JPS6330286B2 - - Google Patents

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 a host-mediated (immunostimulatory effect) function. For this reason,
Although it exhibits antitumor activity in vivo, it does not exhibit direct cell killing activity in vitro. For this reason, in the process of researching green microalgae such as chlorella, the present inventor discovered that certain components contained in the algae have anticancer effects.
Based on this, we conducted intensive research on its components, and found that glycoprotein components with specific molecular weights, isoelectric points, glycoprotein ratios, and protein helical contents were found to be in vivo.
It was discovered that it exhibits antitumor activity both in vitro and has extremely excellent anticancer activity, acting specifically on tumor cells and having no effect on normal cells. In other words, although the anticancer agent of the present invention shows a direct effect on tumor cells. It is a revolutionary anti-tumor drug that does not act on normal cells and solves the non-selective toxicity, 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, Scenedesmus, and Spirulina, which has a molecular weight of 121,000, an isoelectric point (PH) of 8.6, and a glycoprotein ratio of approximately 1:1 and a protein helix content of approximately 19%. It has the following characteristics. 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. Chlorella algae
500g (for fresh chlorella cake, add 5) to 500g of water
After hot water extraction at 95 to 100°C for 20 to 30 minutes, centrifugation was performed at 3000 to 10000 rpm 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, but also dilute acids,
Alternatively, there is no problem with water containing dilute alkali. Through this operation, about 75 g (in terms of powder) of a hot water extract was obtained from 500 g of Chlorella algae. The hot water extract is concentrated under reduced pressure at 50°C, and the total volume is made up to 1 with distilled water. This was passed through a semipermeable membrane and fractionated into a high molecular fraction that did not pass through the semipermeable membrane (hereinafter referred to as A fraction) and a low molecular fraction that passed through the semipermeable membrane (hereinafter referred to as B fraction). .
The yields of A and B were 45 g (in terms of powder) and 30 g (in terms of powder), respectively. Furthermore, 500% of the obtained A fraction
After concentrating to ml under reduced pressure, it was added to a DEAE-cellulose column, and multistage elution was performed while changing the buffer solution stepwise. That is, the fraction eluted with M/50 carbonate buffer
The fraction eluted with M/50 carbonate buffer containing M/10 NaCl was named A ₂ , and the fraction eluted with M/ ₅₀ Carbonate buffer containing 1M NaCl was named A ₃ . The yields of A ₁ , A ₂ , and A ₃ were 13.0 g (in terms of powder), 5.5 g (in terms of powder), and 10 g (in terms of powder), respectively. Fraction _A was concentrated under reduced pressure at 50°C, desalted using a dialysis membrane, and passed through a Sephadex G-150 column.
Fractionation was performed using 15 phosphate buffer (PH7.17), and the obtained fractions were dialyzed and further freeze-dried.
The yield was 140 mg (approximately 0.025% of Chlorella algae). The properties of the substance obtained by the above extraction method are listed below. [] Molecular weight ○A Molecular weight by electrophoresis (calculated value) 128,000 ○B Molecular weight by analytical ultracentrifuge (manufactured by Hitachi, absorption scanning recorder model 282-0060) 121,000 From the above ○A and ○B, the molecular weight is 121,000 It was hot. [] Purity ○A Gel filtration method ○B Sedimentation pattern of analytical ultracentrifuge ○C Confirmed to be a single product by SDS polyacrylamide gel electrophoresis. [] Isoelectric point PH8.6 [] Constituent sugar The obtained substance was hydrolyzed in hydrochloric acid at 100°C for 3 hours, separated by thin phase, paper chromatography and cellulose column chromatography, and then purified using the orcinol sulfuric acid method. The components and amounts were analyzed by . The results are shown in Table 1 below.

【table】

[Table] [] Constituent amino acid residues Automatic amino acid analyzer (manufactured by Hitachi, Ltd.; KLA-3B)
The amino acid components and amounts were analyzed according to the type (type). The results are shown in Table 2 below.

[Table] [] Composition ratio of sugar and protein 490μ/mg: 510μg/mg (approximately 1:1) [] Infrared spectroscopy Infrared spectrometer (manufactured by Hitachi, Ltd.; Model 260)
10 type) [IR r ^kBr _nax cm ^-1 ] to investigate the crystal state. As a result, absorptions attributed to the α-helix amide (), (), and () bands were observed at 1640, 1550, and 1260 nm, and
A random coil structure was observed at 16601530 and 1240nm. In addition, at 870 nm, the C ₁ -position of α-glucan
The angular vibration of equatorial H is at 880nm.
The respiratory vibration of the pyranose ring is at 906 nm, the axial H bending vibration at the C ₁ -position of β-glucan is at 910 nm.
~920nm shows the C-O-C of these pyranose rings.
Furthermore, at 999nm, β
-My husband and I were able to absorb the properties of glucan. [] Argon laser Raman spectroscopic analysis Argon laser Raman spectroscopic analyzer (JEOL
Co., Ltd.; JRS-V1-UV) [r ^H2O(D2O) _nax cm ^-1 ] to investigate the structure in solution. As a result, α-helical amide (I) bands were observed at 1642 nm, α-helical amide () bands at 1309 nm (E ₂ symmetric species), 1294 nm (E ₁ symmetric species), and 1275 nm (A symmetric species), respectively. The latter was confirmed by its disappearance by deuterium conversion with heavy water. In addition, at 1663 nm there is a band of amide (I) with a random coil structure, and at 1257 nm there is an amide (I) band with a random coil structure that disappeared due to deuterium exchange.
There were absorptions belonging to each band. Furthermore, one absorption at 556 nm or 528 nm and
379nm indicates the overlap of symmetric species A or E ₁ and the bending mode of the amide () band, and 350nm
showed the respiratory vibration of a relatively large molecular weight ring in an α-helical structure caused by hydrophobic amino acids. The substances obtained from the above [] and [] maintain an α-helical structure whether in crystal or solution form [] Circular dichroism spectrum (CD curve) Optical rotation dispersion spectrometer (manufactured by Hitachi, Ltd.; UV- 5 type)
(CD aqueous solution PH7.0) to examine the α-helix content. As a result, the CD curve shows a crossover point at 200nm, and at 209nm, [θ) - 9500,
At 222nm, a double minimum was observed at [θ)-7590. From this, the α-helix content is 18.9%
(approximately 19%). The hydrophobic amino acid group, which is more important than the constituent amino acid residues, accounts for about 35% of the amino acid residues, and if sugar components are also taken into account, it accounts for about 20% of the molecule. If the α-helix structure is mainly composed of hydrophobic 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 [Invitro test] (1-1) Measurement using suspension culture method Mouse lymphocytic leukemia culture established strain (L-
1210/V/C), the number of inoculated cells was 5-10 ⁴ cells/
mp. Note that this medium is RPMI 1640 containing 10% FCS.
(GIBCO) with the addition of 100 μg/ml streptomycin and 100 units/ml penicillin to adjust the pH to 7.0. Next, the above medium was incubated at 37°C for 72 hours.
The suspension culture method was used to create each group to which a fixed amount of this substance was added and a control group to which no additive was added. When comparing the number of cells after culture in these groups, it was found that the additive-free control group had 100 cells.
% proliferation rate, whereas the IC-50 (50% growth inhibition concentration) of the group to which this substance was added was 2 μg/ml. (1-2) Measurement by soft agar culture method When the growth inhibitory concentration of this substance for L-1210/V/C was determined according to the standard method (Soft agar cloning analysis method), the IC-50 was 2 μg/C. It was hot in ml. Experimental Example 2 [Invivo test] (2-1) Test using Sarcoma 180 10 ^Six Sarcoma 180 were transplanted intraperitoneally into 5-week-old DD mice (30 mice per group), and once daily from 24 hours after transplantation. 10mg/day of this substance for 5 consecutive days.
The substance was administered intraperitoneally to mice at a rate of 1 kg, 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 ratio (T/C) was 180%. (2-2) Test using P-388 10 ₆ mice of mouse lymphocytic leukemia (P-388) were intraperitoneally transplanted into 5-week-old CDF ₁ mice (30 mice per group), and once every day from 24 hours after transplantation. Once, 10 mg/Kg of this substance was intraperitoneally administered to mice for 5 consecutive days.
The substance was administered, and the ratio of survival days of the mice (ratio to mice not administered with the substance) was determined according to a conventional method. As a result, the life extension rate (T/C) was 160%. (2-3) Test using L-1210 10 ⁵ cases of mouse lymphocytic leukemia (L-1210)
was intraperitoneally implanted into 5-week-old CDF ₁ mice (30 mice per group), and 10 mg/Kg of this substance was intraperitoneally administered to the mice once daily for 5 consecutive days starting 24 hours after implantation.
was administered, and the ratio of survival days of the mice was examined according to a conventional method. As a result, the life extension rate (T/C) was 145%. Experimental Example 3 [Immunological tests, etc.] 33mg/Kg of this substance was administered to rabbits through their ear veins, and an additional 33mg/Kg was administered 10 days later.
No anaphylage-like findings were observed. In addition, when this substance was administered to rabbits at 5 mg/kg through the ear vein every other day for a total of 6 times, and even when administered under the above conditions, a sedimentation line that was distinguishable from normal rabbits was observed in immunoelectrophoresis. was not recognized. For this reason, this substance has low antigenicity and
This means that the antibody titer is also low. Furthermore, no hemolytic coagulation reaction was observed with this substance.

Claims (1)

[Claims] 1. A glycoprotein isolated from green microalgae such as Chlorella, Scenedesmus, and Spirulina, with a molecular weight of 121,000, an isoelectric point (PH) of 8.6, and a glycoprotein ratio of approximately 1:1.
An anticancer drug with a protein helix content of approximately 19%.