Antimalarial agent

Abstract

To provide a new antimalarial agent that achieves a balance between strong antimalarial activity and low cytotoxicity.SOLUTION: An antimalarial agent comprising an ethanol extract and / or a methanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antimalarial drugs. [Background technology]

[0002] Malaria, caused by the malaria parasite Plasmodium, is one of the most common vector-borne infectious diseases. According to a 2018 World Health Organization (WHO) report, malaria caused 405,000 deaths and infected 213 million people worldwide. It has also been reported that 90% of all malaria cases occur in sub-Saharan Africa. Human malaria is typically caused by four species of Plasmodium parasites (P. falciparum, P. vivax, P. malariae, and P. ovale), with the majority of infections caused by P. falciparum. Furthermore, P. falciparum infection is known to cause severe malaria, including severe anemia, cerebral malaria, and acute respiratory failure. Therefore, there has long been a need to combat malaria, and the development of new antimalarial drugs is eagerly awaited. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2022-042163 Summary of the Invention [Problem to be solved by the invention]

[0004] The problem to be solved by the present invention is to provide a novel antimalarial drug that combines high antimalarial activity with low cytotoxicity. [Means for solving the problem]

[0005] Under these circumstances, the present inventors have found, after extensive trial and error, that ethanol and / or methanol extracts of specific plants have both high antimalarial activity and low toxicity to human cells. Therefore, the present invention provides the following: Item 1. An antimalarial drug comprising an ethanol and / or methanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa.

[0006] Item 2. The antimalarial drug according to Item 1, comprising an ethanol and / or methanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, and Boerhavia repens.

[0007] Item 3. The antimalarial drug according to Item 1 or 2, comprising an ethanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa.

[0008] Item 4. The antimalarial drug according to Item 1 or 2, which is an orally administered agent or an externally administered agent. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a novel antimalarial drug that combines high antimalarial activity with low cytotoxicity. [Brief explanation of the drawings]

[0010] [Figure 1]Statistical analysis of parasite reduction and invasion inhibition by ethanol and methanol extracts of Amaranthus spinosus, Ficus hispida, Streblus asper, and Boerhavia repens. (a-b) Error bars represent the mean and standard deviation from three biological replicates. Statistical significance was determined using a student's t-test: * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the negative control. DETAILED DESCRIPTION OF THE INVENTION

[0011] Antimalarial drugs The present invention provides an antimalarial drug containing an ethanol and / or methanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa. The active ingredient of the present invention is an ethanol and / or methanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa. In the present invention, "at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa" may be referred to as plant A. Among the plants A, at least one plant selected from the group consisting of Ficus hispida, Streblus asper, and Boerhavia repens is preferred, and at least one plant selected from the group consisting of Streblus asper and Boerhavia repens is more preferred.

[0012] An extract of plant A can be obtained by soaking plant A or its pulverized product in ethanol and / or methanol. Ethanol and methanol can be used alone or in a mixed solvent of two or more. When using a mixed solvent of ethanol and methanol, the mixing ratio is not particularly limited, but can be, for example, 0.1 to 10 parts by mass, 0.2 to 5 parts by mass, or 0.5 to 2 parts by mass of methanol per 1 part by mass of ethanol. The soaking time for obtaining an extract of plant A is not particularly limited, but can be appropriately set, for example, within the range of 15 minutes to 24 hours, preferably 30 minutes to 1 hour. The soaking temperature is also not particularly limited, but can be appropriately set within the range of 50 to 120°C, preferably 60 to 90°C. In the present invention, the extract of plant A may be used in the form of an extract solution in which the extract is dissolved in the solvent used for the extraction, or an extract purified from the extract solution by a method known per se. Furthermore, in the present invention, the "ethanol and / or methanol extract" of plant A encompasses all of the following: "an ethanol extract of plant A," "a methanol extract of plant A," "an extract of plant A with a mixed solvent of ethanol and methanol," and "a mixture of an ethanol extract of plant A and a methanol extract of plant A." When a mixture of an ethanol extract of plant A and a methanol extract of plant A is used, the mixing ratio is not limited, and can be, for example, 0.1 to 10 parts by mass, 0.2 to 5 parts by mass, 0.5 to 2 parts by mass, or the like, of the methanol extract per 1 part by mass of the ethanol extract.

[0013] When an ethanol extract of plant A is used, at least one selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa is preferred, at least one plant selected from the group consisting of Ficus hispida, Streblus asper, and Boerhavia repens is more preferred, and at least one plant selected from the group consisting of Streblus asper and Boerhavia repens is even more preferred. When a methanol extract of plant A is used, at least one selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, and Amaranthus spinosus is preferred, at least one plant selected from the group consisting of Ficus hispida, Streblus asper, and Boerhavia repens is more preferred, and at least one plant selected from the group consisting of Streblus asper and Boerhavia repens is even more preferred.

[0014] Furthermore, in the present invention, the extract of plant A, which is the active ingredient of the present invention, may be used itself as an antimalarial drug, or may be used as a pharmaceutical composition in combination with various pharmaceutically acceptable carriers (e.g., isotonicity agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc.).

[0015] Examples of isotonicity agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonicity agents can be used alone or in combination of two or more.

[0016] Examples of chelating agents include edetate salts such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, citric acid, etc. These chelating agents can be used alone or in combination of two or more.

[0017] The stabilizer may, for example, be sodium hydrogen sulfite.

[0018] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol. These pH adjusters can be used alone or in combination of two or more.

[0019] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used alone or in combination of two or more.

[0020] Examples of antioxidants include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants can be used alone or in combination of two or more.

[0021] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc. These solubilizing agents can be used alone or in combination of two or more.

[0022] Examples of thickeners include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. These thickeners may be used alone or in combination of two or more.

[0023] Furthermore, the pharmaceutical composition may further contain a compound known to have antimalarial activity in addition to the extract of plant A. Examples of compounds known to have antimalarial activity include artemisinin, quinine, mefloquine, and atovaquone / proguanil combinations. These compounds may be used alone or in combination of two or more. When these compounds are used, the amount used is not particularly limited. For example, 0.001 to 1000 parts by mass, preferably 0.01 to 100 parts by mass, and more preferably 0.1 to 10 parts by mass of the compound having antimalarial activity may be used per part by mass of the extract of plant A. When multiple compounds having antimalarial activity other than the extract of plant A are used, the respective compounds may be used in the above-mentioned proportions, for example.

[0024] In an embodiment of the pharmaceutical composition, the content of the extract of plant A in the composition is not particularly limited and can be appropriately set from conditions such as, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, or 1% by mass or more.

[0025] The dosage form is not particularly limited, and examples thereof include various dosage forms such as oral administration agents such as tablets, pills, capsules, powders, granules, syrups, etc.; injections (intravenous injection, intramuscular injection, local injection, etc.), mouthwashes, drip infusions, topical preparations (ointments, creams, patches, inhalants), and parenteral administration agents such as suppositories. Among the above dosage forms, preferred examples include oral administration agents (tablets, pills, capsules, powders, granules, syrups, etc.), topical preparations (inhalants, ointments, creams, patches, etc.), etc.

[0026] In the present invention, the dosage of the extract of plant A varies depending on the route of administration, the patient's age, weight, symptoms, etc., and cannot be generally defined, but it is sufficient to set the daily dose for an adult to about 5000 mg or less, preferably about 1000 mg or less. There is no particular restriction on the lower limit of the dosage of the extract of plant A, and for example, the daily dose for an adult can be set appropriately within the range of usually 0.1 mg or more, preferably 0.5 mg or more. When administered once daily, this amount should be contained in one formulation, and when administered three times daily, one third of this amount should be contained in one formulation.

[0027] The antimalarial drug of the present invention is administered to patients such as mammals, including humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, and sheep.

[0028] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0029] Materials and Methods 1. Plant collection and identification The plants (Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, Aegle marmelos, Tamarindus indica, Mussaenda corymbosa) were collected from Rajshahi, Bangladesh. Plant identification was confirmed by plant biologists from the Department of Botany, Rajshahi University, Bangladesh. 2. Preparation of Plant Material After collecting the plant samples, the leaf parts were separated. Fresh plant parts were washed thoroughly with tap water 3-4 times and finally with distilled water. They were then dried in the shade at room temperature for 20-25 days. The dried plant parts were then ground into a coarse powder using a grinder, stored in a sealed container at room temperature, and used for subsequent analysis. A portion of the powder was specifically used for crude extraction.

[0030] 3. Plant Extract Preparation Approximately 20 g of dry powder from each sample was mixed with pure ethanol or methanol as the solvent at a weight / volume (w / v) ratio of 1:10. The mixture was filtered through cotton cloth and then Whatman No. 1 filter paper. The extraction process involved shaking in a shaker for 48 hours. The resulting extract was concentrated to dryness in a water bath set at 35°C and stored in sterile bottles at 4-8°C for subsequent analysis. The extraction percentage was calculated using the following formula: Extraction % = weight of extract (g) / weight of plant body (g) x 100 4. Parasite Cultivation and Maintenance The 3D7 strain of parasites was cultured for this study in blood stages using a modified method previously established (Trager W, Jensen JB. Human malaria parasites in continuous culture. Science (1979). 1976;193(4254):673-5. , Mishra K, Dash AP, Swain BK, Dey N. Anti-malarial activities of Andrographis paniculata and Hedyotis corymbosa extracts and their combination with curcumin. Malar J. 2009;8(1).). Briefly, parasites were grown in human red blood cells (RBCs) and maintained in a culture medium consisting of RPMI 1640 (Sigma-Aldrich), 25 mM HEPES, 100 μM hypoxanthine (Wako), 12.5 μg / ml gentamicin (Sigma-Aldrich), 0.5% (w / v) Albumax II (Invitrogen), and 62.5 μg / ml NaHCO3 (Wako). Cultures were maintained at 37°C, 5% O2, 5% CO2, with daily medium changes. Parasite growth and development were monitored by Giemsa staining and microscopy as needed. Optimal conditions for malaria parasite 3D7 culture required a hematocrit of approximately 5% and a parasitemia (the percentage of infected RBCs relative to total RBCs) of 10%. Initial synchronization was achieved by treating early ring-stage parasites with 5% sorbitol sterilized through a 0.45 μm Millipore filter. To obtain highly synchronized mature parasites, gradient centrifugation was performed using 40% to 70% Percoll (GE Healthcare).

[0031] 5.In vitro 293T cell culture Human embryonic kidney (293T) cells were cultured as described in Gene Ther. 2017 Dec 1;24(12):779-86. Briefly, 293T cells were grown in 100 x 20 culture dishes (Thermo Scientific, Nunclo Delta surface) containing DMEM (5 ml) (Nacalai Tesque, Japan), 10% fetal bovine serum (FBS) (Gibco, USA), l-glutamine, 50 IU / ml penicillin, 50 μg / ml streptomycin, and 62.5 μg / ml NaHCO3 (Wako) at 37°C, 5% O2, and 5% CO2. Cell growth was monitored under a microscope. When the cells reached 70%–80% confluence, attached cells were trypsinized (Gibco, USA) and subcultured. Cell numbers were counted using a hemocytometer. Cells were subcultured every 4–5 days (Thermo Scientific, Nunclon Delta surface).

[0032] 6. Plasmodium falciparum (malaria parasite) growth inhibition assay Growth inhibition assays were performed using Nonaka M, Murata Y, Takano R, Han Y, Bin Kabir MH, Kato K. Screening of a library of traditional Chinese medicines to identify anti-malarial compounds and extracts. Malar J. 2018 Jun 25;17(1). Briefly, Plasmodium falciparum (malaria parasites) were synchronized to the ring stage using D-sorbitol or Percoll (GE Healthcare) gradient (70%-40%) centrifugation. Parasites were then allowed to invade fresh red blood cells for 5 hours, treated with 5% filter-sterilized D-sorbitol. After 24 hours of incubation, growth inhibition assays were performed when the parasites reached the trophozoite stage. Uninfected human red blood cells (uRBCs) were added to achieve a parasitemia of approximately 0.3% and a hematocrit of 1%. Ethanol and methanol leaf extracts were dissolved in distilled water and sterilized by filtration through a 0.45 μm Millipore filter to obtain a 20 mg / ml stock solution. Working concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.562 μg / ml were prepared. 147 μl of parasite medium was then seeded into each well of a microtiter plate. 3 μl of extract was added to each well for a total volume of 150 μl. 10 μmol of artemisinin was used as a positive control, and wells containing the same parasite medium but without any drug served as negative controls. Microtiter plates were incubated at 37°C with 5% O2 and 5% CO2. After two days of incubation, 50 μl of culture medium was added to each well. The plates were then incubated at 37°C with 5% O2 and 5% CO2 for an additional two days. After 4 days of incubation, when the parasites were in the vegetative or schizoid stage, parasitemia was observed under a light microscope. The following formula was used to measure GIR (growth inhibition rate): Growth inhibition rate (%) = {1 - [(parasitemia of sample) - (parasitemia of positive control)] / [(parasitemia of negative control) - (parasitemia of positive control)]} × 100 In the above formula, parasitemia indicates the ratio (%) of infected red blood cells to the total number of red blood cells. Half-maximal (50%) inhibitory concentration (IC 50 ) values ​​were calculated using the following formula: I C 50 = 10^ [log(A / B)*(50-C) / (DC) + log(B)] In the formula, A is the minimum concentration at which the inhibition rate exceeds 50%. B is the maximum concentration at which the inhibition rate exceeds 50%. C is the inhibition rate in B. D is the inhibition rate in A.

[0033] 7. Malaria parasite invasion inhibition assay. For the invasion assay, highly synchronized fission yeast-infected erythrocytes were isolated using the Percoll-Sorbitol method according to established protocols. (Jeje TO, Bando H, Azad MTA, Fukuda Y, Oluwafemi IE, Kato K. Antiplasmodial and interferon-gamma-modulating activities of the aqueous extract of stone breaker (Phyllanthus niruri Linn.) in malaria infection. Parasitol Int. 2023 Dec 1;97, Nonaka M, Murata Y, Takano R, Han Y, Bin Kabir MH, Kato K. Screening of a library of traditional Chinese medicines to identify anti-malarial compounds and extracts. Malar J. 2018 Jun 25;17(1)., Kato K, Mayer G, Singh S, Reid M, Miller LH. Domain III of Plasmodium falciparum apical membrane antigen 1 binds to the erythrocyte membrane protein Kx [Internet]. Vol. 12, PNAS April 2005. Available from: www.pnas.orgcgidoi10.1073pnas.0501594102). Briefly, parasites cultured in a 10 ml flask were placed in a 15 ml tube at room temperature and centrifuged at 2,000 rpm for 5 minutes. The supernatant was aspirated, and a 50% hematocrit was adjusted using packed red blood cells. This solution was layered on a 70%-40% Percoll-sorbitol gradient in a 15 ml tube. The tube was centrifuged at 10,000 rpm at room temperature for 15 minutes. A grayish band of mature schizonts was observed at the 40 / 70 gradient interface and carefully collected into a 15 ml tube using a 1 ml pipette.Incomplete medium was added dropwise to the mature schizonts with continuous shaking. Approximately 10 ml of incomplete medium was added and the cells were washed twice by centrifugation at 2,000 rpm for 5 minutes at room temperature. Giemsa-stained smears were examined microscopically to assess the purity of the isolates. Parasite counts were performed using a hemocytometer using the following formula: (Total number of cells counted in the four corners of the hemocytometer) / 4 x 10 4 cells / ml medium. Approximately 1% hematocrit and 2% parasitemia were prepared with fresh red blood cells. 50 The medium (150 μl) containing the concentrations was transferred to a 96-well flat-bottom plate. Mature parasites were then cultured with fresh red blood cells at 37°C under 5% CO2 and 5% O2 for 24 hours.

[0034] 8. Cytotoxicity Assay Cell Count Reagent SF (Nacalai Tesque, Japan) was used for the cytotoxicity assay. 293T cells were cultured in a 96-well microplate, and approximately 5,000 cells were added to each well containing 100 μl of medium. The cells were grown until they reached 70% confluence. The cells were then incubated with various concentrations of the extract (1000, 100, 10, 1, 10). -1 The plates were incubated with 10 μl of Cell Count Reagent SF solution for 96 hours. After 96 hours of incubation, 10 μl of Cell Count Reagent SF solution was added to each well, and the plates were incubated for an additional 1 to 4 hours. The absorbance at 450 nm was then quantified using a microplate reader (Corona Electric, Ibaraki Prefecture). The cell viability (%) was calculated using the following formula: Cell viability (%) = [(absorbance at 450 nm of treated group) / (absorbance at 450 nm of control group)] × 100.

[0035] 9. Selectivity Index The selectivity index (SI) was used to compare the toxicity of the extracts against human cells (293T cells) and malaria parasites. The SI of in vitro toxicity of each extract was estimated as follows: SI (%) = [(IC of 293T cells 50) / (P. falciparum IC 50 )] × 100

[0036] 10.Statistical analysis Data from three biological replicates are presented as the mean ± standard deviation (SD) from three biological replicates. Statistical significance was determined using a paired Student's t-test: * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the negative control.

[0037] result 1. Extraction yield The extraction rate was calculated using the following formula: Extraction yield = weight of extract (g) / weight of plant body (g) × 100 After the drying process, yields were obtained from the ethanolic and methanolic crude extracts of leaves of all nine plants.

[0038] [Table 1]

[0039] 2.In vitro growth inhibition IC of each extract 50 value, 50% cytotoxic concentration (CC 50 ) values ​​and sensitivity index (SI) are shown in Table 2.

[0040] [Table 2]

[0041] Ethanol and methanol leaf extracts of Ficus hispida, Streblus asper, and Boerhavia repens, respectively, showed IC 50The ethanol and methanol extracts of Clerodendrum viscosum, along with the methanol crude leaf extract of Ficus hispida, showed moderate activity, with IC values ​​below 10 μg / ml (ethanol: 9.31, 4.13, 9.63 μg / ml, methanol: 6.63, 7.58 μg / ml). 50 The IC values ​​ranged from 10 to 50 μg / ml (42.43 μg / ml, 27.01 μg / ml, and 15.58 μg / ml, respectively). The ethanol extracts of Amaranthus spinosus and Mussaenda corymbosa showed mild activity, with IC values ​​ranging from 10 to 50 μg / ml. 50 The values ​​were 50-100 μg / ml (59.59 μg / ml, 64.14 μg / ml, 57.09 μg / ml), respectively. The methanol leaf extract of Amaranthus spinosus had IC values ​​of 291.87 μg / ml and 135.19 μg / ml, respectively. 50 The ethanol and methanol leaf extracts of Ficus hispida, Streblus asper, and Boerhavia repens showed higher antiplasmodial activity than the other plants. In particular, with the exception of Boerhavia repens and Clerodendrum viscosum leaves, the ethanol extracts of most plants showed higher activity than the corresponding methanol extracts (Table 2).

[0042] 3. In vitro inhibition of invasion Growth inhibition assays are a standard method for evaluating the overall efficacy of drugs that inhibit parasite invasion, rupture, and / or development. Therefore, we investigated the effects of plant extracts on parasite invasion into erythrocytes. The results showed that administration of the plant extracts inhibited parasite invasion into erythrocytes (Figure 1). Administration of the plant extracts significantly reduced the number of parasites (Figure 1). Most of the affected parasites formed rings, suggesting that the plant extracts did not affect parasite rupture or escape.

Claims

1. 1. An antimalarial drug comprising an ethanol and / or methanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus, and Mussaenda corymbosa.

2. 2. The antimalarial drug of claim 1, comprising an ethanol and / or methanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, and Boerhavia repens.

3. 3. An antimalarial drug according to claim 1 or 2, comprising an ethanol extract of at least one plant selected from the group consisting of Ficus hispida, Streblus asper, Boerhavia repens, Clerodendrum viscosum, Amaranthus spinosus and Mussaenda corymbosa.

4. The antimalarial drug according to claim 1 or 2, which is an oral or topical agent.

Images