JP6967247B1 - Antimalarial - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel antimalarial agent which inhibits the growth of Plasmodium malaria. SOLUTION: This is an antimalarial agent that inhibits the growth of Plasmodium malaria, and contains Chaga and Panax notoginseng as active ingredients. [Selection diagram] Fig. 1

Description

The present invention relates to an antimalarial agent.

Malaria is a globally important infectious disease caused by Plasmodium malaria infection, which causes more than 200 million infected people and more than 430,000 deaths annually, mainly in subtropical and tropical regions (WHO, 2018). ). Plasmodium falciparum (Plasmodium falciparum), Plasmodium malariae (Plasmodium vivax), Plasmodium vivax (Plasmodium vivax), Plasmodium ovale (Plasmodium oval), and Plasmodium knowlesi Five types of (Plasmodium plsmodium) are known. Of these, Plasmodium falciparum (Plasmodium falciparum) is the most pathogenic and causes severe anemia and severe symptoms such as cerebral malaria, resulting in enormous damage.

Chloroquine and sulfadoxin / pyrimethamine have been used as therapeutic agents for malaria, but their use has been restricted or discontinued due to the emergence of drug-resistant protozoans resistant to these agents since the 1950s. .. Therefore, as an alternative treatment method, artemisinin combination therapy in which the antimalarial agent artemisinin is used in combination with other antimalarial agents has been proposed. Artemisinin combination therapy is being promoted specifically as a treatment for Plasmodium falciparum (Plasmodium falciparum). Artemisinin is a compound isolated from sweet wormwood (Artemisia annua), a plant of the genus Artemisia, which has been used as a Chinese herbal medicine for a long time.

Patent Document 1 describes that at least one compound among artemisinin, dihydroartemisinin, artemether, artemether, and artesunate is administered in combination with quinidine or mefloquine. Patent Document 1 describes that the combined use of these antimalarial agents has obtained an antimalarial effect on mice infected with a chloroquine-resistant strain.

European Patent No. 362810

However, in recent years, the appearance of artemisinin-resistant Plasmodium falciparum has been reported in Cambodia, Laos, Myanmar, etc., and there is a strong concern that the therapeutic effect of artemisinin combination therapy will decrease.

The present invention was made based on the new finding that a specific crude drug inhibits the growth of Plasmodium malaria as a result of diligent research by the present researchers. An object of the present invention is to provide a novel antimalarial agent that inhibits the growth of Plasmodium malaria.

The antimalarial agent that solves the above-mentioned problems is an antimalarial agent that inhibits the growth of Plasmodium malaria, and contains Chaga and Panax notoginseng as active ingredients.
In the above configuration, the fraction obtained when the water extract of Chaga and the water extract of rice field seven carrots are subjected to fractionation treatment by high-speed liquid chromatography using an ODS column is contained as an active ingredient. The minutes are the first fraction that is not eluted with 70% methanol and is eluted with 100% methanol, and the first fraction that is not eluted with 100% methanol and is eluted with an elution solvent consisting of chloroform / methanol / water = 6: 4: 1. At least one of the third fractions, which is not eluted with the elution solvent consisting of 2 fractions and chloroform / methanol / water = 6: 4: 1, but is eluted with the elution solvent consisting of a methanol solution of 0.05% trifluoroacetic acid. It is preferably either.

In the above configuration, the Plasmodium falciparum is preferably Plasmodium falciparum.

According to the present invention, a novel antimalarial agent that inhibits the growth of Plasmodium malaria can be obtained.

The graph which shows the malaria protozoan growth inhibition test. (A) and (b) are graphs showing the malaria protozoan growth inhibition rate (%) using the E fraction to the G fraction fractionated by high-speed liquid chromatography using an ODS column as test solutions.

Hereinafter, an embodiment of an antimalarial agent that embodies the present invention will be described.
The antimalarial agent of the present embodiment contains Chaga and Panax notoginseng as active ingredients.
Malaria is affected by the invasion of Plasmodium malaria into the human body by the bite of a female Anopheles mosquito. When a human is sucked into the Anopheles mosquito carrying the Plasmodium malaria, the infectious sporozoite present in the salivary glands of the Anopheles mosquito invades the human blood and rapidly divides in the hepatocytes to produce a large number of merozoites. Merozoite is released into the blood and invades the red blood cells, and divides in the red blood cells while changing their morphology into ring foam (ring), trophozoite (mature nutrient), and schizont (division). When matured at this stage, it destroys red blood cells and releases a large number of merozoites into the blood from inside the mature schizont, invading new red blood cells. By repeating this intraerythrocyte cycle, erythrocyte destruction and merozoite release are stimulated, and symptoms of malaria such as headache, chills / horror, nausea / vomiting appear. As the intraerythrocyte cycle is repeated and the symptoms progress, serious symptoms such as impaired consciousness, coma, convulsions, jaundice, and pulmonary edema appear.

Since the antimalarial agent of the present embodiment inhibits the growth of Plasmodium malaria by the active ingredients Chaga and Panax notoginseng, it exerts an effect of improving the above-mentioned symptoms of malaria.
Chaga is a fungus of the subphylum Basidiomycete belonging to the order Polyporales, Hymenochaetaceae, and Inonotus, and is also known as Chaga mushroom (scientific name: Fuscoporia obliqua). It grows on the trunks of birch trees such as birch and birch, and forms massive and large sclerotia. Chaga may be used in a state where the sclerotium in the form of a mass is washed, dried and crushed, but it is preferable to use it as a powder of Chaga extract obtained by concentrating and drying a water extract of Chaga. The chaga extract powder is, for example, a granular product produced by washing, drying, crushing, fermenting, and water-extracting a massive sclerotium, and then concentrating and drying the water extract. Concentration and drying can be carried out by conventional means such as vacuum evaporation concentration method, spray drying method, freeze-drying method and the like. In addition, a commercially available product can also be used as the chaga extract powder.

Notoginseng is a perennial plant belonging to the genus Panax notoginseng in the family Araliaceae, and is also known as Panax notoginseng Burkill. Notoginseng may be used in a state where underground parts (roots, rhizomes, stems, branch roots, whiskers, lump roots, propagules) collected from nature are washed and dried, and then crushed or cut. It is preferable to use the concentrated and dried water extract of Panax notoginseng as the powder of Panax notoginseng extract. The Panax notoginseng extract powder is, for example, a granular product produced by washing, hot water extraction, and filtering the underground part of Panax notoginseng, then extracting the residue with water, and concentrating and drying the filtrate. .. Concentration and drying in this case can also be performed by conventional means such as a vacuum evaporation concentration method, a spray drying method, and a freeze-drying method. In addition, a commercially available product can also be used as the powder of Panax notoginseng extract.

The mixing ratio of Chaga and Panax notoginseng is preferably 10 to 15 in terms of weight ratio of Chaga extract powder to 1 in Panax notoginseng extract powder, and 37: 3 in weight ratio between Chaga extract powder and Panax notoginseng extract powder. Is more preferable.

Chaga and Panax notoginseng can also be used as purified products that have been fractionated and purified by extracting Chaga extract powder and Panax notoginseng extract powder using one or more known liquid chromatography. As the liquid chromatography, for example, column chromatography can be used, and more specific examples thereof include high performance liquid chromatography (HPLC) and open column chromatography. Examples of the chromatography carrier include ion exchange chromatography, partition chromatography (forward / reverse phase chromatography), adsorption chromatography, molecular exclusion chromatography and the like. As partition chromatography, normal phase chromatography using an adsorbent such as silica gel or alumina as a filler, or an adsorbent on which the surface of silica gel is chemically bonded with an octadecylsilyl group (ODS), an octyl group, a phenyl group, a cyano group, etc. Examples include reverse phase chromatography used as a filler. As the elution solvent, a plurality of types of solvents having the same composition but different mixing ratios may be used in fixed amounts, or solvents having different mixing ratios over time may be continuously used. Further, additives such as organic salts, inorganic salts, buffers, emulsifiers and the like may be dissolved in the elution solvent. Fractionation conditions such as column temperature, elution solvent flow rate, and solvent composition are not particularly limited, and may be appropriately set according to the column to be used and the elution solvent.

As a specific example of liquid chromatography for obtaining a purified product from Chaga extract powder and Panax notoginseng extract powder, it can be carried out by the following method. Using a sample of Chaga extract powder and Panax notoginseng extract powder dissolved in water, it is applied to an ODS column having an inner diameter of 5 mm and a length of 50 mm and adsorbed. When applying the chaga extract powder and the Panax notoginseng extract powder to the ODS column, a certain amount of water is added to the ODS column. Then, a fixed amount of a mixed solvent of methanol and water, a mixed solvent of chloroform, methanol and water, or a mixed solvent of methanol added with trifluoroacetic acid is added to the ODS column to elute. As the mixed solvent of methanol and water, a plurality of mixed solvents in which the mixing ratio of methanol is gradually increased are prepared. Specifically, two kinds of mixed solvents, 70% methanol and 100% methanol, are prepared. As the mixed solvent of chloroform, methanol and water, a mixed solvent of chloroform: methanol: water = 6: 4: 1 is prepared. The mixed solvent in which trifluoroacetic acid is added to methanol is prepared so that the concentration of trifluoroacetic acid is 0.05%. Purified products with anti-malaria activity have a small amount of elution with 70% methanol, and most of them are 100% methanol, a mixed solvent with a mixing ratio of chloroform: methanol: water = 6: 4: 1, and 0.05% with methanol. It is eluted with a mixed solvent containing trifluoroacetic acid.

The purified product eluted by extraction using liquid chromatography may be used as it is, or may be used as a granular product produced by filtration, extraction, concentration, drying, etc. preferable.

When the antimalarial agent of the present embodiment is applied to a drug or a quasi-drug, it is administered by ingestion (oral ingestion), or any administration such as intravascular administration, transdermal administration, and mucosal administration. It is possible to adopt a method, a method of applying to the skin, an article, or the like. The dosage form is not particularly limited, and examples thereof include powders, powders, granules, tablets, capsules, pills, liquids, emulsions, syrups, injections, infusions, coatings, suppositories and the like. ..

In addition to the active ingredients Chaga and Panax notoginseng, the antimalarial agent may contain a pharmaceutically acceptable carrier or additive as long as it does not impair the growth inhibitory effect on Plasmodium malaria. Carriers or additives include excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH regulators, emulsifiers, buffers, stabilizers, solubilizers, fragrances, sweeteners, etc. Examples include surfactants, painkillers, stabilizers and the like.

Next, the action of the antimalarial agent will be described.
Plasmodium that has invaded the human body proliferates in the human body by repeating the intraerythrocyte cycle. Chaga and Panax notoginseng contained in antimalarial agents have an action of inhibiting the growth of such malaria protozoa in erythrocytes, thereby improving the symptoms peculiar to malaria. Chaga and Panax notoginseng particularly effectively inhibit the growth of Plasmodium falciparum.

Next, the effect of the antimalarial agent of this embodiment will be described.
(1) The antimalarial agent contains Chaga and Panax notoginseng as active ingredients.
According to this configuration, it is possible to inhibit the growth of Plasmodium malaria and improve the symptoms peculiar to malaria.

(2) The antimalaria agent contains a fraction obtained when the water extract of Chaga and the water extract of Tanana ginseng are subjected to fractionation treatment by high-speed liquid chromatography using an ODS column as an active ingredient. The fraction is a first fraction that is not eluted with 70% methanol and is eluted with 100% methanol, and an elution solvent consisting of chloroform / methanol / water = 6: 4: 1 that is not eluted with 100% methanol. The second fraction that elutes and the third fraction that does not elute with an elution solvent consisting of chloroform / methanol / water = 6: 4: 1 but elutes with an elution solvent consisting of a methanol solution of 0.05% trifluoroacetic acid. At least one of them.

In this case, the growth of Plasmodium malaria is remarkably inhibited, and the effect of improving the symptoms peculiar to malaria is further remarkably obtained.
(3) Antimalarial agents inhibit the growth of Plasmodium falciparum.

According to this configuration, the serious symptoms peculiar to malaria can be ameliorated.
In addition, this embodiment can be changed and carried out as follows. The present embodiment and the following modified examples can be implemented in combination with each other within a technically consistent range.

-The antimalarial agent can be applied not only to humans but also to drugs, feeds and the like for domestic animals such as livestock and pets.
-The amount and duration of intake of the antimalarial drug are not particularly limited, and are appropriately determined in consideration of the physical function status, age, gender, and other conditions to be applied.

Next, the technical idea that can be grasped from the above embodiment and the modified example will be described.
(B) An antimalarial agent that inhibits the growth of Plasmodium malaria, which contains a water extract of Chaga and a water extract of seven carrots as active ingredients, and the water extract of Chaga and the water of seven carrots. An antimalarial agent characterized in that the blending ratio of the extract is 10 to 15: 1 by weight.

(B) The fraction obtained when the water extract of Chaga and the water extract of rice field ginseng are subjected to fractionation treatment by high-speed liquid chromatography using an ODS column having an inner diameter of 5 mm and a length of 50 mm is used as an active ingredient. The fraction contained above is an elution consisting of a first fraction which is not eluted with 70% methanol and is eluted with 100% methanol and a chloroform / methanol / water = 6: 4: 1 which is not eluted with 100% methanol. The second fraction that elutes with a solvent and the third fraction that does not elute with an elution solvent consisting of chloroform / methanol / water = 6: 4: 1 but elutes with an elution solvent consisting of a methanol solution of 0.05% trifluoroacetic acid. An antimalaria agent characterized by being at least one of the minutes.

(C) A purification step of fractionating and purifying the water extract of Chaga and the water extract of rice field seven carrots by high-speed liquid chromatography using an ODS column having an inner diameter of 5 mm and a length of 50 mm is provided. A method for producing an antimalaria agent, which comprises removing a fraction eluted with 70% methanol.

In the following, the antimalarial effects of Chaga and Panax notoginseng will be specifically described.
<Preparation of Plasmodium malaria culture solution>
As Plasmodium falciparum, a 3D7 strain of Plasmodium falciparum (Plasmodium falciparum) was cultured in a complete medium composed of RPMI1640 medium. As a complete medium, RPMI1640 medium containing 50 mg / L hypoxanthine, 0.05% AlbumaxII, 0.035% NaHCO ₃ , 0.001% gentamicin (containing L-glutamine, Hepes, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) Was adjusted to pH 7.4 to 8.0 with 1N HCl. Erythrocytes were added to the complete medium so that the hematocrit value was 5%, and the cells were cultured in a CO _{2 incubator at 37 ° C. and a 5% CO 2 atmosphere.}

The rate of erythrocytes infected with malaria protozoa (malaria infection rate) was measured by preparing a Giemsa-stained specimen of blood thin layer droplets and measuring the count ratio of infected erythrocytes and uninfected erythrocytes measured under a microscope. The malaria infection rate of the Plasmodium malaria culture solution was 10% or less.

<Preparation of Chaga and Notoginseng>
The Chaga and Notoginseng tested use a formulation developed as a pet supplement (trade name "Ria Nishihaku" (registered trademark), manufactured by Iskra Sangyo Co., Ltd., hereinafter referred to as "Ria Nishihaku"). bottom. "Ria Nishihaku" contains a water extract of Chaga (Chaga extract powder) and a water extract of Panax notoginseng (Panax notoginseng extract powder). Notoginseng extract powder = 37: 3. "Ria Nishihaku" was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg / mL and subjected to the following Plasmodium malaria growth inhibition test. The solution prepared here is referred to as a test stock solution.

<Plasmodium growth inhibition test>
The test stock solution was diluted with the above-mentioned complete medium to a concentration of 500 μg / mL, and then the test solution was diluted 2-fold in 10 steps and dispensed into each well of a 96-well cell culture plate. The final concentration of the test solution after 2-fold dilution is 0.977 μg / mL.

A malaria protozoan culture solution (malaria infection rate 1%, hematocrit value 2.5%) was added to each well, and the cells were cultured in a CO _{2 incubator at 37 ° C. and a 5% CO 2} atmosphere for 72 hours. After 72 hours, the 96-well cell culture plate was left at −30 ° C. overnight, and the malaria protozoan culture solution to which each test solution was added was frozen. Then, the 96-well cell culture plate was returned to room temperature and thawed, and a lysis buffer was added to each well and mixed. The composition of the lysis buffer is 20 mM Tris (pH 7.5), 5 mM EDTA, 0.008% saponin, 0.08% Triton X-100, 0.2 μL / mL SYBR Green I.

96-well cell culture plates were incubated for 1 hour with stirring on a shaker in the dark at room temperature. Then, using a fluorescence multi-plate reader (CytoFluor® Series 4000, manufactured by PerSeptive Biosystems), the fluorescence intensity at an excitation wavelength of 485 nm and a fluorescence wavelength of 530 nm was measured.

Pyrimethamine was used as a positive control. Pyrimethamine was dissolved in DMSO to 25 mM and then dissolved in the above-mentioned complete medium to 2.5 μM as the test solution. For the negative control, the above-mentioned complete medium was used as the test solution.

The Plasmodium malaria growth inhibition test was conducted in triplets for each test solution of "Ria Nishihaku", positive control, and negative control. FIG. 1 is a graph showing the average value of the fluorescence intensity in each well to which the test solution of "Ria Nishihaku" and the negative control group was dispensed. It was found that the test solution of "Ria Nishihaku" had a remarkable growth inhibitory effect on antimalaria protozoa.

<Calculation of _{IC 50>
An IC 50} was calculated based on the results of the Plasmodium malaria growth inhibition test. The IC ₅₀ was calculated by an IC ₅₀ computer (manufactured by AAT Bioquest), and the average value of the triplet in the malaria protozoa growth inhibition test was calculated. As a result, _{IC 50} of "Saihaku TogiA" was 62μg / mL. The results are shown in Table 1.

Although not shown here, test solutions were similarly prepared for water extracts of a plurality of crude drugs other than "Ria Nishihaku" and a malaria protozoan growth inhibition test was conducted. As a result, aqueous extract of any herbal, _{IC 50} was generally about 150 [mu] g / mL. _{It is considered that the IC 50} obtained from the water extracts of a plurality of other crude drugs is also affected by DMSO, which is the solvent of the test stock solution. IC ₅₀ of "Saihaku TogiA" is significantly lower than the test fluid from a plurality of other crude drugs.

<Observation of Plasmodium-infected erythrocytes under a microscope>
At 24, 48, and 72 hours after the start of the malaria protozoan growth inhibition test, "Ria Nishihaku", positive control, and negative control cultures were collected and the malaria infection rate was measured.

Plasmodium malaria gradually deformed and atrophied in each culture medium supplemented with the test solution of "Ria Nishihaku" and the test solution of positive control pyrimethamine, and the malaria infection rate after 72 hours was less than 0.2%. It became almost unobservable. On the other hand, in the negative control, Plasmodium protozoa proliferated, and the malaria infection rate after 72 hours was 2.4%.

<Separation of "Ria Nishihaku">
"Ria Nishihaku" was fractionated by reverse phase high performance liquid chromatography (HPLC). For fractionation, a flash column having an inner diameter of 5 mm and a length of 50 mm filled with octadecylsilyl group-bonded silica gel (ODS) was used. "Ria Nishihaku" dissolved in water was adsorbed on the column and eluted sequentially with an elution solvent to obtain fractions A to G.

As the elution solvent, a mixed solvent of methanol and water was used for the A fraction to the E fraction. As the mixed solvent of methanol and water, 5 types (0%, 20%, 50%, 70%, 100%) in which the content of methanol in the mixed solvent was gradually increased were used. The one eluted with water (0% methanol) is the A fraction, the one eluted with 20% methanol is the B fraction, the one eluted with 50% methanol is the C fraction, and the one eluted with 70% methanol. Was taken as the D fraction, and the one eluted with 100% methanol was taken as the E fraction. The F fraction was eluted using a mixed solvent of trichloromethane, methanol and water (trichloromethane: methanol: water = 6: 4: 1). The G fraction was eluted with a mixed solvent of trifluoroacetic acid (TFA) and MeOH (0.05% TFA in MeOH).

After freeze-drying, each eluate from the A fraction to the G fraction was dissolved in DMSO to 25 mg / mL and used in a malaria protozoan growth inhibition test. The solution prepared here is referred to as a fractionation test stock solution.

<Malaria protozoan growth inhibition test of fraction test solution>
The fraction test stock solution of fractions A to C was diluted with the above-mentioned complete medium so as to be 1250 μg / mL, and then diluted 2-fold in 8 steps as the fraction test solution of a 96-well cell culture plate. Dispensed into each well. The final concentration of the fractionated test solution after 2-fold dilution is 9.8 μg / mL. Similarly, the fraction test stock solution of the D fraction was diluted with the above-mentioned complete medium so as to be 625 μg / mL, and then diluted 2-fold in 10 steps to obtain the fraction test solution. The final concentration of the fractionated test solution after 2-fold dilution is 1.2 μg / mL. Further, the fraction test stock solution of the E fraction to the G fraction was diluted with the above-mentioned complete medium so as to be 250 μg / mL, and then diluted 2-fold in 10 steps to obtain a fraction test solution. The final concentration of the fractionated test solution after 2-fold dilution is 0.49 μg / mL.

The malaria protozoan growth inhibition test of each fraction test solution was carried out in the same manner as the malaria protozoan growth inhibition test of the test solution of "Ria Nishihaku". _{An IC 50} was calculated based on the results of the Plasmodium malaria growth inhibition test. The results are shown in Table 1.

Ｅ画分〜Ｇ画分でのＩＣ_５０が他の画分に比べて低く、顕著なマラリア原虫成長阻害作用を有していることがわかった。

IC ₅₀ of at E fraction ~G fraction is lower than the other fractions were found to have significant malaria parasite growth inhibitory effect.

<Calculation of malaria protozoan growth inhibition rate (%) of fraction test solution>
Based on the results of the malaria protozoan growth inhibition test, the malaria protozoan growth inhibition rate (%) of the fraction test solution of the E fraction to the G fraction was calculated. Here, when the maximum fluorescence intensity in the well containing the fractionation test solution is A and the fluorescence intensity in the negative control well is B, the malaria protozoan growth inhibition rate (%) is ((AB) /. B) It is represented by × 100. The results are shown in FIG. FIG. 2A shows the malaria protozoan growth inhibition rate (%) when the concentration of the fractionated test solution is 62.5 μg / mL, and FIG. 2B shows the concentration of the fractionated test solution of 15.6 μg / mL. It shows the growth inhibition rate (%) of Plasmodium malaria in mL.

In the fraction test solution having a drug concentration of 62.5 μg / mL, the malaria protozoan growth inhibition rate (%) was 60 to 70% in both the E fraction to the G fraction. In addition, the malaria protozoan growth inhibition rate (%) of the G fraction was about 50% in the fraction test solution having a drug concentration of 15.6 μg / mL, which is particularly remarkable among the E fraction to the G fraction. It was found to have a growth inhibitory effect.

<Toxicity evaluation for mammalian cells>
The test solutions of the E to G fractions were evaluated for cytotoxicity to mammalian cells. Cytotoxicity was determined by measuring the IC ₅₀ for mammalian cells. The cells used were Hela cells, which are human cell lines derived from cervical cancer, and P388 cells, which are mouse cell lines derived from lymphoid tumors. The results are shown in Table 2.

顕著なマラリア原虫成長阻害作用を有するＥ画分〜Ｇ画分については、Ｈｅｌａ細胞、Ｐ３８８細胞のいずれに対しても細胞毒性は低かった。

The cytotoxicity of the E to G fractions, which have a remarkable effect of inhibiting the growth of Plasmodium malaria, was low for both Hela cells and P388 cells.

Claims (2)

It is an antimalarial agent that contains Chaga and Panax notoginseng as active ingredients and inhibits the growth of Plasmodium malaria.
The active ingredient contains a fraction obtained when the water extract of Chaga and the water extract of Panax notoginseng are subjected to fractionation treatment by high-speed liquid chromatography using an ODS column.
The fraction is eluted with a first fraction that is not eluted with 70% methanol and is eluted with 100% methanol and an elution solvent consisting of chloroform / methanol / water = 6: 4: 1 that is not eluted with 100% methanol. Of the third fraction, which is not eluted with the elution solvent consisting of chloroform / methanol / water = 6: 4: 1, but is eluted with the elution solvent consisting of a methanol solution of 0.05% trifluoroacetic acid. An antimalaria agent characterized by being at least one of. The antimalarial agent according to claim 1 , wherein the Plasmodium falciparum is Plasmodium falciparum.

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