KR20210018171A - Anti-Toxoplasma Composition Comprising Caenorhabditis elegans fat-1 which Induces Conversion to Omega-3 Fatty Acid - Google Patents

Abstract

The present invention relates to an anti-Toxoplasma gondii composition which has an effect to inhibit the proliferation and survival of Toxoplasma gondii, thereby being able to be usefully used in pharmaceuticals, health food, animal feed, etc. More specifically, the present invention relates to an anti-Toxoplasma gondii composition containing Caenorhabditis elegans fat-1, an enzyme that induces the conversion of omega-6 fatty acids to omega 3-fatty acids, as an active component.

Description

Anti-Toxoplasma Composition Comprising Caenorhabditis elegans fat-1 which Induces Conversion to Omega-3 Fatty Acid}

The present invention relates to an anti-toxoplasma gondii composition that has the effect of inhibiting the proliferation and survival of Toxoplasma gondii and can be usefully used in medicines, health foods, and animal feeds.

Omega-3 polyunsaturated fatty acids (ω3-PUFAs, omega-3 polyunsaturated fatty acids, hereinafter referred to as'omega-3 fatty acids') are essential for maintaining health and body homeostasis, but are not synthesized in the body and must be consumed as food. It is an essential nutrient. Omega-3 fatty acids are present in a very small amount in the human body, but DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) are known to have various physiological activities and are receiving a lot of attention. Except for these, omega-3 fatty acids are known to have very low or no physiological activity. As omega-3 fatty acids, characterized by DHA and EPA, have garnered much attention, clinical and epidemiological studies have shown a variety of mental disorders such as depression, attention deficit hyperactivity disorder, paranoia and dementia, as well as retinal degenerative diseases, autoimmune diseases, kidney and urinary tract. It has been reported that it has a beneficial effect on diseases, android type alopecia, diabetes and hypertriglyceridemia, infant development, cancer, and cardiovascular disease (Patent Publication No. 10-2012-0039521).

Fat1 transgenic mice express the gene Caenorhabditis elegans fat-1, which encodes an omega-3 fatty acid desaturation enzyme that allows the synthesis of omega-3 fatty acids from omega-6 fatty acids. It is known that the contents of DHA, EPA, and alpha linolenic acid (ALA) are high among omega-3 fatty acids produced in the body in Fat1 transgenic mice. This mouse was found to have a high content of omega-3 fatty acids in tissues in a model of mucosal organ damage including acute lung injury, chemically induced colitis, hepatitis and pancreatitis, and the anti-inflammatory response was also stronger. It has also been reported that omega-3 fatty acids have immunomodulatory activity in infectious diseases caused by bacteria and viruses.

Toxoplasmosis is one of the world's most widespread common pathogens. Toxoplasmosis worms use cats as their breeding host and spread to the outside through feces. The human body is infected by directly ingesting water, vegetables, fruits, etc. that are contaminated with the oocyst contained in cat feces, or by eating infected pigs, sheep, and cows as intermediate hosts. It has an infection rate of 10 to 30% worldwide, especially in Europe and North America. In Korea, it is reported that 2 to 8% of the general population are positive for antibodies in serological tests.

The main invading tissues of Toxoplasmosis are lymph glands, retina, brain, etc. If the human body is infected, it can cause retinal degeneration, meningitis, lymphadenitis, etc. However, in the case of a weak poison infection, it can pass for a long time without showing any worrisome clinical symptoms. . However, it causes severe systemic opportunistic infectious diseases in pregnant women, young children, patients with organ transplants and blood transplants, and AIDS patients with immunodeficiency. When a pregnant woman is first infected by Toxoplasmosis in the second or third trimester of pregnancy, the body infects the fetus through the placenta and causes congenital Toxoplasmosis in the fetus. They may remain asymptomatic during the neonatal period, but complications of central nervous system involvement may appear several years later. Some pregnant women may be still born, prematurely or miscarried.

Toxoplasma reticulum has developed various strategies to evade the host's immune defense response so that it can survive and replicate in innate immune cells such as human dendritic cells, macrophages, and neutrophils. For this reason, a satisfactory drug for Toxoplasmosis infection has not yet been known, and improvement is expected by co-administering pyrimethamine, an antimalarial drug, and sulfonamide, a sulfa drug. However, administration of pyrimethamine is forbidden to pregnant women, and potential side effects such as bone marrow failure and hepatotoxicity have been reported, and when administered concurrently, Stevens Johnson syndrome, which is the main symptom of detachment of the epidermis and mucous membrane, may appear as a side effect. This is urgent. Accordingly, for the safer prevention and treatment of Toxoplasmosis infection, the development of drugs using natural substances such as ginger extract (Public Patent No. 10-2012-0017971) and Pseudomonas herb extract (Registration Patent No. 10-1710588) has been actively made. Is losing.

Publication Patent No. 10-2012-0039521 Publication Patent No. 10-2012-0017971 Registered Patent No. 10-1710588

The present invention is an anti-toxoplasmic reticulum composition that can be usefully used as a composition for the prevention and treatment/symptom improvement of diseases caused by anti-toxoplasmosis infection by showing direct antigenic activity against toxoplasmosis while ensuring safety for animals as well as humans. It aims to provide.

The present invention for achieving the above object relates to an anti-toxoplasmic reticulum pharmaceutical composition containing as an active ingredient Caenorhabditis elegans fat-1, an enzyme that induces conversion of omega-6 fatty acids to omega-3 fatty acids.

Omega-3 fatty acids have the effect of inhibiting the proliferation of toxoplasmosis, and thus can be usefully used in the prevention and treatment of Toxoplasmosis Infectious diseases.

Omega-3 fatty acids are essential nutrients that must be consumed from food because they are not biosynthesized in the body. The antitoxoplasmic reticulum pharmaceutical composition of the present invention may contain omega-3 fatty acids themselves, but enzymes that induce the conversion of omega-6 fatty acids to omega-3 fatty acids to enable the biosynthesis of omega-3 fatty acids in the body May be contained as an active ingredient. Alternatively, it may be provided in the form of a gene encoding the enzyme, such as the Caenorhabditis elegans fat-1 gene. The gene includes DNA, RNA or PNA. The gene is contained in an expression vector, and the expression vector may be infected or transduced by various methods known in the art to be introduced into a target cell as an expression type.

The pharmaceutical composition may be prepared and used as a powder, granule, tablet, capsule, or injection by mixing itself or a pharmaceutically acceptable carrier, excipient, diluent, etc. by a method known in the pharmaceutical field. The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount. In the present invention, the'pharmaceutically effective amount' means an amount sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical treatment. Effective dosage levels are the type of disease, the purpose of administration for prevention or treatment, the severity of the disease, age, sex, weight, activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, duration of treatment, and simultaneous use. It may depend on factors including the drug being used and other factors well known in the medical field. The composition of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered simultaneously or sequentially with conventional therapeutic agents. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

The present invention also relates to an anti-toxoplasmosis pharmaceutical composition containing Caenorhabditis elegans fat-1, an enzyme that induces the conversion of omega-6 fatty acids to omega 3- fatty acids, as an active ingredient. The composition of the present invention can be processed in the form of tablets, capsules, pills or liquids, and foods to which the composition of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods. And the like, but are not limited thereto. The effective dose of the health functional food composition of the present invention may be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range. Since the composition of the present invention contains omega-3 fatty acids, which have been ingested through various foods, it can be safely used even when taken for a long time without fear of side effects.

The health functional food composition of the present invention can be used as a health functional food for animals as well as the human body. In particular, in the case of administering the composition of the present invention having anti-toxoplasmosis activity in cats as a breeding host, pigs, cattle, sheep, etc. as intermediate hosts, it is possible to not only reduce diseases of animals, but also to block infections in the human body caused by them. Very useful. When used as a health functional food composition of the present invention, it may be prepared as a feed additive when preparing feed, or prepared as a separate formulation and mixed with feed for administration, or may be provided as a feed top dressing.

As described above, according to the present invention, since omega-3 fatty acids, which have been secured for long-term use as an ingredient contained in food, act as an active ingredient, it can be safely used for pregnant women and children.

In addition, omega-3 fatty acids, which are active ingredients in the composition of the present invention, not only alleviate symptoms of diseases caused by Toxoplasmosis infection by the anti-inflammatory activity, which is the useful activity of omega-3 fatty acids known by the prior art, Since it directly inhibits proliferation, diseases caused by Toxoplasmosis infection can be more effectively prevented and treated.

1 is a confocal micrograph showing the anti-toxoplasmic reticulum activity of DHA in vitro.
Figure 2 is a graph showing the anti-toxoplasmosis activity of DHA in vitro.
3 is an electrophoresis photograph and graph showing the effect of DHA on the expression of proteins and genes related to antitoxoplasmosis.
Figure 4 is a confocal micrograph showing the anti-toxoplasmic reticulum activity of omega-3 fatty acids in vitro.
5 is a graph showing the anti-toxoplasmic reticulum activity of omega-3 fatty acids in vitro.
6 is an electrophoresis photograph and graph showing the effect of omega-3 fatty acids on the expression of proteins and genes related to antitoxoplasmosis.
7 is an electrophoresis photo and graph showing the anti-toxoplasmic reticulum activity of omega-3 fatty acids in an in vivo experiment.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, these drawings and embodiments are only examples for easily explaining the content and scope of the technical idea of the present invention, thereby not limiting or changing the technical scope of the present invention. It will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention based on these examples.

[Example]

Example 1: Preparation of Toxoplasmosis

Toxoplasmosis (GFP-RH strain) at the stage of Tachyzoites, which expresses green fluorescent protein, is Dr. It was provided by Yoshifumi Nishikawa (Obihiro University of Agriculture and Veterinary Medicine, Japan). Tachyzoites of Toxoplasmosis GFP-RH strain were prepared by using DMEM medium containing nutrient mixture F-12, 10% FBA, and 1% Antibiotic-Antimycotic. ) Cells were cultured and maintained in 5% CO ₂ , 37° C.

Example 2: Cell culture and differentiation of bone marrow-derived macrophages

Wild type C57BL/6 mice were purchased from Coretech (Pyeongtaek, Korea), and Fat1 transgenic mice expressing ω3-PUFAs were Dr. It was provided by JX Kang (Harvard Medical School, USA). Macrophages were collected from the bone marrow of each mouse, and macrophage colony-stimulating factor, 10% FBS, 1 mM sodium pyruvate, 50 U/mL penicillin, 50 μg/mL straptoma It was differentiated by culture for 5 to 7 days in a medium containing isin, 5×10 ^-5 M beta-mercaptoethanol.

Example 3: Evaluation of DHA antitoxoplasmosis activity

Bone marrow-derived macrophages (BMDMs) were used as host cells to treat one Toxoplasma oysterworm expressing green fluorescent protein into one bone marrow-derived macrophage for 2 hours at 1 MOI. Then, DHA (Cayman Chemical, Ann Arbor, MI, USA) was treated with 5, 10 or 20 μM, respectively, and cultured.

After 18 hours of DHA treatment, the number of Toxoplasma oysterworms expressing green fluorescent protein infected with bone marrow-derived macrophages was confirmed using confocal micoscopy. 1 and 2 show the results, and it was confirmed that DHA inhibited the proliferation of Toxoplasmosis in a concentration-dependent manner in bone marrow-derived macrophages.

48 hours after DHA treatment, according to the method described in Immunity 2015, 43, 80-91, the expression of the p30 membrane protein TP3 of Toxoplasma chinensis was Western blot, and the expression of SAG1 (surface antifen 1) mRNA was analyzed in real time polymerase chain reaction ( qPCR) confirmed the division and proliferation of Toxoplasmosis. The sequence of primers used for analysis of SAG1 mRNA expression is shown in Table 1 below.

3 shows the results of immunoblot and qPCR analysis. As the concentration of DHA treatment increased, the expression of TP3 protein and SAG1 mRNA decreased, indicating that DHA inhibited the division and proliferation of Toxoplasmic reticulum in cells.

Example 4: Fat1 Transgenic Mice Using Omega-3 Fatty Acids in-vitro Antitoxoplasmosis activity evaluation

Bone marrow-derived macrophages that were collected and differentiated from the bone marrows of Fat1 transgenic mice and wild-type mice were infected with Toxoplasmic reticulum expressing green fluorescent protein at a concentration of 1 MOI. Subsequently, according to the culture time, the number of Toxoplasmic reticulums expressing green fluorescent protein infected with bone marrow-derived macrophages was confirmed with a confocal microscope, and the results are shown in FIGS. 4 and 5. As can be seen in FIGS. 4 and 5, it was confirmed that the number of Toxoplasma oysters was significantly smaller in bone marrow-derived macrophages collected and cultured in Fat1 transgenic mice than in bone marrow-derived macrophages collected and cultured in wild-type mice.

After the infection of Toxoplasmosis worms, division and proliferation of Toxoplasmosis worms was confirmed through immunoblot analysis and qPCR according to the culture time by the same method as in Example 3. 6 shows the immunoblot and qPCR analysis results, and it can be seen that the expression of TP3 protein and SAG1 mRNA was reduced in bone marrow-derived macrophages derived from Fat1 mice compared to the wild type, as in the results of FIGS. 4 and 5. This shows that omega-3 fatty acids inhibit the division and proliferation of Toxoplasmosis in cells.

Example 5: Omega-3 fatty acids using Fat1 transgenic mice in-vivo Antitoxoplasmosis activity evaluation

Intraperitoneal injections of 40 cysts of toxoplasmosis (typw II ME49 strain) were given to 5 wild-type mice and 5 transgenic mice, respectively. After 3 weeks, genomic DNA was extracted from the brain homogenates of each mouse using the G-DEXTM IIc for Cell/Tissue Genomic DNA Extraction Kit (iNtRON) according to the user's manual, and B1, a specific gene of Toxoplasmosis. While the expression of the gene was amplified by PCR using the primers shown in Table 2 below, the number of Toxoplasmic reticulum cysts was confirmed.

7 shows the results, showing that the genomic DNA expression of the B1 gene and the number of cysts of the Toxoplasmic reticulum were significantly reduced in Fat1 transgenic mice compared to wild-type mice.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Anti-Toxoplasma Composition Comprising Caenorhabditis elegans fat-1 which Induces Conversion to Omega-3 Fatty Acid <130> P1120-393 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atcgcctgag aagcatcact 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcgaaaatgg aaacgtgact 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggaactgcat ccgttcatga g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tctttaaagc gttcgtggtc 20

Claims (5)

An anti-toxin spore pharmaceutical composition containing Caenorhabditis elegans fat-1, an enzyme that induces the conversion of omega-6 fatty acids to omega-3 fatty acids, as an active ingredient.
The method according to claim 1,
Antitoxoplasmic reticulum pharmaceutical composition, characterized in that provided in the form of a gene encoding the enzyme.
The method according to claim 2,
Antitoxoplasmic reticulum pharmaceutical composition, characterized in that the gene is provided in the form contained in the expression vector.
An anti-toxoplasmosis health functional food composition containing Caenorhabditis elegans fat-1, an enzyme that induces the conversion of omega-6 fatty acids to omega-3 fatty acids, as an active ingredient.
The method of claim 4,
Health functional food composition, characterized in that the anti-toxin spores health functional food composition for animals.

Images