JPS6328888B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anticancer agent extracted from green microalgae such as chlorella and cenedesmus. The present inventors have discovered that certain components contained in green microalgae such as chlorella, cenedesmus, and spirulina have anticancer effects. That is, the anticancer agent of the present invention is a glycoprotein isolated from green microalgae such as chlorella, cenedesmus, and spirulina, and is a component with a molecular weight of about 120,000 or a component with a molecular weight of about 70,000. 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 carbon source, or a chlorella algae (powder) obtained by spray-drying or freeze-drying this raw cake is prepared as a raw material. Chlorella algae
Disperse 500g (5 for chlorella raw cake) in water 5, extract with hot water at 95-100℃ for 20-30 minutes, centrifuge at 3000-10000 rpm for 20-30 minutes, and remove the supernatant. It 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 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 added to a Sephadex G-25 column, eluted with water, and fractionated into a high molecular fraction (hereinafter referred to as A fraction) and a low molecular fraction (hereinafter referred to as B fraction). A, B
The yields were 45g (in terms of powder) and 30g (in terms of powder), respectively. Furthermore, 500 ml of the obtained A fraction
After concentrating under reduced pressure, it was added to a DEAE-cellulose column, and multistage elution was performed while changing the buffer solution stepwise. That is, fraction A ₁ eluted with M/100 carbonate buffer,
The fraction eluted with M/50 carbonate buffer was divided into A ₂ , M/
The fraction eluted with M/50 carbonate buffer containing 10 NaCl was named _A3 , and the fraction eluted with M/50 Carbonate buffer containing 1M NaCl was named _A4 . The yields of A ₁ , A ₂ , A ₃ , and A ₄ are 10.5 g (powder equivalent), 2.5 g (powder equivalent), 5.5 g (powder equivalent), respectively.
It was 10g (powder equivalent). The _A2 fraction was concentrated under reduced pressure at 50°C, desalted using a dialysis membrane, and then passed through a Sephadex G-150 column.
Fractionation was performed using M/15 phosphate buffer (PH7.17), and the two polymer fractions were separated into A-2-1 A-2-1
It was named -2-2. After concentrating each fraction under reduced pressure at 50°C and desalting with a dialysis membrane,
Lyophilized. The yields of A-2-1 and A-2-2 are as follows:
They were 100mg (powder equivalent) and 90mg (powder equivalent).
The absorption pattern of proteins and sugars in the eluate is shown in Figure 1. Protein was determined by absorbance at 280 nm, and sugar was determined by absorbance at 480 nm using the phenol-sulfuric acid method. Both A-2-1 and A-2-2 fractions were glycoproteins containing about 15 to 35% protein. It was confirmed that it was a glycoprotein by the following method. The presence of sugar was confirmed by the phenol sulfuric acid method, the presence of protein by the acid black method, and the presence of α-L-amino acids by hydrolysis. As shown in Figures 2 and 3, in the infrared absorption spectrum (KBr-tablet method), A-
2-1 and A-2-2 have an amide at 1650 cm ^-1
The presence of a peptide band and an amide band at 1540 cm ⁻¹ prove the presence of a peptide, indicating that it is a protein. When both A-2-1 and A-2-2 were digested with pronase, glucosamine and galactosamine were relatively increased, and aspartic acid, threonine, and serine were also relatively increased. YotsuteN
-Glycosidic bonds and O-glucoxide bonds were suggested. Also, the circular two-color spectrum (CD curve) is 209n.
The presence of a negative maximum in m suggests the presence of α-helix in the protein portion. Since A-2-2 has a very similar absorption pattern to A-2-1, it is considered to be a half-mer of A-2-1. When the molecular weights of A-2-1 and A-2-2 were examined using an analytical ultracentrifuge according to a conventional method, each had a single sedimentation pattern. The molecular weights are approximately 121,000 for A-2-1 and approximately 121,000 for A-2-2.
69,000 and about 70,000. Furthermore, when fractions A-2-1 and A-2-2 were subjected to SDS gel electrophoresis, a single sharp band was obtained as shown in FIG. Thereby, A-2-1 is a pure glycoprotein with a molecular weight of about 121,000, and A-2-2 is about
It was confirmed that it is a pure glycoprotein with a molecular weight of 70,000. The SDS gel electrophoresis experiment shown in FIG. 4 was conducted under the following conditions. That is, the glass tube used for electrophoresis had an inner diameter of 5 mm and a length of 10 cm. Samples were added at 0.03 mg per band. Ten%
The gel was run at 8 mA for 5 hours at a monomer concentration and stained with Acid Black 10B. R _f of A-2-1 is approximately 0.115, and A-2-2
R _f was approximately 0.136. Both A-2-1 and A-2-2 are soluble in water. The anticancer activity of A-2-1 and A-2-2 was confirmed by the following experiment. (Experiment 1) Mouse leukemia L-1210 is cultured at 37°C using a culture solution containing RPMI1640 and 10% FCS. After 24 hours of culture, the drug was added, and after 72 hours, the number of viable cells was counted using the trypan blue dye exclusion method, and the 50% growth inhibitory concentration (IC50) was determined from the ratio to the number of viable cells in the control test tube. Data are shown as the average value of 6 supplementary tests.

[Table] As is clear from the above IC-50 value, the anticancer agent of the present invention has an extremely excellent killing effect on L-1210 cells. (Experiment 2) Cervical cancer-derived Hela-S ₃ monolayer culture strain was cultured at 37°C using a culture solution containing 10% FCS added to Eagle MEM. 72 hours after implantation, from drug inoculation
The 50% growth inhibitory concentration (IC-50) after 48 hours was determined.

[Table] As is clear from the above IC-50 value, the anticancer agent of the present invention has an extremely excellent ability to kill cancer cells. (Experiment 3) 10 ⁶ Sarcoma 180 were intraperitoneally transplanted into 5- to 6-week-old DD mice, and the first group was a control group, the second group was 24 hours after transplantation, and the third group was after transplantation. After 7 days, the fourth group received 25 mg/Kg of A-7 days before transplantation.
2-1 and A-2-2 were administered intraperitoneally at the same site as the tumor implantation site for 5 consecutive days, and the ratio of survival days was observed according to a conventional method.

[Table] Very high survival index was obtained in treatment groups 2 and 3, and fairly strong therapeutic antitumor properties were observed. (Experiment 4) ¹⁰⁶ murine lymphocytic leukemia P388 were transplanted into the peritoneal cavity of 5- to 6-week-old CDF ₁ mice, and 24 hours after transplantation, each group was given the respective concentration. The drug was administered intraperitoneally at the same site as the tumor implantation site for 9 consecutive days, and the survival days ratio was observed according to a conventional method.

[Table] Thus, both A-2-1 and A-2-2 achieved treatment results with an effective survival rate (T/C) of 130% or more.

[Brief explanation of the drawing]

Figure 1 is a fractionation diagram of component A-2 containing the anticancer agent of the present invention measured on a Sephadex G-150 column;
3 and 3 are infrared absorption spectra of the anticancer agent of the present invention, and FIG. 4 is an SDS gel electropherogram of the anticancer agent of the present invention.

Claims (1)

[Scope of Claims] 1. A glycoprotein isolated from green microalgae such as Chlorella, Scenedesmus, Spirulina, etc.
An anticancer drug with a molecular weight of 120,000. 2. A glycoprotein isolated from green microalgae such as chlorella, cenedesmus, and spirulina, with approximately
An anticancer drug with a molecular weight of 70,000.