JPS6328887B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anticancer agent extracted from Chlorella algae and a method for producing the same. The present inventors have discovered that certain components contained in the hot water extract of the unicellular green alga Chlorella have anticancer effects. That is, the anticancer agent of the present invention is a glycoprotein isolated from a hot water extract of Chlorella, and contains a carbohydrate-rich component with a molecular weight of 70,000 or more and a protein-rich component with a molecular weight of 3,000 to 9,000. The above separation can be easily performed using column chromatography. The method for producing the anticancer agent of the present invention will be described below. After outdoor cultivation, 10 g of spray-dried Chlorella algae (manufactured by Chlorella Industries) was suspended in 100 ml of deionized water.
After immersion in boiling water for 20 minutes, it was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was used as a chlorella hot water extract.
This operation was repeated to obtain about 75 g of chlorella hot water extract from 500 g of chlorella powder. The hot water extract was centrifuged and the supernatant was concentrated under reduced pressure at 50°C using a rotary evaporator to 15 ml, and this was applied to a Sephadex G-25 column (2.6 x 40 cm,
Pharmacia) and then eluted with distilled water at a rate of 2 ml/min to obtain a high molecular fraction (hereinafter referred to as fraction A).
and a low molecular weight fraction (hereinafter referred to as B fraction). After fraction A was concentrated to 10 ml under reduced pressure, it was added to a DEAE-cellulose column (2.6 x 32 cm, manufactured by Brown), and elution was performed while changing the buffer solution (developing solution) stepwise. That is, the fraction eluted with M/100 carbonate buffer is A1, the fraction eluted with M/50 carbonate buffer is A2, and the fraction eluted with M/50 carbonate buffer containing M/10NaCl is A3. , and the fractions eluted with M/50 carbonate buffer containing 1M NaCl were named A4. The yields of A1, A2, A3, and A4 were 2.1%, 0.5%, 1.1%, and 2.0%, respectively. Note that the yield was expressed as a percentage obtained by dividing the dry weight of each fraction by the weight of the raw material Chlorella algae. It was clarified by the following two examples that A2 among the above four fractions is effective as an anticancer agent. Example 1 Experimental animal: CDF, mice (6 mice per group) Tumor: P388 Number of transplanted cells: 10 ⁶ cells/mouse Transplantation site: Peritoneal A2 administration number: 9 times from 1 to 9 days A2 administration site: Peritoneal A2 administration Amount: 50mg/Kg, 25mg/Kg, 12.5mg/Kg,
1.5mg/Kg Judgment The average survival days for each of the administration group (T) and control group (C) are determined and expressed as T/C (%). The results are shown in Table 1 below.

[Table] From the above T/C value, it is clear that A2 of the present invention has a survival effect on P388 mice. Example 2 Experimental animals: DD mice (10 mice per group) Tumor: Sarcoma 180 Number of transplanted cells: 10 ⁶ /mouse Transplant site: Peritoneal A2 Number of administrations: 5 times on days 1 to 5 (7 times after tumor transplant) A2 administration site: peritoneal A2 administration amount: 250 mg/Kg Judgment The average survival days of each of the administration group (T) and control group (C) are determined and expressed as T/C (%). T/C is
The rate was 133.3%, and therefore it was determined to be effective.
Among the treated group, two animals were completely cured. The attached drawing shows the infrared absorption spectrum of A2. A2 was fractionated into a carbohydrate-rich component with a molecular weight of 70,000 or more and a protein-rich component with a molecular weight of 3,000 to 9,000.
In this molecular weight measurement, Sephadex G-150
Column (2.6 x 84
Add 1 ml of solution containing about 10 mg of sample to M/cm).
15 phosphate buffer (PH7.19). The markers are Blue dextran (MW2000000) and 4 types of FITC-dextran (NW70000; 40000;
20000; 3000) (manufactured by Pharmacia) was used. The composition of A2 is shown in Table 2 below.

[Table] In Table 2 above, proteins are expressed as serum albumin equivalent values, and carbohydrates are expressed as galactose equivalent values. The molar ratio of the carbohydrate components contained in A2 is shown in Table 3 below.

[Table] In Table 3, the molar ratio is the value when xylose is set to 1.0. The composition of amino acids constituting the protein contained in A2 is shown in Table 4 below.

【table】

[Table] In Table 4 above, 5mg of A2 is added to 6N HCl2
ml, seal the tube, hydrolyze at 100℃ for 24 hours, open the tube, dry under reduced pressure, and add citrate buffer (PH2.6).
It was dissolved in water and subjected to amino acid analysis. The analysis was performed using an automatic amino acid analyzer (manufactured by Mitamura Riken).

[Brief explanation of the drawing]

The drawing shows an infrared absorption spectrum of the anticancer agent of the present invention.

Claims (1)

[Scope of Claims] 1. An anticancer agent which is a glycoprotein isolated from a hot water extract of Chlorella and contains a carbohydrate-rich component with a molecular weight of 70,000 or more and a protein-rich component with a molecular weight of 3,000 to 9,000. 2. An anticancer drug characterized by separating glycoproteins containing a carbohydrate-rich component with a molecular weight of 70,000 or more and a protein-rich component with a molecular weight of 3,000 to 9,000 from a hot water extract of Chlorella using column chromatography. Production method.