Interferon inducer, a process for producing the same and pharmaceutical composition containing the same

An interferon inducer isolated from the plant tissue, having an elemental analysis of H: 8.5-8.7 %, C: 48.8-49 %, N: 6.3-6.5 % and P: 1.0-1.1 % and a molecular weight of from about 100,000 to about 3,000,000 (mainly about 200,000 to about 1,000,000) and containing as main constituents amino acids, sugars and phosphoric acid. This substance is believed to be a homogeneous polymer of protein and sugars, containing phosphoric acid. This substance has an excellent interferon inducing activity and low toxicity and is significantly active against various viruses. The interferon inducer may be produced by extracting a substance having interferon inducing activity from the tissue of a plant of the genus Perilla of the family Labiatae or a variant thereof containing said active substance and recovering the active substance from the extract thereby obtained. Preferably, the extraction may be effected with water and the recovery may be effected by ultrafiltration. Pharmaceutical composition of this invention comprises as active ingredient the interferon inducer of this invention in association with a pharmaceutical carrier or excipient.

BACKGROUND OF THE INVENTION 
This invention relates to interferon inducer, a process for producing the 
same, the use of the same and a pharmaceutical composition containing the 
same. 
Interferon, hereinafter also referred to as IF is a substance capable of 
acting upon animal or human cells to inhibit the growth of a virus and is 
a type of protein liberated from the cell in response to viral infection. 
The activity of IF is specific with respect to an animal species and 
non-specific with respect to a viral species and may vary, with diferring 
conditions used for its induction. It is also known that the growth of 
certain animal tumour type viruses may significantly be inhibited by IF 
under certain conditions. 
A substance capable of acting upon animal or humans cells to induce IF is 
designated as an IF inducer. Thus an IF inducer is of potential interest 
in the prevention and treatment of various human and animal diseases 
caused by viral infection. However, various known IF inducers have never 
been used in practice for such a purpose because of certain serious 
defects. Thus, for example, U.S. Pat. No. 3,583,893 (1971) discloses the 
production of a double-stranded ribonucleic acid as an IF inducer 
originating from a microorganism and describes in its prior art statement 
that many substances including bacteria, viruses, polysaccharides, 
mitogenic agents, endotoxin and the like stumulate interferon formation 
but none is of interest for routine use because of their inter alia 
toxicity, antigenicity and infectiousness. It has thus been believed that 
IF inducers isolated from microorganisms are in general disadvantageous 
for therapeutic use because of their high toxicity. 
Examples of known mitogenic agent isolated from the tissues of higher 
plants include phytohemagglutinin (PHA) [Wheelock, Science, 149:301 (1965) 
and J. Biol. Chem., 212: 607-615 (1955)], pokeweed mitogen [Friedman et 
al, Proc. Soc. Exp. Med., 125:90 (1967) and J. Exp. Med., 124:859-872 
(1966)] and concanavallin A [Willen et al, Cell. Immunol., 6:110 (1973) 
and Methods of Carbohydrate Chemistry, Vol. VI. 108-110 (1972)] 
respectively isolated from the tissues of kidney bean, pokeweed and horse 
bean by extracting with a saline solution or buffer solution, treating the 
extracted solution with an alcohol, followed by purification with column 
chromatography. Due to their extremely low IF-inducing activity, however, 
no successful attempt has been made to use these mitogenic agents for 
preventing and treating various diseases caused by viral infection. 
Other IF inducers isolated from higher plants are also known. That is, 
Kojima et al [Japanese Patent Application as laid open to public 
inspection as Kokai Koho 32107/78] disclosed an IF inducer which is 
believed to be a kind of heteropolymeric saccharide containing as main 
constituents hexose (48%), protein (5%) and uronic acid (40%) and having a 
molecular weight of more than 100,000. This substance is isolated from the 
root of Angelica acutiloba Kitagawa (known in Japan as Toki) by extracting 
the root with hot water to give an extracted solution, subjecting the same 
to dialysis to give a residue, adding acetone to the residue to give a 
precipitate and freeze-drying the same. The extracted solution may, if 
desired, be made up to a suitable quantity by concentrating under reduced 
pressure or by using a Diaflo membrane (MW 10,000), followed by dialysis. 
Subsequently, Kojima and Tamamura [Japanese Patent Application laid open 
to public inspection as Kokai Koho 99313/78] disclosed an IF inducer 
having a molecular weight of more than 20,000 (mainly more than 60,000) 
and containing as main constituents a 1-3 bonded glucose (hexose: more 
than 90%). This IF inducer is produced by extracting the peeling of a 
mulberry e.g. Morus alba L. or M. bombycis Koidzumi with hot water, adding 
an organic solvent to the extracted solution to give a precipitate, adding 
a small amount of water to the same, subjecting the mixture to dialysis to 
give a residue and freeze-drying the same. If desired, the solution after 
extraction or before dialysis may be made up to a suitable quantity either 
by concentrating under reduced pressure or by using a Diaflo membrane. 
These two IF inducers isolated from the tissues of higher plants have high 
IF-inducing activity and low toxicity and may be obtained readily and 
cheaply. However, the cheap and abundant supply of the raw material may 
cease as a result of the continued use of these plants over many years as 
the source of Sino-Japanese traditional drugs. 
SUMMARY OF THE INVENTION 
The present invention is based upon the discovery that a substance which we 
have isolated from the tissue of various plants of the genus Perilla of 
the family Labiatae (known in Japan as the genus Shiso of the family 
Shiso) and variants thereof shows high IF-inducing activity and extremely 
low toxicity. Moreover, the isolation of the active substance may be 
effected readily and simply. 
According to this invention, there is provided a substance having 
IF-inducing activity which is stable in the substantially pure form of an 
amorphous whitish powder and which possesses the following 
physico-chemical characteristics: 
(1) Elemental analysis: H:8.5-8.7%; C:48.8-49%, N:6.3-6.5%, P:1.0-1.1% 
(2) Molecular weight: About 100,000 to about 3,000,000 (mainly about 
200,000-about 1,000,000). 
(3) Melting or decomposing point: Melting point indefinite. Carbonized at 
about 220.degree. C. 
(4) Ultraviolet absorption spectrum: As shown in FIG. 1 (in 0.1 N NaOH 
solution) which is unchanged in water or 1 N NaOH solution. 
(5) Infrared absorption spectrum: As shown in FIG. 2 (by KBr method) 
(6) Solubility in various solvents: Soluble in water, readily soluble in 
aqueous solution of sodium hydroxide, potassium hydroxide and ammonium 
hydroxide, and substantially insoluble in methanol, ethanol, propanol, 
butanol, acetone, chloroform and diethyl ether. 
(7) Color reaction: Positive in ninhydrin reaction, phenol/sulfuric acid 
reation and Dittmer reaction. Negative in Folin's reagent and Elson-Morgan 
reaction. 
(8) Nature: Acidic. 
(9) Main chemical constituents: 
(a) Amino acids (.+-.0.3%): Oxyproline (3.2%), aspartic acid (9.3%), 
threonine (6.1%), serine (4.3%), glutamic acid (7.6%), proline (4.0%), 
glycine (10.0%), alanine (10.3%), valine (6.6%), isoleucine (5.4%), 
leucine (8.6%), phenylalanine (2.0%), lysine (3.9%), arginine (3.4%), 
tyrosine (trace), histidine (1.3%), ammonia (13.4%) 
(b) Sugars (.+-.0.3%): Arabinose (47.09%), galactose (25.66%), glucose 
(20.62%), mannose (4.64%). xylose (1.99%) 
(10) Optical rotation: [.alpha.].sub.D.sup.25 =-75.degree. to -82.degree. 
(-79.degree. in average) (c=0.47% in 0.1 N NaOH). 
The elemental analysis of the active substance of this invention was 
determined by using a Perkin-Elmer Model 240 Elemental Analyzer 
(commercial product of Perkin-Elmer Corpn., U.S.A.). The molecular weight 
of the active substance of this invention was determined by analytical 
ultracentrifugation using a Spinco Model E Analytical Centrifuge 
(commercial product of Beckmann Instrument Inc., U.S.A.), ultrafiltration 
using an Amicon Ultrafilter with XM50, XM100A and XM300 membranes 
(commercial products of Amicon Corpn., U.S.A.) and UK10, UK50 and UK200 
membranes (commercial products of Toyo Roshi K.K., Tokyo) and gel 
filtration using Sephadex G-200 (commercial product of Pharmacia Fine 
Chemicals AB., Sweden). The ultracentrifugation was effected under the 
following conditions and one broad peak having gentle slopes on both sides 
was observed: A sample (0.5-1.0%) of the active substance was suspended in 
a neutral solution of 0.1 M sodium chloride and centrifuged at 20.degree. 
C. at a maximum run of 30,000 or 60,000 r.p.m. Supplementarily, column 
chromatography using gel filtration agents such as e.g. the series of 
Sepharose, Sephacryl (commercial products of Pharmacia Fine Chemicals AB., 
Sweden) and Bio-Gel (commercial products of Bio-Rad Laboratories Ltd., 
U.S.A.) was also used. All the results obtained were compared with the 
control values obtained, for example, by using standard references having 
identified molecular weights such as e.g. blue dextran 2000 
(*2.times.10.sup.6), .alpha..sub.2 -macroglobulin from horse serum 
(*8.times.10.sup.5), thyroglobulin from bovine thyroid 
(*6.69.times.10.sup.5), catalase from bovine lever (*2.1.times.10.sup.5), 
aldolase from rabbit muscle (*1.58.times.10.sup.5) and albumin from bovine 
serum (*6.7.times.10.sup.4) [*.. standard molecular weight] Throughout 
various fractions having different molecular weights, substantailly the 
same elemental analysis and IF-inducing activity (determined by the method 
of hereinafter described Experiment 1) were found. From these results, in 
combination with a broad single band observed by the electrophoresis (cf. 
hereinafter described Experiment 2) and a high recovery ratio (cf. 
hereinafter described Experiment 4), it is found that the active substance 
of this invention is not a mixture but a high molecular weight polymer 
composed of polymers having substantially the same chemical and biological 
characteristics, of which major portion is present in a range of from 
about 200,000 to about 1,000,000 and the minor portion is present in a 
range of from about 100,000-about 200,000 and about 1.000.000-about 
3,000,000. 
The UV and IR absorption spectra were determined respectively by using 
Hitachi 340 Recording Spectrophotometer (commercial product of Hitachi 
Limited,. Japan) and Shimazu Recording Infrared Spectrophotometer IR-27G 
(commercial product of Shimazu Seisaku-sho, Japan). 
The amino acids present were determined by hydrolysis with 6 N HCl at 
110.degree. C. for 48 hours in vacuo, followed by analysis using a 
Technicon Amino Acid Autoanalyzer Type NC-1 (commercial product of 
Technicon Corpn., U.S.A.) and the sugars present were determined by 
hydrolysis with 0.1 N sulfuric acid at 80.degree. C. for 20 minutes and 
with 1 N sulfuric acid at 100.degree. C. for 2 hours respectively, 
followed by analysis using a Technicon Sugar Autoanalyzer Type N-1 
(commercial product of Technicon Corpn., U.S.A.). 
The active substance of this invention also preferably and readily soluble 
in alkaline solutions and substantially insoluble in organic solvents. 
The following tests were effected to ascertain that the active substance of 
this invention represents an IF inducer. 
(1) IF-inducing activity: 
Samples of the active substance of this invention were used to induce IF in 
the cells and serum of test animals and the activity of the resultant IF 
was determined by the methods hereinafter described in Experiment 1. The 
results shown in Table 1 indicates that the IF inducing activity is 
positive. 
TABLE 1 
______________________________________ 
Concentration of sample (.mu.g/ml) 
10 1.0 0.1 0.01 
______________________________________ 
Activity in 
&gt;100 &gt;100 94 &lt;10 
vitro 
______________________________________ 
TABLE 2 
______________________________________ 
Time of collection of blood after 
Activity administration (hour) 
in vivo* 0 1 2 4 6 
______________________________________ 
Rabbit 1 &lt;10 90 240 30 13 
Rabbit 2 &lt;10 25 64 14 10 
______________________________________ 
*Dose = 500 .mu.g/ml 
Table 2 indicates that the results obtained by the method hereinafter 
described in Experiment 1 using two rabbits show the activity reaches its 
maximum 2 hours after administration. It was also confirmed that by the 
method of Experiment 1, IF was induced in the body of the test animals by 
the action of the IF inducer of this invention. 
(2) Stability of the IF inducer: 
Samples (each 1 mg) of the IF inducer of this invention were respectively 
dissolved in water (each 1 ml) and heated at 100.degree. C. for a given 
time or at a given temperature for one hour, and was then treated by the 
method of hereinafter described in Experiment 1 (in vitro method) to 
obtain the results shown in Tables 3 and 4 indicating a high heat 
stability of the IF inducer of this invention. 
TABLE 3 
______________________________________ 
Activity at 
Heating Concentration of sample (.mu.g/ml) 
temperature (C..degree.) 
10 1.0 0.1 0.01 
______________________________________ 
IF activity 
Untreated &gt;100 &gt;100 88 &lt;10 
37 &gt;100 &gt;100 88 &lt;10 
60 &gt;100 &gt;100 80 &lt;10 
80 &gt;100 &gt;100 88 &lt;10 
100 &gt;100 &gt;100 85 &lt;10 
______________________________________ 
Heating time: one hour. 
TABLE 4 
______________________________________ 
Activity at 
Heating time 
Concentration of sample (.mu.g/ml) 
(hour) 10 1.0 0.1 0.01 
______________________________________ 
Untreated &gt;100 &gt;100 96 &lt;10 
1 &gt;100 &gt;100 92 &lt;10 
4 &gt;100 &gt;100 98 &lt;10 
8 &gt;100 &gt;100 95 &lt;10 
24 &gt;100 &gt;100 70 &lt;10 
______________________________________ 
Heating temperature: 100.degree. C. 
From the above-mentioned characteristics, it has been found that the active 
substance of this invention conforms to widely recognized definition of 
any IF-inducer. The induced IF in animal cell or serum in vitro or in vivo 
is inactivated with 0.08% trypsin at 37.degree. C. for 2 hours and morover 
its activity is specific with respect to an animal species and 
non-specific with respect to a viral species. 
In Tables 1-4, the samples used were prepared by the method of Example 1. 
It is thus believed that the active substance of this invention is not only 
a new IF inducer but also a new substance because no such a substance has, 
to our knowledge, ever been reported in the art. For example, mitogenic 
agents such as phytohemagglutinin, pokeweed mitogen and cncanavallin A 
described in the literatures are types of protein having very weak 
IF-inducing activity which is inactivated on heating at 56.degree. C. for 
5 hours, on the contrary to high heat stability and high IF-inducing 
activity of the active substance of this invention. The known IF inducers 
isolated from the root of Angelica actiloba Kitagawa is high molecular and 
its IF-inducing activity is not inactivated on heating at 100.degree. C. 
for one hour. However, its chemical constituents and infrared absorption 
spectrum are different from those of the active substance of this 
invention. The known IF inducer isolated from the peeling of mulberry root 
contains as main constituent a 1-3 bonded glucose and has a different 
molecular weight. Moreover, the mitogenic activity which is found in the 
known IF inducers originating from bactrial endotoxin and higher plants 
such as Angellica actiloba and mulberry is not found in the IF inducer of 
this invention. 
Various plants of the genus Perilla and variants thereof which may be used 
as a source of the product of this invention contain, for example, perilla 
aldehyde, .alpha.-pinene, 1-limonene, perilla ketone, naginata ketone, 
shisonin, p-cumaric acid ester, dihydroperilla alcohol, 1-menthol and the 
like, all of which are low molecular weight substances and, to our 
knowledge, have no IF-inducing activity. The physico-chemical 
characteristics of the known IF inducers [disclosed in U.S. Pat. Nos. 
3,583,893; 3,773,924 and 3,884,845 and Japanese Kokai Koho 121919/78] are 
different from those of the IF inducer of this invention. 
According to another feature of this invention, we provide a process for 
producing an interferon inducer from the plant tissue, comprising 
extracting the said IF inducer with water from the tissue of a plant 
selected from the genus Perilla and variants thereof containing the said 
IF inducer at a temperature from ambient to the boiling point of the 
extraction mixture for a period sufficient to extract the major portion of 
the IF inducer present in the tissue, forming a supernatant from the 
extracted solution, fractionating the supernatant to yield fractions 
containing a major portion of the IF inducer present in the supernatant, 
and recovering the IF inducer therefrom. 
The plants which may be used for the process of this invention include the 
plants of the genus Perilla which are annual herbs grown mainly in e.g. 
India, East Asia and other countries and which are liable to form various 
variants such as mutants and hybrids naturally or artificially. It has 
been found that many plants of the genus Perilla and variants thereof may 
preferably be used for the purpose of this invention. The following plants 
are merely indicated as examples [*Japanese nomenclature]: Shiso* (P. 
frutescens Britton var. crispa Dec. f. purpurea Makino); Aojiso* (P. 
frutescens Britton var. crispa Dec. f. viridis Makino); Katamen-jiso* (P. 
frutescens Britton var. crispa Dec. f. discolor Makino); Chrimen-jiso* (P. 
frutescens Britton var. crsipa Dec. f. crispa Makino); Chrimen-aojiso* (P. 
frutescens Brit. var. crispa Dec. f. viridiscrispa Makino); Toranoo-jiso* 
(P. frutescens Brit. var. hirtella Makino et Nemoto); Egoma* (P. 
frutescens Britton); Lemon-egoma* (P. citriodora Nakai and variants 
thereof. 
[The botanical names are designated with reference to "Genshoku Shokubutsu 
Dai Zukan", vol. I, authored by Murakoshi and Makino and published by 
Hokuryukan, Tokyo (1955), "Yakuyo Shokubutsu Dai Jiten", edited by 
Kariyone and Kimura and published by Hirokawa Shoten, Tokyo (1974) and 
"Saishin Wakan Yakuyo Shikubutsu", authored by Kariyone and Kimura and 
published by Hirokawa Shoten, Tokyo (1978).]. 
It is known that the plants exemplified above have generally low toxicity, 
and for example, Shiso, Aojiso Chirimen-jiso and Lomon-egoma are cultured 
in Japan, China and other countires because their leaves and seeds have 
been used since long as foodstuffs, Sino-Japanese crude drugs, raw 
material for production of perfume and the like. Labiatae is a family with 
many genera containing essential oils such as sage or mint, and to our 
knowledge, it was not previously known that any plant of Labiatae contains 
a substance having IF-inducing activity, as reported, e.g. by Keisetsu 
Otsuka [Kanpo, Geschichte, Theorie und Praxis, 165-189 (1976)], Richard 
Hyatt [Chinese Herbal Medicine, Ancient Art & Modern Science, 136 (1978)] 
and John D. Keys [Chinese Herbs, Their Botany, Chemistry and 
Pharmacodynamics, 256 (1978)]. 
Although any and all the tissues of Perilla plants may be used for the 
process of this invention, the leaves and stems are preferred because the 
seeds contain a weak activity and moreover, additional operation may be 
neccesary for removing the soil from the root of the plant. Both the 
leaves and stems contain substantially the same amount of the active 
substance. The amounts of the active fractions contained in various tissue 
of a plant are substantially unchanged before and after the flowering 
period. 
For better preservation and extraction, it is preferred to use the dried 
plant, although it is possible to use the fresh material if desired. The 
drying method is convenient. If desired, the material may be washed with 
water before use. 
The extraction may preferably effected with water at any convenient 
temperature, e.g. from ambient to the boiling point of the extraction 
mixture. As the active substance of this invention is particularly soluble 
in water under alkaline conditions (e.g. at a pH of 7-10), it is preferred 
to adjust the pH of water before use, for example, with a suitable buffer 
solution, sodium hydroxide, potassium hydroxide, ammonium hydroxide and 
the like. The extraction may be effected over any convenient period of 
time, usually 1-5 days at room temperature, which may be shortened if the 
extraction temperature is raised. Thus, for example, the extraction may be 
effected for a period of time from 30 minutes to 6 hours at 
45.degree.-80.degree. C. with or without stirring. In this manner, it is 
possible to extract a major portion of the active substance contained in 
the starting material (in some cases, more than 90%). However, the use of 
an excessively high temperature should be avoided because the quantity of 
undesired impurities such as e.g. pigments, low molecular weight 
substances and the like appearing in the extract may thereby increase. It 
is also possible to add a suitable antiseptic agent to the extracting 
water. The extraction may be effected continuously or intermittently at 
any convenient ratio of the extracting water to the raw plant. 
It is also possible, if desired to extract the active substance with a 
hydrophilic organic solvent incapable of dissolving the IF inducer of this 
invention such as e.g. methanol, ethanol, propanol, butanol, acetone and 
the like in any convenient amount e.g. 20-80%. In such a case, the 
extraction time and temperature may be convenient e.g. for 4 hours to 2 
days at 40.degree. to 80.degree. C. In such cases, the extraction may be 
effected by using a hydrophilic organic solvent in spite of the 
insolubility of the pure product of this invention in such solvents, for 
example when the extracted solution contains a mixture of complex of 
substances such as e.g. fatty acids, steroids, proteins, mono- or 
polysaccharides and the like. Whilst we do not wish to be bound with 
theoretical considerations, it is believed that extraction using such 
solvents is made possible by buffer action. However, extraction with water 
is most advantageous because it is simpler, cheaper and safer in 
operation. 
The residue of the plant tissue is then removed from the extracted solution 
in conventional manner, for example, by filtration, pressing, 
centrifugation and the like. After this, undesired impurities such as 
pigments, low molecular weight substances are removed from the resultant 
supernatant in order to allow recovery of the active substance of this 
invention. Preferred methods for this purpose are exemplified as follows. 
(A) The supernatant is fractionated by ultrafiltration e.g. using a 
suitable membrane for retatining substances having a molecular weight of 
more than 100,000 because the active substance of this invention is 
present in fractions having a molecular weight of about 100,000 to about 
3,000,000 (mainly about 200,000 to about 1,000,000). The ultrafiltration 
may be effected under suitable pressure, for example, 0.1 to 5 kg/cm.sup.2 
by using a membrane capable of retaining substances having a molecular 
weight of more than 100,000 or 200,000. The active fractions are collected 
and combined, and the combined fractions are freeze-dried to obtain brown 
powders. 
(B) The supernatant is concentrated, if desired, under reduced pressure and 
is treated with a hydrophilic organic solvent (e.g. methanol, ethanol, 
propanol, n-butanol, acetone and the like) at a convenient concentration 
(e.g. 40-70 w/v %) so as to form precipitates containing the active 
substance. The precipitates are freeze-dried to obtain brown powders. 
(C) Instead of the organic solvent, it is possible to add to the 
supernatant an ammonium salt (e.g. ammonium chloride, ammonium sulfate, 
cetylmethylammoniumbromide and the like) or an inorganic metalic salt 
(e.g. zinc chloride, copper chloride and the like) at a convenient 
concentration (e.g. 20 to 50 w/v %) so as to form precipitates which are 
then desalted and freeze-dried to obtain brown powders. 
It is possible to recover the major portion of the active substance 
contained in the starting material (in some cases, more than 90%). 
However, the quantity of impurities contained in the crude powder is 
lowest in the case of method (A), and also method (A) may be effected 
simply. Moreover, it has been confirmed that any significant side effect 
may be avoided even when a large amount of the crude powder obtained by 
(A) is orally administered to humans and animals and thus this crude 
powder may be used for oral administration without further purification. 
If desired, the crude powder thus obtained may further be purified, for 
example, by column chromatography using a suitable agent for gel 
filtration or an ion exchanger. In the former case, the elution may be 
effected with water, although it is possible to use a suitable buffer 
solution. In the latter case, the elution may be effected with a suitable 
buffer solution. Preferable agents for gel filtration are exemplified by 
Sephadex G-50 to G-200, Sepharose 2B to 6B, Sephacryl S-200 or S-300 
(commercial products of Pharmacia Fine Chemicals AB., Sweden), Bio-Gel 
P-30 to P-300, Bio-Gel A (commercial products of Bio-Rad Laboratories 
Ltd., U.S.A.), Sagavac (commercial by QAE-Sephadex A-25 and A-50 (Cl.sup.- 
form), CM-Sephadex C-25 and C-50 (Na.sup.+ form), SP-Sephadex C-25 and 
C-50 (Na.sup.+ form), DEAE-Sephacel (Cl.sup.- form), DEAE-Sepharose CL-6B 
(Cl.sup.- form), CM-Sepharose CL-6B (Na.sup.+ form) (commercial products 
of Pharmacia Fine Chemicals AB., Sweden) and the like. It is also possible 
to use a suitable anion or cation exchange cellulose for the purification. 
The product thus-obtained may contain certain impurities, although its 
IF-inducing activity is sufficient for practical purposes. If desired, the 
amounts of impurities may further be reduced by combining these 
treatments. 
It is apparent moreover that the process of this invention may be applied 
not only to Labiatae but also to all the higher plants such as 
Tracheophyta e.g. Mono- or Dicotyleodoneae when they contains a substance 
having IF-inducing activity and water-solubility. 
According to a further feature of this invention, there is provided a 
process for inducing an IF in the body or cells of human or animal, which 
comprises administering an effective amount of the IF inducer of this 
invention to a human or IF-inducing animal. 
The active substance of this invention may be used without any toxic 
trouble which is inherent to various known IF inducers such as e.g. 
polyI:C, endotoxin and the like. Moreover it has also been found that when 
administered to humans, the activity of the IF inducer of this invention 
is superior to those of the known IF inducers. The IF inducer of this 
invention is capable of not only affording a high antiviral activity but 
also enhancing anti-tumour activity and overall physiological activities. 
It is thus possible to use the IF inducer of this invention not only for 
the prevention and curing of various virally caused diseases of various 
vetebrates such as, for example, humans, mammals (e.g. cattle, horse, pig 
etc.), birds (e.g. fowl, duck etc.), fishes (e.g. rainbow trout etc.) and 
the like but also as anti-tumour agent, agent for improving overall 
healthy conditions for humans and animals. 
The active substance of this invention may be administered e.g. by 
intravenous or intraperitoneal administration, intestinal or oral 
administration, spraying and the like. 
In the case of intravenous administration, it may be possible to administer 
the active substance at a dose of 0.001 to 100 mg/kg/day, calculated as 
the final product. However, such a dose may vary, depending upon, for 
example, the type, age and weight of the host and various other 
conditions, among which the response of the host to IF induction and the 
purpose of the administration are most important. The active substance of 
this invention may preferably be administered to animals, for example, at 
a daily dose of 0.01 to 10 mg/kg (iv.) or 0.1 to 10 mg/kg (ip.) and to 
humans at a daily dose of 0.01 to 1.0 mg/kg (ip.) by injection. In the 
case of oral administration, it is possible to use, for example, more than 
about 10 times of the dose for intravenous administration. When the 
administration is effected topically or in a shorter period of time, a 
larger amount of the active substance may be used. When the dose is 
excessively small, it may be difficult to induce IF in the body or cells 
of the host. However, the use of an excessively large amount of the active 
substance of this invention may, in general, give rise to no serious side 
effect because its toxicity is extremely low. 
It has also been found that when a suitable amount of any IF is 
administered to a host, followed by administering the active substance of 
this invention, the activity of the IF induced by the active substance of 
this invention may significantly be enhanced and for example about 3-10 
folds activity may be obtained thereby. Moreover, it is possible in this 
manner to enhance considerably the response of the host to IF induction 
and also to extend the effective period of time of the IF induced. 
The following samples were used in the following tests: 
Sample A: The crude powder obtained by ultrafiltration by the method of 
Example 1 and freeze-dried. 
Sample B: The finally purified product by the method of Example 1. 
Sample C: The finally purified product by the method of Example 2. 
As test animals, rabbits (same type as that used in hereinafter described 
Experiment 1), mice (weight 25.+-.1 g, ddy-strain, 6 weeks old) and fowls 
(weight about 230 g, White Leghorn, 30 days old) were used. 
(A) IF Induction and Determination of IF Activity 
(I) In vitro Method 
A spleen cell suspension (10.sup.7 cells/ml) was prepared by the method of 
hereinafter described Experiment 1 using an Eagle MEM medium (commercial 
product of Nissui Seiyaku K.K., Tokyo) containing a 10% calf serum (which 
was replaced by a 10% fetal cattle serum in the cases of human tests), 
each of which fraction (1 ml) was added with a given concentration of the 
sample of the active substance and incubated at a given temperature. The 
cultured liquor was centrifuged to separate a supernatant which was used 
for determining the IF activity induced. In the case of humans, the fresh 
spleen was collected from a man (adult deceased by external wounds) and 
used for the preparation of a leucocytes suspension on each occasion, and 
also blood was collected from the vein at the upper arm of a man (adult), 
from which the serum was separated to use for the preparation of a serum 
suspension on each occasion. 
The IF activities were determined by using the cells shown in Table 5 in a 
similar manner to that hereinafter described in Experiment 1. 
TABLE 5 
______________________________________ 
IF induction in vitro 
I II III 
______________________________________ 
Rabbit 3 25 RK-13 
Mouse 30 37 L 
Human spleen 2 25 FL 
Human leucocytes 5 37 FL 
Fowl (White Leghron) 
5 37 Fibroblast 
______________________________________ 
I: Numbers in each group 
II: Cultured for 24 hours at a temperature of (.degree.C.) 
III: Cells used for determination 
The results are shown in Table 6. 
TABLE 6 
______________________________________ 
IF activity in vitro 
Concentration (.mu.g/ml) 
Control* 
Sample 
100 10 1 0.1 0.01 1.0 
______________________________________ 
Rabbit A 200 255 90 &lt;5 &lt;5 
B 240 270 250 140 40 220 
C 260 300 285 165 45 
Mouse A 65 85 30 10 &lt;5 
B 195 225 140 75 20 120 
C 210 250 155 80 10 
Human A 30 35 12 &lt;5 &lt;5 
(spleen) B 34 40 20 8 &lt;5 &lt;5 
C 35 38 21 10 &lt;5 
(leucocytes) 
B 30 20 12 &lt;5 
Fowl A 25 12 &lt;5 
B 74 48 10 &lt;5 
C 80 52 12 &lt;5 
______________________________________ 
*Poly I:C 
(II) In vivo Method: 
(1) Rabbit 
Table 2 shows the IF activities induced in rabbits by the method 
hereinafter described in Experiment 1. 
TABLE 2 
______________________________________ 
Used sample: sample B* 
Time of collection of blood 
IF activity 
after administration of sample 
(in vitro) 
0 1 2 4 6(hour) 
______________________________________ 
Rabbit 1 &lt;10 90 240 30 13 
Rabbit 2 &lt;10 25 64 14 10 
______________________________________ 
*Dose: 500 .mu.g/ml 
Similar treatments were repeated by changing the dose of the sample 
stepwise within a range of 4 to 0.004 mg/kg, and it was found that the IF 
activity induced in the serum reached its maximum 2 hours after 
administration. Also, the maxium was observed about 12 to 15 hours after 
oral administration. 
(2) Mouse 
Mice (each group consisting of 10 mice) were used as test animals and 
treated as follows. 
(a) Each 0.1 ml of a physiological solution of sodium chloride containing a 
given amount of sample B shown in Table 4 was injected into the vein at 
the tail of each mouse which was then allowed to stand for 1, 2, 3 or 5 
hours, or 
(b) Each 0.2 ml of a physiological solution of sodium chloride containing a 
given amount of sample B shown in Table 4 was administered (ip) to each of 
the mice which was then allowed to stand for 2, 4 or 6 hours, or 
(c) Water (0.2 ml) containing a given amount of sample B shown in Table 4 
was orally administered to each mouse which was then allowed to stand for 
2.5, 5, 7.5 or 10 hours. 
On each occasion, the test animal was then sacrificed by cardic puncture. 
Blood was collected from each mouse and used to prepare the serum. The IF 
activity induced was determined in a similar manner to that described 
above. It was observed that the IF activity reached its maximum about 2-3 
hours in the case (a), about 2-4 hours in the case (b) and about 5-8 hours 
in the case (c), after administration. Table 7 indicates the maximum IF 
activities (mean value). A similar tendency was also found when the same 
treatments were repeated by using sample A instead of sample B. 
TABLE 7 
______________________________________ 
Maximum IF activity in vivo (mouse) 
Concentration 
(mg/kg) Intravenous Ip. Oral 
______________________________________ 
4000 33 
400 22 
40 730 280 20 
4 200 85 &lt;5 
0.4 175 53 
0.04 64 20 
0.004 25 &lt;5 
Untreated &lt;5 &lt;5 &lt;5 
______________________________________ 
(3) Humans 
Each 200 mg of sample B was orally administered to each of five men 
(adults, healthy). Blood was collected from the vein at the arm of each 
volunteer after 13 hours from administration and used for the preparation 
of the serum which was then treated in a similar manner to that described 
above to determine the IF activity of about 15 units (mean value). 
(4) Fowl 
Fowls (each group consisting of 10 chickens) were used as test animals. A 
physiological solution of sodium chloride (each 0.2 ml) containing sample 
B (4 mg/kg) was administered into the vein under the wing of each animal. 
After 2 hours on each occasion, the fowl was sacrificed by cardic 
puncture. Similarly, blood was collected for use to determine that the IF 
activity induced in the serum was about 30 units. 
(B) Protection Against Viral Infections 
(1) Mouse (Vaccinia Virus) 
Mice (each group consisting of 20 female) were used as test animals, to 
each of which was administered a physiological solution of sodium chloride 
(each 0.2 ml) containing a given amount of the sampel shown in Table 5 
intravenously, intraperitoneally or orally. After 24 hours, each mouse was 
infected with Vaccinia virus at a dose of of 30 PFD [1 PFD denotes an 
amount of the virus capable of forming the pocks at the tails of 50% of 
the mice used] contained in 0.1 ml of a physiological sodium chloride 
solution by injecting into the vein at the tail. For 9 days after this, 
the numbers of the pocks formed at the tail were compared with the 
corresponding numbers of the pocks found in the untreated mice (39.8 in 
average) to determine the inhibition ratio. A ratio of more than 50% was 
evaluated as an effective ratio. The results are shown in Table 8. 
TABLE 8 
______________________________________ 
Inhibition ratio (%) 
Method of Concentra- Sample 
administration 
tion (mg/kg) 
A B C 
______________________________________ 
Intravenous 40 100 100 100 
4 93 100 100 
0.4 72 100 100 
0.04 61 89 90 
0.004 38 67 65 
Ip 40 87 100 100 
4 68 100 100 
0.4 45 93 95 
0.04 31 75 75 
0.004 18 45 42 
Oral 4000 53 70 67 
2000 42 66 65 
400 28 55 54 
______________________________________ 
(2) Mouse: (Herpes Simplex Virus) 
Similar treatments to those described above were repeated, but Vaccinia 
virus was replaced by Herpes Simplex virus. The sample was administered 
intravenously, orally or ip to the test animals in a similar manner to 
that hereinbefore described in (B,1), followed by intraperitoneal 
infection of the virus. The results were observed for 30 days after 
infection to determine the average survival days which were compared with 
the corresponding days of untreated group. A significant survival effect 
was found. 
(3) Rabbit (Vaccinia Virus) 
(a) Rabbits (each group consisting of 5 rabbits) were used. A physiological 
sodium chloride solution (each 0.1 ml) containing a given amount of sample 
A was injected on each occasion under the skin at the back of the rabbit 
and 24 hours after this, Vaccinia virus (1 ID.sub.50 which denotes an 
amount of the virus capable of forming at least one pock of more than 
6.times.6 mm in size under the skin of 50% of the test rabbits) in 0.1 ml 
of a physiological sodium chloride solution was injected into the same 
site in a similar manner to that used for administration of the sample. 7 
days after this, the numbers of the pocks were counted and compared with 
the corresponding numbers found on the untreated rabbits. The 
concentration of the sample was stepwise changed from 0.02 to 200 
.mu.g/kg, and it was found that a dose of more than 2 .mu.g/kg gave a 100% 
inhibition. 
(b) A similar treatment to that described in (a) was effected except sample 
A was divided into 5 equal parts (100 mg/day) and administered to the 
rabbit on each occasion on the 1st, 3rd, 4th, 6th and 8th days (500 mg/kg 
in total). The virus was divided into 2 parts (10 and 100 ID.sub.50) and 
respectively infected to each rabbit on the 5th day. On the 12th day, no 
pock was found at the nack of each rabbit infected with 10 ID.sub.50 virus 
and weak pocks were found at the back of each rabbit infected with 100 
ID.sub.50 virus. In the latter case, 2 weeks after the infection, the 
pocks disappeared completely. 
(C) Anti-Tumour Effect (Mouse) 
(1) Ehrlich Ascites Tumour 
Mice (each group consisting of 15 mice) were used as test animals. A 
sterilized water (each 0.2 ml) containing Ehrlich ascites tumour cells 
(each 2.5.times.10.sup.6) was transplanted into each animal by injection 
(ip.). 24 hours after this, a given amount of sample B in sterilized water 
(each 0.2 ml) was given to each animal. The administration was effected 
once daily at a dose of 0.2, 1.0 or 5 mg/kg/day (ip.) or 40, 200 or 1000 
mg/kg/day (orally) and continued for 14 days. By administration at a dose 
of 1 or 5 mg/kg/day (ip.), one half of the treated mice were still alive 
and moreover completely cured 60 days after the transplantation. All 
untreated mice deceased on or before the 29th day after the 
transplantation. The results are shown in Table 9. 
TABLE 9 
______________________________________ 
Median survival 
Increased life 
Dose days spun (%) 
______________________________________ 
1 mg/kg/day(ip.) 
&gt;60 &gt;122 
Untreated 27 -- 
______________________________________ 
A similar effect was observed when 5 equal parts (each 1.0 mg/kg) of sample 
B were administered (ip) to the mice at an interval of 3 days. The result 
obtained by continuation of oral administration of sample B (400 or 1000 
mg/kg/day) for 14 days was not superior to the result obtained by the 
abdominal administration. 
(2) S-180 Sarcoma Solid Tumour 
S-180 Sarcoma solid tumour cells (1.times.10.sup.5 cells) were added to a 
sterilized water (0.2 ml). Each mixture was transplanted to each test 
mouse (each group consisting of 15 mice) under the skin at the armpit. 
Samples of the active substance of this invention were administered to the 
animals in a similar manner to that described above. When a dose of 1 
mg/kg/day (ip.) was administered to each of the test animals, the tumours 
of 5 mice were reduced to about 1/2-1/3 in size 40 days after 
administration and 3 mice were completely cured 60 days after the 
administration. By oral administration at a dose of 1000 mg/kg/day/mouse, 
6 mice were alive 60 days after the beginning and their tumours were 
reduced to about 1/3 in size. 60 days after this, 3 mice were completely 
cured. 35 days after the transplantation, no control mouse was alive. 
(D) Combined Use of IF and IF Inducer (Priming Effect) 
(1) A suspension containing lymphoid cells of rabbits (10.sup.7 cells/ml) 
was prepared by the method of hereinafter described Experiment 1 and was 
added with rabbit IF (30 unit/ml). The mixture was treated at 37.degree. 
C. for 6 hours and was then centrifuged to remove the IF. Then an Eagle 
MEM medium (1 ml) and sample B were added to the cells and its activity 
was determined with simultaneous change of the amount of sample B from 
10.sup.-3 to 10.sup.-7 mg/ml. It was found that the activity of the 
induced IF raised to about 3-10 folds and also the response of the cells 
was significantly improved. 
(2) By the method of hereinbefore described (D,1), a suspension of human 
spleen cells (10.sup.7 cells/ml) was prepared and treated with human IF 
(100 unit/ml). The activity of the IF induced raised to about 3-10 folds 
and also the response of the cells was significantly improved. 
(3) Rabbits (each group consisting of 5 rabbits) were administered with 
rabbit IF (10.sup.6 unit) intravenously. 6 hours after this, sample B was 
given to each rabbit by the method of hereinbefore described (A,II,1). It 
was found that the IF activity induced in the serum raised to about 3-6 
folds and the period of time of IF induction was also extended. 
(4) Each of 5 rabbits was administered (ip) with rabbit IF (10.sup.6 units) 
and after 24 hours, an infection test was effected in a similar manner to 
that described in (B,3,b). After 12 days from the infection with the virus 
at a dose of 100 ID.sub.50, no pock was formed at the back of each treated 
rabbit. 
(E) Toxicity 
(1) Acute Toxicity 
A physiological sodium chloride solution containing a given amount of 
sample B was administered to each of the test mice (each group consisting 
of 20 mice; male and female) and test rats (each group consisting of 20 
rats; weight about 95 g; SPF strain; 6 weeks old; male and female) to 
obtain the LD.sub.50 values shown in Table 10. No significant difference 
was found between male and female. 
TABLE 10 
______________________________________ 
Acute toxicity (LD.sub.50) 
Concentration of sample (g/kg) 
Animal Subcutaneous 
Ip Iv Oral 
______________________________________ 
Mouse &gt;2 0.56 0.45 4 
Rat &gt;1 0.6 0.52 &gt;2.8 
______________________________________ 
(2) Subacute Toxicity 
Rats (weight about 95 g; SPF-SD strain; 6 weeks old; each group consisting 
of 20 rats) were used as test animals. Sample A was divided into fractions 
(0.35, 0.7, 1.4 and 2.8 g/kg), each of which was added to each sterilized 
water (from 0.25 to 0.5 ml), and a given amount of the sample was daily 
administered to the test animals compulsively by using a canule. The 
administration was continued for 3 months. In comparison with the 
untreated animals, the healthy conditions of the test animals were 
improved throughout the test period and their body weights increased at a 
remarkably high ratio. All animals were dissected after the end of 3 
months and investigated pathologically. However, it was difficult to 
determine the subacute toxicity reasonably because no significant change 
was observed pathologically. 
(b) Five healthy men (adults) were administered orally with Sample A (200 
mg/day) and the administration was continued for 10 days. As a result, no 
significant side effect was observed. 
For the purpose of administering the active substance of this invention to 
humans and animals with good results, there is provided a pharmaceutical 
composition, which comprises as active ingredient an effective amount of 
the active substance of this invention as hereinbefore defined, in 
association with a pharmaceutical carrier or excipient. 
The composition may be any and all forms adapted to oral, rectal, 
perenteral, percutaneous, intramucous administration and the like. Thus, 
for example, the composition may be solid or liquid for oral 
administeration and may take the forms of powders, syrups, capsules, 
granules, emulsions, suspensions, drops and the like. Such composition 
comprises carrier or excipient conventionally used in the pharmaceutical 
art. Thus, for example, suitable tabletting excipients include lactose, 
potato and soluble starch and magnesium stearate, and for parenteral 
administration, the carrier may be a steril water, physiological solution 
of sodium chloride, armond oil and the like, which may be put in an ampule 
or may be added to the active substance before use. 
The composition may, if desired, further comprises, for example, bonding 
agents, stabilizing agents, emulsifiers, suspending agents, dispersing 
agents, lublicants, antiseptic agents, fillers and the like conventionally 
used in the pharmaceutical art. Such composition comprises carrier or 
excipient conventionally used in the pharmaceutical art. Thus, for 
example, suitable tabletting excipients include lactose, potato and 
soluble starches and magnesium stearate and for parenteral administration, 
the carrier may be a sterile water, physiological solution of sodium 
chloride, armond oil and the like, which may be put in an ampule or may be 
added to the active substance before use. 
The composition may, if desired, further comprise, for example, bonding 
agents, stabilizing agents, emulsifiers, suspending agents, dispersing 
agents, lublicants, antiseptic agents, fillers and the like conventionally 
used in the pharmaceutical art. 
For practical purpose, the composition may be formulated, for example, as 
buccals, troches, eye drops, suppositories and the like for intramucous 
administration, solutions, oils, suspensions and the like for injection 
agents, inhalants, sprays and the like for inhalational administration and 
ointments, plasters, liniments, bathes, sprays and the like for external 
administration. 
Advantageously, the composition may be formulated as dosage units, each 
unit being adapted to supply a fixed dose of active ingredient. Suitable 
dosage unit forms are, for example, tablets, coated tablets, ampules, 
capsules, suppositories and the like. 
The amount of the active ingredient preferably contained in such dosage 
unit forms may, for example, be within a range of from about 4 to about 10 
for oral administration, about 2-3 for subcutaneous administration, about 
1.5-3 for intramuscular administration, about 2-4 for baccals and troches 
and about 5-10 for suppositories, calculated on the basis of the preferred 
amount for intravenous injection. 
The compositions are exemplified as follows. 
______________________________________ 
(1) Parenteral injection: 
Physiological solution of NaCl 
1.0 ml 
Sample B 0.01 g 
packed and sealed in a 2 ml ampule under steril- 
zed conditions. 
(2) Troch: 
White sugar 1 g 
Sample B 0.05 g 
Starch 0.05 g 
(3) Suppository: 
Polyethylene glycol 400 0.8 g 
Liquid polyethylene glycol 1500 
0.2 g 
Sample A 0.2 g 
(4) Syrup: 
CMC-Na 0.2 g 
Simple syrup 20 g 
Ethylparaffin 0.04 g 
Sample B 0.1 g 
(5) Ointment: 
Purified lanolin 5 g 
Yellow wax 5 g 
White vaselin 87 g 
Sample B 3 g 
(6) Liniment: 
Potassium hydroxide 0.3 g 
Glycerin 20 ml 
Ethanol 25 ml 
Sample B 2.5 g 
Make-up water to 100 ml 
______________________________________

PREFERRED EMBODIMENTS 
The following non-limiting examples illustrate the preparation of the 
active substance of this invention. 
EXAMPLE 1 
Dried leaves of Perilla frutescens Britton var. crispa Dec. f. purpurea 
Makino (known in Japan as Shiso) (1 kg) were washed with water and allowed 
to stand in water (20 l) at room temperatureor 3 days to effect 
extraction, followed by centrifugation (6000 r.p.m.) for 20 minutes to 
remove the residue which was washed with water (each 5 l). The washing 
liquid was combined with the supernatant, and the combined solutions were 
fractionated by ultrafiltration using an ultrafilter (Model UD-6, 
commercial product of Bio Engineering K.K., Tokyo) with a UK 200 membrane 
(commercial product of Toyo Roshi K.K., Tokyo) for retaining substances 
having a molecular weight of 200,000 at a pressure of 3 kg/cm.sup.2 to 
give a resiude which was then freeze-dried to obtain a brown powder (8.813 
g). This powder (1.5 g) was dissolved in water (5 ml) and transferred to a 
column (4.5.times.70 cm) packed with Sephadex G-200 (an agent for gel 
filtration, commercial product of Pharmacia Fine Chemicals AB., Sweden). 
The elution was effected with water (600 ml) and the effluent was divided 
into fractions (each 3 ml). Fraction Nos. 27-50 were collected and 
combined and the combined fractions were freeze-dried to obtain a whitish 
powder (117 mg). 
For further purification, this powder (100 mg) was dissolved in a 0.1 M 
tris-HCl buffer solution (5 ml; pH 7.0; I=0.01) and transferred to a 
column (2.5.times.70 cm) packed with DEAE-Sephadex A-50 (an iron 
exchanger, commercial product of Pharmacia Fine Chemicals AB., Sweden). 
The elution was effected with a 0.1 M tris-HCl buffer solution (300 ml; pH 
9.0; containing 0.5 M NaCl). The effluent was divided into fractions (each 
3 ml) and Fraction Nos. 15-25 were collected and combined, and the 
combined fractions were freeze-dried to obtain a whitish powder (58.9 mg) 
containing smaller amounts of impurities and having a sub-whitish powder. 
The physico-chemical characteristics of the final product are as 
hereinbefore described, and its high purity was confirmed by 
ultracentrifugation and electrophoresis. 
For comparison purpose, the IF inducting activities of the substances 
obtained by respective process steps in this example were determined by 
the method hereinafter described in Experiment 1 (in vitro) to give the 
following results. 
TABLE 11 
______________________________________ 
Concentration of sample (.mu.g/ml) 
Step 10 1.0 0.1 
______________________________________ 
(a) Extraction 90 &lt;10 &lt;10 
(b) Ultrafiltration 
&gt;100 85 &lt;10 
(c) Gel filtration 
&gt;100 &gt;100 94 
(d) Ion exchange treatment 
&gt;100 &gt;100 &gt;100 
(final product) 
______________________________________ 
EXAMPLE 2 
A similar treatemnt to that described in Example 1 was effected with the 
exception that Perilla frutescens Britton (known in Japan as Egoma) was 
extracted by allowing the leaves (1 kg) to stand in water at room 
temperature for 2 hours and adding to the water 1 N sodium hydroxide to 
adjust the pH to 8.5, followed by further extraction at 65.degree. C. for 
2 hours. The physico-chemical characteristics of the final product (54.5 
mg) were substantially the same as those of the final product of Example 
1. 
EXAMPLES 3-8 
The leaves, stems and seeds or dried Perilla frutescens Britton var. crsipa 
Dec. f. purpurea Makino (Shiso*); P. frutescens Britton (Egoma*); P. 
frutescens Brit. var. crispa Dec. f. viridis Makino (Aojiso*); P. 
frutescens Brit. var. crispa Dec. f. discolor Makino (Katamen-jiso*); P. 
frutescens Brit. var. crispa Dec. f. crispa Makino (Chirimen-Jiso*): P. 
frutescens Britton var. crispa Dec. f. viridis-crispa Makino 
(Chrimen-aojiso*); P. furtescens Brit. var. hirtella Makino et Nemoto 
(Toranoojiso*) and P. citriodora Nakai (Lemon-egoma*) [*Japanese 
nomenclature] were separately treated in a similar manner to that 
described in Example 1. The physico-chemical characteristics of their 
final products were substantially the same as those of the final product 
of Example 1. 
TABLE 12 
__________________________________________________________________________ 
No. -- Example Number; A -- Seed; B -- Stem; 
C -- Leaf 
Activity at 
(concentration of sample) 
No. 
Plant Tissue 
10 1.0 0.1 .mu.g/ml 
__________________________________________________________________________ 
1. Shiso* (Perilla frutescens 
A 70 
&lt;10 
&lt;10 
Britton var. crispa Dec. f. 
B &gt;100 
&gt;100 
90 
purpurea Makino) 
C &gt;100 
&gt;100 
92 
2. Egoma* (P. frutescens Brit.) 
A 40 
&lt;10 
&lt;10 
B &gt;100 
&gt;100 
90 
C &gt;100 
&gt;100 
85 
3. Aojiso* (P. frutescens Brit. 
A 60 
&lt;10 
&lt;10 
var. crispa Dec. f. viridis 
B &gt;100 
&gt;100 
95 
Makino) C &gt;100 
&gt;100 
90 
4. Katamen-jiso* (P. frutescens 
A 65 
&lt;10 
&lt;10 
Brit. var. crispa Dec. f. 
B &gt;100 
&gt;100 
90 
discolor Makino) 
C &gt;100 
&gt;100 
84 
5. Chirimen-Jiso* (P. frutescens 
A 50 
&lt;10 
&lt;10 
Brit. var. crispa Dec. f. 
B &gt;100 
&gt;100 
90 
crispa Makino) C &gt;100 
&gt;100 
80 
6. Chirimen-aojiso* (P. frutescens 
A 70 
&lt;10 
&lt;10 
Brit. var. crispa Dec. f. 
B &gt;100 
&gt;100 
85 
viridis-crispa Makino) 
C &gt;100 
&gt;100 
80 
7. Toranoo-jiso* (P. frutescens 
A 50 
&lt;10 
&lt;10 
Brit. var. hirtella Makino 
B &gt;100 
&gt;100 
70 
et Nemoto) C &gt;100 
&gt;100 
70 
8. Lemon-egoma* (P. citriodora 
A 50 
&lt;10 
&lt;10 
Nagai) B &gt;100 
&gt;100 
70 
C &gt;100 
&gt;100 
65 
__________________________________________________________________________ 
*By Japanese nomenclature 
EXPERIMENT 1 
Determination of IF induced by IF inducer and IF assay: [Reference: Y. 
Kojima's report in Kitasato Arch. Med., 43:35 (1970)] 
(a) IF Induction in vitro 
A rabbit (weight about 1 kg; New Zealand White; SPF) was sacrificed by 
cardic puncture and its spleen, bone marrow and lymph node cells were 
collected and combined together, from which a cell suspension containing 
the mixed cells (10.sup.7 cells/ml) was prepared using an Eagle MEM medium 
(commercial product of Nissui Seiyaku K.K., Tokyo) containing a 10% calf 
serum. This suspension was divided into fractions (each 1 ml), and 4 
fractions were respectively added with 10, 1.0, 0.1 and 0.01 .mu.g/ml of 
an active substance prepared by the method of Example 1, which was 
incubated at 25.degree. C. for 24 hours, followed by centrifugation to 
obtain each supernatant which was then used to determine the activity of 
the IF induced. 
(b) IF Induction in vivo 
An aqueous solution (2 ml) of the active substance prepared by the method 
of Example 1 (500 .mu.g/ml) was injected into the auticular vein of a 
rabbit (weight about 1 kg; New Zealand White; SPF). 1, 2 4 and 6 hours 
after this, a 2 ml sample of blood was removed from the rabbit on each 
occasion and used to prepare the serum used to determine the IF activity. 
(c) Determination of IF Activity 
In both (a) and (b), the activity of the IF induced was determined in 
reliance with the reduction ratio of plaques in the following manner. 
A monolayer culture of the lined cells RK-13 of rabbit was put in a dish 
and added with a predetermined amount of the solution obtained by the 
method (a) or (b) (suitably diluted). The culture was incubated at 
37.degree. C. overnight of Vesicular stomatitis virus was then added and 
used as the challenge virus. The IF activity is indicated by the reduction 
ratio of plaques and the unit of the IF activity is expressed by the 
reciprocal number of the highest dilution of the sampel required for 
reducing the numbers of plaques to 50%. 
EXPERIMENT 2 
Definition of IF Inducer 
The active substance of this invention represents an IF inducer because the 
samples prepared by the methods (a) and (b) are capable of inhibiting the 
growth of Vesicular stomatitis virus and Vaccinia virus in the lined RK-13 
cells of rabbits of the same animal species, but do not inhibit the growth 
of Vesicular stomatitis virus in L cells of mice i.e. of a different 
animal species, and moreover, their IF activities are inactivated by 
treating with 0.08% trypsin at 37.degree. C. for 2 hours. It has also been 
found that the IF induced by the active substance of this invention is 
stable when dialized against a pH 2 buffer solution at 5.degree. F. for 2 
days, unstable on heating at 60.degree. C. for 2 hours, gives no 
precipitate by centrifugation at 100,000 xg for 2 hours and is non-toxic 
against the cells at the minimum virus-inhibitory level. The IF induced by 
the active substance of this invention may be classified into Type-I IF 
and consists of a complex of .alpha.- and .beta.-types. Also it has been 
observed that IF is induced when the active substance of this invention is 
used, for example, for treating the cells of bone marrow, lymph node, 
spleen and the like in vitro or injected into the body of animals, but no 
IF is induced when applied to treat the primary or continuous cell 
cultures which are known to induce IF by viral infection or treating with 
poly I:poly C. 
EXPERIMENT 3 
In Example 1, the elctrophoresis was effected at 4.degree. C. in 
conventional manner using a commercially available device (Model AE-2, 
product of Toyo Kaguku Sangyo K.K., Tokyo), a polyacrylamide gel plate 
(thickness 3 mm) and a 0.3 M boric acid buffer (pH 8.4). The resultant 
single band indicated that the rest sample had a high putity. 
EXPERIMENT 4 
Determination of Specific IF Inducing Activity 
(A) In a similar manner to that described in Example 1, dried leaves of 
Perilla frutescent Britton var. crispa Dec. f. purpurea Makino (1 kg) were 
extracted with water (20 l) to give an extracted solution (dry weight: 
92.892 g) which was subjected to ultrafiltration using a membrane (UK 200) 
for fractionating substances having a molecular weight of more than 
200,000 to give a residue and filtrate. The residue was freeze-dried to 
give a crude powder (dry weight: 8,813 g) referred to as Fraction No. 1. 
Similarly, the filtrate was treated by using a membrane (UK 10) for 
fractionating substances having a molecular weight of more than 10,000 to 
give a residue and filtrate, both of which were freeze-dried respectively 
to give solid substances referred to as Fraction Nos. II (dry weight: 
10.751 g) and III (dry weight: 71.328 g). 
The IF activities induced by Fraction Nos. I, II and III were individually 
determined by the methods of Experiment 1 to find out that Fraction No. I 
was active and no or little activity was present in II and III. 
Subsequently, Fraction No. 1 (1.5 g) was dissolved in water (5 ml) and 
applied to a column (4.5.times.70 cm) packed with Sephadex G-200 
(commercial product of Pharmacia Fine Chemicals AB., Sweden). Gel 
filtration was effected with water (600 ml) and the effluent was divided 
into fractions (each 3 ml). 0.5 ml of each fraction was used to develop 
the color by the phenol/sulfuric acid method and the absorption at 485 
m.mu. was determined by using a Hitachi Spectrophotometer (Model 40-100, 
commercial product of Hitachi Limited., Tokyo). In reliance upon the peaks 
observed, these fractions were classified and combined to give 7 groups. 
Each group was freeze-dried and used for determining the IF activity 
induced in vitro by the method of Experiment 1. The results are shown in 
the following table, in which Fraction No. I-1 corresponds to Fraction 
Nos. 27-50 described in Example 1. 
TABLE 13 
______________________________________ 
A: Fraction No. (molecular weight) 
B: Yield (%) [100% = dry weight of the starting 
material] 
C: Recovery ratio (%) 
D: IF activity in vitro at a concentration 
of .mu.g/ml 
D(.mu.g/ml) 
A B (%) C (%) 10 1.0 0.1 
______________________________________ 
After ultrafiltration 
All extracts 
9.29 90 &lt;10 &lt;10 
I (&gt;200,000) 
0.88 9.68 &gt;100 85 &lt;10 
II (200,000- 
1.08 11.88 30 &lt;10 &lt;10 
10,000) 
III (&lt;10,000) 
7.13 78.44 &lt;10 &lt;10 &lt;10 
After gel filtration 
I-1 0.085 9.66 &gt;100 &gt;100 94 
I-2 0.055 6.25 60 &lt;10 &lt;10 
I-3 0.017 1.93 &lt;10 &lt;10 &lt;10 
I-4 0.022 2.50 &lt;10 &lt;10 &lt;10 
I-5 0.595 67.61 &lt;10 &lt;10 &lt;10 
I-6 0.020 2.27 &lt;10 &lt;10 &lt;10 
I-7 0.086 9.77 &lt;10 &lt;10 &lt;10 
______________________________________ 
(B) In a similar manner to that described in Example 1, dried leaves of 
Perilla frutescens Britton var. crispa Dec. f. purpurea Makino (50 g) were 
extracted with water (1000 ml) at room temperature for two hours, heated 
at 65.degree. C. for one hour, adjusted to pH 8.5 with addition of 1 N 
NaOH solution (3 ml), heated at 65.degree. C. for two hours, cooled to 
room temperature and centrifuged for 30 minutes (7000 r.p.m.) to remove 
the residue from the supernatant. The residue was washed twice with water 
(each 200 ml) and the washing liquid was combined with the supernatant. 
The combined solutions (dry weight 7.49 g) were subjected to 
ultrafiltration using a membrane (UK-200) capable of fractionating 
substances having a molecular weight of more than 200,000 to give a 
filtrate and residue. The residue was freeze-dried to obtain a crude 
powder (dry weight 0.506 g) which was then freeze-dried to obtain a 
sub-0.506 g) referred to as Fraction I which was then subjected to gel 
filtration and used to determine the IF activity by the method of 
Experiment 1. Separately the filtrate was treated similarly by using a 
membrane (UK-10, commercial product of Toyo Roshi K.K., Tokyo) for 
fractinating substances having a molecular weight of more than 10,000 to 
give a residue and filtrate, both of which were freeze-dried to give 
substances respectively referred to as Fractions II (dry weight 0.042 g) 
and III (dry weight 6.843 g). The IF activities induced by these fractions 
were also determined by the method of Experiment 1. The results are shown 
in Table 14. 
TABLE 14 
______________________________________ 
Fraction IF activity in vitro 
(molecular 
Yield Recovery at a concentration of 
weight) (%)* ratio (%) 10 1.0 0.1 .mu.g/ml 
______________________________________ 
All extracts 
14.98 80 &lt;10 &lt;10 
I (&gt;200,000) 
1.01 6.83 &gt;100 70 &lt;10 
II (200,000- 
0.08 0.54 30 &lt;10 &lt;10 
10,000) 
III (&lt;10,000) 
13.69 92.63 &lt;10 &lt;10 &lt;10 
______________________________________ 
*100% = dry weight of starting material 
(C) From (A) and (B), it is apparent that the IF-inducing activity is most 
prevalent in the fraction having a molecular weight of more than 200,000 
and that impurities contained in the extracted solution were almost 
completely removed by ultrafiltration and gel filtration (more than 98%). 
The amounts of impurities were further reduced by the following ion 
exchange treatment as described in Example 1 and an increase of about 10% 
of the specific activity was observed.