JP6130620B2 - Gene expression promoter - Google Patents

Description

  The present invention relates to a gene expression promoter comprising a guar gum enzyme degradation product.

Mankind has experienced a variety of infectious diseases since ancient times. In other words, viruses, bacteria, molds, protozoa, etc. have invaded the body and have been fighting against adverse effects.
In times when the cause and treatment were not well established, the epidemic of infectious diseases has had an impact on history. Since the second half of the 19th century, the causes of infectious diseases and how to deal with them have been known, and the number of deaths due to infectious diseases has decreased dramatically.
However, since around 1970, it was a new infection that was not known before, “Emerging Infectious Diseases”, and infections that had been reduced in the past due to infectious diseases that had spread in the past, but that reappeared “Re-emerging infections” Is a problem.
Smallpox is the only infectious disease that humanity has eradicated, but in the fifteenth century it was extremely raging.
The plague is called “Black Death” in Europe, and more than 25 million people have been killed in Europe alone.
Influenza, represented by Spanish cold in 1918, Asian cold in 1957, and Hong Kong cold in 1968, is still prevalent every year.
Emerging infectious diseases include AIDS (acquired immune deficiency syndrome, HIV), prion disease, highly pathogenic avian influenza, SARS (severe acute respiratory syndrome), and re-emerging infectious diseases such as tuberculosis and malaria. ing.
An invention that can prevent infectious diseases is desired. Therefore, we considered prevention with foods that can be taken on a daily basis. Development of antibiotics and vaccines is known as an invention relating to the prevention of infectious diseases (see, for example, Patent Documents 1 and 2). However, it is not enough. Antibiotics are very effective against bacteria because they kill bacteria that are harmful to the human body while leaving normal human cells intact. However, there are drawbacks in that bacteria useful for the human body are also killed, and if they are used excessively, the bacteria become resistant. Antibiotics also have no effect on viruses. Viruses are effective in vaccines, which have prevented disease by artificially infecting pathogens that have been weakened in advance by vaccination and creating antibodies in the body. Although it is necessary to develop vaccines that can deal with individual pathogens one by one, there is a drawback that they cannot always be developed. In addition, vaccination may cause side effects such as fever and chills.

The human body has an immune mechanism, and even when a pathogen enters the body, immune cells such as leukocytes and lymphocytes can fight off the pathogen through cooperative play. If immune cells move actively to fight off pathogens, they will not become ill when infected. If the immune cells are weak, they will lose their fight against pathogens and become sick.
Immune cells such as neutrophils such as leukocytes and phagocytic cells such as macrophages generate reactive oxygen species and have a function of killing pathogenic microorganisms. Reactive oxygen species are also generated in nerve cells, cardiomyocytes, vascular endothelial cells, etc., and are considered to function to control cell proliferation and to function as a body oxygen sensor.
Reactive oxygen species are synthesized by enzymes. One of them is known as an enzyme called NOX (NADPH oxidase). NOX reduces oxygen one electron to produce a superoxide anion (O ²⁻ ). On the other hand, there is a dual oxidase or a thyroid oxidase that synthesizes hydrogen peroxide by two-oxygen reduction of oxygen. The existence of Duox1 and Duox2 is known as a gene encoding Dual oxidase. Duox1 mRNA is mainly expressed in the thyroid gland and lung, and Duox2 mRNA is mainly expressed in the thyroid gland and intestine.

2 ', 5'-linked oligoadenylic acid (2-5A) has a structure in which adenylic acids are linked by a 2', 5'-phosphodiester bond. 2-5A activates an enzyme called RNase L that degrades RNA. Activated RNase L degrades viral mRNA and suppresses viral growth. 2-5A is synthesized using 2-5A synthetase (OAS) using ATP as a material. Subtypes such as Oas1, Oas2, and Oas3 are known as genes encoding OAS.
The inflammatory reaction is an action that protects the host from invasion of viruses and the like. Inflammation is necessary for the defense of the living body, but if it goes too far, it can damage the host and become shocked, which can be life-threatening. NLRC is a type of NOD-like receptor (NLR). There are 22 types of NLRs in humans, but many of them are not well understood. Among them, NLRC5 is adjusted so that the expression is increased by the stimulation of INF-γ and LPS to suppress the inflammatory response and not to overdo the reaction.

JP 2010-37294 A (page 1-13) JP 2006-193527 A (page 1-33)

  The present invention aims to solve the problem of infectious diseases and improve the maintenance of human health.

As a result of diligent efforts to solve the above problems, the present inventors have promoted the expression of genes related to the prevention of infectious diseases by orally ingesting a guar gum enzyme degradation product mainly composed of galactomannan polysaccharide. As a result, the present invention has been completed.
That is, the present invention is a gene expression promoter characterized by containing a guar gum enzyme degradation product.

  The gene expression promoter of the present invention has an advantage that it can activate the immune system. Infectious diseases can be prevented by using the gene expression promoter of the present invention.

Hereinafter, the present invention will be described in detail.
The guar gum degradation product in the present invention is a water-soluble dietary fiber, which is made from beans derived from an annual leguminous plant Guar (scientific name: Cyamopsis tetragonoloba) that is edible in India, Pakistan, etc., and is a galactomannan polysaccharide contained in its endosperm It is obtained by hydrolyzing and reducing the molecular weight. The hydrolysis method is not particularly limited, such as an enzymatic decomposition method or an acid decomposition method, but the enzymatic decomposition method is preferred because the molecular weights of the decomposed products are easily uniform. The enzyme used in the enzymatic decomposition method is not particularly limited as long as it is an enzyme that hydrolyzes mannose straight chain, and it is not particularly limited, but it may be β derived from Aspergillus or Rhizopus. -Mannanase is preferred. The average molecular weight distribution of the guar gum degradation product has an upper limit of 1.8 × 10 ⁵ or less, preferably 1.0 × 10 ⁵ or less, and more preferably 2.5 × 10 ⁴ or less. The lower limit of the average molecular weight distribution of the guar gum degradation product is 5 × 10 ² or more, preferably 3.0 × 10 ³ or more, and more preferably 1.0 × 10 ⁴ or more. When the average molecular weight distribution is 5 × 10 ² or less, it becomes impossible to provide the gene expression promoter of the present invention, and when the average molecular weight exceeds 1.8 × 10 ⁵ , there is a disadvantage when the viscosity is high and the product is included in food or drink. Arise. The method for measuring the average molecular weight distribution is not particularly limited. For example, high performance liquid chromatograph using polyethylene glycol (average molecular weight: 2 × 10 ² , 2 × 10 ³ , 2 × 10 ⁴ and 1 × 10 ⁵ ) as a marker. It can be determined by using a method (column: YMC-Pack Diol-120 (manufactured by YMC Co., Ltd., detector: differential refractometer)) or the like for measuring the average molecular weight distribution.

The guar gum degradation product of the present invention is not particularly limited, but those having the average molecular weight distribution of 70% or more, preferably 80% or more are used. Examples of commercially available products include Sun Fiber (manufactured by Taiyo Chemical Co., Ltd.), Fiberlon (manufactured by Dainippon Sumitomo Pharma Co., Ltd.), and Gua Fiber (manufactured by Meiji Food Materials).
EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated concretely, this invention is not limited to these.

Example 1
0.1N hydrochloric acid is added to 900 g of water to adjust the pH to 4.5, and 0.2 g of β-mannanase derived from Aspergillus bacteria (manufactured by Novo Nordisk Bioindustry) and guar gum powder (manufactured by Taiyo Kagaku) 100 g was added and mixed, and guar gum was enzymatically degraded at 40 to 45 ° C. for 24 hours. After the reaction, the enzyme was inactivated by heating at 90 ° C. for 15 minutes. After filtering and separating (suction filtration), the transparent solution obtained by removing insolubles was concentrated under reduced pressure (Yamato evaporator) (solid content: 20% by weight), and then spray-dried [Okawara Kako Co., Ltd. The product was dried to obtain 65 g of a guar gum decomposition product as a powder.

An aqueous solution having a concentration of 0.5 (w / v)% in terms of the amount of the guar gum degradation product obtained by dissolving the guar gum degradation product in water is polyethylene glycol (average molecular weight: 2 × 10 ² , 2 × 10 ³ , 2 × 10 ⁴ And 1 × 10 ⁵ ) as a molecular weight marker, the average molecular weight was determined to be about 20,000 when subjected to high-performance liquid chromatography (YMC-Pack Diol-120, manufactured by YMC Co., Ltd.).

Example 2
0.1N hydrochloric acid is added to 900 g of water to adjust to pH 3, and 0.15 g of β-mannanase derived from Aspergillus bacteria (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder (manufactured by Taiyo Kagaku Co., Ltd.) are added. The mixture was mixed and subjected to enzymatic degradation of guar gum at 40 to 45 ° C. for 24 hours. After the reaction, the enzyme was inactivated by heating at 90 ° C. for 15 minutes. The transparent solution obtained by filtration separation (suction filtration) to remove insolubles was concentrated under reduced pressure (Yamato evaporator) (solid content: 20% by weight) and then spray-dried device [Okawara Kako Co., Ltd. The product was dried to obtain 68 g of a guar gum decomposition product as a powder.

  With respect to the obtained guar gum decomposition product, the average molecular weight was determined in the same manner as in Example 1, and was about 25,000.

Example 3
0.1N hydrochloric acid is added to 900 g of water to adjust to pH 4, and 0.25 g of β-mannanase derived from Aspergillus bacteria (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder (manufactured by Taiyo Kagaku) are added. The mixture was mixed and subjected to enzymatic degradation of guar gum at 50 to 55 ° C. for 12 hours. After the reaction, the enzyme was inactivated by heating at 90 ° C. for 15 minutes. After filtering and separating (suction filtration), the transparent solution obtained by removing insolubles was concentrated under reduced pressure (Yamato evaporator) (solid content: 20% by weight), and then spray-dried [Okawara Kako Co., Ltd. The product was dried to obtain 70 g of a guar gum decomposition product as a powder.

  About the obtained guar gum decomposition product, when the average molecular weight was calculated | required like Example 1, it was about 15,000.

Example 4
A guar gum decomposition product was prepared in accordance with the description of Examples in JP-A-5-117156 (page 4, line 3, to page 4, line 10). The average molecular weight determined in accordance with Example 1 was 5,500.

Example 5
When high performance liquid chromatography was performed for molecular weight measurement in Example 4, a low molecular weight peak was collected. High-performance liquid chromatography was performed again, and the average molecular weight was determined to be about 800.

Test example 1
Experimental animals were BKS. Cg-Dock7 ^m + / + Lepr ^db / J mice (4 weeks old, male) were preliminarily raised on AIN-93G standard diet for 1 week to select individuals without abnormality, and a control group and a guar gum degradation group administration group These were divided into a total of 2 groups (3 per group) and used for the experiment. AIN-93G standard diet (see Table 1 for composition) for the control group and AIN-93G standard diet for the guar gum degradation product administration group from the day when the preliminary breeding was completed until the test end date (28 days intake period) The experimental feed in which 5% of the cellulose powder was replaced with the guar gum degradation product prepared in Examples 1 to 5 was freely ingested.

  The composition of the feed is shown in Table 1. “%” In Table 1 represents “% by weight”.

  After the intake period, the mouse was dissected under anesthesia and the small intestine was removed. After washing the small intestine, it was divided into 3 equal parts, and the site closest to the cecum was cut with scissors. The intestinal mucosal tissue was shaved using a slide glass. Using Isogen (Nippon Gene), total RNA was extracted from mucosal tissue, a part of it was purified with RNeasyMiniKit (QIAGEN), 250 ng of sample was prepared, and hybridized with Affymetrix MouseGene 1.0 STArray (Affymetrix) , And scanned. The scanned data was imported into Partek Genomics Suite (Partek), corrected between arrays (using RMA method), and then subjected to comparative analysis and significant difference test (ANOVA) with the specified combinations.

  The expression level of each gene in the guar gum degradation product administration group was compared with that of the control group, and genes whose expression level was 1.2 times higher in the guar gum degradation product administration group were extracted. As a result, Duox2, Oas1g, Oas3, and Nlrc5 were confirmed as genes whose expression levels increased when the guar gum degradation product was ingested. From the increase in expression of each gene, the strongest effect was confirmed particularly with the guar gum degradation product shown in Example 1. The guar gum degradation products shown in Example 2 and Example 3 showed the strongest effect next to the guar gum degradation product shown in Example 1 (Table 2).

  The gene expression promoter of the present invention can be applied to various foods and can provide foods that can prevent infectious diseases.

Claims (2)

  At least one gene expression promoter selected from the group consisting of Oas1g, Oas3 and Nlrc5, characterized by containing a guar gum degradation product. 2. The gene expression promoter according to claim 1, wherein the average molecular weight of the guar gum degradation product is 5 × 10 ^{2 to} 1.8 × 10 ⁵ .