JP2013189416A - Oyster hot-water extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel superior antitumor substance capable of being easily prepared from natural supply sources.SOLUTION: A composition including an oyster hot-water extract, specifically an oyster hot-water extract obtained under pressurization at 125°C, is provided. The oyster extract is different in molecular weight and physical properties of an active ingredient in comparison with a conventional extract. The composition exhibits such superior effects that NK cells are activated and melanoma can be treated.

Description

  The present invention relates to a composition comprising an oyster hot water extract for treating or preventing a tumor. Preferably, the tumor is melanoma. The present invention also relates to a composition comprising an oyster extract for activating NK cells.

  It is known that an oyster extract mainly composed of a low molecular weight peptide, which is an antioxidant substance fraction, has the ability to inhibit the growth of mouse myeloma cells (Non-patent Document 1). However, an oyster extract containing a sodium periodate-sensitive substance having a molecular weight of about 2000 as an active ingredient and having the ability to activate NK cells is not known. Further, an oyster extract having an antitumor effect on melanoma cells using a sodium periodate-sensitive substance having a molecular weight of about 2000 as an active ingredient is not known.

  The present invention relates to a novel oyster extract having NK cell activation ability and anti-melanoma cell activity.

JP 10-338640 A

  An object of the present invention is to provide a new oyster extract having NK cell activation ability and anti-melanoma cell activity.

  According to the present invention, an oyster extract prepared by a method different from the method of preparing an oyster extract, which has been reported to have antitumor activity, has a sodium periodate-sensitive substance having a molecular weight of about 2000 as an active ingredient, and Unexpectedly, the present invention has been completed by finding that it has NK cell activation ability and anti-melanoma cell activity. The present invention relates to a pharmaceutical composition for treating or preventing a tumor (for example, a pharmaceutical composition for oral administration), an active ingredient for the pharmaceutical composition, a food and beverage composition, and a food and beverage additive Offer things.

The present invention provides, for example:
(Item 1)
A composition for treating or preventing a tumor, comprising an oyster hot water extract, wherein the oyster hot water extract comprises the following steps:
a) A step of hot water extraction of raw oysters at 125 ° C. under pressure for 2 hours,
A composition comprising, as an active ingredient, a sodium periodate-sensitive substance, prepared by a method comprising:
(Item 2)
The composition further comprises:
b) filtering the oyster hot water extract of step (a); and
c) vacuum concentration of the filtrate of step (b),
A composition according to item 1, which is prepared by a method comprising:
(Item 3)
The composition further comprises:
d) sterilizing the concentrate of step (c); and
e) spray drying the sterilized product of step (d),
A composition according to item 2, which is prepared by a method comprising:
(Item 4)
The composition further comprises:
f) adding ethanol to the product spray-dried in step (e) to a final concentration of 30% to prepare a soluble fraction; and
g) adding ethanol to the soluble fraction of step (f) to a final concentration of 50% to prepare an insoluble fraction;
4. A composition according to item 3, which is prepared by a method comprising:
(Item 5)
Item 5. The composition according to items 1 to 4, wherein the tumor is melanoma.
(Item 6)
A composition for activating NK cells, comprising an oyster hot water extract, wherein the oyster hot water extract comprises the following steps:
a) A step of hot water extraction of raw oysters at 125 ° C. under pressure for 2 hours,
A composition comprising, as an active ingredient, a sodium periodate-sensitive substance, prepared by a method comprising:
(Item 7)
The composition further comprises:
b) filtering the oyster hot water extract of step (a); and
c) vacuum concentration of the filtrate of step (b),
7. A composition according to item 6, which is prepared by a method comprising:
(Item 8)
The composition further comprises:
d) sterilizing the concentrate of step (c); and
e) spray drying the sterilized product of step (d),
8. A composition according to item 7, which is prepared by a method comprising:
(Item 9)
The composition further comprises:
f) adding ethanol to the product spray-dried in step (e) to a final concentration of 30% to prepare a soluble fraction; and
g) adding ethanol to the soluble fraction of step (f) to a final concentration of 50% to prepare an insoluble fraction;
9. A composition according to item 8, which is prepared by a method comprising:

  According to the present invention, there is provided a novel oyster extract having NK cell activation ability and anti-melanoma cell activity.

FIG. 1 shows the effect of intraperitoneal administration of Ethanol 50 on NK cytotoxic activity. Ethanol 50 (200 mg / kg) or saline (control) was administered intraperitoneally into C57BL / 6 mice for 3 consecutive days. Total splenocytes were prepared 2 hours after the last dose. FIG. 1 shows the results of determining the cytotoxic activity of NK cells against YAC-1 cells (2 × 10 ⁴ cells / well). Data are shown as mean + standard error (n = 4). ** P <0.01 (Student t test) Comparison with control. FIG. 2 shows the results of inhibition of B16 tumor growth by intraperitoneal Ethanol 50 administration. Ethanol 50 (200 mg / kg) or PBS (control) was administered into the abdominal cavity of C57BL / 6N mice from day -3 to day 14. On day 0, B16 cells (5 × 10 ⁵ cells) were inoculated subcutaneously into mice. Tumor growth was measured with calipers until day 14. Data are shown as mean ± standard error (n = 4 for controls, n = 3 for Ethanol 50). * P <0.05, ** P <0.01 (Student t test) Comparison with controls. FIG. 3 shows the effect of Ethanol 50 administration on Meth A tumor growth. Ethanol 50 (200 mg / kg) or PBS (control) was administered intraperitoneally from BALB / c mice from day -3 to day 14. On day 0, Meth A cells (2 × 10 ⁶ cells) were inoculated subcutaneously into mice. Tumor growth was measured with calipers until day 14. Data are shown as mean ± standard error (n = 4). * P <0.05 (Student t test) Comparison with control. FIG. 4 shows the tumor weight inhibitory effect of Ethanol 50 administration. Ethanol 50 (200 mg / kg) or PBS (control) was administered intraperitoneally from BALB / c mice from day -3 to day 14. On day 0, Meth A cells (2 × 10 ⁶ cells) were inoculated subcutaneously into mice. Tumor weight was measured on day 14. Data are shown as mean + standard error (n = 4). * P <0.05 (Student t test) Comparison with control. FIG. 5 shows the results of the Ethanol 50 fraction inhibiting cell growth of different tumor cell lines. Meth A cells (5 × 10 ³ cells / well), YAC-1 cells (5 × 10 ³ cells / well), CT-26 cells (1 × 10 ³ cells / well), or B16 cells (1 × 10 ³⁾ Various concentrations of Ethanol 50 (0.375 mg / mL, 0.75 mg / mL, or 1.5 mg / mL) were added to cells / well), or cultured for 48 hours without adding Ethanol 50 . Cell proliferation was measured by MTT assay. Data are shown as the mean + standard deviation of triplicate experiments. ** P <0.01, *** P <0.001 (Dunnett's t test), #P <0.05, ## P <0.01 (Dunnett's T3 test) Comparison with controls. FIG. 6 shows Meth A cell proliferation inhibitory activity of each of the oyster extract and ethanol fraction. An MTT assay was used to measure the cell growth inhibitory activity. OE indicates an extract not subjected to ethanol fractionation. OE30i represents an insoluble fraction when the final ethanol concentration is 30%. OE50i indicates an insoluble fraction when the final ethanol concentration is 50% to a soluble fraction when the final ethanol concentration is 30%. OE50s indicates a soluble fraction when the final ethanol concentration is 50%. For Meth A cells (5 × 10 ³ cells / well), oyster extract that is not ethanol fractionated and various ethanol fractions of oyster extract are added (0 mg / mL, 0.75 mg / mL, or , 1.5 mg / mL) for 48 hours. Data are shown as mean of 3 wells + standard deviation. FIG. 7 shows that Ethanol 50 fraction inhibits Meth A cell proliferation. Various amounts of Ethanol 50 fraction were added to Meth A cells (5 × 10 ³ cells / well) (0 mg / mL, 0.375 mg / mL, 0.75 mg / mL, or 1.5 mg / mL). mL), 24 hours and 48 hours, and MTT assay was performed. Data are shown as the mean + standard deviation of triplicate experiments. * P <0.05, *** P <0.001, ### P <0.001 (Dunnett's t test) Comparison with 24-hour or 48-hour culture controls. FIG. 8 shows the effect of Ethanol 50 on early spontaneous apoptosis of Meth A cells using Annexin V expression as an index. Meth A cells (1 × 10 ⁵ cells / mL) were cultured with various concentrations of Ethanol 50 fraction (0 mg / mL, 0.75 mg / mL, or 1.5 mg / mL) for 24 and 48 hours. . Staining was performed using annexin V and PI conjugated with FITC to determine the proportion of early apoptotic cells (total cells vs. annexin V positive PI negative cells). Data are shown as the mean + standard deviation of triplicate experiments. *** P <0.001 and ### P <0.001 (Dunnett's t-test) compared to 24-hour or 48-hour culture controls.

(Preparation of oyster extract)
Typically, oyster extracts are prepared by hot water extraction of raw oysters at 125 ° C. under pressure for 2 hours. In addition to raw oysters, oysters that have been frozen and / or dried and / or cooked may be used. The extraction conditions can be appropriately changed by those skilled in the art. For example, the extraction temperature is 101 ° C to 150 ° C, preferably 110 ° C to 140 ° C, more preferably 120 ° C to 130 ° C, and most preferably. 125 ° C. can be used. The pressure during extraction is not particularly limited, but is typically 1060 hPa to 4900 hPa, preferably 1450 hPa to 3700 hPa, more preferably 2020 hPa to 2750 hPa, and most preferably 2300 hPa to 2400 hPa. The extraction time can be appropriately determined by those skilled in the art. Typically, it is 2 hours, but is not limited to this. In addition, although said Unexamined-Japanese-Patent No. 10-338640 discloses performing extraction for 2 to 3 hours at 80-90 degreeC and atmospheric pressure, as a result, the extract containing a low molecular weight peptide as an active ingredient is obtained. It is different from the extract of the present invention in which the active ingredient is not a peptide.

In the preparation of an oyster extract, the following steps can be added as necessary after the extraction under the above-mentioned high temperature and pressure conditions.
-Filtering the oyster hot water extract;
A step of concentrating the filtrate;
-Sterilizing the concentrate;
-Spray drying the sterilized product;
Adding ethanol to the spray-dried product to a final concentration of 30% to prepare a soluble fraction; and
-A step of adding ethanol to the ethanol-soluble fraction to a final concentration of 50% to prepare an insoluble fraction.

  In the filtration step, any filtration means for removing the solid component in the oyster hot water extract can be used.

  Arbitrary concentration processes can be used for the said concentration, For example, vacuum concentration, heating vacuum concentration, concentration by ultrafiltration etc. can be used, However It is not limited to these. A preferred concentration is vacuum concentration. Those skilled in the art can appropriately determine the degree of vacuum (pressure), temperature, and time used for vacuum concentration.

  The sterilization step is typically performed at 135 ° C. for 15 seconds using a helicoid instantaneous sterilizer, but is not limited to this sterilization method. Spray drying of the sterilized product can be performed by any known method.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

(Example 1: Novel oyster extract having NK cell activation ability)
(A. Experimental materials and methods)
(1. animals)
Female BALB / c and C57BL / 6N mice were purchased from Nippon Charles River Co., Ltd. (Yokohama). Female C3H / HeJ mice were purchased from Nippon SLC Co., Ltd. (Hamamatsu). The room temperature was set to 26 ° C. ± 1 ° C., and water and food were freely fed. In order to recover from stress due to transportation and to adapt to environmental changes, the animals were reared in an SPF environment for 1 week or longer after delivery, and used for experiments at 8-12 weeks of age after acclimatization.

(2. Cells)
YAC-1 cells (mouse lymphoma) were obtained from Riken BRC Cell Bank (Tsukuba City).

(3. Preparation of oyster extract)
The edible portion of raw oysters is extracted with hot water under pressure at 125 ° C and 2364 hPa for 2 hours, filtered, concentrated with a vacuum concentrator, sterilized with a helicoid instant sterilizer, and dried with a spray dryer. And oyster extract powder was obtained. The main components are shown in Table 1.

(4. Preparation of whole splenocytes)
The mouse was sacrificed by cervical dislocation, the spleen was removed from the back, and a splenocyte suspension was quickly prepared. After removing the tissue mass with a stainless steel mesh, erythrocytes were removed by treatment with ACK lysis buffer (0.15M NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM Na ₂ EDTA, pH 7.2). This was washed three times in Eagle's MEM medium, and then suspended in RPMI 1640 medium (Sigma-Aldrich Co.) containing 10% FCS as whole splenocytes.

(5. Purification of NK cells using magnetic bead method)
(5.1. Enrichment of NK cells)
Using a Mouse NK Cell Enrichment Set-DM (BD Biosciences), the procedure was as follows. Mouse whole spleen cells were prepared at 2 × 10 ⁷ cells / mL in ice-cold DMEM medium (without FCS), and biotin-labeled anti-mouse CD4, CD5, CD8a, CD19, CD24, Ly-6G, Ly -6C, TER-119 / Erythroid Cells monoclonal antibody was added and treated on ice for 15 minutes. Subsequently, centrifugal washing was performed, and BD ^™ Imag Streptavidin Particles Plus-DM was added to the cells from which the supernatant had been removed so as to be 20 × 10 ⁷ cells / mL, followed by incubation for 30 minutes in a refrigerator. Thereafter, the cell suspension was transferred to a plastic round bottom test tube, the cells were separated using BD ^™ IMagnet (BD Biosciences), and the negative fraction was used as enriched NK cells.

(5.2. Purification of NK cells)
Using a Mouse NK Cell Separation Set-DM (BD Biosciences), the procedure was as follows. Mouse whole spleen cells were prepared to 2 × 10 ⁷ cells / mL with ice-cooled DMEM medium, and PE-labeled anti-mouse CD49b / Pan-NK Cells monoclonal antibody was added thereto, followed by refrigeration for 15 minutes. Next, centrifugal washing was performed, and BD ^™ Imag Anti-PE Particles 2-DM was added to the cells from which the supernatant had been removed so as to be 20 × 10 ⁷ cells / mL, followed by incubation for 30 minutes in a refrigerator. Thereafter, the cell suspension was transferred to a plastic round bottom test tube, the cells were separated using BD ^™ Imagnet, and the positive fraction was used as purified NK cells.

(6. Cell culture)
RPMI 1640 medium containing 10% FCS, 100 U / mL penicillin G, 100 μg / mL streptomycin and 2 mM L-glutamine was used as the culture medium. All the cultures were performed in a humidified environment under a gas phase of 37 ° C., 5% CO ₂ , 95% air.

(7. Cell number counting method)
0.04% Trypan Blue (Merck) physiological saline solution and an equal volume of the cell suspension were mixed and impregnated into a modified Neubauer type counter with a cover glass adhered. Cells that were not stained with trypan blue under a microscope were counted as viable cells, and the cell viability was calculated.

(8. Measurement of NK cell cytotoxicity)
Whole spleen cells (1 × 10 ⁶ cells / 200 μL / well) or enriched NK cells (1 × 10 ⁵ cells / 200 μL / well) or purified NK cells (1 × 10 ⁵ cells / 200 μL / well) Was cultured with 2-mercaptoethanol (50 μM) and a reagent on a 96-well flat bottom microculture plate (Nunc) for 6 hours, 24 hours or 48 hours. After completion of the culture, the culture plate was washed by centrifugation, and 10% FCS / RPMI 1640 medium (100 μL / well) was added. YAC-1 cells into which Calcein-AM (Wako Pure Chemical Industries, Ltd.) has been incorporated under heating in a carbon dioxide incubator, and 2 × 10 ⁴ cells / 100 μL / well of whole spleen cells, enriched NK cells The purified NK cells were added with 1 × 10 ⁴ cells / 100 μL / well and cultured for 4 hours. Thereafter, the culture plate was washed by centrifugation, 1% -Triton X-100-borate buffer solution was added at 200 μl / well and incubated for 30 minutes to lyse the cells. Then, the fluorescence intensity in the YAC-1 cells was measured with a Fluoroscan Ascent CF. Measurement was performed (excitation wavelength: 485 nm, emission wavelength: 527 nm).

(9. Measurement of the number of living cells (cell proliferation) by the MTT method)
After culturing under the above conditions and removing 150 μL / well of the culture supernatant, a new culture medium was added at 50 μL / well, and MTT solution (5 mg / mL) was added at 25 μl / well and incubated for 2 hours. Thereafter, 100 μL / well of lysing solution (20% SDS / 50% DMF, pH 4.5) was added, and the mixture was allowed to stand overnight at 37 ° C. under light shielding. After lysis, the absorbance at 570 nm was measured with a plate reader.

(10. Measurement of NK cytotoxicity after in vivo administration)
About the powder of the oyster extract prepared in the above “3.”, 30% ethanol-soluble-50% ethanol-insoluble fraction (Ethanol 50) dissolved in 0.2 mL of physiological saline was continuously added for 3 days. It was administered intraperitoneally (200 mg / kg). Total splenocytes were prepared 2 hours after the last intraperitoneal administration, and total splenocytes (1 × 10 ⁶ cells / 100 μL / well) and YAC-1 cells (2 × 10 ⁴ ) incorporating Calcein-AM as described above were prepared. Cells / 100 μL / well) was cultured for 4 hours to measure NK cytotoxicity.

(11. Gel filtration chromatography by Sephadex G-25)
A Sephadex G-25 (Medium) column (PD-10, GE Healthcare Japan, Inc.) was equilibrated with PBS. The oyster extract (4 mg / 2 mL) was applied and then eluted with PBS to collect fractions (1 mL / fraction).

(12. Ethanol fractionation)
Ethanol was added to the H ₂ O solution of the oyster extract to a final concentration of 30%, and the mixture was allowed to stand at 4 ° C. for 24 hours. After centrifugation (4 ° C., 3000 rpm, 10 min), the supernatant was collected in another centrifuge tube, ethanol was added to a final concentration of 50%, and the mixture was allowed to stand at 4 ° C. for 24 hours. After centrifugation, the supernatant was collected in another centrifuge tube. Thereafter, each precipitate and the final supernatant residue were lyophilized.

(B. Results)
(1. Effect of oysters on NK activity in splenocytes)
The effect of oyster hot water extract on NK activity of mouse whole spleen cells was examined. Mouse whole splenocytes were cultured with oyster extract for 24 hours and NK activity was measured. As a result, an NK activity enhancing action was observed. When cytotoxicity against splenocytes was measured by the MTT method, cytotoxicity was not observed with the oyster extract at the concentration used in the experiment, and splenocytes were slightly increased.

(2. Effect of trypsin treatment on NK activity enhancing action of oyster extract)
In order to examine the nature of the active substance in the oyster extract, trypsinization was performed at 37 ° C. for 30 minutes, and the change in NK activity was examined. As a result, the NK activity enhancing action of the oyster extract was not affected at all by the trypsin treatment. In addition, since the oyster extract may contain a substance having trypsin inhibitor activity, it was examined how much the oyster extract inhibits the degradation of azocasein by trypsin. As a result, the oyster extract did not suppress the activity of 0.01% trypsin. Therefore, it was suggested that the active ingredient in the oyster extract is not a protein ingredient.

(3. Ethanol fractionation of NK activity enhancing substance in oyster extract)
The oyster extract was fractionated with 30-50% ethanol, and the NK activity enhancing action of each obtained fraction was measured. As a result, the active substance in the oyster extract had the strongest injury activity in the 30% ethanol-soluble-50% ethanol-insoluble fraction (Ethanol 50). The effect was concentration-dependent and the activity of 1 mg / mL was comparable to IL-2 (10 U / mL), which is known to have an NK activating effect. Further, no cytotoxicity was observed at Ethanol 50 at the concentration used in the experiment. In subsequent experiments, Ethanol 50 was mainly used.

(4. Effects of oyster extract and Ethanol 50 on NK activity over time)
NK activity was measured using splenocytes after culturing for 6, 24 and 48 hours together with oyster extract and Ethanol 50. As a result, oyster extract and Ethanol 50 both enhanced NK activity only slightly during 6 hours of incubation, but increased over time at 24 hours and 48 hours. The degree of activity enhancement from 24 to 48 hours was more pronounced with the oyster extract. In addition, since the same result was observed also in the experiment using the C3H / HeJ mouse having low sensitivity to LPS, it was confirmed that endotoxin was not involved in NK activation. In addition, this NK activation was observed both when enriched NK cells were used and when purified NK cells were used, so that the components in the oyster extract were directly transferred without passing through other types of cells. It was concluded that this was the result of acting on NK cells.

(5. Gel filtration chromatography of Ethanol 50)
In order to measure the molecular weight of the NK activity enhancing substance in Ethanol 50, gel filtration chromatography was performed using Sephadex G-25 (fractionated molecular weight polysaccharide: 100 to 5000, peptide and globular protein: 1000 to 5000). The NK activity of each obtained fraction was examined. As a result, no. 2 to 7 showed an NK activity promoting action. 4. The peak of insulin A chain with a molecular weight of 2531 is No. Between 3 and 4, the peak of Bacitracin with a molecular weight of 1423 is no. Between 4 and 5, it was therefore estimated that the apparent molecular weight of the active ingredient of the oyster extract was about 2000. In addition, as a result of measuring the light absorbency in 280 nm of each fraction, No. Absorption was observed in 2-9.

(6. Effect of carboxypeptidase Y treatment on NK activity enhancing action of Ethanol 50 fraction)
In order to investigate the nature of the active substance in the Ethanol 50 fraction, 0.05% carboxypeptidase Y treatment was performed at 25 ° C. for 30 minutes, and the change in NK activity was examined. As a result, the NK activity enhancing action of the oyster extract was not affected at all by the carboxypeptidase Y treatment. On the other hand, IL-2 enhanced NK activity was inactivated by carboxypeptidase Y treatment. Therefore, it was suggested that the active ingredient in the oyster extract is not a peptide component or a polypeptide component.

(7. Effect of sodium periodate treatment on NK activity promoting action of Ethanol 50 fraction)
Next, in order to examine whether the NK cell activity promoting substance is a carbohydrate, the ethanol 50 fraction was treated with sodium periodate, and the change in the NK cell promoting activity was examined. It is known that sodium periodate cleaves the sugar molecule by cleaving the diol group carbon bond of the sugar molecule by its oxidizing action and converting it to an aldehyde group. Ethanol 50 fraction was treated with sodium periodate dissolved in PBS for 2 days at 4 ° C, reduced with sodium thiosulfate, then subjected to Sephadex G-25 gel filtration chromatography to remove unnecessary ions and perform NK assay. went. As a control group, Ethanol 50 fraction was treated with PBS without sodium periodate for 2 days at 4 ° C. and reduced with sodium thiosulfate, followed by Sephadex G-25 gel filtration chromatography and NK assay. went. As a result, it was recognized that the NK cell activity promoting action was inactivated by sodium periodate treatment. This suggests that the active substance is a carbohydrate.

  Wang et al. (Mar. Drugs 8, 255-268 (2010)) show that oyster peptides produced by hydrolyzing oysters with proteases from the genus Bacillus have an anti-tumor effect via mouse NK cell activation. It was. However, physical properties are different from the oyster extract of the present invention, and therefore, it is considered that the present invention contains different components.

(8. Enhancement of NK activity by in vivo administration of Ethanol 50)
Since Ethanol 50 was found to activate NK cell cytotoxicity in vitro, the effect on NK activity upon in vivo administration was subsequently examined. Ethanol 50 was intraperitoneally administered to mice continuously for 3 days, and splenocytes were prepared 2 hours after the last intraperitoneal administration. Whole spleen cells were cultured with YAC-1 cells for 4 hours, and NK activity was measured. As a result, the NK activity (cytotoxicity) of the Ethanol 50 administration group was significantly enhanced compared to the control group to which physiological saline was administered (FIG. 1). From the above experimental results, the oyster extract obtained by the extraction method of the present invention is an extract containing a novel active ingredient having a molecular weight of about 2000, which is heat-resistant and not a protein or peptide ingredient. Has been demonstrated. The oyster extract of the present invention has been shown to enhance cytotoxicity by directly activating NK cells. Moreover, NK cell activation ability was confirmed also in vivo.

(Example 2: Physical properties and antitumor effect of oyster extract)
(A. Experimental materials and methods)
In addition to Example 1, the following experimental materials and methods were used.

(1. Cells)
B16 cells (mouse melanoma), Meth A cells (mouse fibroblastic sarcoma) and CT-26 cells (mouse colon cancer) were obtained from Riken BRC Cell Bank (Tsukuba City).

(2. In vivo anti-tumor experiment using tumor-bearing mouse model)
Ethanol 50 fraction (200 mg / kg) dissolved in PBS was intraperitoneally administered to C57BL / 6N mice or BALB / c mice every day from 3 days before seeding with B16 cells or Meth A cells to 14 days after tumor seeding. . B16 cells are subcutaneously in the back at 5 × 10 ⁵ cells / 200 μL (RPMI 1640 medium without FCS), and Meth A cells are subcutaneously in the back at 2 × 10 ⁶ cells / 200 μL (RPMI 1640 medium without FCS). Sowing. B16 cells or Meth A cells were seeded on day 0, and PBS was intraperitoneally administered daily from day -3 to day 14 in the control group. From the day when tumor growth was confirmed on the back, the short axis and long axis of the tumor were measured with a caliper. On day 14, C57BL / 6N mice or BALB / c mice were sacrificed by cervical dislocation and tumor weights were measured. Evaluation was performed on the tumor weight and tumor volume of each group. Tumor volume was expressed as 2 minor axes x ² major axes / 2.

(3. Annexin V positive cell measurement)
Annexin V positive cell measurement was performed in accordance with the protocol of Annexin V apoptosis detection kit FITC (eBioscience). 10 × Binding Buffer was diluted with Milli-Q H ₂ O, and the cells were washed with 1 × Binding buffer. A cell suspension was prepared at a concentration of 1 × 10 ⁶ cells / mL, and 5 μL of FITC-annexin V was added and allowed to stand for 10 to 15 minutes. Thereafter, the plate was washed twice with 1 × Binding buffer, 5 μL of PI was added, and FACS measurement was performed.

(B. Results)
(1. Antitumor effect in 50 tumor-bearing mice of Ethanol)
B57 melanoma cells derived from the same species were seeded in C57BL / 6N mice, a tumor-bearing mouse model was prepared, and the antitumor action of Ethanol 50 fraction was examined. Ethanol 50 fraction was intraperitoneally administered to mice in advance (from day -3) (PBS was administered to the control group) to activate NK cells, and then B16 cells were seeded subcutaneously on the back of mice (day 0). Eyes), tumor growth up to day 14 was recorded. Ethanol 50 fraction was administered intraperitoneally every day from day -3 to day 14. Tumor length and breadth from the date of the growth was observed in the tumors were measured with slide calipers, a long diameter × short diameter ^2/2 was examined changes over time as the tumor volume (FIG. 2). In addition, mice were sacrificed 14 days after seeding with B16 tumor cells, and tumor weights were measured. As a result, administration of 50 fractions of Ethanol significantly suppressed the increase in tumor volume and tumor weight. Further, when Ethanol 50 fraction was orally administered (2000 mg / kg) for the same period, a stronger inhibitory effect was observed on the increase in tumor volume and tumor weight.

  This result shows that the oyster extract of the present invention is also effective against melanoma. Jilavenu et al. (Clin. Dermatol. 27, 614-625 (2009)) and guidelines for proper use of anti-cancer drugs (http://nvc.halsnet.com/jhattori/cancer-navi/guideline_Kuidelinez/Guidelinez_Guideline_Kuidelinez_Guidelinez_Guidelinez_Guidelinez_Guideline_Kuidelinez .Htm), the effect of the oyster extract of the present invention that melanoma can be treated is very well anticipated because it is widely recognized that melanoma is difficult to treat. It is an outside effect.

Next, BATH / c mice were seeded with Meth A cells, a tumor-bearing mouse model was prepared, and the antitumor action of oyster extract was examined. Ethanol 50 fraction was intraperitoneally administered to mice in advance (from day -3) (PBS was administered to the control group), and then Meth A cells were seeded in the mouse back skin (day 0), and day 14 Tumor growth to the eye was recorded. Ethanol 50 fraction was administered intraperitoneally every day from day -3 to day 14. Tumor length and breadth from the date of tumor growth was confirmed was measured with slide calipers, a long diameter × short diameter ^2/2 was examined changes over time as the tumor volume. In addition, the mice were sacrificed 14 days after the seeding of Meth A tumor cells, and the tumor weight and spleen weight were measured. As a result, the growth of the tumor volume was suppressed by administration of Ethanol 50 fraction (FIG. 3), and a significant reduction in the tumor volume on the 13th and 14th days was observed. Moreover, suppression of the tumor weight of the 14th day was recognized by administration of Ethanol 50 fraction (FIG. 4).

(2. In vitro antitumor effect of oyster extract)
The direct effect of the Ethanol 50 fraction on tumor cells was examined using in vitro cultured cells. As a result, Ethanol 50 fraction promoted the proliferation of B16 cells contrary to the in vivo effect using tumor-bearing mice. However, Meth A cells (mouse fibroblastoma), YAC-1 cells (mouse lymphoma), CT It suppressed the growth of -26 cells (mouse colon cancer), and among them, the inhibitory effect on Meth A cells was the strongest (FIG. 5).

  Thus, although the Ethanol 50 fraction did not directly inhibit the proliferation of B16 cells in vitro but rather promoted it, it showed an inhibitory effect in vivo using tumor-bearing mice in vivo. It is suggested to suppress tumor growth. It is considered that the Ethanol 50 fraction first activates NK cells and exerts an antitumor effect through it.

  Tomomi Sato (Hirosaki University School of Health Sciences Department of Health Sciences, Graduate School of Sciences, Vol. 2, pp. 297-302, March 2006) is impossible to think that oyster extract activates lymphocytes to show antitumor activity However, the oyster extract of the present invention is a characteristic extract in that it has an NK activity promoting action and an antitumor action.

  In the following experiments, attention was focused on Meth A cells that exhibited a strong growth inhibitory action. First, the growth inhibitory effect of the ethanol fraction of the oyster extract was compared. OE indicates an extract not subjected to ethanol fractionation. OE30i represents an insoluble fraction when the final ethanol concentration is 30%. OE50i represents the insoluble fraction when ethanol is added to the soluble fraction when the final concentration of ethanol is 30% and the final concentration is 50%. OE50s indicates a soluble fraction when the final ethanol concentration is 50%. OE50i (Ethanol 50 fraction), which is an insoluble fraction when the final ethanol concentration is 50%, showed the strongest antitumor action, but OE50i, which is a soluble fraction when the final ethanol concentration is 50%, was also OE50i. Although antitumor activity was not observed, the antitumor activity was observed (FIG. 6).

(3. Antitumor activity over time of Ethanol 50 fraction)
The time course of tumor growth inhibitory activity of the 50 ethanol fractions was evaluated by the MTT method. When Ethanol 50 fraction (0, 0.75, 1.5 mg / mL) and Meth A cells were cultured for 24 hours and 48 hours, a stronger growth inhibitory effect was observed in the culture for 48 hours (FIG. 7). A similar growth inhibitory effect was also observed in the number of viable cells measured by the trypan blue method, but no significant change was observed in cell viability even when the concentration of Ethanol 50 fraction was increased (Table 2).

  Therefore, the Ethanol 50 fraction is considered not to induce Meth A tumor cells to cell death but to suppress their cell growth ability. Further growth inhibition effect was observed when Meth A cells and Ethanol 50 fraction having a smaller number of cells were cultured for a long time (48 hours and 72 hours).

(4. Apoptosis-inducing action of Ethanol 50 fraction)
It is known that phosphatidylserine, usually present inside the cell membrane, appears on the cell surface as the first cell shape change that occurs when cells begin to undergo apoptosis. Since annexin V binds to phosphatidylserine in a calcium-dependent manner, cells in the early stages of apoptosis can be measured by detecting phosphatidylserine seropositive and PI-negative cells. Even when the treatment with the oyster extract of the present invention was performed, no change was observed in the number of dead cells due to apoptosis in the measurement of cell viability in the trypan blue method. May be affected. Therefore, Meth A cells were cultured with Ethanol 50 fraction (0.75, 1.5 mg / mL) for 24 hours and 48 hours, and Meth A cell surface phosphatidylserine was stained with FITC-labeled annexin V, and then FACS. Early apoptotic cells were detected (FIG. 8). As a result, the apoptosis-inducing action of Ethanol 50 fraction on Meth A was recognized in a concentration-dependent manner, and the 48-hour culture showed a stronger action than the 24-hour culture.

(5. Effect of proteinase K treatment on antitumor effect of Ethanol 50 fraction)
In order to examine whether the tumor cell growth inhibitory substance in the Ethanol 50 fraction is a protein, proteinase K treatment was carried out at room temperature for 30 minutes to examine changes in growth inhibitory activity. As a result, the activity of Ethanol 50 fraction was not affected by proteinase K treatment. This suggested that the tumor cell growth inhibitor is not a protein.

(6. Effect of sodium periodate treatment on antitumor effect of Ethanol 50 fraction)
In order to examine whether the tumor cell growth inhibitory substance in the Ethanol 50 fraction is a saccharide, the Ethanol 50 fraction was treated with sodium periodate to examine the change in the growth inhibitory activity. As a result, inactivation of the growth inhibitory action was observed, suggesting that the growth inhibitory component was a carbohydrate.

(7. Gel filtration chromatography of 50 fractions of Ethanol)
In order to measure the molecular weight of the tumor growth inhibitory substance, gel filtration chromatography with Sephadex G-50 (fractionated molecular weight 1500 to 30000) was performed, and the antitumor effects of the obtained fractions were examined using Meth A cells. . As a result, the molecular weight of the active ingredient was about 2000.

  Recently, the anti-tumor effect of glycogen derived from oyster extract has been reported (Nana Kumaki (Anti-tumor activity of oyster extract. Graduate School of Health Sciences, Hirosaki University School of Health Sciences, Vol. 1: 122-128 (2005)), Tomomi Sato. (Analysis of the mechanism of action of antitumor effects in oyster extract. Hirosaki University School of Health Sciences Department of Health Sciences, Graduate School of Science and Technology, Vol. 2: 297-302 (2006)), and Matsuda Yoshikazu et al. (Kassostrea gigas) hot water extraction Of the anti-tumor activity and anti-tumor activity of foods.Micronutrient research 15th: 121-127 (1998)). However, in the oyster extract-derived glycogen in these documents, the active ingredient is a saccharide, as in the oyster extract of the present invention, but the molecular weight of the active ingredient described in these documents is about 4000 kDa, which is a polymer. Different from the active ingredient. Therefore, the oyster extract described in these documents is different from the oyster extract of the present invention.

  According to the present invention, a novel oyster extract having NK cell activation ability and anti-melanoma cell activity is provided.

Claims (9)

A composition for treating or preventing a tumor, comprising an oyster hot water extract, wherein the oyster hot water extract comprises the following steps:
a) A step of hot water extraction of raw oysters at 125 ° C. under pressure for 2 hours,
A composition comprising, as an active ingredient, a sodium periodate-sensitive substance, prepared by a method comprising: The composition further comprises:
b) filtering the oyster hot water extract of step (a); and
c) vacuum concentration of the filtrate of step (b),
The composition of claim 1, which is prepared by a method comprising: The composition further comprises:
d) sterilizing the concentrate of step (c); and
e) spray drying the sterilized product of step (d),
The composition of claim 2, which is prepared by a method comprising: The composition further comprises:
f) adding ethanol to the product spray-dried in step (e) to a final concentration of 30% to prepare a soluble fraction; and
g) adding ethanol to the soluble fraction of step (f) to a final concentration of 50% to prepare an insoluble fraction;
4. The composition of claim 3, which is prepared by a method comprising:   The composition according to claim 1, wherein the tumor is melanoma. A composition for activating NK cells, comprising an oyster hot water extract, wherein the oyster hot water extract comprises the following steps:
a) A step of hot water extraction of raw oysters at 125 ° C. under pressure for 2 hours,
A composition comprising, as an active ingredient, a sodium periodate-sensitive substance, prepared by a method comprising: The composition further comprises:
b) filtering the oyster hot water extract of step (a); and
c) vacuum concentration of the filtrate of step (b),
The composition of claim 6, which is prepared by a method comprising: The composition further comprises:
d) sterilizing the concentrate of step (c); and
e) spray drying the sterilized product of step (d),
The composition of claim 7, which is prepared by a method comprising: The composition further comprises:
f) adding ethanol to the product spray-dried in step (e) to a final concentration of 30% to prepare a soluble fraction; and
g) adding ethanol to the soluble fraction of step (f) to a final concentration of 50% to prepare an insoluble fraction;
9. The composition of claim 8, wherein the composition is prepared by a method comprising: