JP6741429B2 - Composition for treating colorectal cancer and processed food - Google Patents

Description

The present invention relates to a composition for treating colorectal cancer derived from maple syrup.

Colorectal cancer is a disease with a high number of morbidity and mortality worldwide. If the tumor is confined to the mucosal layer, it can be cured completely with an endoscope or surgical operation, but it is often already advanced when it is diagnosed. Chemotherapy with fluoropyrimidine plus oxaliplatin or irinotecan is the standard treatment for advanced colorectal cancer. In recent years, the use of plant-derived preparations such as plant extracts for cancer treatment has become of interest. Natural remedies using plant extracts are expected to have fewer adverse events than conventional cancer therapies.

Maple syrup is a natural sweetener used by people all over the world. Maple syrup contains various substances such as oligosaccharides, organic acids, amino acids, vitamins, minerals such as magnesium and zinc, as well as sucrose and glucose, which are the main components. Furthermore, it has recently been reported that maple syrup contains phenolic compounds such as lignans, coumarins, kebecols and ginarine.

These phenolic compounds have various activities. In vitro studies using butanol extract of maple syrup showed that α-glucosidase has an inhibitory action (Patent Document 1: Non-Patent Document 1), and ethyl acetate extract has antioxidant action and antitumor effect. It was reported to have an action (Non-patent Document 2). In addition, it was reported that ginnarin A has a suppressive effect on the growth of colon cancer cells (Non-Patent Document 3). On the other hand, research on the effect of maple syrup itself was also promoted, and it was revealed that administration of maple syrup to type 2 diabetic model rats significantly suppressed the increase in blood glucose level as compared to rats administered with sucrose solution. Became.

JP, 2006-008523, A

Apostolidis E, Li LY, Lee C and Seem NP: In vitro evaluation of phenotype-encapsulated prep eryth reduct horizontation of inflation. J Funct Foods 3: 100-106, 2011. Legault J, Girard-Lalancette K, Grenon C, Dussault C and Pichette A: Antioxidant activity, inhibition of nitric oxide overproduction, and in vitro antiproliferative effect of maple sap and syrup from Acer saccharum. Journal of medicinal food 13: 460-468, 2010. Gonzalez-Sarrias A, Li Land Seerm NP: Effects of maple (Acer) plant parts extractables on-pronunciation and apo- sol. Phytotherapy research: PTR 26: 995-1002, 2012. Kang JS, Park IH, Cho JS, et al. : Epigalcatechin-3-gallate inhibits collagen production of natural polyp-derivatized fibroblasts. Phytotherapy research: PTR 28: 98-103, 2014. Zhai X, Chi J, Tang W, et al. : Yellow Wine Polyphenolic Compounds Inhibit Matrix Metalloproteinase-2, -9 Expression and Impulse Atherosclerotic Plaque in LDL-Receptor-Knock-out. Journal of pharmacological sciences 2014. Singh T and Katiyar SK: Green tea polyphenol, (-)-epigallocatechin-3-gallate, induces toxicity in human skin cancers by targeting-. Toxicology and applied pharmacology 273: 418-424, 2013. Zhao M, Tang SN, Marsh JL, Shankar S and Srivastava RK: Elective acid inhibitors human pancreatic cancer grow in Balcnu. Cancer letters 337: 210-217, 2013. Tsai CM, Sun FM, Chen YL, Hsu CL, Yen GC and Weng CJ: Molecular mechanics in decompression in vaccinia humorous behaviours. European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 48: 494-501, 2012. Chen Y, Zheng L, Liu J, et al. : Shikonin inhibits prostate cancer cells cells metastasis by reducing matrix metalloproteinase-2/-9 expression via AKT/mTOR and ROS/erw1/2pa. International immunopharmacology 21: 447-455, 2014. Kuo CL, Lai KC, Ma YS, Weng SW, Lin JP and Chung JG: Gallic acid inhibits migration and invasion of SCC4 human oral cancer cells through actions of NFkappaB, Ras and matrix metalloproteinase-2 and -9. Oncology reports 32: 355-361, 2014. Xu Q, Ma J, Lei J, et al. : Alpha-Mangostin supresses the viability and epithelial-mesenchy transition of pancreatic cancer cells by downregulating the pl.K3/A3. BioMed research international 2014: 546353, 2014. Gonzalez-Sarrias A, Ma H, Edmonds ME and Seeram NP: Maple polyphenols, ginnalins A-C, induce S- and G2 / M-cell cycle arrest in colon and breast cancer cells mediated by decreasing cyclins A and D1 levels. Food chemistry 136: 636-642, 2013.

Maple syrup is made by boiling down sap, but its color, aroma and taste change depending on the time of sampling the sap and the temperature. Therefore, in Canada, based on these differences, maple syrup is classified into five grades: grade AA (extra light), grade A (light), grade B (medium), grade C (amber), and grade D (dark). ing.

It has been reported that the color of maple syrup becomes darker as the season progresses, and there is a correlation between the color depth and antioxidant power. This suggests that different grades of maple syrup have different effects on cancer cells.

However, little study has been conducted on the difference in the effect of maple syrup depending on the grade. Therefore, in order to use maple syrup for the treatment of colorectal cancer, it is necessary to evaluate the difference in antitumor effect depending on the grade.

The present invention has been conceived in view of the above requirements, and the effect of using maple syrup was examined by examining the effects on proliferation, migration, and invasion of colon cancer cells by administration of three types of maple syrup with different color tones. The present invention provides a composition for treating large bowel cancer.

Colorectal cancer therapeutic agent according to more specifically the present invention, as a substance derived from a menu over pull syrup, for the treatment of colorectal cancer with as an active ingredient having a molecular weight of 10,000 or more of the proteins contained in the maple syrup grade D It is a composition.

Maple syrup, which is used as a natural sweetener by people all over the world, suppressed the growth and invasion of colon cancer cells by inhibiting the activity of AKT. Therefore, there is a possibility that maple syrup (especially dark one) can be applied as a treatment with fewer adverse events than the conventional treatments for colorectal cancer.

In order to investigate the cytotoxicity of cultured colon cancer cells DLD-1 and SW480 due to a high concentration of sucrose, the sucrose solution used as a control was administered to the culture solution at 0.1-10%, and the cell proliferation at that time was performed. It is a graph which shows the result of having confirmed the ability. It is a graph which shows the result of having investigated the effect of the maple syrup with respect to the growth of a colon cancer cell. 6 is a graph showing the results of examining the effect on the cell proliferation ability using normal colon epithelial cells CCD841CoN in order to evaluate whether maple syrup affects the growth of normal colon cells. It is a graph which shows the result of having examined the effect|action on the cell migration / invasion ability using the Boyden chamber method about the cultured colon cancer cell. 3 is a photograph showing the results of Western blotting that examined the effect on ERK and AKT activation (phosphorylation) when a sucrose solution or Maple Syrup III (grade D) was administered to cells. It is a graph which shows the result of having performed the array analysis by real-time PCR about the kind of mRNA expressed when the maple syrup of grade AA and the maple syrup of grade D were added. 3 is a graph showing the results of a colorectal cancer cell proliferation inhibition test for each fraction of grade D maple syrup divided into fractions having a molecular weight of 10,000 or more and less. It is a graph which shows the experimental result which confirmed the growth inhibitory effect of the colorectal cancer cell which the protein of the fraction of molecular weight 10,000 or more of grade AA and grade D has. It is a photograph which shows the result of electrophoresis of the protein of the fraction of molecular weight 10,000 or more of grade AA and grade D.

Hereinafter, the composition for treating colon cancer and the processed food according to the present invention will be described with reference to graphs. In addition, the following description shows one embodiment of the present invention, and the present invention is not limited to the following embodiment. The following examples can be modified without departing from the spirit of the present invention.

The present invention provides a composition for treating colorectal cancer having a substance derived from maple syrup as an active ingredient. More preferably, this substance is a fraction of Grade D (dark) having a molecular weight of 10,000 or more. (Hereinafter referred to as “grade D high fraction protein”). Therefore, it suffices to include the grade D high fraction protein, not the maple syrup itself. In other words, the substance derived from maple syrup is a composition for treating colorectal cancer (pharmaceutical composition) containing only a grade D high fraction protein as an active ingredient. Of course, the ingredient which has other efficacy may be contained.

The form of the pharmaceutical composition is not particularly limited, and includes capsules, granules, solutions, emulsions, suspensions, powders, tablets, liquids, dips, decoctions, troches, flow extracts, tinctures. , Eye drops, nasal drops, ointments, creams, lotions, injections, suppositories, etc.

Further, it may be a processed food containing a grade D high fraction protein. In particular, it does not need to include maple syrup itself, but may be a processed food in which a grade D high fraction protein and a material other than maple syrup are mixed. That is, this processed food does not need to contain sucrose derived from maple syrup.

Further, as the processed food, for example, candy, gum, jelly, biscuits, cookies, rice crackers, bread, noodles, fish meat/meat paste products, tea, soft drinks, coffee drinks, milk drinks, whey drinks, lactic acid bacteria drinks, yogurt Not only general processed foods such as ice cream, pudding, etc., but also soft extracts, dry extracts, capsules, granules, powders, tablets, liquids, dips, decoctions, troches, flow extracts, tinctures, etc. Alternatively, it may include an alcoholic beverage.

The processed food according to the present invention can be obtained by adding only the separated and purified grade D high fraction protein to the raw materials of these processed foods without adding other components of maple syrup. In other words, as the substance derived from maple syrup in the material of the processed food, only the grade D high fraction protein is contained.

<Material>
Urea used was from GE Healthcare. 3-(3-cholamidepropyl) dimethylammonio-1-propanesulfonate (CHAPS) manufactured by Wako Pure Chemical Industries, Ltd. was used. Thiourea and Triton X-100 used were those manufactured by Nacalai Tesque. Other reagents used were those manufactured by Sigma.

<Maple syrup>
Three types of commercially available maple syrup were used. Classification was performed as follows based on the difference in color tone.
(1) Maple Syrup I: Gold (2) Maple Syrup II: Amber (3) Maple Syrup III: Dark brown In addition, Maple Syrup III is Grade D (dark) and Maple Syrup A extract is Grade A (A). ).

<High performance liquid chromatography (HPLC)>
The concentration of sucrose in the maple syrup was analyzed using LC-10 Advp HPLC (manufactured by Shimadzu) equipped with Shodex RI-71 as a detector. Asahipak NH2P-50 4E (made by Shimadzu) was used for the column. Acetonitrile/water 3:1 (v/v) was used as the mobile phase, and the flow rate was 1 ml/min. 20 μl of sample solution was used for one analysis. The sample solution used was maple syrup diluted 100-fold with water.

<Cultured colon cancer cells>
Cultured colon cancer cells DLD-1 and SW480 and normal colon epithelial cells CCD 841 CoN were purchased from American Type Culture Collection. All cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in a 5% CO ₂ environment.

<Cell proliferation assay>
The cells were dispensed on a 96-well plate at 5×10 ³ cells/well and cultured. On the next day, the culture medium was exchanged for a maple syrup- or sucrose-containing medium, and 24, 48, 72, and 96 hours later, the cell growth ability was measured using WST-8 cell counting reagent (Wako Pure Chemical Industries, Ltd.).

In addition to the WST-8 assay, cell counts were also performed using a Counted Automated Cell Counter (Life Technologies Japan). The cells were dispensed on a 6-well plate at 5×10 ⁴ cells/well and cultured. The next day, the culture medium was exchanged with a medium containing maple syrup or sucrose, and the number of cells was counted after 24, 48, 72, and 96 hours.

<Cell migration and invasion assay>
In vitro cell migration assay was performed using Boyden chamber method. The cells were dispensed into the insert at 1×10 ⁵ cells/insert and incubated in an environment of 37° C. and 5% CO ₂ . A medium containing maple syrup or sucrose was added to the lower layer as a chemoattractant. According to the past literature, the number of cells passing through the insert was counted after 48 hours for DLD-1 and after 20 hours for SW480. All the assays were performed with n=3, and the number of cells present in 5 fields randomly photographed in a blinded manner was counted. Regarding the infiltration ability, the same operation was performed using the insert whose surface was coated with Matrigel.

<Protein extraction>
DLD-1 and SW480 were dispensed and cultured at 5×10 ⁵ cells/100-mm dish. The next day, the culture solution was exchanged for a maple syrup- or sucrose-containing medium, and 72 hours later, the protein was extracted using a protein extract (7M urea, 2M thiourea, 5% CHAPS, 1% Triton X-100). The protein concentration was measured using the Bradford method.

<Western blot>
The extracted protein was subjected to SDS-PAGE under reducing conditions, and then the separated protein was transferred to a PVDF (PolyVinylidene DiFluoride) membrane. The transferred membrane was incubated with an anti-phospho-p44/42 MAPK antibody or an anti-phospho-AKT antibody (Cell Signaling Technology Inc.) overnight at 4°C. After washing the membrane, it was incubated with HRP-conjugated anti-rabbit IgG antibody or HRP-conjugated anti-murine IgG antibody (American Qualex) at room temperature.

After washing, light was emitted using a chemiluminescent reagent, and a luminescence image was photographed using an ImageQuant LAS 500 system (GE health care). In order to confirm whether the same amount of protein was used for comparison, after stripping the same membrane, it was reacted with anti-p44/42 MAPK (Erk1/2) antibody or anti-AKT antibody (Cell Signaling Technology) in the same manner. Was taken.

<Statistical analysis>
All data are shown as mean±standard error. The data were analyzed using Dunnett's test, and were significant when the P value was 0.05 or less. The analysis was carried out using GraphPad Prism version 5 (GraphPad Software). In addition, in the graph, those marked with "*" represent the case where the P value is 0.05 or less.

<Sucrose concentration in maple syrup>
First, the concentration of sucrose, which is the main component of maple syrup, was measured. The results are shown in Table 1. The sucrose concentration of each maple syrup was well controlled and no significant difference was observed among the 3 types of maple syrup. In order to accurately evaluate the effect of maple syrup administration on cancer cells, the sucrose concentration of Maple Syrup II and III was adjusted to be equal to that of Maple Syrup I. A sucrose solution having the same concentration as that of Maple Syrup I was prepared and used as a control solution.

<Cytotoxicity evaluation of sucrose solution on colon cancer cells>
In order to investigate the cytotoxicity of cultured colon cancer cells DLD-1 and SW480 due to a high concentration of sucrose, the sucrose solution used as a control was administered to the culture solution at 0.1-10% (v/v), The cell growth ability at that time was confirmed. The results are shown in Figure 1. In FIG. 1, the horizontal axis represents the culture time (h), and the vertical axis represents the cell growth ability (%) with respect to untreated cells (cells cultured in a medium to which no sucrose solution was added).

Further, FIG. 1(A) shows the case of cultured colon cancer cell DLD-1, and FIG. 1(B) shows the case of cultured colon cancer cell SW480. In both figures, the solid line indicates the untreated, the fine dotted line indicates the culture in the medium containing 0.1% sucrose solution, and the large dotted line indicates the culture in the medium containing 1% sucrose solution. The dotted chain line shows the case of culturing in a medium containing 10% sucrose solution.

Referring to FIG. 1(A), DLD-1 had no effect at other concentrations, but when cultured in a medium containing 10% sucrose solution as compared with untreated cells, 48 and 72 hours. Later, the cell proliferation ability was significantly suppressed. In addition, in the medium containing 10% sucrose, the cell growth ability was significantly suppressed after 48, 72, and 96 hours with respect to SW480 shown in FIG. 1(B). Therefore, in the subsequent studies, it was decided to administer 1% maple syrup and sucrose solution in order to avoid toxicity due to sucrose.

<Effect of maple syrup on proliferation of colon cancer cells>
The effect of maple syrup on the growth of colon cancer cells was examined. The results are shown in Figure 2. FIG. 2(A) shows the results of WST-8 assay of cultured colon cancer cells DLD-1. The horizontal axis represents the culture time (h), and the vertical axis represents the absorption rate at 450 nm. FIG. 2(B) shows the results of the cell count measurement method for cultured colon cancer cells DLD-1. The horizontal axis represents the culture time (h), and the vertical axis represents the number of cells per well (×10 ⁵ cells).

FIG. 2(C) shows the results of WST-8 assay of cultured colon cancer cells SW480. The horizontal axis represents the culture time (h), and the vertical axis represents the absorption rate at 450 nm. FIG. 2D shows the result of the cell count measurement method of the cultured colon cancer cells SW480. The horizontal axis represents the culture time (h), and the vertical axis represents the number of cells per well (×10 ⁵ cells).

In the four figures in FIG. 2, white circles (◯) indicate the case where the cells were cultured in a medium containing 1% sucrose (indicated as “Sucrose”), and black squares (■) contained Maple Syrup I. This is the case when cultured in a medium. The black triangles (▲) indicate the case of culturing in the medium containing Maple Syrup II, and the black circles (●) indicate the case of culturing in the medium containing Maple Syrup III.

Referring to FIG. 2(A), as a result of WST-8 assay, the proliferation ability of DLD-1 cells by administration of maple syrup was 24 hours (Maple Syrup II and III), 48 hours (Maple Syrup III), It was significantly suppressed after 72 hours (Maple Syrup II and Maple Syrup III) (P<0.01, FIG. 2(A)). Further, referring to FIG. 2(B), the proliferation ability of DLD-1 cells was also significantly suppressed after 48 hours (Maple Syrup III) in the cell counting method (P<0.01, FIG. 2). (B)).

Also for SW480, referring to FIG. 2(C), significant in the WST-8 assay after 48 hours (Maple Syrup II and Maple Syrup III), 72 hours (Maple Syrup III), and 96 hours (Maple Syrup III). Cell proliferation was suppressed (P<0.01, FIG. 2(C)). Further, with reference to FIG. 2(D), it was also significantly suppressed after 72 hours (Maple Syrup III) and 96 hours (Maple Syrup III) in the cell count method (P<0.01, FIG. 2). (D)).

<Effect of maple syrup on proliferation of normal colon epithelial cells>
Next, in order to evaluate whether maple syrup had an effect on the proliferation of normal colon cells, the effect on cell proliferation ability was evaluated using normal colon epithelial cells CCD841CoN. Results are shown in FIG. In FIG. 3, the horizontal axis represents the type of medium, and the vertical axis represents the number of cells cultured for 72 hours (×10 ⁴ cells). Although the cell proliferation ability was significantly suppressed in the cancer cells, no difference in the cell proliferation ability of normal colon epithelial cells CCD841CoN was observed 72 hours after administration of maple syrup (FIG. 3).

<Effect of maple syrup on migration and invasion of cancer cells>
Furthermore, the effect on cell migration/invasion ability was examined using the Boyden chamber method. The results are shown in Fig. 4. FIG. 4(A) shows the results of cell migration of cultured colon cancer cells DLD-1, and FIG. 4(B) shows the results of cell migration of cultured colon cancer cells SW480. In both figures, the horizontal axis represents the type of medium, and the vertical axis represents the ratio (%) of the number of migrating cells to the control (“Sucrose” when cultured in a medium containing 1% sucrose).

FIG. 4(C) shows the results of the infiltration ability of cultured colon cancer cells DLD-1, and FIG. 4(D) shows the results of the invasion ability of cultured colon cancer cells SW480. In both figures, the horizontal axis represents the type of medium, and the vertical axis represents the ratio (%) of the number of invaded cells to the control (“Sucrose” when cultured in a medium containing 1% sucrose).

Referring to FIGS. 4(A) and 4(B), no influence of maple syrup on cell migration ability was observed in both DLD-1 and SW480. However, referring to FIG. 4(C), Maple Syrup II and Maple Syrup III significantly suppressed the infiltration ability of DLD-1 (P<0.01). Further, referring to FIG. 4(D), it was confirmed that the infiltration ability of SW480 was also suppressed by Maple Syrup III (P=0.0779).

<Effect of maple syrup on signal transduction pathway of colon cancer cells>
In order to investigate the effect of maple syrup administration on the signal transduction pathway, the effect on ERK and AKT activation (phosphorylation) when sucrose solution or Maple Syrup III was administered to cells was examined. Results are shown in FIG.

FIG. 5(A) is a photograph of a western blot of p-ERK1/2 and ERK1/2 of cultured colorectal cancer cell DLD-1. FIG. 5(B) is a photograph of a western blot of p-ERK1/2 and ERK1/2 of cultured colon cancer cells SW480. Further, FIG. 5(C) is a photograph of a western blot of p-AKT and AKT of cultured colorectal cancer cells DLD-1. FIG. 5(D) is a photograph of a western blot of p-AKT and AKT of cultured colon cancer cells SW480.

In addition, in all the photographs, photographs of Western blotting when 1% sucrose was administered (denoted as “Sucrose”) and when Maple Syrup III was administered are shown.

In FIG. 5(A), in both cases of p-ERK1/2 and ERK1/2, two lines, although thin, can be confirmed upon administration of 1% sucrose and Maple Syrup III. There was no change in the thickness and thickness of each line when sucrose was added and when Maple Syrup III was added.

In FIG. 5(B), in both cases of p-ERK1/2 and ERK1/2, two thick lines can be confirmed in the administration of 1% sucrose and Maple Syrup III. There was no change in the thickness and thickness of each line when sucrose was added and when Maple Syrup III was added.

In FIG. 5(C), in the case of AKT, the line density was almost the same between the administration of Maple Syrup III and the administration of sucrose. However, in the case of p-AKT, the line of Maple Syrup III was thinner than that of sucrose.

Also in FIG. 5(D), in the case of AKT, the line density was almost the same between the case of Maple Syrup III administration and the case of administration of sucrose. However, in the case of p-AKT, the line of Maple Syrup III was thinner than that of sucrose.

Referring to FIG. 5(A) and FIG. 5(B), administration of maple syrup showed no effect on ERK activity. However, referring to FIG. 5(C) and FIG. 5(D), the administration of Maple Syrup III significantly suppressed the activity of AKT for both DLD-1 and SW480.

As described above, the effect of three different types of maple syrup on colon cancer cells was examined. First, in order to accurately evaluate the antitumor effect of maple syrup, we examined whether the high concentration of sucrose contained in maple syrup affects the growth of colon cancer cells. The growth of colon cancer cells was significantly suppressed by sugar (10% administration) (FIG. 1). From this, it was suggested that sucrose contained in maple syrup exhibits cytotoxicity due to its high osmotic pressure.

It has been reported that the growth of prostate cancer (74% suppression), lung cancer (63% suppression), breast cancer (45% suppression), and colon cancer (37% suppression) is suppressed by maple syrup (Non-Patent Document 2). .. In this report, since the study was carried out in a medium containing 5% maple syrup, it was considered that cell growth was possibly suppressed under the influence of high osmotic pressure due to high concentration of sucrose. Therefore, in the present application, a sucrose solution having the same concentration as that of sucrose contained in maple syrup was prepared as a control solution, and the concentration was determined so as not to be cytotoxic by the sucrose solution.

As a result, the cell growth ability of the cells administered with maple syrup was suppressed by the WST-8 assay and the cell counting method, as compared with the colon cancer cells administered with the sucrose solution (FIG. 2). Interestingly, the darker the color of maple syrup, the stronger the growth inhibitory effect. Further, the effect on the growth of normal colon cells was also examined, but the growth inhibitory effect of maple syrup was not found here (FIG. 3). These results suggest that maple syrup acts effectively on fast-growing cells such as cancer cells.

Furthermore, although the cell migration ability was not affected, Maple Syrup II and Maple Syrup III significantly suppressed the cell invasion ability of DLD-1. Moreover, in Maple Syrup III, the cell infiltration ability of SW480 tended to be suppressed (FIG. 4).

From these, it is suggested that maple syrup influences the activity and expression of enzymes such as MMP-2 and MMP-9, which play an important role in infiltration of extracellular matrix such as basement membrane. Was done.

Although there has been no report on the effect of maple syrup on the infiltration ability, it has been recently reported that polyphenols such as epigallocatechin-3-gallate suppress the expression of MMP-2 and MMP-9 ( Non-Patent Documents 4-8). Therefore, it is possible that the expression of MMP-2 or MMP-9 in colorectal cancer cells was suppressed by polyphenols such as kebecol and ginnarin contained in maple syrup.

Next, Maple Syrup III, which had the strongest antitumor effect, was used to examine the phosphorylation status of ERK and AKT, which play important roles in cell proliferation and invasion. Although it did not affect the activity of ERK, administration of Maple Syrup III significantly suppressed the activation of AKT (FIG. 5).

This suggests that induction of apoptosis is involved in the growth inhibitory effect of maple syrup. Further, it has been reported that inhibition of AKT activity is associated with inhibition of cell invasion by suppressing expression of MMP-2 and MMP-9 (Non-patent documents 9-11).

In recent years, it has been reported that Ginnarin AC, which is one of the polyphenols contained in maple syrup, suppresses cell proliferation not by induction of apoptosis but by cell cycle arrest (Non-Patent Document 12). Therefore, it is strongly suggested that maple syrup may contain active ingredients other than polyphenols.

FIG. 6 shows the results of comparison of mRNA expression using real-time PCR. The horizontal axis is the gene name encoding the protein, and the vertical axis is the relative expression level (no unit). Maple syrup grade AA (extra light)-administered cells (the white bar labeled "EXTRA" in the graph) and grade D (dark)-administered cells (indicated as "DARK" in the graph) In the case of (), the expression of each gene is measured. Since "EXTRA" is the standard, the relative expression level of "EXTRA" is 1.0.

Referring to FIG. 6, when dark was administered, the expression of CDKN2A was more than doubled compared to the case where extralite was administered. CDKN2A is a cancer suppressor protein called cyclin-dependent kinase inhibitor 2A, and it has been shown that administration of grade D (dark) causes a large expression of mRNA for producing CDKN2A in cells. From this, it can be seen that the administration of Maple Syrup III has a cancer suppressing effect.

As described above, it was found that Grade D has a cancer suppressing effect. Therefore, the main active ingredients contained in Grade D were investigated next. A 10-fold dilution of Maple Syrup III (30 ml of syrup was diluted to a total volume of 300 ml) was fractionated using an ultrafiltration unit (Amicon (registered trademark) Ultra-15: manufactured by Merck Millipore). A filter having a molecular weight cutoff of 10,000 was used.

The centrifugation conditions were 5000×g (“×” represents multiplication and “g” is gravitational acceleration) for 15 minutes. By centrifugation, it was separated into a fraction having a molecular weight of 10,000 or more and a fraction having a molecular weight of less than 10,000. A fraction having a molecular weight of 10,000 or more is called a "grade D high fraction", and a fraction having a molecular weight of less than 10,000 is called a "grade D low fraction".

The effect of the high-grade D fraction and the low-grade D fraction on colon cancer cells was confirmed by a cell proliferation assay using cultured colon cancer cells DLD-1. The experimental method was in accordance with the explanation in the section <Cell proliferation assay>. Each additive was added to the medium at a concentration of 1% by weight. The culture time is 72 hours.

The results are shown in FIG. 7. Referring to FIG. 7, the horizontal axis represents the type of substance added to the medium. "Non-treatment" means that nothing specially added to the medium.

"Sucrose" indicates sucrose. “Maple Syrup III (MW<10000)” represents the grade D low fraction and “Maple Syrup III (MW>10000)” represents the grade D high fraction. “Maple Syrup III” is “Maple Syrup III” itself including both the grade D low fraction and the grade D high fraction.

Moreover, the vertical axis represents the number of cells (Cells/well (×10 ⁵ )).

From the graph of FIG. 7, it could be said that sucrose and grade D low fraction (Maple Syrup III (MW<10000)) did not significantly inhibit cell growth, as compared to the control (Non-treatment). ..

On the other hand, the grade D high fraction (Maple Syrup III (MW>10000)) and Maple Syrup III had significant cell growth inhibition over the control.

Therefore, it can be said that among the grade D maple syrups, the high grade D fraction having a molecular weight of 10,000 or more has a cell growth inhibitory action on colon cancer cells.

Furthermore, for confirmation, it was confirmed that the proteins of the fractions of extra light and dark having a molecular weight of 10,000 or more have a cell growth inhibitory effect on colon cancer cells. A 10% solution of maple syrup extra light and dark was centrifuged to obtain a fraction having a molecular weight of 10,000 or more. Ammonium sulfate precipitation was performed on these fractions to obtain a protein. The respective proteins are referred to as "grade AA high fraction protein" and "grade D high fraction protein".

To the cultured colon cancer cells DLD-1, 5 μg, 10 μg, and 20 μg of the protein in each fraction were added to the medium (total amount of 2 ml), and cultured for 72 hours.

The results of the cell growth inhibitory action test are shown in FIG. In each of FIGS. 8A and 8B, the horizontal axis represents the type of sample. The grade AA high fraction protein is represented as “GradeAA”, and 5, 10, and 20 included in the category indicate the cases where 5 μg, 10 μg, and 20 μg were added, respectively. Similarly, the grade D high fraction protein is referred to as “Grade D”, and 5, 10, and 20 included in the category indicate the cases where 5 μg, 10 μg, and 20 μg were added, respectively.

The vertical axis of FIG. 8(a) is the cell number (Cells/well (×10 ⁵ )), and the vertical axis of FIG. 8(b) is the cell number as a ratio (%) to the “control”. .. The term "control" refers to the case where the cultured colon cancer cell DLD-1 was cultured in a medium that does not contain the above protein.

With reference to FIG. 8, when the grade AA high fraction protein was added, the number of cells was decreased, but it was not significantly suppressed. On the other hand, with the grade D high fraction protein, it was confirmed that the cell growth was suppressed as the added amount was increased, and that the added amount of 10 μg and 20 μg had a significant inhibitory effect as compared with the control. From the above, it was found that the protein in the high grade D fraction (grade D high fraction protein) has an effect of suppressing the growth of colon cancer cells.

FIG. 9 is a photograph in which the grade AA high fraction protein and the grade D high fraction protein were subjected to SDS-PAGE in a reducing environment, electrophoresed, and visualized by silver staining. The vertical axis represents the molecular weight (kDa). The lane on the left is the grade AA high fraction protein (designated "GradeAA") and the lane on the right is the grade D high fraction protein (designated "Grade D").

As for the grade D high fraction proteins, as shown by the symbols 1 to 5 (the symbols are circled in FIG. 9 ), there are proteins exhibiting a unique expression pattern. On the other hand, among the grade AA high fraction proteins, few proteins were expressed at the same position. Therefore, it can be said that the grade D high fraction protein is an active ingredient for suppressing the growth of colon cancer cells.

The cancer therapeutic agent according to the present invention can be suitably used for the treatment of colorectal cancer and the like.

Claims (2)

A composition for treating colorectal cancer, which comprises as a substance derived from maple syrup, a protein contained in grade D maple syrup having a molecular weight of 10,000 or more as an active ingredient. A processed food for suppressing colon cancer cell growth, which comprises a protein having a molecular weight of 10,000 or more contained in grade D maple syrup as a substance derived from maple syrup.