GB2229919A

GB2229919A - Composition for antiviral medicines comprising lignin derivatives

Info

Publication number: GB2229919A
Application number: GB8922752A
Authority: GB
Inventors: Makoto Machida; Makoto Yashiro; Eiko Takezawa; Sachiko Nanbara; Tetsuya Yoshida
Original assignee: Sanyo Kokusaku Pulp Co Ltd
Current assignee: Sanyo Kokusaku Pulp Co Ltd
Priority date: 1989-03-31
Filing date: 1989-10-10
Publication date: 1990-10-10
Anticipated expiration: 2009-10-10
Also published as: DE4010368C2; CA1335259C; FR2645024B1; DE4010368A1; GB2229919B; GB8922752D0; FR2645024A1

Description

Composition for antiviral medicines The present invention relates to the

use of lignin derivatives for antiviral drugs; in particular, to the prevention and the therapy against AIDS (Acquired Immune Deficiency Syndrome) virus.

The number of patients with AIDS has abruptly increased in recent years; particularly in the U.S.A. and Africa and currently the number of persons affected is about fifty thousand? with virus carriers numbering about a hundred times as manyy all over the world. It is said that almost all virus carriers acquire the disease within a five year period and the disease is fatal. AIDS affects the immune system of living bodies causing it to elapse as the AIDS and destroys virus infectsIthe Helper T cells governing the immune system. As a result. persons infected are prone to malignant tumors or the like and die as a result of this disease.

To date; nucleic acid-based AW(azidothymidine) alone is approved for the therapeutic drug against AIDS. AZT; however; cannot be used for a long term due to the intensive side effects (anaemia etc.).

However; lignin exists abundantly in nature andy after cellulose7 is contained in almost all plants? and is ingested by humans as food. In recent years; the physiological effects(such asintestinal disorders etc.) of vegetable fibers are being investigated.

Moreover7 lignin derivatives are produced particularly in spent liquor in the pulp and paper industry. The potential use of lignin as a medicinal drug has; however7 hardly been investigated and merely its antitumoral property is known.

In accordance with this invention. the physiological effect of ligninsulfonic acid and other lignin derivatives7 in particular; to antiviral effect thereof against NDV (Newcastle Disease Virus) belonging to the paramyxovirus family and RSV (Rous Sarcoma Virus) and HIV (Human Immunodeficiency Virus) belonging to the retrovirus family7 to which AIDS virus also belongs7 has been investigated.

The essence of the invention lies in a composition for anti-AIDS viral and other antiviral medicines comprising spent liquor from pulping and/or processed products as a major constituent.

First, the antiviral activity of sulfite spent liquor was determined to find that the activity is exhibited at about 1.5 mg/ml against NDV and at about 100 pg/ml against RSV. Nexty the sulfite spent liquor was fractionate through ultrafilters (UK type) with fractional molecular weights of 10j000 and 17000. The quantities of sugars7 ashes and ligninsu.rfonic acid in each fraction were determined and the antiviral activity was tested to yield the results as shown in Table 1 below.

Table 1

Ligninsulfonic Anti-NDV Anti-RSV Sugars Ashes acid activity activity Sulfite spent liquor2 24 % 38 % 37 % 1.5 mg/ml 0.1 mg/ml Fractional molecular weight Over 10,000 10 14 75 0.3 0.03 Fractional molecular weight Over 1,000 and under 10,000 14 30 45 2.0 0.3 Fractional molecular weight Under 1.000 33 55 12 7.5 2.0 1 Determined by glycol chitosan method 2 Residual ligin in pulp: 2.0 % 1 1 As indicated abovei it has been found that the substance which exhibits antiviral activity resides in ligninsulfonic acid and in particular that the fraction which exhibits high activity-is of high molecular weight. This fraction has also exhibited potent antiviral activity against HIV. However; this fact does not deny the existence of antivirus-active substances except ligninsulfonic acid in sulfite spent liquor.

The methods of testing antiviral activity in the -invention will be shown below.

In the test for anti-NDV activity, primary culture cells CEF (Chick Embryo Fibroblast) were proliferated on a plate with 96 holes and infected with NW. Then; after 30 minutes7 progressively diluted samples were added and7 24 hours latery the concentration to inhibit the cell fusion caused by virus was judged under the microscope.

In the test for anti-RSV activity; following the proliferation of said CEF on a plate with 96 holes7 this was infected with RSV. After 1 hour; progressively diluted samples were added and. 5 days late; the concentration to inhibit the transformation caused by virus was judged under the microscope.

For the anti-HIV testy MT-4 (human T cells infected by gTLV-1) and HM-III b. were used as the cells and virus7 respectively. The virus medium was prepared from the culture supernatant of Molt 4/ HIV HM-III b cells being 1 z HIV-producing cells. The titer of virus was 1 x 10 6 TCID 50/m' RPMI 1640 (containing antibiotics) was used as a medium. For the culture7 equal quantities of MT-4 cells of 1 x 106 cellsIml and HTLV-III b of 2 x 10 4 TCID 50/M1 were added to each well of a plate with 24 holes, which was cultured for 1 hour to allow to adsorb the virus. Thereafter, sample solutions diluted to various concentrations were added and finally the medium was added to make the volume upto 1 ml, which was cultured for 4 days in a CO 2 incubator at 37 'C. As control? cultures with cells not infected by HTLV-III b and without the addition of a sample were performed. The antiviral effect was judged by two methods; the inhibition of cell degeneration due to HIV and the inhibition of the development of HIV specific antigen onto the surface of cells. First, the cell degeneration effect was judged from the inhibition of the lethal effect on cells through the addition of a sample by counting the number of living cells by trypan blue method for the four-day culture medium both infected and not infected by HIV. Moreover7 for the determination of HIV specific antigen7 after reacting the cells fixed with methanol first with anti-HIV human serum for 40 minutes at 37'C, FITC-labelled anti-human-IgG was added to allow to react for 40 minuties at 370C and then the number of fluorescently - labelled cells was counted using a fluorescent microscope.

Nexty the antiviral activity was determined for various lignin derivatives.

The results are shown in Table 2.

The methods-of preparing the samples in the table are as follows:

- b 1 1 1 Sodium ligninsulfonate Produced by treating calcium ligninsulfonate with sod-Lum., sulfate to exchange the base.

Calcium ligninsulfonate Produced by cooking red pine with cooking liquor of calcium sulfite (CaS03).

Magnesium ligninsulfonate Produced by cooking red pine with cooking liquor of magnesium sulfite (MgSO 3).

Kraft lignin Produced by kraft pulping of red pine to field bleached kraft pulp (residual lignin in pulp-: 2.0 Z). Composition of kraft spent liquor (inorganics 6.2 Z, sugars 2.8 Z, lignin 6.0 Z).

o Dioxane ligin Extracted from wood flour (spruce) treated with alcoholbenzene by heating for 2 hours at 175 'C in a mixed liquor of dioxanewater (1: 1) according to the method of Sakakibara et al. The yield was about 45 Z.

o Thioglycolic acid lignin Prepared according to Brauns et al in a way that degreased wood flour (birch) was added to a mixed liquor of thioglycolic acid with 2N hydrochloric acid to boil for 7 hours, the mass was then separated by filtration and, after washing with water and with ethanol, the residue was extracted with 2 % sodium 4 1 1 hydroxide. The lignin was recovered after precipitated with hydrochloric acid.

o Sulfomethylated product of kraft lignin Prepared in a way that Na2S03 (10 to 20 % based on kraft lignin) and then HCHO (equimols to Na2S03) were added to a slurry (about 25 %) of kraft lignin to treat for 1 to 2 hours at 60 to 80 'C and, after treating further for 2 to 3 hours at 130 to 150 OC, cooling and drying were made.

- e- 1 Table 2

Sample Anti-NDV activity Anti-RSV activity Sodium ligninsulfonate 0.3 mg/ml 0.03 mg/ml Calcium ligninsulfonate 0.4 0.04 Magnesium ligninsulfonate 0.2 0.03 Kraft lignin 1.2 0.1 Dioxane lignin 1.5 0.2 Thioglycolic acid lignin 0.8 0.1 Sulfomethylated product 0.5 0.07 of kraft lignin As shown abovei the antiviral activity was recognized with all lignin derivatives listed in the table and among:.- these.ligninsulfonic acids proved to have a potent antiviral lignin derivatives effect. The/suitable for use in the present invention are not confined to those in the table and any known liqnin derivative may be employed.

In the following; the invention is illustrated by specific examples; but the invention is not confined to these.

<7 1 Example 1

Using dialyzing membrane, 100 nl of sulfite spent liquor shown in Table 1 was dialyzed for 4 days against tap water and for 3 days against deionized water in batch. The liquor having finished the dialysis was concentrated to about 30 ml with rotary evaporator and then freeze-dried to obtain about 3.5 g of fraction rich in ligninsuifonic acid. Both reducing sugar and ash in this fraction decreased to about one tenth compared with those of the original sulfite spent liquor. When determining the anti-NDV and anti-RSV activities, this fraction exhibited the effect at 0.5 ng/ml and 0.04 ng/ml, respectively.

Example 2

After 100 rl of kraft spent liquor shown in Table 2 was adjusted to pH 3.9 to precipitate the portion of kraft lignin, this was centrifuged (10,000 rpm) to collect the precipitated fraction. Then, this was converted to powder by drying under reduced pressure to obtain about 4.0 g of fraction rich in kraft lignin.

suspending this into deionized water, 1N NaOH was added to completely dissolve and a solution with afinal concentration of 1 % was prepared. When determining the anti-NDV and anti-RSV activities using said kraft lignin solution. the effect was seen at 0.8 mg/ml and 0.07 mg/ml, respectively. in terms of solids. -- -.9-- Y -- Example 3

Using a portion of sulfite spent liquor with a fractional molecular weight of over 10,000 shown in Table ii the anti-HIV activity was determined. Results are shown in Table 3.

Table 3.

Concentration Number of Number of Positivit of sample living cells living cells of HIV when not when infected specific infected by by HIV antigen HIV 500 pg/M1 1.36 x 106/M1 1.08 X 106/M1 0 250 1.46 1.41 0 1.57 1.45 0 63 1.53 1.61 0 32 1.58 1.66 0 16 1.56 1.53 0 8 1.58 1.57 0 4 1.59 0.96 20 0 1.61 0.21 90 1 A From the results above, it became clear that the anti-HIV activity of the fraction of sulfite spent liquor with a molecular weight of over 10,000 inhibited completely both the cell degeneration due to virus and the development of HIV specific antigen at a very low concentration of 8 rg/ml and the toxicity against cells was only generated slightly at a high concentration of 500 pg/M1 proving the fraction to be an antiviral agent with very high SI (Selective Index).

1 1

Claims

CLAIMS (1) A composition for anti-AIDS viral and other antiviral medicines

comprising spent liquor from pulping and/or processed products thereof as a major constituent.
(2) A composition according to Claim 1 wherein the spent liquor from pulping is sulfite spent liquor and/ or kraft spent liquor.
(3) A composition according to Claim 2; wherein the sulfite spent liquor is fractionate by ultrafiltration or gel filtration and has a fraction with a molecular weight of not less than about 5j000 as a major component.
(4) Use of a pharmaceutical composition comprising spent liquor from pulping and/or the processed products thereof used as a major constitutent for the prevention or treatment of AIDS or other viral infection.
(5) A composition for anti-AIDS viral and other antiviral medicines comprising a lignin derivative as a major constituent.
(6) A composition according to Claim 5; wherein the lignin derivative is obtained from the lignin in woods or herbs.. as a raw material.
(7) A composition for anti-AIDS viral and other antiviral medicines comprising non-ligneous components in woods and/or herbs andlor derivatives thereof as a major constituent.

Published 1990 at The PatentOMce.StAte House. cc ?l High Rolbo.-n, LondonWC1R4TP.Purthereoples be obtained frorn The PatentOffice tales Brawh, at Mary Cray. Orpington, Kent BR5 3RD. Printed by Multiplex twl=qu" ltdL at Mary Cray, Kent, Con. 1187