JP2583183B2 - Process for producing bioactive glycogen and bioactivity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antitumor agent containing glycogen as an active ingredient.

[0002]

2. Description of the Related Art In today's highly developed fields of medicine and pharmacy, cancer does not try to give way to the number one cause of human death. Therefore, research on cancer treatment is being actively conducted in various fields. As a result, it goes without saying that effective drugs and therapies have been created that have saved many lives. Especially for chemotherapeutic agents, there is no way to know them. However, conventional drugs lack the decisive point due to the toxicity of the substance itself, side effects caused by the mechanism by which the substance acts as a drug, or the narrow range of effective cases. is there. Therefore, a drug with low toxicity and wide spectrum is currently desired.

[0003]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to eliminate the above-mentioned drawbacks, and to administer glycogen obtained using a protease to a cancer patient.
An object of the present invention is to provide a method for curing cancer safely and efficiently.

[0004]

Means for Solving the Problems The present inventors have found that glycogen, which is abundantly contained in scallops, abalones, oysters and other commonly used edible shellfish, and plays a very important role as an energy source for animals. Was extracted by using a proteolytic enzyme, and a study on the antitumor activity of the obtained glycogen was carried out. As a result, it was found that the glycogen had strong antitumor activity. Since this substance has no cytotoxicity, its mechanism of action is different from that of conventional cytotoxic antitumor agents. It is thought that the resistance to the cells is increased, thereby suppressing the growth of the tumor cells and further curing the tumor cells.

In the present invention, glycogen obtained by treating with a protease having the above-mentioned properties is used as a highly active and extremely safe and revolutionary anti-tumor agent for cancer treatment. It is assumed that.

Glycogen is a polysaccharide composed of D-glucose, similar to starch and amylopectin.
-Glucose is glycosidically linked to D-glucose at an alpha 1-6 bond at several places in the main chain where the glucose is glycosidically linked at an alpha 1-4 bond, and D-glucose is further linked to D-glucose at an alpha 1-4 bond. It has a complex dendritic structure having glycoside-linked side chains. Glycogen is a very important compound that is biosynthesized in the liver in animals and is generally involved in energy storage and metabolism. In foods, it is also a substance that forms the center of umami in shellfish such as fish and shellfish, oysters, and abalone. Therefore, the origin of glycogen used in the present invention is considered to be easily available in large quantities and at low cost.

As described above, glycogen is a substance that has been known for a long time, but there is no example in which it is used as an antitumor agent. Conventionally, glycogen has been extracted and structurally studied using a strong alkali or strong acid, and has been used and supplied as a standard. However, according to the extraction conditions, the fine branch structure existing in the living body has been lost.

Therefore, in the present invention, it has been succeeded to make it possible to perform the extraction while maintaining the function in a living body by using a proteolytic enzyme instead of the conventional method of extracting under chemically harsh conditions. did. That is,
Conventionally, contaminants such as proteins which have been removed by treatment with a strong alkali or a strong acid have been made possible under mild conditions using proteolytic enzymes. In the present invention, for the extraction of glycogen, pronase, trypsin and papain are preferably used as proteolytic enzymes. In the extraction of glycogen, a sample that is considered to contain glycogen is degreased and dried by stirring or homogenizing with a hydrophilic organic solvent such as acetone or ethanol as necessary. Or immerse the sample in cold water, hot water, various buffer solutions, etc.
Alternatively, the extract obtained by stirring is desalted and used by dialysis, ultrafiltration, electrodialysis, or the like. The sample subjected to the above treatment is dissolved in a solution adjusted to the optimum hydrogen ion concentration for the enzyme to be used, such as a Tris-HCl buffer, and the enzyme is added thereto.After centrifugation, the supernatant is centrifuged. By adding ethanol, it is possible to extract glycogen in a substantially pure state. Further purification is possible by using gel filtration, ion exchange chromatography or high performance liquid chromatography. Although the antitumor effect can be expected even in the state of the above crude extract, it is preferable to obtain a more efficient and high effect by a state highly purified by gel filtration and anion exchange chromatography. Glycogen is obtained as a white powder, does not deteriorate in a dry state, and retains its activity even by extraction with hot water or high-temperature steam. Long-term storage is possible. Both crude glycogen and purified glycogen are easily soluble in water and various buffer solutions, but do not become completely transparent solutions and become slightly white solutions due to non-filterable white colloidal particles. .

The properties of glycogen extracted, separated and purified by the method of the present invention are as follows.

"Average molecular weight" Fractions A and B in FIG. 1 and glycogen extracted from scallop clam muscle using trichloroacetic acid as a conventional method were used on Sepharose CL-2B gel (trade name, manufactured by Pharmacia). As a result of comparison of molecular weights by gel column chromatography, glycogen obtained by the method of the present invention had a larger average molecular weight than glycogen extracted with trichloroacetic acid and was 100,000 or more (FIG. 2).

"Limit degradation rate of beta-amylase" The limiting degradation rate by beta-amylase was 33% for glycogen extracted with trichloroacetic acid, whereas glycogen obtained by the method of the present invention was 20% or less.

"Unit average chain length" After isoamylase treatment,
By dividing the total sugar content by the reducing terminal sugar content, the unit average chain length was determined. The result was 9.3 for the trichloroacetic acid-extracted glycogen, whereas the glycogen obtained by the method of the present invention was 7. It was 0.

"Proton NMR" is shown in FIG.

"Chemical component analysis values" are shown in Table 1.

[Table 1]

[0015] As a method for treating tumors using glycogen, glycogen can be dissolved in water, physiological saline, or various salt solutions, and then directly injected into the lesion, intravenous administration, or the like. Further, since the substance is originally present in a living body, it has no toxicity. Therefore, the dosage can be controlled in a wide range depending on the disease state. In the present invention, this antitumor activity could be confirmed by the following method. That is, tumor cells were implanted in the abdominal cavity of a mouse, and glycogen was dissolved in physiological saline and administered to the mouse, whereby the tumor could be cured. Since no side effects were observed in the cured mice in this experiment, it was considered that there was no acute toxicity, and it was confirmed that they were very promising substances for tumors. Thus, the present invention was completed.

The antitumor active polysaccharide having such characteristics is beta (1,3) extracted from mushrooms and the like.
(1,6) -Glucan is famous, but the glycogen in the present invention is a material which is completely different because glycoside bonds between sugar residues take reverse alpha bonds. Several other polysaccharides exhibiting antitumor activity have been reported (Whistler et al., Advances in Carbohydrate Chemistry and Biochemistry).
l. , Advances in Carbohydr.
Chem. and Biochem. 32 volumes, 235
２, p. 275, published in 1976). These polysaccharides are widely used even now, but in any case, they are substances whose structure is completely different from the glycogen of the present invention.

Hereinafter, examples of the method for producing glycogen and the method for administering an effective fraction performed in the present invention will be described in more detail.

[0018]

Example 1 (Glycogen extraction)

[Extraction Example 1] (Extraction with scallop using hot water) Raw scallop was immersed in boiling water and boiled for 15 minutes. The supernatant was collected by decantation and filtration with gauze, and concentrated about 10-fold under reduced pressure. The concentrated extract was dialyzed in running water and desalted. This extract is freeze-dried to obtain a desalted dry powder. The desalted dry powder was dissolved in 50 mmol / L Tris-HCl buffer (pH 7.8) containing 2.5 mmol / L calcium chloride, and 1% of the desalted dry powder was added to the solution, followed by addition of 50%. Treated at ℃ for 24 hours. Further, pronase was added again in an amount of 1% of the desalted and dried powder, followed by treatment at 50 ° C. for 24 hours. This enzyme-treated solution is treated at 100 ° C. for 5 minutes to deactivate the enzyme, and then centrifuged to remove solids, add three times the volume of ethanol saturated with sodium chloride, and leave at 4 ° C. As a result, crude glycogen was precipitated. The precipitate was collected by centrifugation and dried to obtain a white powder.

“Extraction Example 2” (Extraction with Hot Steam from Scallops) Raw scallops were steamed with steam at about 120 to 130 ° C. for 15 minutes. Collect the dropped coagulation liquid and reduce
Concentrated by a factor of two. The concentrated extract was dialyzed in running water and desalted. This extract is freeze-dried to obtain a desalted dry powder.
The desalted dry powder is dissolved in 50 mmol / L Tris-HCl buffer (pH 7.8) containing 2.5 mmol / L calcium chloride, and 1% pronase from the desalted dry powder is added. For 24 hours. Again, 1% pronase from the desalted dry powder was added and 50%
Treated at ℃ for 24 hours. This enzyme-treated solution is treated at 100 ° C for 5 minutes.
After inactivating the enzyme, the mixture was centrifuged to remove solids, and ethanol saturated with sodium chloride was added for 3 minutes.
By adding twice the volume and allowing to stand at 4 ° C., crude glycogen was precipitated. The precipitate was collected by centrifugation and dried to obtain a white powder.

[0021]

Example 2 (Example of separation and purification) Crude glycogen was purified by Sephadex G-25 gel (trade name, manufactured by Pharmacia) as an eluent and purified using on-water. Further, the glycogen fraction was fractionated and purified by anion exchange chromatography using DEAE Sephadex A-25 anion exchange gel (trade name, manufactured by Pharmacia). As the elution buffer, a phosphate buffer of 0.1 mol / liter is used.
Separation was performed by changing the concentration of sodium chloride by a linear concentration gradient method of 3 mol / liter. FIG. 1 shows the results.

[0022]

Example 3 (Biological Activity Test) mice (BALB / c, 20 to 30 g) of the abdominal cavity in cultured tumor cells (Meth-A) was transplanted intraperitoneally to a mouse, day 2, day 4, day 6 In each case, 200 per mouse
μg and 50 μg of glycogen were dissolved in 0.5 ml of physiological saline and administered by intraperitoneal injection. The activity was determined on the basis of the healing rate, with mice that did not die from tumor on day 60 being treated as cured mice. The results were as shown in Table 2.

[Table 2]

[Brief description of the drawings]

FIG. 1: Glycogen is DEAE Sephadex A-2
Using a 5-ion exchange column, elution was performed by changing the salt concentration from 0 M and a linear concentration gradient method from 0 M to 0.4 M, and changing the concentration to 2 M. As a result, five fractions of fractions A, B, C, D and E were obtained. It is a figure showing what was obtained.

FIG. 2 is a graph showing the result of comparing the molecular weight of glycogen with glycogen extracted using trichloroacetic acid by subjecting the glycogen to a Sepharose CL-2B column to elute the glycogen. The glycogen obtained in the present invention is trichloroacetic acid. It is a figure which shows that a molecular weight is large compared with the thing extracted using acetic acid.

FIG. 3 shows fractions A to E in FIG. 1 and glycogen extracted using trichloroacetic acid at 270 MHz in heavy water.
It is a figure showing the proton NMR spectrum under the conditions of 70 ° C.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Kazu Matsue 202-4 Ashitani, Aomori City, Aomori Prefecture Aomori Prefecture Industrial Technology Development Center (72) Inventor Jinichi Sasaki 5 Aifucho, Hirosaki City, Aomori Hirosaki University School of Medicine (72) Inventor Kunio Ishida 5 Aimori-cho, Hirosaki City, Aomori Prefecture Hirosaki University School of Medicine (72) Inventor Hidetaka Ichinohe 257-10 Okitsu, Nishidazawa Character, Aomori City, Aomori Prefecture Tomoyanai Aomori (56 References JP-A-53-44614 (JP, A) JP-A-2-268120 (JP, A) JP-A-61-53220 (JP, A)

Claims (1)

(57) [Claims] 1. Treatment and extraction using a proteolytic enzyme
Characterized in that an active ingredient Guriko gain emissions that are
Antitumor agent