JPH0721003B2 - Polysaccharide and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polysaccharide produced extracellularly by a single-celled green alga of the genus Chlorella, and more particularly to a polysaccharide having an antitumor action.

[0002]

2. Description of the Related Art Conventionally, as polysaccharides showing antitumor activity, those derived from mushrooms and bacteria are generally well known, but unicellular green algae of the genus Chlorella are extracellularly produced. It is known that some substances have antitumor activity. For example, Japanese Patent Application Laid-Open No. 4-300898 discloses a glycoprotein complex having an antitumor effect, and Japanese Patent Application Laid-Open No. 61-96992 discloses a muco-heteropolysaccharide which also has an antitumor effect.

An object of the present invention is to provide a polysaccharide which is produced extracellularly by unicellular green alga of the genus Chlorella and which exhibits an antitumor effect, and which contains galactose as its main constituent. Heretofore, a polysaccharide produced extracellularly by a single-celled green alga of the genus Chlorella, which exhibits an antitumor effect and which contains galactose as a main component, has not been known. Therefore, the polysaccharide according to the present invention is a novel polysaccharide.

[0004]

[Means for Solving the Problems] The present inventors have conducted studies on polysaccharides produced by unicellular green algae of the genus Chlorella (hereinafter sometimes simply referred to as “chlorella”) in a culture medium, and found that specific polysaccharides It was found that it exhibits an antitumor effect. When the polysaccharide was examined in more detail, it was found that the polysaccharide is a novel polysaccharide containing 80% by weight of galactose as a constituent sugar in at least a total neutral sugar in a purified state, and the present invention is completed. Came to.

That is, the polysaccharide according to the present invention is a polysaccharide that is produced extracellularly by unicellular green alga of the genus Chlorella, has an antitumor effect, and has a purified content of at least 80% by weight of total neutral sugars. % Galactose is contained as a constituent sugar.

In the method for producing a polysaccharide according to the present invention, a unicellular green alga of the genus Chlorella is cultured in a culture solution, and the supernatant of the culture solution after the culture is purified to have a molecular weight of 15,000 to 25,000.
Is obtained. Hereinafter, the present invention will be described in more detail.

As described above, the polysaccharide of the present invention contains at least 80% by weight of galactose as a constituent sugar in the purified state as a constituent sugar. However, it does not exclude that the polysaccharide contains some impurities, and in that case, the galactose content may be less than 80% by weight. The molecular weight is 15,000 to 25,000.

The polysaccharide of the present invention is produced extracellularly (in a medium) by culturing Chlorella. The medium, culture conditions, etc. used for culturing Chlorella are not particularly limited, and can be performed by a conventional method. For example, as a culture method, autotrophic (Autotrophic),
Heterotrophic or mixed nutrition (Mixo
trophic) can be used. However, considering the efficiency of culture, it is preferable to carry out under heterotrophic conditions or mixed nutritional conditions. The production of the polysaccharide of the present invention is typically carried out as follows.

First, a seed strain stored in an agar slant medium or the like is placed in a Sakaguchi flask and subjected to small-scale liquid culture. Then, using a general chlorella liquid culture medium, culture is carried out for several days to several weeks while gradually increasing the scale. Next, the obtained chlorella culture solution is centrifuged to separate the supernatant and chlorella cells.

The polysaccharide of the present invention is contained in the separated supernatant. Therefore, although the obtained supernatant can be directly used as an antitumor agent, it is preferably purified using ultrafiltration, dialysis, gel filtration chromatography and the like.

When the polysaccharide of the present invention is used as an antitumor agent, a pharmaceutically acceptable additive can be added to the polysaccharide. As the additive, an additive usually used in medicine can be used. For example, when it is used as a tablet or powder, it can be optionally mixed with lactose, sucrose, glucose, starch or the like. When used as an injection, it can be dissolved in physiological saline and housed in an ampoule to prepare a preparation.

When microorganisms other than chlorella are mixed during the culture, the growth and production ability of chlorella may be adversely affected. Therefore, it is desirable to pay sufficient attention to sterilization and handling of each device and culture solution used for culture so that other microorganisms are not mixed.

[0013]

【Example】

 Example 1 Culture of Chlorella and Preparation of Polysaccharide

One platinum loop of a seed strain of Chlorella (Chlorella Industry in-house number CK-206) stored in an agar slant medium was inoculated into a Sakaguchi flask and shake-cultured for 5 days. next,
The culture solution was transferred to a 10-liter jar famentor and cultured for 4 days, and the culture solution was collected. The composition and culture conditions of the medium used here are as follows. Medium composition (per liter of medium) Glucose 40 g Ammonium sulfate 2.2 g Monopotassium phosphate 1.0 g Magnesium sulfate 1.0 g Iron-EDTA 30 mg Arnon A5 solution 6 ml Yeast extract 10 mg Vitamin B1 20 μg Culture conditions pH 7.0 (by NaOH) Adjustment) 30 ℃

The resulting chlorella culture solution was centrifuged at 6000 rpm for 15
After centrifuging for minutes, the supernatant containing the desired polysaccharide and chlorella cells were separated. From this supernatant, PTHK membrane (Mill
After obtaining the polysaccharide fraction by ultrafiltration using ipore Laboratory), this was freeze-dried. The yield was 0.8 to 1.2 g per liter of culture solution. Example 2 Purification of polysaccharide and its chemical properties The polysaccharide fraction obtained in Example 1 was purified by the following method.

First, 5 g of the polysaccharide fraction was dissolved in 0.1 M phosphate buffer (pH 6.81), and this was added to Sephacryl S-300 HR.
It was applied to gel filtration chromatography using (Pharmacia) and the second eluted fraction having high antitumor activity was collected. This fraction was desalted and then lyophilized to obtain 4.2 g of the desired polysaccharide. Next, the following measurements were performed in order to investigate the chemical properties of the obtained polysaccharide. a) General analysis

Phenol / sulfuric acid method for sugar, Lo for protein
The wry method and the lipid were measured by the gravimetric method by extraction with chloroform / methanol. The average values of the results obtained by measuring 10 kinds of samples are shown in Table 1 below. The sugars are shown in terms of galactose and the proteins are shown in terms of bovine serum albumin. Table 1 −−−−−−−−−−−−−−−−− Sugar 942 μg / mg Protein trace Lipid trace −−−−−−−−−−−−−−−−− b) Sugar composition

Neutral sugars were hydrolyzed with 2.5N trifluoroacetic acid at 100 ° C. for 7 hours, and then measured as a trifluoroacetic acid derivative by gas chromatography. 5
The average values of the results obtained by measuring the samples of the seeds are shown in Table 2 below. Table 2 --------------------------- Component% ------------------ Galactose 86.7 Mannose 3.5 Glucose 3. 0 rhamnose 1.0 arabinose traces ----------------------- uronic acid positive ----------------------- For uronic acid, sulfuric acid -The measurement was carried out by the carbazole reaction, but it was not possible to carry out a quantitative measurement due to the strong influence of other sugars.

The ratio of each sugar component in the five samples measured was 81.4-91.9% for galactose,
Mannose is 3.2-3.8%, glucose is 2.8-3.2,
Rhamnose was in the range of 0.9 to 1.1. c) Infrared absorption spectrum The infrared absorption spectrum of the polysaccharide is shown in FIG. The measurement was performed under the following conditions. Equipment used: Nicoret FT-520 KBr tablet method Resolution: 4 cm ^-1 Number of scans: 128 d) Molecular weight From the results of gel filtration chromatography performed under the conditions shown below, the molecular weight was determined using various pullulans of known molecular weight as markers. did. Measurement conditions a) Column: Sephacryl S-300 HR (10 x 460 mm) b) Eluent: 0.1 M phosphate buffer (pH6.81) c) Detector: Differential refractometer

Specifically, a graph (calibration curve) showing the relationship between the molecular weight and the retention time of chromatography was prepared in advance using pullulan of various molecular weights, and the retention time of the polysaccharide was measured under the same conditions. The molecular weight was determined. FIG. 2 is a chart showing the results of gel filtration chromatography of polysaccharides.
3 types of pullulan (P-200 (MW = 186,000), P-5
The calibration curves showing the relationship between the molecular weight and the retention time prepared using 0 (MW = 48,000) and P-5 (MW = 4,800)) are shown in FIG. 3, respectively. The molecular weight of the polysaccharide calculated from these results was 15,000 to 25,000, and the average molecular weight was 20,00.
It was 0. e) Solubility It was easily soluble in water. Example 3 Toxicity test

30 6-week-old female ICR mice, 10 each
Three groups were divided into two groups, each of which was administered with the polysaccharide by intraperitoneal injection and subcutaneous injection, and the remaining one group was non-administered (control) and a toxicity test was conducted. Polysaccharide dose level is 5
mg / ml (physiological saline), 0.2 ml per animal is 2
It was administered once daily and changes in average body weight were measured. The results are shown in Table 3 below. Table 3 ----------------------------------------- Mean body weight Dosage method -------------------- − Start 7 days 14 days −−−−−−−−−−−−−−−−−−−−−−−−−−− Non-administered (control) 21.6 24.9 27.5 Intraperitoneal Injection 21.9 24.9 27.1 Subcutaneous injection 21.1 24.4 27.0 −−−−−−−−−−−−−−−−−−−−−−−−−−−−

As is clear from Table 3, there was no difference in average body weight between the control group and the administration group. No abnormalities were found in the measurement results of average body weight in any of the groups. Therefore, it is clear that the polysaccharide of the present invention has no toxicity. Example 4 Antitumor effect against Sarcoma 180

The antitumor effect against Sarcoma 180 was tested using 8-week-old female ICR mice. The test was 1 x 10 ⁶ Sarcoma 180 Mice inoculated with cells intraperitoneally
The test was carried out by dividing 60 animals into 6 groups of 10 animals, administering the sample to each group by the administration method shown in Table 4 below, and measuring the average survival time after inoculation.

The dose of the sample was 1 mg / ml (physiological saline), and 0.2 ml per mouse was used for 1 day from the day after the tumor transplantation.
Administration was performed once, a total of 10 times. However, for oral administration only, the administration concentration was 10 mg / ml.

At the same time, the content (extract) extracted from the cells of the cultured Chlorella was also tested under the same conditions using 20 female ICR mice inoculated with Sarcoma 180 as described above. The results are shown in Table 4 below. Table 4 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Administration method Average survival days (days) Life extension effect ^* (%) −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Polysaccharide non-administration (control) 1650 Intratumoral injection 34. 4 108.5 Intravenous injection 30.0 81.8 Intraperitoneal injection 25.5 54.5 Subcutaneous injection 22.2 34.5 Oral administration 19.0 15.2 Extract Intratumoral injection 29.9 81.2 Oral Administration 18.2 10.3 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−− * Life-prolonging effect = (mean survival time of test group− Control group mean survival days) x 100 / (control group mean survival days)

As is clear from Table 4, the polysaccharides according to the present invention were found to have a life-prolonging effect by any administration method, and among them, a very high life-prolonging effect was observed in intratumoral injection. In addition, the extract from Chlorella cells also showed a life-prolonging effect, although the activity was weaker than that of the polysaccharide. Example 5 Antitumor effect against Meth A fibrosarcoma

The antitumor effect against Meth A fibrosarcoma was tested using 8-week-old female BALB / c mice. The study tested Meth A fibrosarcoma at 3 x 10 ⁶ Fifty mice inoculated subcutaneously with cells were divided into 5 groups of 10 mice each, and the samples were administered to each group by the administration method shown in Table 5 below.
It was performed by measuring the size of the tumor on the 13th day after inoculation. The dose concentration of the sample was 1 mg / ml (saline), and 0.2 ml per animal was used 2 days after the tumor implantation and 2 days later.
It was administered once. The results are shown in Table 5 below. Table 5 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Administration method Tumor size ^* Tumor growth inhibition rate ^** Complete regression population (mm ² ) (%) (Animal) -------------------------------------------------------------------------------------------------------- 3250 0 Intratumoral injection 71 78.2 5 Intravenous injection 115 64.6 4 Intraperitoneal injection 122 62.4 4 Subcutaneous injection 182 43.6 2 −−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−− * Tumor size = Tumor major axis (mm) x minor axis (mm) after 13 days ** Tumor growth inhibition rate = (Tumor size of control group-of test group) Tumor size) × 100 / (control group tumor size)

As is clear from Table 5, the polysaccharide of the present invention has a tumor growth inhibitory effect in any administration method. In particular, the highest inhibitory effect was observed when it was administered by intratumoral injection, and the number of completely regressing individuals reached half of the whole group.

[0029]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, there is provided a novel polysaccharide which is produced extracellularly by unicellular green alga of the genus Chlorella and has an antitumor effect. Since the polysaccharide of the present invention is produced extracellularly, that is, in the culture medium, it can be easily separated and purified, and in addition, it has high productivity and can be produced at low cost. The culture solution of Chlorella has been conventionally discarded, and the present invention is also useful from the viewpoint of effective utilization of industrial waste.

[Brief description of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of a polysaccharide according to the present invention.

FIG. 2 is a diagram showing the results of gel filtration chromatography of the polysaccharide of the present invention performed using Sephacry S-300 HR as a column and a phosphate buffer solution (pH 6.81) as an eluent.

FIG. 3 is a diagram showing the relationship between molecular weight and retention time in gel filtration chromatography for three types of pullulan as molecular weight markers.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshihiko Sano 1012-6 Kokubun-cho, Kurume-shi, Fukuoka (72) Inventor Masao Okuda 9-64 Kasuga, Kasuga-shi, Fukuoka

Claims (3)

[Claims] 1. A polysaccharide produced extracellularly by a unicellular green alga of the genus Chlorella, which has an antitumor effect and contains galactose in a purified state at least 80% by weight of total neutral sugars.
Polysaccharide contained. 2. A unicellular green alga of the genus Chlorella is cultivated in a culture solution, and the supernatant of the culture solution is purified to have a molecular weight of 15,000 to 2.
The method for producing a polysaccharide according to claim 1, wherein 5,000 fractions are obtained. 3. An antitumor agent containing the polysaccharide according to claim 1 as a main component.