JPH06279292A - Production of glycogen having physiological activity and physiological activity - Google Patents

Abstract

PURPOSE:To extract glycogen in a state of maintaining physiological activity and to use the prepared glycogen as an antitumor agent. CONSTITUTION:Glycogen is extracted by using a protein hydrolyzing enzyme to extract glycogen in a state of maintaining physiological activity. Further, glycogen extracted by this method is administered to a cancer patient to heal a cancer.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antitumor agent containing glycogen as an active ingredient.

[0002]

2. Description of the Related Art Even in the highly developed modern times of the fields of medicine and pharmacy, cancer is not trying to give up the top cause of human death. Therefore, research on cancer treatment is being actively conducted in each field. As a result, it goes without saying that effective drugs and therapies that could save many lives were created. Especially with regard to chemotherapeutic agents, there is no way of knowing how many they are. However, conventional drugs lack a definitive part due to the toxicity of the substance itself, side effects caused by the mechanism of the substance acting 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]

Therefore, the object of the present invention is to eliminate the above-mentioned drawbacks by administering glycogen obtained using a proteolytic enzyme or the like to a cancer patient,
It is to provide a method of safely and efficiently curing cancer.

[0004]

[Means for Solving the Problems] The present inventors have found that glycogen, which is abundant in shellfish such as scallops, abalone, and oysters that are usually used for food, and plays a very important role as an energy source for animals. Was extracted by using a proteolytic enzyme, and as a result of a study on the antitumor activity of the resulting glycogen, it was found that it has a strong antitumor activity. Since this substance does not show cytotoxicity, it has a different mechanism of action from conventional cytotoxic antitumor agents, and tumors can be activated by activating the immune mechanism, that is, the self-defense mechanism that the body should possess. It is thought that the resistance to the cells is increased, and as a result, the growth of the tumor cells is suppressed and the cells are cured.

In the present invention, glycogen obtained by treating with a proteolytic enzyme having the above properties will be used for cancer treatment as an epoch-making antitumor agent having high activity and extremely high safety. It is what

Glycogen is a polysaccharide composed of D-glucose like starch and amylopectin.
-Glucose is glycosidically bonded by alpha 1-4 bond. D-glucose is glycosidic-bonded by alpha 1-6 bond at several places in the main chain, and D-glucose is alpha 1-4 bond by D-glucose. It is characterized in that it has a dendritic complex structure having side chains linked to glycosides. Glycogen is a very important compound that is biosynthesized in the liver and generally involved in energy storage and metabolism in animals. In food, it is also a substance that forms the center of the umami of fish and shellfish, especially scallops, oysters and abalone. Therefore, glycogen used in the present invention has various origins, and is considered to be easily available in large quantities and at low cost.

As described above, glycogen has been a well-known substance for a long time, but there is no example of its use as an antitumor agent. Conventionally, glycogen has been extracted and structurally studied by using a strong alkali or a strong acid, and has been used and supplied as a standard product. However, under this extraction condition, the finely branched structure, which had been present in the living body, was lost.

[0008] Therefore, in the present invention, it has succeeded in enabling extraction in a state in which the function in vivo is maintained by using a proteolytic enzyme instead of the conventional method of extracting under the chemically harsh conditions. did. That is,
Contaminants such as proteins, which were conventionally removed by treatment with a strong alkali or a strong acid, are made possible by the mild conditions of using a proteolytic enzyme. In the present invention, for the extraction of glycogen, pronase, trypsin and papain are preferably used as proteolytic enzymes. For extraction of glycogen, a sample considered to contain glycogen is degreased and dried by stirring or homogenizing it with a hydrophilic organic solvent such as acetone or ethanol as necessary. Alternatively, dip the sample in cold water, hot water, various buffer solutions,
Alternatively, the extract obtained by stirring is desalted by dialysis, ultrafiltration, electrodialysis or the like and used. The sample that has been subjected to the above treatment is dissolved in a solution adjusted to an optimum hydrogen ion concentration for the enzyme to be used, such as Tris-hydrochloric acid buffer solution, the enzyme is allowed to act on the solution, and the supernatant after centrifugation is separated into alcohol, etc., preferably Glycogen can be extracted in an almost pure state by adding ethanol. Further purification is possible by using gel filtration, ion exchange chromatography, or high performance liquid chromatography. The antitumor effect can be expected in the state of the above crude extract, but preferably, the state highly purified by gel filtration and anion exchange chromatography provides an efficient and high effect. Glycogen is obtained as a white powder, does not deteriorate in the dry state, and the activity is retained by extraction with hot water or high-temperature steam, confirming that the child has extremely high thermal stability, It can be stored for a long time. Both crude glycogen and purified glycogen are easily soluble in water and various buffers, but they are not completely transparent solutions and become slightly white solutions due to unfilterable white colloidal particles. .

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

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

"Limitation of Beta-Amylase Degradation Rate" The limit rate of decomposition by beta-amylase was 33% for trichloroacetic acid-extracted glycogen, whereas it was 20% or less for the glycogen obtained by the method of the present invention.

"Unit average chain length" After treatment with isoamylase,
The unit average chain length was determined by dividing the total sugar amount by the reducing end sugar amount. As a result, the glycogen extracted by trichloroacetic acid was 9.3, whereas the glycogen obtained by the method of the present invention was 7. It was 0.

"Proton NMR" is shown in FIG.

"Chemical composition analysis value" is shown in Table 1.

[Table 1]

As a method for treating tumors using glycogen, it is possible to dissolve glycogen in water, physiological saline, or various salt solutions, and then directly administer it to the lesion by intravenous injection or the like. In addition, since it is a substance that originally exists in the living body, it is not toxic, and therefore its dosage can be controlled within a wide range depending on the condition. In the present invention, this antitumor activity could be confirmed by the following method. That is, the tumor could be cured by transplanting tumor cells into the abdominal cavity of a mouse and administering glycogen to this mouse by dissolving it in physiological saline. Since no side effect was observed in the cured mouse in this experiment, it is considered that there is no acute toxicity, and it was confirmed that it is a very promising substance for tumors, and the present invention was completed.

As the antitumor active polysaccharide having such characteristics, beta (1,3) extracted from mushrooms and the like is used.
(1,6) -Glugan is famous, but glycogen in the present invention is completely different in material because the glycosidic bond between sugar residues has an inverse alpha bond. It has been reported that there are several other polysaccharides showing antitumor activity (Whistler et al., Advances in Carbohydrate Chemistry and Biochemistry (Whistler, et al.
l. , Advances in Carbohydr.
Chem. and Biochem. ) Volume 32, 235
, 275, published in 1976), these polysaccharides are widely used even now, but in any case, they are substances having a completely different structure from the glycogen of the present invention.

The method for producing glycogen and the method for administering an effective fraction according to the present invention will be described in more detail below.

[0018]

Example 1 (Glycogen extraction)

“Extraction Example 1” (extraction with hot water from scallop) Raw scallops were immersed in boiling water and boiled for 15 minutes. The supernatant was collected by decantation and filtration with gauze, and concentrated about 10 times under reduced pressure. The concentrated extract was dialyzed in running water to desalt. This extract is freeze-dried to obtain 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% amount of Pronase of the desalted dry powder was added. It was treated at 24 ° C. for 24 hours. Further again, 1% of pronase of desalted dry powder was added and treated at 50 ° C. for 24 hours. The enzyme-treated solution was treated at 100 ° C for 5 minutes to inactivate the enzyme, and then centrifuged to remove solids, add 3 times volume of ethanol saturated with sodium chloride, and leave at 4 ° C. By doing so, crude glycogen was precipitated. A white powder was obtained by collecting the precipitate by centrifugation and drying.

“Extraction Example 2” (extraction with hot steam from scallop) Raw scallops were steamed with steam at about 120 to 130 ° C. for 15 minutes. Collect the dripping coagulant liquid and reduce the pressure to about 13
Concentrated twice. The concentrated extract was dialyzed in running water to desalt. This extract is freeze-dried to obtain desalted dry powder.
The desalted dry powder was dissolved in 50 mmol / l Tris-hydrochloric acid buffer solution (pH 7.8) containing 2.5 mmol / l calcium chloride, and 1% amount of pronase of the desalted dry powder was added, and the mixture was heated to 50 ° C. Treated for 24 hours. Further again, 1% amount of pronase of desalted dry powder was added,
It was treated at 24 ° C. for 24 hours. This enzyme treatment liquid is 5 at 100 ℃
After deactivating the enzyme for 3 minutes, centrifuge to remove solids and add 3 mL of ethanol saturated with sodium chloride.
The crude glycogen was precipitated by adding a double volume and allowing to stand at 4 ° C. A white powder was obtained by collecting the precipitate by centrifugation and drying.

[0021]

Example 2 (Separation and Purification Example) Crude glycogen was purified using Sephadex G-25 gel (trade name, manufactured by Pharmacia) column with deionized water as an eluent. Further, this 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 0.1 mol / liter phosphate buffer is used, and 0 to 0.
Separation was performed by changing the sodium chloride concentration by the linear concentration gradient method of 3 mol / liter. The results are shown in FIG.

[0022]

[Example 3] (Bioactivity test) Mouse (BALB / c, 20 to 30 g) intraperitoneally cultured tumor cells (Meth-A) were intraperitoneally transplanted into a mouse, and the mice were transplanted on the 2nd, 4th and 6th days. And every time, 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 by setting the healing rate as the mouse that did not die of the tumor on the 60th day, and based on the healing rate. The results are shown in Table 2.

[Table 2]

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[Procedure amendment]

[Submission date] July 22, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0022

[Correction method] Change

[Correction content]

[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 And every time, 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 by setting the healing rate as the mouse that did not die of the tumor on the 60th day, and based on the healing rate. The results are shown in Table 2.

[Table 2] ─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 8, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Added

[Correction content]

[Brief description of drawings]

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

FIG. 2 is a diagram showing the results of comparing the molecular weight of glycogen with glycogen extracted with trichloroacetic acid by subjecting glycogen to a Sepharose CL-2B column for elution, and the glycogen obtained in the present invention is trichloro It is a figure which shows that a molecular weight is large compared with what was extracted using acetic acid.

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

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor, Hajime Matsue, Aomori City, Aomori Prefecture, Katsutoshi, Ashiya 202-4, Aomori Industrial Technology Development Center (72) Inventor, Jinichi Sasaki 5 Fufucho, Hirosaki City, Aomori Prefecture Hirosaki University School of Medicine (72) Inventor Kunio Ishida 5 Aki-cho, Aomori Prefecture 5 Fufu-cho, Hirosaki University School of Medicine (72) Inventor Hidetaka Ichinohe Aomori-shi, Aomori Nishidazawa character Okitsu 257-10 Tomoyauchi Aomori Co., Ltd.

Claims (2)

[Claims] 1. An antitumor agent comprising glycogen as an active ingredient. 2. The antitumor agent according to claim 1, wherein glycogen is one that has been treated and extracted by using a proteolytic enzyme.