JPS58128322A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:A carcinostatic agent that contains glycoprotein separated from green microalgae as an active ingredient, thus acting on only tumor cells specifically and causing no effect on normal cells. CONSTITUTION:The objective glycoprotein is separated from green algae such as chlorella, scenedesmus or spirulina, having molecular weight of 121,000, isoelectric point of 8.6pH, glycoside/protein ratio, about 1:1, protein helix content, about 19%. Said glycoprotein shows antitumor activity both in vitro and in vivo, acts only on tumor cells, but does not on normal cells. Said carcinostatic agent is separated by extracting raw cake of chlorella which is cultivated using carbon dioxide, acetic acid, glucose or the like as a carbon source, treating the extract with a semipermeable membrane and fractionating the macromolecular fraction impermeable to the membrane by a variety of column chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anticancer agent extracted from green microalgae such as chlorella, cenedesmus, and spirulina.

All conventional glycoprotein-based antitumor agents have host-mediated (immunostimulating) functions. Therefore, although it exhibits antitumor properties in vivo, it does not exhibit direct cell killing activity in vitro.

Therefore, in the process of researching green microalgae such as chlorella, the present inventor fully investigated that certain components contained in these algae have anticancer effects, and based on this, as a result of intensive research on the components. -4(, a glycoprotein component with a specific molecular weight, isoelectric point, glycoprotein ratio, and protein helix content exhibits antitumor activity both in vivo and in vitro, and acts specifically only on tumor cells. It was discovered that the anticancer agent of the present invention exerts an extremely excellent anticancer effect without affecting normal cells.However, although the anticancer agent of the present invention has a direct effect on tumor cells, it has no effect on normal cells. It is a revolutionary anti-tumor drug that solves the non-selective requirement, which is the biggest problem with today's chemotherapy drugs.

That is, the present invention relates to a glycoprotein isolated from green microalgae such as chlorella, cenedesmus, and spirulina, which has a molecular weight of 12i, ooo, an isoelectric point (pH) of 8.6, and a glycoprotein relative to 1.
:1 protein has the property of containing approximately 19 helices.

Next, an example of a method for separating the anticancer agent of the present invention from chlorella will be described below.

A raw chlorella cake cultured using carbon dioxide, acetic acid, glucose, etc. as a carbonate source, or a chlorella algae (powder) obtained by spray-drying or freeze-drying this raw cake is prepared as a raw material. Disperse 500.9 chlorella algae (5 t in case of chlorella raw cake) in 5 t of water,
After hot water extraction for 20-30 minutes at 3.000-1
The mixture was centrifuged at 0.00 Orpm for 20 to 30 minutes, and the supernatant was used as a chlorella hot water extract. The solvent used for this extraction is not limited to hot water, and water containing dilute acid or dilute alkali may be used without any problem.

Through this operation, about 75 g (in terms of powder) of a hot water extract was obtained from Chlorella algae 5'00II. hot water extract, 5
Concentrate under reduced pressure at 0°C, and add distilled water to bring the total volume to 1 t.

This is passed through a semi-permeable membrane, and the polymer fraction that does not pass through the semi-permeable membrane (
The mixture was fractionated into a fraction (hereinafter referred to as A fraction) and a low molecular weight fraction (hereinafter referred to as 8 fractions) that passed through a semipermeable membrane. The yields of A and H were 45Ii (in terms of powder) and 30F (n in terms of powder), respectively. Furthermore, the obtained fraction A was concentrated under reduced pressure to 500 m/cm, then added to a DEAE-cellulose column, and multistage elution was performed while changing the buffer solution stepwise.

That is, both the fractions eluted with M150 carbonate buffer are AI and M
The fractions eluted with the M2B5 carbonate buffer containing /10 sodium chloride were named A2, and the fraction eluted with the M150 carbonate buffer containing IM food was named A3.

The yield of A I + A2 + AS is 13 each.
．． 0g (powder equivalent), 5.51! (powder equivalent), 10.
9 (in terms of powder).

A, the fraction was concentrated under reduced pressure at 50°C, desalted with a dialysis membrane, passed through a 5ephadex G-150 column, and M/
Fractionation was performed using 15 phosphate buffer (pH 7, 17), and the obtained fractions were dialyzed and further freeze-dried. The yield was 140 m9 (approximately 0.025 h of chlorella raw material).

The properties of the substance obtained by the above extraction method are listed below.

[1] Molecular weight ■ Molecule t for electrophoresis (calculated value) 128,000 @ Analytical ultracentrifuge (Hitachi (■ Ke; Absorption scanning recorder 282-00
60 type) The molecular weight was 121,000 from the above (■) and @.

[11] Purity ■ The purity of the product was confirmed by filtration method @ sedimentation pattern using an analytical ultracentrifuge and OSDS polyacrylamide gel electrophoresis.

[iii 1. Isoelectric point pH 8.6 [1■] Constituent sugar The obtained substance was hydrolyzed in an acid-resistant environment at 100°C for 3 hours,
It was separated by thin phase, filter paper chromatography and cellulose column chromatography, and the components and amounts were analyzed by the orcinol-sulfuric acid method. The results are shown in the first section below.
Shown in the table.

5- Table 1 (V) Constituent Amino Acid Residues Amino acid components and food were analyzed using an automatic amino acid analyzer (manufactured by Hitachi ■; KLA-3B type). The results are shown in the second section below.
Shown in the table.

6- Table 2 [v1] Composition ratio of sugar and protein 490 ttj? Anli: 510 fill/T
nQ (approximately 1:1) [vii] Infrared spectroscopic analysis Infrared spectroscopic analyzer (manufactured by Hitachi ■; Mode 1260 (
Ofja, XI RrkB”cm '1 K J:
Polycrystalline WegaX solid.

As a result, α at 1640, 1550 and 1260 nm
-Amides of helices (1), (II) and (Ill)
Absorption attributed to the band 1660.
A random coil structure was observed at 1530 and 1240 nm.

In addition, at 870 nm, the eq of C1-position of α-glucan
The bending vibration of uatorral H, the respiratory vibration of the pyranose term at 880 nm, and the β vibration at 906 nm.
-The bending vibration of the axial H at the C1-position of glucan is
At 910 and 920 nm, there are asymmetric bending vibrations related to C-0-C of these pyranose terms, and 999 n
Characteristic absorption of β-glucan was observed in M.

(viii') Argon laser Raman spectroscopic analysis Argon laser Raman spectroscopic analyzer (JEOL (made in vermilion; JR
8-Vl -UV ) (γH2αD2°ゝ"cm-"
), the structure in solution was investigated using ILX.

As a result, an α-helical amide (
1), 1309 nm (E2 symmetric species), 1294 nm
(El symmetric species) and 1275 nm (A symmetric species), respectively.
- Helix amide (■) band was observed, and the latter was confirmed by disappearing by deuterium conversion with heavy water.

Further, there was an absorption at 1663 nm that belonged to the amide (1) band with a random coil structure, and an absorption at 1257 nm that belonged to the amide (lit) band with a random coil structure that disappeared by deuterium exchange.

Additionally, one absorption at 556 nm or 528 nm and 37
9 nm indicates the overlapping of the symmetrical species A or E, respectively, and the bending mode of the amide (IV) band, and 350 nm indicates the respiratory vibration of the relatively large molecular weight ring of the α-helical structure due to hydrophilic amino acids. Indicated.

The substance obtained from [vii] and cvii'+] above maintains an α-helical structure whether in crystal form or in solution form.

[IX] Circular dichroism spectrum (CD curve) optical rotation dispersion spectrometer (Hitachi (4!Kami; UV-5 type) CCD water bath liquid pH 7
, 0) was investigated for the content of α-heli and camphorax.

As a result, the CD curve shows a cross-9-over point at 200 nm, and [θ]-950 at 209 nm.
At 0,222 nm, a double minimum was shown at [θ]-7590. From this, the α-helix content is 18.9%
(approximately 19 inches).

The hydrophilic amino acid group, which is more important than the constituent amino acid residues, accounts for approximately 35% of the amino acid residues, and if sugar components are also taken into account, it accounts for approximately 20% of the molecule.

If the α-helix structure is mainly composed of tetrahydric amino acids, the α-helices are thought to be localized at one to several positions in the molecular chain.

The anticancer effect of the substance obtained above was confirmed by the experiment shown below.

Experimental Example 1 (In vitro test) (1-1) Measurement by suspension culture method Mouse lymphocytic leukemia culture established strain (L-121Q7'v
, $ ) to 5 × 10 cells/m
The cells were planted in a medium so that p. In addition, this medium is 1
RPMI 1640 (GIBCO) with 0% FC8
to 100 μl of steref') mycin and 100 un
The pH was adjusted to 7.0 by adding it/lJ of penicillin.

10- Next, the above medium was incubated at 37° C. for 72 hours according to the 5 suspension culture method to create each group to which a fixed amount of this substance was added and a control group to which no addition was made. When we compared and judged the number of cells after culture in these groups, we found that the non-additive control group showed a 100% proliferation rate, while the IC-50 (50%) growth rate in the group to which this substance was added.
The growth inhibitory concentration) was approximately 2 μg.

(1-2) Measurement using soft agar culture method When the growth inhibitory concentration of L-1210/V/C of this substance was determined according to the standard method (5 of agar cloning analysis method), IC-5.0 was determined. It was 2μgfinl.

Experimental Example 2 [: Invivo test] (2-1) Test using Delcoma 180 106 Delcoma 180 were transplanted into the peritoneum of 5-week-old DD mice (30 mice per group), and once every day from 24 hours after transplantation. The substance was intraperitoneally administered to mice at a rate of 101101n for 5 consecutive days, and the ratio of survival days of the mice (ratio to mice to which the substance was not administered) was determined according to a conventional method. As a result, the life extension ratio (T/C') was 180% (2-2) P-3
In a study conducted by 88, 106 mice of murine lymphocytic leukemia (P-388) were implanted intraperitoneally into 5-week-old CDF1 mice (30 mice per group), and this substance was administered once daily for 5 consecutive days starting 24 hours after implantation. 110m9A was administered intraperitoneally to mice, and the ratio of survival days of the mice (ratio to mice to which this substance was not administered) was determined according to a conventional method.

As a result, the life extension rate (T/C) was 160chi.

(2-3) Test using L-1210 10 mouse lymphocytic leukemia L-1210 were tested in 1 group with 30
Transplanted intraperitoneally into 5 week 4 CDF1 mice, transplantation 2
Starting from 4 hours later, 10171≠g of this substance was intraperitoneally administered to mice once a day for 5 consecutive days, and the ratio of survival days of the mice was determined according to a conventional method. As a result, the life extension rate (T/C
) was 145%.

Experimental Example 3 [Immunological tests, etc.] When p 33 vAg of this substance was administered to rabbits through their ear veins, and 331 vAg was further administered 10 days later, no anaphylage-like findings were observed.

In addition, when C5m9A9 was administered to rabbits through the ear vein a total of 6 times at intervals of one day, and even when administered under the above conditions, a sedimentation line that was distinguishable from normal rabbits was observed in immunoelectrophoresis. was not recognized.

From these facts, it can be seen that this substance has low antigenicity, that is, the antibody titer is also low. Furthermore, no hemolytic coagulation reaction was observed with this substance.

=13- Director General of the Japan Patent Office Shima 1) Haruki Tono 1, Indication of the case Patent Application No. 116/1982 2, Name system of invention Cancer drug 3, Relationship with the amended person case Patent application Chlorella Industries Co., Ltd. 4, Agent 5, Voluntary amendment 6, Specification subject to amendment 7, Contents of amendment (1) In box 4, line 19 of the specification, the phrase ``■ Molecular weight as determined by electrophoresis'' was replaced with ``■ ■Molecular weight determined by electrophoresis.''

(2) In the 7th line of page 5 of the specification, the phrase ``pure product'' has been corrected to ``single product.''

(3) Table 1 on page 6 of the specification is corrected as follows.

Record Chapter 1 Table (4) Table 2 on page 7 of the specification box is corrected as follows.

Record Table 2

Claims (1)

[Claims] A glycoprotein isolated from green microalgae such as chlorella, cenedesmus, and spirulina, with a molecular weight of 121,000.
, an isoelectric point (pH) of 8.6, a glycoprotein ratio of 1:1, and a protein helix content of approximately 19%.