Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITION FOR ANTIVIRAL MEDICINES
BACKGROUND OF THE INVENTION
The present invention relates to the use of lignin
derivatives for antiviral drugs, in particular, to the
prevention of and the therapy against AIDS (Acquired Immune
Deficiency Syndrome) virus.
The number of patients with AIDS has abruptly increased
recently, centering on the U.S.A. and Africa, and currently it
amounts to about fifty thousand persons, the virus carriers
existence being about a hundred times as many, all over the
world. It is said that almost all virus carriers come down
with the disease within five years and the death rate reaches
about 100%. AIDS causes the immune system of living bodies to
collapse through the fact that the AIDS virus infects the
helper T cells governing the immune system and destroys them.
As a result, persons succumb to opportunistic infections,
malignant tumors or the like, and die with this disease.
To date, nucleic acid-based AZT (azidothymidine) alone is
approved as a therapeutic drug for use against AIDS. The AZT,
however, cannot be used for a long term, due to its intensive
side effects (anemia, etc.).
On the other hand, lignin exists abundantly in nature,
after cellulose, and is contained in almost all plants, and has
been ingested by human beings as a part of their food.
Recently, the physiological effects (for intestinal disorders,
etc.) thereof are attracting attention as a vegetable fiber.
Moreover, the production of lignin derivatives is centered on
spent liquor in the pulp and paper industry. The potential
thereof as a medicinal drug, however, has been hardly
investigated and merely its antitumoral property is known.
Thus, the physiological effects of ligninsulfonic acid and
other lignin derivatives and, in particular, its antiviral
3~ effect against NDV (Newcastle Disease Virus) belonging to the
PAT 13979-1
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paramyxovirus family and RSV (Rous Sarcoma Virus) and HIV
(Human Immunodeficiency Virus) belonging to the retrovirus
family, to which the AIDS virus also belongs, has been
investigated, leading to the completion of the invention.
SUMMARY OF THE INVENTION
The gist of the invention lies in a composition for anti-
AIDS viral and other antiviral medicines having spent liquor
from sulfite pulping and/or processed products as the major
constituent.
DETAILED DESCRIPTION OF THE INVENTION
First, the antiviral activity of sulfite spent liquor was
determined to find that activity is exhibited at about 1.5
mg/ml against NDV and at about 100 ,ug/ml against RSV. Next,
the sulfite spent liquor was fractionated through ultrafilters
(UK type) with fractional molecular weights of 10,000 and
1,000. The quantities of sugars, ashes and ligninsulfonic acid
in each fraction were determined and the antiviral activity was
tested to obtain the results as shown in Table 1 below.
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It has been found that the substance which exhibits the
antiviral activity resides in ligninsulfonic acid and, in
particular, the fraction which exhibits high activity has a
molecular weight on the high side. This fraction 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 for antiviral activity are set out
below.
In a test for anti-NDV activity, primary culture cells
CEF (Chick Embryo Fibroblast) were proliferated on a plate
with 96 holes and infected with NDV. Then, after 30 minutes,
progressively diluted samples were added and, 24 hours later,
the concentration needed to inhibit cell fusion caused by the
virus was determined under a microscope.
In a test for anti-RSV activity, following the
proliferation of said CEF on a plate with 96 holes, the CEF
was infected with RSV. After 1 hour, progressively diluted
samples were added and, 5 days later, the concentration
required to inhibit the transformation caused by the virus was
determined under a microscope.
For the anti-HIV test, MT-4 (human T cells infected by
HTLV-1) and HTLV-IIIb were used as the cells and virus,
respectively. The virus medium was prepared from the culture
supernatant of Nolt 4/HIVHTLV_IIIb cells being HIV-producing
cells. The titer of virus was 1 x 106 TCIDso/ml. RPMI 1640
(cont~i n ing antibiotics) was used as a medium. For the
culture, equal quantities of MT-4 cells of 1 x 106 cells/ml
and HTLV-IIIb of 2 x 104 TCIDso/ml were added to each well of
a plate with 24 holes, which was cultured for 1 hour to allow
adsorbtion of the virus. Thereafter, sample solutions diluted
to various concentrations were added and, finally, the medium
was added to make up the volume to 1 ml, which was cultured
3~ for 4 days in a CO2 incubator of 37 C. As the references,
PAT 13979-1
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cultures with cells not infecte by HTLV-IIIb and without the
addition of 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 sample by
counting the number of living cells using the trypan blue
method for the four-day culture medium, both infected and not
infected by HIV. For the determination of HIV specific
antigen, 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 and allowed to react for 40 minutes at
37 C and then the number of fluorescently labelled cells were
counted using a fluorescent microscope.
Next, the antiviral activity was determined for various
lignin derivatives, also.
Results are shown in Table 2.
The methods of preparing samples in said table are as
follows:
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o Sodium ligninsulfonate
Produced by treating calcium ligninsulfonate with sodium sulfate
to exchange the base.
o Calcium ligninsulfonate
Produced by cooking red pine with cooking liquor of calcium
sulfite (CaS03).
o Magnesium ligninsulfona~e
Produced by cooking red pine with cooking liquor of magnesium
sulfite (MgS03).
o Kraft lignin
Produced by kraft pulping of red pine to field bleached kraft
pulp ~residual lignin in pulp : 2.0 %).
Composition of kraft spent liquor (inorganics 6.2 %, sugars
2.8 %, lignin 6.0 %).
o Dioxane ligin
Extracted from wood flour (spruce) treated with alcohol-benzene
by heating for 2 hours at 175 C in a mixed liquor of dioxane-
water (1 : l) according to the method of Sakakibara et al.
The yield was about 45 %.
o Thioglycolic acid lignin
Prepared according to Brauns et al in a way that degreased
wood flour (birch) was added to 2 mixed liquor of thio~lycolic
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
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hydroxide. The lignin was recovered after being precipitated
with hydrochloric acid.
o Sulfomethylated product of kraft lignin
Prepared in a way that Na2SO3 (10 to 20 % based on kraft lignin)
and then HCHO (equimols to Na2SO3) 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 C, cooling and drying.
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
Th~oglycolic acid lignin 0.8 0.1
Sulfomethylated product 0 5 0 07
of kraft lignin
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As noted above, the antiviral activity was recognized with
all lignin derivatives listed in the table and thereamong
ligninsulfonic acids were proved to have a very potent
antiviral effect. The useful lignin derivatives are not
confined to those in the table and any one currently known will
serve.
In the following, the invention will be exemplified, based
on the examples, but the invention is not confined to these.
Example 1
Using a dialyzing membrane, 100 ml 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 finish-
ed the dialysis,was concentrated to about 30 ml with arotary evapo-
rator and then freeze-dried to obtain about 3.5 g of fraction
rich in ligninsulfonic 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 mg/ml and 0.04 mg/ml, respectively.
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Example 2
After 100 ml of kraft spent liquor shown in Table 2 was
adjusted to pH 3.0 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, lN NaOH was added
to completely dissolve and a solution with a final 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.
Example 3
Using a portion of sulfite spent liquor with a fractional
molecular weight of over 10,000 shown in Table 1, the anti-HIV
activity was determined. Results are shown in Table 3
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Table 3
Concentration Number of living Number of living Positivity of
of sample cells when not in- cells when infected HIV specific
fected by HIV by HIV antigen
5500 ~g/ml 1.36 x 106/ml 1.08 x 106/ml 0 %
250 1.46 1.41 0
125 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 1.5~ 0
4 1.59 0.96 20
0 1.61 0.21 90
2~ 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 degenera-
tion due to virus and the development of HIV specific antigen at
a very low concentration of 8 ~g/ml and the toxicity against cells
was only generated slightly at a high concentration of 500 ~g/ml
proving the fraction to be an antiviral agent with very high
SI (Selective Index).
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