JP2022042163A - Agent for preventing plasmodium from invading erythrocytes - Google Patents

Abstract

To provide a novel agent for preventing Plasmodium from invading erythrocytes.SOLUTION: An agent for preventing Plasmodium from invading erythrocytes contains extract from Phyllanthus ninuri.SELECTED DRAWING: None

Description

The present invention relates to an inhibitor of Plasmodium erythrocyte invasion into erythrocytes.

Malaria is one of the most common vector-borne infections caused by the Plasmodium genus Plasmodium. A 2018 World Health Organization (WHO) report estimates that 405,000 people have died and 213 million people are infected with malaria worldwide. It is also reported that 90% of all cases of malaria occur in sub-Saharan Africa. Human malaria is usually caused by four species of Plasmodium protozoa (P.falciparum, P.vivax, P.malariae and P.ovale), but the majority of infections are caused by P.falciparum. In addition, P. falciparum infection is known to cause severe malaria such as severe anemia, cerebral malaria, and acute respiratory failure. Therefore, there has been a long-standing need to combat malaria. No malaria vaccine has been approved so far, but several effective antimalarial drugs have been developed. For example, quinine is extracted from the cincona tree and artemisinin is found in the Chinese plant Artemisia annua. Thus, antimalarial drugs from medicinal plants contribute to malaria control.

However, in recent years, the emergence of drug-resistant protozoans has caused serious problems, and WHO recommends artemisinin-based combination therapy (ACT) for the treatment of malaria. Most of the existing anti-malaria drugs target protozoans in erythrocytes (RBC), but since anti-malaria treatment results in long-term exposure of the drug to the protozoans, merozoite is targeted for erythrocytes as a therapeutic development target. The step of intrusion has received a lot of attention.

However, few antimalarial drugs target protozoan erythrocyte invasion. For example, in Non-Patent Document 1, as a result of screening a library of 400 kinds of compounds containing compounds having antimalaria activity, 15 kinds of compounds that suppressed the release of Plasmodium erythrocytes from erythrocytes and the invasion of erythrocytes of Plasmodium malaria were suppressed. It is reported that there were 24 types of erythrocytes. Therefore, the development of new antimalarial drugs targeting the invasion of protozoans into erythrocytes is still eagerly awaited.

Int J Parasitol. 2020 Mar; 50 (3): 235-252. Southeast Asian J Trop Med Public Health. 2007 Jul; 38 (4): 609-15 J Nat Prod. 2005 Apr; 68 (4): 537-9 J Exp Med 216, 1733-1748, doi: 10.1084 / jem.20182227 (2019). mBio 9, doi: 10.1128 / mBio.01738-18 (2018). Phytother Res 31, 980-1004, doi: 10.1002 / ptr.5825 (2017). Interdiscip Toxicol 4, 206-210, doi: 10.2478 / v10102-011-0031-9 (2011). . Antimicrob Agents Chemother 57, 1455-1467, doi: 10.1128 / aac.01881-12 (2013). PLoS Pathog 8, e1002401, doi: 10.1371 / journal.ppat.1002401 (2012).

An object to be solved by the present invention is to provide a novel Plasmodium erythrocyte invasion inhibitor.

Under such circumstances, the present inventors have investigated a wide variety of components and found that the extract of Phyllanthus ninuri has an effect of suppressing the invasion of Plasmodium malaria into erythrocytes. The present invention is based on such novel findings.

Accordingly, the present invention provides the following sections:
Item 1. Inhibitors of Plasmodium malaria into erythrocytes, including an extract of Phyllanthus ninuri.

Item 2. A combination drug for preventing or treating malaria, which comprises an extract of Phyllanthus ninuri and an antimalarial drug.

Item 3. A prophylactic or therapeutic agent for malaria, which comprises an extract of Phyllanthus ninuri, for use in combination with an antimalarial drug.

According to the present invention, a novel agent for inhibiting the invasion of Plasmodium into erythrocytes is provided by using an extract of Phyllanthus ninuri, which has not been known for its effect of inhibiting the invasion of Plasmodium into erythrocytes, as an active ingredient. be able to.

Here, it was known that the extract of Phyllanthus ninuri, which is the active ingredient of the present invention, has antimalarial activity (Non-Patent Documents 2 and 3). However, as described above with reference to Non-Patent Document 1, most of the compounds having antimalarial activity do not have the effect of suppressing erythrocyte invasion of Plasmodium malaria. In addition, there are still many unclear points about the mechanism of inhibition of protozoan growth or development, the mechanism of inhibition of protozoan erythrocyte invasion, etc. of each drug having antimalarial activity. Therefore, a great deal of trial and error is required to confirm which compound has the erythrocyte invasion inhibitory activity of the protozoan among the various components reported to have antimalarial activity. Therefore, the surprising effect of the present invention is unpredictable from the prior art.

Treatment with Phyllanthus niruri extract (PE) inhibits the growth of Plasmodium falciparum in vitro. (A) Direct image and schematic extraction procedure of P. niruri. A photo of P. niruri was taken at a collection point in a town in southwestern Nigeria. After air-drying, the mixture was boiled at a ratio of 1:10 (weight per amount of distilled water of the plant sample) for 1 hour for extraction. (B) After treatment with PE for 24 hours, the amount of lactate dehydrogenase (LDH) released from HFF into the medium was measured. (C) Infected erythrocytes were untreated or treated with PE for 96 hours before counting paracitemia. Artemisinin (ART) was used as a positive control drug. (D) Effective concentration that inhibits the growth of 50% (IC ₅₀ ) of parasites. The indicated concentrations of PE were prepared and used to treat infected red blood cells for 96 hours. The percentage of paracitemia obtained was plotted against that concentration using the inverse logarithm of the concentration. The values shown are the average of ± sd. (E) The state of infected erythrocytes 24 hours after PE or ART treatment was confirmed by Giemsa staining. (All data were performed in 3 independent experiments, with 3 sample iterations in each experiment) (BD). ** p <0.01; NS No significant difference (Student's t-test). PE affects the invasion of Plasmodium falciparum into erythrocytes. (A) Image of infected red blood cells after or without treatment with 200 ug / ml PE. Well-synchronized cultures of P. falciparum were grown to the late vegetative stage and cultured for 24 hours with or without treatment with PE. Smear specimens were prepared from each group and observed under a microscope. (B) Purified schizont-infected erythrocytes were mixed with fresh erythrocytes and medium to give 2% paracitemia and 1% hematocrit. After culturing for 24 hours with PE treatment, ART treatment or untreated, smears were prepared and the infection rate was measured. (C) Invasion inhibition assay using washed erythrocytes. (D) It was measured whether PE treatment could inhibit the growth of Plasmodium in erythrocytes. The values shown are the average of ± s.d. (All data were subjected to 3 independent experiments, with 3 sample iterations in each experiment) (B-D). ** p <0.01, *** p <0.001; N.S. No significant difference (Student's t-test). PE exerts an antimalarial effect on Plasmodium berghei in vivo. (A-D) Untreated or PE-treated mice were infected with P. berghei. (A) Paracitemia was evaluated by Giemsa staining. (B) Survival rate was analyzed. (C) The effect on the blood-brain barrier (BBB) was measured by Evans blue (EB) perfusion. Uninfected mice were used as controls. (D) The amount of IFN-γ in serum was evaluated by ELISA. The values shown are the average of ± s.d. (All data were performed in 3 independent experiments, with 3 sample iterations in each experiment) (A, C, D). ** p <0.01, *** p <0.001; N.S. No significant difference (Logrank test or Student's t-test).

Plasmodium erythrocyte invasion inhibitor The present invention provides an erythrocyte invasion inhibitor of Plasmodium malaria, which comprises an extract of Phyllanthus ninuri. The active ingredient of the present invention is an extract of Phyllanthus ninuri. Phyllanthus niruri L. (Euphorbiaceae) is a widespread tropical plant known as one of the medicinal plants and is used to treat malaria in some endemic areas. In addition, the above-mentioned Non-Patent Documents 2 and 3 describe that the extract of Phyllanthus niruri has antimalarial activity. Specifically, Non-Patent Document 2 describes that the P. niruri extract using each of the three solvents (water, MeOH, CHCl ₃ ) has antimalarial activity. In addition, Non-Patent Document 3 describes that three kinds of compounds contained in P. niruri extract using boiling water exhibit antimalarial activity. However, it is not known by what mechanism Phyllanthus niruri and its extract bring about antimalarial effect, and no erythrocyte invasion inhibitory activity of protozoans is known.

An extract of Phyllanthus niruri can be obtained by immersing Phyllanthus niruri or a pulverized product thereof in a solvent. Examples of the solvent include an aqueous solvent and a non-aqueous solvent, and an aqueous solvent can be preferably used. Examples of the aqueous solvent include water, methanol, ethanol, acetone and the like. Examples of the non-aqueous solvent include chloroform, hexane, dichloromethane and the like. As these solvents, one kind alone or two or more kinds can be used as a mixed solvent. The parental immersion time for obtaining the extract of Phyllanthus niruri is not particularly limited, but can be appropriately set in the range of, for example, 15 minutes to 24 hours, preferably 30 minutes to 1 hour. The temperature at the time of immersion is not particularly limited, but can be appropriately set in the range of 50 to 120 ° C, preferably 60 to 90 ° C. In the present invention, even if the extract of Phyllantus niruri is used in the state of an extract in which the extract is eluted in the solvent used for the above extraction, the extract is purified from the extract by a method known per se. You may use the thing.

In the present invention, examples of malaria protozoa include Plasmodium falciparum, Plasmodium falciparum, Plasmodium malaria, Oval malaria, and Plasmodium falciparum. Will be.

Further, in the present invention, even if the extract itself of Phyllanthus niruri, which is the active ingredient of the present invention, is used as an inhibitor of the invasion of Plasmodium malaria into erythrocytes, various pharmaceutically acceptable carriers (for example, isotonic agents) are used. , Chelating agent, stabilizer, pH adjuster, preservative, antioxidant, solubilizing agent, thickening agent, etc.) may be used as a pharmaceutical composition.

Examples of the tonicity agent include saccharides such as glucose, trehalose, lactose, fructose, mannitol, xylitol and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol and propylene glycol, sodium chloride, potassium chloride and calcium chloride. Examples include inorganic salts. These isotonic agents can be used alone or in combination of two or more.

Examples of the chelating agent include edetates such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, calcium edetate, ethylenediaminetetraacetic acid salt, nitrilotriacetic acid or a salt thereof, and hexametallin. Examples thereof include acid soda and citric acid. These chelating agents can be used alone or in combination of two or more.

Examples of the stabilizer include sodium bisulfite and the like.

Examples of the pH adjuster include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, and also alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and alkali metal carbonates or hydrogen carbonates such as sodium carbonate. Examples thereof include salts, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as tromethamol. These pH regulators can be used alone or in combination of two or more.

Examples of preservatives include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate, chlorhexidine gluconate, benzalkonium chloride, and chloride. Examples thereof include quaternary ammonium salts such as benzethonium and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylenebiguanide, chlorhexidine and the like. These preservatives can be used alone or in combination of two or more.

Examples of the antioxidant include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like. These antioxidants can be used alone or in combination of two or more.

Examples of the solubilizing agent include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, macrogol, D-mannitol and the like. These dissolution aids can be used alone or in combination of two or more.

Examples of the thickening agent include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, chondroitin sodium sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol and the like. These thickening agents can be used alone or in combination of two or more.

In addition to the extract of Phyllanthus niruri, the pharmaceutical composition may further contain a compound alleged to have antimalarial activity. Compounds known to have antimalarial activity include, for example, artemisinin, quinine, mefloquine, atovaquone / proguanil mixture and the like. 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, but for example, 0.001 to 1000 parts by mass, preferably 0.01 to 100 parts by mass, a compound having antimalaria activity is used with respect to 1 part by mass of the extract of Phyllantus niruri. More preferably, 0.1 to 10 parts by mass can be used. When a plurality of compounds having antimalarial activity other than the extract of Phyllanthus niruri are used, for example, each compound can be used at the above usage ratio.

In the embodiment of the pharmaceutical composition, the content of the extract of Phyllanthus niruri in the composition is not particularly limited, and 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. It can be appropriately set from conditions such as% or more, 5% by mass or more, and 1% by mass or more.

The form of the preparation is not particularly limited, and for example, oral administration agents such as tablets, rounds, capsules, powders, granules and syrups; injections (intravenous injection, intramuscular injection, local injection, etc.), gargling agents, drip agents. , Various formulations such as external preparations (ointments, creams, patches, inhalants), parenteral administrations such as suppositories, and the like can be mentioned. Among the above-mentioned pharmaceutical forms, for example, oral administration agents (tablets, pills, capsules, powders, granules, syrups, etc.), external preparations (inhalants, ointments, creams, patches, etc.) and the like are preferable. Can be mentioned.

In the present invention, the dose of the extract of Phyllantus niruri varies depending on the route of administration, the age, body weight, symptoms, etc. of the patient and cannot be unconditionally defined, but the daily dose for adults is usually about 5000 mg or less, preferably about 1000 mg. The amount may be as follows. The lower limit of the dose of the extract of Phyllanthus niruri is not particularly limited, and for example, the daily dose for an adult can be appropriately set in the range of usually 0.1 mg or more, preferably 0.5 mg or more. When it is administered once a day, it is sufficient that this amount is contained in one product, and when it is administered three times a day, it is sufficient that this amount is contained in one third amount.

The inhibitor of the invasion of Plasmodium malaria into erythrocytes of the present invention is administered to a patient such as a mammal. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep and the like.

Combination drug, preventive or therapeutic agent for malaria The extract of Phyllanthus niruri, which is the active ingredient of the present invention, has an effect of inhibiting the invasion of Plasmodium malaria into erythrocytes. Therefore, the extract of Phylllanthus niruri is combined with an antimalaria drug other than the extract of Phylllanthus niruri, particularly an antimalaria drug having a different point of action from the extract of Phylllanthus niruri (for example, an antimalaria drug having no effect of inhibiting invasion into erythrocytes). It is useful to use. Therefore, in one embodiment, the present invention provides a combination pharmaceutical product for preventing or treating malaria, which comprises an extract of Phyllantus ninuri and an antimalarial drug. Also provided in another embodiment is a prophylactic or therapeutic agent for malaria comprising an extract of Phyllanthus ninuri for use in combination with an antimalarial agent.

In these embodiments, the method for producing the extract of Phyllanthus niruri, the method for using it, and the like are the same as those described in the above-mentioned "Inhibitor for invasion of Plasmodium malaria into erythrocytes".

Also, in these embodiments, "combination" is not only an embodiment containing both an extract of Phyllanthus ninuri and other antimalarial drugs in one dosage form such as one tablet, capsule, etc., but also separately. Various embodiments that may be used in combination therapy may be included, such as embodiments in which an extract of Phyllanthus ninuri and other antimalarial agents are each formulated in a dosage form.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

material and method
Mice, cells, parasites
Six-week-old female C57BL / 6N mice were obtained from the Charles river. All animal experiments were conducted with the approval of the Animal Research Committee of Tohoku University. MOLT-4 cells were maintained in RPMI (Nacalai Tesque) containing 10% heat-inactivated FBS, 100 U / ml penicillin, 0.1 mg / ml streptomycin. P. berghei ANKA and P. falciparum HB3 clones were obtained from the Malaria Research and Reference Reagent Resource Center (MR4; American Type Culture Collection, Manassas, VA). Parasites were cultured in medium containing RPMI 1640, 25 mM HEPES, 100 uM hypoxanthine, 12.5 ug / ml gentamicin sulfate 5% (w / v) Albumax I, and 62.5 ug / ml NaHCO3.

Preparation of plant materials and extracts
Fresh samples of the entire Phyllanthus ninuri plant were collected from Ado-Ekiti, Nigeria. The whole plant was air dried and crushed with a mortar and pestle.

reagent
Antibodies to NF-kB p65 (sc-8008) were obtained from Santa Cruz Biotechnology.

P. falciparum GIA
The antimalaria protozoan activity of P. ninuri extract against Plasmodium falciparum HB3 was evaluated. The extract was weighed, dissolved in distilled water and sterilized by a filter (0.45 μm millipore filter) to prepare a stock solution having a concentration of 5000 μg / ml. Working solutions with a concentration of 200 μg / ml were prepared by diluting with complete parasite medium. 150 microliters of P. falciparum parasite culture suspension pre-synchronized to the ring stage with 5% sorbitol were equally divided into wells on a 96-well microtiter plate with a final hematocrit of 2% and parasitemia of 0.5%. It was adjusted with fresh red blood cells (AB type) so as to be. Cultures were incubated at 37 ° C. with 5% CO ₂ and 5% O ₂ . 48 hours after incubation, 5 ul of complete medium was added to each culture. Artemisinin was used as the positive control and wells containing no drug but cultured in the same paracitemia and hematocrit were used as the negative control. Evaluation of in vitro GIA results was performed after 96 hours of incubation using microscopy.

P. falciparum invasion assay Purified schizont-infected erythrocytes for invasion inhibition assay were prepared by the Percoll sorbitol method. Briefly, old medium was removed from the parasite culture by centrifugation at 2000 rpm for 5 minutes at room temperature. The packed erythrocytes were then resuspended in 50% hematocrit and applied with a gradient made from 70% and 40% Percoll-Sorbitol solutions. The gradient was then centrifuged on an SS-34 rotor at 10,000 rpm for 20 minutes at 20 ° C. without brake. Infected erythrocytes were collected from interface 40/70 and washed twice with incomplete medium. Smear specimens were prepared to calculate infection rates using a hemocytometer. Purified schizont was mixed with complete medium to obtain 1% hematocrit and fresh ABO erythrocytes were added to bring total paracitemia to 2%. The culture (150 ul) was transferred to a 96-well plate and the extract was added appropriately to a final concentration of 200 ug / ml. Cultures were incubated at 37 ° C. for 24 hours at 5% CO ₂ and 5% O ₂ . The culture supernatant was aspirated, the cell pellet was smeared and stained with Giemsa stain. The rate of red blood cells infected with ring-stage protozoans was measured (about 24 hours after incubation).

P. falciparum development assay
In the P. falciparum development assay, purified schizont-infected erythrocytes were prepared by the Percoll sorbitol method as described above. Fresh ABO erythrocytes were incubated with plant extract for 1 hour and then washed 3 times. Purified schizont was mixed with complete medium to give 1% hematocrit and washed erythrocytes were added to form 2% paracitemia. The untreated group and the group not washed after the treatment were used as the control group. The culture (150 ul) was transferred to a 96-well plate and the extract was added appropriately to a final concentration of 200 ug / ml. Cultures were incubated at 37 ° C. for 24 hours at 5% CO ₂ and 5% O ₂ . The culture supernatant was aspirated, the cell pellet was smeared and stained with Giemsa stain. Paracitemia measured the rate of red blood cells infected with ring-stage protozoans (approximately 24 hours after incubation).

Evaluation of P. berghei's Paracitemia Blood smear was taken daily from all infected mice and stained with Giemsa. Paracitemia was assessed by counting at least 5 × 10 ³ red blood cells.

Evaluation of Blood-Brain Barrier Disorder Evans Blue Vascular Leakage was measured according to the method described in Non-Patent Document 4. Mice were infected with 5 × 10 ⁶ P. berghei infected erythrocytes, and on the 5th day of infection, 200 μl of 2% Evans blue (Nacalai Tesque) was intravenously injected. Mice were euthanized and dissected 1 hour after Evans Blue injection, the brain was separated and photographed, followed by brain weight measurements. The brain was then placed in 2 ml formamide at 37 ° C. for 48 hours. Evans blue concentration was measured at 620 nm with an ELISA reader using 100 μl of solution. Evans blue concentration was calculated using a standard curve starting at 200 μg / ml. The content of Evans blue is represented in Figure 3C as "μg Evans blue / g brain".

Quantification of cell viability (LDH assay)
The PE-induced cytotoxicity was measured according to the method described in Non-Patent Document 5. Cell viability was assessed by measuring lactate dehydrogenase (LDH) leakage into the culture medium using the CytoTox96 Non-Radio Cytotoxicity Assay Kit (Promega). Adherent cells HFF were seeded on plates in appropriate culture volumes and incubated at 37 ° C., 5% CO ₂ and 5% O ₂ . After 24 hours, the old medium was aspirated and replaced with new medium containing 200 ug / ml extract. The cells and extracts were then incubated at 37 ° C. with 5% CO ₂ and 5% O ₂ for various times as shown. Culture supernatants were collected, centrifuged at 3000 rpm for 5 minutes, and then the activity of LDH in the cell-free supernatant was measured according to the manufacturer's instructions. Cell-free medium was used as a negative control. Culture supernatants of cells treated with Triton X-100 (0.1%) to kill all cells were used as positive controls.

PE IC ₅₀ decision
To calculate the IC50 of PE for P. falciparum, a GIA assay was performed using 12.5, 25, 50, ₁₀₀ , 200 μg / ml. The number of parasites in PE-treated cells is shown in the figure as the rate of suppression of parasites standardized with untreated parasites.

IFN-γ enzyme-linked immunosorbent assay (ELISA) analysis A mouse IFN-γ ELISA kit (RayBiotech) was used according to the manufacturer's instructions. Briefly, 100 ul of serum from mice and standards were added well to 96-well microplates coated with anti-mouse IFN-γ (immobilized antibody). They were incubated at room temperature (RT) for 2.5 hours with gentle shaking. After washing 4 times, 100 ul of prepared biotinylated anti-mouse IFN-γ was added and incubated for 1 hour at room temperature with gentle shaking. Wells were washed 4 times, 100 ul of HRP-conjugated streptavidin solution was added and incubated for 45 minutes with gentle shaking. After washing it, it was reacted with a one-step substrate reagent (3,3,5,5-tetramethylbenzidine) in the dark with gentle shaking. After 30 minutes of incubation, 50 ul of 2N sulfuric acid was added to stop the reaction and the color intensity at 450 nm was measured with a microplate reader. A curve was created and the absorbance at 450 nm was plotted against the concentration of IFN-γ in the standard well. The concentration of IFN-γ in the sample was determined by comparing the absorbance of the sample with this curve.

Statistical analysis All statistical analyzes were performed using Prism 7 (GraphPad). One point shown in each figure shows the mean value of 3 samples by biological repetition, and 3 independent experiments were performed. The statistical significance of the mean difference was analyzed using the unpaired two-sided student's t-test. P-values below 0.05 were considered statistically significant. The statistical significance of the difference in mouse survival time between the two groups was analyzed by the Kaplan-Meier survival analysis logrank test.

Reference example PE treatment has little known detailed effect of P. niruri on the growth of Plasmodium falciparum parasites, which inhibits the growth of Plasmodium falciparum under in vitro culture conditions . Therefore, lyophilized aqueous P. niruri extract (PE) was prepared to test whether P. niruri has the ability to have antiparasitic effects (Fig. 1A). It has been reported that PE did not show toxicity both in vitro and in vivo (Non-Patent Documents 6 and 7). It was confirmed that no significant cytotoxicity was observed at a concentration of 100 μg / ml PE for primary human foreskin fibroblasts (HFF) (Fig. 1B). Next, to test the antiparasitic activity of PE, Plasmodium falciparum was cultured in 100 μg / ml PE for 96 hours and a growth inhibition assay (GIA) was performed (Fig. 1C). As previously reported, PE treatment significantly reduced the number of parasites compared to the untreated state (Figures 1C and 1E). Furthermore, the _IC50 value of PE for P. falciparum inhibition was found to be 35.11 μg / mL (Fig. 1D). Previous studies used a methanol extract of P. ninuri with higher antimalarial activity (IC ₅₀ : 2.9-4.1 μg / ml) compared to this result (IC ₅₀ : 35.11 μg / ml). It is shown. This difference in activity is considered to be due to the difference in antimalarial activity due to the difference in extraction method, but this method was selected because the water extract prepared by the simple decoction method is useful for evaluation in line with the actual situation.

Example 1 PE affects erythrocyte invasion of Plasmodium falciparum.

GIA is commonly used to measure the comprehensive ability of a drug to inhibit parasite invasion, rupture, and / or differentiation / proliferation (Non-Patent Document 9). Therefore, in order to test whether PE affects the differentiation and proliferation of parasites in erythrocytes, synchronized ring-shaped parasites (immature nutrients) are cultured in a culture medium containing PE for 24 hours. The morphology of parasites (schizonts) was compared (Fig. 2A). There is no observable effect of PE on parasite morphology (Fig. 2A) and parasitemia (Fig. 2D), indicating that the point of action of PE is not the differentiation and proliferation of parasites in erythrocytes. Next, to investigate whether PE affects erythrocyte invasion of parasites, synchronized schizonts were cultured with PE for 24 hours and parasites were compared (Fig. 2B). PE treatment significantly reduced the number of parasites (Fig. 2B). In addition, the majority of infected parasites are in the ring phase (data not shown) rather than in the schizont stage, indicating no effect of PE on parasite rupture and release. Since parasite erythrocyte invasion requires interaction between the host receptor and the parasite ligand, we evaluated whether PE affects erythrocyte surface receptors. Red blood cells were treated with PE for 1 hour. The erythrocytes were then washed and cultured with infected erythrocytes for 24 hours (Fig. 2C). As shown in Figure 2C, pre-washed erythrocytes lose their ability to inhibit parasite invasion, thus indicating that PE affects the parasite ligand or parasite invasion process rather than the erythrocyte surface receptors. Suggests. Since most antimalarial drugs such as artemisinin are known not to inhibit protozoan erythrocyte invasion, the protozoan erythrocyte invasion inhibitory effect shown in FIG. 2C above is peculiar to PE.

Example 2 PE has multiple antimalarial effects in vivo on mouse malaria P. berghei Next, it was tested whether PE treatment could control the development of cerebral malaria in vivo. P. berghei ANKA, a mouse malaria protozoan, was used to infect C57BL / 6 (B6) mice, which are known to be excellent models of experimental cerebral malaria (Non-Patent Document 9). When mock or PE-treated mice were challenged with P. berghei-infected erythrocytes, PE-treated mice showed reduced parasitemia (Fig. 3A) and higher survival (Fig. 3B). In addition, PE-treated mice were found to have significantly reduced blood-brain barrier damage (Fig. 3C). In addition, serum IFN-γ levels in P. berghei-infected mice were significantly reduced in the presence of PE (Fig. 3D). Taken together, these results show that PE has multiple effects in vivo and protects against the progression of cerebral malaria through suppression of parasite growth and suppression of blood-brain barrier damage associated with excessive inflammation. ing.

Claims (3)

Inhibitors of Plasmodium malaria into erythrocytes, including an extract of Phyllanthus ninuri. A combination drug for preventing or treating malaria, which comprises an extract of Phyllanthus ninuri and an antimalarial drug. A prophylactic or therapeutic agent for malaria, which comprises an extract of Phyllanthus ninuri, for use in combination with an antimalarial drug.

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