WO2018225847A1

WO2018225847A1 - Retroviral proliferation inhibitor, retroviral infection prophylactic drug containing same, and retroviral infection onset prophylactic drug

Info

Publication number: WO2018225847A1
Application number: PCT/JP2018/021962
Authority: WO
Inventors: 伊波　匡彦; 誠友利; 勇悦田中; 卓也福島; 恵美宮良; 直樹今泉
Original assignee: 株式会社サウスプロダクト; 国立大学法人琉球大学
Priority date: 2017-06-08
Filing date: 2018-06-08
Publication date: 2018-12-13
Also published as: JPWO2018225847A1; JP7201180B2

Abstract

Provided is an agent that: inhibits the proliferation of retroviruses involved in retroviral infections such as acquired immunodeficiency syndrome, HTLV-1-associated myelopathy, and adult T cell leukemia; prevents retroviral infection; and prevents the onset of retroviral infection. A retroviral proliferation inhibitor characterized by containing metal-bonded fucoidan in which one or more metals selected from the group consisting of potassium, calcium, magnesium, zinc, and selenium are bonded to the sulfate groups of fucoidan, the zinc content in the metal-bonded fucoidan being 0.005% or higher per fucoidan 1 in terms of mass when the metal is zinc; a retroviral infection prophylactic drug containing the retroviral proliferation inhibitor; and a retroviral infection onset prophylactic drug.

Description

Retrovirus growth inhibitor, retroviral infection preventive agent containing the same, and retroviral infection onset preventive agent

The present invention relates to a retrovirus growth inhibitor, a retrovirus infection preventive drug containing the same, and a retrovirus infection preventive drug.

A retrovirus is a spherical virus having a single-stranded RNA as a gene. This retrovirus is divided into several subfamilies such as oncovirus, lentivirus, and spumavirus.

Among these retrovirus subfamilies, HIV-1, 2 etc. lentiviral subfamilies are associated with acquired immunodeficiency syndrome (AIDS), HTLV-1, 2 oncovirus subfamilies are HTLV-1-related myelopathy ( HAM), adult T cell leukemia (ATL), and HTLV-1 associated uveitis (HU) are known.

To date, antiretroviral therapy using multiple antiviral drugs has been the mainstream for the treatment of diseases related to retroviruses (for example, combination therapy of one integrase inhibitor and two nucleic acid reverse transcriptase inhibitors). ), Maintenance of compliance and cost are high, and there is a problem in effectiveness, and it is not widely used (Non-patent Document 1).

An object of the present invention is to provide means for suppressing the growth of retroviruses by a simple method.

As a result of diligent research to solve the above-mentioned problems, the present inventors have found that the counter ion of the sulfate group of fucoidan is made into a specific metal ion, thereby suppressing the growth of retrovirus and completed the present invention. I let you.

That is, the present invention contains a metal-bonded fucoidan in which one or more metals selected from the group consisting of potassium, calcium, magnesium, zinc and selenium are bound to the sulfate group of fucoidan,
When the metal is zinc, the retrovirus growth inhibitor is characterized in that the content of zinc in the metal-bonded fucoidan is 0.005% or more with respect to fucoidan 1 in terms of mass.

The present invention is also a retroviral infection preventive agent or a retroviral infection preventive agent containing the retrovirus growth inhibitor.

Furthermore, the present invention is a metal-bonded fucoidan characterized in that one or more metals selected from the group consisting of potassium, calcium, magnesium and selenium are bonded to the sulfate group of fucoidan.

The retrovirus growth inhibitor of the present invention can inhibit retrovirus growth because it suppresses capsid protein production of retroviruses. In addition, the retrovirus growth inhibitor of the present invention can also prevent cells infected with retroviruses from becoming syncytia.

Therefore, the retroviral growth inhibitor of the present invention can be used as a retroviral infection preventive agent or a retroviral infection onset preventive agent.

It is a figure which shows the criteria of the presence or absence of syncytium formation in Example 1 (A: syncytium formation negative, B: syncytium formation positive). The results of measuring the amount of p24 produced two days after adding and administering fucoidan obtained by changing the metal binding to the sulfate group obtained in Example 1 to ATL-056i cultured cells at a final concentration of 10 μg / ml. FIG. It is a figure which shows the result of having measured the amount of p24 production 2 days after adding and administering the selenium-binding type fucoidan obtained in Example 12 to ATL-056i cultured cells at a final concentration of 10 μg / ml.

The active ingredient of the retroviral growth inhibitor of the present invention is a metal-bonded fucoidan (hereinafter referred to as “sulfate group”) in which one or more metals selected from the group consisting of potassium, calcium, magnesium, zinc and selenium are bound. It may be simply referred to as “metal-bonded fucoidan”).

The metal content in this metal-bonded fucoidan is not particularly limited when the metal is selected from the group consisting of potassium, calcium, magnesium and selenium. For example, potassium, calcium, The metal selected from the group consisting of magnesium and selenium is 0.005% or more, preferably 0.01% or more, more preferably 0.1% or more, particularly preferably 0.5% or more, and particularly preferably 1% or more. It is. Although the upper limit of these metal amounts is not specifically limited, For example, it is 5% or less.

Further, in this metal-bonded fucoidan, when the metal is zinc, the metal content is different from the zinc content contained in normal fucoidan as will be described later. Is 0.005% or more, preferably 0.01% or more, more preferably 0.1% or more, particularly preferably 0.5% or more, and particularly preferably 1% or more. Although the upper limit of these metal amounts is not specifically limited, For example, it is 5% or less.

The origin of fucoidan is not particularly limited. (Sargassum fulvellum) and other brown algae-derived seaweeds. Among these fucoidans, Okinawa mozuku fucoidan derived from Okinawa mozuku is preferable.

As shown in the following formula, Okinawa Mozuku Fucoidan has α1,3-linked fucose as the main chain, one molecule of glucuronic acid bonded to six fucose molecules, and half of fucose is sulfated. .

The fucoidan described above is commercially available from, for example, South Product Co., Ltd., Takara Bio Co., Ltd., Funakoshi Co., Ltd., etc., and literature (M. 16: 19-26, 1999) can be used without particular limitation. In addition, fucoidan may be one having a molecular weight adjusted by hydrolysis with an acid such as hydrochloric acid.

Usually, nothing is bound to the sulfate group of fucoidan, or sodium or a small amount (about 0.0001 to 0.002 mass%) of zinc is bound, but instead of this, potassium, calcium, magnesium, In order to bond one or more metals selected from the group consisting of zinc and selenium, a solution containing fucoidan is prepared, and these metals are bonded to the sulfate group of fucoidan by ion exchange.

The solution containing fucoidan used above is not particularly limited. For example, a solution obtained by dissolving already purified fucoidan in water or the like, or a brown algae containing fucoidan is extracted with a weakly acidic solution or the like, and insoluble matter is extracted. And the like prepared by removing. The content of fucoidan in this solution is not particularly limited, but is, for example, 0.1 to 5% by mass, preferably 1 to 3% by mass.

In the above, the method of ion exchange is not particularly limited. For example, a solution containing fucoidan is dialyzed or hydrofiltered against an acidic solution, and then from the group consisting of potassium, calcium, magnesium, zinc and selenium. A method of neutralizing with an alkaline aqueous solution containing one or more selected metals (hereinafter sometimes simply referred to as “metals”) (neutralization method), 1 of the metal salt in a solution containing fucoidan It can be carried out by a method of adding seeds or two or more species and further adding ethanol for precipitation (precipitation method), a method using an ion exchange resin, or the like.

The acidic solution used in the neutralization method is not particularly limited, and can be obtained, for example, by adding an acid such as hydrochloric acid to water to adjust the pH to 1 to 5, preferably 2.5 to 3.5. It is done.

The method for dialysis of a solution containing fucoidan against an acidic solution is not particularly limited. For example, a solution containing about 3% by mass of fucoidan is placed in a dialysis membrane and placed in an acidic solution of about 10 times volume or more. Stir overnight. In addition, if an ultrafiltration membrane is used instead of a dialysis membrane, hydrofiltration can be performed.

The alkaline aqueous solution containing one or more of the metals used above is selected from the group consisting of potassium compounds, calcium compounds, magnesium compounds, zinc compounds and selenium compounds that exhibit alkalinity when dissolved in water. It is prepared by adding one type or two or more types. Examples of the potassium compound that exhibits alkalinity when dissolved in water include potassium hydroxide, potassium carbonate, and potassium acetate. Examples of calcium compounds that exhibit alkalinity when dissolved in water include calcium hydroxide, calcium carbonate, and calcium acetate. Examples of magnesium compounds that exhibit alkalinity when dissolved in water include magnesium hydroxide and magnesium carbonate. Examples of the zinc compound that exhibits alkalinity when dissolved in water include zinc hydroxide, zinc carbonate, and zinc gluconate. Examples of the selenium compound that exhibits alkalinity when dissolved in water include sodium selenite. What is necessary is just to add the quantity of the potassium compound, calcium compound, magnesium compound, zinc compound, and selenium compound which are contained in alkaline aqueous solution so that the quantity of potassium, calcium, magnesium, zinc, and selenium with respect to fucoidan may become the above-mentioned quantity. The alkaline aqueous solution thus prepared is brought into contact with an acidic aqueous solution containing fucoidan prepared as described above. This contact takes place in an aqueous solution. Further, the amount of metal in the alkaline aqueous solution is not particularly limited, and may be appropriately adjusted according to the amount of metal to be bound to fucoidan.

The solution containing fucoidan used in the precipitation method may be the same as that used in the neutralization method. One or more of the metal salts are dissociated as cations when dissolved in water. Examples of the potassium compound include potassium chloride, potassium hydroxide, potassium carbonate and the like. Examples of calcium compounds include calcium chloride, calcium carbonate, calcium sulfate and the like. Examples of the magnesium compound include magnesium chloride and magnesium sulfate. Examples of the zinc compound include zinc chloride and zinc acetate. Examples of the selenium compound include selenium tetrachloride and sodium selenite. The amount of the metal salt added to the solution containing fucoidan is not particularly limited, and may be appropriately adjusted according to the amount of metal to be bound to fucoidan. After adding one or more of the above metal salts to the fucoidan-containing solution, an approximately equal amount of alcohol such as ethanol is further added and allowed to stand.

After performing ion exchange by the above method, washing, drying, and purification may be performed as appropriate.

As described above, a metal-bonded fucoidan in which one or more metals selected from the group consisting of potassium, calcium, magnesium, zinc and selenium are bonded to the sulfate group of fucoidan is obtained. In these metal-bonded fucoidans, the fact that potassium, calcium, magnesium, zinc and selenium are bound to the sulfate group of fucoidan and the amount thereof can be measured by an atomic absorption photometer or ion chromatography.

A metal-bonded fucoidan in which one or more metals selected from the group consisting of potassium, calcium, magnesium, zinc and selenium are bonded to the sulfate group of fucoidan described above, preferably potassium, calcium, A metal-binding fucoidan to which one or more metals selected from the group consisting of magnesium and selenium are bound can suppress retrovirus growth. Here, the suppression of retrovirus growth means suppression of conjugation between infected cells and non-infected cells, and suppression of production of structural proteins such as p24 and other capsid proteins.

Therefore, the metal-bonded fucoidan can be used as a retrovirus growth inhibitor. Here, HTLV-1 or HIV-1 is preferable as the retrovirus.

The retrovirus growth inhibitor of the present invention only needs to contain the above-mentioned metal-bound fucoidan, but the component that does not impair the effect of the metal-bound fucoidan in an amount of 100 mg or more, preferably 500 mg of fucoidan per day. At the same time, the powder, granule, liquid, or gel may be formed according to a conventional method, and these may be in the form of beverages, tablets, soft capsules, or the like.

In addition, since the metal-binding fucoidan, which is an active ingredient of the retrovirus growth inhibitor of the present invention, is conventionally derived from seaweeds of edible brown algae, it may be contained in a conventionally known food or drink as it is.

The retrovirus growth inhibitor of the present invention can suppress the production of capsid proteins of retroviruses, and can also prevent cells infected with retroviruses from becoming syncytia.

Specifically, when the retrovirus growth inhibitor of the present invention is used as a preventive agent for retrovirus infection, a person who is not infected with HTLV-1 or HIV-1 takes about 4000 mg of metal-binding fucoidan in the above form per day. Up to 1 to 3 times a day for 1 month or longer.

Furthermore, when the retrovirus growth inhibitor of the present invention is used as a preventive agent for the onset of retrovirus infections, it is anti-HTLV-1 or HIV-1 antibody positive and clinically in a carrier state, but ATL and HAM, HU, AIDS Those who have not developed symptoms may take the metal-bonded fucoidan in the above form in an amount of up to about 4000 mg per day, divided into 1 to 3 times per day for one month or longer.

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

Example 1
Preparation of metal bonded fucoidan:
(1) Preparation of Fucoidan (Sodium Bonded Type) Raw Okinawa mozuku was added to water and stirred. The pH was adjusted to 3.0 using 6N HCl, and the mixture was extracted at 90 ° C. for 1 hour. After centrifugation, the supernatant was desalted and concentrated with a UF membrane (fractionated molecular weight 10,000). Neutralization (pH 5.5) with 25% NaOH. It was freeze-dried to obtain fucoidan (molecular weight of 10,000 or more).

(2) Preparation of fucoidan with different metals (a) Preparation of zinc-binding fucoidan 1 g of fucoidan obtained in (1) above was dissolved in 100 mL of milli-Q water and diluted using a dialysis membrane with a molecular weight cut off of 8,000. Dialyzed overnight against 1 L of hydrochloric acid (pH 3.0). After completion of dialysis, it was confirmed that the pH was 3.0 or less. To this was added zinc hydroxide to pH 5.5. Insoluble zinc hydroxide was removed by centrifugation. The supernatant was freeze-dried to obtain a zinc-binding fucoidan.
(B) Preparation of calcium-binding fucoidan 1 g of fucoidan obtained in (1) above was dissolved in 100 mL of milli-Q water, and dialysis membrane with a molecular weight cut off of 8,000 was used to dilute dilute hydrochloric acid (pH 3.0). And dialyzed overnight. After completion of dialysis, it was confirmed that the pH was 3.0 or less. To this was added calcium hydroxide to pH 5.5. Insoluble calcium hydroxide was removed by centrifugation. The supernatant was freeze-dried to obtain a calcium-binding fucoidan.
(C) Preparation of sodium-binding fucoidan 1 g of fucoidan obtained in (1) above was dissolved in 100 mL of milli-Q water, and a dialysis membrane with a molecular weight cut off of 8,000 was used to dilute dilute hydrochloric acid (pH 3.0) to 1 L. And dialyzed overnight. After completion of dialysis, it was confirmed that the pH was 3.0 or less. To this was added sodium hydroxide to pH 5.5. Insoluble sodium hydroxide was removed by centrifugation. The supernatant was freeze-dried to obtain sodium-binding fucoidan.
(D) Preparation of Magnesium Bonded Fucoidan 1 g of fucoidan obtained in (1) above was dissolved in 100 mL of milli-Q water, and a dialysis membrane with a molecular weight cut off of 8,000 was used to dilute 1 L of diluted hydrochloric acid (pH 3.0). And dialyzed overnight. After completion of dialysis, it was confirmed that the pH was 3.0 or less. To this was added magnesium hydroxide to pH 5.5. Insoluble magnesium hydroxide was removed by centrifugation. The supernatant was freeze-dried to obtain a magnesium-binding fucoidan.
(E) Preparation of potassium-binding fucoidan 1 g of fucoidan obtained in (1) above was dissolved in 100 mL of milli-Q water, and a dialysis membrane with a molecular weight cut off of 8,000 was used to dilute dilute hydrochloric acid (pH 3.0) to 1 L. And dialyzed overnight. After completion of dialysis, it was confirmed that the pH was 3.0 or less. To this was added potassium hydroxide to pH 5.5. Insoluble potassium hydroxide was removed by centrifugation. The supernatant was lyophilized to obtain potassium-binding fucoidan.

The fucoidan prepared above was named as shown in Table 1. Table 1 also shows the results of measuring the substituted metal content contained in each fucoidan with an atomic absorption photometer.

Potassium-bonded fucoidan contained 0.15% of potassium relative to fucoidan 1 in terms of mass. The calcium-binding fucoidan contained 2.07% of calcium relative to fucoidan 1 in terms of mass. The magnesium-bonded fucoidan contained 1.50% of magnesium with respect to fucoidan 1 in terms of mass. The zinc-bonded fucoidan contained 1.26% of zinc relative to fucoidan 1 in terms of mass.

Example 2
Measurement of p24 production:
The fucoidan obtained by changing the metal binding to the sulfate group obtained in Example 1 was added to and administered to ATL-056i cultured cells at a final concentration of 10 μg / ml, and the amount of p24 produced after 2 days was measured. The production amount of p24 was measured by the following procedure using an HTLV-1 p24 antigen capture antibody and an HRP-labeled p24 detection antibody. The antibody is described in the literature (Tanaka Y., et al., Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human T cell leukemia virus type-1 envelope gp46. AIDS Res. Hum. Retroviruses, 30, 542-552, 2014.). The results are shown in FIG.

<Measurement reagents and instruments>
Cassette: 16 wells for ELISA (8 wells x 2)
p24 antigen capture antibody solution: 200-fold solution of p24 antigen capture antibody, dissolved in PBS containing 0.2% Triton-X100, 0.2% BSA, 0.01% thimerosal Blocking solution: 1% casein / PBS, Antigen dilution containing 0.01% thimerosal: dissolved in PBS containing 0.2% Triton-X100, 0.2% BSA, 0.01% thimerosal p24 standard stock solution: recombinant HTLV-1 p24 500 ng / ml
HRP-labeled p24 detection antibody solution: 200-fold solution of HRP-labeled p24 detection antibody, dissolved in PBS containing 0.2% Triton-X100, 0.2% BSA, 0.01% thimerosal Washing solution: 0.05% Tween PBS containing -20
Substrate coloring solution: citrate buffer containing 0.01% hydrogen peroxide as temperament and TMB as coloring agent Stop solution: 2N sulfuric acid

<Measurement procedure>
Put 50 μl of p24 antigen capture antibody solution into each well, let stand for 1 hour, then put 100 μl of blocking solution into each well, let stand for 10 minutes, and repeat washing operation 5 times, cassette coated with p24 antigen capture antibody Was made. 50 μl of antigen dilution is added to each well of a cassette coated with p24 antigen capture antibody. 50 μl of p24 standard stock solution was placed in a well and serially diluted with a diluent. Add 50 μl of sample to each well. 50 μl of an HRP-labeled antibody p24 detection antibody solution was added, sealed, and reacted at room temperature for 1 hour. After the reaction, the reaction solution is discarded and washed 5 times with a washing solution. Next, 80 μl of the substrate coloring solution is added to each well, and is then shielded from light and allowed to stand at room temperature for 10 to 15 minutes to confirm color development. Thereafter, 50 μl of the stop solution is added to each well. Finally, the absorbance is measured with a plate absorptiometer at a wavelength of 450 nm (control wavelength 540 nm or 630 nm), and the p24 antigen in the sample is quantified from a calibration curve prepared in advance.

From the above results, metal-bonded fucoidan (Fuco-2, Fuco-3, Fuco-4, Fuco-5) in which potassium, calcium, magnesium, or zinc is bound to the sulfate group of fucoidan is converted to fucoidan (sodium-bonded). In comparison, it was found that the production of HTLV-1 capsid protein (p24) was remarkably suppressed.

Example 3
Presence or absence of syncytia formation:
The fucoidan in which the metal binding to the sulfate group prepared in Example 1 was changed was administered to ATL patient-derived HTLV-1-infected cells and uninfected human T cell line Jurkat at various concentrations, and after 8 hours and 24 hours, a microscope was used. The state of the cells was observed. In the state of A in FIG. 1, it was determined that the formation of syncytia was inhibited, and in the state of B, it was determined that the formation of syncytia was not inhibited. The concentration at which it was judged that the formation of syncytia was completely inhibited after 8 hours and 24 hours was defined as the complete inhibition concentration, which is shown in Table 2.

Based on the above results, metal-binding fucoidan (Fuco-2, Fuco-3, Fuco-4, Fuco-5) in which potassium, calcium, magnesium or zinc is bound to the sulfate group of fucoidan is fucoidan (sodium-bonded). It was found to inhibit the formation of syncytia compared to. In particular, the formation inhibitory effect of metal-bonded fucoidan (Fuco-2, Fuco-3, Fuco-4) in which potassium, calcium or magnesium was bound to the sulfate group of fucoidan was remarkable.

Example 4
Beverages:
A container obtained by dissolving 1 g of fucoidan (sodium-bound type) obtained in Example 1 (1), 125 mg of citric acid, and 25 mg of sucralose in 50 ml of water was obtained.

Example 5
Administration study:
The beverage obtained in Example 4 was distributed to the subjects (HTLV-1 carrier: age 20 years old or older: 10 people) and allowed to drink once a day, 3 times a day for 6 months. The body weight and blood pressure were measured every month from the time of drinking until 6 months after the end of drinking, and 10 ml of venous blood was collected. Venous blood was stored at −80 ° C. after separating PBMCs by specific gravity centrifugation. DNA was extracted from stored PBMCs according to a conventional method, and the amount of provirus was measured by the following real-time PCR method. The results are shown in Table 4.

<Real-time PCR method>
The number of copies of HTLV-1 provirus and human β chain globin gene in case DNA was measured by real-time PCR, and 1 copy of HTLV-1 provirus per cell and 2 copies of β chain globin gene in 100 PBMCs. The number of copies of HTLV-1 provirus was calculated. For the measurement, LightCycler 480 (Roche Diagnostics) was used, and the TaqMan probe method was used. For detection of HTLV-1, the pX region, which is well conserved in HTLV-1, was targeted. The probe and primer sequences used (SEQ ID NOs: 1 to 6) are shown in Table 3. The reaction solution was 1 × LightCycler 480 Probe Master (Roche Diagnostics), 0.5 μM sense primer, 0.5 μM antisense primer, 0.1 μM TaqMan probe, and a total amount of 20 μl of template DNA. Calibration curves were prepared with 1 × 10 ¹ to 10 ⁶ copies of HTLV-1 plasmid DNA and 2.5 × 10 ¹ to 10 ⁴ copies of healthy human DNA. The reaction conditions were the first heat denaturation at 95 ° C. for 5 minutes, 50 cycles of 95 ° C. for 10 seconds and 60 ° C. for 30 seconds.

―: Not measured

The amount of HTLV-1 provirus (copy number / 100 PBMCs) was significantly lower after drinking 6 months than before drinking (median 12.3 vs 10.0, Willcoxon signed rank test, p = 0.021).

As described above, the HTLV-1 carrier was able to reduce the amount of HTLV-1 provirus by drinking fucoidan (sodium-binding type). Therefore, one or more metals selected from the group consisting of potassium, calcium, magnesium, and zinc were bound to the sulfate group of fucoidan, which was significantly more effective than fucoidan (sodium-binding type) in the syncytium formation test. When metal-bound fucoidan is taken by HTLV-1 carriers, it is expected to significantly reduce the amount of HTLV-1 provirus.

Example 6
Preparation of metal bonded fucoidan:
In Example 1 (1), fucoidan is prepared in the same manner except that raw Okinawa mozuku is replaced with raw gagome kelp. About this fucoidan, operation of Example 1 (2) can be performed and the fucoidan from which a metal differs can be prepared.

Example 7
Preparation of metal bonded fucoidan:
In Example 1 (1), fucoidan is prepared in the same manner except that raw Okinawa mozuku is replaced with raw wakame. About this fucoidan, operation of Example 1 (2) can be performed and the fucoidan from which a metal differs can be prepared.

Example 8
Beverages:
One of each metal-bonded fucoidan obtained in Example 1 (2) was dissolved in 1.5 g of citric acid, 125 mg of citric acid, and 25 mg of sucralose in 50 mL of water, and the beverage was obtained.

Example 9
Beverages:
One of each metal-bonded fucoidan obtained in Example 1 (2), 0.5 g, citric acid 125 mg, and sucralose 25 mg dissolved in 50 mL of water was filled into a container to obtain a beverage.

Example 10
Confirmation of cytotoxicity:
For each metal-bonded fucoidan (0, 62.5, 125, 250, 500 or 1000 μg / mL) obtained in Example 1 (2), the literature (Haneji K., et al., Fucoidan extracted from Cladosiphon okamuranus Tokida Induced apoptosis of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells. Nutr. Cancer., 52, 189-201, 2005.). As a result, no cytotoxicity was observed up to a concentration of at least 1000 μg / mL.

Example 11
Safety check:
Regarding the zinc-binding fucoidan (0, 250, 500 or 833 mg / kg / rat) obtained in Example 1 (2) (a), the literature (Li N. et al., Toxicological evaluation of fucoidan extracted from Laminaria japonica in Wistar rats. Food Chem Toxicol. 43, 421-426, 2005.) As a result, no problem was observed until 28 days at a concentration of at least 833 mg / kg / rat.

Example 12
Preparation of metal bonded fucoidan:
5 g of fucoidan obtained in (1) of Example 1 was dissolved in 100 mL of distilled water. Separately, 2.2077 g of selenium tetrachloride was dissolved in 100 mL of distilled water. After mixing these, 200 mL of ethanol was added and allowed to stand at room temperature. Centrifugation (10000 rpm, 10 minutes) was performed, and the precipitate was washed with ethanol three times and dried with an evaporator (70 ° C.). The ethanol precipitate was dissolved in milli-Q water and dialyzed. After dialysis, lyophilization was performed to obtain a selenium-binding fucoidan. When the content of the substituted metal contained in the selenium-bonded fucoidan was measured with an atomic absorption spectrophotometer, the selenium-bonded fucoidan contained selenium at 177.1 ppm (the selenium-bonded fucoidan converted selenium into fucoidan 1 in terms of mass). 1.77%).

Example 13
Presence or absence of syncytia formation:
The selenium-binding fucoidan obtained in Example 12 was examined for the syncytium formation inhibitory effect by the same method as in Example 3. As a result, the syncytium formation complete inhibitory concentration was 1.25 mg / mL.

Example 14
Measurement of p24 production:
With respect to the selenium-binding fucoidan obtained in Example 12, the amount of p24 produced two days after addition / administration to the ATL-056i cultured cells at a final concentration of 10 μg / mL was measured by the same method as in Example 2. The results are shown in FIG. Selenium-linked fucoidan was found to significantly suppress p24 antigen production.

The retrovirus growth inhibitor of the present invention can prevent retrovirus infection or prevent the onset of retrovirus infection.

Claims

Containing a metal-bonded fucoidan in which one or more metals selected from the group consisting of potassium, calcium, magnesium, zinc and selenium are bonded to the sulfate group of fucoidan;
When the metal is zinc, the content of zinc in the metal-bound fucoidan is 0.005% or more with respect to fucoidan 1 in terms of mass, and the retrovirus growth inhibitor.
The retrovirus growth inhibitor according to claim 1, wherein the retrovirus is HTLV-1 or HIV-1.
3. The retrovirus growth inhibitor according to claim 1 or 2, wherein the fucoidan is Okinawa mozuku fucoidan.
The retrovirus growth inhibitor according to any one of claims 1 to 3, which suppresses the production of structural proteins.
The retrovirus growth inhibitor according to any one of claims 1 to 4, which inhibits syncytium formation.
A retroviral infection preventive agent comprising the retroviral growth inhibitor according to any one of claims 1 to 5.
A retroviral infection preventive agent containing the retroviral growth inhibitor according to any one of claims 1 to 5.
A metal-bonded fucoidan, wherein one or more metals selected from the group consisting of potassium, calcium, magnesium and selenium are bonded to the sulfate group of fucoidan.
The metal bonded fucoidan according to claim 8, wherein the fucoidan is Okinawa Mozuku fucoidan.