Prophylactic and / or therapeutic agent for colon cancer containing mannose derived from coffee beans and / or coffee extraction residue, and method for producing same

Abstract

To provide a prophylactic and / or therapeutic agent for colon cancer containing mannose derived from coffee beans and / or coffee extraction residues, and a method for producing the same.SOLUTION: The present invention provides a prophylactic and / or therapeutic agent for colorectal cancer containing mannose derived from coffee beans and / or a coffee extraction residue because mannose is contained in a hydrolysate of coffee beans and / or a coffee extraction residue and the hydrolysate is effective for the prevention and / or treatment of colorectal cancer. Since the raw material is coffee beans existing in nature, it can be safely ingested or administered as compared with chemically synthesized mannose. It also leads to effective utilization of coffee extraction residues which have been incinerated or treated as industrial wastes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a preventive and / or therapeutic agent for colon cancer (hereinafter sometimes simply referred to as "therapeutic agent") containing mannose derived from coffee beans and / or coffee extract residue, and a method for producing the same. More specifically, the present invention relates to a therapeutic agent containing a mannose-containing hydrolysate of coffee beans and / or coffee extract residue, and a method for producing the same. [Background technology]

[0002] According to the National Cancer Registry Incidence and Rate Report (Reiwa 2, prepared by the Cancer and Disease Control Division, Health and Environmental Health Bureau, Ministry of Health, Labour and Welfare), colorectal cancer is the second most common cancer site for both men and women, accounting for approximately 15-16% of all cancers. With changes in dietary habits, the number of colorectal cancer patients is expected to continue to increase, creating a need for therapeutic drugs to prevent and / or treat colorectal cancer.

[0003] On the other hand, it is well known that cellular metabolism is altered in cancer cells, and cancer cells, in particular, have the property of consuming large amounts of sugar compared to normal cells. Because many cancer cells exhibit enhanced glucose uptake, research into their responses to monosaccharides other than glucose has been ongoing. For example, it has been found that mannose is taken up by cancer cells via the same transporter as glucose, but accumulates intracellularly as mannose-6-phosphate, inhibiting further glucose metabolism in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, and glycan synthesis. As a result, it has been reported that mannose suppresses the growth of several types of cancer cells and promotes cell death in response to major chemotherapeutics (see, for example, Non-Patent Document 1).

[0004] There are various methods currently in use for producing mannose, including a chemical synthesis method in which an aqueous solution containing D-glucose and a metal such as molybdic acid as a catalyst is heated and epimerized to produce a mannose-containing sugar solution (see, for example, Patent Document 1), and a method in which mannose is produced by hydrolyzing natural products such as fruits and peels.

[0005] Coffee extract residue is said to contain a large amount of polysaccharides, mainly composed of mannose (see, for example, Non-Patent Document 2). Reported methods for producing mannose from coffee extract residue include adding sulfuric acid to produce mannan oligomers (see, for example, Patent Document 2) and adding acetic acid or formic acid to produce a mannooligosaccharide-containing substance (see, for example, Patent Document 3). Furthermore, mannooligosaccharides extracted from coffee extract residue have recently been reported to have a variety of functions and effects, including the effects of improving taste, improving serum lipids, inhibiting the increase in lipid peroxides, and promoting the growth of beneficial intestinal bacteria such as bifidobacteria. They have also been reported to promote lymphocyte proliferation (see, for example, Patent Document 4) and inhibit cancer cell proliferation (see, for example, Patent Document 5). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 63-12072 [Patent Document 2] Special Publication No. 5-52200 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-132187 [Patent Document 4] Japanese Patent Application Laid-Open No. 2004-051582 [Patent Document 5] Japanese Patent Application Laid-Open No. 2015-189731 [Non-patent literature]

[0007] [Non-Patent Document 1] Pablo Sierra Gonzalez, et. al., “Mannose impairs tumor growth and enhances chemotherapy” NATURE, 29 November 2018, vol 563. [Non-patent document 2] Allan GW Bradbury, Don J. Halliday, J. Agric. Food Chem., 1990, 38(2), pp 389-392. Summary of the Invention [Problem to be solved by the invention]

[0008] Chemically synthesized mannose is inexpensive and is therefore often used in experiments. Chemically synthesized mannose is also used in Non-Patent Document 1. However, the method using molybdic acid as a catalyst, which is a commonly used method for producing chemically synthesized mannose and which is described in Patent Document 1, has the problem that it is difficult to use the produced mannose for food, pharmaceuticals, etc. from the viewpoint of safety.

[0009] As for mannose derived from natural products, Patent Documents 2 and 3 describe methods for producing mannose derived from coffee extract residue, but do not describe its effectiveness against cancer. Furthermore, Patent Document 4 describes that mannose-based oligosaccharides have immunostimulating effects, and Patent Document 5 describes that mannopolyosylfructose has an anticancer effect against mouse leukemia cells, but neither document focuses on mannose alone. Furthermore, neither document describes that hydrolysates of coffee beans and / or coffee extract residue containing mannose alone are effective against colon cancer.

[0010] Therefore, an object of the present invention is to provide a preventive and / or therapeutic agent for colon cancer containing mannose derived from coffee beans and / or coffee extract residue, and a method for producing the same. [Means for solving the problem]

[0011] That is, the present invention has been made to solve the above-mentioned problems, and embodiments of the present invention may include at least some of the configurations listed below. (1) A preventive and / or therapeutic agent for colon cancer, which contains mannose derived from coffee beans and / or coffee extract residue. (2) A preventive and / or therapeutic drug for colon cancer, which contains a hydrolysate of coffee beans and / or coffee extract residue containing mannose. (3) adding an enzyme to coffee beans and / or coffee extract residue to subject them to hydrolysis treatment, thereby preparing a liquid composition containing mannose; filtering the liquid composition to prepare a filtrate; and combining the filtrate to form a formulation. A method for producing a preventive and / or therapeutic drug for colon cancer. [Effects of the Invention]

[0012] The preventive and / or therapeutic agent for colon cancer of the present invention contains mannose derived from coffee beans and / or coffee extract residue and can be used for the prevention and / or treatment of colon cancer. Because the source of the mannose used in the present invention is naturally occurring coffee beans, it can be safely ingested or administered and can serve as an alternative to the conventionally known chemically synthesized mannose. Furthermore, coffee extract residue cannot be used as animal feed due to its high lipid content, and most of it has been incinerated or disposed of as industrial waste. However, using coffee extract residue to produce mannose in the present invention is preferable because it also leads to a reduction in industrial waste. [Brief explanation of the drawings]

[0013] [Figure 1] Reactivity of roasted coffee bean-derived mannose to colon cancer spheroids [Figure 2] Photographic images showing the difference in reactivity of coffee concentrate with and without enzyme treatment against colon cancer spheroids (HC73T) [Figure 3] A graph showing the difference in reactivity of coffee concentrate with and without enzyme treatment against colon cancer spheroids (HC73T) [Figure 4] Photographic images showing the difference in reactivity of coffee concentrate with and without enzyme treatment against colon cancer spheroids (HC6T) DETAILED DESCRIPTION OF THE INVENTION

[0014] The preventive and / or therapeutic agent for colorectal cancer of the present invention contains mannose derived from coffee beans and / or coffee extract residue. In other words, the therapeutic agent of the present invention contains a hydrolysate of coffee beans and / or coffee extract residue containing mannose.

[0015] The therapeutic agent of the present invention focuses on the "monosaccharide" mannose, and it is preferable that the therapeutic agent of the present invention does not contain oligosaccharides. Here, "oligosaccharide" refers to a compound in which approximately 2 to 20 monosaccharides are linked together by glycosidic bonds, and includes not only those consisting of a single type of oligosaccharide, but also mixtures (compositions) of multiple types of oligosaccharides with different degrees of polymerization. In this specification, A to B refer to A or more and B or less unless otherwise specified.

[0016] <Method for producing mannose derived from coffee beans and / or coffee extract residue> Mannose exists in D- and L-forms, but since the L-form does not exist in nature, D-mannose is referred to as mannose in this specification.

[0017] Coffee beans and / or coffee extract residue contain large amounts of polysaccharides consisting of arabinogalactan (galactan) and galactomannan, and it is known that mannose is released from galactomannan. In the present invention, mannose is produced using coffee beans and / or coffee extract residue, which are distributed and consumed in large quantities worldwide, and used for pharmaceutical purposes. Coffee extract residue is easily available, and its use also leads to the effective use of industrial waste.

[0018] The coffee beans used in the present invention are not limited by their type or origin, and can be any coffee beans, such as Arabica, canephora, or liberica, or a variety native to each country. Furthermore, coffee beans from any origin, such as Ethiopia, Brazil, or Colombia, can be used. A single type of bean may be used alone, or a blend of two or more types of beans may be used. Even low-quality coffee beans or small-sized coffee beans that would normally be discarded as having no commercial value can be used. Green beans or roasted coffee beans may be used. The roasting method (direct flame, hot air, far-infrared, charcoal, etc.), roasting level (light roast, medium roast, dark roast, etc.), and extraction under normal pressure or pressure, and the degree of extraction, are not important.

[0019] Since increasing the surface area by grinding coffee beans increases the mannose yield, it is preferable to use finely ground beans. The grinding method (such as a large grinder or mill) is not critical as long as the beans are finely ground. Finely ground coffee beans refer to coffee bean powder with an average particle size of 0.1 mm or more and less than 0.6 mm, medium-fine ground coffee beans with an average particle size of 0.6 mm or more and less than 0.8 mm, medium ground coffee beans with an average particle size of 0.8 mm or more and less than 1.2 mm, and coarse ground coffee beans with an average particle size of 1.2 mm or more and less than 3.0 mm. The average particle size herein refers to the particle size corresponding to 50% (d50) of the cumulative undersize percentage by mass, as measured by the sieving test method described in JIS 8815-1994.

[0020] The coffee extraction residue in the present invention refers to the so-called used coffee grounds that remain after roasting coffee beans, grinding them in a mill or the like, and adding an appropriate amount of water or hot water (e.g., 90 to 98°C) to the resulting coffee powder during the preparation of a coffee beverage. As mentioned above, the type and origin of the coffee beans, whether they are blended or not, their quality, and whether they have been roasted or not are not important. The degree of grinding is also not important, although as mentioned above, finer grinding results in a higher mannose yield than coarse grinding.

[0021] Methods for producing mannose from coffee beans and / or coffee extract residue include hydrolysis of coffee beans and / or coffee extract residue. Hydrolysis methods include, but are not limited to, methods using acid and / or heat, enzymes, and microbial fermentation. In the present invention, hydrolysis using enzymes is preferred because it produces fewer by-products, has fewer limitations on equipment required for the hydrolysis step, and places less strain on the environment.

[0022] <Enzyme hydrolysis treatment> The enzymatic hydrolysis treatment will now be described in detail. It is said that approximately 50% of the components of coffee beans and / or coffee extract residue are carbohydrates, and that approximately 30% (w / w) or more of these carbohydrates is hemicellulose, a major component composed of galactose, arabinose, and mannose. Therefore, the enzyme used is not particularly limited as long as it has the activity to act on the main component, hemicellulose, and liberate monosaccharides. For example, enzymes that act on the main chain of hemicellulose to liberate monosaccharides, such as mannanase, galactomannanase, glucomannanase, mannosidase, α-xylidase, xyloglucanase, arabinanase, β-xylosidase, xylanase, α-arabinofuranosidase, and cellulase, are preferred. Among these, mannan-degrading enzymes such as mannanase, mannosidase, and galactomannanase are particularly preferred. Before the enzymatic hydrolysis, delignification and degreasing may be carried out, which can increase the yield of mannose.

[0023] A preferred hydrolysis treatment method is, for example, to suspend coffee beans and / or coffee extract residue in water, add the enzyme, and suspend the mixture with stirring. The amount of enzyme to be added can be selected as desired based on factors such as reaction conditions, but is preferably 1 to 10,000 units, and more preferably 10 to 2,000 units, per gram of coffee beans and / or coffee extract residue (substrate). The optimal reaction conditions for the enzyme can be selected according to the selected enzymatic reaction. The reaction temperature is preferably set so that the enzyme is not inactivated, for example, at 30 to 70°C, preferably 40 to 60°C, and more preferably 50 to 60°C. The reaction time depends on conditions such as the amount of enzyme used, but is typically preferably 3 to 72 hours, more preferably 12 to 60 hours. Furthermore, the reaction pH depends on the type of enzyme used, but is preferably 2 to 9, more preferably 2.5 to 8, and even more preferably 3 to 6. Organic or inorganic acids can be added to adjust the optimal pH. Examples of organic acids include oxalic acid, acetic acid, propionic acid, formic acid, lactic acid, fumaric acid, and citric acid. Examples of inorganic acids include hydrochloric acid, sulfuric acid, and phosphoric acid.

[0024] If necessary, enzymes such as glucosidase and galactosidase, which liberate glucose and galactose present in the branched side chains of hemicellulose, can be used in combination with enzymes acting on the main chain. Furthermore, the monosaccharide yield can be increased by mixing two or more enzymes with different activities. The enzymes used may be any fraction of the culture of the bacterial strain from which the enzyme originates, which has the activity of liberating monosaccharides. Furthermore, fractions containing these enzymes can be purified or partially purified by conventional methods before use, if necessary.

[0025] After the enzyme reaction is complete, the enzyme is inactivated by heating under any conditions that sufficiently inactivate the added enzyme, such as by stirring in a hot bath at about 90 to 95°C for 10 to 20 minutes or by boiling.

[0026] Thereafter, filtration is preferably carried out to remove insoluble solids, thereby obtaining a filtrate. The material of the filter paper used for filtration, its retention particle size, and the like are not particularly limited. A preferred material is filter paper made of cellulose fiber. The retention particle size (the particle size that can retain 90% or more when a seven-type powder dispersion specified in JIS Z 8901 is naturally filtered) is preferably 0.1 μm to 5 μm, and more preferably 0.5 μm to 2 μm. Alternatively, the enzyme reaction may be stopped by removing the enzyme by filtration.

[0027] In the present invention, the mannose concentration in the filtrate is preferably 0.05 to 2.0 g / L, more preferably 0.1 to 1.5 g / L. The mannose concentration in the filtrate is preferably measured using a D-mannose analysis kit (K-MANGL) manufactured by Japan Biocon Co., Ltd. The measurement method in this case follows the instruction manual for the analysis kit.

[0028] The filtrate is preferably purified to further increase the purity of mannose. Purification can be performed by known methods, such as decolorization using bone charcoal, activated carbon, carbon dioxide, adsorption resin, magnesia method, etc., and desalting and deacidification using ion exchange resin, ion exchange membrane, electrodialysis, etc. The combination of purification methods and purification conditions can be appropriately selected depending on the amounts of pigments, salts, acids, etc. in the reaction solution containing monosaccharides, as well as other factors.

[0029] The form of the mannose of the present invention is not particularly limited, and examples include an aqueous solution containing mannose, a powder, etc. To obtain a powder, known techniques such as solid-liquid separation, resin purification, membrane purification, concentration, spray drying, freeze drying, and crystallization can be used. From the viewpoint of ease of handling, a powder is preferred. From the viewpoint of ease of preparation, an aqueous solution is preferred.

[0030] <The present invention's method for preventing and / or treating colorectal cancer and its manufacturing method> The preventive and / or therapeutic agent for colon cancer of the present invention contains the mannose-containing filtrate. More specifically, the method for producing a therapeutic agent of the present invention includes the steps of: a step of adding an enzyme to the coffee beans and / or coffee extract residue to subject them to hydrolysis treatment, thereby preparing a liquid composition containing mannose; filtering the liquid composition to prepare a filtrate; and combining the filtrate to form a formulation.

[0031] The therapeutic agent of the present invention can be used for at least one of preventing the onset of colorectal cancer, inhibiting the progression of cancer in patients with colorectal cancer, inhibiting the growth of colorectal cancer or tumors in the colorectum, and preventing the recurrence of colorectal cancer. Colorectal cancer in the present invention includes colon cancer, colon adenocarcinoma, and rectal cancer.

[0032] The therapeutic agent of the present invention can be administered to humans and non-human mammals for whom treatment of colorectal cancer is desired or required. Non-human mammals include, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, and hamsters, including pet animals, livestock, and laboratory animals. A preferred example of the therapeutic agent is humans.

[0033] The dosage of the therapeutic agent of the present invention can be appropriately adjusted taking into consideration the purpose of use, the recipient, the recipient's gender, age, weight, and stage of cancer progression, etc. This dosage varies depending on various conditions, and therefore a dosage or frequency of administration lower than the above range may be sufficient, or a dosage or frequency of administration exceeding the above range may be required. The administration regimen may be a single administration or multiple administrations, but multiple administrations are preferred because the effect is sustained with continued administration. The concentration of mannose to be administered is preferably 0.001 mM to 100 mM, more preferably 0.01 mM to 50 mM, and even more preferably 0.01 mM to 10 mM.

[0034] The dosage form of the therapeutic agent of the present invention is not particularly limited, and may be an oral administration formulation (tablets, coated tablets, powders, Examples of the formulation include granules, capsules, liquids, etc.), formulations for airway administration, formulations for intraperitoneal administration, formulations for intravenous administration, injections, suppositories, patches, ointments, etc., but oral formulations, formulations for airway administration, and formulations for intravenous administration are preferred. Examples of formulations for intravenous administration include intravenous injection formulations and intravenous drip injection formulations. For humans, oral formulations and formulations for intravenous administration are preferred.

[0035] For formulation, the therapeutic agent of the present invention may be mixed with non-toxic, inert, pharmaceutically acceptable excipients, such as solid, semi-solid, or liquid diluents, dispersants, fillers, and carriers, as needed. Furthermore, the therapeutic agent of the present invention may contain additives such as stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, flow enhancers, flavoring agents, colorants, fragrances, antiseptics, media, physiological saline, and other medicinal agents, provided that the effects of the present invention are not impaired.

[0036] The therapeutic agent of the present invention may be administered in combination with other cancer therapeutic agents or therapies. Treatment methods that can be used in combination with the therapeutic agent of the present invention include chemotherapy, radiation therapy, chemoradiotherapy, immunotherapy, hormone therapy, surgery, or stem cell therapy. Chemotherapy is preferred, and preferred chemotherapeutic agents for use in chemotherapy include, but are not limited to, platinum chemotherapy agents, anthracycline therapeutic agents, or alkylating chemotherapy agents. Among these, cisplatin is preferred as a platinum chemotherapy agent, and doxorubicin is preferred as an anthracycline therapeutic agent. Concomitant administration refers to administration simultaneously with the administration of the therapeutic agent of the present invention, or before or after the administration of the therapeutic agent of the present invention. Alternatively, the therapeutic agent of the present invention and the other cancer therapeutic agent can be mixed to form a single formulation. [Example]

[0037] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples. <Preparation of mannose derived from coffee beans and / or coffee extract residue> (Production of mannose derived from green coffee beans) Unroasted coffee beans (Yirgacheffe G1 from Ethiopia) were ground in a coffee mill (RANCILIO MD 40) to produce coffee powder (3 g). The coffee beans were classified as Arabica, but contained a mixture of Ethiopian native species. The beans were ground into coarse and medium grinds. 10 mL of water was added and stirred to prepare an aqueous coffee powder solution (an IKA VORTEX3 was used for stirring; the same applies below). 0.1 g of mannanase (cellulosin GM5, HBI Corporation, unit count: 10,000 units / g) was dissolved in 1 mL of water, and 0.25 mL of this solution was added to the aqueous coffee powder solution and stirred to initiate the enzymatic reaction. The mixture was left to stand at 60°C for 48 hours in a constant temperature dryer (ETTAS OFW-300V) to allow the enzyme reaction to occur, and the resulting liquid composition was then subjected to suction filtration using a dry aspirator (DRY ASPIRATOR DAS-01) equipped with filter paper (quantitative filter paper No. 5C, retention particle size 1 μm, manufactured by ADVANTEC) to prepare a mannose-containing hydrolysate of coffee beans (green beans).

[0038] (Manufacturing of mannose derived from coffee beans (roasted beans)) Coffee beans (Yirgacheffe G1 from Ethiopia) roasted in a roasting oven (FUJI ROYAL R-110) were medium-ground in a coffee mill (RANCILIO MD 40) to produce coffee powder (3 g). The same procedure as described above was then used to prepare a coffee bean (roasted bean) hydrolysate containing mannose.

[0039] (Manufacturing of mannose derived from coffee extract residue) Coffee beans (Ethiopian Yirgacheffe G1) roasted in a roasting oven (FUJI ROYAL R-110) were medium-ground in a coffee mill (RANCILIO MD 40) to produce coffee powder (3 g). Hot water was added to the coffee and extracted using a paper drip filter. The coffee grounds remaining on the paper were used as coffee residue. 10 mL of water was added to the coffee and the procedure was repeated as described above to prepare a coffee residue hydrolysate containing mannose.

[0040] <Mannose concentration measurement> The mannose concentration in each filtrate obtained above was measured using a D-mannose analysis kit (K-MANGL) manufactured by Japan Biocon Co., Ltd. (n=1 for each). The results are shown in Table 1. Using finely ground coffee beans resulted in a hydrolysate with a higher mannose concentration. The hydrolysate from coffee extract residue had the highest mannose concentration. [Table 1]

[0041] <Reactivity of mannose derived from roasted coffee beans to colon cancer spheroids> Using a colon cancer spheroid line, we performed image analysis to assess the reactivity of roasted coffee bean-derived mannose (a mannose-containing hydrolysate of roasted coffee beans) and, as a comparison, commercially available mannose (chemically synthesized mannose) as a reagent to colon cancer spheroids. All experiments were performed four independent times.

[0042] Colon cancer spheroids were derived from colon cancer patients. In the present invention, HC73T (hereinafter sometimes referred to as "73T") owned by Kyoto University was used. The cryopreserved spheroids were thawed and cultured to the appropriate cell volume. The culture medium used was Advanced DMEM / F12 (Thermo Fisher), 5% FBS, 10 μM Y27632, 1 μM SB431542, 50 ng / ml EGF, 100 ng / ml FGF-basic, 100 units / ml penicillin, 0.1 mg / ml streptomycin, 2 mM L-glutamine, and 5 μg / ml Plasmocin (Invivogen, San Diego, CA). Cells for drug sensitivity testing were seeded at 800 cells / well in a 96-well plate (TPP_96F).

[0043] Mannose derived from roasted coffee beans (a mannose-containing hydrolysate of roasted coffee beans) obtained by the method described above and reagent mannose (Fujifilm, Wako Pure Chemical Industries) were dissolved in the medium, and additive solutions were prepared so that the mannose concentrations were 125 mM, 25 mM, 5 mM, 1 mM, 0.2 mM, and 0.04 mM, respectively, and each concentration was added to 4 wells.

[0044] The day after seeding was designated Day 0, and on Day 3, images were taken using a Cell3 imager duos (SCREEN Holdings, model CC-8000, software ver. 1.6 Rev. 2.0.1). Using manual focus mode, the central 61% of the well was set as the imaging range (a size that would fit the entire gel within the range), and continuous cross-sectional images were acquired at 15 points every 50 μm along the z-axis, and output as fully in-focus images. The reaction was initiated on Day 0 by changing the medium. After that, culturing continued without changing the medium until the end of the experiment. The imaging intervals were kept approximately constant.

[0045] The Measure function of the analysis software [SCREEN Holdings, Software ver. 1.6 Rev. 2.0.1] was used to apply the program to the spheroid image data and calculate the area value. Furthermore, volume data was calculated from the calculated area value using the program software R. When calculating the volume data, the program calculated the radius from the area, the volume of the sphere from the radius value, and calculated the total volume per well. The volume ratio was calculated from the volume values ​​of Day 3 / Day 0, and the ratio of each concentration was calculated and compared, assuming the control to be 1.

[0046] From the volume data obtained, the volume ratio (Day 3 / Day 0) of each data point was calculated and a standard curve was created. A t-test was used to test for significant differences from the control group. A p-value of less than 0.05 was considered significant. The results are shown in Figure 1. Note that the vertical axis in Figure 1, GEI, stands for growth effect index.

[0047] As is clear from Figure 1(A), coffee bean (roasted coffee bean)-derived mannose (a mannose-containing hydrolysate of roasted coffee beans) exhibits a very high inhibitory effect on colon cancer cell proliferation at a low mannose concentration of 1 mM. Figure 1(B) confirms that the effect is already apparent at a concentration of at least 0.2 mM.

[0048] <Difference in reactivity of coffee concentrate with or without enzyme treatment against colon cancer spheroids> 0.1 g of the aforementioned mannanase (cellulosin GM5) was weighed and added to 40 mL of ion-exchanged water in a plastic jar with a lid. The mixture was stirred to dissolve and then kept in a constant temperature bath at 58°C. Coffee beans (Yirgacheffe G1 from Ethiopia) roasted in a roasting oven (FUJI ROYAL R-110) were ground in a coffee mill (RANCILIO MD 40) to produce coffee powder (12 g), which was then placed in the plastic jar with a lid. The enzyme reaction was carried out at 58°C for 48 hours. The mixture was then stirred in a water bath at 90°C to 95°C for 10 minutes to inactivate the enzyme. The mixture was centrifuged at 12,000 rpm for 20 minutes, and the supernatant was collected. An additional 20 mL of ion-exchanged water was added to the precipitate and stirred. The mixture was centrifuged again at 12,000 rpm for 20 minutes, and the supernatant was collected. The supernatants from the two centrifugations were combined and filtered through a Millipore 1 μm filter. The filtrate was concentrated using an evaporator, and ion-exchanged water was added to adjust the weight of the concentrate to 10 g. This coffee concentrate was designated "enzyme-treated coffee concentrate." The mannose concentration of this liquid was 78.8 mg / g (0.437 mmol / g). The mannose concentration was measured using the HPLC method described below.

[0049] <Liquid chromatography test conditions> Equipment: Differential refractive index detector (SHIMADZU Prominence) Column: Aminex HPX-87P (BIO RAD, 300 mm L x 7.8 mm ID) Mobile phase: water, flow rate: 0.6 ml / min, column temperature: 60°C Sample volume: 10 μL

[0050] A "coffee concentrate (untreated with enzyme)" was prepared in exactly the same manner as above, except that mannanase (cellulosin GM5) was not added. The mannose concentration of this concentrate was 0 mg / g.

[0051] The "enzyme-treated coffee concentrate" was dissolved in the aforementioned medium to prepare sample solutions with mannose concentrations of 5 mM, 1 mM, 0.5 mM, 0.2 mM, and 0.04 mM. In addition, "coffee concentrate (untreated with enzyme)" was dissolved in culture medium, and the reagent mannose (Fujifilm Wako Pure Chemical Industries) was added to prepare sample solutions with mannose concentrations of 5 mM, 1 mM, 0.5 mM, 0.2 mM, and 0.04 mM. Using the above sample solution, the reactivity to colon cancer spheroids (73T) was examined by the method described above.

[0052] The results are shown in Figures 2 and 3. Figure 3 is a quantification of the image in Figure 2. Figure 2 shows image data of the spheroids on Day 3, with the day after seeding the colon cancer spheroids (73T) being Day 0. Figures 2 and 3 show that the enzyme-treated hydrolysate group exhibited a significant inhibitory effect on colon cancer cell proliferation compared to the enzyme-untreated group to which the reagent mannose had been added. In particular, when the coffee concentrate (enzyme-treated) contained 1 mM or more of mannose, a significant difference in the inhibitory effect on colon cancer spheroid proliferation was observed compared to the enzyme-untreated group (see Figure 3).

[0053] The results when HC6T colon cancer spheroids were used instead of HC73T colon cancer spheroids are shown in Figure 4. Even when HC6T was used, the enzyme-treated hydrolysate group showed a significant inhibitory effect on colon cancer cell proliferation compared to the enzyme-untreated group to which the reagent mannose was added.

[0054] It has been shown that mannose-containing hydrolysates obtained by hydrolyzing coffee beans and / or coffee extract residue have an inhibitory effect on colon cancer cell proliferation that cannot be achieved by simply adding mannose as a reagent. This is thought to be because the enzyme treatment enhances the inhibitory effect on colon cancer cell proliferation by allowing various physiologically active substances other than mannose that are presumed to be contained in the hydrolysates to have a synergistic effect with mannose.

[0055] Thus, because the raw material for the therapeutic agent of the present invention is coffee beans, which exist in nature, it can be ingested or administered more safely than chemically synthesized mannose. Furthermore, the mannose-containing hydrolysate obtained by the present invention can be used as a functional food, a nutritional supplement, or a food for specified health uses. For example, the mannose-containing hydrolysate can be used as a functional coffee beverage, or the hydrolysate can be freeze-dried or powdered and used as a stick-type health food. This also leads to the effective use of coffee extraction residue, which has previously been incinerated or disposed of as industrial waste.

Claims

1. A preventive and / or therapeutic agent for colon cancer, comprising mannose derived from coffee beans and / or coffee extract residue.

2. A preventive and / or therapeutic agent for colon cancer, comprising a hydrolysate of coffee beans and / or coffee extract residue containing mannose.

3. a step of adding an enzyme to coffee beans and / or coffee extract residue to subject them to hydrolysis treatment, thereby preparing a liquid composition containing mannose; filtering the liquid composition to prepare a filtrate; and combining the filtrate to form a formulation. A method for producing a preventive and / or therapeutic drug for colon cancer.

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