JPS6011892B2 - Method for producing interferon inducer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides interferon (
The present invention relates to a method for producing an promoting agent (hereinafter referred to as IF).

The present invention is based on the knowledge that a substance having excellent IF-inducing activity is contained in the tissues of various plants belonging to the genus Safflower of the Asteraceae family, or its variants, and that this active substance can be simply isolated at a low cost. It is based. Therefore, an object of the present invention is to provide a method for producing an IF inducer that has excellent IF inducing activity and low toxicity and can be produced simply and at low cost.

The present invention provides a substance that is stable in the form of an amorphous white powder, has IF-inducing activity, and has the following physicochemical properties.

Physical and chemical properties [1] Elemental analysis H: 6.54 ± 0.3%, C: 41.9 ± 0.3%, N
:2.39±0.3%, P:0.28±0.03% [2
'Molecular weight: approximately 100,000, approximately 3,000,000 (mainly approximately 50,000)
10,000 to 1,000,000) [Ultracentrifugation method using Spinco Model E analytical ultracentrifuge (manufactured by Beckman, USA), Amicon ultrafurnace filtration machine and XM50, XMIOOA and XM30
Ultrafurnace filtration method using G door filter membrane (manufactured by Amicon, USA) UKI0, UK50 and UK20 door filter membrane (manufactured by Toyo Paper),
and measured by gel filtration method using Sephadex G-200 (manufactured by Pharmacia Fine Chemical AB, Sweden)] {3} Melting point or decomposition point Melting point unclear.

Carbonizes at about 220 qo. ■ Ultraviolet absorption spectrum as shown in Figure 1 (measured in 0.1N NaOH, but did not change in water or INNaOH) showing shoulders at about 250 and 280 degrees.

(5- Infrared absorption spectrum as shown in Figure 2 (KBr method) ■ Solubility in various solvents Dissolves in water, particularly well in alkaline aqueous solutions such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide.

Methanol, ethanol, fu. Low Solubility: Slightly soluble in butanol, acetone, chloroform, and ether.Positive to ninhydrin reaction, phenol/sulfuric acid reaction, phorin reagent, and ditzmer reaction.

Negative for the Gelson-Morgan test.

[8} Acidic nature■ Main chemical composition...amino acids oxyproline (15.61±0.4%) aspartic acid (5.51±0.4%) threonine (10.90±0.4%) serine
(9.64 soil 0.4%) glutamic acid
(3.96 soil 0.4%) Proline
(4.27±0.4%) Glycine (9
．． 09±0.4%) Alanine (8.2
4 soil 0.4%) Valine (4.04±0
．． 4%) Isoleucine (5.59 soil 0.4
%) Leucine (2.56±0.4%) Phenylalanine (1.32±0.4%) Lysine
(2.18 soil 0.4%) arginine
(1.24±0.4%) Tyrosine
(1.94±0.4%) Histidine
(0.93±0.4%) Ammonia (12.
98 Soil 0.4%) (Hydrolyzed with isohydrochloric acid at 110 qC for 4 hours under reduced pressure and analyzed with Technicon Amino Acid Auto Analyzer Model NC-1 manufactured by Technicon, USA) 'o} Sugar arabinose (9 .38±0.6%) Galactose (20.98±0.6%) Glucose
(64.98±0.6%) Mannose
(3.26±0.6%) xylose
(1.40±0.6%) (After hydrolysis with 0.1N sulfuric acid at 80°C for 20 minutes and IN sulfuric acid at 100°C for 2 hours, analysis was performed using Technicon Sugar Autoanalyzer Model N-1, manufactured by Technicon, USA. ) Skin Specific rotation [Q] Volume = 1630 to 169o average + 66o (concentration 0.
49% in 0.1 N NaOH) {B} Biological properties m m Induced activity A sample of the IF inducer according to the present invention was used to induce IF in the cells and serum of a test animal, and its activity was measured in the test example below. The results measured by the method described are shown in Tables 1 and 2, and IF-transferring superactivity was observed.

The results obtained using the method described in the test example below using the two rabbits in Table 1 are as shown in Table 2, and the maximum activity was reached in both rabbits 2 hours after administration.

It was confirmed that IF was induced in the test animals' bodies by the action of the IF-inducing agent according to the present invention by the method described in the Test Examples below in Table 2.

2) Stability of poly-induced activity A sample of the poly-inducing agent according to the present invention (1 serving each) was dissolved in water (1 serving each), and the mixture was incubated at 100°C for a predetermined time or at a predetermined temperature.
After heating for each time, the activity of each sample was measured according to the in vitro method described in Test Example 1. The results are shown in Tables 3 and 4. It was found to be stable.

Table 3 (Note) Heating time: 1 hour Table 4 (Note) Heating temperature 100°C (3) Acutely toxic male and female mice (ddy strain, 5 weeks old, weight 20
(10 animals in each group) were used as test animals.

LD5 as a result of intraperitoneal or oral administration of the IF inducer of the present invention dissolved in physiological saline to mice. Value is >1
．． 5 g/kg (peritoneal) and >10 g/kg (oral), with no significant difference between females and males. [4] Anti-tumor active mice (ddy strain, 5 weeks old, body weight 20 ± 1 night, 10 in each group
S-180 Sarcoma solid tumor (
2 x 2 x 2 sails) or Erich ascites cancer (2.5 x 1
After 24 hours, an aqueous solution of the IF inducer of the present invention (containing 0.2 females) was orally administered to each mouse once a day for 14 days.

Antitumor properties were observed. From the above characteristics, the substance according to the present invention has a molecular weight of about 1
It is thought to be a complex of sugar and protein containing phosphoric acid and having a polymer of 0,000 to about 3,000,000 molecules (mainly about 500,000 to about 100 molecules).

Furthermore, IF induced in animals or in vitro by this substance is not only inactivated by trypsin (0.08%, 370, 2 hours), but also has animal species-specificity and virus species-nonspecificity. It was found that this substance falls under the generally accepted definition of an m-inducing agent. Since no substance is known that has similar physicochemical and biological properties to the substance according to the invention, this substance is a new substance and a novel affinity-inducing agent.

That is, known plant agglutinins such as phytohemagglutinin, pokeweed mitogen, and concanavalin A are proteins with a molecular weight of 10 or more, and when heated at 5600 for 1 hour or 6000 for 5 hours, they induce IF. Not only do they lose activity, but their IF-induced activity is very weak. In contrast, the IF inducer according to the present invention has a different chemical composition, 1000
It is distinguished from plant agglutinins in that it is stable even when heated at zero for several hours, and that it has high IF-inducing activity. Next, the known IF inducer obtained from Toki root is a polymer substance with a molecular weight of more than 100,000 and does not become inactive even when heated at 100 qo for 1 hour, but its chemical composition (hexose, 48%, uronic acid, 40%
%, protein 5%) and infrared absorption spectra distinguish it from the IF inducers according to the invention.
Furthermore, since the IF inducer obtained from the root bark of mulberry has a molecular weight of 20,000 or more (mainly 60,000 or more) and is mainly composed of 1-3 linked glucose (containing 96% hexose), the IF inducer according to the present invention can be distinguished from agents.

Cartamine, cartamone, neocartamine, etc. contained in chrysanthemum plants of the genus Safflower, which are the raw materials for producing the IF inducer according to the present invention, are low-molecular substances that do not have IF-inducing activity and have different physical and chemical properties from the IF inducer according to the present invention. ing.
The IF inducer according to the present invention has superior IF inducer activity than known IF inducers isolated from plant tissues, has very low acute toxicity when orally administered to animals, and has antitumor activity. Therefore, it is expected to be used for the prevention and treatment of viral infections such as carcinogenic viruses in humans and animals.

The method for producing an IF inducer provided by the present invention is directed to
The method is characterized in that the above-mentioned active substance is extracted from the tissue of a plant that belongs to the IF-inducing active substance (Female) or its mutants and contains the IF-inducing active substance and is recovered from the extract.

The plants used in the method of the present invention are plants that are abundantly cultivated or grow naturally in various countries around the world, and tend to naturally or artificially form mutants or variants such as miscellaneous walls.

It has been found that many plants of the genus Safflower and their variables from various countries can be used for the purpose of the present invention. The plants listed below are illustrative only. Safflower tinct
oriusLinne) Careham (Careham)
Order lanat order Linne) Canham order arbo
reSCenSCartham low baetjC Satoshi N
Yman safflower has been cultivated in various countries for many years as a raw material for yellow and red pigments and as a raw material for edible oil, and is used as a medicinal herb in Japan and China.

Fresh plants may be used as raw materials for the method of the present invention, but from the standpoint of preservation and extraction efficiency, dried plants may also be used. . Extraction can generally be carried out with water at any temperature (eg, from room temperature to boiling of the extraction mixture).

The substances according to the invention can be used in alkaline aqueous solutions (e.g. pH 7-
10), it is recommended to adjust the pH of the water during extraction using a known buffer solution, sodium hydroxide, potassium hydroxide, ammonium hydroxide, etc. Although the extraction time is arbitrary, it is usually 1-5 days at room temperature. If the extraction temperature is high, the extraction time will be shortened (e.g. 40-100 pm).
(30 minutes to 6 hours). By the method of the present invention, most of the active ingredients contained in plant tissues (90% or more in some cases) can be extracted. If desired, a suitable preservative can be added during extraction. Extraction may be continuous or batchwise. The ratio of extraction water to raw material is arbitrary. Alternatively, the active substance may be extracted from the plant tissue with a suitable concentration (e.g. 20-80%) of a hydrophilic organic solvent such as methanol, ethanol, propanol, butanol, dimethyl sulfoxide, acetone. In this case, the temperature and time may be arbitrary (for example, 4 hours to 2 days at 40-80 qo).The plant residue is removed from the extract by a conventional method such as filtration, squeezing, or centrifugation. The active ingredients are recovered by removing inactive ingredients such as low molecular weight substances and pigments from the extract. Examples of practical methods for this purpose are as follows.The active ingredients of the IF inducer according to the present invention are: Since it exists in the part containing substances with a molecular weight of about 100,000 to about 3 million (mainly about 500,000 to 1,000,000), an extraocular furnace using an appropriate membrane that can separate substances with a cutoff molecular weight of 100,000 or more is necessary. Treat the upper light solution using the method.

The pressure in the ultrafurnace can be, for example, 0.1-5k9/ground. The active moieties thus obtained are collected and lyophilized to yield a brown powder.

'B} The extract is concentrated under reduced pressure if desired, and a hydrophilic organic solvent (e.g., methanol, ethanol, propanol, butanol, acetone, etc.) is added to the extract or its concentrate at an appropriate concentration (e.g., 40-7%). ), a precipitate containing the active ingredient is formed, which is dried under reduced pressure to obtain a brown powder.

Instead of the organic solvents mentioned above, ammonium salts such as ammonium chloride, ammonium sulfate, cetyltrimethylammonium bromide or inorganic metal salts such as zinc chloride, steel chloride can be added at appropriate concentrations (e.g. 20-5 skin/v).
%), a precipitate containing the active ingredient is formed, so the precipitate is desalted by, for example, dialysis with a cellulose tube or filtered in an ultra-furnace through a membrane with a molecular weight cutoff of 10,000, and then dried. A brown powder is then obtained.

By treating the extract as described above, most of the active ingredients in the raw materials (90% or more in some cases) can be recovered.

However, the content of inert ingredients in the dry coarse powder obtained is the lowest in the worst method. In addition, the wind method 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 the method was orally administered to animals. This crude powder is then purified by conventional methods such as column chromatography using gel filtration agents or ion exchange agents.

When a gel filtration agent 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. Examples of practical gel filter agents are Sephadex G-50 to G-200,
From Sef Arrows 2B to 8B, Sef Acrylic S-20
0 or S-300 (manufactured by Pharmacia Fine Chemical AB, Sweden), Biogel P-30 (manufactured by Pharmacia Fine Chemical AB, Sweden) to P-300, Biogel A (manufactured by Biorad, USA)
Made by Laboratories>, Sagavac (UK, Cerabac)
(manufactured by Laboratories), etc. Practical examples of ion exchangers are DEAE Cephadex A-25 and A-50.
(CI-type), QAE Cephadex A-25 and A
-50 (CI-type), CM Cephadex C-25 and C-50 (N type), SP Cephadex C-25 and C-50 (Na+ type), DEAE Cephacel (CI
- type), DEAE Sepharose CL-ship (CI-type),
CM Sepharose CL general (Na type 10) (manufactured by Pharmacia Fine Chemicals AB, Sweden), etc. The crude powder can also be purified using a suitable anion or cation exchange cellulose.

Although the product thus obtained contains some impurities, it can be put to practical use as an IF inducer. If desired, impurities can be further removed by combining the above purification steps. Example 1 Dried safflower (Canhamus tinctor)
ius Linne) flowers (2k9) with water, then water (
Extracted by leaving it in a 40-day bath at room temperature for 3 days,
This was centrifuged (600 m.p.m., 20 minutes) to separate the extract and the residual liquid, and the isosasa was washed twice with water (10 min each), and the washing liquid was combined with the extract. .

The extract obtained in this way was passed through a UD-6 type ultrafurnace filter (Bio Engineering K.K., Tokyo) using a UK-200 ultrafurnace membrane (molecular weight cut off: 20, manufactured by Toyo Roshi Co., Ltd.). It was then passed through an ultra-furnace (at a pressure of 3k9'). The residue was collected and lyophilized to give a brown powder (89.46 min.). This powder (
2) was dissolved in water (5), and the aqueous solution was poured into a column (4.5 x 70
The eluate was added to the skin) and eluted with water (600 mm), the eluate was divided into 3 sections each, and the sections from 25 mm to 5 mm were combined and freeze-dried to form a white powder (155 mm). 04 female) was obtained. For further purification, this powder (100 yen) was added to 0.01 M Tris-HCl buffer (pH 7.0, 1=0.
01) (5') and DEAE Sephadex A
-50 (Pharmacia Huain Chemical AB, Sweden) was added to a column (2.5 x 70 arcs) packed with 0.1 M Tris-HCl buffer (pH 9.0, 0.9
The eluate was divided into 3 upper sections, and the 1st to 27th sections were combined. This solution was filtered through a UK-1 filter membrane (molecular weight cut off: 10,000,
After desalting by ultrafiltration using Toyo Roshi Co., Ltd.), the mixture was freeze-dried to obtain an amorphous white powder (72.3-9). The physicochemical and biological properties of this product are as described above. Comparing this to the first white powder, the IF-inducing activity was approximately the same, but the amount of impurities was reduced. High purity was confirmed by ultracentrifugation and aerophoresis. 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 Table 5.

Table 5 Example 2 Dried safflower (Ca. hamusla)
After washing the flowers (lk9) of the ship Linne, 20 minutes of water was added thereto, left at room temperature for 2 hours, and then heated and extracted at 10,000 for 2 hours.

Thereafter, it was purified according to the method of Example 1. The physicochemical and biological properties of this purified product (70.3-9) were not significantly different from those obtained in Example 1.
Test Example 1 Method of IF induction using IF inducer and measurement of IF activity (Reference: Y. Kojima, Kitasato-C
h. , Exp. , Med. , 43:3il970)'a
``How to induce ``by in vitro method'' Rabbit (body weight approx.
kg, New Zealand White breed, SPF) were whole bled and sacrificed, corner pigs, bone marrow and lymph node cells were collected, a cell suspension containing 107 mixed cells/secretion was made, and in each suspension section (1 secretion), After adding 10, 1, 0.1, and 0.01 muta/ml of the IF inducer obtained by the method described in Example 1 of the present invention, and culturing at 25°C for 2 hours, each culture solution was centrifuged. Then, remove the light solution and use it for activity measurement.

(b) Method for inducing IF by in vivo method Aqueous solution of IF inducer obtained by the method described in Example 1 (500 muta/co) Blood was collected at 2 sites 1, 2, 4, and 6 hours after the injection into the ear vein, and the serum was used for IF activity measurement.

'c} Measurement of IF activity In both the above {a} (b-methods), the produced 'activity is measured by the 50% black half-life method using rabbit kidney established cell line (RK-13).

First, on a monolayer culture of the above-mentioned cannons prepared in advance in a Petri dish, the appropriately diluted sample solution obtained by the above [Table B] method was added, and after culturing overnight at 37°C, A virus (Vesic arstomatis viridis) was used as a challenge virus, and the IF activity was measured using the black reduction rate as an index. Note that the unit of IF activity is I
F is expressed as the reciprocal of the dilution representing 50% of the black number in untreated cells. Test Example 2 Method for proving that it is an IF inducer The IF sample produced by the method of 'Autumn b' above inhibits the proliferation of varicella stomatitis virus on rabbit RK-1 grand follicles of the same animal species. , vaccinia virus (Vaccinia virus)
iavims), but does not inhibit the proliferation of varicella stomatitis virus in L cells of mice with different animal bacteria.

Furthermore, when 0.08% tribucin is applied at 370° C. for 2 hours, the IF activity is inactivated. Test Example 3 Dew air phoresis and spectrophoresis were performed using a device (AE-Z) manufactured by Toyo Kagaku Sangyo (Tokyo).
The test was carried out using a polyacrylamide gel plate with a thickness of 3 cm and 0.3 M borate buffer (Book 8.4).

As a result, a single band was observed, and it was confirmed that the material of the present invention was electrolytically uniform.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the material according to the invention, and FIG. 2 shows the infrared absorption spectrum. Figure 2 Figure 1

Claims (1)

[Scope of Claims] 1. Compositae, genus Safflower (Ca
1. A method for producing an interferon-inducing agent, which comprises extracting the active substance from a tissue of a plant or a variant thereof belonging to the genus P. rthamus and containing the interferon-inducing active substance, and recovering the active substance from the extract. 2. A method according to claim 1, in which the active substance is recovered by adding a hydrophilic organic solvent to the extract. 3. A method according to claim 1, in which the active substance is recovered by adding an ammonium salt or an inorganic metal salt to the extract. 4. A method according to claim 1 for recovering active substances from an aqueous extract by ultrafiltration.