KR102498802B1 - Pharmaceutical composition for preventing or treating malaria comprising fucoidan - Google Patents

Abstract

The present invention relates to a composition containing fucoidan as an active ingredient, and more particularly, to a pharmaceutical composition containing fucoidan as an active ingredient for preventing or treating malaria, and a health functional food containing fucoidan as an active ingredient for preventing or improving malaria. It's about the composition.
The composition containing 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 malaria parasites from entering red blood cells through adsorption without side effects, and can overcome conventional therapeutic agents. When mixed with phosphorus chloroquine at a predetermined ratio, the effect is further enhanced, and thus it can be usefully used as a new antimalarial drug.

Description

Pharmaceutical composition for preventing or treating malaria comprising fucoidan as an active ingredient

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

41% of the world's population lives in malaria-endemic regions such as Africa, Asia, the Middle East, Central and South America, Hispaniola and Oceania. Malaria affects 350 to 500 million people each year, of which more than one million patients die, most of them young children in sub-Saharan Africa. In addition, malaria is known to account for about 10.7% of all deaths among children in developing countries.

Malaria is an acute and often chronic infectious disease caused by the presence of protozoan parasites within red blood cells. Malaria, caused by unicellular parasites of the genus Plasmodium, is transmitted from person to person by the bite of a female mosquito, and 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 sporozoites in the mosquito's saliva are transferred to the blood and then to the liver. In the liver, sporozoites divide rapidly, then enter the bloodstream and invade red blood cells. Inside these red blood cells, merozoites multiply rapidly until they rupture, releasing new generations of merozoites into the bloodstream, which then infect other red blood cells.

Malaria-related symptoms usually involve the rupture of red blood cells, which leak waste materials, toxins, and other debris into the blood, which can cause extreme fever, anemia, severe headaches, convulsions, and confusion. In addition to symptoms such as etc., it leads to death in severe cases.

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

Accordingly, synthetic chemicals similar to quinine, such as chloroquine and mefloquinone, have been developed and used as antimalarial drugs. As reported, there is an urgent need to develop a new malaria treatment that can overcome this.

In addition, recently, interest in drugs using natural products has increased according to the trend of naturalism and well-being, and accordingly, functional foods and drugs using natural products have been developed. However, the development of a natural product-based pharmaceutical composition or a raw material thereof, which has excellent antimalarial effects while having a low possibility of side effects and tolerance, has yet to be developed.

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

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

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

The composition containing 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 malaria parasites from entering red blood cells through adsorption without side effects, and can overcome conventional therapeutic agents. When mixed with phosphorus chloroquine at a predetermined ratio, the effect is further enhanced, and thus it can be usefully used as a new antimalarial drug.

1 is a chloroquine-sensitive strain, 3D7 strain (Fig. 1 (a)) and a chloroquine-resistant strain, Dd2 strain (Fig. 1 (b)), inoculated into normal red blood cells, distilled water, chloroquine 100 nM (about 51.5 ng / ml) or 200 After treatment with nM (about 103 ng/ml), it is a graph showing the results of measuring the infection rate of malaria for 48 hours at 24 hour intervals.
Figure 2 is a chloroquine-sensitive strain 3D7 strain (Fig. 2 (a)) and chloroquine-resistant strain Dd2 strain (Fig. 2 (b)) inoculated into normal red blood cells, distilled water, chloroquine 200 nM (about 51.5 ng / ml), fucoidan This is a graph showing the results of measuring the malaria infection rate for 48 hours in 24 hour increments after 20 μg/ml or 200 nM of chloroquine and 20 μg/ml of fucoidan (mixed at a volume ratio of 1:1).
3 is a chloroquine-sensitive strain 3D7 strain and a chloroquine-resistant strain Dd2 strain in distilled water, chloroquine 200 nM (about 51.5 ng / ml) or chloroquine 200 nM and fucoidan 20 μg / ml after complex treatment (mixing at a volume ratio of 1: 1), It is a graph confirming the statistical significance between each group after 48 hours.
Figure 4 is a graph showing the results of measuring the change in the number of protozoa according to the mixing ratio (volume ratio) of chloroquine and fucoidan during cultivation of the chloroquine-resistant strain Dd2 strain.
Figure 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) when culturing the chloroquine-resistant strain Dd2 strain (measured after 48 hours) is a graph showing
Figure 6 is a graph showing the measurement results of red blood cell viability according to the concentrations (10 nM, 100 nM, 1000 nM) of chloroquine (100 nM), fucoidan (20 μg / ml) and histidine when culturing the chloroquine-resistant strain Dd2 strain.
Figure 7 shows chloroquine (100 nM), fucoidan (20 μg / ml), and histidine concentrations (10 nM, 100 nM, 1000 nM) in addition to treatment according to histidine concentration (10 nM) when culturing Dd2 strain, a chloroquine-resistant strain. , 100 nM, 1000 nM) is a graph showing the results of measuring the change in the number of protozoa (measured after 48 hours) according to the treatment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating malaria comprising fucoidan as an active ingredient.

From another point of view, the present invention relates to a health functional food composition for preventing or improving malaria containing fucoidan as an active ingredient.

As used herein, the term "composition" is intended to include any product made directly or indirectly by combining the specific components, as well as products containing the specific components.

As used herein, the term "treatment" includes (a) inhibition of the development of a disease, condition or condition; (b) alleviation of disease, illness or symptoms; or (c) eliminating the disease, disorder or condition. The composition of the present invention inhibits the effects of malaria infection by improving the survival rate of malaria-infected cells, or suppresses the development of diseases or symptoms caused by malaria infection by inhibiting the generation, proliferation and replication of malaria parasites, It serves to eliminate or alleviate it. Therefore, the composition of the present invention may be used as a treatment composition for malaria infection by itself or may be administered together with other anti-malarial drugs to be applied as a therapeutic adjuvant to suppress malaria infection. The term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

As used herein, the term “prevention” means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed with, but is likely to have, the disease or condition.

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

In the present invention, the pharmaceutical composition may be characterized by containing a pharmaceutically acceptable carrier, and the carrier may include ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer substances, water, salt, At least one selected from the group consisting of electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and wool. can

In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, buccal, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It may be characterized in that it further contains at least one adjuvant selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity adjusters, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized by having 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, the patient's weight, age, sex, administration method and administration time, and usually a skilled physician is effective in the treatment or prevention desired. The dosage can be easily determined.

In the present invention, health functional food refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body according to Act No. It refers to food consumed for the purpose of obtaining useful effects for health purposes such as regulation or physiological action.

The health functional food composition of the present invention can be formulated into a conventional health functional food formulation known in the art, and can be formulated into granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, and drinks. , It can be made into alcoholic beverages, etc., and there is no particular limitation on the type of health functional food.

The health functional food composition of the present invention is any herbal form suitable for administration to the human body or animal body, more specifically, any form conventional for oral administration, such as food or feed, food or feed additives and supplements, Solid forms such as fortified foods or feeds, tablets, pills, granules, capsules and effervescent formulations, or liquid forms such as solutions, suspensions, emulsions, beverages, pastes, etc., nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, It may contain preservatives and the like, and these components may be used independently or in combination.

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

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

In the present invention, the composition may further include histidine in addition to chloroquine.

At this time, the concentration ratio of chloroquine and histidine included 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 diseases, 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 a subject of treatment, observation, or experimentation, 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, as considered by a researcher, veterinarian, physician or other clinician, which An amount that induces relief of the symptoms of the disease or disorder being treated is included. It is obvious 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 dose 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 the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, and general health condition , Gender and diet, administration time, administration route and secretion rate of the composition, treatment period, can 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 are intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Example

experimental material

The malaria protozoan 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 the method of Trager and Jensen (1976). modified 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 μg/ml gentamycin to RPMI 1640. After the prepared culture medium was divided into T-25 flasks, normal red blood cells and each strain of 3D7 and Dd2 were inoculated. 5 mg of commercially available diphosphate chloroquine (Sigma) and 2 mg of fucoidan (Sigma) were dissolved in distilled water or phosphate buffered saline (PBS), and then diluted appropriately to prepare chloroquine and fucoidan dissolved stock solutions. Alanine, arginine, histidine, lysine and valine purchased from Sigma were then prepared at a concentration of 100 μM. The prepared flask was injected with a mixed 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 with the Wright stain method and confirmed under a microscope.

Chloroquine Resistance confirmation and malaria infection rate measurement

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

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

As shown in Figure 1, as a result of the experiment, the chloroquine-sensitive strain 3D7 strain showed a significant decrease in parasitemia depending on the treatment concentration of chloroquine after 48 hours, whereas in the case of the chloroquine-resistant strain Dd2 strain, even after 48 hours of chloroquine treatment, A concentration-dependent decrease in parasitemia was not observed.

fucoidan or with fucoidan Chloroquine Measurement of malaria infection rates according to multiple treatments

Experiments were conducted by dividing 3D7, a chloroquine-sensitive strain of P. malaria, and Dd2, 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 combined treatment of chloroquine and fucoidan, the same volume ratio (1:1) of chloroquine and fucoidan stock solution was used.

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

As shown in Figure 2, it was found that parasitemia was significantly reduced compared to the control group after 48 hours when fucoidan alone was treated in 3D7 and Dd2, and the degree of parasitemia reduction was further improved when chloroquine and fucoidan were combined. , it was confirmed that the infection inhibition ability was very excellent.

Statistical significance measurement of parasite blood reduction in 3D7 and Dd2 strains by combined treatment with fucoidan and chloroquine

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

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

with fucoidan Chloroquine according to the mixing ratio Dd2 of strain protozoa measure change

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. In the mixing ratio experiment, the volume ratio of chloroquine and fucoidan stock solution was 10:1 (187.6ng/ml : 3.64μg/ml), 5:1 (171.9ng/ml : 6.7μg) within 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 (34.4ng/ml: 33.3μg/ml), 1:10 (18.8ng/ml: 36.4μg/ml).

As shown in FIG. 4, it was found that the number of protozoa was effectively reduced regardless of the mixing volume ratio during complex treatment with chloroquine and fucoidan. It was confirmed that the effect of reducing the number of protozoa during treatment was very excellent.

Measurement of infection rate of chloroquine-resistant falciparum malaria according to combined treatment with chloroquine and amino acids

When culturing the chloroquine-resistant Dd2 strain, chloroquine (200 nM) and each amino acid (alanine, arginine, histidine, lysine, and valine) (1000 nM) were treated in combination and the change in the number of protozoa was measured 48 hours later. It was screened, and the results are shown in Figure 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 hour) to 2.9% (48 hours) when histidine was combined. Considering that the 8.8% protozoan infection rate at 0 hours in the group treated with 200 nM chloroquine alone decreased to 5.9% after 48 hours, the combined treatment of chloroquine and histidine resulted in a more than two-fold decrease in protozoan infection rate confirmed.

with fucoidan Chloroquine and erythrocytes according to histidine complex treatment. survival rate and Chloroquine tolerance tropical fever Malaria infection rate measurement

Following the previously confirmed complex treatment of chloroquine (200 nM) and fucoidan (20 μg/ml), the experiment was conducted by additionally treating histidine by concentration in order to increase the efficacy of the drug by reducing the concentration of chloroquine and 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 parasite, a chloroquine-resistant falciparum malaria, and cultured in a flask for 48 hours. did After the start of the culture, the culture medium was collected at 0 hour, 24 hours, and 48 hours to measure the survival rate of red blood cells and the infection rate of protozoa, and the results are shown in FIGS. 6 and 7 below.

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

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

Having described specific parts of the present invention in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the invention will be defined by the appended claims and their equivalents.

Claims (12)

Contains fucoidan, chloroquine and histidine as active ingredients,
The volume ratio of the chloroquine and the fucoidan is 5:1 to 1:2 and 1:10,
The pharmaceutical composition for preventing or treating malaria, characterized in that the concentration ratio of the chloroquine and the histidine is 1:0.1-10. delete delete delete delete delete Contains fucoidan, chloroquine and histidine as active ingredients,
The volume ratio of the chloroquine and the fucoidan is 5:1 to 1:2 and 1:10,
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. delete delete delete delete delete

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