JP2007254338A - Physiologically active substance, and immunomodulating activator, allergy control activator, anti-cancer activator, active oxygen scavenging activator, food, beverage, supplement and medicine using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a physiologically active substance derived from sorghum, which is effective for treating and preventing diseases such as immunological disease, cancer, etc., and is safe. <P>SOLUTION: An extract obtained by immersing the whole grain flour of white sorghum and sorghum after removal of bran part in a phosphate buffer solution has IgM antibody production accelerating activity as shown in a graph of figure A. An extract of white sorghum with a phosphate buffer solution has various physiological activities such as IgE antibody production inhibitory activity, anticancer activity to colon cancer, active oxygen scavenging activity, etc. The physiologically active substance is applicable to foods, beverages, supplements, medicines, etc. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a physiologically active substance, an immunomodulating active agent, an allergy suppressing active agent, an anticancer active agent, an active oxygen scavenging active agent, foods, beverages, supplements and pharmaceuticals using the same.

  Sorghum is a cereal with high production in countries such as the United States, India, China, Mexico and Nigeria. Sorghum is an annual grass of the gramineous family and is also called sorghum, kaolyan, milo, etc. Sorghum is roughly divided into four types: grain sorghum, sweet sorghum, bloom sorghum and glass sorghum. Recently, white sorghum that does not contain gluten has attracted attention as a cereal for combating wheat allergies. White sorghum is a kind of grain sorghum, and has the characteristic that it contains essential amino acids and is rich in minerals, vitamin B group, dietary fiber and unsaturated fatty acids in addition to the characteristic of not containing gluten. In view of these characteristics, foods using white sorghum have been developed. Examples of foods using white sorghum include puffed snacks (Patent Document 1), noodles (Patent Document 2), sparkling beverages (Patent Document 3), and syrup (Patent Document 4).

JP 2004-135530 A JP 2004-337095 A Japanese Patent Laid-Open No. 2005-40045 JP 2005-323555 A

  Many conventional white sorghum utilization methods have focused on their nutritional characteristics, but it cannot be said that a technique for fully exploiting the performance of white sorghum has been developed. On the other hand, development of safe and effective pharmaceuticals, health foods, supplements and the like is required for diseases such as immune diseases and cancer.

  Accordingly, an object of the present invention is to provide a sorghum-derived physiologically active substance that is effective and safe for treatment and prevention of diseases such as immune diseases and cancer.

  In order to achieve the above object, the physiologically active product of the present invention comprises at least one selected from the group consisting of a part of sorghum, all of sorghum and a sorghum extract.

  In order to achieve the above-mentioned object, the present inventor has conducted a series of studies and found that sorghum has various physiological activities such as immune preparation activity, allergy suppression activity, anticancer activity and active oxygen scavenging activity. The present invention has been reached. Since the physiologically active substance of the present invention uses sorghum that has been used for food for many years, it is excellent in safety. The present invention is also effective for the treatment and prevention of various diseases such as immune diseases and cancer. In addition, the physiologically active substance of the present invention is not limited to the above-described four physiological activities, and may have other physiological activities.

  In the present invention, the extract is preferably one obtained by extracting a part of sorghum and / or all of sorghum with a water-containing solvent.

  In the present invention, the sorghum is not particularly limited, and may be various sorghums such as grain sorghum, sweet sorghum, bloom sorghum and glass sorghum. Among them, white sorghum is preferable.

  In the present invention, the water-containing solvent is preferably at least one of water and a buffer solution. The buffer is not particularly limited, and for example, a buffer excellent in safety to a living body is preferable. From this viewpoint, a phosphate buffer or the like is preferable.

  The immunomodulating active agent of the present invention is characterized by containing the physiologically active product of the present invention. The physiologically active substance preferably contains at least one of bran part-removed sorghum and water-containing solvent extract of bran part-removed sorghum. The immunomodulating activity is, for example, an activity that promotes the production of IgM antibodies. The IgM antibody production promoting factor in the physiologically active substance is, for example, a protein having a molecular weight of 10,000 or more, inactivated by trypsin treatment, and not completely inactivated by heat treatment at 100 ° C.

  The allergy-inhibiting active agent of the present invention is characterized by containing the physiologically active product of the present invention. The physiologically active substance preferably contains at least one of a sorghum bran portion and a water-containing solvent extract of the sorghum bran portion. The allergy inhibitory activity is, for example, an inhibitory activity on IgE antibody production. The IgE antibody production inhibitory factor in the physiologically active substance is, for example, a high molecular factor having a molecular weight of 10,000 or more or a low molecular factor having a molecular weight of 1,000 or less.

  The anticancer active agent of the present invention comprises the physiologically active product of the present invention. The physiologically active substance preferably contains at least one of a bran portion of sorghum and a water-containing solvent extract of the bran portion of sorghum. The anticancer activity is, for example, anticancer activity against colorectal cancer. The anti-colon cancer active factor in the physiologically active substance is, for example, a high molecular factor having a molecular weight of 10,000 or more or a low molecular factor having a molecular weight of 1,000 or less, and is not inactivated by trypsin treatment, and is completely inactivated by heat treatment at 100 ° C. It is a non-protein substance that does not deactivate.

  The active oxygen scavenging activator of the present invention comprises the physiologically active product of the present invention.

  The food of the present invention comprises the physiologically active product of the present invention, the immunomodulating activator of the present invention, the allergy suppressing activator of the present invention, the anticancer activator of the present invention, and the active oxygen scavenging activator of the present invention. It includes at least one selected from the group consisting of.

  The beverage of the present invention comprises the physiologically active product of the present invention, the immunomodulating activator of the present invention, the allergy suppressing activator of the present invention, the anticancer activator of the present invention, and the active oxygen scavenging activator of the present invention. It includes at least one selected from the group consisting of.

  The supplement of the present invention comprises the physiologically active substance of the present invention, the immunomodulating activator of the present invention, the allergy suppressing activator of the present invention, the anticancer activator of the present invention, and the active oxygen scavenging activator of the present invention. It includes at least one selected from the group consisting of.

  The medicinal product of the present invention includes the physiologically active product of the present invention, the immunomodulating activator of the present invention, the allergy suppressing activator of the present invention, the anticancer activator of the present invention, and the active oxygen scavenging activator of the present invention. It includes at least one selected from the group consisting of.

  In the present invention, as described above, the entire sorghum may be used, a part thereof, or an extract thereof may be used.

  Examples of the sorghum include a bran portion, a sorghum excluding the bran portion, an outer skin of the bran portion, and an endothelium of the bran portion.

  The sorghum extract can be obtained, for example, by immersing a part or all of the sorghum in water or a buffer solution for a certain period of time. In the immersion, it is preferable to stir the immersion liquid. When the physiologically active product of the present invention is heat stable, the water or buffer solution may be heated and immersed.

  The form of the physiologically active product or the various active agents of the present invention is not particularly limited, and can take various forms such as a liquid, a sol, a gel, a solid, a granule, and a powder. In addition, the physiologically active product of the present invention or the various active agents may contain various additives that are acceptable for pharmacological or food safety, if necessary.

  Next, examples of the present invention will be described. However, the present invention is not limited to the following examples.

  This example relates to the immunomodulating activity of the physiologically active product of the present invention.

  5 g of the sample powder from which the whole grains of white sorghum and bran have been removed are suspended in 20 mL of 10 mM phosphate buffer (pH = 7.4) (hereinafter referred to as “NaPB”), and stirred at 4 ° C. overnight, then insoluble. Objects were removed by centrifugation to obtain a soluble fraction. A part of the obtained soluble fraction was dialyzed against 10 mM NaPB. Thus obtained (a) whole grain (with dialysis), (b) whole grain (without dialysis), (c) bran removal (white: with dialysis) (d) bran removal (white: no dialysis) The effect on human hybridoma cell line HB4C5 cells on IgM antibody production was confirmed for a total of 4 samples. HB4C5 cells were cultured in ERDF medium (Kyokuto Pharmaceutical Co., Ltd.) supplemented with 10 μg / ml insulin, 20 μg / ml transferrin, 20 μM ethanolamine, and 25 nM sodium selenite. The immunoregulatory activity was evaluated by measuring the amount of IgM secreted into the medium after 6 hours of culture by the enzyme antibody method. The amount of protein was measured by the Lowry method. These results are shown in graphs (A) and (B) of FIG. In the figure, (A) shows the results of the samples (a) and (b), and (B) shows the results of the samples (c) and (d).

  As shown in the graphs (A) and (B) of FIG. 1, the promotion of IgM antibody production was confirmed in all the samples, and the activity was particularly high in the samples (c) and (d) from which the bran was removed. . In the bran-removed samples (c) and (d), the amount of antibody produced by HB4C5 cells was promoted by 3.8 times when added at about 200 μg / ml in terms of protein. Further, it was confirmed that there was no influence of dialysis on the activity, and the molecular weight of the active substance was confirmed to be about 10,000 or more molecules.

  Next, the bran-removed (white: no dialysis) sample (d) was treated at each temperature of 30, 37, 50, 60, 70, 85, 95, and 121 ° C. for 20 minutes, and then HB4C5 cells. The effect of promoting IgM production was confirmed. The result is shown in the graph (A) of FIG. As shown in the drawing, the activity of the sample (d) was completely maintained even after the heat treatment at 85 ° C., and a slight decrease in activity was observed after the 95 ° C. treatment. From this, it was confirmed that the active factor is a heat-stable substance. However, it was deactivated by the 121 ° C. treatment.

  Next, the sample (d) heat-treated at 100 ° C. for 30 minutes and the non-heated sample (d) were added to the medium at various concentrations, and the influence on the IgM antibody production of HB4C5 cells was confirmed. The result is shown in the graph (B) of FIG. As shown in the figure, the heated sample (d) was found not to be completely deactivated, although a decrease in activity was observed with respect to the non-heated sample (d).

  Next, the sample (d) was treated with trypsin to examine the effect of trypsin on the IgM production promoting effect. That is, equal amounts of various concentrations of trypsin solution were added to a white sorghum (white) extract (sample (d)) and reacted at 37 ° C. for 15 minutes. Thereafter, trypsin was inactivated by heating at 100 ° C. for 2 minutes, and then quickly cooled on ice to examine IgM production promoting activity. The result is shown in the graph (C) of FIG. As shown in the figure, as the concentration of trypsin increased, the IgM production promoting effect decreased, and the IgM production promoting activity was inactivated at a trypsin concentration of 100 μg / mL.

  Summarizing the results of this example, there is an immunomodulatory active factor in white sorghum, which is abundantly contained in bran-removed white sorghum, which is thermostable and active by dialysis. Since it did not decrease, the molecular weight of the factor was 10,000 or more, and since it was inactivated by trypsin treatment, it was confirmed that the factor was a protein.

  This example is an example relating to the allergy suppressing activity of the physiologically active substance of the present invention.

  5 g of the sample powder from which the whole grains of white sorghum and bran were removed was suspended in 20 mL of 10 mM NaPB, stirred at 4 ° C. overnight, and then insoluble matter was removed by centrifugation to obtain a soluble fraction. A part of the obtained soluble fraction was dialyzed against 10 mM NaPB. Thus obtained (a) whole grain (with dialysis), (b) whole grain (without dialysis), (c) bran removal (white: with dialysis) (d) bran removal (white: no dialysis) The effect of human myeloma cell line U266 cells on IgE antibody production was confirmed for a total of 4 samples. U266 cells were cultured in ERDF medium (Kyokuto Pharmaceutical Co., Ltd.) supplemented with 10 μg / ml insulin, 20 μg / ml transferrin, 20 μM ethanolamine, and 25 nM sodium selenite. Allergy-inhibiting activity was determined by measuring the amount of IgE secreted into the medium after incubation for 24 hours by the enzyme antibody method. The amount of protein was measured by the Lowry method. The results are shown in graphs (A) and (B) in FIG. In the figure, (A) shows the results of the samples (a) and (b), and (B) shows the results of the samples (c) and (d).

  As shown in the graph (A) of FIG. 3, the IgE production inhibitory effect was confirmed in the samples (a) and (b) which are whole grain samples. On the other hand, as shown in the graph (B) of FIG. 3, the samples (c) and (d), which are the bran-removed samples, had no suppressing effect. From this result, it can be said that the IgE production inhibitory factor exists in the bran portion. Further, since the activity is not affected by dialysis, it can be said that the active factor is a polymer having a molecular weight of 10,000 or more.

  Next, 5 g of white sorghum bran powder (endothelium / outer skin) powder was suspended in 20 mL of 10 mM NaPB, stirred at 4 ° C. overnight, insolubles were removed by centrifugation, and two samples of the soluble fraction were obtained. Obtained. In the sample preparation, dialysis is not performed. About the obtained sample, the IgE production inhibitory effect was confirmed by the above-mentioned method. The result is shown in the graph (A) of FIG.

  As shown in the figure, IgE production inhibitory effect was observed in both endothelium and outer skin samples. When compared by activity per protein amount, the skin extract sample showed higher specific activity.

  Next, changes over time in IgE production of U266 cells were confirmed in a medium supplemented with an endothelial extract sample (425 μg / ml) and an outer skin extract sample (180 μg / ml). The result is shown in the graph (B) of FIG. As shown in the figure, it was confirmed that the addition of the endothelial extract sample almost completely suppressed IgE production after the first day of culture. At this time, when the cell viability was measured, the cell viability was almost the same level as that of the control. Therefore, the decrease in IgE production of U266 cells by the treatment with the factor in white sorghum bran was It was confirmed that this was not due to cytotoxicity to the cells, but an effect of suppressing IgE productivity.

  Next, when the outer skin extract was fractionated with an ultrafiltration membrane having a molecular weight cut off of 1000, IgE production inhibitory activity was also observed on the filtrate side (molecular weight of 1000 or less).

  Summarizing the results of this example, the white sorghum bran portion contains a factor that suppresses IgE production, and its abundance is greater in the endothelium than in the outer skin. As the factor, a high molecular weight factor having a molecular weight of 10,000 or more It was confirmed that there were 1000 or less low molecular factors.

  This example relates to the anticancer activity of the physiologically active product of the present invention.

  5 g of white sorghum bran powder (endothelium / outer skin) powder was suspended in 20 mL of 10 mM NaPB and stirred overnight at 4 ° C., and then insoluble matter was removed by centrifugation to obtain two types of samples of the soluble fraction. In the sample preparation, dialysis is not performed. The anticancer activity of the obtained extract sample was confirmed using mouse colon cancer cell line Colon26 cells. Colon 26 cells were cultured in ERDF medium (Kyokuto Pharmaceutical Co., Ltd.) supplemented with 5% fetal calf serum. Two days after culturing, the absorbance was determined by dye reduction using WST-8 (Kishida Chemical Co., Ltd.). WST-8 is 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazoleum, monosodium salt, and is an intracellular dehydrogenase It is a tetrazolium salt that is reduced by water to produce water-soluble formazan. By measuring the absorbance of the produced formazan at 450 nm, the number of viable cells can be measured. The number of cells and the amount of formazan produced are in a linear proportional relationship. The result is shown in the graph (A) of FIG.

  As shown in the figure, the concentration-dependent decrease in the number of viable cells of Colon 26 cells was confirmed in both endothelium and outer skin samples. In the comparison of the activity using the amount of protein as an index, no clear difference in activity was recognized between the endothelial extract sample and the envelope extract sample.

  Next, Colon26 cells were cultured in the medium supplemented with an endothelial sample (1000 μg / ml) and an outer skin sample (500 μg / ml), and the influence over time on cell proliferation was confirmed by the same method as described above. The result is shown in the graph (B) of FIG. As shown, both samples strongly inhibited colon cancer cell growth.

  Next, when the outer skin extract sample was fractionated with an ultrafiltration membrane having a molecular weight cut off of 1000, anticancer activity was also observed on the filtrate side (molecular weight of 1000 or less). On the other hand, the anti-cancer activity was confirmed when the fraction not filtered through the ultrafiltration membrane was dialyzed against 10 mM NaPB and then the anti-cancer activity was measured. From these results, the presence of a high molecular factor having a molecular weight of 10,000 or more and a low molecular factor having a molecular weight of 1,000 or less were confirmed as anticancer active factors. Next, in the same manner as in Example 1, each of the high molecular weight active factor and the low molecular weight active factor was digested with trypsin, a proteolytic enzyme, and then the absorbance was measured as described above to obtain anticancer activity. evaluated. The result is shown in the graph (C) of FIG. As shown in the figure, no decrease in anticancer activity due to trypsin treatment was observed in both the high molecular weight active factor and the low molecular weight active factor. In addition, in order to examine the thermal stability of each of the high molecular weight active factor and the low molecular weight active factor, each was treated at each temperature of 30, 37, 50, 60, 70, 85, 95, and 121 ° C. for 20 minutes. Thereafter, the anti-cancer activity was evaluated by measuring the absorbency as described above. The result is shown in the graph (D) of FIG. As shown in the figure, there was no reduction in anticancer activity due to heat treatment.

  Summarizing the results of this example, the white sorghum bran portion contains an anti-cancer active factor against colorectal cancer, and as the factor, there are a high molecular weight factor having a molecular weight of 10,000 or more and a low molecular factor having a molecular weight of 1,000 or less. These factors were confirmed to be non-protein and thermostable.

  This example relates to the active oxygen scavenging activity of the physiologically active product of the present invention.

5 g each of white sorghum bran powder and white sorghum bran powder were suspended in 20 mL of 10 mM NaPB, stirred overnight at 4 ° C., and then insolubles were removed by centrifugation to remove the soluble fraction from the bran. Samples and bran skin samples were obtained. The bran skin sample was fractionated with an ultrafiltration membrane having a molecular weight cut off of 1000 to obtain a filtrate (molecular weight of 1000 or less), which was used as a low molecular weight sample. For the sample thus obtained, superoxide dismutase (SOD) -like activity was measured using SOD Assy Kit-WST (manufactured by Dojindo Laboratories) according to the instruction manual. The results are shown in Table 1 below.

(Table 1)
SOD-like activity per 1 mg of extracted protein (unit / mg)
Bran removal sample 50.7
Bran skin sample 46.9
Small molecule sample 1.1

  As shown in Table 1, high SOD-like activity was confirmed in both the bran removed sample and the bran skin sample. On the other hand, since the activity of the low molecular weight sample was low, it can be said that the SDO-like active factor is a polymer.

  Summarizing the results of this example, it can be said that SDO-like activators exist in both the bran portion and the bran portion of white sorghum, and the active factor is a polymer.

  As described above, the physiologically active product of the present invention has various activities such as immunomodulating activity, allergy suppressing activity, anticancer activity, and active oxygen scavenging activity, and is safe. Therefore, according to the present invention, foods, beverages, supplements, pharmaceuticals, and the like useful for the prevention or treatment of immune diseases and diseases such as cancer can be provided, and the application fields thereof are not limited and are wide.

FIG. 1 (A) is a graph showing the immunomodulating activity of one example of the present invention. FIG. 1 (B) is a graph showing the immunomodulating activity of the Example. FIG. 2A is a graph showing the thermal stability of the example. FIG. 2B is a graph showing the thermal stability of the example. FIG. 2C is a graph showing the results of the trypsin treatment of the example. FIG. 3 (A) is a graph showing the allergy suppressing activity of other examples of the present invention. FIG. 3 (B) is a graph showing the allergy suppressing activity of the other examples. FIG. 4A is a graph showing the allergy suppressing activity of the other examples. FIG. 4 (B) is a graph showing the allergy suppressing activity of the other examples. FIG. 5 (A) is a graph showing the anticancer activity of yet another example of the present invention. FIG. 5 (B) is a graph showing the anticancer activity of the other examples. FIG. 5C is a graph showing the results of the trypsin treatment of the other examples. FIG. 5D is a graph showing the thermal stability of the other examples.

Claims (18)

A physiologically active product comprising at least one selected from the group consisting of a part of sorghum, all of sorghum and a sorghum extract. The physiologically active substance according to claim 1, wherein the extract is obtained by extracting a part of sorghum and / or all of sorghum with a water-containing solvent. The physiologically active product according to claim 1 or 2, wherein the sorghum is white sorghum. The physiologically active product according to any one of claims 1 to 3, wherein the water-containing solvent is at least one of water and a buffer solution. An immunomodulatory active agent comprising the physiologically active product according to any one of claims 1 to 4. The immunomodulating active agent according to claim 3, wherein the physiologically active substance contains at least one of bran part-removed sorghum and a water-containing solvent extract of bran part-removed sorghum. The physiologically active product according to claim 5 or 6, wherein the immunomodulating activity is an activity of promoting production of IgM antibody. An allergy-suppressing active agent comprising the physiologically active product according to any one of claims 1 to 4. The allergy-suppressing active agent according to claim 8, wherein the physiologically active substance contains at least one of a bran portion of sorghum and a water-containing solvent extract of the bran portion of sorghum. The allergy inhibitory activity according to claim 8 or 9, wherein the allergy inhibitory activity is an inhibitory activity of IgE antibody production. The anticancer active agent containing the bioactive substance as described in any one of Claims 1 thru | or 4. The anticancer active agent according to claim 11, wherein the physiologically active substance comprises at least one of a bran portion of sorghum and a water-containing solvent extract of the bran portion of sorghum. The anticancer active agent according to claim 11 or 12, wherein the anticancer activity is anticancer activity against colorectal cancer. An active oxygen scavenging activator comprising the physiologically active substance according to any one of claims 1 to 4. The physiologically active product according to any one of claims 1 to 4, the immunomodulating active agent according to any one of claims 5 to 7, and the allergy suppression according to any one of claims 8 to 10. A food comprising at least one selected from the group consisting of an active agent, the anticancer active agent according to any one of claims 11 to 13, and the active oxygen scavenging active agent according to claim 14. The physiologically active product according to any one of claims 1 to 4, the immunomodulating active agent according to any one of claims 5 to 7, and the allergy suppression according to any one of claims 8 to 10. A beverage comprising at least one selected from the group consisting of an active agent, the anticancer active agent according to any one of claims 11 to 13, and the active oxygen scavenging active agent according to claim 14. The physiologically active product according to any one of claims 1 to 4, the immunomodulating active agent according to any one of claims 5 to 7, and the allergy suppression according to any one of claims 8 to 10. A supplement comprising at least one selected from the group consisting of an active agent, the anticancer active agent according to any one of claims 11 to 13, and the active oxygen scavenging active agent according to claim 14. The physiologically active product according to any one of claims 1 to 4, the immunomodulating active agent according to any one of claims 5 to 7, and the allergy suppression according to any one of claims 8 to 10. A pharmaceutical comprising at least one selected from the group consisting of an active agent, the anticancer active agent according to any one of claims 11 to 13, and the active oxygen scavenging active agent according to claim 14.

Images