JPS6152806B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to interferon (hereinafter referred to as IF)
Regarding inducing agents. As is well known, if
is a substance produced in cells or bodies of humans or animals, and has the effect of suppressing the proliferation of viruses specifically for animal species and non-specifically for virus species. An IF inducer is a substance that has the effect of inducing IF when administered into the body or cells of a human or animal. Therefore, IF inducers, like IF, are expected to be useful in the prevention and treatment of viral infections in humans and animals. However, since conventionally known IF inducers generally have drawbacks such as high toxicity, they have never been put to practical use for this type of purpose. With the aim of providing an IF inducer that has excellent IF-inducing activity and low toxicity and can be easily produced, the present inventor has developed a plant or a variant thereof that belongs to the family Asteraceae and contains an IF-inducing active substance. invented an IF-inducing active substance and its manufacturing method, which consists of a process of extracting the above-mentioned active substance from the tissue of the patient and recovering it from the extract (Patent Application No. 1540 of 1971;
(See No. 160089). Examples of plants used in this method are: Artemisia princeps Pamp., A. capillaris Thun., A. capillaris Thun.
absinthium L.), A.maritima L.
), wormwood (A.kurramensis Qaz.), sagebrush (A.montana Pamp.), wormwood (A.
feddei Le′v.et Van.), wormwood (A.
japonica Thun.), Japanese mugwort (A.keiskeana)
Miq.), Japanese mugwort (A. stolonifera)
Komar.), A. monophylla
Kitam.), white mugwort (A. stelleriana Bess.),
A. vulgaris L., A. abrotanum L., A. campestris
L., A.vallesiaca All., A.molinieri Que′z, A.
dracunculus L., A.ludoviciana Nutt., A.
arbuscula Nutt., A. tridentata Nutt., A.
filifolia Torr., A. caudata Michx., A.
lindleyana Bess., A.frigida Willd., A.biennis
Willd. The plants illustrated here have low toxicity. The leaves and seeds of certain Artemisia plants have been used for many years, for example, as food, medicinal herbs, etc. Although all plant tissues can be used as raw materials for producing the active substance, it is practically preferable to use leaves and stems, and from the viewpoint of preservation and extraction efficiency, it is preferable to use dry raw materials. Extraction of the raw material is generally carried out with water at any temperature (for example from room temperature to the boiling of the extraction mixture) and preferably under alkaline conditions (for example PH 7-10) for any time. For example, it is usually 1 to 5 days at room temperature, and 30 minutes to 6 hours at 45 to 80°C with stirring. Extraction may be continuous or batchwise, and the ratio of extraction water to raw material may be arbitrary. or a suitable concentration of a hydrophilic organic solvent such as methanol, ethanol, propanol, butanol, dimethyl sulfoxide, acetone (e.g. 20
-80%) at any temperature (eg, 20 to 80°C) for an appropriate time (eg, 4 hours to 2 days). After removing plant residues from the extract by conventional methods such as filtration, squeezing or centrifugation, the active ingredients are recovered, for example, by the following method. (A) Treat the supernatant by ultrafiltration using an appropriate membrane that can fractionate substances with a molecular weight cutoff of 100,000 or more.
The ultraviolet pressure is, for example, 0.1 to 5 kg/cm ² . The resulting active portion is collected and lyophilized to yield a brown powder. (B) The extract is optionally concentrated under reduced pressure and treated with a hydrophilic organic solvent (e.g. methanol, ethanol,
propanol, butanol, acetone, etc.) to the extract or its concentrate at an appropriate concentration (e.g.
40-70w/v%), a precipitate containing the active ingredient is formed, and when this is dried under reduced pressure, a brown powder is obtained. (C) Instead of the above organic solvents, use ammonium salts such as ammonium chloride, ammonium sulfate, and cetyltrimethylammonium bromide, or inorganic metal salts such as zinc chloride and copper chloride at appropriate concentrations (e.g., 20-50 w/v%). When added in such an amount, a precipitate containing the active ingredient is formed, and when the precipitate is desalted and dried, a brown powder is obtained. By processing the extract as described above, most of the active ingredients in the raw materials (90% in some cases) can be removed.
above) can be recovered. However, the content of inert ingredients in the obtained dry coarse powder is the lowest in method (A). In addition, method (A) is easy to operate, inexpensive, and can be carried out in a short time. Moreover, it was found that no significant side effects were observed even when a large amount of the coarse powder obtained by method (A) was orally administered to animals. This crude powder is then purified by conventional methods such as column chromatography using gelatinizers or ion exchange agents. When a gelatinizer is used, elution may be performed with an appropriate buffer, but usually water may be used for elution. If an ion exchange agent is used, elute with an appropriate buffer. Since the active ingredient is a water-soluble acidic substance, various gelling agents, which are commonly used for the purification of this kind of substance,
Purification can be achieved using ion exchange agents or anion or cation exchange cellulose. Although the product thus obtained contains some impurities, it can be used practically as an IF inducer. If desired, impurities can be further removed by combining the above purification steps. Physical and chemical properties The final product obtained by this method is stable in the form of an amorphous white powder and has the following physical and chemical properties. (1) Elemental analysis H: 7.4±0.4%, C: 45.6±0.4%, N: 13.5±0.4%, P: 2.8±0.3% (2) Molecular weight About 100,000 to about 3 million (mainly about 500,000 to 100 [Ultracentrifugation method using Spinco Model E analytical ultracentrifuge (manufactured by Beckman, USA), Amicon ultracentrifuge and XM50, XM100A and XM300
Ultrafiltration method using membranes (manufactured by Amicon, USA) UK10, UK50, and UK200 membranes (manufactured by Toyo Paper), and Cefdex G-200 (manufactured by Pharmacia Huain Chemical, Sweden)
(3) Melting point or decomposition point Melting point is unclear. Carbonizes at approximately 220℃. (4) Ultraviolet absorption spectrum As shown in Figure 1 (measured in 0.1N NaOH, but did not change in water or 1N NaOH) (5) Infrared absorption spectrum as shown in Figure 2 (KBr method) (6) Various types Solubility in solvents: Soluble in water, particularly well in alkaline aqueous solutions such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide. methanol, ethanol, propanol, butanol, acetone,
Slightly soluble in chloroform and ether. (7) Color reaction Positive for ninhydrin and deutstomer reactions. Negative for furin reagent and Elson-Morgan reaction. (8) Properties Acidic (9) Main chemical composition (a) Amino acids (±0.6%) Aspartic acid (9.5%) Threonine (4.9%) Serine (4.7%) Glutamic acid (8.4%) Proline (3.1%) Glycine (10.2 %) Alanine (11.2%) Valine (6.9%) Isoleucine (4.6%) Leucine (7.8%) Tyrosine (trace amount) Phenylalanine (2.9%) Lysine (6.2%) Histidine (1.8%) Arginine (5.3%) Ammonia ( 12.1%) (After hydrolysis with 6N hydrochloric acid at 110℃ for 48 hours under reduced pressure, it was analyzed using the Technicon Amino Acid Auto Analyzer Model NC-1, manufactured by Technicon, USA) (b) Sugar Not detected (at 80℃ with 0.1N sulfuric acid) for 20 minutes and with 1N sulfuric acid
After hydrolysis at 100℃ for 2 hours, it was analyzed using Technicon Sugar Auto Analyzer Model N-1 (manufactured by Technicon, USA) (10) Specific rotation [α] ²⁶ _D = +73° to +79° Average +76° (Concentration 0.47 % in 0.1N NaOH) From the above characteristics, the substance according to the present invention has a molecular weight of about 100,000 to about 300, mainly composed of amino acids and phosphoric acid.
It is thought to be a type of protein that contains phosphoric acid and has a polymer of about 1,000,000 (mainly about 500,000 to about 1,000,000). In addition, IF induced in animals or in vitro by this substance was induced by trypsin (0.08%,
This substance is not only inactivated at 37°C for 2 hours, but also has animal species specificity and virus species non-specificity, making it a new substance that falls under the generally accepted definition of an IF inducer. It turned out to be. Furthermore, in order to provide a practical method for using this active substance, we conducted various tests as described below and investigated its biological properties, and as a result, we invented a method for using this active substance. IF can be induced by administering an effective amount of the active substance into the body or cells of a human or an animal capable of inducing IF. With known IF inducers such as poly I:C and endoxin, toxicity is a problem, but with this active substance, there is no such concern, and moreover, the inducing activity in humans is higher than that of known IF inducers. is also excellent. It was found that this active substance not only provides high antiviral activity but also improves antitumor and physiological effects. Therefore, this active substance can be used in humans, mammals (cows, horses,
It is used not only for the prevention and treatment of viral infections in various vertebrates such as pigs (pigs, etc.), birds (chickens, ducks, etc.), fish (rainbow trout, eel, yellowtail, etc.), but also as an anti-tumor agent, physiological action improvement, etc. It can also be used as a health promoter. The active substance can be administered by intravenous injection, intraperitoneal injection, intraintestinal, oral, transdermal, spray, and other methods. Appropriate dosage (final purified product, daily dose)
is 0.001-100mg/Kg for intravenous administration,
Conditions such as host type, age, weight, etc., especially IF
It is appropriately selected depending on the response to induction, the purpose of administration, etc. Practically speaking, in animals intravenous administration 0.01
~10 mg/Kg, intraperitoneally 0.1 to 10 mg/Kg, intravenous 0.01 to 1.0 mg/Kg for humans, and about 10 times or more for oral administration. Larger amounts can be used for short-term or topical administration. If the dose is too low, IF will not be induced, but if the dose is too high, there will be no particular problem as the toxicity is low. Appropriate amount as desired
According to the method of administering the active substance after administering IF (priming), the biological activity value of the active substance can be increased approximately 3 to 10 times compared to when administered alone, and at the same time, the biological activity of the active substance can be increased by 3 to 10 times. It can improve responsiveness and extend the effective time of IF. In the following explanation, as samples, the dried crude powder of the ultrafiltration fraction of Production Example 1 and its final purified product, the final purified product of Same Example 2, and foreign-grown Artemisia vulgaris were treated by the method of Production Example 1. The final purified products (hereinafter referred to as samples A, B, C, and D) were used as test animals, in addition to rabbits of the same species used in Test Example 1, mice (body weight 25
g, ddy strain, 6 weeks old), chicken (female, weight approx.
230g, white leghorn, 30 days old) was used. (A) Measurement of IF induction and IF activity () In vitro method Eagle containing 10% calf serum (fetal bovine serum for humans) according to the method of Test Example 1
Using MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), a suspension of spleen cells (10 ⁷ cells/ml) from the test animal was prepared, and a sample of the active substance was added to each section (1 ml) and cultured at a predetermined concentration and temperature. The supernatant obtained by centrifuging the culture solution was used for IF activity measurement. However, in the case of humans, in addition to using the spleens of two male adults who died from trauma, the blood of five healthy male adults was taken from the superior gingival vein, and the white blood cells separated from this were used to separate the blood. I made a floating liquid. The IF activities listed in Table 2 were measured according to Test Example 1 using the cells for measurement shown in Table 1.

【table】

【table】

[Table] () In vivo method (1) Rabbit The rabbit IF activity obtained in Test Example 1 is shown in Table 3.

[Table] Change the dosage of sample B from 4mg/Kg to 0.004mg/
When the test was carried out in the same manner by changing the Kg range, the maximum value of IF activity in serum was observed 2 hours after administration. In addition, when administered orally, the maximum value appeared approximately 10 to 13 hours later. (2) Mice Using 10 mice per group, (a) Add sample B in an amount to give the concentration shown in Table 4 to physiological saline (0.1 ml each), inject into the tail vein of each mouse, , 2, 3, 5 hours later, or (b) similar amounts of sample B were added to physiological saline (each
(0.2 ml) and intraperitoneally administered to each mouse, and 2, 3, 4, 6 hours later, or (c) Add sample B to water (0.2 ml each) in an amount to achieve the concentration listed in Table 4. was orally administered to each mouse, and 2.5, 5, 7.5, 10, and 13 hours later, whole blood was collected from the heart of each mouse to prepare serum, and IF activity was measured according to the method described above. In the case of (a), (b), and (c), the maximum value of IF activity is about 2, about 3-4, and about 7.5, respectively.
Appeared after ~10 hours. Table 4 shows the maximum IF activity values (average values). The same tendency was observed when sample A was used.

[Table] (3) Human: 200 mg each of sample B to 5 healthy male adults.
was orally administered, blood was collected 13 hours later, and the IF activity was examined according to the method described above, and an average of 14 units of activity was observed in the serum. (4) Chickens Sample B was applied to the underwing veins of 10 chickens per group.
Physiological saline (0.2 ml) containing 4 mg/Kg of each was injected, whole blood was collected from the heart 2 hours later, and the IF activity in the serum was examined according to the above method, and it was found to be about 26 units. (B) Virus infection prevention test (1) Mice (vaccinia virus) Mice (female, 20 mice per group) were injected with physiological saline containing the active substance in the amount shown in Table 5 (each
0.2 ml) was administered intravenously, intraperitoneally, or orally, and 24 hours later, a physiological saline solution containing vaccinia virus at 30 PFD ₅₀ (30 times the amount of virus that causes 50% pox on the tail of a mouse) was administered. The prevention rate of pox was calculated by injecting 0.1 ml into the tail vein and comparing the number of pox that appeared in the tail over the next 9 days with that of the untreated group (No. 5
table). It was judged to be effective if the inhibition rate was 50% or more.

[Table] (2) Mouse (herpes simplex) Herpes simplex virus was used instead of vaccinia virus. After administering the sample intravenously orally or intraperitoneally in the same manner as in Test (B) (1) above, the virus was inoculated intraperitoneally for testing. After observing the mice for 30 days after infection, we determined the average survival time of the mice and compared them with the untreated group.
It was found to have a life-prolonging effect. (3) Rabbits (vaccinia virus) (a) Physiological saline (0.1 ml each) containing sample A was injected intradermally on the backs of rabbits (5 rabbits per group), and 24 hours later, vaccinia virus 10 ID was injected into the same area. ₅₀ (the amount of virus inoculum that causes 50% of smallpox sizes of 6 x 6 mm or larger to appear within the skin of rabbits is called 1 ID ₅₀ ), and 7 days later, local injections are administered. investigated for smallpox. Samples were serially diluted 10 times.
A range of 0.02 to 200 μg was used. When the dose was 2 μg or more, the pox inhibition rate was 100%. (b) Using rabbits (5 rabbits per group), each rabbit was given a sample on the 1st, 3rd, 4th, 6th, and 8th day (5 times in total).
A dose of 100 mg/day was administered orally, and 10 ID ₅₀ and 100 ID ₅₀ of vaccinia virus were inoculated intradermally on the back on the 5th day. By day 12, no pox was observed in the rabbits inoculated with 10ID ₅₀ , and slight pox was observed in the rabbits inoculated with 100ID ₅₀ , which completely disappeared by approximately 2 weeks post-infection. (C) Antitumor effect (mouse) (1) Ehrlitsu ascites cancer 15 mice per group, 2.5x cancer cells
Sterile water ( ^0.2 ml) containing Sample B was intraperitoneally implanted into each mouse, and 24 hours later, sterile water (0.2 ml) containing Sample B was administered to each mouse continuously for 14 days (once a day). . The dose is i.p.
Oral doses were 0.2, 1.0 or 5 mg/Kg/day and 40, 200 or 1000 mg/Kg/day per animal. When administered intraperitoneally at 1.0 or 5 mg/Kg/day, the median survival time of mice was 33 days.
One-third of the patients survived 50 days after transplantation and were completely cured, but all patients in the untreated group died by 29 days after transplantation. A similar effect was also obtained when 1.0 mg/Kg was administered intraperitoneally 5 times every 3 days. The results of oral administration (1000 mg/Kg/day) were similar to those of intraperitoneal administration. (2) S-180 solid tumor Sterile water containing 1 x 10 ⁵ tumor cells (0.2 ml)
was subcutaneously transplanted into the axilla of each mouse (15 mice per group), and Sample B was administered in the same manner as above. With intraperitoneal administration (5 mg/Kg/day), the median survival time was 40 days, and in 5 animals, the tumors had shrunk to about 1/2 by 40 days after transplantation. Oral administration (400 or 1000
mg/Kg/day) was inferior to intraperitoneal administration. All animals in the control group died by day 35. (D) Combination use of this active substance and IF (priming) (1) Prepare a cell suspension containing rabbit lymphoid cells (10 ⁷ cells/ml) according to the method of Test Example 1, and add 30 units/ml to this. ml of rabbit IF was added and treated at 37°C for 6 hours. centrifuge the liquid
Remove IF and add Eagle's MEM medium (1
ml) and Sample B were added to induce IF according to the method of Test Example 1, and the activity was measured. Amounts of sample B were used ranging from 10 ^-3 to 10 ^-7 mg/ml. The induced IF activity value was approximately 3 to 10 times higher, and the responsiveness of the cells was also improved. (2) A human spleen cell suspension was prepared according to the method described in (D) () above, and treated with 100 units of human IF. The induced IF activity value increased approximately 3 to 6 times, and the responsiveness of the cells also improved. (3) One million rabbits (5 rabbits per group)
Six hours after the intravenous injection of IF, Sample B was administered according to the method in (A) () (1). IF in serum
It was found that the activity value increased approximately 3 to 10 times and that the IF induction time was extended. (4) Next, the rabbits (5 rabbits per group) were given 1 million units.
IF was injected intravenously, and the infection prevention test described in (B) (3) (b) above was conducted the next day. Smallpox was completely inhibited in areas inoculated with 10 ID ₅₀ viruses. but
A small amount of smallpox was observed in 1/3 of the inoculated areas where ₅₀ 100ID were inoculated, but all of it disappeared by two weeks after infection. (E) Toxicity (1) Acute toxicity Physiological saline containing sample B was administered to mice (male and female, 20 animals per group) and rats (male and female, weight approximately 95 g, SPF-SD strain, 6 weeks old, 1
The ID ₅₀ measured by administration to each group of 20 animals is shown in the table below, and there was no significant difference between males and females.

[Table] (2) Subacute toxicity (a) Rat (body weight approx. 95 g, SPF-SD strain, 6
(20 weeks old, 20 animals per group) were used. Sample A
0.35, 0.7, 1.4, and 2.8 g/Kg (rats) were added to 0.25 to 0.5 ml of sterile water, and a fixed amount was forcibly administered to each group every day for 3 months using a sonde. Compared to the group, their general health was better and their weight gain was higher. Three months later, the animal was dissected and pathologically examined, and no abnormalities were found. Therefore, it was not possible to confirm accurate subacute toxicity values. (b) Sample A was given to five healthy adults (male) every day.
When 200 mg was administered orally for 10 consecutive days, no significant side effects occurred, general health status improved, and the patient felt refreshed. On the other hand, plants of the genus Artemisia, which are the active substances of the present invention, generally have low toxicity, and certain types have been cultivated for food in Japan, China, etc. since ancient times, and are also widely used as medicinal herbs and raw materials for the production of fragrances. On the other hand,
The above test confirmed that the toxicity of this active substance is extremely low. Therefore, when administering the active substance orally to humans and animals, it is practical to
It is recognized that there are no significant side effects due to toxicity even if the crude product after ultraviolet lapse is continuously administered in large quantities. In order to administer the active substance to humans or animals, a pharmaceutical composition containing the active substance as an active ingredient and a pharmaceutically acceptable carrier or excipient is provided. The composition may be in a form suitable for oral, rectal, parenteral, transdermal, or intramucosal administration. For example, for oral administration they may be solid or liquid and may be in the form of powders, capsules, granules, tablets, coated tablets, syrups, emulsions, suspensions, or drops. Compositions of this type include carriers or excipients commonly used in pharmaceuticals. Examples of suitable tablet excipients are lactose, potato starch or soluble starch, and magnesium stearate; examples of carriers for parenteral administration are sterile water, saline, or almond oil; It may be placed in an ampoule or the active substance may be added immediately before use. These compositions may optionally contain binders, stabilizers, emulsifiers, suspending agents, dispersants, lubricants, preservatives, fillers, etc. commonly used in pharmaceuticals. Other examples of practical dosage forms are for mucous membranes, such as vacuoles, troches, eye drops, suppositories, etc., for injections, such as aqueous solutions, oil solutions, suspensions, etc., and for inhalation, inhalants. For external use, there are ointments, plasters, liniments, bath preparations, sprays, etc. Advantageously, these compositions are prepared as dosage units shaped to supply a fixed amount of active substance. Suitable examples thereof are tablets, coated tablets, ampoules, capsules, suppositories. The practical amount of active substance contained in a dosage unit is, for example, about 4 to 10 times that of an intravenous solution, and about 2 to 3 times that of a subcutaneous injection.
For intramuscular injections, it should be about 1.5 to 3 times, for troches and troches, it should be about 2 to 4 times, and for suppositories, it should be about 5 to 10 times. Formulation Example (1) Injection Physiological saline 1.0 ml Sample B 0.01 g Aseptically seal in a 2 ml ampoule. (2) Lozenge 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 Single syrup 20 g Cyclamine Acid Na 0.1 g Ethylparaben 0.04 g Sample B 0.1 g (5) Ointment Purified Lanolin 5 g Beeswax 5 g White vaseline 87 g Sample B 3 g (6) Paint Potassium hydroxide 0.3 g Glycerin 20 ml Ethanol 25 ml Sample B 2.5 g Water An example of a manufacturing method to add 100 ml is as follows. Production example 1 After washing dried mugwort leaves (1 kg) with water, extract by leaving them in water (20) at room temperature for 3 days, centrifuging (6000 rpm, 20 minutes), and extracting the extract. The residue was washed twice with water (5 portions each), and the washings were combined with the extract. The extract thus obtained was subjected to ultrafiltration (pressure 3Kg/ ^cm2 ). The residue was collected and lyophilized to give a brown powder (19.676g). This powder (1.5 g) was dissolved in water (5 ml), and the aqueous solution was dissolved in a column (4.5 x 70 cm) packed with Cephadex G-200 (Pharmacia Fine Chemicals AB, Sweden). It was added to a column (4.5 x 70 cm) packed with G-200 (Farmacia Huain Chemical AB, Sweden), eluted with water (600 ml), and the eluate was divided into sections of 3 ml each. Freeze-drying was performed according to the classification above to obtain a white powder (220 mg). For further purification, this powder (100 mg) was added to 0.01 M Tris-HCl buffer (PH
7.0, = 0.01) (5 ml) and packed with DEAE Sephadex A-50 (Pharmacia Huain Chemical AB, Sweden).
x 70cm) and elute with 0.1M Tris-HCl buffer (PH9.0, containing 0.5M NaCl, 300ml). Divide the eluate into 3ml sections each. Combined. This solution was desalted and freeze-dried to obtain an amorphous white powder (62.7 mg). The physicochemical and biological properties of this product are as described above. Comparing this with the first white powder,
The IF-induced activity was approximately the same, but the amount of impurity was reduced. High purity was confirmed by ultracentrifugation and electrophoresis. For comparison, the IF-inducing activity of the substances obtained in each step was measured by the in vitro method described in Test Example 1 below, and the results are shown in the table below.

[Table] Production example 2 After washing dried Kawara mugwort leaves (1 kg) with water, add 20% of water to it and leave it at room temperature for 2 hours.
The pH was adjusted to 8.5 by adding 1N sodium hydroxide. Next, this was heated and extracted at 65°C for 2 hours. Thereafter, it was purified according to the method of Production Example 1. The physicochemical and biological properties of this purified product (60.2 mg) were not significantly different from those obtained in Example 1. Production Example 3-26 The dried leaves, stems and seeds of the plants shown in Table 8 were treated separately according to the method described in Production Example 1,
The IF-inducing activity of the products obtained in the second step of all production examples was measured by the method (in vitro method) described in Test Example 1 below. The results are shown in Table 8.

【table】

【table】

[Table] Test example 1 IF induction method and IF activity measurement method (Reference: Y.
Kojima, Kitasato Arch., Exp., Med., 43 :
35, 1970) (a) In vitro method for inducing IF Rabbits (approximately 1 kg in weight, New Zealand White breed, SPF) were killed by blood sampling, spleen, bone marrow and lymph node cells were collected, and calf serum was collected. A cell suspension containing 10 ⁷ /ml of mixed cells was prepared using Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10%, and each cell suspension (1 ml) was filled with cells obtained by the method described in Production Example 1 of the present invention. IF inducer 10, 1, 0.1,
After adding 0.01 μg/ml to each and culturing at 25° C. for 24 hours, each culture solution was centrifuged and the supernatant was collected and used for IF activity measurement. (b) Method for inducing IF by in vivo method 2 ml of the aqueous solution (500 μg/ml) of the IF inducer obtained by the method described in Production Example 1 was added to a rabbit (approximately 1 ml body weight).
Kg, New Zealand White breed, SPF) was injected into the ear vein, blood was collected (2 ml) 1, 2, 4, and 6 hours later, and the serum was used for IF activity measurement. (c) Measurement of IF activity In both methods (a) and (b) above, the produced IF activity was measured using rabbit kidney established cell line (RK-13).
It is performed using the 50% plaque method. First, on a monolayer culture of the above cells prepared in advance in a shear dish, the appropriately diluted IF obtained by the above methods (a) and (b) was added.
After adding the sample solution and incubating at 37° C. overnight, IF activity was measured using Vesicular stomatitis virus as a challenge virus and the plaque reduction rate as an index. The unit of IF activity is the number of plaques in IF-untreated cells.
It is expressed as the reciprocal of the dilution indicating 50%. Test Example 2 Method for proving that it is an IF inducer The IF sample produced by the methods (a) and (b) above inhibits the growth of vesicular stomatitis virus on rabbit RK-13 cells of the same animal species. In addition, it also suppresses the proliferation of Vaccinia virus, but it does not suppress the proliferation of vesicular stomatitis virus in L cells of mice of different animal species. Also 0.08% trypsin
The IF activity is inactivated when allowed to act at 37°C for 2 hours.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the active substance of the present invention, and FIG. 2 shows the infrared absorption spectrum.

Claims (1)

[Claims] 1. The above-mentioned active substance recovered from the tissue extract of a plant belonging to the Asteraceae family, Artemisia genus, and containing an interferon-inducing active substance or its variants, as an active ingredient, and a pharmaceutically acceptable carrier or excipient. A composition for inducing interferon, which is made to coexist with.