WO2021060862A1 - Pharmaceutical composition for preventing or treating malaria, containing fucoidan as active ingredient - Google Patents

Abstract

The present invention relates to a composition containing fucoidan as an active ingredient and, more specifically, to: a pharmaceutical composition for preventing or treating malaria, containing fucoidan as an active ingredient; and a dietary supplement composition for preventing or alleviating malaria, containing fucoidan as an active ingredient. The composition containing fucoidan as an active ingredient, according to the present invention, is a natural substance derived from brown algae, and prevents Plasmodium from invading, through adsorption, red blood cells without side effects, thereby being capable of overcoming chloroquine resistance and low sensitivity, and if mixed in a predetermined ratio with chloroquine, which is a conventional treatment agent, the effect thereof further increases, and thus the present invention can be effectively used as a novel agent for treating malaria.

Description

A pharmaceutical composition for preventing or treating malaria containing fucoidan as an active ingredient

The present invention relates to a composition comprising fucoidan as an active ingredient, and more particularly, a pharmaceutical composition for preventing or treating malaria comprising fucoidan as an active ingredient, and a health functional food for preventing or improving malaria comprising fucoidan as an active ingredient It relates to the composition.

41% of the world's population lives in malaria-transmitted areas such as Africa, Asia, the Middle East, Central and South America, Hispaniola and Oceania. There are 350 to 500 million malaria cases each year, of which more than one million dies, most of which are young children in sub-Saharan Africa. In addition, malaria is known to account for about 10.7% of deaths in children in developing countries.

Malaria is an acute and often chronic infectious disease caused by the presence of protozoan parasites in red blood cells. Malaria caused by single-celled parasites in the genus Plasmodium is transmitted from person to person by biting by female mosquitoes. The transmission of malaria is initiated when a female mosquito bites a person already infected with the malaria parasite. When an infected mosquito bites another person, the sporozoite in mosquito saliva passes into the blood and then into the liver. In the liver, sporozoites rapidly divide, then enter the bloodstream and invade red blood cells. Inside these erythrocytes, merocite proliferates rapidly until it ruptures the red blood cells, thereby releasing a new generation of merozite into the bloodstream, which subsequently infects other red blood cells.

Malaria-related symptoms are generally associated with rupture of red blood cells.The destruction of red blood cells leaks waste materials, toxins, and other debris into the blood, which leads to extreme fever, anemia, severe headaches, convulsions, and mental confusion. In addition to symptoms such as back, severe cases lead to death.

As a malaria treatment, quinine, an antimalarial compound extracted from the bark of the South American cinchona tree, has been used for a long time. However, quinine is short-acting and does not prevent recurrence of the disease, and has a disadvantage in that it causes side effects such as dizziness and hearing loss.

Thus, synthetic chemicals similar to quinine, such as chloroquine and mefloquinone, have been developed and used as treatments for malaria, but according to the US CDC, resistance to chloroquine, mefloquine, quinine, etc. in tropical fever protozoa and triday fever protozoa, which are human malaria parasites. As reported, the development of a new malaria treatment that can overcome this is urgent.

In addition, in recent years, according to the trend of naturalism and well-being, interest in drugs using natural products is increasing, and accordingly, development of functional foods and drugs using natural products is continued. However, the development of a natural product-based pharmaceutical composition or raw material thereof, which has a low possibility of causing side effects and resistance, and has excellent malaria treatment effect, is still insufficient.

The present invention has been devised to solve the above-described problems, and in the present invention, it is intended to provide a pharmaceutical composition for preventing or treating malaria comprising fucoidan as an active ingredient.

In the present invention, it is also intended to provide a health functional food composition for preventing or improving malaria comprising fucoidan as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating malaria comprising fucoidan as an active ingredient.

The present invention also provides a health functional food composition for preventing or improving malaria comprising fucoidan as an active ingredient.

The composition comprising fucoidan according to the present invention as an active ingredient is a natural substance derived from brown algae, and can overcome resistance and low sensitivity to chloroquine by preventing invasion into red blood cells through adsorption of malaria protozoa without side effects. When mixed with phosphorus chloroquine in a predetermined ratio, the effect is further increased, and thus it can be usefully used as a new malaria treatment.

Figure 1 is a chloroquine sensitive strain 3D7 strain (Figure 1 (a)) and chloroquine resistant strain Dd2 strain (Figure 1 (b)) inoculated into normal red blood cells, distilled water, chloroquine 100 nM (about 51.5 ng/ml) or 200 After nM (about 103 ng/ml) treatment, a graph showing the result of measuring the malaria infection rate for 48 hours in units of 24 hours.

Figure 2 is a chloroquine sensitive strain 3D7 strain (Figure 2 (a)) and chloroquine resistant strain Dd2 strain (Figure 2 (b)) inoculated into normal red blood cells, distilled water, chloroquine 200 nM (about 51.5 ng/ml), fucoidan A graph showing the result of measuring the malaria infection rate for 48 hours in units of 24 hours after a combination treatment of 20 μg/ml or 200 nM of chloroquine and 20 μg/ml fucoidan (mixed in a volume ratio of 1:1).

Figure 3 is a chloroquine sensitive strain 3D7 strain and chloroquine resistant strain Dd2 strain distilled water, chloroquine 200 nM (about 51.5 ng / ml) or chloroquine 200 nM and fucoidan 20 ㎍ / ml complex treatment (mixed in a volume ratio of 1: 1) after, This is a graph confirming the statistical significance between each group after 48 hours.

4 is a graph showing the result of measuring the change in the number of protozoa according to the mixing ratio (volume ratio) of chloroquine and fucoidan during culture of the Dd2 strain, which is a chloroquine resistant strain.

5 is a result of measuring the change in the number of protozoa according to the combination of chloroquine (200 nM) and each amino acid (alanine, arginine, histidine, lysine and valine) (1 μM) during culture of the Dd2 strain, a chloroquine-resistant strain (measured after 48 hours) It is a graph showing.

Figure 6 is a graph showing the results of measuring red blood cell survival rate according to treatment of chloroquine (100 nM), fucoidan (20 μg/ml) and histidine concentrations (10 nM, 100 nM, 1000 nM) when culturing the Dd2 strain, a chloroquine resistant strain.

7 is a chloroquine-resistant strain Dd2 strain incubation of chloroquine (100 nM) and fucoidan (20 μg/ml) and histidine concentrations (10 nM, 100 nM, 1000 nM) in addition to the concentrations of histidine (10 nM). , 100 nM, 1000 nM) is a graph showing the result of measuring the change in the number of protozoa according to the treatment (measured after 48 hours).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

The present invention relates to a pharmaceutical composition for the prevention or treatment of malaria comprising fucoidan as an active ingredient in one tube.

The present invention relates to a health functional food composition for preventing or improving malaria comprising fucoidan as an active ingredient from another perspective.

As used herein, the term "composition" is considered to include not only products containing certain ingredients, but also any products made directly or indirectly by the combination of certain ingredients.

The term "treatment" as used herein refers to (a) inhibition of the development of a disease, disease or condition; (b) alleviation of the disease, disease or condition; Or (c) to eliminate a disease, disease or condition. The composition of the present invention inhibits the effect of malaria infection by improving the survival rate of malaria-infected cells, or inhibits the generation, proliferation and replication of malaria protozoa, thereby inhibiting the development of a disease or symptoms thereof, which causes malaria infection, or It serves to eliminate or alleviate it. Accordingly, the composition of the present invention may itself be a therapeutic composition for a malaria infection disease, or may be administered together with another therapeutic agent for malaria and applied as a therapeutic adjuvant for suppressing malaria infection. The term "treatment" or "therapeutic agent" includes the meaning of "treatment aid" or "treatment aid".

The term “prevention” as used herein refers to suppressing the occurrence of a disease or disease in a subject that has not been diagnosed with a disease or disease, but is likely to have such a disease or disease.

As used herein, the term "administering" or "administering" refers to directly administering a therapeutically effective amount of a composition of the present invention to a subject so that the same amount or an amount corresponding thereto is formed in the body of the subject.

In the present invention, the pharmaceutical composition may be characterized in that it contains a pharmaceutically acceptable carrier, wherein the carrier is an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, Electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and wool paper. I can.

In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It may be characterized in that it further contains any one or more adjuvants selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity modifiers, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized in that it has a formulation selected from the group consisting of gels and powders.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, weight, age, sex, mode of administration and time of administration of the patient, and generally skilled physicians are effective in the desired treatment or prevention. The dosage can be easily determined.

In the present invention, the health functional food refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No. 6722, and contains nutrients for the structure and function of the human body. It refers to food consumed for the purpose of controlling or obtaining beneficial effects for health purposes such as physiological effects.

The health functional food composition of the present invention may be formulated in a formulation of a general health functional food known in the art, and granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, and drinks. , Alcoholic beverages, etc., and there is no particular limitation on the kind of the health functional food.

The health functional food composition of the present invention is in any herbal form suitable for administration to an animal body including the human body, more specifically any form conventional for oral administration, for example, food or feed, food or feed additives and adjuvants, It may be in a solid form such as fortified food or feed, tablets, pills, granules, capsules and foam formulations, or in liquid form such as solutions, suspensions, emulsions, beverages, pastes, etc., and nutrients, vitamins, electrolytes, sweeteners, coloring agents, organic acids, It may contain a preservative or the like, and these ingredients may be used independently or in combination.

In the present invention, the composition may be characterized in that it further comprises chloroquine.

At this time, the volume ratio of chloroquine and fucoidan contained in the composition is preferably 10:1 to 1:10, and 5:1 to 1:2 and 1:10, as can be seen from the results of the following examples. More preferable.

In the present invention, the composition may be characterized in that it further comprises histidine in addition to chloroquine.

At this time, the concentration ratio of chloroquine and histidine contained in the composition is preferably 1:0.1-10, as can be seen from the results of the following examples.

In addition, the present invention provides a method for preventing or treating malaria or malaria infectious disease comprising administering a therapeutically effective amount of the pharmaceutical composition to a mammal.

In this case, the term "mammal" refers to a mammal that is the object of treatment, observation or experiment, and preferably refers to a human.

In addition, the term "therapeutically effective amount" refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human thought by a researcher, veterinarian, doctor or other clinician, which Includes an amount that induces relief of symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health condition of the patient. , Sex and diet, administration time, administration route and secretion rate of the composition, treatment period, it may be adjusted according to various factors including drugs used simultaneously.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments and the like. However, these examples and the like are intended to describe the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example

Experiment material

The malaria protozoa P. falciparum 3D7 and Dd2 strains used in this experimental example were collected from patients infected with each strain and subcultured in a laboratory-adapted manner, and the culture method was Trager and Jensen's method (1976). It was transformed and used. That is, it was prepared by adding 0.25% AlbuMAX, 0.05% hypoxanthine, 25 mM HEPES buffer, 0.225% sodium bicarbonate, and 10 ㎍/ml gentamycin to RPMI 1640. The prepared culture solution was divided into a T-25 flask, and then normal red blood cells and 3D7 and Dd2 strains were inoculated. Commercially available diphosphorylated chloroquine (Sigma) 5 mg and fucoidan (Sigma) 2 mg were dissolved in distilled water or phosphate buffered saline (PBS) and diluted appropriately to prepare a stock solution for dissolving chloroquine and fucoidan. Subsequently, alanine, arginine, histidine, lysine and valine purchased from Sigma were prepared at a concentration of 100 μM. The prepared flask was injected with a mixture gas composed of each treatment drug and 5% O ₂ , 5% CO ₂ and 90% N ₂ , sealed, and cultured in an incubator at 37°C. During the experiment, parasite blood was stained by Wright stain method and confirmed under a microscope.

Confirmation of chloroquine resistance and measurement of malaria infection rate

Each dissolution stock solution prepared above was appropriately diluted, and a final concentration of 100 nM or 200 nM of chloroquine was treated in a flask inoculated with 3D7 and Dd2, and the malaria infection rate was measured for 48 hours every 24 hours, and the results are as follows. It is shown in Figure 1.

Figure 1 is a chloroquine sensitive strain 3D7 strain (Figure 1 (a)) and chloroquine resistant strain Dd2 strain (Figure 1 (b)) inoculated into normal red blood cells, distilled water, chloroquine 100 nM (about 51.5 ng/ml) or 200 After nM (about 103 ng/ml) treatment, a graph showing the result of measuring the malaria infection rate for 48 hours in units of 24 hours.

As shown in FIG. 1, the experimental results showed that the 3D7 strain, a chloroquine-sensitive strain, significantly decreased parasitemia after 48 hours depending on the treatment concentration of chloroquine, whereas the Dd2 strain, a chloroquine-resistant strain, was treated with chloroquine after 48 hours. No concentration-dependent reduction of parasites was observed.

Measurement of malaria infection rate by fucoidan or fucoidan and chloroquine complex treatment

The experiment was conducted by dividing the 3D7 strain, which is a tropical malaria chloroquine sensitive strain, and the Dd2 strain, which is a chloroquine resistant strain, into a control group, a chloroquine 200 nM treatment group, a fucoidan 20 μg/mL treatment group, and a chloroquine and fucoidan complex treatment group. In the case of the complex treatment of chloroquine and fucoidan, the same volume ratio (1:1) of the stock solution of chloroquine and fucoidan was used.

Figure 2 is a chloroquine sensitive strain 3D7 strain (Figure 2 (a)) and chloroquine resistant strain Dd2 strain (Figure 2 (b)) inoculated into normal red blood cells, distilled water, chloroquine 200 nM (about 51.5 ng/ml), fucoidan A graph showing the result of measuring the malaria infection rate for 48 hours in units of 24 hours after a combination treatment of 20 μg/ml or 200 nM of chloroquine and 20 μg/ml fucoidan (mixed in a volume ratio of 1:1).

As shown in FIG. 2, when fucoidan alone was treated for 3D7 and Dd2, parasitic congestion was significantly reduced compared to the control group after 48 hours, and when chloroquine and fucoidan were combined, the degree of parasitic congestion was further improved. , It was confirmed that the infection inhibitory ability was very good.

Fucoidan Chloroquine According to complex treatment 3D7 And Dd2 Statistical Significance of Strain Reduction in Parasitic Congestion

Figure 3 is a chloroquine sensitive strain 3D7 strain and chloroquine resistant strain Dd2 strain distilled water, chloroquine 200 nM (about 51.5 ng / ml) or chloroquine 200 nM and fucoidan 20 ㎍ / ml complex treatment (mixed in a volume ratio of 1: 1) after, This is a graph confirming the statistical significance between each group after 48 hours.

3A is a result of confirming the statistical significance between the treated chloroquine-sensitive strain 3D7 strain and the chloroquine-resistant strain Dd2 strain, and it was confirmed that there was a significant difference in parasitic congestion results in the chloroquine-treated group. Figure 3 (b) is a result of confirming the statistical significance between distilled water, chloroquine or chloroquine and fucoidan complex treatment group in the 3D7 strain, which is a chloroquine sensitive strain, as a result of parasitic congestion between chloroquine alone treatment group and chloroquine and fucoidan complex treatment group. There was no difference, and FIG. 3 (c) is a result of confirming the statistical significance between distilled water, chloroquine or chloroquine and fucoidan complex treatment group within the Dd2 strain, which is a chloroquine resistant strain, as a result of confirming the statistical significance between the chloroquine treatment group and the chloroquine and fucoidan complex It was confirmed that there was a significant difference in parasitic congestion results between treatment groups.

Measurement of changes in the number of protozoa of the Dd2 strain according to the mixing ratio of fucoidan and chloroquine

The change in the number of protozoa of the Dd2 strain according to the mixing ratio (volume ratio) of fucoidan and chloroquine was measured, and the results are shown in FIG. 4 below. The mixing ratio experiment was performed by setting the volume ratio of chloroquine and fucoidan stock solution to 10:1 (187.6ng/ml: 3.64μg/ml), 5:1 (171.9ng/ml: 6.7μg) in the same total amount (200 μl) of 1:1 volume ratio. /ml), 2:1(137.6ng/ml: 13.32μg/ml), 1:1(103.2ng/ml: 20μg/ml), 1:2(68.7ng/ml: 26.7μg/ml), 1: 5 (34.4 ng/ml: 33.3 μg/ml) and 1:10 (18.8 ng/ml: 36.4 μg/ml) were adjusted to proceed.

As shown in Figure 4, it was found that the number of protozoa was effectively reduced regardless of the mixing volume ratio during the complex treatment of chloroquine and fucoidan, and in particular, chloroquine and fucoidan were mixed in a volume ratio of 5:1 to 1:2 and 1:10. Therefore, it was confirmed that the effect of reducing the number of protozoa during treatment was very excellent.

Chloroquine According to the complex treatment of amino acids Chloroquine tolerance Tropical heat Malaria infection rate measurement

When culturing the Dd2 strain, a chloroquine-resistant strain, chloroquine (200 nM) and each amino acid (alanine, arginine, histidine, lysine and valine) (1000 nM) were combined with each other and measured the number of protozoa after 48 hours. Screening was performed, and the results are shown in FIG. 5 below.

As shown in FIG. 5, unlike the group treated with alanine, arginine, lysine, and valine, the protozoal infection rate was significantly reduced from 8.9% (0 hours) to 2.9% (48 hours) when combined with histidine. Considering that the 8.8% protozoan infection rate at 0 hours decreased to 5.9% after 48 hours in the group treated with chloroquine 200 nM alone, the protozoal infection rate decreased by more than two times through the combination treatment of chloroquine and histidine. Was confirmed.

Fucoidan Chloroquine And histidine complex treatment of red blood cells Survival rate And Chloroquine Measurement of resistant tropical fever malaria infection rate

Following the above-identified complex treatment of chloroquine (200 nM) and fucoidan (20 μg/ml), the experiment was conducted by additional treatment of histidine by concentration in order to increase the efficacy of the drug by reducing the concentration of chloroquine and to increase the viability of red blood cells. Specifically, chloroquine (100 nM), fucoidan (20 μg/ml), and various concentrations of histidine (10 nM, 100 nM, 1000 nM) were treated with PfDd2 strain protozoa, a chloroquine-resistant tropical malaria, and cultured in a flask for 48 hours. I did. After the start of the culture, the culture medium was collected at 0, 24, and 48 hours to measure the survival rate and protozoal infection rate of red blood cells, and the results are shown in FIGS. 6 and 7 below.

As shown in Figure 6, the combination treatment by concentration of chloroquine (100 nM), fucoidan (20 μg/ml) and histidine was compared to the negative control group (distilled water treatment group) compared to the survival rate of red blood cells (number of red blood cells / number of red blood cells after 48 hours / number of red blood cells) ) Was found to increase significantly. Specifically, in the distilled water treatment group, 66% of red blood cells survived compared to 0 hours, and the chloroquine-fucoidan-histidine complex treatment group was 77%, 75%, and 81 in the combined treatment groups with histidine concentrations of 10 nM, 100 nM and 1000 nM, respectively. % Was found to have a very good red blood cell survival rate. In addition, the red blood cell survival rate was 77% in the chloroquine 100 nM, fucoidan 20 μg/ml and histidine 10 nM combination treatment group compared to the chloroquine 100 nM treatment group, which showed a 70% red blood cell survival rate, confirming that the red blood cell survival rate was significantly increased. I did.

In addition, as shown in Figure 7, chloroquine (200 nM) and fucoidan (20 μg / ml) complex treatment group showed a protozoal infection rate of 1.32%, chloroquine (100 nM), fucoidan (20 μg / ml) and histidine Complex treatments by concentration showed 2.58%, 1.79%, and 1.71% protozoal infection rates, respectively, after 48 hours of cultivation. Through these results, when the combination treatment of chloroquine (100 nM), fucoidan (20 μg/ml) and histidine (100 nM or 1000 nM) was equivalent to the combination treatment of chloroquine (200 nM) and fucoidan (20 μg/ml). It was confirmed that the protozoal infection rate was shown, and through this, it was confirmed that the excellent effect of reducing the number of protozoa can be exhibited even under a low concentration of chloroquine when histidine is additionally included.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

The composition of the present invention can overcome resistance and low sensitivity to chloroquine, and when mixed with chloroquine, which is a conventional therapeutic agent, in a predetermined ratio, the effect is further increased, and thus it can be usefully used as a new malaria treatment.

Claims (12)

A pharmaceutical composition for preventing or treating malaria comprising fucoidan as an active ingredient. The method of claim 1, The composition is a pharmaceutical composition for preventing or treating malaria, characterized in that it further comprises chloroquine. The method of claim 2, A pharmaceutical composition for preventing or treating malaria, characterized in that the volume ratio of the chloroquine and the fucoidan is 10:1 to 1:10. The method of claim 3, A pharmaceutical composition for preventing or treating malaria, characterized in that the volume ratio of the chloroquine and the fucoidan is 5:1 to 1:2 and 1:10. The method of claim 2, The composition is a pharmaceutical composition for preventing or treating malaria, characterized in that it further comprises histidine. The method of claim 5, A pharmaceutical composition for preventing or treating malaria, characterized in that the concentration ratio of the chloroquine and the histidine is 1:0.1-10. A health functional food composition for preventing or improving malaria comprising fucoidan as an active ingredient. The method of claim 7, The composition is a health functional food composition for preventing or improving malaria, characterized in that it further comprises chloroquine. The method of claim 8, A health functional food composition for preventing or improving malaria, characterized in that the volume ratio of the chloroquine and the fucoidan is 10:1 to 1:10. The method of claim 9, A health functional food composition for preventing or improving malaria, characterized in that the volume ratio of the chloroquine and the fucoidan is 5:1 to 1:2 and 1:10. The method of claim 8, The composition is a health functional food composition for preventing or improving malaria, characterized in that it further comprises histidine. The method of claim 11, The health functional food composition for preventing or improving malaria, characterized in that the concentration ratio of the chloroquine and the histidine is 1:0.1-10.

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