JP2022111399A - Composition for inhibiting ubiquitin ligase, and preventing/or treating composition containing the same for improving cachexia induced by cancer - Google Patents

Abstract

To provide a novel composition for inhibiting ubiquitin ligase, and preventing and/or treating a composition containing the same for improving cachexia induced by cancer.SOLUTION: The composition for inhibiting ubiquitin ligase includes (A) a guar gum decomposition product having an average molecular weight of 1.8×103 to 2.0×105, of which 70 mass% or more falls within the average molecular weight and (B) guar bean protein, and has a viscosity of 50 mPa s or less of a 1 mass% aqueous solution measured at 25°C and 60 rpm by means of a Brookfield viscometer; the guar gum decomposition product is obtained by hydrolyzing galactomannan polysaccharide contained in albumen derived from guar using β-mannanase derived from microorganisms to be depolymerized, and (A) : (B) falls in a range of 25 to 1000.SELECTED DRAWING: Figure 1

Description

The present invention relates to a ubiquitin ligase inhibitory composition, and a preventive and/or therapeutic composition containing the same for ameliorating cancer-induced cachexia.

Ubiquitin is a small protein consisting of 76 amino acids, so named because it is ubiquitous. Ubiquitin is used to modify other proteins and is involved in biological phenomena such as protein degradation, DNA repair, translation regulation, and signal transduction. It is evolutionarily highly conserved and has almost the same amino acid sequence in all eukaryotes.
Ubiquitin ligases are proteins that associate with ubiquitin-linked E2 ubiquitin conjugating enzymes, recognize protein substrates, and assist in the transfer of ubiquitin from E2 to the substrate or directly catalyze this reaction. The ubiquitin ligase confers substrate specificity on the E2 enzyme by interacting with both the target protein and the E2 enzyme. Ubiquitination by ubiquitin ligase regulates various activities such as cell migration, DNA repair, and signal transduction, and is recognized as extremely important in cell biology.
A substance that inhibits ubiquitin ligase can exert various actions such as muscle atrophy/bone atrophy, osteoporosis, muscle fatigue, and muscle reduction by inhibiting the action of ubiquitin ligase (Patent Document 1).

JP 2007-314469 A

However, no satisfactory ubiquitin ligase inhibitory composition has been obtained yet, leaving room for further research.
The present invention has been made in view of the above problems, and its object is to provide a novel ubiquitin ligase inhibitory composition, and a preventive and/or therapeutic agent containing the same for improving cancer-induced cachexia. It is to provide a composition.

The present inventor found that a composition containing a specific guar gum hydrolyzate and guar bean protein has ubiquitin ligase inhibitory activity, and basically completed the present invention.
Thus, the ubiquitin ligase inhibitory composition according to the present invention is composed of (A) guar gum degrading material having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ and containing 70% by mass or more of those having an average molecular weight within the range of 70% by mass or more. and (B) guar bean protein, and the viscosity of a 1% by mass aqueous solution when measured using a Brookfield viscometer at 25 ° C. and 60 rpm is 50 mPa s or less (more preferably 20 mPa s or less) wherein the guar gum hydrolyzate is contained in guar-derived endosperm and contains a galactomannan polysaccharide having a content ratio of galactose and mannose (galactose:mannose) in the range of 1:1.5 to 1:2.1. It is obtained by hydrolyzing with a microorganism-derived β-mannanase to reduce the molecular weight, and has a dietary fiber content of at least 70% by mass as determined by the enzyme-HPLC method, and contains oligosaccharides of guar gum decomposition products. is 15% by mass or less, and of the amino acid composition in the containing protein, (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids ≥ 0.4, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine) ≥ 4, (cystine + tyrosine)/total amino acids ≥ 0.1, and (valine + histidine + proline + leucine)/total amino acids ≤ 0.12, and the range of (A): (B) is in the range of 25-1000.

A composition for preventing and/or treating cachexia according to another invention is characterized in that it contains the ubiquitin ligase inhibitory composition and is for improving cachexia induced by cancer.
Further, food, drink and feed according to another invention are characterized by containing the ubiquitin ligase inhibitory composition. By ingesting this food and drink, cachexia induced by cancer can be improved, so it is good for cancer patients.
In the present invention, it is possible to provide foods, beverages and pharmaceuticals containing the composition of the present invention. Food and beverages include food and beverages, such as dietary supplements, health foods, foods for specified health uses, foods with function claims, diet therapy foods, general health foods, supplements, tea beverages, coffee beverages, and juices. , soft drinks, drinks, cooked rice, bread, noodles, dairy products, processed eggs, processed marine and livestock foods, confectionery, oils and fats and processed foods, seasonings, side dishes, and the like. Pharmaceuticals include pharmaceuticals or quasi-drugs, and are preferably oral formulations or enteral formulations, and can be in the form of liquids, tablets, granules, pills, syrups, and the like.
Feed in the present invention refers to food to be fed to organisms other than humans, and its form is not particularly limited. Organisms to which the feed can be applied are not particularly limited, but examples include farmed animals and pet animals. Farmed animals include livestock such as horses, cattle, pigs, sheep, goats, camels, and llamas, experimental animals such as mice, rats, guinea pigs, and rabbits, and poultry such as chickens, ducks, turkeys, and ostriches. be. Pet animals include, for example, dogs and cats.

INDUSTRIAL APPLICABILITY According to the present invention, a novel ubiquitin ligase inhibitory composition and a cachexia-preventing and/or therapeutic composition containing the same for improving cancer-induced cachexia can be provided.

1 is a graph showing changes in body weight of mice in each group when a bioactivity test of guar gum hydrolyzate was conducted. It is a graph which shows the change of the food amount of the mouse|mouth of each group. FIG. 10 is a graph showing the results of measuring the body weight of mice in each group on day 21 (final day). FIG. Fig. 10 is a graph showing the results of measuring the body weight of mice in each group when tumors were removed after dissection on day 21 (final day). It is a graph which shows the result of having measured the muscle weight (right-and-left bilateral gastrocnemius muscle weight) after dissecting on the 21st day (last day). FIG. 10 is a graph showing the results of examining the expression level (mRNA) of ubiquitin ligase (Astrogin) in skeletal muscle after dissection on the 21st day (last day). FIG. 10 is a graph showing the results of examining the expression level (mRNA) of skeletal muscle ubiquitin ligase (MuRF-1) after dissection on the 21st day (last day).

Next, embodiments of the present invention will be described with reference to the drawings. can be implemented in
Guar gum is a water-soluble natural polysaccharide obtained from the endosperm (more precisely, the cotyledons) of guar beans, and has two molecules of mannose linked in a straight chain and one molecule of galactose as a side chain. It is a polysaccharide and has an average molecular weight of about 2.0×10 ⁵ to 3.0×10 ⁵ . Guar gum is known to have physiological effects such as blood sugar level elevation inhibitory effect, cholesterol lowering effect, and bowel movement improving effect. In the present invention, the guar gum hydrolyzate is derived from beans derived from the annual leguminous plant guar (scientific name: Cyanopsis tetragoloba), which is used as food in India, Pakistan, etc., and hydrolyzes the galactomannan polysaccharide contained in the endosperm. and water-soluble dietary fiber obtained by reducing the molecular weight. The method for hydrolyzing guar gum is not particularly limited and may be an enzymatic decomposition method, an acid decomposition method, or the like.

The enzyme used in the enzymatic decomposition method is not particularly limited as long as it is an enzyme that hydrolyzes linear mannose chains, but it is preferable to use β-mannanase derived from bacteria belonging to the genus Aspergillus or Rhizopus. The upper limit of the average molecular weight distribution of the guar gum decomposition product is 2×10 ⁵ or less, preferably 1.0×10 ⁵ or less, more preferably 2.5×10 ⁴ or less. The lower limit of the average molecular weight distribution of the guar gum decomposition product is 1.8×10 ² or more, preferably 3.0×10 ³ or more. When the average molecular weight distribution is smaller than 1.8×10 ² , it becomes difficult to sufficiently exhibit ubiquitin ligase inhibitory activity, and when the average molecular weight exceeds 2×10 ⁵ , the viscosity is too high to include in food and drink. becomes difficult. ^The method for ^measuring the ^{molecular weight} distribution is not particularly limited. There is a method using a liquid chromatography method, and the like.
The guar gum decomposition product of the present invention contains 70% by mass or more, preferably 80% by mass or more of those having the average molecular weight within the above range.

Galactose is a type of monosaccharide classified as aldohexose, and has a molecular formula of C ₆ H ₁₂ O ₆ and a molecular weight of 180 (both of which are the same as glucose). The configuration is 2-position (second from the top in the Fisher projection formula), -OH at 5-position is in the same direction, 3-position and 4-position are in the opposite direction, and the configuration at 5-position of D-galactose is D-glycerol Same as aldehyde.
Mannose is a type of monosaccharide classified as aldohexose, and has a molecular formula of C ₆ H ₁₂ O ₆ and a molecular weight of 180 (both of which are the same as glucose). The steric configuration is such that the --OH at the 2- and 3-positions are in the same direction and the 4- and 5-positions are in the opposite direction, and the steric configuration at the 5-position of D-mannose is the same as that of D-glyceraldehyde. Mannose is poorly metabolized in humans and hardly enters the glycolysis system when orally ingested.
The oligosaccharide content was 6 to 15% by mass (15% by mass or less) according to the high performance liquid chromatograph analysis chart.
Amino acid composition in proteins can be measured by a known method using, for example, HPLC. Asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively, during protein hydrolysis in pretreatment for amino acid analysis, and asparagine and aspartic acid, and glutamic acid and glutamine cannot be distinguished during measurement. Therefore, these are collectively quantified as aspartic acid and glutamic acid.
Cachexia is a pathological condition that is accompanied by loss of muscle fibers and presents with anorexia, weight loss, general weakness, and the like. Diseases that cause cachexia include organ failure such as heart failure, chronic obstructive pulmonary disease, liver failure, and renal failure, chronic infectious diseases such as AIDS and malaria, cancer, and rheumatoid arthritis.
The present invention is particularly effective in improving cachexia induced by cancer.
The ubiquitin ligase inhibitory composition of the present invention can be orally ingested as it is or mixed with foods, beverages, and the like. The dose of the ubiquitin ligase inhibitory composition when taken orally is not particularly limited, but is 0.5 g to 70 g (preferably 3 g to 30 g, more preferably 6 g to 18 g) per adult per day.

<Preparation of guar gum decomposition product>
Example 1
After adjusting the pH to 4.5 by adding 0.1N hydrochloric acid to 900 g of water, 0.2 g of β-mannanase derived from bacteria belonging to the genus Aspergillus (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder were added and mixed. The mixture was reacted at 40° C.-45° C. for 24 hours. After the reaction, the enzyme was deactivated by heating at 90°C for 15 minutes. The reaction solution was separated by suction filtration, and the clear solution obtained by removing insoluble matter was concentrated under reduced pressure (Yamato evaporator). A solid content of 20% by mass was obtained. This was dried with a spray dryer (manufactured by Okawara Kakoki Co., Ltd.) to obtain 65 g of a guar gum decomposition product as a powder.
A 0.5 (w/v) % concentration aqueous solution was obtained by dissolving the guar gum decomposition product in water. Using polyethylene glycol (average molecular weight: 2×10 ² , 2×10 ³ , 2×10 ⁴ and 1×10 ⁵ ) as a molecular weight marker, high performance liquid chromatography (column: YMC-Pack Diol-120, detector A differential refractometer) was used to determine the average molecular weight, which was about 20,000. Those having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ were contained in an amount of 85 mass % or more.

Moreover, when the viscosity of the 1 mass % aqueous solution was measured using a Brookfield viscometer at 25° C. and 60 rpm, it was 8 mPa·s.
When the content ratio of galactose and mannose (galactose:mannose) was measured, it was 1:1.7.
The dietary fiber content was measured by enzyme-HPLC method and found to be 90% by weight.
The oligosaccharide content was 7.3% by mass.
In addition, amino acid analysis of the protein contained in the guar gum decomposition product was performed, and the results were as shown in Table 1. From these results, (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine), (cystine + tyrosine) / total amino acids, (valine + histidine) + proline + leucine)/total amino acids were 0.47, 5.0, 0.11 and 0.10, respectively.
Further, the ratio of guar gum hydrolyzate to guar bean protein was found to be guar gum hydrolyzate:guar bean protein=279:1.

Example 2
After adjusting the pH to 3 by adding 0.1N hydrochloric acid to 900 g of water, 0.15 g of β-mannanase derived from bacteria belonging to the genus Aspergillus (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder were added and mixed. The mixture was reacted at 40° C.-45° C. for 24 hours. After the reaction, the enzyme was deactivated by heating at 90°C for 15 minutes. The reaction solution was separated by suction filtration, and the clear solution obtained by removing insoluble matter was concentrated under reduced pressure (Yamato evaporator). A solid content of 20% by mass was obtained. This was dried with a spray dryer (manufactured by Okawara Kakoki Co., Ltd.) to obtain 68 g of guar gum decomposition product as a powder.
When the average molecular weight of the guar gum decomposition product was determined in the same manner as in Example 1, it was about 2.5×10 ⁴ . According to the results of the HPLC chart, 85% by mass or more of those having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁴ were contained.
Moreover, when the viscosity of the 1 mass % aqueous solution was measured using a Brookfield viscometer at 25° C. and 60 rpm, it was 10 mPa·s.
When the content ratio of galactose and mannose (galactose:mannose) was measured, it was 1:1.8.
The dietary fiber content was measured by enzyme-HPLC method and found to be 89% by weight.
The oligosaccharide content was 10% by mass.
In addition, amino acid analysis of proteins contained in guar gum decomposition products revealed that (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine). , (cystine+tyrosine)/total amino acids, and (valine+histidine+proline+leucine)/total amino acids were 0.56, 22.3, 0.16 and 0.03, respectively.
Further, the ratio of the guar gum decomposition product to the guar bean protein was found to be guar gum decomposition product:guar bean protein=418:1.

Example 3
After adjusting the pH to 4 by adding 0.1N hydrochloric acid to 900 g of water, 0.25 g of β-mannanase derived from bacteria belonging to the genus Aspergillus (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder were added and mixed. The mixture was reacted at 50° C.-55° C. for 12 hours. After the reaction, the enzyme was deactivated by heating at 90°C for 15 minutes. The reaction solution was separated by suction filtration, and the clear solution obtained by removing insoluble matter was concentrated under reduced pressure (Yamato evaporator). A solid content of 20% by mass was obtained. This was dried with a spray dryer (manufactured by Okawara Kakoki Co., Ltd.) to obtain 70 g of a guar gum decomposition product as a powder.
When the average molecular weight of the guar gum decomposition product was determined in the same manner as in Example 1, it was about 1.5×10 ⁴ . According to the results of the HPLC chart, 86% by mass or more of those having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ were contained.

Moreover, when the viscosity of the 1 mass % aqueous solution was measured using a Brookfield viscometer at 25° C. and 60 rpm, it was 9 mPa·s.
When the content ratio of galactose and mannose (galactose:mannose) was measured, it was 1:2.0.
The dietary fiber content was measured by enzyme-HPLC method and found to be 88% by weight.
The oligosaccharide content was 9% by mass.
In addition, amino acid analysis of proteins contained in guar gum decomposition products revealed that (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine). , (cystine+tyrosine)/total amino acids, and (valine+histidine+proline+leucine)/total amino acids were 0.48, 9.6, 0.17 and 0.05, respectively.
Further, the ratio of guar gum hydrolyzate to guar bean protein was found to be guar gum hydrolyzate:guar bean protein=357:1.

Example 4
A guar gum hydrolyzate was prepared according to the examples of JP-A-5-117156 (page 4, line 3 to page 4, line 10). When the average molecular weight was determined according to Example 1, it was 5.5×10 ³ .

<Biological activity test>
1. Test Method In order to investigate the bioactivity of the guar gum hydrolyzate prepared as described above, the following test was conducted.
32 male mice (BALB/c, 7 weeks old) were randomly divided into the following 4 groups of 8 mice each. Group 1: Group with tumor inoculation and with guar gum degradation product.
As a feed for rats and mice, AIN93G (manufactured by CLEA Japan, Inc., excluding 5% by mass of cellulose) to which 5% by mass of guar gum decomposition product was added was used and bred.
Group 2: Group with tumor inoculation and no guar gum degradation product.
As a feed for rats and mice, AIN93G (manufactured by CLEA Japan, excluding 5% by mass of cellulose) to which 5% by mass of sucrose was added was used.
Group 3: No tumor inoculation, group with guar gum degradation product.
As a feed for rats and mice, AIN93G (manufactured by CLEA Japan, Inc., excluding 5% by mass of cellulose) to which 5% by mass of guar gum decomposition product was added was used and bred.
Group 4: no tumor inoculation, no guar gum degradation product group.

As a feed for rats and mice, AIN93G (manufactured by CLEA Japan, excluding 5% by mass of cellulose) to which 5% by mass of sucrose was added was used.
From the 14th day before tumor inoculation (day-14), each group was fed with the above feed. On day 0 (day 0), mice in groups 2 and 4 were administered 5×10 ⁶ colon cancer cells (colon26) by subcutaneous injection into the right flank. The mice were dissected on the 21st day (day21) after administration of the cancer cell line.
From the 14th day to the 21st day, body weight, food intake, and tumor size were measured appropriately. After dissection on the 21st day, body weight, tumor size, muscle weight (left and right gastrocnemius), muscle atrophy markers (blood LPS, inflammatory cytokines, TGF-β family, SCFA, zonulin) were measured, and cecal contents (SCFA , fresh stool) and intestinal preservation (Carnoy fixation, immunostaining, intestinal epithelium) were performed. In addition, the expression levels (mRNA) of ubiquitin ligase in skeletal muscle were compared.

2. Test results <body weight, food amount, muscle mass>
FIG. 1 shows changes in body weight of mice in each group. Groups not inoculated with colon cancer cells (groups 3 and 4) showed a gradual weight gain from 14 days before to 21 days.
On the other hand, in the groups inoculated with colon cancer cells (groups 1 and 2), no weight gain was observed from around the 5th day, and on the 21st day, compared to the group not inoculated with colon cancer, were significantly underweight. Further, on the 21st day, Group 2 (without guar gum hydrolyzate) showed significant weight loss compared to Group 1 (with guar gum hydrolyzate).
FIG. 2 shows changes in food intake of mice in each group. In the group not inoculated with colon cancer cells, no significant change in food intake was observed throughout the entire test period.
On the other hand, in the colon cancer cell-inoculated group, a significant decrease in the amount of food was observed after the seventh day. Moreover, on the 21st day, a significant reduction in the amount of food was observed in the second group compared to the first group.

Figure 3 shows the body weight of the mice in each group on day 21, Figure 4 shows the body weight of the mouse after removing the tumor weight after dissection, and Figure 5 shows the muscle weight after dissection (both left and right sides). gastrocnemius muscle weight) are shown respectively. The groups inoculated with colon cancer cells (groups 1 and 2) showed a significant decrease in muscle mass compared to the groups not inoculated with colon cancer cells (groups 3 and 4). Between the groups not inoculated with colon cancer cells (groups 3 and 4), no change in muscle mass was observed due to the presence or absence of guar gum decomposition products. On the other hand, among the groups inoculated with large intestine group cells, group 1 (with guar gum hydrolyzate) was significantly (P = 0.035) higher in muscle mass than group 2 (without guar gum hydrolyzate). rice field.
Although not shown in the figure, no significant difference in tumor size was observed between groups inoculated with colon cancer cells (between groups 1 and 2).

<Measurement of ubiquitin ligase inhibitory activity in skeletal muscle>
Among the ubiquitin ligases, the expression levels of Atrogin and muscle RING finger 1 (MuRF-1) in skeletal muscle were examined. FIG. 6 shows Astrogin mRNA in each group, and FIG. 7 shows MuRF-1 mRNA in each group. In both graphs, the expression level of intraskeletal muscle ubiquitin ligase was low in the group not inoculated with colon cancer cells, regardless of the administration of the guar gum hydrolyzate (no significant difference between groups 3 and 4). .
On the other hand, in the colon cancer cell-inoculated group (group 2) without the guar gum degradation product, the expression level of ubiquitin ligase was significantly increased. However, in the colon cancer cell-inoculated group with the guar gum degradation product (group 1), the expression level of ubiquitin ligase was significantly lower than in the group 2. As a result, it was found that the guar gum degradation products have ubiquitin ligase inhibitory activity.
Thus, in an animal model in which cancer-induced cachexia was caused by subcutaneous transplantation of colorectal cancer cells, administration of guar gum degradation products resulted in decreased skeletal muscle weight, decreased body weight, and increased tumor-free body weight. significantly suppressed the decrease. In addition, guar gum degradation products were found to suppress the enhancement of ubiquitin ligase expression in skeletal muscle.

Thus, according to the present embodiment, a novel ubiquitin ligase inhibitory composition and a cachexia-preventing and/or therapeutic composition containing the same for improving cancer-induced cachexia can be provided.

Claims (7)

(A) a guar gum decomposition product having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ and containing 70% by mass or more of those within the average molecular weight range, and (B) guar bean protein, and B A ubiquitin ligase inhibitory composition having a viscosity of 50 mPa s or less in a 1% by mass aqueous solution when measured at 25° C. and 60 rpm using a viscometer,
The guar gum hydrolyzate is contained in the guar-derived endosperm, and the content ratio of galactose and mannose (galactose:mannose) is in the range of 1:1.5 to 1:2.1. and has a dietary fiber content of at least 70% by mass and an oligosaccharide content of 15% by mass of the guar gum hydrolyzate as determined by the enzyme-HPLC method. The following is the amino acid composition in the contained protein: (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids ≥ 0.4, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine) ≥ 4, (cystine + tyrosine) / total amino acids ≥ 0.1 and (valine + histidine + proline + leucine) / total amino acids ≤ 0.12, and the range of (A): (B) is 25 to 1000 A range of ubiquitin ligase inhibitory compositions. A composition for preventing and/or treating cachexia, comprising the ubiquitin ligase inhibitory composition according to claim 1, for ameliorating cachexia induced by cancer. A food or drink containing the ubiquitin ligase inhibitor according to claim 1 . A pharmaceutical product containing the ubiquitin ligase inhibitor according to claim 1. A medicament containing the cachexia preventive and/or therapeutic composition according to claim 2. A feed containing the ubiquitin ligase inhibitor according to claim 1 . A feed containing the composition for preventing and/or treating cachexia according to claim 2.

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