JP6113800B2 - Method for producing antitumor agent, method for producing antitumor food, and method for producing antitumor drug - Google Patents

Description

The present invention relates to a method for producing an antitumor agent, a method for producing an antitumor food, and a method for producing an antitumor drug .

  Various methods for producing substances having antitumor activity have been studied. Patent Document 1 relates to, as an example, a method for extracting a polysaccharide, which is an active ingredient having antitumor activity originally contained in a plant of the genus Panax, from the leaf or stem of the plant.

JP 2005-53576 gazette

  In Patent Document 1, an active substance is extracted from a plant body. For this reason, the yield of the active substance depends on the amount of the active substance originally present in the plant body. On the other hand, the amount of the active substance contained in the plant body is considered to largely depend on the conditions for each growing group and the conditions for each individual, such as the growth environment of the plant and individual differences. Therefore, it is difficult to obtain a certain amount of active substance by the method of Patent Document 1. Moreover, since the raw material is the plant itself, it is relatively difficult to keep the quality of the raw material constant. Unless the quality of the raw material is kept constant, it is difficult to keep the quality of the active substance extracted therefrom. As described above, according to the method of Patent Document 1, there is a possibility that a substance having an antitumor action cannot be stably produced in terms of quality and production amount.

An object of the present invention is to provide a method for producing an antitumor agent, a method for producing an antitumor food, and a method for producing an antitumor drug that are easily produced stably in terms of quality and production amount.

The antitumor agent of the present invention is a method for producing an antitumor agent comprising a composition comprising a fraction obtained by subjecting glucose to heat treatment, wherein the heating temperature in the heat treatment is 160 ° C. or higher and 200 ° C. Is less than. The present inventors have found that a substance having antitumor activity can be generated by directly heating a saccharide such as a monosaccharide such as glucose or fructose, a disaccharide such as sucrose, or a polysaccharide such as cellulose, not a plant body. Saccharides are easily supplied with a constant quality by industrial production or the like, and are easy to store. By directly heating sugar of a certain quality, it is easy to stably produce a substance having antitumor activity at a certain quality and in a certain amount. Therefore, it is a composition comprising a fraction of the resultant product obtained by heat-treating saccharides, and contains a substance having anti-tumor activity as a composition, so that it can be stably produced in terms of quality and production amount. An antitumor agent can be obtained .

Moreover, when applied to reverse phase high performance liquid chromatography, the composition is preferably eluted in a methanol aqueous solution having a concentration of 40 to 70% by mass. When extracting the said composition, the substance which has anti-tumor activity was able to be acquired when 40-70 mass% methanol aqueous solution was used as a solvent with a high performance liquid chromatography column.
Moreover, it is preferable that the molecular weight of the said composition is 5000 or more. According to this, about the composition which consists of a fraction of the result which heat-processed glucose, the molecular weight of the composition is 5000 or more, and antitumor activity is high.

The method for producing an antitumor food according to the present invention is a method for producing an antitumor food comprising a composition comprising a fraction obtained by subjecting glucose to heat treatment, wherein the heating temperature in the heat treatment is 160 ° C. or higher. And it is less than 200 degreeC.

The method for producing an antitumor drug of the present invention is a method for producing an antitumor drug comprising a composition comprising a fraction obtained by subjecting glucose to heat treatment, wherein the heating temperature in the heat treatment is 160 ° C. or higher. And it is less than 200 degreeC.

It is a figure showing the result of the sugar heat processing test by sucrose and glucose. It is a figure showing the result of having performed the sugar heat processing thing test after fractionating a test liquid using a gel filtration chromatography. It is a figure showing the result of having performed the sugar heat processing thing test after fractionating a test liquid using a gel filtration liquid chromatography and a reverse phase high performance liquid chromatography. It is a figure showing the result of the sugar heat processing test by commercially available caramel.

  The antitumor agent according to one embodiment of the present invention will be described below. The composition having antitumor activity contained in the present antitumor agent consists of a fraction of the resulting product obtained by heat-treating saccharides. The molecular weight of the composition is 5000 or more. Glucose is preferable as the saccharide used as the raw material of the composition, but it may be fructose, other monosaccharides, disaccharides such as maltose, or polysaccharides such as cellulose. Multiple types of these saccharides may be included. The basic structure of the saccharide is preferably a pentose such as D-ribose or a hexose such as glucose or cellulose. More preferred is hexose. Moreover, multiple types of saccharides among these may be used as a raw material of a composition. The composition may be a commercially available caramel fraction produced by subjecting sugars to heat treatment.

  A method for producing a composition having antitumor activity (antitumor active substance) in the antitumor agent will be described. First, water is added at an appropriate ratio to the saccharide, and heated for a predetermined time under a predetermined temperature condition. Water may or may not be added. For example, in the case of glucose, water is added to glucose at a ratio of 10 ml (milliliter) of water to 1 g of glucose, and heated at 160 to 200 ° C. for 1 to 4 hours. Preferably, heating is performed at 180 ° C. for 2 hours. After the heat treatment, a composition having a molecular weight of 5000 or more is fractionated by gel filtration chromatography. For the fractionation, a conventionally known method may be used. For example, a fractionation method by centrifugation or the like may be used. Next, reverse-phase high performance liquid chromatography analysis is performed on the obtained fraction, and a methanol aqueous solution is used as a developing solvent to obtain a fraction eluted in a developing solvent having a methanol concentration of 40 to 70% by mass. The solute is removed from this fraction and used as an antitumor agent.

  Examples of the food according to one embodiment of the present invention include foods for specific health, functional display foods, and other so-called health foods containing the composition having antitumor activity. In particular, foods for specified health use and functional labeling foods among them, that is, health function foods, are foods for specific uses that have been institutionalized as foods that are expected to have specific insurance purposes. Specific forms of food may include supplements, beverages, soups, breads, confectionery, and the like or additives thereof. The manufacturing method of these foods is not specifically limited, A conventionally well-known method can be used for each use.

  The food or medicine according to one embodiment of the present invention is prepared by various solid preparations such as granules, capsules, tablets, powders, etc. containing the composition having antitumor activity, or liquids, It can be prepared in the form of a liquid preparation such as a suspension or syrup.

  Capsules or tablets can be produced using known additives that are acceptable as pharmaceuticals or foods, and can be applied with conventional formulation techniques employed in the field of pharmaceuticals or foods. For example, a tablet can be prepared by adding and blending each component according to the formulation, crushing, granulating, drying, sizing and mixing, and tableting the resulting preparation mixture.

  Examples of additives for formulation include a disintegrant, a binder, a fluidizing agent, a lubricant, and a coloring agent. Moreover, when making it into the form of a liquid agent, thickeners, such as pectin, can be mix | blended. You may prepare in another form by a conventionally well-known method.

  The antitumor agent, food and medicine of this embodiment are mainly used by oral ingestion. As shown in the Examples described later, it exerts an inhibitory effect on tumor growth. Therefore, this embodiment is implemented as an antitumor agent, food, and pharmaceutical for specific use that suppresses tumor growth. In addition, what mixed the composition with the said multiple types of said anti-tumor activity from which the saccharide | sugar used as a raw material differs may be used for the antitumor agent which concerns on this embodiment, a foodstuff, or a pharmaceutical. In addition, the food or medicine contains not a composition that is a fraction of a product obtained by subjecting a saccharide to heat treatment, but a composition that is composed of the resultant product itself that is obtained by subjecting a saccharide to heat treatment. You may let them.

[Example]
Hereinafter, examples according to the above-described embodiment will be described. This example relates to a test (hereinafter, referred to as a sugar heat-treated product test) using a sugar-heat-treated product related to the above embodiment and a fraction of the heat-treated product.

<Administration of administration test, measurement of tumor and evaluation of tumor>
Heat treatment was performed under predetermined heating conditions to 1 g of various sugars added with 10 ml (milliliter) of water. A test solution was obtained by adding water to a result of this treatment (hereinafter referred to as a sugar heat-treated product) and mixing. This test solution was used as a liquid to be administered to mice. The liquid to be administered to mice is hereinafter referred to as a test liquid to be administered. For conditioning, 5 ml of female BALB / cAJcl-nu / nu mice purchased from Clea Japan Co., Ltd. were administered with 5 ml of water as drinking water for 1 week. Next, some mice were subcutaneously injected with 1 × 10 ⁵ W14 malignant transformant cells added to physiological saline. The W14 malignant transformant cell is a cell produced by transferring the H-ras oncogene into rat fetal fibroblasts (Sato N, Torigoe T, Yagihashi A, Okubo M, Takahashi S, Takahashi N, Enomoto K, Yamashita T, Fujinaga K and Kikuchi K, Tumor Res, 22, 15-26, 1987). This cell has CD44 known as a surface marker for pancreatic cancer stem cells and breast cancer stem cells as a surface marker (Kanki K, Torigoe T, Hirai I, Sahara H, Kamiguchi K, Tamura Y, Yagihashi A, Sato N, Microbiol). .Immunol., 44 (12), 1051-61, 2000). Next, 5 ml per day of the test liquid to be administered was administered as drinking water over a period of about 3 weeks to mice subcutaneously injected with W14 malignant transformant cells. On the other hand, among the mice conditioned for drinking, 5 ml of water was administered as drinking water to mice not injected subcutaneously with W14 malignant transformant cells. This mouse was used as a control.

The volume of the tumor formed in each mouse was measured while subcutaneously injecting the W14 malignant transformant cells and then administering the administration test solution or water to the mice. The volume of the tumor is (calculation) volume = (minor axis) ² × (when the width in the longitudinal direction of the tumor when viewed from the front is the major axis and the width in the direction perpendicular to the longitudinal direction is the minor axis. Calculated by (major diameter) × 0.5. The above-described operation was performed on a plurality of mice for each saccharide serving as the basis of the test solution to be administered, and also on a plurality of control mice. Then, at the stage where the tumor volume in the control mouse just exceeded 1000 mm ³ (a stage about 3 weeks after the subcutaneous injection), the tumor of the mouse administered with the test liquid to be administered was evaluated. In the evaluation, for each saccharide that is the basis of the administration target test solution, an average value of tumor volumes of a plurality of mice administered with the administration target test solution corresponding to each saccharide is calculated, and a plurality of individuals corresponding to the control are calculated. The average value of the tumor volume of the mice was calculated. The tumor growth inhibition rate was calculated as follows. The above is the flow of the sugar heat-treated product test.
(Tumor growth inhibition rate) = (1− (mean value of tumor volume of mice administered test solution to be administered) / (mean value of control tumor volume)) * 100

<Tumor growth inhibition rate by each saccharide>
As a test on monosaccharides and disaccharides, glucose (manufactured by Nacalai Tesque) and sucrose (granulated sugar) were each dissolved in water at a ratio of 10 ml of water to 1 g, and heat-treated at 180 ° C. for 2 hours. The sugar heat-treated product was used, and the above-mentioned sugar heat-treated product test was performed by adjusting the administration target test solution so that the concentration of the sugar heat-treated product was 200 μg / ml. As a result, as shown in FIG. 1, the tumor growth inhibition rate by the sugar heat-treated product of glucose was 100%, and the tumor growth inhibition rate by the sugar heat-treated product of sucrose was 20%. In addition, as a result of the above sugar heat-treated product test under the same conditions for fructose (manufactured by Nacalai Tesque Co., Ltd.) and maltose (manufactured by Sigma Aldrich Japan GK), the tumor growth inhibition rate of fructose is 30%. Maltose had a tumor growth inhibition rate of 24%.

  In addition, as a polysaccharide test, a sugar heat-treated product obtained from cellulose (manufactured by Nacalai Tesque Co., Ltd.), xylose (manufactured by Sigma Aldrich Japan GK), fucoidan (manufactured by Sigma Aldrich Japan GK), and pentose sugar test As described above, the sugar heat-treated product test was performed using sugar heat-treated products obtained from 2-deoxy-D-ribose (manufactured by Sigma Aldrich Japan GK) and D-ribose (manufactured by Sigma Aldrich Japan GK). It was. In the heat treatment, 2-deoxy-D-ribose was heated at 160 ° C. for 2 hours, and fucodane was heated at 140 ° C. for 2 hours until it became dark brown. The other saccharide heating conditions were the same as the above heating conditions for glucose. As a result, the tumor growth inhibition rate by the sugar sugar heat-treated product and the tumor growth inhibition rate by the D-ribose sugar heat-treated product are both 50%, and 2-deoxy-D-ribose and xylose sugar heat-treated product. The tumor growth inhibition rate by -20% was -21% and -14%. A negative tumor growth inhibition rate indicates that tumor growth was promoted relative to the control. Moreover, the tumor growth inhibition rate by the heat-treated product of fucoindan was 38%. From the above, it was found that each heat-treated product of glucose, fructose, sucrose, maltose, cellulose, D-ribose and fucoidan has antitumor activity.

<Fractionation of tumor active substance: Part 1>
Next, as described below, the test liquid was fractionated using gel filtration chromatography, and the sugar heat-treated product test was performed on the fraction. Glucose was dissolved in water at a ratio of 10 ml of water with respect to 1 g, heat treatment was performed at 180 ° C. for 2 hours, and the test solution was adjusted so that the concentration of the sugar heat-treated product in water was 200 mg / ml. Next, the test solution was subjected to gel filtration chromatography under the following conditions. As the column, a glass column having a length of 30 cm and an outer diameter of 2.4 cm was used. The glass column was packed with a Sephadex (registered trademark) G-25 column (Pharmacia). The size of the filling part is 26 cm long and 2.2 cm inside diameter. After 1 ml of test solution was applied to the top of the column, gel filtration separation was started. Development was performed with distilled water at a flow rate of 0.5 ml / min and 5 ml per fraction. FIG. 2 shows the results of measuring the absorbance of each fraction at a wavelength of 230 nm (nanometer). 230 nm corresponds to the wavelength having the highest absorbance as a result of measuring the absorbance spectrum of the test solution before fractionation in advance. As shown in FIG. 2, the sugar heat-treated product in 1 ml of the test solution is fractionated to about fraction numbers 5 to 15. Since 5 ml per fraction, the sugar heat-treated product was dispersed in 50 ml of distilled water. Since the Sephadex G-25 column has an exclusion limit molecular weight of 5000, the fractions (fraction numbers 6 and 7 in FIG. 2) that first flowed out of the column are molecules having a molecular weight of 5000 or more. Fractions Nos. 6 and 7 were brown. Fraction numbers 8 and 9, fraction numbers 11 and 12, and fraction numbers 13 and 14 were light yellow.

<Tumor growth inhibition rate by each fraction>
Fraction test solution A was prepared by adding water to the mixture of fractions 6 and 7 and diluting it 50 times. Moreover, the fraction test solution B was obtained by adding water to the mixture of fractions 13 and 14 and diluting it 50 times. The final concentration of the fraction test solution A was estimated to be approximately 1 mg / ml, and the final concentration of the fraction test solution B was estimated to be 0.4 mg / ml. The final concentration was estimated based on the assumption that the area of the region between the portion corresponding to each fraction and the horizontal axis in the absorbance line graph of FIG. 2 is approximately proportional to the concentration of each fraction. Fractionation test solutions A and B were each independently administered as test solution, and the sugar heat-treated product test was performed. As a result, as shown in FIG. 2, the tumor growth inhibition rate of the fraction test solution A was 85%, and the tumor growth inhibition rate of the fraction test solution B was −5%. Therefore, it was discovered that the antitumor activity was high in the fraction corresponding to a molecular weight of 5000 or more among the fractions of the sugar heat-treated product.

<Fractionation of tumor active substance: 2>
Next, high-performance liquid chromatography was applied to fractions 6 and 7 collected by gel filtration liquid chromatography to specify the tumor active substance in more detail. The amount of liquid per application to which high performance liquid chromatography can be applied is small. Therefore, a large amount of the above fractions 6 and 7 is collected by gel filtration liquid chromatography, and a dry substance is obtained by freeze-drying the fraction, and dissolved in water to obtain a liquid to be subjected to high performance liquid chromatography. It was decided to make it easier to adjust the concentration of the. Therefore, in the gel filtration liquid chromatography, a 100 ml syringe having a large inner diameter was used instead of the glass column. This syringe was filled with Sephadex G-25 so that the short diameter of the filling portion was 4 cm and the length was 8 cm. As a result, 5 ml of the test solution can be injected at a time during gel filtration liquid chromatography. Distilled water was used as a developing solvent, and this was poured with a pipette into the blank area at the top of the syringe. In order to improve separation in fractionation, the flow rate of the developing solvent was adjusted by adjusting the diameter of the injection needle of the syringe. In this example, a 0.80 mm × 38 mm injection needle was used.

  The test solution was fractionated as follows using the above gel filtration liquid chromatography with an increased injection volume per one time. Glucose was dissolved in water at a ratio of 10 ml of water with respect to 1 g of glucose, and this was subjected to heat treatment at 180 ° C. for 2 hours, and a test solution was prepared using the resulting heat-treated product. This test solution was subjected to the above gel filtration liquid chromatography using a syringe. Thereby, the brown phase having a molecular weight equal to or higher than the exclusion limit molecular weight of Sephadex G-25, which was first eluted from the syringe, was fractionated. Since this column is relatively short, the brown phase could be eluted in a short time of about 15 minutes. This brownish-brown phase was freeze-dried and the resulting dry substance was dissolved in distilled water to a concentration of 100 mg / ml. This was made into the test liquid applied to a high performance liquid chromatography.

  Reversed phase high performance chromatography was performed under the following conditions. As the column for analysis, a TSKgel ODS-100V 5 μm column (manufactured by Tosoh Corporation) having an inner diameter of 4.6 mm and a length of 15 cm was used. The flow rate of the developing solvent was 0.5 ml / min. As a developing solvent, an aqueous methanol solution was used while changing the concentration of methanol. Specifically, the concentration of methanol was changed as follows. 10% by mass at the start of chromatography, 10% to 100% by mass at 0-15 minutes, and 100% by mass at 15-25 minutes, 100% at 25-30 minutes. The amount was reduced in inverse proportion to the time from 10% to 10% by mass, and 10% by mass at 30 to 35 minutes. Thus, 35 minutes was set as one cycle, 20 microliters of the said test liquid was inject | poured into the column, and the reverse phase high performance liquid chromatography was performed.

  The absorbance curve in FIG. 3A shows the result of measuring the absorbance at a wavelength of 254 nm for the liquid eluted at each elution time in the reverse phase high performance liquid chromatography. The voltage on the vertical axis is proportional to the absorbance. The absorbance at a wavelength of 254 nm is based on the fact that the absorbance was highest at a wavelength of 250 to 260 nm as a result of measuring the absorbance spectrum of the fractions 6 and 7. Then, the respective fractions of a mountain region 1 appearing at an elution time of 4 minutes, a mountain region 2 appearing at an elution time of 6 to 8 minutes, and a mountain region 3 appearing at an elution time of 8 to 10 minutes were obtained. Methanol was removed from each fraction and water was added to prepare a test solution to be administered, which was administered to mice in the same manner as in the sugar heat-treated product test. Each fraction contains a component derived from the dry substance. The dosage of the dry substance-derived component administered to each mouse was estimated as follows. 5 μg for mice administered with the test liquid for administration corresponding to region 1, 35 μg for mice administered with the test liquid for administration corresponding to region 2, and 15 μg for mice administered with the test liquid for administration corresponding to region 3 It is. This estimation was made based on the process that the area below the absorbance curve of the regions 1 to 3 is approximately proportional to the concentration of the above components in each fraction of the regions 1 to 3. As a result, as shown in FIG. 3B, the tumor growth inhibition rate of the mice administered with the administration target test solution corresponding to region 1 was −20%. The tumor growth inhibition rate of the mice administered with the test solution to be administered corresponding to region 2 was 97%. The tumor growth inhibition rate of the mice administered with the administration target test solution corresponding to region 3 was 60%. Therefore, the substance having antitumor activity can be obtained by the fractionation of regions 2 and 3. Regions 2 and 3 are fractions of liquid that elutes between 5 and 10 minutes elution time. That is, among the components derived from the dry substance, a component eluted in a methanol aqueous solution having a concentration of 40 to 70% by mass has antitumor activity.

<Whether water is added to glucose>
When heat treatment was performed at 180 ° C. for 2 hours without adding water to glucose, it became brown as in the case of adding water, and the pattern of the absorbance curve was the same when high performance liquid chromatography was performed. From this, it is possible to obtain a sugar-heat-treated product containing a component having antitumor activity both when the heat treatment is performed by adding water to the saccharide and when the heat treatment is performed without adding water to the saccharide. There was found.

  According to the present embodiment and examples described above, a substance having antitumor activity can be generated by directly heating a saccharide that is easy to be supplied with a certain quality by industrial production or the like, and that is easy to preserve, not a plant body. Therefore, an antitumor agent that can be easily produced stably in terms of quality and production amount can be obtained by using as a composition a substance having antitumor activity that is a fraction of the resultant product obtained by subjecting saccharides to heat treatment.

  Moreover, it is possible to provide the composition which consists of a fraction of the result obtained by heating saccharides and has antitumor activity as a foodstuff containing it. Furthermore, it is also possible to provide a pharmaceutical product containing a resultant product obtained by heating saccharides or a composition thereof having antitumor activity.

  The above is a description of a preferred embodiment of the present invention, but the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope described in the means for solving the problem. It is possible. Hereinafter, modified examples according to the above-described embodiment will be described. Further, description of portions common to the above-described embodiment will be omitted as appropriate.

<Heating conditions>
With respect to the production of a sugar heat-treated product of glucose, the heating conditions were variously changed and tested. As a result of adding water and heating at 180 ° C. while changing the heating time, it was found that a component having antitumor activity began to be obtained in a heating time of 1 hour, and a large amount of component was obtained in about 2 hours. On the other hand, as a result of heating at 180 ° C. while changing the heating time without adding water, a component having antitumor activity began to be obtained in about 30 minutes when water was not added, and a large amount of component was obtained in about 1 hour. It turned out to be obtained. In addition, as a result of heating for 2 hours after changing the heating temperature, an effective component can be obtained at about 160 to 170 ° C. with or without adding water, but such a component can be suitably obtained at 180 ° C. found. Moreover, since it became easy to be scorched when heating temperature became 200 degreeC or more, it became clear that it was not preferable.

<Caramel>
When the above sugar heat-treated product test was performed using a commercially available caramel (manufactured by Fujii Pharmaceutical Co., Ltd.) as a sugar heat-treated product, the tumor growth inhibition rate was 86% as shown in FIG. Commercially available caramel is also obtained by heat-treating sucrose with water. For this reason, it was found that a substance having antitumor activity can be obtained even when a commercially available sugar heat-treated product (or a fraction thereof) is used.

<Others>
The sucrose (manufactured by Nacalai Tesque Co., Ltd.) different from the sucrose was subjected to the sugar heat-treated product test. As a result, the tumor growth inhibition rate of the sucrose sugar-treated product was 88%. This may be due to differences in the degree of purification of sucrose.

Claims (5)

  A method for producing an antitumor agent comprising a composition comprising a fraction obtained by subjecting glucose to heat treatment, wherein the heating temperature in the heat treatment is 160 ° C. or more and less than 200 ° C. A method for producing an antitumor agent. When applied to a reverse phase high performance liquid chromatography, wherein the composition, manufacturing method of the antitumor agent according to claim 1, characterized in that eluted methanol solution of 40 to 70% strength by weight. The method for producing an antitumor agent according to claim 1 or 2 , wherein the composition has a molecular weight of 5000 or more. A method for producing an antitumor food comprising a composition comprising a fraction obtained by subjecting glucose to heat treatment, wherein the heating temperature in the heat treatment is 160 ° C. or higher and lower than 200 ° C. A method for producing an anti-tumor food. A method for producing an antitumor drug comprising a composition comprising a fraction obtained by subjecting glucose to heat treatment, wherein the heating temperature in the heat treatment is 160 ° C. or higher and lower than 200 ° C. A method for producing an antitumor drug.