JPS59164725A - Antitumor-active substance atf 1011, its preparation and use - Google Patents

Abstract

NEW MATERIAL:An antitumor-active substance ATF1011 separated from aloe and having the following characteristics. Molecular weight, 64,000+ or -3,000 (gel- filtration, concentration:5mg/ml); isoelectric point, pI 4.0-5.5; solubility, soluble in water and insoluble in methanol, ethanol and acetone; qualitative analysis of protein, positive to copper-Folin reaction and biuret reaction; tumor cell agglutination activity, none; activity for forming lymphocyte blast, none. USE:An antitumor agent. PREPARATION:The crude protein separated from aloe is purified by ion exchange chromatogaphy. In the above process, the elution is carried out under the gradient of the salt concentration from 0 to 0.4M. Using a solution obtained by adding sodium choride to 0.05M phosphate buffer solution, and the fraction having an electrical conductivity of 15-23mOMEGA/cm is collected to obtain the objective active substance.

Description

[Detailed description of the invention]

The present invention provides a novel antitumor active substance A that can be isolated from aloe vera.
TF 1011, its manufacturing method and its uses. Aloe has been widely used as a folk medicine since ancient times, and the Japanese Pharmacopoeia also lists aloe powder or aloe extract. The medicinal ingredients in aloe that have so far been identified are mainly low-molecular substances, and there are only a few cases in which high-molecular substances have been clarified as medicinal ingredients. Recent research has found that alloctin A, a glycoprotein isolated by Meiki et al., has antitumor activity (Japanese Patent Application Laid-Open No. 54
See Publication No. 113'l13. ). The present inventors tried to separate the amount of mouth water using one aloe and one protein. Results 1. IN, 1-to the glycoprotein alloctin (
, r different proteins? ■New anti-pulmonary tumor active substance ATFIOI
+ was sent to the prefecture, and the present invention was not completed)) 1, -. That is, the present invention provides an antitumor active substance ATF+011 which has the following 1.1 properties and can be isolated from aloe vera, a method for producing the same, and an anticancer agent containing this as an active ingredient in a content of 1. (1) Molecule I (gel filtration method, sample concentration 5 before 5)/
me): 64000±3000 (2) Isoelectric point: p + 4.0 to 5.5 (3) Solubility P1: Soluble in water. Insoluble in methanol, ethanol, and acetone. (4) Protein qualitative reaction Copper-furin reaction, positive biuret reaction: Positive (5) Experimental tumor cell aggregation activity Yes ((1)
No light hemagglutination activity 7) Lymphocyte blastogenesis activity None 1- Alloctin is a glycoprotein and has light hemagglutination activity and 10 lymphocyte blastogenesis activity, whereas the present invention material

It is clear that the substance of the present invention is different from alloctin A because it has the protein 1 and P which do not have these effects. The uninvented substance 'r' has been shown to have significant antitumor activity in the syngeneic lung, and is also expected to suppress cancer metastasis, making it useful in the prevention of cancer in medicine.5. , its use as a therapeutic agent is f! The substance of the present invention has the above-mentioned characteristics, but also has the following anti-resistance properties: (1) Appearance: white to grayish white powder (2)
Elemental analysis Carbon 42-53 Hydrogen 6-8 Nitrogen 14-18% (3) Ultraviolet absorption spectrum 2B, characteristic absorption peak of protein at 0 ntnl (4) Content of constituent amino acids Amino acid content (IrI'f%) ) Aspartic acid 12-20 threonine
3-6 Seri 7 4-7 Glutamic acid 9-14 P1 Choline 2
~71 Glycine 5-9 Alanine 3-5 Insutin 1-2 Valine 4-7 Methionine 2-4 Inleucine 2-50 Isine
9～】4 chironn
4-7 Phenylalanine 2-4 Lysine 1-3 Histidine 1-4 Arginine 5-9 Note that tryptophan was not quantified. (5) Infrared absorption spectrum + 650cm 'F near mid I vibration (C-0 stretching)
The characteristic absorption band due to
Characteristic absorption band due to T bending and (-N stretching) Yes (6) The content of neutral sugar (in terms of mannose) does not exceed 1 part of the present invention riH, crude protein obtained from aloe is subjected to ion exchange chromatography can be separated by 0.05 M in eluate and 17! When adding sodium chloride to the J acid buffer and performing elution with a concentration gradient from 0 to 0.4M, the electrical conductivity was 15 mυ/α to 23 mV.
It has been found that it is sufficient to separate the two parts such that the ratio is /cm. A person skilled in the art would be able to check the presence or absence of antitumor activity from the ion-exchange chromatography fraction 1 using the elution conditions described in -1- as a standard, even if the conditions are variously changed.
．． Therefore, the substance of the present invention can be easily separated. Therefore, in order to separate the fraction 1 and the corresponding fractions in the elution step 1, a buffer other than phosphate buffer or a salt other than common salt was used.11. Tari, /I
:11 ([Since the substance of the present invention can be purified for 1 minute by slightly changing the gradient and electrical conductivity, this method is also applicable to the production of the present invention as long as the above-mentioned fractions are separated by ion-exchange chromatography.) Method 1 is included.Alternatively, (9t1) the substance of the present invention is obtained by converting the above-mentioned phase protein into gel chroma I.
・It can also be fractionated by graphic fractionation. It was found that when the test sample was loaded at a concentration of 3 mg/mlV or higher, it was sufficient to fractionate both the sample and the sample having a molecular weight of 64,000±3,000. Is it necessary for a person skilled in the art? From this, the presence or absence of antitumor activity can be checked, and both fractions having the indicated molecular weight can be easily separated by gel chromatography fractionation. Of course, by combining the ion exchange chromatography method and this gel chromatography method, the substance of the present invention can be produced more easily or in a more purified form. The substance of the present invention is extracted and separated from aloe by fractionating the crude protein separated by a conventional method using ion exchange chromatography and gel chromatography, and separating both components that have antitumor activity. J-L. Of course, here is V! ! The substances listed in No. 11 have the characteristics of the above-mentioned substances of the present invention. To separate crude protein from aloe vera, for example, it is preferable to crush the aloe vera finely, remove solid matter, and apply the ammonium sulfate fraction to the filtrate. Ion exchange chromatography, for example DF, AE-
One piece of cellulose column chromatography is sufficient. It is sufficient to check both fractions showing antitumor activity using the fractionation filter of ion exchange chromatography and separate the active fraction, but it is more convenient to add salt to 0.05M phosphate buffer and When performing elution with a concentration gradient from O to 0.4 M, the electrical conductivity is 15 mU/cm.
It is sufficient to separate both parts of the range of -23mtJ//cnl. Next, gel chromatography may be performed using, for example, cross-linked dextrin gel column chromatograph f-1. Check the gel chromatography fractions 1, 2, and 2 fractions showing antitumor activity.
'me and L te f', + charge, molecule rf4H64o
o o l 3n n. It is sufficient to shift the fraction within the range by N [. The aloe may be any plant belonging to the genus Aloe of the Liliaceae family, such as Aloe aloe.
arhorescens MILL), Softla Aloe (Aloe perryi BAKER), Curacao Aloe (Aloe barbadensis MI)
LLER), and its aboveground parts such as leaves, trunks, roots, etc.
It can be separated from any underground part. The substance of the present invention is non-toxic or has low toxicity and can be used as an anticancer agent, that is, for the prevention and treatment of cancer in humans and other warm-blooded animals. When used as an anticancer agent, various formulations are possible depending on the route of administration. For example, oral preparations include capsules, tablets, granules, syrups, etc.;
It can be administered locally in one formulation, such as an IJ agent and an ointment. Among the formulations listed in L, it is particularly effective to use as an injection. When used as an injection, for example, 40
A physiological saline solution of HH77Mp may be used. In addition, from the results of animal experiments, the amount of tj- cast for the first eye is usually 0.1 to 2 f+ 00. It is expected to be around mg. The present invention will be explained in detail below with reference to Examples and Test Examples. . Example Aloe arborescens M
After thoroughly crushing 2 tons of leaves of ILL), first, filter impurities and coarse substances using a centrifugal dehydrator, and then continuously centrifuge the filtrate using a high-speed centrifuge to remove fine solid substances.
The supernatant was separated. Add supernatant liquid + 500 tF ammonium sulfate to saturation of 40 tons, stir overnight at 5C, and centrifuge for #1. From this, 3.9 kg (wet) of precipitate was collected. 0.0% of this ammonium sulfate precipitate. Q5M! I phosphate buffer (p
H7, 5) + 2tr, put 1 tube of Union Carbide -10- cellulose, and use the same buffer as an external solution.
Dialysis was performed for days. After adjusting the pH of the dialysis solution to 7.5t, the insoluble portion was removed using centrifugal bone # (12000G, 20 minutes).1. Ta. And the obtained jji'r + e z
DEAE-cellulose (Whatman If) equilibrated with 0.05 M phosphate buffer (pH 7,5)
E52-1) Column (25tynΦX 50 ('r
nll) f ko negative 6: f shita. The column was thoroughly washed with the same buffer for 1 time to remove non-adsorbed substances, and then eluted with the same phosphate buffer containing 0.4M sodium chloride. Twenty-two hours of this eluate was concentrated with Amicon Follow Fiber 1 (1r'5) and 1g of excess A:(). into a cellulose tube, dialyzed for 30 minutes using deionized water as an external solution, and then
[1 DEΔIQ-cellulose carano, black 1 thornfish (10 cmφ x 83 tynH) tc was investigated. Konokara 1. After washing with the same phosphate buffer, F7 was eluted using a linear concentration gradient method of sodium chloride (from 0 to 0.4 M, with a gentle gradient from 0.15 M in the middle).
Figure 1). Antitumor activity fraction], i.e., common salt: 0 at lH
．． From 1 M (1,1! IM, expressed as electrical conductivity, 1
Fraction E ranging from 5 m1j/nn to 23 mV/m was observed (antitumor activity test example 1). This fraction 1 was concentrated using the ultrafiltration method described above. . (11% L2 18% as Rt I 50 me / E - / 2. CHI Z -) after dialysis for 30 hours using deionized water as an external solution 3 times 1-1 r
lEΔE-cellulose column chroma!・Graphy (1)
0CmΦx! 12 ctnH) As with this column I, , 2+'i11n, after washing with phosphate buffer for 1 to 10 minutes, it was eluted using a linear concentration gradient method (0 to 0.3M) of 1 to 7 minutes (Figure 2). ). Fraction 1 was carried out according to the above-mentioned criteria, and the antitumor activity test was carried out using ultrafiltration, filtration, and further concentration using a cellulose tube. 31]. Powder obtained by freeze-drying the treated solution (3.43)
Dissolve 714 fl mg of 2 in 0.05 M phosphate buffer (pH 8,0) i-1. Loaded onto a column (44 mm Φ x 75 α IT'l) and eluted with the same buffer (Figure 3).The antitumor active fraction was concentrated by ultrafiltration, and then transferred to a cellulose tube. After dialysis for 30 hours using deionized water as an external solution, lyophilization was performed.
1011.260 mg of the anti-lung tumor active substance ATF of the present invention was prepared. T! 7. The substance has the following properties. (1) Appearance White to grayish white powder (2+
Dissolved M'('L Soluble in water. Dissolved in one volume of organic solvent such as methanol, ethanol, acetone, etc.) Elemental bone 1i Carbon Hydrogen Nitrogen 47.65% 7.05 15.57tfA (4)
Protein and 1. Properties (aqueous solution) ■ Copper-folin reaction positive 1 no 1 (2) Biuret reaction positive ■ 280 old in ultraviolet absorption spectrum in? 3. First, place the sample in a vacuum tube at 6N)! Ili point hydrochloric acid total hydrochloric acid, -
13-++Or), and was hydrolyzed for 24 hours. The amino acid content of the hydrolyzate was determined by the method of Spackman et al.
D, T(, + '5tein, W, H, l
andmoore, S. (1958) Anal
, Chem, 30 +11! ln, ~1206)
So, Amino Acid Analyzer ([1st 835) (1j)
This is the analytical calculation. The results are shown in Table 1. It is characterized by a high content of 1'l7 amino acids such as aspartic acid and glutamic acid. Table 1 -14- (ol Sugar Determination 1i (0) The total Ii1 of () monosaccharides in neutral sugars can be determined by the ortho-now sulfuric acid method (l?
, ,I, Winzler+ ” Method
or Hiochemical Analysis+”
ed, by I), GIick+ In1crsc
iencc+NewYork+Vat, L
p27 9 + + 05F1 Year reference 1.
), k and the phenol-sulfuric acid method (,1,E, F(o
r1ge+ B, T, Hofreiter+ ”M
ethodin Carbohydrate Chem
iSl,ry+”erl, by R,L. Wll!1iller+ M, L, Wolfrom
, Academic Press+Vol1. I,
See p388 + 1962. )? I asked for it from here. As a result of the analysis, for both methods, the mannose conversion was 0.
．． Values of 4-0.7% were obtained. □■ Aminosugars The total amount of aminosugars can be determined by a reaction combining N-acetylation and Morgan-Elson reaction (G, A. LeVVy+ A, McAllan+Biochem
, J, +73 ++27. See (+959). ) t. As a result of the analysis, a value of 0.3% tJ1·゛ was obtained in terms of glucosamine. (7) Molecule: 111 Molecular part & 1 Calculated from gel filtration method. The measurement was performed by equilibrating with 0.05M phosphate buffer (pHs, o) in advance. Cross-linked dextran gel (Pharmacia 1 Sephadex G-+00J) column (15 cm ψ x 90>
) l) is used. The sample was dissolved in the same 5-phosphate buffer to a concentration of 6 mg/ml and loaded with 1-1111-1. Elution was performed with the same phosphate buffer. Molecule 1 or 4th
As shown in Figure t, the relationship between the logarithm of the molecular weight and the elution amount for a standard with a known molecular weight (1 < white) was determined by calculating the standard curve. As a result of the analysis, the molecule m, A value of 3000 was obtained. (8) Spectrum I・Ru ■ Ultraviolet absorption spectrum The ultraviolet absorption spectrum measured by dissolving in water is shown in Figure 5. ■ Infrared absorption spectrum K l (r Measured by tablet method) The infrared absorption spectra are not shown in Figure 6. (9) Isometric points 5 Electrical points Fj S Polyacrylamide gel isoelectric focusing method 1 The measurements were carried out using a thin layer gel isoelectric focusing device. This was carried out using Multifor (LK 8).Also, polyacrylamide gel brake 1.
6.5, and the isoelectric point marker was Pharmacia pl.
Calibration kit 2.5-6.5 was used. As a result, values of p14.3 to 5.2 were obtained. (Ill Tumor cell aggregation activity Tumor cell aggregation activity was investigated using a microtiter method.@The cancer cells were mouse tumor cells EL1, M
M46 (3×10′ cells/ml) was used. The sample is
At EL4, from a concentration of 1 o vtg/wrl,
In addition, in the case of MM46, starting from a concentration of 1 mg/me, successive 2-fold dilutions were performed for measurement. As shown in Table 2, aggregation activity was observed. -17- Table 2 Tumor cell agglutination activity '011 Hemagglutination activity Hemagglutination activity was investigated by microtiter method in the same manner as tumor cell agglutination activity. The red blood cells used were human type 0 red blood cells (2 x 10 cells/go) purchased from Flora, USA.
The samples were sequentially diluted 2-fold from the initial concentration 1OIIg/: , , */ and measured. As a result, no hemagglutinating activity was observed in the product of the present invention. Oa Lymphocyte blastogenic activity Lymphocyte blastogenic activity is widely used ("H)
- Investigated by thymidine incorporation experiment. That is, first, spleen cells of LB/C mice were added to B in culture medium +1-M E
Mt suspension (10X106-18-eel Is/me) 200 lll and sample 81
Mix 10 lll of '1 solution on a microplate,
Incubate for 72 hours. Next, add 501tl ("TI)-thymidine (
After 8 hours, the lymphocytes were filtered and purified. The release ability of the incorporated (8H:l-thymidine) was measured using a liquid perisotylation counter (1) to determine the presence or absence of activity. No bulging activity was observed. (1,1 Antitumor 1 Reno, Active Invention Substance ATF + 011 has Strong Antitumor Restoration 1'l ~ 1 ~ 1 ~ 1 ~ 1 ~ 1 ~ 1 ~ 1 ~ 1 ~ 1 ~ 1 Antitumor 1 Reno, 1 + 'l using mice: 1 tumor iM I'l test 1 Test Example 1 Ion exchange chroma I of ?fl 2 +'1-it r+
- In the graph (Fig. 1) there are two fractions, fraction 1 and fraction ■? After getting stuck, the following method was used to obtain anti-tumor activity 1.
I didn't save 1. MM I 02 tumor cells (3 50 m9/kg was injected into the lung tumor twice on the 16th day and on the 6th week day. The test results are shown in Table 3. Tumor growth 1tll +l: rate (@ stands for Calculated using the formula. Antitumor activity was observed.Test Example 2 The results of the antitumor activity test of the present substance ATFIOI+ are shown in Table 41.The test method was the same as Test Example 1.
The antitumor effect was investigated by changing the dosage and treatment. Furthermore, after tumor cell transplantation, 14F10t and F11 were subcutaneously transplanted to the opposite site to examine tumor establishment. Table 4 As is clear from Table 4, the substance of the present invention has a strong anti-1
The flt bL activity is 1~. Moreover, due to the administration of the substance of the present invention, the re-implanted tumor was found to be on the gold side, and no colonization was observed. Also, the substance of the present invention? As a result of conducting a mouse acute toxicity test, the LD, o value was 272 mg/lcg or more.
1-''- (intravenous injection). This figure shows the determination results of molecules mI'1TII related to the substance 1 that was removed from the cellulose column.
8000), (b) double egg white albumin (45000),
0 gimotrizosinogen A (25000), O-mu c (
+2500). Fig. 5 is the ultraviolet absorption spectrum of the substance obtained in 1r Zheng diagram +: t, , Ime', - example, and Fig. 6 is its infrared absorption spectrum. Co., Ltd. -22- outσO ', 0TX) I + da (-m-) picture / Ω Beigun 1 Haruka Tomomi / ) @ @ tσO <--=ro / Ωtuai*iyo Y Tomomi () 01! , [Q Song - Re-Uta Kui Sen (Ling) Procedural Amendment 1, Indication of Case 1981 Special Application No. 38460 2, Title of Invention Anti-tumor active bamboo material △TF1011, its 'N manufacturing method and its Use 3: Relationship with the case of the person making the amendment Patent applicant A 1 location 571t Kyobashi-chome, Chuo-ku, Tokyo 8Q4
, Date of amendment order Voluntary issue 7, Contents of amendment As shown in the attached sheet, Amendment content 1, Page 19 of the specification, "10μt" in the first line is followed by r
Add (0,01 mg/mfJ)-l. , the following statement is added between lines 9 and 10 on page 19 of the specification. [In addition, in the above-mentioned measurement of lymphocyte immaturity, concanavalin (CONA) was used as a control substance in 1),
Con's activity was measured in Nokui and Fuchimaro, which exhibits pannolymphocyte rejuvenating activity. Therefore, substances exhibiting activity under the above measurement conditions are not included in the substances of the present invention. On the other hand, substances that do not exhibit activity under the above measurement conditions are included in the substances of the present invention as long as their physical properties match those of the substances of the present invention. "that's all

Claims (1)

[Claims] 1. An antitumor active substance ATF 1011° that has the following properties and can be isolated from aloe (1) Molecular weight (gel filtration method; sample concentration 5erg/
yml) 64000±3000 (2) Isoelectric point pl 4.0-5.5 (31 Solubility Soluble in water. Methanol, ethanol and acetone t
This is insoluble. . (4) Protein qualitative reaction Copper-Folin reaction Positive Biuret reaction Positive (11) Tumor cell aggregation activity Yes (6)
Human hemagglutinating activity No 7) Lymphocyte blastogenic activity No 2. When separating and purifying the crude protein isolated from aloe using ion exchange chromatography, 0.05 M! Acid buffer? This is a solution containing common salt from 0 to 0.4. When performing elution with a concentration gradient up to M, the electrical conductivity is 15 mVα ~ 23
A method for producing an anti-tumor active substance ATFIOII, which comprises separating both components such that my/l:m. 3. An anticancer agent containing the antitumor active substance ATFIOII as an active ingredient.