A beta-1,3-glucan of sulfated curdlan having a sulfur content of 12.4 to 17% and an average molecular weight of 27,000 to 330,000 daltons. Sulfate curdlan exhibits strong HIV inhibitory activity with little anticoagulant activity and little toxicity.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to an anti-viral agent containing a modified 
and low-molecular weight .beta.-1,3-glucan having a very potent anti-human 
immunodeficiency virus (anti-HIV) activity. 
2. Description of the Background 
It is known that inorganic ion-containing organic high polymer compounds 
are effective for the prevention and treatment of retroviral infectious 
diseases. See, for example, Japanese Patent Application Laid-Open No. 
62-215529. In particular, sulfates of polysaccharides are also known to be 
effective therefor. See, for example, Japanese Patent Application 
Laid-Open No. 63-45223. However, the anti-HIV activity of these compounds 
is not well understood. Moreover, the necessary structure thereof appears 
to be correlated to a blood anti-coagulating activity which is also 
manifested. This anti-coagulating activity is, of course, undesirable in 
the use of the compounds. Unfortunately, practically useful sulfated 
polysaccharides, without the above side effects, are not presently 
available. 
Further, with respect to the method of sulfating polysaccharides, and the 
method of isolating the sulfated polysaccharides, a detailed investigation 
has been conducted with a dextran sulfate of .alpha.-1,6-glucan 
(Carbohydrate Research, 21 (1972), 420-426), but a detailed investigation 
on .beta.-1,3-glucan has not yet been conducted. 
Thus, at present, a need continues to exist for compounds which exhibit a 
more potent anti-HIV activity, but which also have a reduced 
anti-coagulating activity. It would also be desirable to develop such 
compounds having fewer impurities than are found in conventional agents 
for HIV therapy. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the present invention to provide a 
substance which has excellent anti-HIV activity. 
It is also an object of the present invention to provide a substance having 
reduced anti-coagulating activity, and having fewer impurities contained 
therein. 
The above objects and others are provided by a modified and low molecular 
weight .beta.-1,3-glucan having a sulfur content of from about 
14.5.+-.2.5% and a weight average molecular weight by gel filtration of 
from about 20,000 to 330,000.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the present invention, it has been discovered that the 
above activities are obtained by a modified and low molecular weight 
.beta.-1,3-glucan of a sulfated curdlan or lentinan, having, in general, a 
sulfur content of at least 12% or more and having a weight average 
molecular weight by gel filtration of at least 20,000 or more. The 
.beta.-1,3-glucans of the present invention have excellent anti-HIV 
activity and weak anti-coagulant activity. 
In particular, the present invention provides: 
(A) a modified and low molecular weight .beta.-1,3-glucan from curdlan as a 
raw material, which has a sulfur content of about 14.5% +2.5% and a weight 
average molecular weight of from about 20,000 to 330,000, and 
(B) a modified and low molecular weight .beta.-1,3-glucan from lentinan as 
a raw material, which has a sulfur content of about 14.5% .+-.2.5% and a 
weight average molecular weight of from about 20,000 to 330,000. 
The above-mentioned compound (A) may be obtained from a known curdlan as a 
raw material. As curdlan has a low solubility, it is important that the 
curdlan first be activated so as to elevate the solubility. The activating 
treatment to be applied to curdlan for this purpose entails placing the 
curdlan in a suspension in a polar organic solvent and then a non-polar 
organic solvent step-by-step in order. Further, in sulfation, even a 
slight amount of water in the reaction system would substantially 
interfere with the reaction. Accordingly, as curdlan is highly 
hygroscopic, it is preferable to dry the curdlan to lower the water 
content thereof to about 2% or less for the purpose of effecting the 
reaction quantitatively and in a stable manner. Dimethylsulfoxide 
(hereinafter referred to as "DMSO") and a sulfating agent such as 
piperidine sulfate are also fully dried and then used in the sulfation 
reaction, whereby the low molecular weight production and sulfation are 
effected simultaneously. As curdlan is hardly soluble in DMSO, it is 
preferred to stir the same in DMSO for several hours to overnight at room 
temperature or for several hours at 60.degree. C. to 90.degree. C. for 
complete dissolution. Next, the resulting solution is reacted with a 
sulfating agent such as piperidine sulfate, which is used in an amount of 
from about 1 to 3 equivalents per glucose residue, whereupon the reaction 
temperature is about 60.degree. C. to 90.degree. C. and the reaction time 
is from about 1 hour to 24 hours. When stronger conditions than the above 
are used, for example, 3 equivalents or more of piperidine sulfate, a 
reaction temperature of higher than 90.degree. C. or a reaction time of 
longer than 24 hours, the reaction is often accompanied by a side-reaction 
with coloration, excess low-molecularization or release of sulfate 
radical. 
In order to isolate the sulfated curdlan from the reaction solution, the 
reaction mixture may be neutralized with, for example, sodium hydroxide or 
sodium hydrogen carbonate and then subjected to dialysis so as to isolate 
the sulfated curdlan. However, as the dialysis method requires a long time 
and removal of impurities is insufficient by this method, it is preferred 
that the product be isolated from the reaction solution in the form of a 
precipitate and then separated from the DMSO or other solvents used. For 
precipitation of the product, which is a high molecular weight polymeric 
substance, adding only the organic solvent to the reaction mixture is 
inadequate. Thus, in accordance with the present invention, it has been 
found that the addition of a salt together with an organic solvent is 
effective for efficient precipitation of the product in the form of its 
sodium salt. The salt to be used for the purpose may be an inorganic salt 
such as sodium chloride. However, as the salt is an impurity, and should, 
therefore, be present in the smallest amount in view of purification of 
the product, the salt is desirably an organic solvent-soluble salt which 
can be washed and removed with an organic solvent, for example, sodium 
acetate. However, other organic solvent-soluble salts may be used. 
The precipitate obtained, which contains the product and the salt, is 
washed several times with an organic solvent and then is dissolved in 
water. The washing may be batchwise effected by repeated washing and 
treatment with a centrifuge. However, use of a ceramic membrane is more 
preferred as the washing can be effected continuously. 
The aqueous solution containing the product can be de-salted by passing the 
same through an ultrafiltration membrane, whereupon a ceramic membrane can 
also be used. The resulting aqueous solution containing only the product 
may be directly freeze-dried. Where the product is obtained in the form of 
a powder, a proper amount of an organic solvent-soluble salt may be added 
to the aqueous solution and the intended product can be precipitated with 
an organic solvent. 
The above-mentioned compound (B) is obtained from a known lentinan as a raw 
material. In the sulfation of lentinan, even a slight amount of water in 
the reaction system would substantially interfere with the reaction. 
Accordingly, as lentinan is highly hygroscopic, it is preferable to dry 
the same to lower the water content thereof to 2% or less prior to the 
reaction for the purpose of effecting the reaction quantitatively and 
stably. DMSO and a sulfating agent such as piperidine sulfate are also 
fully dried and then used in the sulfation reaction. As lentinan is hardly 
soluble in DMSO, it is important that lentinan be stirred in DMSO for 
several hours at room temperature for complete dissolution. 
In order to isolate the product from the reaction solution, the same 
process may be used as that for obtaining the product when using curdlan 
as a raw material. 
In the above process, acetone is an example of a polar solvent which may be 
used, and diethyl ether is an example of a non-polar solvent which may be 
used. However, other polar and non-polar solvents may be used, and in view 
of the present disclosure, such other solvents are readily known to those 
of ordinary skill in the art. 
Having described the present invention, reference will now be made to 
certain examples which are offered solely for the purpose of illustration 
and are not intended to be limitative. 
Example 1: Preparation of Modified Low-Molecularized .beta.-(1,3)-glucan 
from Curdlan as a Raw Material 
1 g of curdlan was suspended in 50% acetone for 24 hours and then suspended 
in 75% acetone for 24 hours, in 100% acetone for 24 hours and in 100% 
ether for 24 hours in that order. Afterwards, the precipitate formed was 
dried overnight under reduced pressure over phosphorus pentoxide. 1 g of 
the thus activated dry curdlan was added to 100 ml of DMSO as previously 
dried with Molecular Sieve 4A and stirred overnight at room temperature, 
whereby the former was dissolved in the latter. 2.5 g of piperidine 
sulfate was added thereto and heated up to 85.degree. C., whereupon the 
reaction was continued for 1 hour under this condition. After completion 
of the reaction, the reaction mixture was cooled with water and 500 ml of 
methanol solution of 5% sodium acetate was added thereto, whereupon the 
product was precipitated out. The resulting precipitate was washed twice 
with 100 ml of methanol and then dissolved in 100 ml of water, and the 
resulting solution was desalted through an ultrafiltration membrane with a 
cutoff for compounds having a molecular weight of 10,000. 
To 100 ml of aqueous solution of the thus desalted product was added 400 ml 
of methanol solution of 5% sodium acetate, whereby the product was 
precipitated out. The precipitate was washed twice with 100 ml of methanol 
and then dried under reduced pressure to obtain 1.7 g of the intended 
product of modified and low-molecularized .beta.-(1,3)-glucan (Compound 
No. CS-1). 
S-content (Elementary Analysis):14.4% 
Weight Average Molecular Weight (Gel Filtration Method): 113,000 
In the same manner as above, other modified and low-molecularized 
.beta.-1,3-glucans of Compound Nos. CS-2 to CS-11 were obtained. 
Example 2: Preparation of Modified Low-Molecularized .beta.-1,3-glucan from 
Lentinan as a Raw Material: 
1 g of lentinan as dried overnight under reduced pressure over phosphorus 
pentoxide was added to 100 ml of DMSO as previously dried with Molecular 
Sieve 4A and stirred for 2 hours at room temperature and dissolved 
therein. After 2.3 g of piperidine sulfate was added thereto, the whole 
was heated up to 85.degree. C. and then reacted for 1 hour under this 
condition. After completion of the reaction, the reaction mixture was 
cooled with water, and 50 ml of 30% sodium acetate and then 600 ml of 
acetone were added thereto to precipitate the product. The resulting 
precipitate was washed several times with 200 ml of acetone and then 
dissolved in 100 ml of water and then de-salted through a 0.14 .mu.m 
ceramic membrane. To 100 ml of aqueous solution of the thus de-salted 
product was added 400 ml of methanol solution of 5% sodium acetate whereby 
the product was precipitated out. The precipitate was washed twice with 
100 ml of methanol and then dried under reduced pressure to obtain 1.8 g 
of the intended product of modified and low-molecularized 
.beta.-1,3-glucan (Compound No. LS-1). 
S-content (Elementary Analysis): 14.2% 
Weight Average Molecular Weight (Gel Filtration Method): 158,000 
In the same manner as above, other modified and low-molecularized 
.beta.-(1,3)-glucans of Compound Nos. LS-2 to LS-8 were obtained. 
Example 3: Determination of Anti-HIV Activity and Anti-Coagulating Activity 
of Modified Low-Molecularized .beta.-(1,3)-Glucans: 
MT-4 cells were infected with HTLV-III at moi=0,002. The specimen to be 
examined was diluted with a medium in a determined concentration and mixed 
with the infected cells in a ratio of 1/1, and 30.times.10.sup.4 cells/ml 
were incubated. 6 days after incubation, the number of living cells and 
the antigen-positive ratio were determined. 
The specimen to be examined was blended with 50 parts by volume of rat 
plasma and 1 part by volume of a solution of the test substance as 
stepwise diluted with a physiological salt solution and the activated part 
thromboplastic time (APTT) of the specimen was determined. The 
concentration of the test substance to prolong APTT twice was obtained and 
this was compared with the concentration of sodium heparin solution (whose 
titer of anti-coagulating activity is known) to prolong APTT of the 
control specimen twice, whereby the anti-coagulating activity of the test 
substance was represented as the titer of sodium heparin by unit/mg. The 
results were summarized in the following Table. 
__________________________________________________________________________ 
3.3 .mu.g/ml 
10 .mu.g/ml 
Weight 
Anti- 
Percen- 
Percen- 
Percen- 
Percen- 
Average 
Coagula- 
tage of 
tage of 
tage of 
tage of 
Molecular 
ting Living 
Positive 
Living 
Positive 
S-Content Weight 
Activity 
Cells 
Antigen 
Cells 
Antigen 
(%) (.times. 10.sup.4) 
(.mu./mg) 
(%) (%) (%) (%) 
__________________________________________________________________________ 
DS-1 
17.9 1.7 38.6 2.1 &gt;90 93 &lt;1 
DS-2 
17.9 12.2 71.9 1.5 &gt;90 93 &lt;1 
LS-1 
14.2 15.8 9.0 107 &lt;1 100 &lt;1 
LS-2 
15.2 7.3 15.0 95 &lt;1 84 &lt;1 
LS-3 
12.5 14.9 8.9 121 n.a. 103 n.a. 
LS-4 
13.4 11.6 11.2 90 &lt;1 99 &lt;1 
LS-5 
15.2 30.5 11.1 89 &lt;1 103 &lt;1 
LS-6 
12.8 26.3 11.2 77 &lt;1 101 &lt;1 
LS-7 
11.7 11.4 n.a. 3.2 &gt;90 46 &gt;90 
LS-8 
10.7 17.4 10.6 1.6 &gt;90 3.2 &gt;90 
CS-1 
14.4 11.3 10.9 104 &lt;1 102 &lt;1 
CS-2 
12.4 33.0 16.5 112 &lt;1 103 &lt;1 
CS-3 
14.0 4.5 9.5 113 &lt;1 99 &lt;1 
CS-4 
13.1 10.2 n.a. 107 &lt;1 93 &lt;1 
CS-5 
12.0 7.2 n.a. 1.9 &gt;90 14 &gt;90 
CS-6 
11.7 13.1 n.a. 0 &gt;90 4.5 &gt;90 
CS-7 
16.2 2.7 15.3 100 &lt;1 103 &lt;1 
CS-8 
14.7 15.2 16.2 115 &lt;1 114 &lt;1 
CS-9 
12.8 22.9 12.7 104 &lt;1 110 &lt;1 
CS-10 
16.8 6.5 11.2 94 &lt;1 97 &lt;1 
CS-11 
14.9 2.0 5.8 0.6 &gt;90 0.6 &gt;90 
__________________________________________________________________________ 
D.S.: Dextran Sulfate 
L.S.: Modified LowMolecularized Lentinan 
C.S.: Modified LowMolecularized Curdlan 
n.a.: not analyzed 
Thus, in accordance with one aspect of the present invention, a ceramic 
membrane is used to effect the purification of sulfated polysaccharides, 
in particular, the purification of the .beta.-1,3-glucans of the present 
invention. 
In accordance with another aspect of the present invention, a modified and 
low molecular weight .beta.-1,3glucan of a sulfated curdlan or lentinan is 
provided having an excellent anti-HIV activity, and a greatly reduced or 
weaker anti-coagulating activity as compared to dextran sulfate.