JP3983514B2 - Apoptosis inducer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apoptosis-inducing substance extracted from plum pulp, plum meat or plum extract and a method for producing the same, and is useful as a cancer preventive and therapeutic agent obtained from plum pulp, plum or plum extract as a raw material. Concerning substances.
[0002]
[Prior art]
Currently, cancer is the top cause of death and people are very interested in its prevention and treatment. In addition, many methods for cancer prevention and treatment that can be taken on a daily basis have been proposed.
[0003]
In recent years, research on apoptosis, that is, cell self-destruction, has been actively conducted in the field of cancer research. Apoptosis not only plays an important role in the formation of tissues and organs, and the maintenance and defense of living body homeostasis in the ontogeny of living organisms, but it has been elucidated that it is closely related to the occurrence of many diseases.
[0004]
Abnormal cell control by apoptosis is considered to be one cause of cancer formation. It is said that if a cell that should be killed survives without apoptosis, that is, cell self-destruction, the cell undergoes various stimuli and undergoes mutations in the chromosome, eventually becoming a cancer cell. Cancer cells can grow only after they have acquired resistance to the apoptotic mechanism. In other words, cancer is a disease caused by a decline in the apoptotic mechanism. Therefore, the process of canceration of cells with various gene mutations is related to the process of acquiring resistance to apoptosis.
[0005]
In order for apoptosis to function normally, factors that stimulate apoptosis induction are necessary. By this factor, it is supposed that apoptosis is finally activated by activating the execution process of cell death.
[0006]
It is known that many anticancer agents are factors that induce apoptosis in cancer cells. However, it is difficult to optimally set the amount of use and the period of use of anticancer agents, and the side effects are a heavy burden on cancer patients.
[0007]
[Problems to be solved by the invention]
The investigation of factors that induce apoptosis induction is very important for the prevention and treatment of cancer.
[0008]
An object of the present invention is to provide a substance that serves as an apoptosis-inducing stimulus.
[0009]
[Means for Solving the Problems]
In order to solve the above problems, the present invention provides an apoptosis-inducing substance obtained by extracting plum pulp, plum meat or plum extract in an organic solvent.
[0010]
The apoptosis-inducing substance is prepared by adding an organic solvent to plum pulp, plum meat or plum extract and extracting the components contained therein, and then centrifuging to collect the supernatant and concentrating the supernatant. Can be obtained.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The present invention extracts plum fruit (raw), plum meat (treated with salting, etc.) or plum extract (plum pulp or hot water extract of plum meat) in an organic solvent for apoptosis. An inducer is obtained, and the growth of cancer cells is suppressed by the action of an apoptosis inducer.
The apoptosis inducer of the present invention obtained by extracting plum pulp, plum meat or plum extract in an organic solvent is considered to be a low molecular weight lipid / steroid fraction having a molecular weight of 1000 or less.
[0012]
Extraction is performed using an organic solvent in order to extract components in plum pulp, plum meat or plum extract. The organic solvent is preferably a fat-soluble one such as chloroform or alcohol, and it is particularly desirable to use chloroform, methanol, ethanol or the like alone or as a mixture thereof.
[0013]
The method for producing an apoptosis-inducing substance of the present invention will be described with reference to the process diagram of FIG.
Add organic solvent to plum pulp, plum meat or plum extract (step 1), crush plum pulp or plum meat (plum extract if necessary) (step 2), constant while heating Stir for hours (step 3). As a result, the components of plum pulp, plum meat or plum extract are extracted in the organic solvent. The pulverization, heating and stirring may be performed simultaneously.
Subsequently, the organic solvent containing the extract is centrifuged (step 4), and the supernatant is recovered (step 5). Next, the obtained supernatant is concentrated under reduced pressure using an evaporator (step 6). The concentration method is not limited to this.
After concentration, if necessary, dilution with a solvent (step 7) and filter sterilization (step 8) can be performed.
By the above steps, an organic solvent extract of plum pulp, plum meat or plum extract is obtained (step 9).
[0014]
The resulting organic solvent extract of plum pulp, plum meat or plum extract contains an apoptosis-inducing substance. By ingesting this extract, normalize and activate the function of apoptosis. Can do.
An extract containing an apoptosis-inducing substance can be processed for beverages and ingested on a daily basis in the same manner as sports drinks and teas, or added to foods as health foods. In addition, it can be ingested after being processed into capsules, tablets, syrups and the like like vitamin preparations.
[0015]
【Example】
In order to confirm the effect of the apoptosis inducer obtained from plum pulp, plum meat or plum extract, the following experiment was conducted. In the experiment, plum meat or plum extract was used as a raw material.
[0016]
(Example 1)
90 ml of a 50% chloroform / methanol equivalent liquid was added to 10 g of plum meat and homogenized at room temperature for 5 minutes. Next, it stirred for 5 hours, keeping temperature at 50-60 degreeC. The obtained solution was centrifuged at 4 ° C. for 10 minutes under a centrifugal acceleration of 9000 × g, and the supernatant was collected. The collected supernatant was concentrated under reduced pressure with an evaporator at 50 ° C. for 3 hours, and the resulting concentrate was sterilized by filtration through a Millipore filter (0.22 μm).
[0017]
In this example, an equal volume of 50% chloroform / methanol mixture is used as the organic solvent, but for extraction of components in plum meat or plum extract, the mixing ratio of chloroform and methanol is 20:80 to An 80:20 chloroform / methanol mixture can be used.
[0018]
The following experiment 1 and experiment 2 were conducted in order to examine the cancer cell growth inhibitory action by the action of an apoptosis inducer contained therein using the plum / methanol extract of plum meat obtained as described above. In the experiment, human gastric cancer cells (KATO III cells), which are cancer cells derived from human gastric cancer patients, were used.
[0019]
(Experiment 1)
Human gastric cancer cells (KATO III cells) were cultured in RPMI 1640 medium containing 10% bovine serum, penicillin G (50 IU / ml) and streptomycin (50 μg / ml).
50% chloroform / methanol (control sample (1) (2)), plum meat 50% chloroform / methanol extract (experimental sample) in a suspension containing 4-5 × 10 ⁵ cells per ml of cultured human gastric cancer cells (1) (2)) was added respectively. The experimental samples (3) and (4) were prepared so that the plum meat extract was 2 mg / ml and 4 mg / ml with respect to the human gastric cancer cell suspension.
The obtained sample was allowed to stand in 37 ° C., 95% air-5% CO ₂ and cultured for 3 days.
Three days later, the number of human gastric cancer cells in each sample (1) to (4) was counted with a hemocytometer, and the cell growth inhibition rate was calculated. The results are shown in FIG.
[0020]
Referring to FIG. 2, all of the experimental samples (3) and (4) to which the extract of plum meat was added compared to the control samples (1) and (2) to which the extract of plum meat was not added. It can be seen that is greatly reduced. In particular, it can be seen that the experimental sample (4) to which a large amount of plum extract was added was able to kill about 70% of human gastric cancer cells. On the other hand, no killing of human gastric cancer cells was observed in the control sample (1) (2).
From the above, it can be confirmed that the chloroform / methanol extract of plum meat contains a component that suppresses the growth of human gastric cancer cells, that is, an apoptosis inducer. In addition, it can be seen that as the amount of the apoptosis-inducing substance added to human gastric cancer cells is increased, the growth inhibitory action can be improved.
[0021]
(Experiment 2)
To a suspension containing 4-5 × 10 ⁵ cells per ml of human gastric cancer cells (KATO III cells), the following solvent or plum extract was added, and cultured for 3 days under the same conditions as above. Sample (1) ~ (4) was obtained. (1) and (2) are control samples, and (3) and (4) are experimental samples.
(1) Add 50% chloroform / methanol to the suspension of human gastric cancer cells to prepare 10 μl / ml.
(2) Add 50% chloroform / methanol to the suspension of human gastric cancer cells to prepare 20 μl / ml.
(3) Plum meat chloroform / methanol extract was added to human gastric cancer cell suspension to prepare 2 mg / ml.
(4) Plum meat chloroform / methanol extract was added to the suspension of human gastric cancer cells to prepare 4 mg / ml.
[0022]
Each cultured sample was centrifuged (centrifugal acceleration 3000 × g, 5 minutes), and the cells remaining after removing the supernatant were collected. The cells were washed once with PBS (−). 20 μl of cell lysis buffer was added to the resulting cell pellet to lyse the cells.
Next, an RNase A solution was added and reacted at 50 ° C. for 2.5 hours, and then proteinase K solution was added and reacted at 50 ° C. for 2.5 hours to extract DNA fragments.
2 to 3 μl of gel loading solution was mixed with 10 μl of the obtained DNA extract, added to the well of a 2% agarose gel plate, and electrophoresed at 100V. After electrophoresis, the gel was immersed in water, and DNA emitting ethidium bromide fluorescence was detected with a UV transilluminator. A photograph was taken of the distribution state of the detected DNA, and the photograph is schematically shown in FIG. In addition, M shown in FIG. 3 is a DNA molecular weight marker.
[0023]
Referring to FIG. 3, when compared with the DNA molecular weight marker M, DNA fragmentation is not observed in the control sample (1) (2), but the experimental sample (3) (4) is indicated by an arrow in the figure. In addition, DNA fragmentation was observed. That is, it was confirmed that the organic solvent extract of plum meat has an effect of inducing apoptosis.
[0024]
(Example 2)
Next, Example 2 was carried out using plum meat extract as a raw material. The same as Example 1 except that the raw material was changed to the plum extract. In order to investigate the growth inhibitory action of human gastric cancer cells (KATO III cells) using the chloroform / methanol extract of the obtained plum meat extract, the following Experiment 3 and Experiment 4 were performed.
[0025]
(Experiment 3)
The same method as in Experiment 1 was performed. The control samples (1) and (2) used in the experiment were the same as those in Experiment 1, and experimental samples (3) and (4) were prepared by adding plum extract to human gastric cancer cell suspension at 4 to 5 × 10 ⁵ cells / ml. 50% chloroform / methanol extract is added. Experimental samples (3) and (4) were prepared so that the chloroform / methanol extract of plum meat extract was 1.75 mg / ml and 3.5 mg / ml with respect to the suspension of human gastric cancer cells. Is. The experimental results are shown in FIG.
[0026]
Referring to FIG. 4, the experimental samples (3) and (4) to which the extract of plum meat extract was added were all compared to the control samples (1) and (2) to which the extract of plum meat extract was not added. It can be seen that human gastric cancer cells are decreased. In particular, it can be seen that the experimental sample (4) to which a large amount of the extract of plum meat extract was added was able to kill about 50% of human gastric cancer cells compared to the control sample. On the other hand, no killing of human gastric cancer cells was observed in the control sample (1) (2).
From the above, it can be confirmed that the chloroform / methanol extract of plum meat extract contains a component that suppresses the growth of human gastric cancer cells, that is, an apoptosis inducer. In addition, it can be seen that as the amount of the apoptosis-inducing substance added to human gastric cancer cells is increased, the growth inhibitory action can be improved.
[0027]
(Experiment 4)
The same method as in Experiment 2 was performed. The control sample (1) (2) used in the experiment was the same as in Experiment 2, and the experimental sample (3) was prepared by adding 1.75 mg / mL of plum gastric extract chloroform / methanol extract to human gastric cancer cell suspension. The sample prepared so as to be ml, and the experimental sample (4) were prepared by adding chloroform / methanol extract of plum meat extract to 3.5 mg / ml. The experimental results are shown in FIG.
[0028]
Referring to FIG. 5, when compared with the DNA molecular weight marker M, DNA fragmentation is not observed in the control sample (1) (2), but the experimental sample (3) (4) is indicated by an arrow in the figure. In addition, DNA fragmentation was observed. That is, it was confirmed that the organic solvent extract of plum meat extract has an effect of inducing apoptosis.
[0029]
In the said Example 1 and Example 2, although the plum meat and the plum extract were used as a raw material, the same effect is acquired even if it uses the plum pulp as a raw material. The same effect can be obtained by extracting an apoptosis-inducing substance from a mixture of two or three kinds of plum pulp, plum meat or plum extract.
[0030]
Mumefural contained in plum extract, structural isomers of Mumefural, or hydroxymethylfurfural also have an apoptosis-inducing action, but when the same experiment as described above was conducted, the effect was very weak compared to the present invention. Was confirmed.
[0031]
【The invention's effect】
Since the apoptosis inducer of the present invention is made from plum pulp, plum meat, or plum extract obtained from plum fruit that is safe as food, it can be easily ingested with the same sense as food. It is ideal for daily and continuous cancer prevention and treatment because it does not suffer from side effects and other damage.
In addition, since the apoptosis-inducing substance of the present invention is made from plum pulp, plum meat, and plum extract, it can be obtained at a relatively low cost and can contribute to human health through cancer prevention and treatment.
[0032]
The above description of the embodiments is for explaining the present invention, and should not be construed as limiting the invention described in the claims or reducing the scope thereof. Moreover, each part structure of this invention is not restricted to the said Example, A various deformation | transformation is possible within the technical scope as described in a claim.
[Brief description of the drawings]
FIG. 1 is a schematic process diagram showing one embodiment of the present invention.
FIG. 2 is a graph comparing the growth inhibitory effects of human gastric cancer cells in Experiment 1.
FIG. 3 is a schematic diagram comparing the DNA distribution states of human gastric cancer cells in Experiment 2. FIG.
FIG. 4 is a graph comparing the growth inhibitory effects of human gastric cancer cells in Experiment 3.
FIG. 5 is a schematic diagram comparing the DNA distribution states of human gastric cancer cells in Experiment 4.

Claims (1)

Plum pulp, containing apoptosis inducing substance obtained by extracting plum or plum extract in an organic solvent, apoptosis-inducing agents on human gastric cancer cells.

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