JPH0222231A - Composition for antiviral drug - Google Patents

Abstract

PURPOSE:To obtain a composition for anti-AIDS virus and other antiviral drugs comprising waste liquor of pulp digestion and/or a treated and processed material thereof as main constituent element. CONSTITUTION:The title composition containing waste liquor of sulfite pulp digestion or waste liquor of kraft pulp digestion and/or a treated and processed material thereof such as a fraction obtained by fractionating waste liquor of sulfite pulp digestion by ultrafiltration or gel filtration and having >=5,000 molecular weight as a main constituent element. Lignin sulfonates contained in the digested waste liquor show strong antiviral actions and fractions having high activity exist especially in a polymer side. Lignin derivatives using lignin in wood or trees and plants have the same antiviral action.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention uses lignin derivatives as antiviral agents, especially AIDS (AIDS).
Personquited In++nune Delicien
c7Syndtom) virus prevention and treatment.

[Problems to be solved by conventional technology and inventions] The number of AIDS patients has increased rapidly in recent years, mainly in the United States and Africa.Currently, there are approximately 50,000 patients worldwide, and the number of virus carriers is approximately 50,000. There are 100 times more. Will carriers are 5
Most of these patients develop the disease within a year, and the mortality rate is said to be approximately 100%. AIDS is a disease in which the AIDS virus infects and destroys helper T cells that govern the immune system, causing the immune system of the body to collapse, resulting in death from opportunistic infections, malignant tumors, etc.

To date, the only drug to treat AIDS is the nucleic acid AZT.
(Azidothymidine) is the only approved drug, and AZT is also unsuitable for long-term use due to severe side effects (such as anemia).

On the other hand, lignin is the second most abundant substance in nature after cellulose, is present in almost all plants, and has been ingested by humans as part of food since ancient times. In recent years, as a type of plant fiber, its physiological effects (intestinal regulating effect, etc.) have been attracting attention.

In addition, lignin derivatives are mainly produced from wastewater from the paper and pulp industries. However, its potential as a drug has hardly been investigated, and only a small amount of antitumor activity is known.

[Means for Solving the Problems] Therefore, regarding the physiological effects of lignin derivatives such as lignin sulfonic acid, NDV, which belongs to the Paramyxoviridae family, was studied. 5eV
irus) and RSV (Rous Sa+comx), which belongs to the same family of retroviruses as the AIDS virus.
Virusl and HIV (Hu+axn i+
The present invention was developed by studying the antiviral effect against the virus (An+unedeliciency viIus).

The gist of the present invention resides in anti-AIDS virus and other antiviral pharmaceutical compositions containing pulp cooking effluent and/or processed products thereof as main components.

[Action and effect of the invention]

First, the antiviral activity of sulfite pulp effluent was examined, and it showed activity against NDV at about 1.5++g/ml and against R3V at about 100++g/ml. Next, the sulfite pulp waste liquid was treated with a molecular weight cutoff of 10,000,
and was fractionated using a +000 × outer filtration membrane (UK type). The sugar content, ash content, and lignin sulfonic acid content of each fractionated component were quantified and the antiviral properties were tested, and the results shown in Table 1 below were obtained.

As described above, we have discovered that there is a fraction of the main body of genin sulfonic acid that exhibits antiviral activity, particularly on the polymer side, that exhibits high activity. This fraction also showed strong antiviral activity against HIV. However, this does not deny the existence of substances exhibiting antiviral activity other than ligninsulfonic acid in the sulfite pulp wastewater.

The antiviral activity testing method in the present invention is shown below.

The anti-NDV activity test was performed using primary cultured cells CEF (Chick
Embry7o Fib+oblasl) was grown on a 96-well plate, then infected with NDV, 30 minutes later, serially diluted samples were added, and 24 hours later, the concentration that suppressed cell fusion caused by the virus was determined using a microscope. did.

In the anti-R3V activity test, the aforementioned CEF were grown in a 96-well plate, then infected with R3V, and 1 hour later, serially diluted samples were added, and after 5 hours, the concentration that suppressed the transformation caused by the virus was determined. Judgment was made using a speculum.

The anti-HIV test used MT-4 (HTLV-1-infected human T cells) as cells and HTLV-m as the virus. The virus fluid is Mol+ 4 which is HIV producing cells.
/HI was prepared from the culture supernatant of VHTLV-wb cells. Virus titer is 1 x 10' TCI D5o
/ml. In addition, RPMI+640 was used as a medium.
(containing antibiotics) was used.

Culture at 1x106 cells/ml in each well of a 24-well plate.
of MT-4 cells and 2 X 10' TCI k+/11
An equal amount of HTLV-m was added and cultured for 1 hour to adsorb the virus. After that, the samples diluted to each concentration were added, and the medium was finally added to make a volume of 1 ml.
Culture in a 02 incubator for 4 hours. H as a control
TLV-m.

Cultures were performed with uninfected cells and without sample addition.

The antiviral effect was evaluated using two methods: inhibition of cell degeneration caused by HIV and inhibition of expression of HIV-specific antigen on the cell surface. First, the cytopathic effect was determined by counting the number of viable cells using the Trivan Blue method for HrV-infected and non-infected 4-H culture fluids, and by suppressing the cell-killing effect by adding the sample. To measure HIV-specific antigen, cells fixed with methanol were first reacted with anti-HIV human serum at 37°C for 40 minutes, then FITC-labeled anti-human IgG was added and reacted at 37°C for 40 minutes. The number of cells was counted using a fluorescence microscope.

Next, the antiviral activity of various lignin derivatives was also measured.

The results are shown in Table 2.

The preparation method of the samples in the table is as follows.

0 Sodium ligninsulfonate Calcium ligninsulfonate (Na2SQl)
) and base replacement.

O Calcium lignin sulfonate Manufactured by steaming red pine with calcium sulfite (CaSO3) cooking liquor.

Manufactured by cooking O-ligninsulfonate magnesium red pine with magnesium sulfite (MgS03) cooking liquor.

0 kraft lignin Manufactured by cooking red pine into bleached kraft pulp (residual lignin in pulp: 2.0%).

Kraft pulp effluent composition (inorganic matter 6.2%, sugar 2.8%
, lignin 60%) 0 dioxane lignin According to the method of Sakakibara et al., alcohol/benzene treated wood flour (
spruce) in a dioxane/water (1:1) mixture at 175°C.
, heated extraction for 2 hours. Yield was approximately 45%.

According to the method of Brauns et al., defatted wood flour (birch) was added to a mixture of thioglycolic acid and 2N hydrochloric acid, boiled for 7 hours, separated, and the residue was washed with water and ethanol. Extracted with % sodium hydroxide. It was recovered after precipitation with hydrochloric acid.

0 Kraft lignin sulfomethylated product Kraft lignin slurry (approximately 25%) Na 23
01 (IQ ˜20% vs. Kraft Rikunin).

Then HCHO (3 equimolar Na2SQ) was added and 6
After treatment at 0 to 80°C for 1 to 2 hours, further 13 (1 to 15
Treat at 0°C for 2-3 hours and then cool.

Dry.

Table 2 All of the lignin derivatives shown in the table above were found to have antiviral activity, and among them, lignin sulfonic acids were found to have a strong antiviral activity. The lignin derivatives are not limited to those shown in the table, and any currently known lignin derivatives may be used.

〔Example〕

EXAMPLES Hereinafter, the present invention will be illustrated by examples, but the present invention is not limited thereto.

"Example 1" 100 ml of the sulfite pulp wastewater shown in Table 1 was dialyzed against tap water for 4 days and against deionized water for 3 days in batches using a dialysis membrane. The dialysis-completed solution was concentrated to about 30...1 using a rotary evaporator and then freeze-dried to obtain about 3.5 g of a fraction rich in ligninsulfonic acid. This fraction has a reducing sugar and ash content of approximately 171% compared to the original sulfite pulp wastewater.
It had decreased to O. For this fraction, anti-NDV, anti-R3
When V activity was measured, each was 0.5 mg/m
It was effective at 0.04 mg/ml.

Example 2 100 ml of the kraft pulp wastewater shown in Table 2 was adjusted to pH 3.
, 0 to precipitate the kraft lignin portion, the precipitated fraction was collected by centrifugation (10,000+pm), and then powdered by vacuum drying to obtain about 4.0 g of a kraft lignin-rich fraction.

This was suspended in deionized water and 1N NaOH was added to completely dissolve it, resulting in a final concentration of 1% solution. When the anti-NDV and anti-RSV activities of this kraft lignin solution were measured, they were 0.8 mg/ml in terms of solid content.
An effect was observed at 0.07 mg/ml.

[Example 3] Fractionated molecules J of sulfite pulp wastewater shown in Table 1! tlO,
Anti-HIV activity was measured for the portion above QOO. The results are shown in Table 3.

From the above results, the sulfite pulp waste liquid molecule m10.000
The anti-HIV activity of the above fractions was shown to be effective against viral cell degeneration and H
Both S, I, (Selecl
It has become clear that it is an antiviral agent with a very high index (Iye Index).

Claims (7)

[Claims] (1) Anti-AIDS and other antiviral pharmaceutical compositions containing pulp cooking effluent and/or processed products thereof as main components. (2) Pulp cooking effluent is sulfite pulp cooking effluent and/or
The composition according to claim 1, which is a kraft pulp cooking effluent. (3) The composition according to claim 2, wherein the sulfite pulp cooking effluent is fractionated by ultrafiltration, gel filtration, etc. and the main component is a fraction having a molecular weight of about 5,000 or more. (4) A method for preventing and/or treating AIDS virus and other viral infections, characterized by using pulp cooking effluent and/or processed products thereof as a main component. (5) Anti-AIDS virus and other antiviral pharmaceutical compositions containing a lignin derivative as a main component. (6) The composition according to claim 5, wherein the lignin derivative is derived from lignin in wood or herbs. (7) Anti-AIDS virus and other antiviral pharmaceutical compositions containing non-lignin components and/or derivatives thereof in wood and/or herb components as main components.