JPS6069024A - Interferon-inducing substance - Google Patents

Abstract

NEW MATERIAL:An interferon (IF)-inducing substance having following properties: description: stable in amorphous white powder; elementary analyses in %: H 6.54+ or -0.3, C 41.9+ or -0.3, N 2.39+ or -0.3, P 0.28+ or -0.3; molecular weight: about 100,000-3,000,000; properties: acidic; specific rotatory power: [alpha]D<26>+63-69 (0.49% in 0.1N NaOH); solubility: readily soluble in aqueous alkali, hard soluble in methanol, acetone; color reaction: positive to ninhydrin reaction, negative to Elson-Morgan reaction. USE:Antitumor, antiviral agent: Its activity is higher than known IF inducer originating from plants with lower toxicity. Further it is obtained throgh simple operations at lower costs. PREPARATION:A plant of safflower in Asteraceae is extracted in an alkali aqueous solution at pH 7-10 and 40-100 deg.C for 30min to 6hr. Then the extract is subjected to ultrafiltration to collect the fractions corresponding to its molecular weight and purified in usual manners to obtain the objective IF-inducing substance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an interferon (hereinafter referred to as 1F)-inducing active substance isolated from plant tissues.

The present invention provides that a substance having excellent F-inducing activity is contained in each plant belonging to the Asteraceae family, Safflower lineage, or in the tissue of its Da mobilization, and that this active substance can be easily and inexpensively produced. I am thrilled with the knowledge that I can do this.

Therefore, an object of the present invention is to provide an IF-inducing active substance that has excellent IF-inducing activity and low susceptibility and can be easily and inexpensively prepared.

The present invention provides a substance that is stable in the form of an amorphous white powder and that destroys IP-induced activity and the following physical and chemical properties.

(Al physical and chemical properties (1) cumulative analysis H: 6.54 ± 0.3%, C: 41.9 ± 0.3
%.

N2 2.39±0.3%, P: 0.28
±0.034 Nu11 (2) Molecules approximately 100,000 to 3,000,000 (mainly approximately 1,000 to 10,000
00,000) [Ultracentrifugation method using Spinco Model E analytical ultracentrifuge (manufactured by Peckman, USA), Amicon ultrafilter Oyohi XM50. XM 100 A and xy
300 Filtration membrane (manufactured by Amicon, USA) UKIQ, UK
Ultrafiltration method using 5o Oyobi TJK 200 F filtration membrane (manufactured by Toyo Roshi) and Sephadex G-200 (
Sweden, Pharmacia Fine Chemicals AB
(3) Melting point or decomposition point Melting point uncertain. Carbonizes at about 220″C.

(4) The ultraviolet rice grain absorption spectrum in Figure 1 shows shoulders at about 250 and 280 mμ (measured in 0,1 HNaOH, but did not change in water or IN NaOH).

(5) Infrared absorption method as shown in Figure 2 (KBr method) (6) Solubility in various solvents Dissolved in water, sodium hydroxide, potassium hydroxide, water 1
It dissolves particularly well in alkaline aqueous solutions such as ammonium zotide.

methanol, ethanol, propatool, butanol,
Slightly soluble in acetone, chloroform, and ether.

(7) Positive for color reaction ninhydrin reaction, phenol/sulfuric acid reaction, Folin's reagent and Dittmer reaction.

There is a gap in Elsin Morgan's reaction.

(8) G acidic (9) Main conjugated bile formation (I1 Amino acid ogiciproline (15,61±0.4) As/R ragic acid (5,51±0.4) Threonine (10,
90±0.4th) serine (9,64±0.4th) glutamic acid (3,96±0.4th) proline (
4,27±0.4th) Glycine (9,09±0.4th) Alanine (8,24±0.4th) Valine (4,04±0.4th) Isoleucine
(5,59±0.4cm) Leucine (2,56cm),
4%) Phenylalanine (1,32±0.4%)
Arginine (2,18± 0.4%) Arginine (1,24
±0.4%) Tyrosine (1,94±0.4%) Histidine (0,930-4%) Ammonia (12,98±0.4%) (1 with 6N hydrochloric acid
After hydrolysis under reduced pressure at 10"C for 48 hours, Technicon Amino Acid Auto Analyzer NC manufactured by Technicon, USA
Arabinose (9,38±0.6qIJ) Galactose (20,98±0.6%) Glucose (64,9
8 ± (), 6%) Mannose (3,26 ± 0.6%) After hydrolysis for 2 hours at °C, Technicon, Inc., USA, Technicon Sugar Autoanalyzer N-
1f-%'z)+101 Specific rotation [α] 6-+63°~+696 Average+(56°(f
ft W 0.49%, g, IN 1iaOJ (medium) fBl Biological properties (1) I P'-induced activity In the cells and serum of test animals using the sample of the F-prone substance according to the present invention. IP was induced and its activity was measured by the method described in the test examples below. The results are shown in Tables 1 and 2, and IFM activation was observed.

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

Table 2 (F activity, in-vide method) Time at 2 hours after administration (hours) Rabbit 1 15 80 470 175 50 Rabbit 2
15-90 500 280' 70 It was confirmed that EF was induced in the body of the test animal by the action of the IF inducer according to the present invention by the method described in the test example below.

(2) Effect of F-inducing activity Samples of the F-XPvj addictive substance according to the present invention (1M9 each)
were dissolved in water (1 ml each) and heated at 100°C for a predetermined time or at a predetermined temperature for 1 hour, and the activity of each sample was measured according to the in vitro method described in Test Example 1. 1 according to the present invention, as shown in Table 4.
The F-activated substance was found to be thermally stable.

Table 3 Heating and moistening sample concentration (μt/ml untreated >100 >100 90 <1037 >1
00 >100 95 <1060 >100 >10
0 90 <1080 >100 >100 92 <
10100 > 100 > 100 93 < 10
(E) Heating time: 1 hour Table 4 (F activity) Untreated > 100 > 100 92 < 101
>100 >100 95 <104 >100 >1
00 93 <108 >100 >100 65 <
1024 >100 75 35 <1.0 (Note) Heating temperature 100°C (3) Acute toxicity Male and female mice (aay strain, 5 weeks old, body M2o
±1? , 10 animals in each group) were used as test animals. Dissolved in physiological saline! r, L The engineered fat-addicting substance according to the present invention,
As a result of intraperitoneal or oral administration to mice, the LDso value was >
1. sr/kg (AU cavity) and > 10? 7kg
(Oral) 6. D. There was no significant interaction between females and males.

(4) Anti-II! l! Calorie active mice (cLdy strain, 5 years old, weight gain ±11, 10 in each group)
8-180 Sarcoma solid tumor (
2x 2
After hours 5t (0,2η
The same aqueous solution (containing) was orally administered to each mouse once every day for 14 days, and antitumor activity was observed.

Due to the above properties, the substance according to the present invention has amino acids, sugars,
It has about 100,000 to about 3,000,000 macromolecules (mainly about (capital) to about 1,000,000) to molecules mainly composed of phosphoric acid, and is thought to be a complex of sugar and protein containing phosphoric acid. .

In addition, trypsin (0.08%, 37"C
In addition to being inactivated within 12 hours), it also has animal fIII specificity and virus virus non-specificity, so the immaterial is a substance that falls under the generally accepted definition of an F inducer. I understand.

Since there are no known substances having the same physicochemical and biological properties as the active substance according to the present invention, the present substance is a new substance and a novel IF-inducing active substance. Namely, the known phytohemagglutinin,
According to known literature, plant agglutinins such as mitogenes and concanavalin A are proteins with a molecular weight of more than 10,000,000, and when heated at 56°C for 1 hour or at ω"C for 5 hours, they induce F9 activation activity. In addition, these F-inducing activities are very weak.On the other hand, the F-inducing addictive substance according to the present invention has a different chemical composition and a 100'C
It is distinguished from plant agglutinins in that it is stable even when heated for several hours, and that it has high FR-activating activity.

Next, the known IPB agent obtained from the root of the tree is a polymer substance with a molecular weight of more than 100,000 and does not become inactive even when heated at 100°C for 1 hour, but its chemical composition (hexose, 48%, uron) 1340%, protein 5115 receptor] and infrared absorption spectrum, it is distinguished from the IF-inducing active substance according to the present invention.

Furthermore, since the IF rust-inducing agent obtained from the root bark of mulberry is mainly composed of 1-3 bonded glucose (containing 96 molecules of hexose) with a molecular weight of 20,000 or more (mainly 60,000 or more), the present invention Engineering P' luci propensity 1 is distinguished from biological cost.

Cartamine, which is of course contained in Asteraceae safflower, which is the raw material for producing the F-addictive substance No. 11 according to the present invention;
Cartamon and Neocartaminzaki are low-molecular substances that do not have IF-inducing activity, and have the physical and chemical properties of the pigeon-addicting substance according to the present invention.

The IF-stimulated seedling 1 organism T, 1 according to the present invention has a better IF-inducing propensity than known IP inducers isolated from plant tissues, and when orally administered to animals, the acute Very low, anti-III! Because it has tumor activity, it is expected to be used in the prevention and treatment of viral infections such as oncogenic viruses in humans and animals.

The method for producing Compositae safflower paper (Ca
rthamus) i or its variants from the tissue of a plant containing kAl and a biogenic substance, above ¥1-
% to extract the active substance and recover this from the extract.
It is a sign.

The plants used in the method of the present invention are plants that are abundantly cultivated or grow wild in various countries around the world, and tend to naturally or artificially form variants such as mutants and hybrids. It has been found that many safflower plants from various countries and their variables can be used for the purpose of the present invention. The plants listed below are illustrative only.

Safflower (Carthamus tinctorius)
Linne) Carthamus
1anatus Linne) Carthamus
arborescens Linne Cartham
Us baeticus Nyman safflower has been cultivated in various countries for many years as a raw material for yellow and purple sardines and as a raw material for edible oil, and is used as a medicinal herb in Japan and China.

Although fresh plants may be used as raw materials for the method of the present invention, it is advantageous to use dried plants from the viewpoint of preservation and extraction efficiency. The drying method is optional and may be natural drying or hot air drying. Use 1: You may wash the IJ with water.

Extraction can generally be carried out with water at any temperature (for example, at the temperature of the extraction mixture).

The substances according to the invention can be used in alkaline aqueous solutions (e.g. pH 7-
10), it is advisable to adjust the pH of the water during extraction using a known buffer solution, sodium hydroxide, potassium hydride, ammonium hydroxide, etc. Although the extraction time is arbitrary, it is usually 1-5 days at room temperature. Higher extraction temperatures will shorten the extraction time (e.g. 40-100"C)
(30 minutes to 6 hours).

By the method of ``Kinei 19'', it is possible to extract most of the active ingredients contained in plant tissues (more than 90% in some cases). If desired, a suitable bird control agent can be added during extraction. Extraction may be rapid-fire or patch-based. The ratio of extraction water to raw material is arbitrary.

The solution is mixed with an appropriate degree of hydrophilic organic solvent (e.g. -8()%) such as methanol, ethanol, pro/ethanol, butanol, dimethyl sulfoxide, acetone.
The active substance may be extracted from plant tissue. The temperature and time for this restriction may be arbitrary (for example, 4 hours to 2 days at 40-80'C).

Plant residues are removed from the extract by conventional methods such as filtration, squeezing or centrifugation, and the resulting extract is free of inactive components such as low molecular weight substances and pigments, and the active components are recovered. . An example of a practical method for this is as follows.

(Aj The IF-inducing active ingredient according to the present invention has a molecular weight of about 1
00,000 to about 3,000,000 (mainly about 10,000 to 100,000)
10,000) Since it exists in the part containing the substance, it is a fractionated molecule! 10
The supernatant liquid is processed by ultrafiltration using a suitable membrane that can separate more than 10,000 substances. The pressure of ultrafiltration is, for example, 0.1
-5 kg7cm'1.

The active moieties thus obtained are collected and lyophilized to yield a brown powder.

(Bl) Concentrate the extract under reduced pressure if desired, and add a hydrophilic organic solvent (e.g. methanol, ethanol, propatool, butanol, acetone, etc.) to the extract or its concentrate at an appropriate concentration (e.g. 40-70 w/w). v%)
If you add it so that it is, a precipitate containing the active ingredient will form, so remove this under reduced pressure. Upon drying, a brown powder is obtained.

(C) Instead of the above organic solvents, ammonium chloride, ammonium sulfate, cetyltrimethylammonium bromide, or inorganic gold M salts such as zinc chloride, steel chloride, each at an appropriate concentration (e.g.
/v%), a precipitate containing the active ingredient is generated, so the precipitate is desalted by, for example, dialysis with a cellulose tube or ultrafiltration with a membrane with a molecular fraction i4.10,000. After drying, a brown powder is obtained.

By treating the extract in the manner described above, most of the active ingredients in the raw materials (90% or more using a lifting platform) can be recovered. However, method (A) has the lowest inert ingredient content in the obtained dry coarse powder. Also(
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 the coarse powder obtained by method (A) was orally administered to animals.

The 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 with a suitable buffer solution -C may be used, but it is usually sufficient to elute with water. If an ion exchange agent is used, elute with an appropriate buffer.

Examples of practical gel filtration agents are Sephadex G-■ to G-2001, Sepharose 2B to 6B, Sephacryl S-200 or S-300 (Pharmacia Fine Chemicals ABff, Sweden),
Biogel P-30 to 'P-3004, such as Biogel A (manufactured by Biorad Laboratories, USA) and Sagapac (manufactured by Ceravac Laboratories, UK). Common examples of ion exchange agents are DEI Sephadex A-25 and A-Shame (C1-type), QA Sephadex A-25 and A-Shame (Ct-type), CM
Sephadex C-25 and C-50 (Na” M
), "'Cef 7 Dex C-5O and C-5Q (Na
+ type), DffiARi Sephacel (cL- type), D
IAIC-1=770-s CL-5B (Ct-9)
, CM Cef 7o-S CL-6B (Na+ type) (Sweden, Pharmacia Fine Chemicals A B
(manufactured by)

The crude powder can also be purified using a suitable anionic or cationic parent cellulose.

Although the product thus obtained contains a slight amount of impurity, it can be used as a P' inducer. If desired, impurities can be further removed by combining the above purification steps.

Disaster Example 1 Dried safflower (Carthamus tincto)
riuθLinne) flowers (2 kg) were washed with water,
The 9A residue was extracted by leaving it in water (40 t) at room temperature for 3 days, and the fish core was treated (600 Or, p, m-1 addition), separated into the extract and the residual stream, and the 9A residue was poured into water (40 t). 1OL each
), and the washing liquid was combined with the extract liquid. The extract obtained in this way was treated with a UD-6 type ultracentrifuge (K, Toki, Nomiio Engineering), and was heated to tlK-200.
Ultrafiltration was carried out using an ultra 1p filtration membrane (fraction molecule short cut, manufactured by Toyo P Paper Co., Ltd.) (pressure 3, 9.7α2). Freeze-drying was carried out under special conditions to obtain a brown powder (89,469). This powder (22) was dissolved in water (5 ml), and the aqueous solution was poured into a column (4
, 5X 70an) K was added and eluted with water (600d>), and the eluate was divided into 3 ml portions.
The fractions up to 5:l'i' were combined and freeze-dried to obtain a white powder (155.04 my). For further purification, this powder (100 capes) was dissolved in 0.01 M) Lis-HCl buffer (pH 7.0, I-0.01) (5 ml) and 1cAB Sephadex A-50 (Pharmacia. Fine Chemical AB.

(Sweden) packed column (2,5x 70cr
n) and 0.1 M) Lissu salt lS2 buffer (
1) Add H9-0,0,5M salt, 300 yd)
Divide the eluate into 3-parts, starting from No.
After desalting and desalting, the mixture was subjected to ultrafiltration using a 10,000-yen paper (manufactured by Toyo P Paper Co., Ltd.), and then freeze-dried to obtain an amorphous white powder (72.3 cm). The physicochemical and biological properties of this product are as described above. Comparing this with the white powder of Choi, the F-induced activity was approximately the same, but the amount of impurities 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.
It was as shown in the table.

Table 5 After extraction > ioo < lo < i.

After ultrafiltration > 100 95 < 10 After gel filtration > 100 > 10 + 1 964 > 9
After 1 > 100 > 100 > 100 seeds 1M Example 2 Dried Carthamua 1an
AtausLinne) flowers (1ki9) were washed, water was added to them, and the mixture was left at room temperature for 2 hours.
The physicochemical and biological properties of this a product (70,311F) were obtained by !lv Example 1. There was no significant difference between the percentage of

Test Example I Method of inducing IFF using IP inducer and measurement of IP activity (References: Y, Kojima, Kitasato
Arch.

EXI), MG (1,, 43: 35.1970
) (al In vitro method of inducing F
JB? [, Bone marrow and lymph node cells were collected, a cell suspension containing mixed cells 107/me was prepared, and the cell suspension containing the cell suspension (1tlLl) was filled with the engineered F inducer obtained by the method described in Example 1 of the present invention. Addictive substance 1
After adding 0.1, 0.1 and 0.01 μf/me and culturing at 5°C for 24 hours, each culture solution was centrifuged and the supernatant was collected and used for IP activity measurement.

(b) Method for inducing F-F by in-bidet method Aqueous solution of F-F-inducing addictive substance obtained by the method described in Example 1 (500
μt/! ILe) 2 Place a rabbit at the foot of the mountain (approximately 1 kg in weight)
, New Sealant White Yuzu, 8PF) was injected into the ear vein, and blood was collected (2 mJ) 1, 2, 4, and 6 hours later, and the serum was used for IP activity measurement.

(C) Measurement of IF activity In both the above (altbl method) and the measurement of the produced 1F activity, the produced 1F activity is carried out by the addition black half-life method using rabbit kidney cell line (RK-13). First, prepare it in a petri dish in advance. The above (al (1)
Add the appropriately diluted engineering F sample solution obtained by method 1, and
After overnight incubation at 7°C, varicella stomatitis virus (Vesi
cular 'stomatitis virus)
was used as the attacking virus, and the activity of F was measured using the black reduction rate as an index. Note that the unit of IP activity is engineering F.
It is expressed as the reciprocal of the dilution representing 5)% of the black number in untreated cells.

Test Example 2 The IF sample produced by the method of (IL) (b) above was used to induce hydric stomatitis V9 on rabbit RK-13 cells from the same animal, Yuzu. In addition to suppressing virus proliferation, vaccinia virus (V
accinia virus), but
L cells from mice of different animal species do not inhibit the proliferation of varicella stomatitis virus. Also, add 0.08% trypsin to 3
After 2 hours at 7°C, the IF activity is inactivated.

Test Example 3 Electrophoresis Electrophoresis was performed using an apparatus (AE
-Z type), a polyacrylamide gel plate with a thickness of three heads, and a 0.3 M borate buffer (PH8,4). As a result, a single band was observed, indicating that the substance of the present invention was electrophoretically 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. 'il [! 1 220 300 400 Wavelength (nm)

Claims (1)

[Claims] (1) Stable in the state of amorphous white powder,
A substance having interferon-inducing activity and the following chemical properties. (a) Elemental analysis H: 6.54±0.3%, C: 41.9±0.3. N: 2.39±0.3%, P:0.28±0.03
(b) Molecular weight from about 100,000 to about 3,000,000 (mainly from about 1,000,000 to about 1,000,000) (c) Pixel point or decomposition point and melting point are unclear. Ashes at approximately 220°C. b) Ultraviolet absorption spectrum as shown in Figure 1 (measured in 0,1 N NaOH) (e)
Infrared pure absorption spectrum (Figure 2) (KBr method) (f) Solubility in solvents Dissolves in water, particularly well in alkaline aqueous solutions such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, etc. Slightly soluble in methanol, ethanol, pronoRol, butanol, acetone, chloroform, and ether. (g) Positive for color reaction, phosphorus reaction, phenol/(jtt& reaction, Folin reagent and Dittmer reaction. Negative for Ersin-Morgan reaction. Oxyproline, (15,61±0.4th) Aspartic acid (5,510,,1%) Threonine (10
,90±(),4%) Serine (9,64±0.4
%) Glutamic acid (3,96±0.4%) Proline (4
,27±0.4%) Glycine (9,09±0.4%) Alanine (8,24±0.4%) Valine (4,04±0.4) Inleucine (5,59± 0.4%) Leucine (2
, 56 ± 0.4 width) Phenylalanine (1,32 ± 0.4 width) Rishi 2
(2,18±0.4 da6) Arginine (1
,24±0.4%) Tyrosine (1,94±0.4%) Histidine (0,93±0.4%) Ammonia (12,98±0.4%) (Technicon
(Measured with Amino Acid Autoanalyzer IC-1 model) (1) Monosaccharide arabinose (9,38±〇, 6ti) galactose
(20,98±0.6%) Glucose (64,98±
(), 6%) Mannose (3,26±0.6 ratio) Xylose (1,40+: 0.6%) (measured with Technicon Sugar Auto Analyzer Model N-1) ■) Specific optical rotation [α] = +63″～+696 Average +66°(at
11□, Section 49, 0. IN NaOH)