JPH10338640A - Antitumor agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine having decreased adverse effect by formulating an alcohol-treated product obtained by bringing an alcohol into contact with oyster meat extract with hot water. SOLUTION: The objective medicine is obtained by mixing a low molecular fraction [the brown top clear layer part which is obtained in the final concentration of 40 (wt./wt.)% or more by bringing the above-stated extract into contact with an alcohol and possesses a molecular weight of 5,000 or less; low molecular weight peptides and salts] obtained by bringing an alcohol (ethanol) into contact with an oyster meat hot water (50-90 deg.C) extract, an antioxidation substance fraction (the deep brown precipitate part obtained in the final concentration of 60 wt.% or more by further bringing the low molecular weight fraction into contact with an alcohol; low molecular weight perptides) and particularly a high molecular weight fraction [the brown precipitate part which is obtained in the final concentration of 40 wt.% or more by bringing the above-stated extract into contact with an alcohol and possesses a molecular weight of 5,000 or more; γmax (aqueous solution) in the UV absorption spectrum: 265.5 nm, 198 nm; ninhydrin reaction: positive; high molecular weight proteins, glycogen and zinc].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antitumor agent derived from oyster meat.

[0002]

2. Description of the Related Art Cancer is the leading cause of death among Japanese people, and it is still a major problem even with the rapid progress of medicine. Various substances have been developed and synthesized in anticipation of their antitumor activity. In addition, oncogenes (ras, src, myc, etc.) and tumor suppressor genes (R
B, p53, etc.), the mechanism of canceration is being elucidated, and substances that specifically act on these factors involved in canceration are required. At present, substances derived from natural products, which can be expected to have new effects and further reduce side effects, are attracting attention.

[0003] By the way, oyster meat is also called sea milk,
It contains taurine, glycogen, proteins, vitamins, minerals, amino acids, low molecular peptides, nucleic acid-related substances such as adenosine monophosphate, etc., and is extremely rich in nutritional value.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an antitumor agent derived from a natural product.

[0005]

Means for Solving the Problems The present inventor has conducted various studies on the components of oyster meat, and as a result, found an antitumor activity in the alcohol-treated oyster meat hot water extract, thereby completing the present invention. Reached. That is, the present invention is as follows.

(1) An antitumor agent comprising, as an active ingredient, an alcohol-treated product obtained by bringing alcohol (preferably at a final concentration of 40 (w / w)% or more) into contact with a hot water extract of oyster meat. (2) The above-mentioned (1), wherein the alcohol-treated product is a high molecular fraction, a low molecular fraction or an antioxidant fraction, particularly a high molecular fraction obtained by bringing alcohol into contact with the hot water extract of oyster meat. Antitumor agent. (3) The antitumor agent according to the above (2), wherein the high molecular fraction has the following characteristics i) to v). i) A brown precipitate obtained by contacting an alcohol having a final concentration of 40 (w / w)% or more with a hot water extract of oyster meat and containing a large amount of those having a molecular weight of 5000 or more ii) UV absorption spectrum: λ _max (aqueous solution): 265.5 nm, 198 nm iii) Ninhydrin reaction: positive iv) Main component: polymer protein, glycogen, zinc v) Solubility: water-soluble. (4) The antitumor agent according to the above (2), wherein the low molecular fraction has the following characteristics i) to v). i) A brown supernatant obtained by bringing an alcohol having a final concentration of 40 (w / w)% or more into contact with the hot water extract of oyster meat and containing a large amount of those having a molecular weight of 5000 or less ii) UV absorption spectrum : Λ _max (aqueous solution): 267.5 nm, 201 nm iii) Ninhydrin reaction: positive iv) Main component: low molecular weight peptide, salts v) Solubility: water soluble. (5) The antitumor agent according to (2), wherein the antioxidant fraction has the following characteristics i) to v). i) a dark brown precipitate obtained by further contacting the low molecular weight fraction with an alcohol having a final concentration of 60 (w / w)% or more and having a high molecular weight of 1,000 to 5,000 ii) UV absorption spectrum : Λ _max (aqueous solution): 267.5 nm, 198 nm iii) Ninhydrin reaction: positive iv) Main component: low molecular weight peptide v) Solubility: water soluble. (6) The above (1) to wherein the alcohol is ethanol
The antitumor agent according to (5).

[0007] Oyster meat used as a raw material of the present invention is
The shape is not particularly limited as long as the antitumor activity is not inactivated, that is, raw, frozen or oyster meat made into a powder form can be used. The hot water extract in the present invention can be obtained, for example, as a supernatant fraction obtained by adding hot water to oyster meat and removing precipitates. The temperature of the hot water is usually 50 to 90 ° C, preferably about 70 to 80 ° C, and the extraction time is usually 2 to 3 hours.

In the present invention, the hot water extract is brought into contact with an alcohol (particularly at a final concentration of 40 (w / w)% or more, more preferably about 40 to 80 (w / w)%), that is, subjected to alcohol fractionation. However, by giving some modification to the original oyster meat component, an excellent antitumor activity can be obtained.

As the alcohol used for alcohol fractionation, ethanol is preferably used because of its polarity and low toxicity. The fractionation time is usually 1 to 24 hours, preferably 5 to 12 hours. It is preferable that the solid content of the hot water extract is adjusted to 20 to 40 (w / w)% before the alcohol fractionation. The preparation may be performed by a method known per se such as concentration.

[0010] The alcohol-treated oyster meat hot water extract is
It is possible to distinguish between high molecular fraction and low molecular fraction,
The antioxidant fraction can be further separated from the low molecular fraction. The alcohol-treated product may be used as it is as the active ingredient of the present invention, or a further fraction of the alcohol-treated product may be used as the active ingredient of the present invention. Among them, a polymer fraction having strong antitumor activity is particularly preferred.

The high molecular fraction used as an active ingredient of the antitumor agent of the present invention can be obtained as a precipitate fraction of the alcohol-treated product. That is, when the oyster meat hot water extract is subjected to alcohol fractionation (particularly, a final concentration of 40 (w / w)% or more, more preferably about 40 to 80 (w / w)%),
A precipitate is formed, and this precipitate can be used as a high molecular fraction. More specifically, for example, Japanese Patent Application No. 59-18
2430 (Japanese Patent Publication No. 4-63672). The polymer fraction has the following properties. i) A brown precipitate obtained by contacting an alcohol having a final concentration of 40 (w / w)% or more with a hot water extract of oyster meat and containing a large amount of those having a molecular weight of 5000 or more ii) UV absorption spectrum: λ _max (aqueous solution): 265.5 nm, 198 nm iii) Ninhydrin reaction: positive iv) Main component: polymer protein, glycogen, zinc v) Solubility: water-soluble.

The low-molecular fraction used as an active ingredient of the antitumor agent of the present invention can be obtained as a supernatant fraction of the alcohol-treated product. That is, when the oyster meat hot water extract is subjected to alcohol fractionation (particularly, a final concentration of 40 (w / w)% or more, preferably about 40 to 80 (w / w)%), precipitation occurs. Can be used as a low-molecular fraction. The low molecular fraction has the following properties. i) A brown supernatant obtained by bringing an alcohol having a final concentration of 40 (w / w)% or more into contact with the hot water extract of oyster meat and containing a large amount of those having a molecular weight of 5000 or less ii) UV absorption spectrum : Λ _max (aqueous solution): 267.5 nm, 201 nm iii) Ninhydrin reaction: positive iv) Main component: low molecular weight peptide, salts v) Solubility: water soluble.

The antioxidant fraction used as an active ingredient of the antitumor agent of the present invention can be obtained by further subjecting the above low-molecular fraction to alcohol fractionation. As a method of obtaining an antioxidant fraction, for example, Japanese Patent Application No. 8-11 / 1990
The method can be performed according to the method described in No. 5135. That is, it can be manufactured as follows. After the low molecular fraction is concentrated by a method known per se until the solid content becomes 30 to 45 (w / w)%, the concentrated solution is adjusted to pH 3 or less, preferably about pH 3. After pH adjustment, alcohol was added to the solution at a final concentration of 55 to 70 (w / w).
%, And centrifuged to obtain a precipitate as an antioxidant fraction. It is preferable to use ethanol for the alcohol fraction for obtaining the antioxidant fraction as described above. The antioxidant fraction has the following properties. i) a dark brown precipitate obtained by further contacting the low molecular weight fraction with an alcohol having a final concentration of 60 (w / w)% or more and having a high molecular weight of 1,000 to 5,000 ii) UV absorption spectrum : Λ _max (aqueous solution): 267.5 nm, 198 nm iii) Ninhydrin reaction: positive iv) Main component: low molecular weight peptide v) Solubility: water soluble.

In order to confirm the characteristics of each of the above fractions,
Each known method is used. That is, the molecular weight is estimated from the behavior of the presence or absence of precipitate formation when subjected to alcohol fractionation. The UV absorption spectrum can be measured using a commonly used absorbance meter. The solubility in water is observed according to the method prescribed in the Japanese Pharmacopoeia. Further, as a method for confirming the main component, a color reaction, a qualitative reaction, or a quantitative reaction specific to each component, which is usually used, can be used. For example, the presence of protein can be confirmed by the Kjeldahl method, glycogen can be confirmed by the modified Somogi method, zinc can be confirmed by atomic absorption spectrometry, peptide can be confirmed by high performance liquid chromatography, and salts can be confirmed by atomic absorption spectrometry.

The alcohol-treated oyster meat hot water extract used as an active ingredient of the antitumor agent of the present invention, and the high molecular fraction, low molecular fraction and antioxidant fraction of the alcohol-treated product are precipitated. Although it can be used as it is, further drying steps (for example, methods such as drum drying, spray drying, freeze drying, etc.),
Alternatively, powdering, tableting, and the like may be performed together with a known excipient. The method of administering the antitumor agent according to the present invention is a general method in the field, that is, oral (in the form of tablets, capsules, granules, syrups, etc.), injection such as intravenous injection, arterial injection and the like. , Administration by infusion or the like is generally applied. The dose may be an amount capable of exhibiting antitumor activity, and may vary depending on, for example, the subject, the type of tumor, the administration route, the dosage form, and the like. Yes, it is administered once or several times in expectation of maintenance in the body.

Examples of carriers for pharmaceutical preparations include shellac, gelatin, gum arabic, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, methylcellulose, carboxymethylcellulose, talc, mannitol, sorbitol, starch, sucrose ester, dextrin, sucrose, Examples thereof include solid additives such as lactose, glucose, calcium carbonate, and calcium phosphate, and liquid additives such as water, physiological saline, and ethanol.

[0017]

The present invention will be described in more detail with reference to the following Examples, Experimental Examples and Formulation Examples of the antitumor agent of the present invention, but the present invention is not limited thereto.

Example 1 50 g of water was added to 50 g of frozen raw oysters,
Extract with hot water for ~ 3 hours and centrifuge (1500 rpm, 10 minutes, manufactured by Hitachi, Ltd.). The supernatant was concentrated by heating to a solid content of 30 (w / w)%. Ethanol was added to the obtained concentrated solution to a final concentration of 40 (w / w)%, left standing for 12 hours, and then centrifuged at 3000 rpm (manufactured by Hitachi, Ltd.) for 10 minutes to separate the precipitate and the supernatant.
Obtained as a high molecular fraction and a low molecular fraction, respectively. The properties of the obtained high molecular fraction and low molecular fraction were examined. As a result, both the high molecular fraction and the low molecular fraction had the same characteristics as those shown above.

Example 2 The supernatant (low-molecular fraction) obtained in Example 1 was concentrated by heating under reduced pressure at 50 ° C. using a rotary evaporator until the solid content became 37 (w / w)%. did. After adjusting the pH to 3 by adding 0.1 M hydrochloric acid to the obtained supernatant,
Ethanol was added to a final concentration of 60 (w / w)%, left to stand for 12 hours, and then centrifuged at 3000 rpm (manufactured by Hitachi, Ltd.) for 10 minutes to obtain a precipitate as an antioxidant substance fraction. The properties of the obtained antioxidant fraction were examined. As a result, it had the same characteristics as those shown above.

Experimental Example 1 Effect on Cell Proliferation 1 (In the Case of Myeloma Cells) The high-molecular fraction, low-molecular fraction, and alcohol-treated oyster meat hot water extract, which is an active ingredient of the antitumor agent of the present invention, The effect of the antioxidant fraction on the proliferation ability was examined using mouse myeloma cells (myeloma cells). 1) Test method Preparation of cells Mouse myeloma cells (P3-X63-Ag-6,5,5)
3: Dainippon Pharmaceutical) was suspended in a culture solution (containing penicillin A (final concentration 100 U / ml) and streptomycin (final concentration 100 μg / ml)) at a concentration of 1 × 10 ⁵ / ml. The solution was dispensed into a microtiter plate (Falcon). The cell number was calculated using a hemocytometer and a microscope. The cells were cultured for 24 hours in a CO ₂ incubator (37 ° C., 5% CO ₂ ). Addition of Samples The hot water extract of oyster meat, the high molecular weight fraction and the low molecular weight fraction of the alcohol-treated product of hot water extract of oyster meat prepared in Example 1 and the antioxidant substance fraction prepared in Example 2 were respectively used. The solid content was adjusted to be 37 (w / w)% and used as a sample of the stock solution. Here, the solid content is measured as Brix (sugar content) by a handheld refractometer manufactured by Atago. The cells prepared as described above were prepared by diluting the stock solution of each sample 10-fold with physiological saline so that the solution volume was 10 μl per well and the concentration was 10, 100, 1000, 10000-fold dilution of the sample stock solution, respectively. Was added. The treated cells were returned to a CO ₂ incubator (37 ° C., 5% CO ₂ ) and cultured for another 36 hours. Untreated cells were also prepared as a control. Measurement of Cell Number The number of cells was counted 36 hours after treatment with each sample. The cells in each well are washed several times with PBS (-) and completely recovered. The collected cell suspension is centrifuged (1000 rpm,
The cells were collected and again calculated using a hemocytometer and a microscope. 2) Results The results are shown in Table 1.

[0021]

[Table 1]

As shown in Table 1, the myeloma cells to which the high-molecular-weight fraction, the low-molecular-weight fraction and the antioxidant-fraction, which are alcohol-treated oyster meat hot water extracts, were inhibited from growing. Fraction showed strong antitumor activity.

Experimental Example 2 Effect on Cell Proliferation 2 (A3
1 cell and SV-T2 cells) The high molecular weight fraction, low molecular weight fraction and antioxidant fraction of the alcohol-treated oyster meat hot water extract, which is the active ingredient of the antitumor agent of the present invention, were normal. As the cells, A31 cells (Balb / c 3T3 which is a normal fibroblast derived from a mouse)
Clone) as SV-T2 cells (A
31 cells were transformed with cancer virus SV40) to examine the effect on the proliferation ability. 1) Test method Preparation of cells A31 cells (ATCC: CCL163) and SV-T
2 cells (ATCC: CCL163.1) at 2 × 10 ⁴ /
ml of culture solution (penicillin A (final concentration 100 U /
ml) and streptomycin (final concentration 100 μg /
ml), and 0.1 ml was dispensed into a 96-well microtiter plate. The cells were cultured for 24 hours in a CO ₂ incubator (37 ° C., 5% CO ₂ ). Addition of Samples The hot water extract of oyster meat, the high molecular weight fraction, the low molecular weight fraction of the alcohol-treated oyster meat hot water extract prepared in Example 1, and the antioxidant substance fraction prepared in Example 2 were respectively used. Experimental example 1
In the same manner as described above, the solid content was adjusted to 37 (w / w)% to prepare a sample stock solution. 1 liquid per well
The solid content per well was 0.2, 0.1, 0.05, 0.025, 0.
The stock solution of each sample was serially diluted 2-fold with physiological saline so as to be 0125 and 0%, and added to the cells prepared above. The treated cells were again transferred to a CO ₂ incubator (37
(5 ° C., 5% CO ₂ ) and further cultured for 24 hours. Measurement of Cell Number The number of cells 24 hours after treatment with each sample was measured using an MTT assay. The MTT assay is an MTT
The reagent produces purple crystals, but when the MTT reagent is added to cells, the cells are taken up in the cells when they are alive and active, utilizing the fact that crystals are produced in the cells. In this method, the degree of cell proliferation is measured by measuring the amount of the crystals by an absorbance method. The specific assay procedure is as follows.

Each of the cells cultured for 24 hours after the treatment
By 10μl per well MTT reagent (Wako Pure Chemical Industries, Ltd., PBS (-) in the preparation to 5 mg / ml) was added, after shaking gently, the plates again CO ₂ incubator (37 ° C., 5% CO ₂ ) And incubated for 4 hours. After incubation, centrifuge the whole plate (2
000 rpm, manufactured by Hitachi, Ltd. for 10 to 15 minutes), and the supernatant is removed. Isopropanol-0.04N hydrochloric acid is dispensed at 100 μl per well to the cells remaining on the plate and shaken until the crystals in the cells are sufficiently dissolved. Subsequently, 20 μl of a 3% SDS solution was dispensed per well, and the plate was shaken gently.
(Bio-Rad) and measured at OD595-655. The growth rate (%) was calculated from the absorbance of each sample, assuming that the absorbance of the well to which the sample was not added (0% well) was 100%. The results are shown in FIG. 2) Results As shown in FIG. 1, the low-molecular-weight fraction and the antioxidant fraction of the alcohol-treated oyster meat hot water extract have a higher tumor cell growth inhibitory effect than the hot water extract. However, it has a large effect on the growth of normal cells. On the other hand, the high molecular weight fraction of the alcohol-treated oyster meat hot water extract did not show a growth inhibitory effect on normal cells (rather, a growth promoting effect was observed), but showed a selective growth inhibitory effect on tumor cells. It turns out that it has. These results suggest that among the alcohol-treated oyster meat hot water extracts, the high-molecular fraction is particularly effective as an active ingredient of the antitumor agent with less side effects. In addition, the low-molecular fraction and the antioxidant fraction have a strong tumor cell growth inhibitory effect, although they have an effect on normal cells.

EXPERIMENTAL EXAMPLE 3 Life Prolonging Effect of Tumor Cell-Transplanted Mice The high-molecular fraction, low-molecular fraction and antioxidant substance fraction of the alcohol-treated oyster meat hot water extract which is the active ingredient of the antitumor agent of the present invention Were examined for survival benefit in mice transfected with tumor cells. 1) Sample The high-molecular fraction, low-molecular fraction and antioxidant fraction of the hot-water extract of oyster meat obtained in Examples 1 and 2 and the alcohol-treated product of the hot-water extract were used as samples. The dose of each sample was 100 per kg of mouse body weight in terms of solid content.
mg, the amount of the administration solution was 10 ml per kg of mouse body weight, and physiological saline was used for dilution. Further, as a control, physiological saline alone was administered in an amount of 10 ml per kg of mouse body weight. 2) Test method 10 ⁶ cells /0.1ml P338D ₁ murine lymphocytic leukemia cells (Dainippon Pharmaceutical) Crj: were transplanted (i.p.) in CDF ₁ mice (Charles River Japan). On the day after transplantation, the above samples were treated as 10 animals in each administration group (day 1).
From 20 to 20 days, continuous administration (intraperitoneal administration) three times a day for 20 days (administration was not performed after the 21st day), and the number of surviving days was measured. However, if animal weakness is observed within the administration period, administration is discontinued at that point. The mean value ± standard deviation of the number of days alive in each administration group was determined, and a significant difference from the control group (saline administration group) was examined. Table 2 shows the results.

[0026]

[Table 2]

3) Results In the case where the high-molecular fraction of the alcohol-treated oyster meat hot water extract was administered, a significantly longer life was observed as compared with the control group. These results suggest that the high molecular weight fraction of the alcohol-treated oyster meat hot water extract has high antitumor activity.

Experimental Example 4 Effect on Cell Proliferation 3 (MK
In the case of N-28 cells and MKN-45 cells) For the high molecular weight fraction of the alcohol-treated oyster meat hot water extract, which is the active ingredient of the antitumor agent of the present invention, using the gastric cancer cell line MKN cells The effect on its proliferation ability was examined. P is a tumor suppressor gene derived from a moderately differentiated adenocarcinoma
MKN-28 cells having 53 mutant types and MKN-45 cells derived from a poorly differentiated adenocarcinoma and having a wild type of p53 which is a tumor suppressor gene were used. 1) Test Method Preparation of Cells MKN-28 cells and MKN-45 cells (both obtained from Dainippon Pharmaceutical) were each cultured at a concentration of 5 × 10 ⁴ cells / 2 ml (RPMI containing 10% fetal bovine serum).
(1640 medium), a total volume of 2 ml was transferred to a 35 mm-diameter culture dish (manufactured by Falcon), and the cells were cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 48 hours. Addition of sample The polymer fraction of the alcohol-treated oyster meat hot water extract prepared in Example 1 was serially diluted with a culture solution to prepare solutions having solid contents of 100, 250, 500, and 1000 µg / ml, respectively. did. The cells prepared above were added to PBS (-)
After washing with, various concentrations of polymer fraction solutions were added. The treated cells were again transferred to a CO ₂ incubator (37
C., 5% CO ₂ ) and cultivation was continued for another 72 hours. Untreated cells were also prepared as a control. Measurement of Cell Number and Observation of Cells The number of cells 72 hours after the treatment with each sample was measured by a trypan blue exclusion test, and the state of the cells when further treated with a 1000 μg / ml high-molecular fraction solution was measured using a phase contrast microscope. Was observed. 2) Results FIG. 2 shows the results of counting the number of cells by the trypan blue elimination test. As shown in FIG. 2, the growth of MKN-45 cells was inhibited in a concentration-dependent manner by treating the MKN-45 cells with the polymer fraction, which was an alcohol-treated oyster meat hot water extract. On the other hand, it did not affect the proliferation ability of MKN-28 cells. This result is consistent with observation with a phase contrast microscope.

EXPERIMENTAL EXAMPLE 5 Effect on Cell Cycle From Experimental Example 4, it was found that the high molecular weight fraction, which is an alcohol-treated oyster meat hot water extract, inhibited the growth of MKN-45 cells, but the proliferation of MKN-28 cells. Has no effect. Then, in order to investigate the effect on the proliferation in more detail, the effect of the polymer fraction on the cell cycle was examined. 1) Test method Preparation of cells MKN-28 cells and MKN-45 according to Experimental Example 4
Cells were seeded in culture dishes 35mm diameter, 37 ℃, 5% CO ₂
The cells were cultured in the presence for 48 hours. Addition of Sample The polymer fraction of the alcohol-treated oyster meat hot water extract prepared in Example 1 was serially diluted with a culture solution to prepare solutions having solid contents of 100, 500, and 1000 μg / ml, respectively. After the cells prepared above were washed with PBS (-), the polymer fraction solutions at each concentration were added. The treated cells were again cultured at 37 ° C. in the presence of 5% CO ₂ for another 24 hours. Untreated cells were also prepared as a control. Analysis by flow cytometry (flow-cytometric analysi
s) Cells treated 24 hours after treatment with each sample were trypsinized and detached from the culture dish, and the cells were collected as a precipitate by centrifugation. The resulting precipitate was fixed by suspending in 70% ethanol. Rehydrate in HBSS buffer,
The cells were collected again as a precipitate by centrifugation. To facilitate the removal of low molecular weight DNA from apoptotic cells, the resulting precipitate was 192 parts 0.2 MN.
a ₂ HPO ₄ and 8 parts of 0.1 M citric acid were suspended in 40 μl of a phosphate-citrate buffer (pH 7.8) and left at room temperature for 30 minutes (Gong J. et al., Anal. Bioche).
m., 1994, 218, 314-319). Add the above cell suspension to 5
The solution was filtered through a nylon mesh of 0 μm, and 50 μg / ml of propidium iodide (Calbiokim) and 0.1 μm / ml
g / ml RNAse (Sigma) was added. The DNA content in the stained nucleus was analyzed by FACScan (Becton Dickinson). The number of stained nuclei in each phase was measured by FACScan S-fit program (Becton Dickinson). 2) Results Regarding MKN-45 cells, treatment with the high-molecular-weight fraction of the alcohol-treated oyster meat hot water extract significantly increased the concentration of S-phase cells in a concentration-dependent manner (cells in G1 phase (Significant decrease), but no change was observed in the cell cycle of MKN-28 cells. As an example, FIG. 3 shows the results when a high molecular fraction of 1000 μg / ml was added.

Experimental Example 6 Influence on Cell Death In order to further examine the inhibition of the growth of MKN-45 cells in detail, next, the function of the high molecular fraction in cell death of MKN-45 cells was examined. . Cell death is morphologically classified into apoptosis and necrosis. Apoptosis is a phenomenon that cells actively trigger themselves under physiological conditions without causing inflammation.
Does not affect surrounding cells. On the other hand, necrosis is caused by the deterioration of the environment, and the contents of the cells are released, causing inflammation and affecting surrounding cells.
Apoptosis is characterized by chromosomal aggregation of the nucleus, fragmentation of the nucleus, loss of cell surface microvilli, and condensed cytoplasm. Necrosis is characterized by mitochondrial swelling and nucleus shrinkage. 1) Test method Preparation of cells MKN-45 cells were seeded on a 35 mm-diameter culture dish according to Experimental Example 4, and cultured at 37 ° C in the presence of 5% CO ₂ for 48 hours. Addition of Sample The polymer fraction of the alcohol-treated oyster meat hot water extract prepared in Example 1 was diluted with a culture solution to prepare solutions having solid contents of 500 and 1000 μg / ml, respectively. After the cells prepared above were washed with PBS (-), the polymer fraction solutions at each concentration were added. The treated cells were again
Cultivation was further performed for 72 hours at 5 ° C in the presence of 5% CO ₂ . Untreated cells were also prepared as a control. Observation of cell nuclei by double staining using Hoechst 33342 and propidium iodide As an index of apoptosis, chromosome aggregation of cell nuclei and fragmentation of cell nuclei, which are features of the apoptosis, were examined. Hoechst 33342 and propidium iodide were used as staining solutions for cell nuclei (Nakajima H. et al., J. Exp. Med., 1995, 18).
1, 1037-1046). Both are fluorescent dyes that specifically bind to DNA, but propidium iodide is excreted in living cells, but Hoechst 33342 is not excreted. The double staining of Hoechst 33342 and propidium iodide utilizing this feature makes it possible to distinguish between living cells and dead cells and observe the state of their nuclei with a fluorescence microscope. MK to which the polymer fraction solution prepared above was added
N-45 cells were treated with 10 μg / ml Hoechst 33342.
(37 ° C., 15 minutes) and incubated with 10 μg / ml propidium iodide (37 ° C., 10 minutes) and stained. The free cells and the cells adhering to the culture dish were detached by trypsinization, collected, washed with HBSS buffer, and centrifuged (300 × g). The obtained precipitate was resuspended in 200 µl of HBSS buffer, a part thereof was placed on a slide glass, and then observed under a microscope. 2) Results Condensation of nuclear chromatin and fragmentation of nuclei were observed in some of the MKN-45 cells treated with the polymer fraction of the alcohol-treated oyster meat hot water extract. This result indicates that the high molecular fraction may have caused apoptosis.

Formulation Example 1 The polymer fraction of the alcohol-treated oyster meat hot water extract, which is the active ingredient obtained in Example 1, was drum-dried to powder. To 1 kg of the obtained powder, 200 g of lactose is added and mixed, and 15 to 20% of water is added and kneaded. The kneaded product is granulated by a granulator, and then dried and tableted. At this time, the powder of the active ingredient in one tablet is 200 to 300.
mg. In tableting, sugar-coated tablets were prepared by syrup coating as usual to prevent moisture and oxidation.

[0032]

The alcohol-treated product of the hot water extract of oyster meat has a remarkable antitumor activity, and in particular, its high molecular fraction can suppress its growth ability specifically in tumor cells.
By providing the antitumor agent of the present invention, oyster meat conventionally known as a nutritional food is found to have new utility, and it is possible to obtain an antitumor agent having a new effect and reduced side effects.

[Brief description of the drawings]

FIG. 1 shows normal cells (A31: -O) at various concentrations of a high-molecular fraction, a low-molecular fraction and an antioxidant fraction of a hot water extract of oyster meat and an alcohol-treated oyster meat hot water extract.
-) And a graph showing the effect on the growth of tumor cells (SV-T2:-■-).

FIG. 2: MKN-45 cells (wild-type p53:-○-) and MKN-28 cells (mutant p5) at various concentrations of the high-molecular fraction of the alcohol-treated oyster meat hot water extract.
3: A graph showing the effect of-●-) on proliferation.

FIG. 3 is a graph showing the effect of the high molecular weight fraction (1000 μg / ml) of the alcohol-treated oyster meat hot water extract on the cell cycle of MKN-45 cells and MKN-28 cells.

Claims (8)

[Claims] 1. An antitumor agent comprising as an active ingredient an alcohol-treated product obtained by bringing alcohol into contact with a hot water extract of oyster meat. 2. The antitumor agent according to claim 1, wherein the alcohol-treated product is a high-molecular fraction, a low-molecular fraction or an antioxidant fraction obtained by bringing alcohol into contact with the hot water extract of oyster meat. 3. The antitumor agent according to claim 1, wherein the alcohol-treated product is a high molecular fraction obtained by bringing alcohol into contact with a hot water extract of oyster meat. 4. The antitumor agent according to claim 2, wherein the high molecular fraction has the following characteristics i) to v). i) A brown precipitate obtained by contacting an alcohol having a final concentration of 40 (w / w)% or more with a hot water extract of oyster meat and containing a large amount of those having a molecular weight of 5000 or more ii) UV absorption spectrum: λ _max (aqueous solution): 265.
5 nm, 198 nm iii) Ninhydrin reaction: positive iv) Main component: high molecular protein, glycogen, zinc v) Solubility: water soluble. 5. The antitumor agent according to claim 2, wherein the low molecular weight fraction has the following characteristics i) to v). i) A brown supernatant obtained by bringing an alcohol having a final concentration of 40 (w / w)% or more into contact with the hot water extract of oyster meat and containing a large amount of those having a molecular weight of 5000 or less ii) UV absorption spectrum : Λ _max (aqueous solution): 267.
5 nm, 201 nm iii) Ninhydrin reaction: positive iv) Main component: low molecular weight peptide, salts v) Solubility: water soluble. 6. The antitumor agent according to claim 2, wherein the antioxidant fraction has the following characteristics i) to v). i) a dark brown precipitate obtained by further contacting the low molecular weight fraction with an alcohol having a final concentration of 60 (w / w)% or more and having a high molecular weight of 1,000 to 5,000 ii) UV absorption spectrum : Λ _max (aqueous solution): 267.5 nm, 198 nm iii) Ninhydrin reaction: positive iv) Main component: low molecular weight peptide v) Solubility: water soluble. 7. The final concentration of alcohol contacted with the hot water extract of oyster meat is 40 (w / w)% or more.
The antitumor agent according to any one of the above. 8. The method according to claim 1, wherein the alcohol is ethanol.
8. The antitumor agent according to any one of to 7 above.