Geranium thunbergii extract with antimalarial properties

Abstract

To provide a Geranium thunbergii extract having antimalarial properties. [Solution] This invention relates to the antimalarial effect of an extract obtained by reacting Geranium thunbergii with an organic solvent and then fractionating the resulting mixture with another organic solvent. It has been confirmed that this extract exhibits an even superior antimalarial effect compared to conventional antimalarial agents such as chloroquine and artemisinin. Therefore, it is expected that using the Geranium thunbergii extract of this invention will enable the development of novel antimalarial therapeutic agents with improved antimalarial effects.

Description

[Technical Field]

[0001] This invention relates to a Geranium thunbergii extract having antimalarial properties. Specifically, it is obtained by reacting Geranium thunbergii with an organic solvent and fractionating the resulting solution, and is characterized by exhibiting superior antimalarial effects compared to conventional antimalarial materials. [Background technology]

[0002] Malaria is an acute febrile infectious disease caused by infection with Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum, all belonging to the genus Plasmodium. Malaria treatments include chloroquine, mefloquine, primaquine, and artemisinin. Chloroquine is typically used as a first-line treatment for malaria. However, its effectiveness has been halved due to the rapid spread of chloroquine-resistant malaria. Artemisinin is recommended as a first-line treatment for chloroquine-resistant malaria. However, since malaria resistant to artemisinin is currently emerging and spreading, there is a growing need for the development of novel antimalarial agents that can replace artemisinin.

[0003] Geranium thunbergii is a medicinal material used in traditional Chinese medicine. Because it contains tannins, it is used as an antidiarrheal, diuretic, laxative, intestinal regulator, and tonic. Registered Korean Patent No. 10-1762216 discloses that Geranium thunbergii extract is used as a pharmaceutical composition exhibiting antiplatelet activity. Published Korean Patent No. 10-2023-0091289 describes that Geranium thunbergii extract is used in combination with Litsea japonica fruit extract as an obesity treatment. Published Korean Patent No. 10-2024-0146756 discloses that Geranium thunbergii extract is used as a composition for improving or treating menstrual pain. However, no efficacy related to malaria has been confirmed for the aforementioned Geranium thunbergii extract.

[0004] All documents, including patent documents and references cited herein, are incorporated here by reference, with each document clearly and specifically indicated, and their entire contents are incorporated here to the same extent as they are described herein. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Republic of Korea Registered Patent No. 10-1762216 Gazette [Patent Document 2] Republic of Korea Publication Patent No. 10-2023-0091289 [Patent Document 3] Republic of Korea Publication Patent No. 10-2024-0146756 [Overview of the project] [Problems that the invention aims to solve]

[0006] In the present invention, screening is performed in multiple stages to discover materials with anti-malaria activity from a variety of natural products, and the anti-malaria activity according to the extraction method is analyzed. It has been experimentally proven that the extract obtained by extracting geranium with an organic solvent has anti-malaria activity superior to that of conventional anti-malaria substances.

[0007] Therefore, an object of the present invention is to provide a geranium extract for anti-malaria produced using an organic solvent. Specifically, after reacting geranium powder with an organic solvent to produce a reaction product, the reaction product is fractionated with an organic solvent to produce a geranium extract for anti-malaria.

[0008] Other objects and technical features of the present invention will be more specifically disclosed by the following detailed description of the invention, claims, and drawings.

Means for Solving the Problems

[0009] The present invention provides a geranium (Geranium thunbergii) extract for anti-malaria produced using an organic solvent.

[0010] ​​​​​​​​​​​​​The present invention relates to the anti - malaria effect of an extract obtained by reacting Geranium thunbergii with an organic solvent and then fractionating it with an organic solvent. It has been confirmed that it exhibits an anti - malaria effect superior to that of chloroquine and artemisinin, which are conventional anti - malaria agents. Therefore, it is expected that by using the Geranium thunbergii extract of the present invention, a novel anti - malaria therapeutic agent with improved anti - malaria effect can be developed.

Brief Description of the Drawings

[0013] [Figure 1] The process of manufacturing a natural product fraction from a natural product extract sample of the present invention is shown. [Figure 2] The method for screening the anti - malaria effect of the natural product extract of the present invention is shown. [Figure 3] The results of the primary screening for confirming the anti - malaria effect against the natural product extract of the present invention are shown. [Figure 4] The results of analyzing the anti - malaria effect IC50 for 15 natural product fractions selected using the secondary screening of the present invention are shown. [Figure 5] The results of analyzing the anti - malaria effect IC50 of the Geranium thunbergii extract of the present invention are shown.

Modes for Carrying Out the Invention

[0014] The present invention provides a Geranium thunbergii extract for anti - malaria manufactured using an organic solvent, wherein the organic solvent is methanol, ethanol or chloroform. The Geranium thunbergii extract for anti - malaria of the present invention is characterized by being obtained by fractionating a Geranium thunbergii reaction product produced by reacting Geranium thunbergii powder with methanol or ethanol with methanol, ethanol or chloroform.

[0015] According to embodiments of the present invention, the antimalarial Geranium thunbergii extract is produced by a method comprising: a first step of grinding Geranium thunbergii to an 80-mesh size to produce Geranium thunbergii powder; a second step of mixing the Geranium thunbergii powder with a first organic solvent and reacting them at room temperature for 6 hours to produce a Geranium thunbergii reaction product; a third step of filtering the Geranium thunbergii reaction product to obtain a Geranium thunbergii reaction solution, and freeze-drying it to produce a Geranium thunbergii extract; and a fourth step of mixing the Geranium thunbergii extract with distilled water to produce an aqueous solution of Geranium thunbergii extract, mixing it with a second organic solvent, and then obtaining an organic solvent fraction using a separatory funnel. The first organic solvent is methanol or ethanol, and the second organic solvent is chloroform. Preferably, the first organic solvent is ethanol, and the second organic solvent is chloroform.

[0016] According to another embodiment of the present invention, the antimalarial Geranium extract is produced by a method comprising a fourth step in which an aqueous layer is obtained from a separatory funnel, the aqueous layer is mixed with a third organic solvent, and then a fifth step in which an organic solvent fraction is obtained using a separatory funnel. The third organic solvent is ethyl acetate.

[0017] In short, the antimalarial Geranium extract of the present invention may be a chloroform fraction obtained by reacting Geranium powder with methanol and then adding chloroform, a chloroform fraction obtained by reacting Geranium powder with ethanol and then adding chloroform, an ethyl acetate fraction obtained by reacting Geranium powder with methanol and then adding chloroform to the aqueous layer obtained by adding ethyl acetate, or an ethyl acetate fraction obtained by reacting Geranium powder with ethanol and then adding chloroform to the aqueous layer obtained by adding ethyl acetate. Preferably, it is an ethyl acetate fraction obtained by reacting Geranium powder with ethanol and then adding chloroform to the aqueous layer obtained by adding ethyl acetate.

[0018] According to embodiments of the present invention, it was confirmed that the aqueous layer does not have the ability to suppress malaria activity.

[0019] The Geranium thunbergii extract for antimalarial use of the present invention exhibits malaria activity inhibitory capacity IC 50 The present invention is characterized by having an IC50 concentration of 15-70 μg / mL, and preferably, the antimalarial Geranium thunbergii extract of the present invention has a malaria activity inhibitory capacity IC50. 50 The present invention is characterized by having an IC50 concentration of 15-40 μg / mL, and more preferably, the antimalarial Geranium thunbergii extract of the present invention has a malaria activity inhibitory capacity IC50. 50 It is characterized by having a concentration of 15-19 μg / mL.

[0020] The present invention will be described in detail below with reference to examples.

[0021] Examples 1. Experimental Method 1) Preparation of natural product extracts In this invention, 292 natural product extracts (CBF-1 to 292) and fractions were prepared, and their antimalarial efficacy (malaria activity inhibitory ability) was evaluated in vitro. The 292 natural product extracts and fractions were prepared by the following method.

[0022] First, 1 kg of dried natural product was ground to an 80-mesh size using a pulverizer (Daesung Artlon DA280-S, Paju, South Korea) to produce natural product powder. The natural product powder was added to methanol (≧99.8%) (Sigma-Aldrich, USA), mixed at room temperature, and reacted for 6 hours. The reaction product of the natural product powder and methanol was filtered using Whatman No. 2 filter paper to obtain a liquid, which was then concentrated using a rotary evaporator (Rotavapor® R-220SE, Buchi Labortechnik AG, Switzerland) and dried using a freeze-dryer (Ilshinbiobase Co., Ltd, Yangju, South Korea) to produce a natural product extract.

[0023] Figure 1 shows the process of producing a fraction from a natural product extract according to the present invention. Panel A) schematically shows the steps of producing a fraction from a natural product extract, and Panel B) shows the process of obtaining the fraction using a separatory funnel.

[0024] 100.0 g of the natural product extract was mixed with 1.0 L of distilled water, and then stirred every 10 minutes for 1 hour to produce an aqueous solution of the natural product extract. The aqueous solution of the natural product extract and chloroform (≧99.0%) (Sigma-Aldrich, USA) were mixed in a 1:1 volume ratio, and the mixture was stirred every 30 minutes for 3 hours at room temperature using a separatory funnel to separate the chloroform layer (methanol-chloroform fraction) and the first aqueous layer.

[0025] The first aqueous layer was mixed with ethyl acetate (≧99.3%) (Sigma-Aldrich, USA) in a 1:1 volume ratio, and then separated into an ethyl acetate layer (methanol-ethyl acetate fraction) and a second aqueous layer (methanol-distilled water fraction) by stirring every 30 minutes for 3 hours at room temperature using a separatory funnel.

[0026] The chloroform layer (methanol-chloroform fraction), ethyl acetate layer (methanol-ethyl acetate fraction), and second aqueous layer (methanol-distilled water fraction) obtained as described above were concentrated using a rotary evaporator (Rotavapor® R-220SE, manufactured by Buchi Labortechnik AG, Switzerland) and dried using a freeze-dryer (manufactured by Ilshinbiobase Co., Ltd, Yangju, South Korea).

[0027] Furthermore, in the process of producing the aforementioned natural product fraction, methanol, which is the organic solvent used to react with the natural product powder, was replaced with ethanol to produce a natural product extract. The chloroform layer obtained from this was named the ethanol-chloroform fraction, the ethyl acetate layer obtained from the first aqueous layer was named the ethanol-ethyl acetate fraction, and the second aqueous layer was named the ethanol-distilled water fraction.

[0028] The fraction obtained from the chloroform layer averaged 1.25 g, the fraction obtained from the ethyl acetate layer averaged 11.75 g, and the fraction obtained from the second aqueous layer averaged 57.32 g. In the present invention, the methanol-ethyl acetate fraction is used in a screening experiment, and if necessary, the antimalarial effect IC of the fraction is determined. 50 We analyzed it.

[0029] 2) Preparation for Plasmodium falciparum malaria To analyze the antimalarial effect of the natural product extract of the present invention, red blood cells and Plasmodium falciparum (P. falciparum, strain 3D7) were prepared.

[0030] First, to prepare red blood cells, blood was centrifuged at 1,500 rpm for 5 minutes at room temperature to separate the serum layer from the red blood cell layer. Then, white blood cells were separated from the red blood cell layer using an Acrodisc® white blood cell filter to obtain only red blood cells.

[0031] To prepare a culture medium for Plasmodium falciparum malaria, 1 pack of RPMI (Roswell Park Memorial Institute) 1640 medium powder, 2 g of D-glucose, 2.3 g of sodium bicarbonate, and 0.1 mM hypoxanthine were added to 1 L of distilled water and mixed. The pH was then adjusted to 7.4, and the mixture was filtered through a 0.45 μm filter. To the solution obtained after filtration, 10% AlbuMAX™ I solution and gentamicin (10 mg / mL) were added to prepare the Plasmodium falciparum malaria culture medium.

[0032] To activate the malaria pathogen, Plasmodium falciparum (3D7 strain), tropical malaria cells (red blood cells infected with Plasmodium falciparum) stored at -80°C were dissolved in a water bath, and then a 12% sodium chloride (NaCl) solution was added and the mixture was allowed to stand at room temperature. Subsequently, a 1.6% NaCl solution was added, the supernatant was removed by centrifugation, and then a 0.9% NaCl solution was added and the supernatant was removed by centrifugation. The infected red blood cells and the separated red blood cells were then cultured in malaria-specific medium at 37°C and 5% CO2 with a final hematocrit value of 2% and 1% parasitic blood (parasitemia). During the culture period, parasitic blood was confirmed and the medium was changed.

[0033] 3) Screening of natural products with antimalarial effects Figure 2 shows a method for screening the antimalarial effect of the natural product fraction of the present invention. Panel A) shows 1% (hematocrit value: 2%) of Plasmodium falciparum parasite blood at the ring stage, prepared to confirm the antimalarial effect of the natural product fraction of the present invention, and Panel B) shows a 96-well plate coated with the natural product fraction and Plasmodium falciparum parasite blood to confirm the antimalarial effect of the natural product fraction of the present invention.

[0034] After coating a 96-well plate with 1 mg / mL of the prepared natural product fraction (methanol-ethyl acetate fraction), ring-stage Plasmodium falciparum parasite blood was added at a concentration of 1% (hematocrit value: 2%) and incubated for 48 hours. As a positive control, infected red blood cells containing 10 μM chloroquine and an equal amount of dimethyl sulfoxide (DMSO), a representative antimalarial drug, were used, and as a negative control, uninfected red blood cells were used. After incubation, a mixture of 4X lysis buffer SYBR® green (2,500X) was added, and fluorescence intensity (485 nm: excitation / 530 nm: fluorescence) was measured using a microplate reader. In the screening process, natural product extracts showing even higher antimalarial efficacy compared to the positive control were selected (primary screening), and these were subjected to secondary screening using the same method.

[0035] The malaria activity inhibitory activity IC of the natural product fractions selected by the aforementioned natural product screening 50 The following was calculated: Malaria activity inhibitory capacity IC 50 To calculate the concentration, a 400 μg / mL natural product fraction was diluted 12 times by a factor of 0.5 to a concentration of 0.195 μg / mL and repeatedly inoculated into a 96-well plate three times. 100 μL of 1% parasite blood (2% hematocrit) was added and the mixture was incubated for 48 hours. Subsequently, a mixture of 4X lysis buffer SYBR® green (2,500X) was added, and the fluorescence intensity (485 nm: excitation / 530 nm: fluorescence) was measured using a microplate reader.

[0036] 2. Experimental Results 1) Results of natural product screening Figure 3 shows the results of the primary screening to confirm the antimalarial effect of the natural product fractions of the present invention. Panel A) shows the results of the primary screening of the antimalarial effect on a total of 292 natural product fractions of the present invention, and Panel B) shows the results of selecting 72 natural product fractions that show a higher antimalarial effect than the positive control from the results of the primary screening in Panel A). The red regions in Panels A) and B) of Figure 3 mean natural product fractions that show a better antimalarial effect than the positive control (CQ), the red dots mean natural product fractions that show hemolysis, the red dotted line means the normalized mean fluorescence intensity of the chloroquine positive control, and the blue dotted line shows the normalized mean fluorescence intensity of DMSO. Secondary screening was performed on the selected 72 natural product fractions, and the antimalarial effect IC 50 was analyzed.

[0037] Figure 4 shows the results of analyzing the antimalarial effect IC 50 for 15 natural product fractions selected using the secondary screening of the present invention.

[0038] As a result of the experiment, it was confirmed that the fraction of the natural product CBF-124 had an IC 50 = 0.154 ng / mL, which has been proven through previous research. The fraction of the natural product CBF-150 had an IC 50 = 5.31 μg / mL confirmed, the fraction of the natural product CBF-012 had an IC 50 = 7.57 μg / mL confirmed, the fraction of the natural product CBF-015 had an IC 50 = 8.18 μg / mL confirmed, the fraction of the natural product CBF-151 had an IC 50 = 9.32 μg / mL confirmed, the fraction of the natural product CBF-053 had an IC 50 = 10.36 μg / mL confirmed, and the fraction of the natural product CBF- :011 had an IC50 It was confirmed that the concentration was 10.67 μg / mL, and the fraction of natural product CBF-0111 was IC 50 It was confirmed that the concentration was 12.26 μg / mL, and the fraction of natural product CBF-154 was IC 50 It was confirmed that the concentration was 16.54 μg / mL, and the fraction of natural product CBF-059 was IC 50 It was confirmed that the concentration was 19.11 μg / mL, and the fraction of natural product CBF-060 was IC 50 It was confirmed that the concentration was 26.66 μg / mL, and the fraction of natural product CBF-054 was IC 50 It was confirmed that the concentration was 26.83 μg / mL, and the fraction of natural product CBF-105 was IC 50 It was confirmed that the concentration was 32.48 μg / mL, and the fraction of natural product CBF-034 was IC 50 It was confirmed that the concentration was 35.30 μg / mL, and the fraction of natural product CBF-096 was IC 50 It was confirmed that the concentration was 64.71 μg / mL. The natural product fraction with the most outstanding antimalarial effect was confirmed to be natural product CBF-150, and it was confirmed that natural product CBF-150 is a fraction obtained from the ethanol layer of Geranium thunbergii, and natural product CBF-151 is a fraction obtained from the methanol layer of Geranium thunbergii.

[0039] 2) Analysis of the antimalarial effect of Geranium thunbergii fraction As is clear from the aforementioned natural product screening, among the 292 natural products, it was confirmed that the Geranium thunbergii extract (CBF-150 natural product fraction), excluding CBF-124, which has already been studied for its antimalarial effects, exhibited the best antimalarial effect. In this invention, in order to determine which fraction of the Geranium thunbergii fraction exhibits the best antimalarial effect, the methanol-chloroform fraction (MeOH-CHCl3), ethanol-chloroform fraction (EtOH-CHCl3), methanol-ethyl acetate fraction (MeOH-SiO), ethanol-ethyl acetate fraction (EtOH-SiO), methanol-distilled water fraction (MeOH-DDW), and ethanol-distilled water fraction (EtOH-DDW) of Geranium thunbergii were subjected to the above method to determine the antimalarial effect IC 50 We analyzed it.

[0040] Figure 5 shows the antimalarial effect of the Geranium thunbergii extract of the present invention. 50 The results of the analysis are shown below. Panel A) shows the antimalarial effect IC of methanol-chloroform fraction (MeOH-CHCl3) of Geranium thunbergii. 50 The results of the analysis are shown, and panel B) shows the antimalarial effect of methanol-ethyl acetate fraction (MeOH-SiO) of Geranium thunbergii. 50 The results of the analysis are shown, and panel C) shows the antimalarial effect of methanol-distilled water fraction (MeOH-DDW) of Geranium thunbergii. 50 The results of the analysis are shown, and panel D) shows the antimalarial effect of the ethanol-chloroform fraction (EtOH-CHCl3) of Geranium thunbergii. 50 The results of the analysis are shown, and panel E) shows the antimalarial effect of the ethanol-ethyl acetate fraction (EtOH-SiO) of Geranium thunbergii. 50 The results of the analysis are shown, and panel F) showed the antimalarial effect of ethanol-distilled water fraction (EtOH-DDW) of Geranium thunbergii. 50 The results of the analysis are shown below. The analysis revealed that the methanol-chloroform fraction (MeOH-CHCl3) of Geranium thunbergii was IC2. 50It was confirmed that the IC20 concentration was 27.92 μg / mL, and the methanol-ethyl acetate fraction (MeOH-ELISA) of Geranium thunbergii was IC20. 50 It was confirmed that the concentration was 60.60 μg / mL, and the methanol-distilled water fraction (MeOH-DDW) of Geranium thunbergii was IC2. 50 It was confirmed that the concentration was 283.5 μg / mL. The ethanol-chloroform fraction of Geranium thunbergii (EtOH-CHCl3) showed IC50. 50 It was confirmed that the IC20 concentration was 79.21 μg / mL, and the ethanol-ethyl acetate fraction (EtOH-SiO2) of Geranium thunbergii was IC20. 50 It was confirmed that the concentration was 16.38 μg / mL, and the ethanol-distilled water fraction (EtOH-DDW) of Geranium thunbergii was IC2. 50 It was confirmed that the concentration was 885.1 μg / mL.

[0041] 3. Conclusion In this invention, out of a total of 292 natural products, 72 natural antimalarial materials exhibiting high antimalarial activity against malaria parasites were identified. Among these, 15 natural antimalarial materials with excellent efficacy were identified, and Geranium thunbergii was identified as the material with the most excellent efficacy among them. Geranium thunbergii extract was produced from the aforementioned Geranium thunbergii using methanol or ethanol, and the Geranium thunbergii extract was extracted with chloroform, ethyl acetate, or distilled water. As a result, it was confirmed that the Geranium thunbergii extract extracted with ethanol and ethyl acetate (ethanol-ethyl acetate fraction of Geranium thunbergii) showed the best antimalarial effect, and this was confirmed to be even better than the conventional antimalarial agents chloroquine and artemisinin. Therefore, it is recognized that by using the Geranium thunbergii extract of the present invention, more specifically the ethanol-ethyl acetate extract of Geranium thunbergii, it is possible to develop a novel antimalarial therapeutic agent with improved antimalarial effect.

[0042] The specific embodiments described herein are representative of preferred aspects or examples of the present invention and do not limit the scope of the invention. It will be apparent to those skilled in the art that variations and other uses of the present invention do not depart from the scope of the invention as described in the claims herein.

Claims

1. An antimalarial extract of Geranium thunbergii, prepared using an organic solvent.

2. The antimalarial extract of Geranium thunbergii according to claim 1, characterized in that the organic solvent is methanol, ethanol, or chloroform.

3. The antimalarial Geranium thunbergii extract according to claim 1, characterized in that it is obtained by fractionating a Geranium thunbergii reaction product, which is produced by reacting Geranium thunbergii powder with methanol or ethanol, with methanol, ethanol, or chloroform.

4. The aforementioned antimalarial extract of Geranium thunbergii exhibits malaria activity inhibitory activity (IC). ５０ The antimalarial extract of Geranium thunbergii according to claim 1, characterized in that the concentration is 15 to 70 μg / mL.

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