JPS625407B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to interferon (hereinafter referred to as IF)-inducing active substances isolated from plant tissues. The present invention is based on the knowledge that a substance with excellent IF-inducing activity is contained in the tissues of various plants belonging to the Lamiaceae family, the genus Lamiaceae, or its variants, and that this active substance can be isolated easily and inexpensively. It is based. Therefore, an object of the present invention is to provide an IF-inducing active substance that has excellent IF-inducing activity and low toxicity and can be easily produced 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. (A) Physical and chemical properties (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 Approximately 100,000 to approximately 3 million ( Mainly about 500,000 to 1,000,000) [Ultracentrifugation method using Spinco Model E analytical ultracentrifuge (manufactured by Beckman, USA), Amicon ultrafilter 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 reaction, phenol/sulfuric acid reaction, and deittomer reaction. Negative for furin reagent and Elson-Morgan reaction. (8) Properties Acidic (9) Main chemical composition (a) Amino acid oxyproline (3.2±0.3%) Aspartic acid (9.3±0.3%) Threonine (6.1±0.3%) Serine (4.3±0.3%) Glutamic acid (7.6±0.3%) Proline (4.0±0.3%) Glycine (10.0±0.3%) Alanine (10.3±0.3%) Valine (6.6±0.3%) Isoleucine (5.4±0.3%) Leucine (8.6±0.3% ) Tyrosine (trace amount) Phenylalanine (2.0±0.3%) Lysine (3.9±0.3%) Histidine (1.3±0.3%) Arginine (3.4±0.3%) Ammonia (13.4±0.3%) (110% with 6N hydrochloric acid After hydrolyzing under reduced pressure at ℃ for 48 hours, it was analyzed using the Technicon Amino Acid Auto Analyzer Model NC-1 manufactured by Technicon, Inc. (b) Sugar arabinose (47.09 ± 0.3%) Galactose (25.66 ± 0.3%) Glucose (20.62 ±0.3%) Mannose (4.64±0.3%)
After hydrolysis at 100°C for 2 hours, it was analyzed using a Technicon Sugar Auto Analyzer Model N-1 manufactured by Technicon, USA) (10) Specific rotation [α] ²⁵ _D = -75° to -82° Average -79° (Concentration 0.47%, in 0.1N NaOH) (B) Biological properties (1) IF-inducing activity A sample of the IF-inducing active substance according to the present invention was used to induce IF in the cells and serum of test animals, and its activity was measured. The results of measurement using the method described in the test examples below are shown in Tables 1 and 2, and IF-inducing activity was observed.

[Table] Table 2 shows the results obtained using the method described in Test Example 1(b) below using two rabbits, and the maximum activity was reached in both rabbits 2 hours after administration.

[Table] According to the method described in the test example below,
It was confirmed that IF was induced in the test animal's body by the action of the IF-inducing agent. (2) Stability of IF-induced activity Samples of IF-induced active substances according to the present invention (1 each
mg) in water (1 ml each), heated at 100°C for a specified time or at a specified temperature for 1 hour, and then treated each sample according to the in vitro method described in Test Example 1. The results are shown in Table 3. As shown in Table 4, the induced active substance according to the present invention was found to be stable to heat.

【table】

[Table] (3) Acute toxicity Male and female mice (ddy strain, 5 weeks old,
Test animals (body weight 20±1 g, 10 animals in each group) were used as test animals.
As a result of intraperitoneal or oral administration of the IF inducer of the present invention dissolved in physiological saline to mice, the LD50 value was 560 mg/Kg (peritoneal) and >4.
g/Kg (oral), and there was no significant difference between females and males. (4) Antitumor activity Mice (ddy strain, 5 weeks old, body weight 20 ± 1 g, 10 mice in each group) were used as test animals, and S-180 Sarcoma solid tumor (2 x 2 x 2 mm) or Ehritz ascites carcinoma (2.5 ×10 ⁶ pieces) were transplanted into the axilla or abdominal cavity of mice, and 24 hours later, an aqueous solution of the IF inducer of the present invention (containing 0.2 mg) was given to each mouse once daily.
After oral administration for 14 days, antitumor activity was observed. From the above characteristics, the substance according to the present invention has a molecular weight of about 100,000 to about
It has 3 million macromolecules (mainly about 500,000 to about 1 million) and is thought to be a complex of sugar and protein containing phosphoric acid. Furthermore, IF induced in animals or in vitro by this substance is not only inactivated by trypsin (0.08%, 37°C, 2 hours), but also has animal species specificity and virus species nonspecificity. This substance has a generally accepted IF
It was found that the substance falls under the definition of an inducer. Since no substance is known that has similar physicochemical and biological properties to the active substance according to the invention, this substance is a new substance and a novel IF-inducing substance. Namely, known phytohemagglutinin,
According to known literature, plant agglutinins such as pokeweed mitogen and concanavalin A are proteins with a molecular weight of over 100,000 and a temperature of 1 at 56°C.
After heating at 60° C. for 5 hours, not only do they lose their IF-inducing activity, but their IF-inducing activity is very weak. In contrast, the IF according to the present invention
Inducers are distinguished from plant agglutinins in that they have a different chemical composition, are stable even when heated at 100°C for several hours, and have 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 its chemical composition (hexose, 48%,
40% uronic acid, 5% protein) and different infrared absorption spectra.
Distinguished from IF-inducing active substances. 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 It is distinguished from Perylaldehyde, α-
Pinene, l-limonene, perilla ketone, naginata ketone, shisonin, p-coumaric acid ester, dihydroperyl alcohol, l-menthol, etc.
It is a low-molecular substance that does not have IF-inducing activity, and has different physical and chemical properties from the IF-inducing active substance according to the present invention. Next, the known method described in U.S. Pat.
The IF inducers are not isolated from plant tissues and their physicochemical properties are different from those of the IF-inducing active substances according to the invention. The IF-inducing active substance according to the present invention has an IF-inducing activity that is superior to known IF-inducing agents isolated from plant tissues.
It has inducing activity, has very low acute toxicity when orally administered to animals, and has antitumor activity, so it is expected to be used for the prevention and treatment of viral infections such as carcinogenic viruses in humans and animals. be done. The method for producing the IF-inducing active substance provided by the present invention is based on
or extracting the above-mentioned active substance from the tissue of a plant belonging to that variable and containing the IF-inducing active substance,
It is characterized by recovering this from the extract. The plants used in the method of the present invention are annual plants that are abundantly grown around the world, and tend to naturally or artificially form variants such as mutants and hybrids. It has been found that many plants of the genus Perilla and their variables from various countries can be used for the purpose of the present invention. The plants listed below are illustrative only. Perilla (P.frutescens Britton var.crispa Dec.f.
purpurea Makino), P.frutescens Britton var.crispa
Dec.f.viridis Makino), P.frutescens Britton var.crispa
Dec.f.discolor Makino), P.frutescens Britton var.crispa
Dec.f.crispa Makino), P.frutescens Britton var.
crispa Dec.f.viridis−crispa Makino), P.frutescens Britton var.
hirtella Makino et Nemoto), Perilla (P.frutescens Britton), Lemon perilla (P.citriodora Nakai) (scientific name: Michio Murakoshi, Tomitaro Makino, Illustrated encyclopedia of primary color plants, Hokuryukan, 1955, Tatsuo Karimi, Koichi Kimura, Encyclopedia of Medicinal Plants, Hirokawa Shoten, 1974 and Tatsuo Karime,
Yushiro Kimura, Latest Japanese and Chinese Medicinal Plants, Hirokawa Shoten, 1978
by). The plants exemplified here have low toxicity, such as perilla, perilla, chili mesojiso, and lemon perilla, which are cultivated in Japan, China, and other countries, because their leaves and seeds are used, for example, in foods, herbal medicine, and fragrances. This is because it has been used as a raw material for many years. Since all tissues of plants belonging to the Lamiaceae family and containing the IF-inducing active substance according to the present invention can be used in the method of the present invention, there is no problem in providing abundant raw materials at low cost. It is practical to use leaves and stems because seeds are less active and roots require more effort to remove soil. The amount of active ingredient contained in the leaves and stems is almost the same. The amount of active ingredient contained in each part of the tissue does not change significantly between before and after flowering of the plant. Although fresh raw materials may be used, it is advantageous to use dried raw materials from the viewpoint of preservation and extraction efficiency. The drying method is optional and may be natural drying or hot air drying. May be washed with water before use. Extraction can generally be carried out with water at any temperature (eg, from room temperature to boiling of the extraction mixture). Since the substance according to the present invention is highly soluble 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. Higher extraction temperatures shorten the extraction time (e.g. 30 minutes to 6 hours at 45-80° C. with stirring). By the method of the invention, the majority (in some cases up to 90%) of the active ingredient contained in the plant tissue is
above) can be extracted. However, as the extraction temperature increases, the amount of unnecessary components such as pigments extracted also increases, so it is necessary to avoid raising the temperature too high. 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. Plant residues are removed from the extract by conventional methods such as filtration, squeezing, or centrifugation, and inactive components such as low-molecular substances and pigments are removed from the resulting extract, and active components are recovered. An example of a practical method for this is as follows. (A) The IF-inducing active ingredient according to the present invention has a molecular weight of approximately
100,000 to about 3 million (mainly about 500,000 to
1,000,000), the supernatant liquid is treated by ultrafiltration using an appropriate membrane that can separate substances with a molecular weight cutoff of 100,000 or more. The ultraviolet pressure can be, for example, 0.1-5 kg/cm ² . The active moieties thus obtained are 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.
40-70 w/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 solvent, use ammonium salts such as ammonium chloride, ammonium sulfate, and cetyltrimethylammonium bromide, or inorganic metal salts such as zinc chloride and copper chloride at an appropriate concentration (for example, 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 a gelatin or ion exchange agent. If a gel-filtration agent is used, elution may be performed with an appropriate buffer, but
Usually, it is sufficient to elute with water. If an ion exchange agent is used, elute with an appropriate buffer. An example of a practical gelling agent is Cephadex
From G50 to G-200, from Seph Arrows 2B to 6B, Seph Acrylic S-200 or S-300 (Sweden, Pharmacia Huain Chemical AB)
), Biogel P-30 to P-300, Biogel A (manufactured by Biorad Laboratories, USA), and Sagaback (manufactured by Serabak Laboratories, UK). Practical examples of ion exchangers are DEAE Cephadex A-25 and A-50
(Cl ^- type), QAE Sephadex A-25 and A-
50 (Cl ^- type), CM Sephadex C-25 and C
-50 (Na ⁺ type), SP Cephadex C-25 and C-50 (Na ⁺ type), DEAE Cephacel (Cl ^- type),
DEAE Cepharose CL-6B (Cl ^- type), CM Cepharose CL-6B (Na ⁺ type) (manufactured by Pharmacia Huain Chemical AB, Sweden), etc. The crude powder can also be purified using a suitable anionic or cationic ion exchange cellulose. Although the product thus obtained contains a slight amount of impurity, it can be used as an IF inducer. If desired, impurities can be further removed by combining the above purification steps. Example 1 After washing dried perilla leaves (1Kg), extract by leaving in water (20) at room temperature for 3 days, centrifuging (6000 rpm, 20 minutes), and extracting liquid. 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 using a UD-6 type ultrafilter (Bio Engineering KK, Tokyo) using a UK200 ultrafiltration membrane (molecular weight cut off: 200,000, manufactured by Toyo Paper Co., Ltd.) (pressure 3 kg/ ^cm2 ). The residue was collected and lyophilized to give a brown powder (8.813g). This powder (1.5 g) was dissolved in water (5 ml), the aqueous solution was added to a column (4.5 x 70 cm) packed with Cephadex G-200 (Pharmacia Huain Chemical AB, Sweden), and water (600 ml) was added. Divide the eluate into sections of 3 ml each, and
The above sections were combined and freeze-dried to obtain a white powder (117 mg). For further purification, this powder (100 mg) was added to 0.01 M Tris-HCl buffer (PH
7.0, I=0.01) (5 ml) and packed with DEAE Sephadex A-50 (Pharmacia Huain Chemical AB, Sweden) (2.5
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 (58.9 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] Example 2 After washing dried perilla leaves (1 kg) with water, 20 g of water was added thereto and left at room temperature for 2 hours, then 1N sodium hydroxide was added to adjust the pH to 8.5. Next, this was heated and extracted at 65°C for 2 hours. Thereafter, it was purified according to the method of Example 1. The physicochemical and biological properties of this purified product (54.5 mg) were not significantly different from those obtained in Example 1. Example 3-8 Seeds, leaves, and stems of perilla, perilla, perilla, perilla, perilla, chilimen perilla, perilla perilla, perilla perilla, and lemon perilla were dried separately, and then treated according to the method described in Example 1 to obtain Table 6 shows the results of measuring the IF-inducing activity of the products obtained in Example 1 and the products of Examples 1 and 2 by the method described in Test Example 1(a) (in vitro method).

【table】

[Table] The physicochemical and biological properties of each product obtained were substantially the same as those of the product obtained by the method of Example 1. Test example 1 IF induction method using IF inducer and measurement of IF activity (References: Y. Kojima, Kitasato Arch.,
Exp., Med., 43:35, 1970) (a) Method for inducing IF by in vitro method Rabbits (weighing approximately 1 kg, New Zealand White breed, SPF) were killed by collecting whole blood, and the spleen, bone marrow, and lymph nodes were killed. Collect the cells, make a cell suspension containing 10 ⁷ /ml of mixed cells, and divide each suspension (1 ml) into
IF obtained by the method described in Example 1 of the present invention
10, 1, 0.1, and 0.01 μg/ml of inducers were added, respectively, and after 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 Example 1 was added to a rabbit (approximately 1 ml body weight).
Kg, New Zealand White breed, SPF) was injected into the ear vein, and blood was collected 1, 2, 4, and 6 hours later (2
ml) 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 carried out by the Plack half method. First, place the above on a monolayer culture of the above cells prepared in advance in a shear dish.
After adding the appropriately diluted IF sample solution obtained by methods (a) and (b) and incubating at 37℃ overnight, Vesicular stomatitis virus was added to the cells as a challenge virus, and the cells were incubated at 37℃ for 1 hour. After overnight culture, IF activity was measured using the plaque reduction rate as an index.
Note that the unit of IF activity is expressed as the reciprocal of the dilution representing 50% of the number of plaques in IF-untreated cells. 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. It also inhibits the proliferation of Vaccinia Virus, but does not inhibit the proliferation of Vesicular Stomatitis Virus in L cells from mice of a different species. Furthermore, when treated with 0.08% trypsin at 37°C for 2 hours, the IF activity is inactivated. Test Example 3 Electrophoresis For electrophoresis, an apparatus (AE-Z type) manufactured by Toyo Kagaku Sangyo Co., Ltd. (Tokyo) was used.
8.4). As a result, a single band was observed, indicating that the substance of the present invention was electrophoretically uniform.

[Brief explanation of the drawing]

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

Claims (1)

[Scope of Claims] 1. A substance that is stable in the form of an amorphous white powder and has interferon-inducing activity and the following physical and chemical properties. (a) Elemental analysis H: 8.5-8.7%, C: 48.8-49%, N: 6.3-6.5%, P: 1.0-1.1% (b) Molecular weight From about 100,000 to about 3 million (mainly from about 500,000 to about 3 million) (up to approximately 1 million) (c) Melting point or decomposition point Melting point is unclear. Carbonizes at approximately 220℃. (d) Ultraviolet absorption spectrum As shown in Figure 1 (Measured in 0.1N NaOH) (E) Infrared absorption spectrum As shown in Figure 2 (KBr method) (F) Solubility in solvents Dissolves in water, especially potassium hydroxide It dissolves well in alkaline aqueous solutions such as , sodium hydroxide, and ammonium hydroxide. Slightly soluble in methanol, ethanol, propanol, butanol, acetone, chloroform, and ether. (g) Color reaction Positive for ninhydrin reaction, phenol/sulfuric acid reaction, and deutstomer reaction. Negative for furin reagent and Elson-Morgan reaction. (H) Properties Acidic (L) Main chemical composition (a) Amino acid oxyproline (3.2±0.3%) Aspartic acid (9.3±0.3%) Threonine (6.1±0.3%) Serine (4.3±0.3%) Glutamic acid (7.6±0.3%) Proline (4.0±0.3%) Glycine (10.0±0.3%) Alanine (10.3±0.3%) Valine (6.6±0.3%) Isoleucine (5.4±0.3%) Leucine (8.6±0.3% ) Tyrosine (trace amount) Phenylalanine (2.0±0.3%) Lysine (3.9±0.3%) Histidine (1.3±0.3%) Arginine (3.4±0.3%) Ammonia (13.4±0.3%) (Technicon Amino Acid Auto analyzer
(measured with NC-1 type) (b) Sugar arabinose (47.09±0.3%) Galactose (25.66±0.3%) Glucose (20.62±0.3%) Mannose (4.64±0.3%) Xylose (1.99±0.3%) (Technicon sugar auto (measured with Analyzer N-1 model) (nu) Specific optical rotation [α] ²⁵ _D = -75° to -82° Average -79° (Concentration 0.47%, in 0.1N NaOH)