JPS625128B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new substance, alloctin A, which can be isolated from aloe vera. The present inventor isolated alloctin A from aloe vera, which has the following properties, and found that this new substance has various physiological activities and can be expected to be used as a medicine for treating cancer, inflammation, burns, skin diseases, etc. Heading: The present invention has been completed. (1) Molecular weight: 1.8×10 ⁴ (2) Structure: Glycoprotein with a protein to sugar ratio of approximately 8:2 (weight) (3) Single band in polyacrylamide gel electrophoresis in the presence of sodeime dodecyl sulfate When the S--S bond is cleaved with 2-mercaptoethanol, two bands are obtained. (4) Lymphocyte mitogenic activity
and has the effect of promoting lymphocyte membrane fluidity. (5) Has red blood cell and tumor cell agglutination activity. (6) It has the effect of reacting with certain serum proteins to form sedimentation lines on an agarose plate. (7) Has a complement third component activation effect. Aloe has been known as a medicinal plant since ancient times, and the Japanese Pharmacopoeia includes aloe powder and aloe extract. The main components of these substances are low-molecular substances, and the alloctin A of the present invention is a glycoprotein, which is a high-molecular substance different from these known low-molecular substances. On the other hand, the plant component concanavalin A
(ConcanavalinA) and other known lectins
The alloctin A of the present invention differs from any of the above in terms of molecular weight, sodium dodecyl sulfate (hereinafter referred to as SDS)-polyacrylamide gel electrophoretic properties, and the physiological activity of the alloctin A of the present invention. Furthermore, although it is known that aloe contains components that have anticancer activity, no examples have been isolated of them in pure form, and glycoproteins have never been isolated. The alloctin A of the present invention is applied to plants belonging to the genus Aloe of the Liliaceae family, such as Aloe aloe.
arborescens MILL), Socotra Aloe (Aloe
perryi BAKER), Aloe
barbadensis MILLER), Cape Aloe (Aloe
forox MILLER) (herein referred to as Aloe), and can be isolated from both above-ground and underground parts such as leaves, stems, and roots. An example of the separation method will be described below. After thoroughly crushing 1 kg of Aloe arborescens MiLL leaves, the young leaves were separated using gauze. Contaminants and coarse substances were precipitated and removed by centrifugation at 10,000 rpm for 30 minutes, and the supernatant was separated. 0-
The precipitate was separated by 40% ammonium sulfate fractionation, added with 0.05M carbonate-bicarbonate buffer (PH9.5), dialyzed and desalted, and then concentrated by freeze-drying. The dialyzed product obtained in this way (hereinafter referred to as
AS-40) had hemagglutination activity. Dissolve AS-40 in carbonate-bicarbonate buffer (PH
9.5) and then by adding 1M acetic acid, the pH is
Adjusted to 4.4. Centrifugation (10,000 rpm, 20 minutes) separates the supernatant (called acidic Sup) and precipitate (acidic
Ppt) was separated. More acidic than acidic Ppt
Higher hemagglutination activity was found in Sup.
Lymphocyte blastogenic activity was observed only in Ppt. After freeze-drying the acidic Ppt, add it to 0.05M phosphate buffer (PH
8.0), and coat Pharmacia's cross-linked dextran gel, Sephadex G-200, on a column (1.5
x 25 cm), equilibrated with the same buffer solution, and then passed through this solution for elution. Elution was performed using the same buffer solution at 4°C and a flow rate of 2 ml/h, and fractions were collected in 1.3 ml units. The protein elution curve (A280mμ) is shown in Figure 1a. Hemagglutination activity is indicated by diagonal lines. V ₀ indicates void volume. Hemagglutination activity and lymphocyte blastogenesis activity were found only in the P-2 fraction, which is a fraction of 27.3 ml to 44.2 ml after the start of outflow, and in the P-1 and P-3 fractions before and after that. No such physiological activity was found. P-
Two fractions are fractions containing alloctin A of the present invention. For the P-1 fraction, repeat Sephadex G-
200 gel chromatography was performed to further purify the product to obtain P-1. 10.4ml~18.2 in Figure 1a
Concentrate the P-1 fraction and fill a 1.5 x 25 cm column with Sephadex G-200 equilibrated with 0.05 M phosphate buffer (PH 8.0), and place the concentrate on top of it. , 0.05M phosphate buffer (PH
8.0). Elute with the same buffer as above,
Aliquots of 1.3 ml were collected at 4°C at a flow rate of 2 ml/h. The results are shown in Figure 1b. V ₀ indicates void volume. No hemagglutinating activity was found in this fraction. The P-2 fraction was also subjected to Sephadex G-
200 gel chromatography was performed for further purification. The 27.3 to 44.2 ml P-2 fraction collected in Figure 1a was transferred to a Spectrum membrane tube (Spectrum Medical Industries, Inc-Calf).
Sephadex G concentrated (1.2 mg) in USA) and equilibrated in advance with 0.05 M phosphate buffer (PH8.0).
-200 was packed into a 1.5 x 25 cm column, the concentrate was placed thereon, and the column was eluted with 0.05M phosphate buffer. 4
℃, and aliquots of 1.3 ml were collected at a flow rate of 2 ml/h. The results are shown in Figure 1c. Hemagglutination activity is indicated by diagonal lines. The substance P-2 purified here is alloctin A of the present invention. On the other hand, acidic Sup was lyophilized and then dissolved in 4 ml of 0.05M phosphate buffer (PH8.0).
Sephadex G-100 (crosslinked dextran gel manufactured by Pharmacia) equilibrated with phosphate buffer (PH8.0) was packed into a 2.0 x 30 cm column, the above solution was poured onto it, and eluted with 0.05M phosphate buffer. Elution was performed using the same buffer solution at 4°C and a flow rate of 5 ml/h, and 2.5 ml aliquots were collected. The results are shown in Figure 2. Hemagglutination activity is indicated by diagonal lines. This aggregation activity was found only in the S-1 fraction, and this physiological activity was not found in the S-2 fraction. The S-1 fraction was purified by gel chromatography again in the same manner to obtain the active substance S-1 (alloctin B). From the above experiments, alloctin A of the present invention can be isolated by repeating gel chromatography using Sephadex G-200, using the physiological activity as an indicator, if necessary. In addition, alloctin A can also be conveniently obtained by affinity column chromatography using sugar or protein. The above experimental results are summarized and shown in Table 1.

[Table] It can be understood from the following experiment that alloctin A of the present invention exhibits specificity in SDS-polyacrylamide gel electrophoresis. In addition, SDS-polyacrylamide gel electrophoresis analysis is performed using sodium dodecyl using a 10% gel.
Weber and Osborn method using sulfate (Weber, K., and Osborn.M (1969) J.Biol.
Chem. 244 , 4406-4412). The gel is Coomassie brilliant blue (Coomassie
Perform protein staining with 10% acetic acid and 10%
% isopropanol mixture. SDS-polyacrylamide gel electrophoresis analysis experiment: SDS-polyacrylamide gel electrophoresis analysis was performed on P-1, P-2, and S-1.
10% in 0.1M phosphate buffer (PH7.2) containing 0.1% SDS
Gel was used. The samples used were those treated with 2-mercaptoethanol and those not treated, and 1%
5 in 0.01M phosphate buffer (PH7.2) containing SDS, 10% glycerol and 0.001% bromophenol.
Heated to 100°C for minutes. The obtained pattern is shown in FIG. a is P-1
is shown for those treated with 2-mercaptoethanol and those not treated, which gave the same results with and without treatment. b is P-2 2-
Not treated with mercaptoethanol, c is P-
2 treated with 2-mercaptoethanol, d
"e" indicates S-1 not treated with 2-mercaptoethanol, and "e" indicates S-1 treated with 2-mercaptoethanol. As is clear from FIG. 3, P-1, P-2 and S-1 gave a single band without treatment with 2-mercaptoethanol. On the other hand, when treated with 2-mercaptoethanol, P-1 gave the same single band, whereas P-2 gave two separate bands. The substances corresponding to these two bands are both peptides and have different molecular weights, with the smaller one being α and the larger one being β. 2- for S-1
The mercaptoethanol treated product gave a single band like the untreated one, but a smaller band (γ) than the untreated one. One of the means for specifying the alloctin A of the present invention is to use SDS-polyacrylamide and
Gel analysis was performed and gave a single band, but 2-
Check whether mercaptoethanol treatment gives two bands. SDS as above
A polyacrylamide gel electrophoresis analysis experiment is performed on a certain substance, and the obtained results are compared with the results of an SDS-electrophoresis analysis experiment of alloctin A under the same conditions as this experiment to check whether they are the same or not. If they are the same, the substance can be determined to be alloctin A. Of course, if you conduct an experiment on a certain substance under exactly the same conditions as the SDS-polyacrylamide gel electrophoresis analysis experiment and compare it with the result of P-2 in Figure 3, you can determine that the substance is alloctin A. can. The molecular weight of alloctin A of the present invention was determined from the results of a molecular weight measurement experiment using SDS-polyacrylamide gel electrophoresis analysis. Molecular weight measurement experiment: Sample not treated with 2-mercaptoethanol (P
From the SDS-polyacrylamide gel electrophoresis analysis of -1, P-2 and S-1), a standard curve was obtained by plotting the relationship between the logarithm of the molecular weight and the mobility for a standard protein of known molecular weight, as shown in Figure 4a. decide. The SDS-polyacrylamide gel electrophoresis analysis experiment was conducted in the same manner as described in FIG. 3 above. The relationship between the mobility and the logarithm of the molecular weight was plotted using the following standard protein preparation (molecular weight is shown in parentheses) and is shown in FIG. 4a. Cytrome C (12500), Chymotrypsinogen A (25000), Ovalbumin (45000), Bovine Serum Albumin (67000). Next, we use the method of Segrest et al. (Jere P.Segrest and
Richard L. JacKson Methods in Enzymology
28 , 54-63, 1972), molecular weight measurement experiments were carried out using polyacrylamide gel electrophoresis analysis using SDS using gels with various concentrations. The results are shown in Figure 4b. In FIG. 4b, it is as follows. ●-●: P-2 ○-○: α ▲-▲: P-1 (β) ■-■: S-1 □-□: γ Based on the above results, the molecular weight was determined as follows. D-1: 1.05×10 ⁴ P-2: 1.8×10 ⁴ (Alloctin A) S-1: 2.4×10 ⁴ (Alloctin B) P-2 and S treated with 2-mercaptoethanol
The results obtained in the SDS-polyacrylamide gel electrophoresis analysis experiment for P-1 are the same as those for P-2.
The molecular weights of α, β corresponding to two bands, and γ corresponding to one band for S-1 were similarly determined. α: 7.5×10 ³ β: 1.05×10 ⁴ γ: 1.2× ¹⁰ Structure of 4 subunits: P-1 shows the same single band in SDS-polyacrylamide gel electrophoresis analysis regardless of whether it is treated with 2-mercaptoethanol or not. gave. Therefore, P
-1 has no subunits formed by SS bonds. In SDS-polyacrylamide gel electrophoresis analysis of P-2 untreated with 2-mercaptoethanol, it gives a single band with a molecular weight of 1.8 x 10 ⁴ , while the 2-mercaptoethanol treated product gives a single band with a molecular weight of 7.5. Two bands corresponding to the smaller (α) and larger (β) peptides were given at positions corresponding to ×10 ³ and 1.05 × 10 ⁴ . this is
A P-2 (alloctin A) molecule with a molecular weight of 1.8×10 ⁴ has one α-subunit and one β-subunit.
This shows that it is composed of subunits, and that these two subunits constitute alloctin A through SS bonds. Furthermore, the results of the Ouchterlony test using antisera #513 and #533 (see Figures 5a and 5b), P-
It can be seen that 1 and P-2 have the same antigenicity.
Therefore, P-1 is considered to be a β-subunit chain that exists independently from P-2 in plants. In SDS-polyacrylamide gel electrophoresis analysis without 2-mercaptoethanol treatment, S-1
shows a single band with a molecular weight of approximately 2.4×10 ⁴ , whereas 2-mercaptoethanol-treated S-1
Electrophoretic analysis of the sample gave a single band at a position corresponding to a molecular weight of approximately 1.2×10 ⁴ . This shows that the S-1 molecule, which has a molecular weight of approximately 2.4×10 ^{4 ,} is composed of S--S bonds from two γ-subunits, each of which has a molecular weight of approximately 1.2×10 ⁴ . It was confirmed that alloctin A and alloctin B are glycoproteins because the ninhydrin reaction was positive, and the neutral sugar color reaction, for example, the Moritschille-Pearl reaction, was positive, and in amino acid analysis, both alloctin A and alloctin B were confirmed to be glycoproteins. N-
Acetylglucosamine was proven. From the quantitative analysis results, P-1, P-2 and S-1
It was confirmed that sugars constituted 24.9%, 18.3%, and 50% or more, respectively. Therefore, the alloctin A of the present invention has a protein to sugar ratio of approximately 8:2 (weight).
It can be seen that it is a glycoprotein. The following amino acid analysis experiment results show that alloctin A contains large amounts of acidic amino acids such as aspartic acid and glutamic acid as constituent amino acids. Amino acid analysis: P-1 (β), P-2 (αβ) and S-1 (γ
Table 2 shows the amino acid composition of ₂ ). Tryptophan was not quantified. It is characterized by a high content of acidic amino acids such as aspartic acid and glutamic acid, and a low proportion of methionine and histidine.

[Table] The molecular weight of β calculated from SDS-polyacrylamide electrophoresis is 10,500. On the other hand, the analytical values obtained by amino acid analysis are as shown in Table 2. Now, if the number of methionine and histidine, which are the minimum constituent amino acids of β, is 1, the number of other amino acids is the 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 9392, which roughly matches 10500, and it can be said that the integer value of this amino acid and the molecular weight of 10500 are correct. Similarly, the molecular weight of P-2 calculated by SDS-polyacrylamide electrophoresis is 18,000, and the molecular weight of P-2 calculated from the number of each amino acid obtained with methion as 1 and its molecular weight is 17,038, which is a good match. do. In the amino acid composition thus obtained, both α and β have two half-cystines in one molecule. It can be understood that this is combined through an S-S bond to form P-2 (αβ). In the above analysis experiment, the sample was hydrolyzed using distilled HCI in a vacuum-sealed tube at 110°C for 20 hours. The amino acid content of the hydrolyzate was determined by the method of Spackman et al. (Spackman, DH, Stein, WH,
and moore, S. (1958) Anal.Chem. 30 , 1190−
1206) and Hitachi Aminosan Analyzer (KLA-
3B) was analyzed and calculated. Cysteine and cystine were obtained by the method of Liu et al. (Liu, TY, and Inglis AS,
“Methods in Enzymologry” ed, by Hirs, C.
HW, and TimaSkeff, SN, Acad, Press;
New York, New York, 25, P. 55, 1972). The total amount of neutral sugars was determined using the orcinol-sulfuric acid method (Winzler, RJ, “Method of Biochemical
Analysis”ed. by Glick, D., Interscience,
New York, New York, Vol., P.279, 1956
(year). The above experiment is not necessarily accurate as it did not analyze all amino acids, but it can be explained consistently with other results, and this value is within the error range of plus or minus an integer value of about 10% of the given integer value. It is thought that there is. Ouchterlony test: Ouchterlony test was performed using two antisera, antiserum #513 and antiserum #533, for the identification of P-1, P-2 and S-1. Figure 5a shows the pattern of Ouchterlony immunodiffusion gel using antiserum #513, with a hole in the center:
Antiserum #513, 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). The P-1 and P-2 sedimentation lines are both fused into each other. No sedimentation line was detected in either S-1 or S-2. That is, antiserum #513 reacts with P-1 and P-2 but does not react with S-1. Figure 5b shows the pattern of Ouchterlony immunodiffusion gel using antiserum #533, with a hole in the center:
Antiserum #533, 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) The sedimentation lines of P-1 and P-2 are completely fused, but the sedimentation line of S-1 is spurred out from that of P-2. No sedimentation line was detected for S-2.
That is, antiserum #533 is P-1, P-2 and S-
1 reacted. These results show that P-1 and P-2 have common antigenicity, which is different from that of S-1. One antiserum was made in rabbits against AS-40, and the other antiserum was made in rabbits against the sedimentation line formed between AS-40 and normal rabbit serum on an agarose gel plate. Rabbits were administered approximately 1 mg of AS-40 emulsion every week for two months and an emulsion that cut out the sediment lines after washing them with saline. Both emulsions are completely Freund
Contains Adju Band (manufactured by Kyokuto Pharmaceutical Co., Ltd.). The obtained antisera were designated as anti-AS-40 (antiserum #533) and anti-PL (antiserum #513), respectively. The immunoelectrophoresis method is the method of Hirschfeld (Hirschfeld, J. (1960) Sci.Tools, 7 ,
1825), and the immunodiffusion method is the Ouchterlony method (Ouchterlony, O. (1953) Acta Path.MiCrobiol
Scand 32 , 231-240). It was confirmed by the following test that alloctin A of the present invention has hemagglutinating activity. Hemagglutination activity test: Red blood cell specimens from humans, sheep, rabbits, etc. are tested in P-2 and S-
1 agglutinated and showed no A-BO-O blood group specificity in the hemagglutination activity test in human systems. The main experiments were conducted using human red blood cells.
When red blood cells were treated with 0.1% trypsin, P-2 and P-1 increased their agglutination by approximately five times.
The minimum amount of red blood cell aggregation for the trypsin-treated human red blood cells is shown in Table 1. S-1 showed strong hemagglutination activity, and P-2 showed approximately one-fourth the hemagglutination activity of S-1. Subunit β of P-1 and P-2 did not show hemagglutination activity. Hemagglutination titration is performed using microtiter equipment (Cooke Laf.Prod.,
Virginia, USA) using a 2% red blood cell suspension in 2% saline. 0.14MNaCl
Sample dissolved in 0.01M phosphate buffer containing (1.0
mg/ml) were diluted stepwise. After 60 minutes of incubation, the degree of aggregation increased to a level that was visible to the naked eye. Titer values were measured as timer/mg protein. It was confirmed through the following experiment that alloctin A of the present invention has tumor cell aggregation activity. If the agglutination when a certain concentration of P-2 is added to a certain number (1 x ¹⁰⁵ ) of human red blood cells treated with trypsin is defined as (+), the agglutination value of each cell determined visually and under a microscope, P -2 S-1 Human red blood cells 〓 〓 Mol+4B 〓 〓 〓 P3HR-1 〓 〓 HNG-100 〓 〓 〓 NC-37 〓 〓 Ehritzch ascites liver cancer 〓 〓 AH-130 〃 〓 〓 Alloctin A of the present invention has a lymphocyte inducing effect This was confirmed in the following experiment. Lymphocyte blastogenesis: Pure human lymphocytes are almost always prepared by the method of Kawaguchi et al. (Kawaguchi, T., Matsumoto, I., and
Osawa, T. (1973) J.Biol.Chem. 249 , 2786−
2792). Human venous blood was placed in a heparinized syringe, and the heparinized blood was transferred to a glass cylinder, allowing the red blood cells to sink to the bottom. The leukocyte-rich plasma was removed and placed over an equal volume of the following solution in a glass centrifuge tube. 9.1 g of Ficoll (manufactured by Pharmacia), 25 ml of urographine solution (60%, manufactured by Schering), 125 ml of double-distilled water, centrifuged for 20 minutes with 400 g of water. The white interface layer between the serum layer and its urographine-ficoll layer was removed by aspiration. The average percentage of lymphocytes in this layer is over 98%, and the viability is 90% and 100% by trypan blue exclusion test.
It was found that it was between %. Lymphocytes then
0.25% Bovine Serum Albumin
The cells were washed with 0.15M NaCl-5M phosphate buffer (PH=7) and used for a lymphocyte regeneration test. The lymphocyte blastogenesis activity was measured as follows. Morphological tests were performed by calculating the percentage of transformed cells from Giemsa-stained specimens by counting approximately 1000 cells per test specimen. In the [ ³ H] thymidine uptake experiment, 1 × 10 ⁶ lymphocytes were placed in each test tube, [ ³ H] thymidine (2.0 μC, Radiochemical Center, UK) was added, and samples (P-1, P- 2, S-1, etc.) and incubated for 60 hours. The cells were then collected and washed three times with isotonic phosphate buffer (PH7.2) at 4°C. Then, 1.5 ml of chilled 5% TCA (trichloroacetic acid) followed by 2 ml of chilled methanol was added to the cell pellet, followed by 3% bovine serum albumin (BSA) as the carrier protein.
Added a drop of. Precipitate “Manifold”Multiple
It was washed on a membrane (CF/C 2.5 cm; Watmann) in a sample collector (Millipore). After drying, the membrane was placed in a scintillation vial. After incubation at 37°C with addition of 0.3 ml of solbilizer (Soluene TM 100, Packard) or 2 h incubation at 55°C, 5 ml of scintillation solution (5 g
PPO and 0.1 g POPOP/toluene) were added to each vial. Vials were kept at 4°C overnight and then counted on a Beckman LS-200B liquid scintillation counter. In culture of human lymphocytes with P-2 and acidic Ppt, large lymphocytes with morphologically transformed features were found in other fractions, P-1,
Compared to the culture system of S-1 and cells cultured without additives, the number of cells was increased. When approximately 1000 cells were counted, approximately 70% of the lymphocytes were observed to be transformed after 72 hours of culture. This rate of transformation was almost the same as PHA-W, which was used as a positive control. In order to demonstrate the function of the rejuvenating substance under test in a [ ³ H]thymidine incorporation experiment, lymphocytes were incubated with acidic Ppt and Ppt.
-1, P-2, S-1, S-2, etc., in different amounts. As can be seen from Table 1, lymphocyte priming activity was detected in both acidic Ppt and P-2, but P-
1, S-1 and S-2. It was confirmed through the following experiment that alloctin A of the present invention has an effect of promoting lymphocyte membrane fluidity. P-2 and FITC (Fluorecene.iso thiocyanate)
Next, in the cells at 37℃ for 5 minutes, 10 minutes, and 30 minutes.
Perform Incubat. The patch-cap Formation was observed and photographed under a fluorescence microscope.

[Table] It was confirmed through the following experiment that alloctin A of the present invention has the effect of reacting with certain serum proteins to form a sedimentation line on an agarose plate. Reactivity test with serum proteins: The reactivity of the crude extract of aloe was tested in various animals (human, human,
Tested on serum from rabbits, goats, dogs, cats, horses, pigs, rats, fetal rats, cows, fetal cows, snakes, and stupa. Of these sera, 10 sera and egg yolk showed positive Ouchterlony test reactions. Aloe extract P-2 reacted not only with mammalian serum but also with egg yolk, fish, amphibian, and reptile serum. Of all the samples, only egg white did not react. Furthermore, two or more sedimentation lines were detected for almost all serum and egg yolks. Regarding P-1, P-2, S-1 and S-2,
The same experiment as above was performed. FIG. 5c is a pattern diagram of the reaction results of P-2 and serum proteins of non-immunized animals by immunodiffusion plate. 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 sedimentation lines are observed indicating a sedimentation reaction between P-2 and human serum proteins. That is, only P-2 reacted with human serum proteins, and two sedimentation lines were detected. Immunoelectrophoresis was performed using human serum to determine which types of serum proteins react with P-2. FIG. 5d is an immunoelectrophoretic pattern diagram of α ₂ -macroglobulin in human serum. It was performed using 1.4% agarose in veronal buffer (PH=8.6, μ=0.05). The right side is the anode and the left side is the cathode. The sedimentation lines are rabbit anti-human serum (top), rabbit anti-human _α2 -macroglobulin (bottom), and P-2.
(middle, arrow) formed against. The immunoelectrophoretic pattern shows that the electrical mobility of the human serum protein precipitation line for P-2 (arrow) completely matches that of α ₂ -macroglobulin. Diagram from top to bottom: Holes 1 and 2, human serum; groove a, rabbit anti-human serum; groove b, P-2 solution (300 μg/ml) in 0.05M phosphate buffer; groove c, rabbit anti-human serum; α ₂ -macroglobulin. That is, α ₂ -macroglobulin was one of the serum proteins that reacted with P-2. (See Figure 5d). It was confirmed by the following test that alloctin A of the present invention has an activating effect on the third component of complement ( _C3 component). Serum complement component (third complement component,
C3) Activation test: When the C3 component or C3 proactivator is activated, changes in immunoelectrophoretic mobility are observed.
The C3 component (β1A/β1C) moves to the β region. C3
The proactivator immunoprecipitation line is seen in the β region in agarose plate electrophoresis, and when activated, it migrates to the γ region. Figures 5e and 5f show the immunoelectrophoretic mobilities of the C3 component and C3 proactivator of human serum incubated with P-2 for 1 hour at 32°C. FIG. 5e is a pattern diagram of immunoelectrophoresis of C3 in human serum incubated with P-2. In this experiment, the test method was the same as that described in FIG. 5d except that the right side was the cathode and the left side was the anode. The precipitation reaction was performed using rabbit anti-human C3 (β
1C/β1A) serum. Each hole described contains 1 ml of human serum and 1 ml of 300 μg/ml P-2 solution.
(hole 1), and 0.85% saline (hole 2). FIG. 5f is an immunoelectrophoretic pattern diagram of C3 proactivator in human serum incubated with P-2. Experiments were performed in a similar manner as described in Figure 5e. Precipitation reactions were performed against rabbit anti-human C3 proactivator. Holes 1 and 2 contain the same samples as described in holes 1 and 2 in FIG. 5e, respectively. In the case of human serum pretreated with P-2, use anti-human C3.
A sedimentation line for the proactivator (rabbit) was seen in the γ-region. In control serum not pretreated with P-2, sedimentation lines were seen only in the β-region. The sedimentation line for anti-human C3 (rabbit) is α
- seen in the area. A sedimentation line was seen in the β-region for control serum complement components. Complement system is classical pathway or alternative
activated via either pathway. And only Mg ⁺⁺ is required for the alternative pathway, while both Mg ⁺⁺ and Ca ⁺⁺ are required for the classical
It is known that it is essential for the pathway. The following experiment determined whether P-2 is activated via the classical pathway or alternative pathway regarding the complement system. P-2 suspension (1 mg/
ml) 0.1 ml was added. After reacting the mixture at 37℃ for 1 hour, centrifugation (5000 rpm, 30 minutes) under cooling.
I performed the operation. Immunoelectrophoretic analysis was performed using the supernatant to test complement activity. A sedimentation line for the C3 proactivator converted in the presence of EGTA was seen in the γ region, whereas no sedimentation line was seen in the γ region for the C3 proactivator converted in the presence of EDTA. Activation of complement components was also dependent on temperature. These results suggest that activation of complement components by P-2 is an alternative.
It can be seen that this occurs via a pathway. It was confirmed through actual experiments that alloctin A of the present invention has strong anticancer activity. Anticancer activity test: For the anticancer activity test, Dongli rats weighing about 100 g were used. Figure 6 shows the carcinogenicity of AH-130 cells after intraperitoneal transplantation of 5 x ¹⁰³ to 2 x ¹⁰⁶ cells into rats 30 to 40 days old.
It was shown to. All animals died of cancer between 8 and 27 days. AH-130 cancer cells 7 days after transplantation, 0.5ml
Ascites (containing 5 x ¹⁰³ cancer cells) was intraperitoneally transplanted into rats. The effects of aloe extracts administered at different times before and after cancer cell transplantation on cancer growth were tested. The results are shown in FIG. a: After intraperitoneal administration of 2 mg (2 mg protein)/rat/day of rat acidic Ppt for 7 hours daily, 5x
10 ³ AH-130 cells were transplanted 7 days after the last administration of acidic Ppt. b: control. Rats were transplanted with 5×10 ³ AH-130 cells without administering any aloe extract. From FIG. 7, it can be seen that acidic Ppt has anticancer activity. Further examination results P-1 and P-2
Among them, only P-2 was confirmed to have anticancer activity. Next, P corresponding to the survival time of cancer cell-implanted animals
An experiment was conducted to determine the dosage of -2. Various amounts of P-2 were administered daily for 7 days, 14 to 7 days before transplantation of 5 x ¹⁰³ AH-130 cancer cells. 80 results
Data were recorded for 54 days after the death of all control animals due to cancer, and are shown in Figure 8. From FIG. 8, the number of surviving cancer-free animals, that is, the anticancer activity of P-2 changes depending on the dose. Each percentage value represents the average value for 25-30 rats from three experiments. Furthermore, in an experiment in which 50 μg of P-2 was administered daily for 7 days, and 1 x 10 ⁵ AH-130 cells were again transplanted to 10 rats that survived 120 days after cancer cell transplantation, the cancer completely regressed in all rats. . On the other hand, a group of 5 male and female JCL-1CR mice (weighing approximately 30 g) was subcutaneously implanted (2 x 10 ⁵ cells) into the right shoulder of the mice with Sarcoma 180 maintained in the ascites form to form a solid tumor. I conducted an experiment as Alloctin daily after Sarcoma 180 transplant
When A30γ/1 animal (dissolved in phosphate buffer, pH 7.2 made isotonic with saline) was administered for 2 weeks, the inhibition ratio (tumor inhibition rate) after 60 days was
The tumor regression rate was 80%, and the tumor had completely regressed in 2 out of 10 animals. Since the aloe extract of the present invention, alloctin A, is non-toxic, no special treatment is required after the cancer is completely regressed. From the above experimental results, it is understood that the anticancer activity of alloctin A in particular is not a direct cytotoxic effect on cancer cells, but an immune effect due to a reaction mediated by host cells.

[Brief explanation of the drawing]

1a-c and FIG. 2 are gel chromatography elution pattern diagrams in an example of a method for separating alloctin A, and FIG.
Figures 4a and 4b are SDS-polyacrylamide gel electrophoresis pattern diagrams, and Figures 4a and 4b show the molecular weight measurement results for alloctin A, and Figures 5a to c
5 is a pattern diagram of an Ouchterlony immunodiffusion gel for alloctin A, and FIGS. 5d-f are immunoelectrophoresis patterns for alloctin A. Figures 6 to 8 show the results of anticancer activity tests regarding alloctin A.

Claims (1)

[Scope of Claims] Alloctin A which has the following properties and can be isolated from aloe vera. (1) Molecular weight: 1.8×10 ⁴ (2) Structure: Glycoprotein with a protein to sugar ratio of approximately 8:2 (weight) One band is given, and when the S--S bond is cleaved with 2-mercaptoethanol, two bands are given. (4) It has a lymphocyte blastogenic effect and a lymphocyte membrane fluidity promoting effect. (5) Has red blood cell and tumor cell agglutination activity. (6) It has the effect of reacting with certain serum proteins to form sedimentation lines on an agarose plate. (7) Has a complement third component activation effect.