JP2008195668A - Composition for preventing infection of protozoan, and method for preventing infection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition preventing the infection of a protozoan. <P>SOLUTION: The composition for preventing the infection of the protozoan, especially Trypanosoma brucei contains an aqueous solvent extract of humus soil as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a composition for protecting against infection with protozoa, particularly against Trypanosoma brucei, and a method for protecting against infection with Trypanosoma brucei.

  It is known that soil called humus soil is obtained from wetlands and the seabed, and has been humused to a state where flora and fauna of plankton are deposited and its structural shape is not seen. Humus is rich in fulvic acid, humic acid and various minerals. Moreover, it is known that the extract extracted from humus soil has a bactericidal action, and it was conventionally used as a bactericidal deodorant etc. (for example, patent documents 1-6).

In addition, it is known that a composition containing an aqueous solvent extract of humus has anti-cancer properties (Patent Document 7) and can enhance infection defense against bacterial, fungal and viral diseases in fish (Patent Document 8). It has been.
JP 7-492 A Japanese Patent Laid-Open No. 10-59861 JP-A-11-29423 JP-A-5-192666 JP-A-6-87752 JP-A 63-84618 JP 2006-111537 A JP 2006-109709 A

  The present inventors have now administered a composition comprising an aqueous solvent extract of humus soil to a mouse and then challenged the mouse with a protozoan, particularly Trypanosoma brucei, so that the protozoa The present invention was completed by finding out that it does not infect animals.

Therefore, the present invention provides a composition for protecting against protozoa, particularly Trypanosoma brucei, comprising an aqueous solvent extract of humus as an active ingredient.
The present invention also provides an infection protection method for protozoa, particularly Trypanosoma brucei, comprising administering the above composition to animals other than humans.

  By using the composition of the present invention, infection of protozoa, particularly Trypanosoma brucei, and thus infection caused by infection of the protozoa can be suppressed.

  The composition of the present invention is a composition for protecting against protozoa, particularly Trypanosoma brucei. Trypanosoma brucei includes Trypanosoma brucei brucei and Trypanosoma brucei gambiense.

In the present invention, the aqueous solvent extract of humus soil is obtained by extracting the humus soil with an aqueous solvent.
The humus soil is mainly composed of humic substances produced by depositing dead bodies of animal and phytoplankton and being decomposed by microorganisms. This humic substance is formed by repeated decomposition and metabolism by microorganisms, and is known to contain various organic substances and minerals.
Although the humus used in this invention is not specifically limited, The soil which exists in 5-30m from the soil surface of a wetland, the soil which exists in 0.5-250m from the seabed surface, etc. can be used preferably.

  Examples of the aqueous solvent include water; an acidic aqueous solution such as dilute hydrochloric acid; and an aqueous alcohol such as aqueous ethanol. Water from which chlorine is removed is preferable. The method for removing chlorine is not particularly limited, and examples thereof include a method of leaving at room temperature for several days, a method using a reducing agent such as ascorbic acid, and a method using an adsorbent such as activated carbon. Although the pH of an aqueous solvent is not specifically limited, The thing of the range of pH 6.0-8.0 is preferable, More preferably, it is the range of pH 6.3-7.5.

Although the method to obtain the aqueous solvent extract of humus soil is not specifically limited, The method containing the following process (1)-(3) is preferable.
(1) The mixture of humus soil and an aqueous solvent is repeatedly stirred and allowed to stand until the pH is 2.8 or less and the electric conductivity is 400 mV or more.
(2) remove solids,
(3) The obtained supernatant is allowed to stand at ambient temperature for a long time.

In the step (1), first, a suspension is obtained by adding 5 to 10 times (weight ratio) of an aqueous solvent to the humus soil while stirring in a container such as a water tank.
The resulting suspension is then treated at ambient temperature. In the present specification, the ambient temperature is a temperature of 20 to 45 ° C, preferably 25 to 40 ° C, more preferably 28 to 35 ° C.
The above treatment is preferably performed by repeating stirring and standing. Stirring may be performed to such an extent that the humus deposited at the bottom of the container is diffused into the aqueous solvent, and may be either continuous or intermittent. This treatment is preferably performed until the supernatant obtained by allowing the suspension to stand still has a pH of 2.8 or less, an electric conductivity of about 400 mV or more, and a temperature of 28 ° C. or more for one week or more. . Such a state can usually be obtained about 3 to 5 weeks after mixing the humus soil and the aqueous solvent. In this step, nutrient sources such as a nitrogen source and a phosphorus source can be added as desired.

  The method for removing the solid matter in the step (2) is not particularly limited as long as it is a method capable of separating the humus soil and the liquid and removing the humus soil. it can. In the method of standing, the suspension is allowed to stand at 25 to 35 ° C. for about 150 to 240 hours to precipitate humus soil, and then only the supernatant is obtained. In order to remove the solid matter more reliably, the suspended matter may be further removed using a cloth or the like. The supernatant thus obtained has a pH of about 2.5 to 3.0.

  Step (3) is performed by leaving the obtained supernatant at ambient temperature for a long period of time. The long term may be about 180 to 500 days, preferably about 300 to 400 days. During this standing, the temperature may be constant or may vary. Usually, when it hold | maintains for 30 to 60 days at the temperature of 25-35 degreeC, and holds for 30 to 60 days at the temperature of 5-15 degreeC, the aqueous-solvent extract of humus soil more preferable to use for animal feed other than a human is obtained. be able to.

  The liquid obtained by the above standing may be further filtered. Filtration may be performed to such an extent that suspended matters in the liquid can be removed. For example, the filtration can be performed using two types of membrane filters having a pore diameter of 40 to 100 μm and 10 to 40 μm.

The aqueous solvent extract of humus obtained by the above method is a light brown, substantially odorless liquid having a pH of 2.0 to 3.0 and an electric conductivity of 350 to 500 mV.
The aqueous solvent extract of humus soil has a weight after freeze-drying of 0.5 to 1 g per 100 ml of the aqueous solvent extract of humus soil, and is adsorbed through a DAX-8 resin column (fulvic acid). The weight after freeze-drying is 0.001 g or less per 100 ml of the aqueous solvent extract of humus.

The aqueous solvent extract of the above humus soil preferably contains the following components in 1 liter.
Calcium 200-300ppm
Sulfur 1000-1500ppm
Iron 10-50ppm
Zinc 1-8ppm
Copper 0.1-1ppm
Sodium 80-100ppm
Manganese 5-10ppm
Magnesium 80-100ppm
Aluminum 300-400ppm
Silicon 30-40ppm
Nickel 0.1-1ppm
Strontium 0.5-5ppm
Yttrium 0.1-1ppm
Lithium 0.1-1ppm

Furthermore, the aqueous solvent extract of the above humus can contain a trace amount of boron, lanthanum, scandium, selenium and the like.
Each of these components can be measured using a high frequency induction analyzer (ICP).

  The aqueous solvent extract of the above humus can be used after being produced by the above method and then concentrated to an appropriate volume. The degree of concentration is not particularly limited as long as moisture remains. For example, it can be used after being concentrated to 90% or less, preferably 80% or less, more preferably 70% or less of the volume before concentration. .

  The aqueous solvent extract of the above humus can be concentrated as it is or diluted with a suitable medium to obtain the composition of the present invention. The medium used for dilution is not particularly limited as long as it is harmless to the human body and does not impair the effects of the composition of the present invention, and water is preferably used.

  To the composition of the present invention, additives such as preservatives (eg, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid), coloring agents, and fragrances that are commonly used in foods and pharmaceuticals are added. Can do.

  The amount of intake of the composition of the present invention is not particularly limited because the composition is harmless to animals including humans, but if the composition is ingested about 5 to 10 mL per kg of body weight per day, the protozoan Can protect against infection and maintain health.

  When the composition of the present invention is used as a medicine, it can be used in the form of a liquid preparation as it is, after being concentrated or diluted with a suitable medium.

  Said pharmaceutical formulation may contain the additive added to a normal pharmaceutical solution. Such additives include pharmaceutically acceptable additives such as suspending agents (eg sorbitol, syrup, methylcellulose, glucose syrup, gelatin edible fat), emulsifiers (eg lecithin, sorbitan monooleate or Gum arabic), non-aqueous excipients (eg, almond oil, fractionated coconut oil or oily esters of alcohols such as glycerin, propylene glycol or ethyl alcohol), preservatives, colorants or fragrances.

The food containing the composition of the present invention is not particularly limited, and other than normal foods, foods intended for health, health maintenance and promotion in a more positive sense than normal foods, such as health foods, functions Foods, dietary supplements, supplements or foods for specified health use.
Examples of such foods intended for health and health maintenance / promotion include liquid or semi-solid or solid products, specifically powders, granules, tablets, capsules, etc. Liquid form is preferred.

Examples of normal food include beverages (soft drinks such as coffee, juice and tea drinks,
Milk drinks, lactic acid bacteria drinks, carbonated drinks, etc .; Fermented foods (bread, miso, etc.); Noodles (udon, pasta, etc.); Confectionery (jelly, yokan, yogurt, etc.); Processed goods (soup, curry, beef bowl, Miscellaneous cooking, miso soup, etc.); seasonings (dressing, umami seasonings, etc.).
An ordinary food containing the composition of the present invention can be produced by adding the composition of the present invention by mixing, coating, spraying or the like in the above-described food production process or to the final product.

The composition of the present invention can protect against infection with protozoa, particularly Trypanosoma brucei, by administering to animals. Therefore, a method for protecting against protozoa, particularly Trypanosoma brucei, comprising administering the above composition to an animal is also one aspect of the present invention.
Examples of the method for administering the above composition include oral administration and intraperitoneal administration, and oral administration is preferred.

  In the method of the present invention, an animal is intended to mean mammals including humans (for example, humans, mice, rats, guinea pigs, rabbits, horses, cows, dogs, cats, etc.), birds and fish.

  In the above method, the above composition is preferably administered, for example, at about 5 to 10 mL / day per kg of body weight.

<Method for producing aqueous solvent extract of humus soil>
PH 6.5 dechlorinated water was added to humus soil collected at a point 20 m from the soil surface in Nagasaki Prefecture with stirring, and the mixture was allowed to stand at ambient temperature for 30 days with stirring twice a day. Thereafter, the suspension was allowed to stand to obtain a supernatant, which was filtered with a cloth to remove suspended matters, and allowed to stand at ambient temperature for 365 days. In this period, the maximum ambient temperature was 30 ° C., and the minimum temperature was 10 ° C. The obtained supernatant was filtered through a housing type filter (manufactured by Cuno) having a pore size of 50 μm and 25 μm to obtain an aqueous solvent extract of humus soil. The pH of the aqueous solvent extract of the obtained humus soil is 2.8 ± 0.05 (measured with a pH meter model 9620 manufactured by Horiba Seisakusho), and the electrical conductivity is 400 mV or more (electric conductivity produced by Horiba Seisakusho). Degree measurement meter 9382-10D type). Moreover, the lyophilized weight per 100 ml of the aqueous solvent extract of the obtained humus soil was 0.65 g, and the weight after lyophilization of the component adsorbed on the DAX-8 resin column (manufactured by Spelco) was 0.0002 g. Met.
Table 1 shows the results of measuring the aqueous solvent extract of this humus soil with a high-frequency induction analyzer (ICP, manufactured by PerkinElmer).

Example 1
The aqueous solvent extract of humus obtained as described above (hereinafter referred to as humus soil extract) is diluted to 1, 2, 3, 4 or 6% by volume in chlorine-free tap water. As a control, chlorine-free tap water (tap water) to which no humus soil extract was added was used.
Eight-week-old BALB / c mice (6 to 20 mice per group, the same number of males and females, average body weight 20 g) were allowed to freely ingest a diluted solution of humus soil extract or tap water, and were bred for 21 days.
Each group of mice was challenged by intraperitoneal injection of 10 Trypanosoma brucei brucei (1.0 × 10 ¹ /0.1 mL-phosphate physiological saline sucrose (PSG) buffer). The survival of mice in each group was observed for 20 days. During the observation period, a diluted solution of humus soil extract or tap water was freely ingested.

The survival rate of each group of mice 20 days after challenge with trypanosomes is shown in FIG. Significant differences between the control group and the humus soil extract diluted solution administration group at each concentration were calculated by chi-square test.
In addition, Table 2 shows the average days of death of mice in the control group and the humus soil extract diluted solution administration group of each concentration.

  From the results of FIG. 1 and Table 1, it can be seen that administration of Trypanosoma brucei brucei in mice could be prevented by administering a diluted solution of humus soil extract.

Comparative Example 1
We investigated whether infection with Trypanosoma brucei brucei could be prevented when mice were administered fulvic acid, which is known to be a component of humus soil.
Purified fulvic acid dissolved in phosphate buffered saline (prepared by Prof. Miyajima, Faculty of Science and Engineering, Saga University) at 200 μg per administration, 8 weeks old BALB / c mice (8 mice per group, same sex, An average body weight of 20 g) was given by intraperitoneal injection.
The administration of purified fulvic acid was performed 6 days before and 2 days after the challenge by intraperitoneal injection of 10 Trypanosoma brucei brucei (1.0 × 10 ¹ /0.1 mL-PSG buffer), and 2 days after the challenge. And on the 6th day (FIG. 2). Control group mice (n = 10) were similarly administered phosphate buffered saline without fulvic acid.

After challenge with Trypanosoma brucei brucei, the mice of each group were observed for life and death. The result of calculating the survival rate is shown in FIG.
From this result, it can be seen that administration of purified fulvic acid does not show the effect of preventing infection with Trypanosoma brucei brucei in mice.

Example 2
Experiments similar to Example 1 were performed using Trypanosoma brucei gambiense as the protozoan to attack. That is, as in the experiment of Example 1, mice (n = 6, same sex) were ingested with a 3% diluted solution of humus soil extract for 21 days. Control mice (n = 6) received tap water. The mice were then challenged by intraperitoneal injection of 10 Trypanosoma brucei gambiense (1.0 × 10 ¹ /0.1 mL-PSG buffer).

  The survival rate of each group of mice after challenge with trypanosomes is shown in FIG. The average number of days of death of mice given a 3% dilution of the humus soil extract was 22.4 days (p <0.01 compared to the control group), and the average number of days of death of mice in the control group was 7.3 days. Met.

Example 3
In order to investigate the effect of the period of giving a diluted solution of humus soil extract to mice on the survival rate of mice, the following experiment was conducted.
Experiments were conducted in the same manner as in Example 1 except that mice were given a 3% diluted solution of humus soil extract 21 days before, 10 days, 5 days, or 0 days before the challenge by intraperitoneal injection of Trypanosoma brucei brucei. Went. Control mice received tap water from 21 days prior to challenge by intraperitoneal injection of Trypanosoma brucei brucei.

  The survival rate of each group of mice is shown in FIG. It can be seen that the survival rate of the mice given the 3% dilution of the humus soil extract from 21 days before the challenge was the highest.

  From the above, it can be understood that trypanosoma infection can be prevented by administering a composition containing a humus soil extract to animals.

It is a graph which shows the survival rate of 21 days after the challenge with the trypanosomes of the mouse | mouth (n = 6-20) which administered the humus soil extract or the tap water for 21 days. 4 is a dosing schedule in the administration experiment of the purified fulvic acid of Comparative Example 1. FIG. It is a graph which shows the survival rate for 12 days after the challenge with the trypanosomes of the mouse | mouth which administered purified fulvic acid (n = 8) or tap water (n = 10). It is a graph which shows the survival rate in 20 days after the challenge with the trypanosomes of the mouse | mouth (n = 6) which administered the humus soil extract or the tap water for 21 days. It is a graph which shows the influence which the period which gives a humus soil extract to a mouse | mouth before the attack with trypanosoma has on the survival rate of a mouse | mouth.

Claims (6)

  A composition for protection against protozoa, particularly Trypanosoma brucei, comprising an aqueous solvent extract of humus as an active ingredient.   The composition according to claim 1, wherein the Trypanosoma brucei is Trypanosoma brucei brucei or Trypanosoma brucei gambiense.   The aqueous solvent extract of humus soil has a pH of 2.0 to 3.0, contains calcium, iron, sodium, magnesium, aluminum and silicon as minerals and 1000 to 1200 ppm of sulfur. Composition. An aqueous solvent extract of humus soil
(1) The mixture of humus soil and aqueous solvent is repeatedly stirred and allowed to stand until the pH is 2.8 or less and the electric conductivity is 400 mV or more.
(2) remove solids,
(3) The composition according to any one of claims 1 to 3, which is obtained by a step of leaving the obtained supernatant at ambient temperature for a long period of time.   The composition according to any one of claims 1 to 4, comprising 1 to 20% by weight of an aqueous solvent extract of humus, based on the composition.   A method for protecting against infection with protozoa, particularly Trypanosoma brucei, comprising administering the composition according to any one of claims 1 to 5 to an animal other than a human.

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