JP3216056B2 - Immunostimulating substance, water-soluble paramylon and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunopotentiator, a water-soluble paramylon used for improving human immunity, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, paramylon extracted from Euglena obtained an immunostimulatory effect as it is or by alkali treatment, chemical modification, cross-linking and / or a combination thereof. In addition, although paramylon has a β-1,3-glucan bond, its action was unknown because it was insoluble in water.

[0003]

The above-mentioned conventional paramylon-treated product is inferior in water solubility, has a low activation effect, is improved by chemical modification, and is effective even in animal experiments. There were problems such as lack of sex.

[0004]

According to the present invention, a water-soluble paramylon derivative having a partially or completely modified hydroxyl group can be obtained by reacting paramylon, a natural product, with glycidol. Paramylon can accumulate in cells of Euglena by culturing it in a medium mainly composed of glucose. Paramylon in Euglena cells can be easily removed by crushing the cells, and can be purified by treatment with alcohol or toluene. By reacting the thus purified paramylon with glycidol, a water-soluble paramylon derivative can be obtained. Paramylon is insoluble in most organic solvents, but is soluble in aqueous alkaline solutions and some organic solvents.

In the present invention, the reaction is carried out in a 0.1 to 2N aqueous sodium hydroxide solution. In this case, the amount of paramylon can be reacted up to 5 to 10%. Paramylon is dissolved in an aqueous sodium hydroxide solution, glycidol is added, and the mixture is stirred and reacted. It was recognized that the amount of glycidol to be added was preferably 4 to 5 times the amount of paramylon.
The reaction temperature in this case is 35 ° C. to 45 ° C., and a reaction time of 3 hours is sufficient.

Hereinafter, the present invention will be described in detail with reference to examples.

[0007]

Example 1 Euglena gracilis Z cells cultured using the Oda culture medium were collected by centrifugation. The cells were washed twice with a 50% ethanol solution, then washed with water, and collected by centrifugation again.
The suspension was suspended in an aqueous M urea solution and subjected to ultrasonic crushing. This was centrifuged and the precipitate was collected. The precipitate was suspended again in a 6 M aqueous urea solution, stirred for 1 hour, and centrifuged to collect the precipitate. The precipitate was prepared from toluene: water (1:
The mixture was suspended in the mixture of 4), stirred for 3 hours, and the precipitate was collected by centrifugation. By performing this operation three times, a white paste was obtained. This paste is mixed at 70 ° C for 3
By drying for a time, paramylon as a white powder could be obtained.

2 g of paramylon obtained as described above was completely dissolved in a 1N aqueous solution of sodium hydroxide to prepare a 5% paramylon solution. Glycidol was added dropwise to this solution to a final concentration of 15%, and reacted at 40 ° C. for 3 hours. After the completion of the reaction, the pH is 6.5 to 7.
Adjusted to zero. To this reaction solution, three times the volume of water was added, and the mixture was stirred well, and dialyzed for 16 hours using a cellophane tube with a molecular fraction of 5000.

After completion of the dialysis, centrifugation (10000)
g, 30 minutes), and the supernatant was freeze-dried under vacuum to obtain 2.5 g to 2.9 g of white glycidol-treated paramylon.

As an analysis of the structure of the glycidol-treated paramylon, an attempt was made to measure an infrared absorption spectrum. In the structures of paramylon before and after treatment with glycidol, absorption of hydroxyl groups derived from sugars at 1100 to 1400 cm ^-1 is hardly observed after the treatment, and the hydroxyl group is replaced by glycidol. It is shown that it is. In addition, the other absorption spectra showed little change,
The structure of β-1,3-glucan, which is the basic structure of paramylon, is maintained (see the infrared chart in FIG. 1).
Table 1 shows the weight percentages of C (carbon), H (hydrogen), and N (nitrogen) of the glycidol-added paramylon derivative produced by this method.

[0011]

[Table 1]

[0012]

Example 2 (Synthesis Example of Paramylon G) Paramylon (Lot. No. 901)
(029) 2.011 g was dissolved in 40 ml of 1N caustic alkali, and 8 ml of 2.3-epoxy-1-propanol was added thereto.
hr ultrasonic (38 KH ₂ ) treatment, reaction liquid yellow, 0.5
Neutralization with N acetic acid, dialysis using a cellulose tube (Visking Co), lyophilization, and a yield of 2.976 g (after drying under reduced pressure on P ₂ O ₂ , weighing). [Α] _D -3.4 ± 0.40 (c = 0.515%) (Measured value of substitution rate) Paramylon G was converted to N with 2NH ₂ SO ₄ .
100 ° C. ₂ gas sealed tube, 10h heated to hydrolyze, Bac
After neutralization with O ₃ , the aqueous solution was freeze-dried, further dried under reduced pressure on P ₂ O ₅ , aliquoted, trimethylsilylated with trimethyltyrosilane, and subjected to gas chromatography mass spectrometry. 6-position substitution of paramylon 51-52%,
2,4 and 4,6 substitutions were less than 3%.

[0013]

As described above, the water-soluble glycidol-added paramylon of the present invention has an effect of exhibiting an immunopotentiating effect.

[0014]

[Experimental Example 1] A 10% (vol / vol) sheep erythrocyte suspension 0.2 m as an antigen was injected into 6-8 week old CFE ₁ , male mice.
1 (2 × 10 ⁸ sheep erythrocytes) was injected into the tail vein for immunization. From 2 days before the antigen sensitization, an aqueous solution of glycidol-added paramylon was added in an amount of 1 mg, 10 mg / kg of mouse body weight.
mg, 50 mg and 100 mg were administered intraperitoneally once a day for 5 days.

Four days after the immunization, the number of antibody-producing cells in the spleen was measured by a modified Cunningham method. As a result, 5
In the 0 mg / kg and 100 mg / kg administration groups, significant antibody production activating effects were observed, respectively (see FIG. 2).

[0016]

[Experimental example 2] Microphage cytotoxicity enhancing action.

[0017] were administered to a C ₃ H / HeSle male mice of 6-8 weeks old glycidol addition paramylon aqueous body weight 1kg per 10mg and 50mg in 5 days intraperitoneally. Twenty-four hours after the final administration, the mice were exsanguinated and killed, and the macrophages exuded from the peritoneal cavity were aseptically recovered, washed, lysed with Tris ammonium chloride solution, further washed, and washed with 5%
The suspension was adjusted to 10 ⁶ cells / ml after suspension in a medium supplemented with fetal calf serum. As target cells, mouse leukemia cells P388
Peritoneal exudate macrophage: 1 P388 cells
0: 1 and 5: 1. 5 × 10 ⁴ / hole, 5: 1 for 96: 1 flat bottom plate
Then, macrophages exuded from the abdominal cavity were added at 2.5 × 10 ⁴ cells / well and cultured at 37 ° C. under 5% carbon dioxide for 2 hours. The culture supernatant was discarded, and 100 μl of a fresh medium was added to adjust to 5 × 10 ⁴ cells / ml. 100 μl / well of the P388 cell suspension was added and cultured for 17 hours. To this, 10 μl of tritium thymidine was added, and the culture was further continued for 4 hours, and the radioactivity incorporated into the cells was measured. As a result, in all the administration groups, the necrosis effect on tumor cells was enhanced (see FIG. 3).

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum graph of paramylon of the present invention.

FIG. 2 shows 1 mg of water-soluble glycidol-added paramylon before and after immunization with 6 to 8 week old CFE ₁ , male mice in sheep erythrocytes in an experiment for confirming the effect of the product of the present invention.
/ Kg, 10mg / kg, 50mg / kg, 100mg
The figure which shows the ability of the anti-sheep erythrocyte plaque forming cell at the time of / kg administration.

FIG. 3 is a graph showing a comparison of the ability of sheep erythroid plaque forming cells when water-soluble glycidol-added paramylon is administered and when schizophyllan is administered.

────────────────────────────────────────────────── (5) References JP-A-4-346931 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C08G 37/00 CA (STN) REGISTRY (STN) )

Claims (2)

(57) [Claims] (1) Glycidol is a natural product of paramylon .
(2,3-epoxy-1-propanol),
By partially or completely modifying the hydroxyl group of the paramylon
The obtained C (carbon): 41.87% by weight, H (hydrogen):
7.26% by weight, N (nitrogen): 0.00% by weight water-soluble
Glycidol-added paramylon derivatives . 2. The method of claim 1, wherein the natural product paramylon is glycidol.
(2,3-epoxy-1-propanol),
By partially or completely modifying the hydroxyl group of the paramylon
The obtained C (carbon): 41.87% by weight, H (hydrogen):
7.26% by weight, N (nitrogen): 0.00% by weight water-soluble
Immunity based on glycidol-added paramylon derivative
Activating substance.