocytic leukemia Molt 4B cells was counted using a hemocytometer, and the cell growth inhibition rate was determined. The result is shown in FIG. The experimental sample was prepared so that the extract of sake lees was 2 mg / ml with respect to the human lymphoid leukemia Molt 4B cell suspension, and the experimental sample was 4 mg / ml.

The human lymphocytic leukemia Molt 4B
The cells are cancer cells derived from human acute lymphoblastic leukemia.

(Experiment 2) Human lymphocytic leukemia Molt
The following were added to 4-5 × 10 ⁵ cells / ml of 4B cells, respectively, and cultured for 3 days to obtain samples. And are control samples, and and are experimental samples. 50% ethanol was added to the human lymphocytic leukemia Mol
t4B cells were added to the suspension and adjusted to 10 μl / ml. 50% ethanol was added to the human lymphocytic leukemia Mol
t4B cells were added to the suspension and adjusted to 20 μl / ml. The ethanol extract of sake lees was used for the human lymphocytic leukemia M
What was added to the suspension of olt 4B cells and adjusted to 2 mg / ml. The ethanol extract of sake lees was used for the human lymphocytic leukemia M
What was added to the suspension of olt 4B cells and adjusted to 4 mg / ml.

The samples from each of the cultured
After centrifugation (3000 × g, 5 minutes), the cells remaining after removing the supernatant were collected. The cells were washed once with PBS (-). 20 μl of a cell lysis buffer was added to the cell pellet to lyse the cells. RNase A
The solution was added, reacted at 50 ° C. for 2.5 hours, and then proteinase K solution was added, and reacted at 50 ° C. for 2.5 hours.
The DNA fragment was extracted. 10 µl of the DNA extract and 2-3 µl of the gel loading solution were mixed, added to the wells of a 2% agarose gel plate, and electrophoresed at 100V.
After electrophoresis, the gel is immersed in water, and then DNA that emits ethidium bromide fluorescence with a UV transilluminator.
Was detected. Thereafter, a photograph was taken, and the photograph is schematically shown in FIG. Note that M shown in FIG. 3 is a DNA molecular weight marker.

From Example 1, it is understood that the ethanol extract of sake lees contains a component that suppresses the proliferation of human lymphocytic leukemia Molt 4B cells, that is, an apoptosis-inducing substance. In addition, the larger the amount of the apoptosis-inducing substance added to human lymphocytic leukemia Molt 4B cells, the higher the growth inhibitory effect.

(Example 2) Next, Example 2 was performed using fermented rice bran extract lees as a raw material. Example 2 was performed in the same manner as in Example 1 except that the raw material was fermented rice bran extract cake. Experiments 3 and 4 were performed to examine the growth inhibitory effect of human lymphocytic leukemia Molt 4B cells using the obtained ethanol extract of the rice bran fermented extract cake.

(Experiment 3) The experiment was performed in the same manner as in Experiment 1.
The sample used for the experiment, and the control sample were the same as in Experiment 1, and the experimental sample was 4
~ 5 × 10 ⁵ cells / ml human lymphocytic leukemia M
It is a rice bran fermented extract lees 50% ethanol extract added to olt 4B cells. In addition, in the experimental sample, the ethanol extract of the rice bran fermented extract lees was 1.7 with respect to the human lymphocytic leukemia Molt 4B cell suspension.
5 mg / ml, and the experimental sample was 3.
It was prepared to be 5 mg / ml. FIG. 4 shows the experimental results.

(Experiment 4) The experiment was performed in the same manner as in Experiment 2.
For the samples used, and
The same control sample as above or the ethanol extract of rice bran fermented extract lees was used for the human lymphocytic leukemia Molt 4B
The one added to the cell suspension to a concentration of 1.75 mg / ml is the one obtained by adding the ethanol extract of the rice bran fermented extract lees to a concentration of 3.5 mg / ml. FIG. 5 shows the experimental results.

From Example 2 above, the ethanol extract of the rice bran fermented extract lees was used to prepare human lymphocytic leukemia Mol.
The effect of suppressing the growth of t4B cells is confirmed.

In other words, the effect of the apoptosis-inducing substance contained in the ethanol extract of the rice bran fermented extract lees to suppress the proliferation of human lymphocytic leukemia Molt 4B cells is as follows:
It is confirmed in the same manner as when the sake lees shown in Example 1 is used.

Although the sake lees or the rice bran fermented extract lees were used alone in Examples 1 and 2, the sake lees and the rice bran fermented extract lees may be mixed and used.

[0031]

As shown and described above, according to the apoptosis-inducing substance comprising the sake lees or the fermented rice bran extract lees of the present invention and the method for producing the same, the sake lees obtained at the stage of producing liquor and the like, which are safe as food, are obtained. Or, using rice bran fermented extract lees as a raw material, it can be easily and safely ingested with the same feeling as food, and there is no damage such as side effects, so daily and continuous prevention and treatment of cancer Ideal for

Further, by using the above-mentioned sake lees and fermented rice bran extract lees, which were conventionally discarded, it is possible to newly obtain an apoptosis-inducing substance which can be used for the prevention and treatment of cancer. And contribute to waste management.

[Brief description of the drawings]

FIG. 1 is a schematic process drawing showing one embodiment of the present invention.

FIG. 2 is a graph showing the cancer cell growth inhibitory effect of the ethanol extract of sake lees in Experiment 1.

FIG. 3 is a schematic diagram showing a distribution state of DNA fragmented by an ethanol extract of sake lees in Experiment 2.

FIG. 4 is a graph showing the cancer cell growth inhibitory effect of the ethanol extract of the rice bran fermented extract lees in Experiment 3.

FIG. 5 is a schematic diagram showing a distribution state of DNA fragmented by an ethanol extract of rice bran fermented extract lees in Experiment 4.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Koichiro Muramatsu 5 Maruyama, Shimomachi, Nishio-shi, Aichi Pref. Aioi Fermentation Co., Ltd. Reference) 4B018 MD91 ME08 MF01 MF06 4B028 AC06 AG01 AP22 AS15 4C088 AB74 AC04 AC14 BA08 BA10 BA11 CA06 CA08 CA11 CA12 NA14 ZB26 ZC41

Claims (2)

[Claims] 1. An apoptosis-inducing substance comprising sake lees or rice bran fermented extract lees, comprising an organic solvent extract of sake lees or fermented rice bran extract lees. 2. A sake lees or a rice bran fermented extract lees, comprising adding an organic solvent to the sake lees or the rice bran fermented extract lees, stirring and extracting, and then centrifuging to obtain a supernatant, and concentrating the supernatant. For producing apoptosis-inducing substances.