JPH02262524A - Aids virus growth-inhibitory agent - Google Patents

Abstract

PURPOSE:To provide an AIDS virus multiplication-inhibiting agent by containing a water-soluble lignin eluted and separated from the tissue of a plant or a salt thereof as an active ingredient. CONSTITUTION:An AIDS virus multiplication-inhibiting agent contains a water- soluble lignin or its salt, preferably lignosulfonic acid or its alkali salt or alkaline earth salt as a main active ingredient. The water-soluble lignin is prepared by a method such as (i) a method wherein the tissue of a plant is heated in an aqueous solution of a bisulfite salt and a sulfite under an elevated pressure to sulfonate the lignin and the resultant water-soluble lignin is eluted, (ii) a method wherein the tissue of a plant is heated in an aqueous solution of sodium sulfide or hydrogen sulfide to produce water-soluble thiolignin, which is eluted, or (iii) a method wherein the tissue of a plant is treated with alcohol, phenol, mercaptan, thioglycolic acid, etc., to solubilize the lignin, followed by separating the resultant soluble lignin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an AIDS virus growth inhibitor whose active main ingredient is water droplet lignin obtained from plant tissue or a salt thereof.

“Conventional technology” AIDS virus (Humai Immunodefinition)
The science virus, HIVJ, is a type of retrovirus that infects human T lymphocytes.
As a result, the patient's immune system is weakened, opportunistic infections and cancers occur that are not seen in large numbers, and the patient becomes immunodeficiency leading to death.

Since AIDS was first identified in 1981, WIIO
The cumulative number of cases reported in 1988 was 12
More than 77,000 people died in the United States, and more than half of them have already died. AIDS has a long incubation period of about 8 years on average, and if the 1 million to 1.5 million people infected with the former V infection in the United States begin to develop the disease in a few years, the number of patients is expected to increase rapidly in the future. The cumulative number of patients is estimated to reach 365,000 in 1992.

The AIDS virus has a two-molecule RNA genome in its core, a glycoprotein envelope surrounding it that carries antigenicity, and it adsorbs to cells. Then, it takes off its envelope and enters the cell, where the DNA is reverse transcribed from the RNA genome by the action of reverse transcriptase, and the DNA is integrated into the cell nucleus. Viral RNA is synthesized from this DNA, proteins are synthesized, and virus particles are formed and bud from the cell membrane. If this process occurs rapidly, it may cause cell collapse.

Currently, azidothymidine 1AZT is used as an AIDS treatment drug.
AZT is known to have the effect of inhibiting viral reverse transcriptase and terminating DNA synthesis, and is said to be effective in reducing opportunistic infections and prolonging the survival period of patients.

In addition, sulfated polysaccharides such as dextran sulfate have anti-HIV
It has been reported that it has an effect (JpnJ, C
ancer Res, (Gann), 78.
1164-+168: November, 1987), and clinical trials are underway as a therapeutic agent.

In addition to these, many substances having anti-old V effects have been reported, and research is continuing as a therapeutic agent for AIDS.

On the other hand, JP-A-52-13583, JP-A-53-641
No. 1, JP-A-54-154507, JP-A-57-10
No. 6624, JP-A-57-130919, etc. disclose antitumor substances and antiviral agents containing lignin isolated from plants and processed products thereof as active ingredients.

``Problem to be solved by the invention'' AIDS therapeutic agents that have been proposed so far include, for example, AZ
T, which inhibits reverse transcriptase and suppresses virus proliferation, and dextran sulfate, which inhibits HIV
There are various types of infection, including suppression of cell-to-cell infection (suppression of virus adsorption) through the formation of syncytia.

However, since AZT is toxic to the bone marrow, long-term administration causes strong side effects.

Furthermore, other AIDS therapeutic agents such as dextran sulfate are still in the clinical trial stage, and it is unclear whether sufficient therapeutic effects can be obtained.

On the other hand, JP-A-52-13583, JP-A-53-641
No. 1, JP-A-54-154507, JP-A-57-10
No. 6624, JP-A-57-130919, etc. disclose that lignin and its processed products have anti-tumor and anti-viral effects. Nothing is suggested.

Therefore, an object of the present invention is to provide an AIDS virus growth inhibitor that has an excellent growth-inhibiting effect on the AIDS virus and has few side effects.

[Means for solving the problem] As a result of intensive research to achieve the above object, the present inventors have discovered that water-soluble lignin or its salts eluted and separated from plant tissue has an excellent effect of inhibiting the proliferation of the AIDS virus. This discovery led to the completion of the present invention.

That is, the present invention provides an AIDS virus growth inhibitor whose active main ingredient is a water-soluble ligton or a salt thereof that has been eluted and separated from plant tissues.

The present invention will be explained in more detail below.6 In the present invention, various plants rich in lignin, such as coniferous trees, broad-leaved trees, and plants of the genus family, are used as raw materials.8 These plants are After being prepared as a tissue rich in lignin, such as wood, straw, bagasse, etc., it may be used as the raw material of the present invention.

Lignin, along with cellulose and hemicellulose, is a main component that forms the skeleton of plants, and constitutes an intermediate membrane between cell membranes.

The content of lignin reaches 15 to 30% in wood, herbs, etc., and is the second most abundant organic substance after cellulose. Since lignin exists in plant tissues in a form tightly bound to other components, it is difficult to separate natural lignin from plants in its original form. For this reason, lignin separated from plant matter is denatured in some way.

Various methods are known for separating lignin from plants, but they can be broadly divided into methods that elute lignin and methods that leave lignin as a residue.6 The AIDS virus growth inhibitor of the present invention is The former method targets lignin eluted and separated from plant tissue. Examples of methods for eluting and separating lignin from plant tissues include the following methods.

■A method in which plant tissue is heated under pressure in an aqueous solution of bisulfite and sulfite to sulfonate the lignin, making it water-soluble and eluting it.

This method is used when wood is pulped using the sulfide method, and the lignosulfonate produced in large quantities during this process is currently discarded without being effectively utilized.

■A method in which plant tissue is heated with an aqueous solution of sodium sulfide or hydrogen sulfide to generate and elute water-soluble thiolignin.

This method is well known as the main reaction in the Kraft valve manufacturing method.

■ Plant tissues are treated with alcohol, phenol, mercaptan, thioglycolic acid, hydrotropic reagents (e.g.
(sodium xylene sulfonate) etc.
A method to solubilize and separate lignin.

In this case, it is also possible to further perform organic solvent extraction from the eluate to separate water-soluble lignin and fat-soluble lignin.

■A method in which plant tissues are heated under pressure in a caustic soda solution to make them water-soluble and elute.

In the present invention, water-soluble lignin which has been eluted and separated from plant tissue by various methods is used. The lignin thus separated may be chemically modified, such as gutsulfonate or thiolignin, or may be in the form of a salt. As the salt, alkali or alkaline earth salts are preferably employed. .

Furthermore, in the present invention, the lignin or its salt thus eluted and separated may be chemically or enzymatically treated to hydrolyze it or have a substituent introduced thereinto.

Thus, in the present invention, water-solubilized lignin, particularly lignosulfonic acid or a salt thereof, which is eluted and separated by the sulfide method, is preferably employed.

Lignin is an aromatic compound with a complex three-dimensional structure produced by the random polymerization of radicals generated by enzymatic dehydrogenation of three types of monoglycols, namely coniferyl alcohol, cinavir alcohol, and P-coumaryl alcohol. It is a compound. For this reason, the phenylpropane units and positions constituting lignin are not formed by one type of unit bond, but by various different C--C and ether bonds. In addition, the chemical structure of lignin is
It varies depending on the type of plant used as a raw material and the elution method.

For example, calcium gutsulfonate, which is eluted and separated from coniferous wood by the sulfide method, has a weight average molecular weight of 10.000, a methoxyl group of 10.77%, and a sulfonic acid group f-SO, H1 of 0.00% per phenylpropane unit. The purity of lignin is 86%, and it also contains impurities such as sugar.6 The estimated structure of lignosulfonic acid has already been reported (see Figure 1).
It is known that lignin is a modified lignin in which a sulfonic acid group is mainly introduced into the α-position of the side chain of lignin. In addition, its molecular structure in aqueous solution has a large number of sulfonic acid groups on the outside of a network-like hydrophobic core.

Similarly, sodium sulfur phosphate derived from the coniferous sulfide cooking method has a purity of 85% or 90% and an average molecular weight of 6.000 or 18.000.

Furthermore, the toxicity of calcium lignosulfonate was tested and the results are as follows.

That is, various concentrations of calcium sulfonate were dissolved in physiological saline and filtered through a 0.45 gm Millipore filter.Thirdly, 6-week old Wister rats (weighing about 200 g, 3 rats in - group) were prepared. The above solution was injected into the tail vein of the animal, and the survival or death was determined for 5 days.

As a result, when calcium lignosulfonate was intravenously injected in an amount of 70 n+g or more per animal, all the animals died the next day, but when the amount of calcium lignosulfonate was 50 mg or less per animal, they survived the next day. Therefore, LDi in intravenous administration of calcium lignosulfonate.

was found to be between 250 and 350 mg/kgO. In addition, the LD in oral administration of lignosulfonate to rats is reported to be 40 g/kg (D, K, Luscombe et al.: Fd
, (:osmet.

Toxicol, 11.229 (197'3)).

"Action and Effect" As mentioned above, the AIDS virus proliferation inhibitor of the present invention has the following effects:
The active ingredient is water-soluble lignin or its salts that have been eluted and separated from plant tissue using various methods. As is clear from the examples described below, this AIDS virus proliferation inhibitor exhibits a remarkable effect of inhibiting AIDS virus proliferation in culture experiments using MT-4 (cultured human T cell line) infected with HIV. 6 Therefore, by administering the AIDS virus proliferation inhibitor of the present invention to the human body, for example, by oral administration, intravenous or intravenous injection, it is possible to suppress the proliferation of the AIDS virus and obtain an AIDS therapeutic effect. It is expected that the

In addition, although the mechanism of action of the AIDS virus growth inhibitor of the present invention is not clear, it does not have a direct effect on DNA, etc., so it is believed to have fewer side effects than other AIDS therapeutic agents. Conceivable.

Furthermore, lignosulfonate, which is produced in large quantities when wood is pulped using the sulfide method, is not effectively utilized in many cases (currently it is discarded), and the present invention It is also possible to effectively utilize industrial waste.

"Example" Example 1 (a) Production method Five times the amount of distilled water was added to the waste liquid obtained by pulping softwood by acid sulfide cooking to bring the solid content concentration to 10.
% solution and added dropwise to 10 times the amount of ethanol with stirring, and the resulting pale brown precipitate was collected by centrifugation and freeze-dried to obtain a lignosulfonate. The obtained gutu sulfonate is a calcium salt type and has a lignin content of 86
%, and other substances such as sugar were included. The calcium gutsulfonate obtained here had an average molecular weight of about 10,000.

(bl anti-old V test Multiplicity of infection (M
, 0.1. After infection with lO,001 and adsorption of virus to cells for 1 hour at 37°C, cells were infected with RPMI-164.
The cells were washed with 0 medium, inoculated into individual wells of a 24-well tissue culture plate at 10'/ml, and cultured at 37°C in humidified air with 5% CO2. Calcium lignosulfonate was added to each concentration at the time of inoculation and compared with a control group without additives. The medium was replaced every 4 days, and calcium lignosulfonate was added to the medium each time.

The number of old V antigen-positive cells was measured every 4 days by indirect fluorescent antibody method using a fluorescence microscope and compared with the control group. At the same time, cell viability was measured by trypan blue dye exclusion method.

As a result, the control group had 90 antigen-positive cells on day 4 of culture.
% or more, and the number of viable cells also decreased to 22%. On the other hand, the calcium lignosulfonate addition group was 50LLg/m
1 suppressed antigen-positive cells to 1% and the number of viable cells to 96%.
On the 8th day of culture, the control group had more than 95% antigen-positive cells, but the calcium lignosulfonate added group had
It was suppressed to 1-2% at 504z g/ml and 1.100 tLg/ml, respectively.

However, 500μg/calcium lignosulfonate
When ml was added, toxicity to MT-4 cells was observed.

Next, HIV persistent producing cells (MOLT-4/1 (IVI)
Then, the mixture was mixed with HIV-uninfected cells (MOLT-41 in a ratio of 1:1) to perform a test for inhibition of giant cell formation (infected cells fuse due to the virus and efficiently induce multinucleated giant cells).

Briefly, the above cell mixture was adjusted to 5 X 10'/ml and seeded into individual wells of tissue culture plates;
Calcium lignosulfonate was added at various concentrations and cultured at 37°C in humidified air with 5% CO2. In addition, as a Tomei group, lignosulfate was similarly cultured without the addition of calcium fluoride.

After culturing, the cells were observed with a microscope over time, the number of living cells was counted, and the cell fusion inhibition rate of the group to which calcium lignosulfonate was added was calculated.

As a result, after 20 hours of culture, more than 95% cell fusion was observed in the control group to which calcium lignosulfonate was not added, but in the group to which calcium lignosulfonate was added.
lOLLg/ml inhibited cell fusion by 6.6%, 50u
g/ml 1.100 u g/ml showed 92-94% inhibition of cell fusion.

Example 2 To the calcium gutsulfonate obtained in Example 1,
After adding 72% sulfuric acid and incubating at 30°C for 1 hour, water was added and autoclaved at 120°C for 1 hour. Add NaOH solution to the obtained distillate to make it neutral,
The generated precipitate was redissolved and 5ephadex G-1
After desalting with a 0 column, it was freeze-dried. The resulting gutsulfonic acid has a purity of approximately 96% after removal of sugars.
% of lignosulfonic acid, Example 1
When testing the anti-old V effect in the same manner as 50Ii
At concentrations higher than g/ml, the appearance of IIIV antigen-positive cells by MT-4 cells was suppressed to 1% or less.

In addition, in the giant cell formation inhibition test using MOLT-4 cells conducted in the same manner as in Example 1, 10 ug/ml
31.3% at 50ug/ml, 83.4% at 100
A cell fusion inhibition rate of 92.8% was obtained at ug/ml.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the estimated molecular structure of gutsulfonic acid, which is the active ingredient of the AIDS growth inhibitor of the present invention.

Claims (4)

[Claims] (1) An AIDS virus growth inhibitor whose main active ingredient is water-soluble lignin or its salts eluted and separated from plant tissues. (2) The AIDS virus proliferation inhibitor according to claim 1, which contains lignosulfonic acid or a salt thereof as an active main ingredient. (3) The AIDS virus proliferation inhibitor according to claim 1 or 2, wherein the salt is an alkali or alkaline earth salt. (4) The AIDS virus proliferation inhibitor according to any one of claims 1 to 3, which is eluted and separated from a coniferous tree, a broadleaf tree, or a plant belonging to the family Araceae.