KR810001442B1 - Process for preparing anticancer substance(p-2) - Google Patents

Abstract

Novel glycoprotein of mol. wt. 1.8x104 with a ratio of protein to sugar 8 to 2 is isolated from aloe. The substance reflecting nontoxic anticancer and antiinflammatory actions is effective for burns and skin diseases. Thus, aqueous agent from aloe is treated with salt to give precipitate from which alkali soluble glycoprotein(P-2) with anticancer action is extracted. It yields only a single band on SDS polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment and yields 2 discrete bands on electrophoresis with the treatment.

Description

Manufacturing method of anticancer substance (P-2)

FIG. 1 and FIG. 2 are diagrams of a gellographic elution pattern in one embodiment of the method of the present invention.

3 is a diagram of SDS-polyacrylamide, gel electrophoresis pattern for P-1 and P-2,

4 shows the molecular weight measurement results for P-2,

5a to 5d show the results of the reaction test between the substance obtained according to the present invention and serum,

6 and 8 show the results of the anticancer activity test on the material obtained according to the present invention.

The present invention relates to a method for preparing an anticancer substance.

The present inventors produce a precipitate from the liquid in the aloe (leaf, stem, root) of the liliaceae plant, separates the substance which is soluble in alkaline aqueous solution and insoluble in acidic aqueous solution, and they are erythrocyte agglutinating activity and lymphocyte glazing activity. With various physiological activities such as cancer, inflammation, burns, skin diseases and the like can be expected to be used to come to complete the present invention.

If necessary for the method of the present invention, the anticancer substance can be purified again by, for example, combining gel chromatography.

As a substance contained in the liquid substance of aloe vera, all the substances which precipitate generate | occur | produce by heat processing and are easy to melt | dissolve in alkaline aqueous solution are all substances obtained by the method of this invention.

Aloe used in the starting material of the present invention is a plant of the genus Aloe of the family Liliaceae. Examples include Aloe arborescens MILL, Aloe perryi BAKER, Aloe bactadensis MILLER, and Aloe ferox MILLER. Aloe can be harvested from above ground and underground areas such as leaves, stems, and roots.

The liquid contained in the aloe used in the present invention includes a liquid obtained by directly separating an aloe, for example, using a mixer, from which the precipitate is removed by centrifugation, if necessary, or deionized, such as ion exchange resin treatment. Suitable addition of pretreatment and purification means such as decolorization treatment is included.

The method of producing a precipitate by salt treatment is good according to the salting operation of the ordinary method, for example, it can be performed using ammonium lactate. In the case of ammonium lactate, it is necessary to attach it to the salting operation of 0 to 40% to obtain a good purity material, and to the salting operation of 0 to 70% when the quantity is important.

In the present invention, as a method of separating a substance that is easy to dissolve in an alkaline aqueous solution, for example, pH 7 to 11, preferably, a solution of pH 8 to 10 is dissolved in a buffer (for example, 0.05 carbonic acid-bicarbonate), followed by centrifugation. For example, the method of removing the precipitate which generate | occur | produces in 10,000 revolutions, about 20 to 30 minutes can be used.

In the present invention, a method of separating a substance which does not dissolve in an acidic aqueous solution is, for example, pH 3 to 7, preferably 4 to 5 of the aqueous solution (for example, using 1M acetic acid), the centrifugation operation described above. This can be done by separating the precipitates.

The substance obtained in the present invention has anticancer activity and can be used as an anticancer agent, for example, by injection or oral administration. Practically enough, the present invention is sufficient, but if it needs to be purified again, it may be applied to gel chromatography as described later.

When the substance obtained in the present invention has such anticancer activity, at least a certain amount of hemagglutination activity and preferred one have lymphocyte glazing activity as described above. In addition, one of the features of the present invention is the discovery of such a fact.

The present invention will be described in detail by the following examples.

Example 1

1 kg of leaves of Aloe arborescens MILL were crushed well and then separated by a mixer, for example. Centrifugation at 10,000 revolutions for 30 minutes allowed to precipitate and remove the coarse, coarse, large material and to separate the clear water above. Attached to the 0-40% hyaline fraction, the precipitate was aliquoted, 0.05M carbonic acid-bicarbonate buffer pH 9.5 was added, dialysis, desalted, then freeze dried and powdered.

In addition, the after-treatment obtained in this way (hereinafter referred to as AS-40) had erythrocyte aggregation activity. The pH was adjusted to 4.4 by dissolving AS-40 in carbonate-bicarbonate buffer (pH 9.5) and then adding acetic acid. The supernatant (called acid sup) and the precipitate (called acid precipitate (ppt)) were separated by centrifugation (10,000 revolutions 20 minutes). Higher hemagglutination activity was found in acid sup than acid precipitates. Lymphocyte glazing activity was only recognized for acidic precipitates.

After freeze-drying the acidic precipitate was dissolved in 0.05M phosphate buffer (pH 8.0), and the Palmer sash crosslinked dextrangel, Sephadex G-200 was filled in a column (1.5 × 25 cm) and flattened with the same buffer solution as The sample was placed and passed through this solution and eluted.

The same buffer was used, and the mixture was eluted at 4 ° C under a flow rate of 2 ml / h, and fractionated in 1.3 ml units.

The elution curve of the protein (A280 mμ) is shown in FIG. Erythrocyte aggregation activity is indicated by diagonal lines. VO represents the void mass.

Erythrocyte agglutination activity and lymphocyte glaze activity were found only in the P-2 fraction, which was an aliquot of 27.3 to 44.2 ml after the initiation of glaze, but the physiological activity was not found in the P-1 and P-3 fractions before and after the glaze.

The P-1 fraction was subjected to gel chromatography again with Sephadex G200, and purified again to obtain P-1. In Fig. 1, 10.4 ml to 18.2 ml of the fraction fraction P-1 fraction was concentrated, and the Sephadex G-200, previously flattened with 0.05 M phosphate buffer (pH 8.0), was filled in a column of 1.5 × 25 cm and concentrated on the column. Water was added and eluted with 0.05 M phosphate buffer (pH 8.0).

Elution was carried out in the same buffer as above, and 1.3 ml were aliquoted at a flow rate of 4 ° C. and 2 ml / h. No fractions of the hemagglutination activity were found in this fraction.

The P-2 fraction was again subjected to gel chromatography using Sephadex G-200 and purified again. 2.73-44.4 mL P-2 fractions aliquoted in FIG. 1 were placed in a Spectrmn Medical Industrie, Inc Calb (USA), placed in a tube, dehydrated with polyethylene glycol (1.2 mg), and 0.05 in advance. Sephadex G200 leveled with M phosphate buffer (pH 8.0) was charged to a 1.5 × 25 cm column and the concentrate was loaded and eluted with 0.05 M phosphate buffer.

1.3 ml were aliquoted at 4 degreeC and the flow rate of 2 ml / h. The results are shown in FIG. Erythrocyte aggregation activity is indicated by diagonal lines.

In this way, purified substance P-2 (sugar protein) can be separated.

The above experimental results are summarized and shown in Table 1.

TABLE 1 Summary of Tablets (1000 g of Alto Leaf)

^{* 1} : Minimum aggregation amount of human red blood cells

^{* 2} : Minimum amount required for 1 × 10 ⁶ lymphocytes to enclose 20,000 cpm [methyl-3H] pyridine

In addition, it was confirmed that the purified substance P-2 was a glycoprotein, indicating that the nin-hydrin reaction, xanthine protein reaction, Mauritsch woodranski reaction, andron reaction, ferring reaction, elson-molecular reaction, and peroxonic acid-schiff reaction were positive. . N-acetyl gluconamine was demonstrated in P-2 in amino acid analysis.

The quantitative analysis confirmed that 18.3% constituted sugar. Therefore, it was found that the ratio of P-2 protein to sugar was about 8 to 2 (weight) glycoprotein.

Elemental Analysis Values:

C 42.0-54.4%, H 5.7-7.0%

N 13.4-16.4%, O 20.2-24.6%

Qualitative absorption spectrum:

13 C-nm R spectrum (chemical shift):

Optical intensity

The linearity in wavelength 470nm was measured using the cell of length 1cm. The P-2 solution in 7.7 mg / ml of 0.05 M phosphate buffer (pH 8.0) in terms of bovine-rub albine showed negative photoluminescence at 25 ° C.

The material obtained by the process of the invention was separated from altoe. Purified glycoprotein (P-2) or a substance containing it, but P-2 has the following properties.

(1) Molecular weight 1.8 × 10 ⁴

(2) The ratio of protein to sugar is about 8 to 2 (weight).

(3) Polyadorinamide in the presence of sodium dodecyl sulfate (SDS). Give a single band by gel electrophoresis and give 2 bands when the S-S bond is cleaved by 2-mercaptoethanol.

(4) It has lymphocyte glazing action and lymphocyte fluid flow promoting action.

(5) It has red blood cell and tumor cell aggregation activity.

(6) Reacts with certain types of serum proteins and forms sedimentary glands on agarose plates.

(7) It has a complement third component activation.

Glycoprotein P-2 is SDS-polyacrylamide. It can be understood in the following experiment that the gel electrophoretic phase specificity is shown. SDS, polyacrylamide. Gel electrophoresis analysis was performed in accordance with Ueva and Osborn method (Weber. K., and Osborn. M (1969) J. Biol. Chem. 244, 4406-4412) using sodium-dodecylsulfate with a 10% gel. It was. Gels were protein stained with Coomassie brillant blue and bleached with a mixture of 10% acetic acid and 10% isopropanol.

SDS-Polyacrylamido. Gel electrophoresis assay: SDS-polyacrylamide and gel electrophoresis analysis were performed on P-1 and P-2. A 10% gel in 0.1 M phosphate buffer (pH 7.2) containing 0.1% SDS was used. Samples were heated at 100 ° C. for 5 min in 0.01 M phosphate buffer (pH 7.2) containing 1% SDS, 10 glycerol and 0.001% bromine phenol, using and without treatment with 2 mercapto ethanol.

The obtained pattern is shown in FIG. a indicates that P-1 was treated with or without 2-mercaptoethanol, and this gave the same result as treated or untreated. b indicates that P-2 was not treated with 2-mercapto ethanol, and C indicates that P-2 was treated with 2-mercapto ethanol.

As apparent from FIG. 3, P-1 and P-2 gave a single band without 2-mercapto ethanol treatment. In the case of 2-mercapto ethanol treatment, P-1 gave the same single band, whereas P-2 gave two separate bands. Any substance corresponding to these two bands is a peptide, and the molecular weight is different. The smaller one is α and the larger one is β.

The molecular weight of Arokcin A of the present invention is SDS-polyacrylamide. Obtained from the results of molecular weight measurement by gel electrophoresis.

Molecular weight test

Molecular weight measurement experiments were performed in gel electrophoresis with various concentrations ranging from 5% to 20% according to Segrest et al. (Jere P. Segrest and Richard L. Jackson, Methods in Enzymoloqy 28, 54-63 (1972)). As the molecular weight standard, the bobbinsi-rupalbmin ovalbumin group modolibsi 1-gen A and cytochrome C were used, and the results are shown in Fig. 4. In Fig. 4, as follows.

From the above results, the molecular weight was determined as follows.

1.8 × 10 ⁴

SDS-polyacrylamide of P-2 treated with 2-mercapto ethanol. From the results obtained in the gel electrophoresis assay, the molecular weights of α and β corresponding to the two bands were determined to be the same.

α: 7.5 × 10 ³ β: 1.05 × 10 ⁴

Structure of a sub unit

P-1 is 2-mercapto ethanol treated, untreated SDS-polyacrylamide. Gel electrophoresis gave the same single band. Therefore, P-1 does not have a subunit constituted by S-S bonds.

P-2 is SDS-polyacrylamide of 2-mercapto ethanol untreated. In gel electrophoresis, a single band having a molecular weight of 1.8 × 10 ⁴ was given. However, as a 2-mercapto ethanol treatment, small (α) and large sides at positions corresponding to the molecular weight of 7.5 × 10 ³ and 1.05 × 10 ⁴ , respectively Two bands corresponding to the peptide of (β) were given. This is a P-2 molecule having a molecular weight of 1.8 × 10 ⁴ composed of one α-subunit and one β-subunit. Again, these two subunits indicate that they constitute P-2 in SS combination.

Again, the octarona test using antiserum # 513 and # 533 showed that P-1 and P-2 had the same satellites. Therefore, P-1 is considered to be a β-subunit chain which exists independently of P-2 in plants.

The fact that glycoprotein, P-2 is glycoprotein, was confirmed by the positive ninhydrin reaction and neutral color reaction, for example, Morishupial reaction, and N-acetyl glucosamine was proved by glycoprotein P-2 in amino acid analysis. .

The results of amino acid analysis experiments shown below indicate that P-2 has a high content of acidic amino acids such as aspartic acid and glutamic acid as constituent amino acids.

Amino Acid Analysis:

The amino acid compositions of β and P-2 (αβ) are shown in Table 2. Tryptophan was not quantified. It is characterized by a high content of acidic amino acids such as aspartic acid glutamic acid and a low ratio of methionine and histatin.

TABLE 2

a: Calculate the molecular weight of P-2 as 1.8x10 ⁴

b: integer value

In SDS-polyacrylamide electrophoresis, the calculated molecular weight of β is 10,500. On the other hand, the analytical values obtained by amino acid analysis are shown in Table 2. If the number of methionine and histidine, the minimum constituent amino acids of β, is 1, the number of amino acids is an integer value in parentheses in Table 2, and the molecular weight of β calculated from this integer value and the molecular weight of each amino acid is 9.392, which corresponds to approximately 10,500. The integer value of this amino acid and the molecular weight of 10,500 are justified. Similarly, the molecular weight of P-1 calculated from the molecular weight of 18,000 obtained by SDS-polyacrylamide electrophoresis of P-2 and methionine and the molecular weight of P-2 calculated from the molecular weight is 17.038, and the value of the molecular weight is correct. I knew that.

In the amino acid composition thus obtained, two anticystines, such as α and β, are contained in one molecule. It can be understood that this binds by S-S binding and makes P-2 (αβ). In the analytical experiment, the sample was hydrolyzed at 110 ° C. for 20 hours using distilled Hcl in a vacuum sealed tube. The amino acid content of hydrolysis is determined by the method of Speckman et al. (Spackman, D.H., Stein, EW.H., and moore, S. 1958) Anal. Chem. 30, 1190-1206) and calculated by Hitachi Amino Acid Analyzer (KLA-3B). Methods of cystine and cysteine (Liu, TY, and lnglis AS, Methods in Enzymology ed, by Hirs, CHW, and Timaskeff, SN, Acad, Preess, New York, New York, 25, P. 55, 1972) It was analyzed as S-sulfocysteine by.

The total amount of neutral sugars was determined by the Olcinol lactic acid method (Winzler, R.J., method of Biochemical Analysis ed. By Glick, D., Interscience New York, New York, Vol, II, P. 279, 1956). The above experiment is not necessarily accurate for all amino acids, so it is not necessarily accurate, but it can be explained without contradicting other results, and it is considered that the value is within the margin of error by adding and subtracting (±) an integer value of about 10% of a given integer value. .

Octa-taroni-test:

Because of the identification of P-2, the O-kartroni-test using two antiserums, antiserum # 513 and antiserum # 533, showed that both antiserum # 513 and # 533 reacted with P-2. . Antiserum # 533 immunizes rabbits with AS-40 as an antigen to make serum. Antiserum # 513 cleaves the sedimentation line formed between AS-40 and normal rabbit and serum on agaro-Sgel plate and uses it as antigen. Made by immunity.

The obtained antiserum was made into anti-AS-40 (antiserum # 533) and anti-PL (antiserum # 513), respectively. Immunohistophoresis was performed by Hilschfeld, J. (1960) Sci. Tools. 7, 1852) and immunodiffusion was performed by the Ouchterlouy, 0, (1953) Acta Path.Microbiol Scand 32, 231. -240).

It was confirmed by the following test that P-2 has erythrocyte aggregation activity.

Erythrocyte agglutination activity test:

P-2 aggregated erythrocyte standards such as humans, sheep and rabbits and did not show A-B-0 blood group specificity in the erythrocyte aggregation activity test in the human system. Major experiments were carried out using human red blood cells. P-2 and P-1 increased cohesiveness approximately 5 times when red blood cells were treated with 0.1% trypsin. Table 1 shows the minimum amount of hemagglutination of the trypsin-treated erythrocytes.

Erythrocyte aggregation, titration was carried out using a 2% erythrocyte suspension of 2% physiological saline with micro titer-amplification (Cooke Laf. Prod., Virginia, USA). Samples (1.0 mg / ml) dissolved in 0.01 phosphate buffer containing 0.14 M Nacl were serially diluted. The degree of aggregation after 60 minutes of incubation was visually determined. Titration was measured using titration / mg protein.

Tumor cell aggregation activity of P-2 was compared with Concanapharin A (Con A) by the same method as hemagglutination-titration, and the results shown in the table were obtained.

[Tumor cell aggregation appropriate value]

* Treated with a mixture of 0.02% trypsin and 00.02% EDTA.

** treated with 0.02% EDTA

It was confirmed by the following experiment that P-2 has lymphocyte glazing activity.

Lymphocyte Glazing Activity:

The preparation of pure human lymphocytes is approximately by Kawaguchi et al. (Kawaguchi. T., Matsumoto, I., and Osawa, T. (1973) J. Biol. Chem. 249, 2786-2792). Heparin-coated human venous blood was centrifuged lightly, red blood cells were removed, leukocyte-rich plasma was picked up, and placed in an equal amount of glass on a glass disc.

9.1g picol (made by Palmasha)

25 ml of urographin solution (60% se-ring company)

125 ml twice distilled water

Centrifugation was performed at 400 g for 20 minutes.

The white inter phase layer between the serum layer and its urographine picol layer was removed with aspiration. The average rate of lymphocytes in the bilayer was more than 98% and the cell survival rate was 90% and 100 deaths by trypan [fire-eki school-Johnrest. Lymphocytes were then rinsed with 0.15 M NaCl-5 M phosphate buffer (pH-7) of 0.25% bovine vine syrup albamine and used for lymphocyte glazing testing.

The measurement of lymphocyte glazing activity was performed as follows.

Morphological testing was performed by calculating the percentage of transformed cells in the specimen stained by calculating approximately 10000 cells per test sample.

In the thymidine collection experiment, 1 × 10 ⁶ lymphocytes were added to each test tube, [3H] thymidine (2.0 μC, Radio Chemical Center-England) was added, and again, samples (P-1, P-2, etc.) were added. Time incubated. The cells were collected and washed three times with 4 ° C phosphate buffer (pH 7.2). Then, 1.5 ml of cooled 5% TCH (trichloroacetic acid) followed by 2 cooled methanol was added to the cell pellet, followed by a drop of 3% bovine vinecy-rubalmin (BSA) as a protein of the carrier. The precipitate was washed on Menbran (CF / C2.5 cm: manufactured by Wattman Co., Ltd.) in a “Manifold” Multiple sample collector (Millipore). The membrane was dried and placed in a scintillation vial. 0.3 ml of soluene (100. Packard) was added, followed by incubation at 37 ° C. or incubation at 55 ° C. for 2 hours, and 5 ml of scintillation solution (5 g PPO and 0.1 g POPOP / L toluene) was added to each vial. Was added.

The vial was held 4 ° overnight and then counted with a Backman-LS-200B liquid cinchration counter.

In the culture of human lymphocytes with P-2 and acid precipitates, large lymphocytes with morphologically modified forms were more frequently compared with the culture systems of cells cultured with other fractions P-1 and no addition.

When approximately 1000 cells were counted, about 70% of lymphocytes were observed to deform after 72 hours of culture. The rate of this modification was almost the same as PHA-W used as a positive control. In order to indicate the function of the glaze provided in the test in the collection experiment of [3H] thymidine, lymphocytes were treated with standard amounts of acidic ppt P-1, P-2 and the like.

As shown in Table 1, lymphocyte glazing activity was detected in both acidic ppt and P-2, but not in P-1.

It was confirmed in the following experiment that P-2 has lymphocyte fluidity promoting activity.

P-2 and (Fluorecene. Iso thiocyanate) were combined. (Cebra, JJ Goldstein, G (1965) J. lmmunol., 95, 230-245). FITC-P-2- was added to 2x10 <^7> cells, and it immersed at 37 degreeC for 40 minutes. After stopping the reaction with 10 mM NaN ₃ , the cells were washed well with PBS and cap formation was observed under fluorescence microscopy.

The results compared with Con A are shown in the following table.

Peripheral lymphocytes of normal people

**rat

It was confirmed in the following experiment that P-2 reacts with certain kinds of proteins of serum and has a function of forming a sedimentation line on agarose-plate.

Reaction test with serum proteins:

Reactivity of P-2 was measured by serum diffusion tests (octanei test) of various animals (humans, rabbits, goats, dogs, cats, horses, pigs, rats, fetuses, cattle, fetuses, snakes, and rats). Tested against.

All of these sera and egg yolk indicated positive octane-test response.

That is, P-2 reacted not only with serum of mammals but also with serum of egg yolk fish, benign species and reptiles. Of all the samples, only 1 million did not react. Again, more than one sedimentation line could be detected for all subserum yolks.

5 is a pattern diagram of the result of the reaction by immunodiffusion plate between P-2 and serum proteins of non-immunized animals.

Center hole normal rabbit serum: hole 1, P-1 (1 mg / mL), hole 2, P-2 (1 mg / mL), hole 3, S-1 (1 mg / mL), hole 4, S- 2 (1 mg / ml). Two sedimentary lines are observed, indicating sedimentation reactions in P-2 and human serum proteins. Only P-2 reacted with human serum protein and two sedimentary glands were detected.

Immunoelectrophoresis was performed using human scaffolds to determine what kind of scaffold proteins reacted with P-2. 5 is an immunoelectrophoretic pattern of α ₂ (macroglobulin) of human serum. 1.4% positive electrode and negative electrode on the left side of the veronal buffer solution (pH = 8.6, pH = 0.05). -Human serum (upper), rabbit anti-human α ₂ -macroglobulin (lower) and P-2 (center, arrow), according to immunoelectrophoresis pattern human serum protein for P-2 (arrow) It was found that the electrical mobility of the sedimentation line of was completely in agreement with that of α ₂ -macroglobulin.

The figure shows the holes 1 and 2 human serum, groove (groove) a rabbit anti-human serum: groove b, P-2 solution dissolved in 0.05 M phosphate buffer (300 μg / ml): groove C, anti-human α ₂ Macrogloburin serum (rabbit) or α ₂ -macroglobulin was one of the serum proteins that reacted with P-2 (see also FIG. 5B).

It was confirmed by the following test that P-2 has a complement third component (C ₃ component) activating action.

Serum complement component (complement third component C ₃ ) by P-2

Activation Test:

Changes in immunoelectrophoretic mobility were seen when the complement third component (C ₃ ) or C ₃ proactivator was activated. That is, sacred C ₃ (β ₁ C) B is in the region but when activated, C ₃ (β ₁ A) moves to the α region. In addition, the immunoelectrophoretic sedimentation line of the C ₃ proactivator is seen in the β region in agarose-plate electrophoresis and moves to the α region when activated.

5c and 5d show the immunoelectrophoretic mobility of the C ₃ component and C ₃ proactivator of P-2 incubated at 32 ° C. for one hour.

FIG. 5C is a pattern diagram of immunoelectrophoresis of C ₃ in human serum incubated with P-2. FIG.

In this experiment, the test method was carried out by the same method as described in FIG. 5B, with the right side being the negative electrode and the left positive electrode. The precipitation reaction was performed on rabbit anti-human C ₃ (β ₁ C / β ₁ A) serum. In a hole 1, 5 µl of 0.9 ml of human serum, 0.1 ml of physiological saline and a mixed solution, and in hole 2, a mixture of 0.9 ml of human serum and 0.1 ml of 300 µg / ml P-2 solution at 37 ° C. 5 microliters were added each in soaking for 1 hour. As shown in the figure, in the reaction with anti C ₃ (β ₁ C / β ₁ A) of human serum not treated with P-2, the sedimentation line was seen only in the β region. In the reaction with and C ₃ , the sedimentation line is seen not only in the β region but also in the α region.

5D is a pattern diagram of immunoelectrophoresis of proactivators in human serum incubated with P-2. The experiment was conducted in the same manner as described in FIG. Settling reactions were performed on rabbit anti-human C ₃ proactivator. Holes 1 and 2 each include the same sample as described for hole 1 and 2 in FIG. 5C.

For human serum not pretreated with P-2, the sedimentation line for anti-human C ₃ proactivators (rabbits) is shown in the β-region, and for human serum pretreated with P-2, the sedimentation line is for the β-region. It was also seen in the α-region as well.

The complement system is activated via either the classical pathway or the alternative pathway. And it is known that only mg ⁺⁺ is needed for the improved pathway and that both mg ⁺⁺ and Ca ⁺⁺ are essential for the classical pathway.

Activation by P-2 was determined by the following experiments, whether by classical or improved pathways.

0.1 ml of P-2 suspension (1 mg / ml) was added to 0.9 ml of fresh human serum. The mixture was reacted for one hour at 37 DEG C, cooled and centrifuged (5,000 revolutions, 30 minutes).

In order to test the complement activity, the supernatant was again used for immunoelectrophoresis analysis. The sedimentation line of the combined C ₃ proactivator in the presence of EDTA is shown in the r region. On the other hand, experiments performed in the presence of EDTA were not shown in the r region. The activation of complement components was also dependent on the temperature. Again, P-2 is C ₁ , C ₂ , and C ₄ by methods such as Takada, Y. Arimoro, Y. Mineta, H., and Takada, A. (1978) Immunology 34, 509-515. Has been confirmed not to activate. From these results, it was confirmed that the activation of the complement component by P-2 occurred via an improved route.

It was confirmed by the following experiment that the substance and its tablets obtained in the present invention have a strong anticancer activity.

Anticancer activity test:

For the anticancer activity test, about 100 g of weight-don rats were used.

Carcinogenicity of AH-130 cells after intraperitoneal transplantation of 5 × 10 ³ −2 × 10 ⁶ cells to mice at 30 to 40 days of age is shown in FIG. All animals died of cancer for 8 to 27 days.

Rabbits ascites 0.5 ml (containing 5 × 10 ³ cancer cells) 7 days after AH-130 cancer cell phagocytosis was transplanted intraperitoneally of mice. The effects of acidic precipitates administered prior to cancer cell transplantation on cancer growth were tested. The results are shown in FIG.

a: Rats were administered 2 mg (2 mg protein) / rat / day of acidic precipitate intraperitoneally for 7 hours daily, and then 5 × 10 ³ AH-130 cells were transplanted 7 days after the last administration of acid.

b: Control mice were transplanted with 5 × 10 ³ AH-130 cells without any aloe extract.

In FIG. 7, it was found that there is anticancer activity against acidic precipitates. As a result of further examination, it was confirmed that P-2 alone has anticancer activity between P-1 and P-2. Next, experiments were conducted to determine the dose of P-2 corresponding to the survival of cancer cell transplanted animals.

Various amounts of P-2 were administered daily for 7 days, 14-7 days prior to 5 × 10 ³ AH-130 cancer cell transplantation. The results were recorded for 80 days ie 54 days after death of all of the control animals and are indicated in FIG. In FIG. 8, it was found that the anticancer activity of the number of surviving animals without cancer, that is, P-2, was changed by the dose. Each% value represents the mean for 25-30 rats in three experiments.

In another experiment, 50 μg of P-2 daily for 14-7 days before cancer cell transplantation and 10 mice surviving 120 days after cancer cell transplantation were again transplanted with 1 × 10 ⁵ AH-130 cells. Completely withdrawn.

On the other hand, JCL-ICR mice (about 30g in weight) each of the five males were grouped and zalcoma 180 in plural form was subcutaneously implanted on the right shoulder of the mouse (2 × 10 ⁵ cells) and solid tumors. The experiment was performed. P-2 30r / one (dissolved in phosphate buffer pH 7.2 due to salt isotonic use) was administered daily for two weeks after coma 180 transplantation, and the inhibition ratio after 60 days was 80% and heat Two of them had completely degenerated.

Ten 30 g JCL-ICR mice were transplanted and maintained intraperitoneally of allogeneic ICR mice, and 2 × 10 ⁵ S-180 tumor cells 7 days after transplantation were implanted under the right shoulder volume of ICR mice. From day 7 post implantation, acidic precipitates (15 μg / one) and AS-40 (1 mg / one) were injected intraperitoneally daily for two weeks.

As a treatment, LCR mice were implanted with 2 × 10 ⁵ S-180 tumor cells on their shoulders, and the acid precipitates and AS-40 were not administered. Three animals were killed at 60 days after implantation in the right shoulder, and tumor weight was compared with the untreated group (three) and tumor inhibition rate was calculated.

Tumor inhibition rate Acid precipitation rate: 84%

AS-40: 72%

For the remaining seven animals, survival days were compared to the untreated group, with acidic precipitates and the AS-40-administered group surviving 20 to 30 days long. There was no complete degeneration.

P-2 and acidic precipitates obtained in the present invention were nontoxic to the extent used in anticancer experiments and were not found to be particularly sequelae after the cancer was completely retreated.

In addition, it is understood that the anticancer activity of P-2 is not a direct cellular effect on cancer cells but an immune effect by a reaction via a host cell.

Claims (1)

It is possible to separate precipitated glycoproteins from the liquid in aloe by salt treatment and to separate the glycoproteins dissolved in the alkaline aqueous solution or to generate precipitates by salt treatment in the liquid aqueous solution in aloe, and to separate the glycoproteins soluble in the alkaline aqueous solution and insoluble in the acidic aqueous solution. The manufacturing method of anticancer substance (P-2) characterized by the above-mentioned.

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