KR102292131B1 - A composition for treating or preventing toxoplasmosis comprising Oleanolic acid - Google Patents

Abstract

In the present invention, oleanolic acid has not previously been reported to have an antitoxoplasmosis or toxoplasmosis, which is one of zoonoses.
In the present invention, it was confirmed that the test substance has an anti-Toxoplasmosis effect or activity indicating survival inhibition or mitosis inhibition and growth inhibition on Toxoplasmosis, and toxoplasmosis (Toxoplasmosis) using the test substance. ), it was confirmed that it can be used as a composition for the treatment, improvement or prevention of.
The present invention relates to a pharmaceutical composition for treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient, a health food composition for prevention or improvement, a feed composition for prevention or improvement, a veterinary pharmaceutical composition for treatment, improvement or prevention, and prevention Or it relates to a quasi-drug composition for improvement. The pharmaceutical composition, health food composition, feed composition, veterinary drug composition, and quasi-drug composition according to the present invention have an anti-toxoplasma gondii effect or activity indicating survival or mitosis inhibition and growth inhibition on Toxoplasmosis. It can be usefully used as a composition for the treatment, improvement or prevention of symptoms.

Description

TECHNICAL FIELD [0001] A composition for treating or preventing toxoplasmosis comprising Oleanolic acid as an active ingredient

The present invention relates to a pharmaceutical composition for the treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient, a health food composition for prevention or improvement, a feed composition for prevention or improvement, It relates to an animal pharmaceutical composition for treatment, improvement or prevention, and a quasi-drug composition for prevention or improvement.

More specifically, the single substance of olreahnol acid (Oleanolic acid) are zoonotic infectious diseases of the parasite Toxoplasma Toxoplasma gondii inducing plasma increase: survival direct inhibitory action, and insecticidal action or cleavage of Toxoplasma gondii for (Toxoplasma gondii T. gondii) Because it has an effect of inhibiting proliferation and growth, a pharmaceutical composition for treatment, improvement or prevention of toxoplasmosis, a health food composition for prevention or improvement, a feed composition for prevention or improvement, an veterinary pharmaceutical composition for treatment, improvement or prevention, and prevention or improvement It is useful as a quasi-drug composition for use.

Hundreds of years ago, communicable diseases such as tuberculosis, flu, plague, smallpox and cholera were high-risk contagious diseases that caused rapid death to all of humanity, including children and adults. These communicable diseases have killed millions of people around the world over the centuries, and the prevention, treatment and suppression of some communicable diseases are still difficult. Among various studies for the suppression of these infectious diseases, the discovery of penicillin, an antibiotic, was a turning point that caused great changes in the inhibition or treatment of infectious or pathogenic microorganisms by antibiotics, disease suppression, health and health improvement, and human lifespan extension. However, the damage caused by infectious microorganisms, which are still pathogenic, crosses national borders and causes various problems and diseases in the economic, environmental, medical and public health fields.

Toxoplasma gondii (Toxoplasma gondii: T. gondii ) is a pathogen that infects the central nervous system of AIDS patients, infants, immunocompromised or immunocompromised adults and patients, causing toxoplasmosis or Toxoplasmosis. The main cause is parasites. Toxoplasma gondii or Toxoplasma gondii is a very small parasitic organism compared to animal cells and is a crescent-shaped parasitic organism with a length of 8 ㎛ and a diameter of 2 ㎛.

Toxoplasma gondii (Toxoplasma gondii ) The internal organs constituting the single cone, apicoplast, mitochondria, ribosomes, nucleus, Golgi apparatus, lob tree, dense granules and micronemes (Micronemes) and the like. Toxoplasmosis has three stages of infection, including tachyzoites, bradyzoites, and sporozoites, and these stages carry out the parasitic life cycle necessary for the proliferation and growth of Toxoplasmosis. make up Also, when Toxoplasmosis is transmitted, it is transmitted through two stages (cyst and ooccyst). Humans can become infected by placental infection, which refers to the transmission of Toxoplasma gondii from mother to fetus, and can be transmitted by eating uncooked or undercooked food or through raw meat infected with Toxoplasma gondii. It can be transmitted through spores present in water (oocysts) or through cat feces. The tachyzoite phase refers to a stage of rapid division and proliferation in the host cell and replication. When Toxoplasma gondii is infected with animal cells, Toxoplasma gondii proliferates inside the cell, forming a parasitephorous vacuole (PV), and the parasitephorous vacuole (PVM) Toxoplasma gondii acquires nutrients from the host cell through the membrane and divides and proliferates in the PVM to differentiate. The parasitophorous vacuole membrane (PVM) is an indigestible vacuole that provides nutrients and protects Toxoplasmosis from acidification.

Until recently, studies on Toxoplasmosis have shown that various micro-organs and substances, including mycones, rhoptries, Apicoplasts, conoids, and dense granules of Toxoplasmosis, are closely related to the parasitic function of Toxoplasmosis. reported to be strongly related. Each organelle releases an associated protein that triggers a different function during each step of the Toxoplasmosis invasion into host cells. Myconems are involved in the attachment of Toxoplasmosis to host cells, and Loptree plays an important role in PVM formation and interaction with host cells. is reported to be

Currently, there is no commercially available effective vaccine for treating toxoplasmosis, and sulfadiazine and pyrimethamine are being used as antitoxoplasmosis (antitoxoplasmosis) treatment agents. To date, there is no effective and satisfactory drug, and it is used for the treatment of toxoplasmosis by co-administration of pyrimethamine, an antimalarial drug, and sulfonamide, a sulfa drug, and pyrimethamine, sulfadiazine or spiramycin. Currently, a combination of pyrimethamine-sulfamethoxazole is commercially available, but the administration of pyrimethamine to pregnant women requires careful diagnosis and prescription. In addition, if there is a retinal disease, the clindamycin-sulfadiazine combination may show good progress, and steroids may be used together to reduce the inflammatory reaction. However, recently, as side effects are caused due to various toxicity and resistance of the above drugs, research on the development of drugs that cause effects on the inhibition and insecticidal effect of Toxoplasmosis by using various natural resources is being actively conducted. In this respect, R&D drugs for the development of new anti-Toxoplasma gondii drugs should be effective in disease treatment, non-toxic or insignificant to humans and animals, and should strongly and effectively inhibit or kill Toxoplasma gondii, the source of infection.

Accordingly, the present inventors have found that oleanolic acid directly inhibits the survival of Toxoplasma gondii (T. gondii ), which is a parasite that causes Toxoplasmosis, a zoonotic disease, or mitosis propagation for Toxoplasmosis. And, as well as having a growth inhibitory effect, it was confirmed that it is effective in Toxoplasmosis and Toxoplasmosis-infected cells by inhibiting the survival of infected cells infected with Toxoplasmosis and inhibiting the proliferation and growth of Toxoplasmosis in infected cells. completed.

An object of the present invention is to provide a pharmaceutical composition for the treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient.

Another object of the present invention is to provide a health food composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient.

Another object of the present invention is to provide a feed composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient.

Another object of the present invention is to provide an veterinary pharmaceutical composition for the treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient.

In addition, another object of the present invention is to provide a quasi-drug composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient.

As one aspect for achieving the above object, the present invention provides a pharmaceutical composition for the treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient.

As used herein, the term "Oleanolic acid" is "Oleanic acid" or "CAS Number 508-02-1" or IUPAC nomenclature "(4aS, 6aR, 6aS, 6bR, 8aR, 10S, 12aR, 14bS)- 10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene- It is called "4a-carboxylic acid" and exhibits light yellow crystals or white powder at room temperature or refrigeration (2-8℃), the chemical formula is C ₃₀ H ₄₈ O ₃ and molecular weight is 456.70 g/mole am.

In the present invention, after dissolving oleanolic acid in DMSO or ethanol, solutions were prepared for each concentration using a medium, and the antitoxoplasmosis effect or activity of the test substance against Toxoplasmosis was measured using the MTT assay.

In one embodiment of the present invention, it was confirmed that oleanolic acid directly inhibits or reduces the survival rate of Toxoplasmosis, and thus it was confirmed that oleanolic acid exhibits anti-toxoplasmosis activity or anti-toxoplasmosis-related effect (FIG. 1). to 2).

In the present invention, after dissolving oleanolic acid in DMSO or ethanol, a solution is prepared for each concentration using a medium, and the antitoxoplasmosis activity or antitoxoplasmosis effect of the test substance on Toxoplasmosis infection-infected cells using the MTT measurement method. was measured.

In an embodiment of the present invention, it was confirmed that oleanolic acid effectively inhibits or reduces the survival rate of Toxoplasmosis-infected cells, and oleanolic acid exhibits anti-toxoplasmosis activity or anti-toxoplasmosis-toxoplasmosis-to-toxoplasmosis-to-sporophyte effect on cells infected with Toxoplasmosis. This was confirmed. Due to the characteristics of Toxoplasmosis, when Toxoplasmosis invades cells and infects cells, the resistance of Toxoplasmosis becomes strong and causes resistance of infected cells. cannot be played back. The present inventors confirmed by administering Oleanolic acid to infected cells infected with Toxoplasmosis. As a result, Oleanolic acid selectively kills Toxoplasmosis in cells infected with Toxoplasmosis, thereby inhibiting the proliferation of Toxoplasmosis in the cells and effectively killing infected cells. As a result, it was confirmed that toxoplasmosis can be treated by killing ( FIG. 3 ).

As used in the present invention, the term "toxoplasmosis" is called "toxoplasmosis" and is a parasite-derived zoonotic infectious disease caused by infection of Toxoplasma gondii, a type of protozoa, and a woman is pregnant. Infection with Toxoplasmosis during pregnancy causes miscarriage and infertility and causes malformations and congenital Toxoplasmosis in the fetus through "placental infection". The main symptoms are hydrocephalus, retinochoroiditis, and convulsions , cerebral calcification, and psychomotor retardation.

In addition, acquired Toxoplasmosis infection may cause symptoms such as lymphadenopathy, fever, headache, myalgia, splenomegaly and erythematous rash, myocarditis or meningitis, and atypical pneumonia.

Toxoplasma gondii is found anywhere in the world where its definitive host cat or feline wild animal lives, and all mammals, including humans, pets and livestock, and birds and other animals are known to act as intermediate hosts, and in the United States More than 60 million people are reported to be infected with Toxoplasmosis.

In addition, "Toxoplasmosis" is a disease caused by infection by Toxoplasmosis. When Toxoplasmosis becomes infected with the human body, it parasitizes in muscle tissue. It can invade and cause various symptoms.

Toxoplasmosis has various symptoms depending on the organ infected with Toxoplasmosis. Toxoplasma gondii grows slowly using host nutrients and can spread to various organs, causing inflammation. When a person is infected with Toxoplasmosis, the normal tissues of the body are destroyed at a very slow rate by the inflammatory reaction with the immune cells within the tissues, and granulomas may occur around them.

As confirmed in the Examples of the present invention, the oleanolic acid of the present invention inhibits and inhibits the survival of Toxoplasmosis, thereby preventing, improving, and treating toxoplasmosis caused by Toxoplasmosis infection. The composition of the present invention It may be provided as a pharmaceutical composition for the prevention, improvement or treatment of toxoplasmosis comprising as an active ingredient.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of toxoplasmosis by ingestion or administration of the composition containing oleanolic acid according to the present invention.

As used herein, the term "treatment" refers to any action that improves or benefits the symptoms of the disease by ingestion or administration of the composition containing oleanolic acid according to the present invention.

As used herein, the term "improvement" refers to the ingestion or administration of a composition containing oleanolic acid as an active ingredient to alleviate the onset or symptoms of the disease, or to inhibit the survival of Toxoplasmosis, and to inhibit or reduce growth and proliferation. It means the effect of alleviating the symptoms of toxoplasmosis.

As used in the present invention, the term "anti-toxoplasma gondii effect" or "anti-toxoplasma gondii activity" refers to the effect of resisting toxoplasmosis, more specifically, the survival inhibitory action of toxoplasmosis by drugs, physiologically active substances, or known chemicals. As that, for the purpose of the present invention, it means the inhibition of survival or proliferation inhibition and growth inhibition of Toxoplasmosis.

In the present invention, the therapeutic, ameliorating or preventive effect of toxoplasmosis of the pharmaceutical composition may be achieved by having an anti-toxoplasmosis effect or activity indicating the survival inhibition or mitosis inhibition and growth inhibition of Toxoplasmosis. In an embodiment of the present invention, treatment and improvement of toxoplasmosis by confirming that oleanolic acid has an anti-toxoplasmosis effect or activity indicating the survival or mitosis inhibition and growth inhibition of Toxoplasmosis through the MTT measurement method Or it was confirmed that a preventive effect can be achieved ( FIGS. 1 to 3 ).

The oleanolic acid of the present invention is not limited thereto, but may preferably be a synthetic material obtained by chemical synthesis or a material derived from plants or natural products. Oleanolic acid can be obtained using general synthetic methods known in the art, extraction methods derived from animals, plants, or natural products, separation methods, or purification methods. In addition, the extraction method or separation method is not limited thereto, but preferably organic solvent extraction, boiling water extraction, hot water extraction, cold extraction, steam reflux extraction, reflux cooling extraction, ultrasonic extraction, vacuum concentration or freeze drying, ultracentrifugation Separation and the like can be used.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, but is not limited thereto, but is preferably as follows.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, poly vinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, mineral oil, and the like. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations include one or more compounds and at least one excipient, for example, starch, calcium carbonate, sucrose or lactose. (lactose), gelatin, etc. can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, amino acids, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used, and the form that can be manufactured is not limited by the above examples.

In addition, the pharmaceutical composition of the present invention may include tablets, pills, powders, granules, capsules, sustained release formulations, sublingual tablets, suspensions, internal solutions, emulsions, injections, spirits, transdermal absorption agents, mucosal absorption agents, oral disintegrants, Patches, films, coatings, patches, inhalants, syrups, emulsions, gels, ointments, eye drops, sterile aqueous solutions, non-aqueous solutions, freeze-dried preparations, suppositories or aerosols may have any one formulation selected from the group consisting of And, the form of the formulation that can be prepared by the above example is not limited.

The pharmaceutical composition of the present invention has no particular restrictions on its dosage, and may vary depending on absorption, body weight, patient's age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. have. The pharmaceutical composition of the present invention is to be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way can be prepared using a specialized dosing method according to the judgment of an expert and individual needs, or several times at intervals of a certain time, if necessary. can be administered or ingested.

When using the pharmaceutical composition of the present invention, the composition may be used as it is or may be used in combination with other pharmaceutical ingredients or other therapeutic agents, and may be used singly or in multiples. The content of the active ingredient may be appropriately used according to a conventional method in consideration of the effective amount range, and may be appropriately determined according to the purpose of use (prevention, improvement or therapeutic treatment), but is not limited thereto.

In another aspect, the present invention provides a health food composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient. The health food composition may be preferably achieved by having oleanolic acid has an anti-toxoplasmosis effect or activity indicating survival or mitosis inhibition and growth inhibition on Toxoplasmosis, as described above. same.

When the composition of the present invention is used as an additive to food, the composition may be added as it is or used together with other health foods or components thereof, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be suitably determined according to the purpose of use, and the food composition of the present invention has no great limitation on the mixing amount.

The type of food of the present invention is not particularly limited, and examples of health food to which the composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. There are dairy products, various soups, beverages, teas, juices, drinks, alcoholic beverages, and vitamin complexes, and may include both health food and functional health food in a conventional sense. It may also include food used as feed for animals, and the form of food that can be manufactured is not limited by the above example.

In addition, when the health food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, natural carbohydrates, etc. as an additional component like a conventional beverage. In addition to the above, the health food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit drinks, fruit juice drinks and vegetable drinks. The composition of the present invention may be prepared in the form of tea, juice, and drink for drinking, or may be ingested by granulation, tableting, encapsulation and powdering, but the form that can be prepared by the above example is not limited.

As another aspect, the present invention provides a feed composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient. The feed composition may be preferably achieved by oleanolic acid having an anti-Toxoplasmosis effect or activity indicating survival or mitosis inhibition and growth inhibition on Toxoplasmosis, as described above. .

When the composition of the present invention is used as a feed additive, the composition may be added as it is or mixed with other feed ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be suitably determined according to the purpose of use, and the feed composition of the present invention has no major limitation on the mixing amount.

There is no particular limitation on the type of feed of the present invention, and the composition includes various auxiliary substances such as amino acids, inorganic salts, vitamins, antioxidants, microbial agents, etc. as auxiliary components, and vegetable protein feed such as milled or crushed wheat, barley, and corn, blood meal, In addition to the main ingredients such as animal protein feed such as meat meal and fish meal, animal fat and vegetable fat, it can be used together with nutritional supplements, growth promoters, digestion and absorption promoters, and disease prevention agents.

The dosage form, method, and formulation of the feed may be varied, and the form that can be manufactured is not limited by the above example.

The feed composition of the present invention can be applied to a number of animal diets including mammals, poultry, fish and crustaceans, that is, feed. It can be used for mammals including cattle, pigs, horses, deer and goats, which are important as commercial livestock, zoo animals such as elephants, camels, giraffes and monkeys, and pets such as dogs and cats, chickens, ducks, geese , can be used for poultry such as pigeons and commercially raised fish and crustaceans such as crucian carp, carp, trout, mullet, and shrimp, but is not limited thereto.

Further, as another aspect, the present invention relates to an veterinary pharmaceutical composition for the treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient. That is, the composition of the present invention can be used as an veterinary pharmaceutical composition for the purpose of treating, improving or preventing toxoplasmosis.

The composition may be preferably achieved by oleanolic acid having an anti-Toxoplasmosis effect or activity, which exhibits survival or mitosis inhibition and growth inhibition on Toxoplasmosis, as described above.

When the composition of the present invention is used as a veterinary pharmaceutical composition, the composition may be used as it is or may be used together with other pharmaceuticals or quasi-drug ingredients, and may be appropriately used according to a conventional method, but is not limited thereto. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, improvement or therapeutic treatment).

In another aspect, the present invention relates to a quasi-drug composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient. That is, the composition of the present invention may be added to a quasi-drug composition for the prevention or improvement of toxoplasmosis.

The composition may be preferably achieved by oleanolic acid having an anti-Toxoplasmosis effect or activity, which exhibits survival or mitosis inhibition and growth inhibition on Toxoplasmosis, as described above.

When the composition of the present invention is used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method, but is not limited thereto. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or improvement). There is no limitation on the type of the quasi-drug composition of the present invention, but preferably disinfectant cleaner, shower foam, gargrin, wet tissue, detergent soap, spray, hand wash, ointment, liniment, cream, cataplasma, humidifier filler, cosmetics, shampoo, It can be used to manufacture quasi-drugs such as coatings, detergents, feminine hygiene products, sanitary pads, sanitary napkins, condoms, bands, filter fillers, sanitary containers, sanitary bags or masks, and the form that can be manufactured is not limited by the above examples.

The pharmaceutical composition, health food composition, feed composition, veterinary medicine composition or quasi-drug composition according to the present invention has an anti-toxoplasma gondii effect or activity indicating survival or mitosis inhibition and growth inhibitory action against Toxoplasmosis. It can be usefully used for the treatment, improvement or prevention of symptoms, and can be used for various purposes.

1 shows the results of measuring the antitoxoplasmosis activity of each concentration of the test substance against Toxoplasmosis by applying oleanolic acid to the MTT measurement method. In addition, "+" indicates the antitoxoplasmosis activity of the test substance against Toxoplasmosis.
2 shows the results of measuring the antitoxoplasmosis effect or activity of each concentration of the test substance on Toxoplasmosis by the MTT measurement method after administering the test substance to each concentration of Toxoplasmosis.
Figure 3 shows the results of measuring the anti-toxoplasmosis effect of each concentration of the test substance on Toxoplasmosis-infected cells by applying the MTT measurement method after administering each concentration of the test substance to Toxoplasmosis-infected cells infected with Toxoplasmosis. .

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

: ingredient

RPMI 1640 media and FBS (Fetal Bovine Serum, bovine serum) were purchased from Gibco (USA), and Oleanolic acid, Sulfadiazine, MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H- Tetrazolium bromide; Thiazolyl Blue Tetrazolium Blue; Methylthiazolyldiphenyl-tetrazolium bromide) and DMSO (Dimethyl Sulfoxide) were purchased from Sigma-Aldrich Co. LLC, St. Louis, MO, USA. Ethanol and 96 well plates were purchased from Dongin Biotech Co., Ltd. Other chemical agents, reagents, and consumables were purchased from Dongin Biotech Co., Ltd., Darwin Bio Co., Ltd., BD KOREA, Merck Chemicals Co., Ltd. (Germany), and Sigma-Aldrich Co., Ltd. (USA).

: Toxoplasmosis and cell culture

Toxoplasma gondii , Toxoplasma gondii RH strain) was purchased from ATCC (USA), and Hela cells were purchased from Seoul National University's Korea Cell Line Bank and used after culture. was used. Hela cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 unit/mL penicillin and 100 μM streptomycin at 37° C., 5% CO _{2 in air condition.} Hela cells were infected with Toxoplasmosis in a ratio of 1:5 (cell: Toxoplasmosis = 1:5) and used for the test.

: MTT assay method

(1) Measurement of the antitoxoplasmosis effect on Toxoplasmosis

The direct measurement of the antitoxoplasmosis effect or activity of the test substance against Toxoplasmosis is as follows. First, Toxoplasma gondii was grown in the abdominal cavity of BALB/c mice, then Toxoplasmosis was collected, inoculated with Toxoplasma gondii (1 × 10 ⁷ /well) in a 24 well plate, and cultured. mL) and incubated for 24 hours at 37° C., 5% CO ₂ condition, and the experimental group administered with Sulfadiazine as a drug control group was also cultured to compare the effects of the test substance.

24 hours after administration of the test substance, add "(3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide or Thiazolyl Blue Tetrazolium Blue)" solution and shield the 24 well palte with foil After incubation at 37°C, 5% CO _{2 in} air conditions for 3 hours. After incubation, DMSO was added, followed by mixing (shaking) at room temperature for 20 minutes, transferring the solution to a 96 well plate, and measuring at 570 nm with an ELISA Reader (Microplate Reader) to determine the antitoxoplasmosis effect or activity of the test substance against Toxoplasmosis. It was confirmed (FIGS. 1 to 2).

(2) Measurement of effects on Toxoplasmosis-infected cells

: Hela cells (1 × 10 ⁵ ) were cultured in a 24 well plate at 37° C. and 5% CO _{2 in} air. After 24 hours, the cells were infected with Toxoplasmosis and oleanolic acid was administered by concentration. After 24 hours, add the "(3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide or Thiazolyl Blue Tetrazolium Blue)" solution and shade the 24 well palte with foil. ℃, and incubated for 3 hours under the condition of 5% CO _{2 in air.} After incubation, DMSO was added to dissolve Formazan formed, followed by mixing at room temperature for 20 minutes (shaking), transferring the solution to a 96 well plate, and measuring at 570 nm with an ELISA Reader (Microplate Reader) measuring instrument. The anti-toxoplasma gondii effect was confirmed (FIG. 3). The proliferation rate (PR) of Toxoplasmosis-infected cells was calculated as (OD test group/OD control group) × 100%.

As a result, it was found that the test substance, oleanolic acid, exhibits an anti-toxoplasmosis effect or activity, which exhibits a concentration-dependent effect on survival or proliferation inhibition and growth inhibition on Toxoplasmosis through the antitoxoplasmosis effect by the MTT measurement method. was confirmed (FIGS. 1 to 3). In addition, it was confirmed that oleanolic acid not only directly induces the survival inhibitory action of Toxoplasmosis, but also effectively inhibits the survival and apoptosis of Toxoplasmosis-infected cells, thereby exhibiting the anti-toxoplasma gondii effect.

These results mean that oleanolic acid can be used and usefully used as an anti-toxoplasmosis composition for the treatment, improvement or prevention of toxoplasmosis because oleanolic acid has an antitoxoplasmosis effect or activity against toxoplasmosis.

As described above, the measurement results of the test material for the present invention are shown in FIGS. 1 to 3 .

The pharmaceutical composition, health food composition, feed composition, veterinary drug composition, and quasi-drug composition according to the present invention have an anti-toxoplasma gondii effect or activity indicating survival or mitosis inhibition and growth inhibition on Toxoplasmosis. It can be usefully used for the treatment, improvement or prevention of symptoms, and can be used for various purposes. The composition according to the present invention can be usefully used in industrial fields of pharmaceutical industry, food industry, agriculture and fishery industry and manufacturing industry.

In FIG. 1, "+" indicates the anti-toxoplasmosis activity or effect of the test substance against Toxoplasmosis.

Claims (6)

A pharmaceutical composition for the treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient.
According to claim 1, wherein the composition is Toxoplasma gondii ( Toxoplasma gondii ) The composition characterized in that it has an anti-Toxoplasma gondii effect or activity indicating a survival inhibitory action or a proliferation inhibition and growth inhibition action.
A health food composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient.
A feed composition for preventing or improving toxoplasmosis comprising oleanolic acid as an active ingredient.
An animal pharmaceutical composition for the treatment, improvement or prevention of toxoplasmosis comprising oleanolic acid as an active ingredient.
A quasi-drug composition for the prevention or improvement of toxoplasmosis comprising oleanolic acid as an active ingredient.

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