JPH07300424A - Antitumor agent composition - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent composition showing excellent cytocydal activity and/or proliferation suppression of a tumor cell, especially a leukemia cell and an adjust T cell leukemia cell. CONSTITUTION:This antitumor agent composition is extracted from a plant belonging to a bamboo or a bamboo grass. The composition is positive in ninhydrin reaction, has about 10,000-30,000 average-molecular weight by get filtration chromatography, shows pH about 4.0-7.0, is slightly soluble in an organic solvent such as benzene or chloroform, soluble in an alcohol, an acidic or an alkaline aqueous solution and has ultraviolet light absorption approximately at 250nm (acidity) and approximately at 280nm (alkalinity) in the aqueous solution. The composition is obtained by pulverizing rhizome of a bamboo or a bamboo grass, compressing by a press, passing the pulverized rhizome and the liquid obtained in the compressing through a cloth having a rough mesh to remove a solid fiber component and a foreign matter and further filtering the filtrate by using a filter paper and/or an ultrafilter.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent composition.

[0002]

2. Description of the Related Art Currently, as antitumor agents, there are synthetic substances represented by platinum complexes and alkylating agents. These generally exhibit a cytocidal effect (antitumor activity) by cross-linking with guanine of DNA in permeated cells.

[0003]

As an antitumor agent, sufficient antitumor agents have not been obtained in terms of selectivity with respect to normal cells and cell killing effect. At present, the mainstream of antitumor agents are synthetic substances, and their activity is often a cytocidal effect formed by cross-bonding with guanine of DNA.

However, various factors such as tumor state, type, compatibility between drug and patient itself, nature of tumor cell, and state of complications are involved, and these antitumor agents may be developed in various ways. It is anticipated and there is a need for it.

At present, in addition to synthetic new medicines, there are Chinese medicines, which are mainly extracts of plants and the like.

[0006] By the way, in the cells of plants, there are protoplasms essential for life support and other contents. Substances extracted from plants include starch, fat particles, carbohydrates,
Known are pigments, various acids and salts (eg, malic acid, calcium and magnesium oxalate, etc.), proteins, hormones, glycoproteins, sugars, inulin, gums, tannins, alkaloids, and proteolytic enzymes. . These include waste products provided they have nutrients for metabolism. Generally, in plants, waste products are not excreted outside the body but accumulate in cells. Therefore, it can be said that the plant extract contains various physiologically active substances.

[0007] Kumazasa and other bamboo grasses or bamboos have been used for medicinal purposes since ancient times, but none have been directly used for antitumor agents and antiviral agents.

The present inventors have investigated the use of physiologically active substances extracted from bamboo or bamboo grass as an antitumor agent, and have found a novel antitumor agent composition.

[0009]

[Means for Solving the Problems] That is, the present invention provides an antitumor agent composition that can be extracted from a plant belonging to bamboo or bamboo grass, wherein the composition has a positive ninhydrin reaction and an average molecular weight by gel filtration chromatography. Is about 10,000 to about 3
It exhibits a pH of about 4.0 to about 7.0, is sparingly soluble in organic solvents such as benzene and chloroform, soluble in alcohol, soluble in alkaline or acidic aqueous solutions, and absorbs UV-visible light in the aqueous solutions. Around 250 nm (acidic) and 280
The antitumor agent composition is characterized in that it has a wavelength in the vicinity of nm (alkaline).

The method of extracting the composition of the present invention from bamboos or bamboo grasses can be performed by any method.

For example, first, the rhizome is crushed. Any ordinary method can be applied to this crushing. That is, although it may be hit with the back of a kitchen knife, it is preferable to use a pressing machine in consideration of prevention of foaming and minimizing the contact time with oxygen in the air. The finely ground rhizomes and the liquid obtained at the time of pressing are filtered through a rough cloth such as gauze to remove solid fiber components and foreign substances, and further filtered using a filter paper and / or an ultrafilter. Thus, the composition of the present invention is obtained. The evaporation residue per 1 ml of this extract is about 0.1 to about 1
mg.

Alternatively, the composition of the present invention can be obtained by extracting bamboos or bamboo grass with an organic solvent such as alcohol, preferably methanol or ethanol, and then distilling off the solvent. The composition is then obtained in the form of a pale yellow solid or candy.

The composition of the present invention can be further purified before use. Such purification methods include, but are not limited to, chromatography including gel filtration, electrophoresis, dialysis, ultrafiltration, salting out, and precipitation with an organic solvent.

More specifically, examples of the chromatography include chromatography with a column packed with dextran or polyacrylamide or alumina.

Examples of dialysis include dialysis using a semipermeable membrane such as a collodion membrane and a cellophane membrane.

Examples of the ultrafiltration include filtration using a porous membrane made of an inorganic material and a resin.

As the aggregating agent for salting out, for example, ammonium sulfate, sodium chloride, barium carbonate, etc. can be used, and for precipitation treatment with an organic solvent, for example, methanol, ethanol, acetone, etc. can be used. It is preferable to carry out under the condition that the active ingredient in the product is not modified.

When the composition of the present invention is extracted from bamboos or bamboo grasses, it is preferable to use the rhizome of the bamboos or bamboo grasses. The rhizome refers to the rhizome of bamboos or bamboo grasses, but in the present invention, it is not limited to the range of names according to the scientific classification of plant tissues.

The bamboos or bamboo grasses from which the substance of the present invention can be extracted are selected, for example, from the following various types, but are not limited thereto.

Single-axis bamboos 1. Pleurotus ostreatus, sardine, oyster mushroom,
Amberjack, Shipotiku, Astragalus, Kisimatake,
1. White oyster mushrooms, bee honey, black bee squirrels, unmonchik, yellow squirrels, sesame mushrooms, kabrochik, shihamachiku, hoteichiku, ginmeihachiku, shimahoteichiku and kechiki. 2. Pleurotus cornucopiae Pleurotus cornucopiae, Pleurotus cornucopiae, Pleurotus cornucopiae, Pleurotus cornucopiae, Pleurotus cornucopiae and P. mellifera Tochiku genus Touchiku and Suzukonarihira 4. 4. Citrus spp. Oxameza genus Oxamexasa axis-type bamboos 6. Bambusa multiplex genus Bambusa multiplex, Hououchiku, Houshouchiku, Suhouchiku and Taizanchiku bamboo grass 7. 7. Pleurotus genus Kanzantiku, Taimenchik, Ryukyuchiku, Medake, Hagarari medaka, Hakonedake, Azmanezasa, Himeshimatake, Kenezasa, Oshiromachiku, Akebonozasa, Kamuroza and Iyosare 8. Sasa genus Miyakosasa, Kumasasa, Bamboo shoots, Rakkyochiku, Nemagaritake, Pleurotus chinensis and Kintaisasa Also, the commonly known bamboo shoots are enlarged spores of the rhizomes of Mosouchi. Bamboo shoots are also included in the bamboos and bamboo grasses referred to in the present invention.

Further, even in the composition extracted from the same type of bamboo or bamboo grass, some variations in antitumor activity were observed depending on their living areas. Factors considered to be this are rocks in the habitat, soil, bacteria in the soil, temperature, humidity, rainfall, location conditions (sunlight hours, wind), symbiotic plants, and animals living in the living area. Bamboo mushrooms that grow in the north and cold regions are preferable. In particular, those in the Tohoku region are suitable, and the metal species and concentration contained in the bedrock,
In addition, the species and amount of bacteria in the soil are considered to be a major factor.

The compositions of the present invention are at least in vivo
It has antitumor activity in tro. The antitumor agent comprising the composition of the present invention is a tumor cell, particularly leukemia,
A mode in which a remarkable activity is observed against tumor cells derived from adult T-cell leukemia, and the response from the administration of the drug to the expression of the effect (antitumor activity) is specific, and the mechanism of action is different from that of conventional antitumor agents Was presented.

That is, after administration of the drug, there is a period in which its effect cannot be confirmed, but a strong cell killing effect is exhibited from a certain point. Moreover, although the tumor cell nucleus was reduced, there was no disintegration image, and balloon-like cells observed in conventional antitumor agents were hardly seen.

These indicate that the mechanism of action of the antitumor agent by the composition of the present invention may be different from the conventional ones, and antitumor activity is expressed by acting on protein or RNA. The possibility is estimated.

That is, the composition of the present invention can be expected to have a cytocidal effect not only on tumor cells but also on virus-infected cells such as adult T-cell leukemia.

Accordingly, the composition of the present invention comprises hepatitis B, non-A
It may be used as an antiviral agent against diseases such as non-hepatitis B and AIDS.

When the composition is used as an antitumor agent, it can be administered in any dosage form and administration route.

The dosage form includes, for example, tablets, capsules, syrups and the like, and may further contain pharmacologically acceptable excipients, auxiliaries and stabilizers.

The route of administration includes, but is not limited to, subcutaneous injection, intramuscular injection, intravenous injection, and oral administration.

The dose of the composition of the present invention is 1
1 to 6 kg, about 0.001 mg to about 10 g per day
Administer in divided doses.

However, since the dose varies depending on the animal species (including human), age, medical condition, drug susceptibility, etc., the dose outside the above range may occur. In that case, a person skilled in the art will set the optimal dose.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

[0033]

【Example】

[Example 1] Preparation of Rhizome Extract of Bamboo Shoots (Bamboo) (1-1) Red rhizomes that were densely present in the roots of the uniaxial type Bamboo sp. It was crushed using a pressing machine, the squeezed liquid and the crushed granules were taken in gauze, and this was squeezed to obtain a bamboo shoot rhizome squeezed liquid. By filtering this bamboo shoot rhizome pressing liquid using a filter paper (Toyo Roshi Kaisha, Ltd.), a colorless viscous liquid containing the composition of the present invention was obtained. This was named Solution A. The pH of the liquid A exhibited an acidity of about 5.4.

The composition of the present invention was soluble in water in acidity and alkalinity, but turbidity was generated in the vicinity of neutrality (when the solution A was diluted with water).

Distilled water (9.0 ml) was added to the liquid (A) (1.0 ml) to make the total amount 10.0 ml. 1 ml of this 10-fold diluted solution was collected and 9.0 ml of 0.1N NaOH solution was added to make the total volume 10.0 ml. When the ultraviolet absorption of this solution was measured, absorption was observed at 240 nm, 278 nm and 330 nm. Similarly, 1 ml was taken from the 10-fold diluted solution, and 9.0 ml of 0.1N HCl solution was added to bring the total volume to 1
It was 251 when the ultraviolet absorption of the solution of 0.0 ml was measured.
Absorption was observed at nm and 323 nm.

The ninhydrin reaction of the liquid A was positive, and the average molecular weight by gel filtration chromatography was about 10,000-
It was about 30,000.

(1-2) The finger-shaped rhizomes closely packed in the roots of the uniaxial Bamboo genus Bamboo genus Mosouchi were collected at 3 to 4 cm, and the fibrous portion covering the outside was removed. The white flexible strands that were present inside were obtained and collected. These were crushed using a pressing machine, the squeezed liquid and the crushed granules were taken in gauze, and this was squeezed to obtain a bamboo shoot root squeezed liquid. This bamboo shoot rhizome squeezing liquid is a filter paper (Toyo Filter Paper Co., Ltd.)
Was naturally filtered to obtain a colorless viscous liquid containing the composition of the present invention, which was designated as solution B.

Distilled water (9.0 ml) was added to the solution (B) (1.0 ml) to make the total amount 10.0 ml. 1 ml of this 10-fold diluted solution was collected and 9.0 ml of 0.1N NaOH solution was added to make the total volume 10.0 ml. When the ultraviolet absorption of this solution was measured, absorption was observed at 275 nm and 328 nm.
Similarly, 1 ml was taken from the 10-fold diluted solution,
The UV absorption of a solution in which 9.0 ml of N HCl solution was added to make the total volume 10.0 ml was 250 nm and 32.
Absorption was observed at 0 nm.

The ninhydrin reaction of solution B was positive, and the average molecular weight by gel filtration chromatography was about 10,000-
It was about 30,000. In addition, about 1 mg of an evaporation residue was obtained by evaporating and drying 1 ml of the liquid B to dryness, and the residue was hardly soluble in organic solvents such as benzene and chloroform and soluble in alcohol.

[Example 2] Preparation by alcohol extraction from Rhododendron japonicus (2-1) Crushed Rhododendron japonicum rhizome 33.64
g in a 1 liter round bottom flask,
l of ethanol was added, and the mixture was extracted on a water bath for about 3 hours while warm, and the insoluble matter was filtered off to obtain a yellow ethanol solution. Next, the extracted solution was concentrated to dryness under reduced pressure, the obtained solid residue was dissolved in a small amount of ethanol at a warm time, an appropriate amount of water was added, and the mixture was left to stand, and a pale yellow solid precipitate came out. This substance was ninhydrin-positive, soluble in methanol and ethanol, sparingly soluble in water, soluble in warm water, exhibited pH 4.8 (aqueous solution), and exhibited antitumor activity.

Furthermore, 100 ml of the ethanol extraction residue was added.
Was similarly extracted with methanol for 3 hours, filtered and then concentrated to dryness under reduced pressure to obtain a pale yellow solid. This substance showed similar physical properties and biological activity to those obtained by ethanol extraction.

(2-2) Crushed Pleurotus cornucopiae 45
g in the same manner as in the above (2-1), 150 ml of ethanol,
Then, after extraction with 100 ml of methanol at a warm temperature, the combined extract was passed through a column packed with activated alumina to adsorb the target substance. It was eluted with hydrochloric acid having a pH of 4.0, the eluate was neutralized, concentrated under reduced pressure and evaporated to dryness to obtain a powdery pale yellow solid. Furthermore, the target substance was recovered by passing methanol through alumina of a hydrochloric acid eluate. Total amount about 50m
g of a pale yellow solid was obtained, and this substance showed the same physical properties and biological activity as those obtained in the above (2-1).

[Example 3] Measurement of antitumor activity (3-1) Culture of tumor cells Human lymphoblastic leukemia cell line (adult T cell leukemia cell line; RPMI-8402 strain) or mouse leukemia cell line (L 1210) was cultivated for 6 days under normal culture conditions using a non-selective medium (normal medium).

(3-2) Method for adjusting cell number Adult T-cell leukemia cells (RPMI-8) in a non-selective medium.
402) or mouse leukemia cell (L-1210) culture medium was added to adjust the cell number to 10 ⁵ cells / ml.

(3-3) Measurement of antitumor activity-1 0 μl of solution B sterilized by filtration with Millipore filter
(Control), 0.5 μl, 5 μl, 50 μl and 180 μl were collected and added (administered) to 5 ml of the above-mentioned adult T cell leukemia cell culture medium. Furthermore, the fluorescent dye DAP was added so as to be 1 μg / ml, and the chromosome of tumor cells was colored. Then, every 24 hours, B
0.5 ml from the tumor cell culture solution containing broth and DAP
Was collected, and images magnified 5500 times by a fluorescence microscope were continuously recorded using a dynamic observation device (VTR monitor). From this recorded image, the total number of tumor cells and the number of deaths were counted. Here, the number of deaths of tumor cells refers to "cell death of tumor cells" when the tumor cells meet the following conditions "release of nuclear fragments to the outside of the cell", "cell lysis" or "liquid outflow of nuclear material". It was considered and measured.

The above procedure was continued for 6 days, and the antitumor activity of solution B was calculated as the tumor cell killing rate and the total cell number (growth inhibition rate). The time course of the cell killing rate is shown in FIG. Fig. 2 shows the cell killing rate and total cell number (growth suppression rate) on day 2.
It was shown to.

The present dynamic observation method is capable of confirming the cell morphology in real time, and considers not only the manner in which a physiologically active substance is damaged by a cell, but also the changing process of the cell, and the previous action of the physiologically active substance. It is possible.

Regarding the change in cell morphology after administration of the composition of the present invention, no image of tumor cell disintegration was observed,
The cell nuclei shrank over time, and the cytoplasm collapsed. A wide variety of changes in cell morphology, such as cell ballooning, found in common anticancer agents have not been confirmed.

(3-4) Measurement of antitumor activity-2 0 μl (control), 10 μl from solution A sterilized by filtration
1, 50 μl and 150 μl each are taken, and the adult T
Antitumor activity was measured in the same manner as in (3-3) except that each was added to 5 ml of cell leukemia cell culture medium. The time course of the cell killing rate of solution A calculated from this is shown in FIG. Also, regarding the change in cell morphology, the same behavior as in (3-3) was confirmed.

(3-5) Measurement of antitumor activity-3 Two drops of Tween-80 were added to about 1 mg of the alcohol extract (pale yellow solid) obtained in (2-1) of [Example 2], and further added. Sterilized water was added to make a total volume of 3 ml, and after filter sterilization, 0 μl (control), 10 μl, 50 μl, and 150 μl were collected, and 10 ⁵ cell / ml of adult T
Cell leukemia cells (RPMI-8402) or mouse leukemia cells (L-1210) were added to 5 ml of a culture solution,
The antitumor activity was measured in the same manner as in -3). The time course of the cell killing rate of the substance of the present invention calculated from this is shown in FIG.
And shown in FIG. As is clear from the figure, for adult T-cell leukemia cells, 150 μl gave about 100
%, About 90% of the cell killing effect was obtained on the 4th day with 50 μl, and with L-1210 cells, 1 μl was obtained with 150 μl.
A cell killing effect of about 100% on the day and about 90% at 70 hours with 50 μl was obtained.

Regarding the change of cell morphology (3-
The same behavior as 3) was confirmed.

The same test was performed on the pale yellow solid obtained in (2-2) of [Example 2], and the same cell killing activity was confirmed for both tumor cells.

Example 4 Preparation Example of Antitumor Agent Composition According to the Present Invention 100 mg of the solution B obtained in [Example 1] was placed in a 10 ml ampoule tube, and nitrogen was sealed therein, and then the ampoule was closed. From this, 10 ampoules were created.

[0054]

INDUSTRIAL APPLICABILITY Tumor cells, particularly leukemia and adult T-cell leukemia cells, exhibit remarkable cytocidal activity and / or growth inhibitory activity and are useful as antitumor agents.

[Brief description of drawings]

FIG. 1 Adult T-cell leukemia cells (RPMI-8402)
5 shows the time-dependent change in the tumoricidal cell rate of the composition of the present invention (solution B) against. In the system added with 50 μl or more, remarkable cytotoxicity is observed from the 4th day after administration.

FIG. 2 Adult T-cell leukemia cells (RPMI-8402)
Antitumor activity at each concentration of solution B on day 6 after administration to. In the figure, the white bar represents the total cell number (right vertical axis), and the black bar represents the cell killing rate (left vertical axis). Remarkable cytotoxicity is observed in the system added with 50 μl or more. In addition, a decrease in total cell number (inhibition of tumor cell growth) is also observed in the system containing 0.5 and 5 μl.

FIG. 3 Adult T-cell leukemia cells (RPMI-8402)
5 shows the time-dependent change in the tumoricidal cell rate of the composition of the present invention (Solution A). In the system added with 150 μl or more, remarkable cytotoxicity is observed from the 4th day after administration.

FIG. 4 shows the time course of the tumoricidal cell rate of the composition of the present invention (alcohol extract of (2-1) of [Example 2]) against mouse leukemia cell line (L-1210).

FIG. 5: Adult T-cell leukemia cells (RPMI-8402)
2 shows the time-dependent change in the tumoricidal cell rate of the composition of the present invention (alcohol extract of (2-1) in [Example 2]) against.

Claims (2)

[Claims] 1. An antitumor agent composition which can be extracted from a plant belonging to bamboo or bamboo grass, wherein the composition has a positive ninhydrin reaction and an average molecular weight by gel filtration chromatography of about 10,000 to about 30,000. It has a pH of 4.0 to about 7.0, is poorly soluble in organic solvents such as benzene and chloroform, soluble in alcohol, soluble in acidic or alkaline aqueous solution, and has an ultraviolet-visible absorption in the aqueous solution of around 250 nm. An antitumor agent composition having (acidic) and around 280 nm (alkaline). 2. The plant belonging to the bamboo or sasa genus is moss of the genus Physcomitrella patens, Kikkochiku, Madake, Kinmeichiku, Ogongonchi, Shipotchiku, Konshimadake, Kisimatake, Kakurodoke, Hachiku, Kurochiku, Unmonchiku,
Yellow-tailed moss, sycamore, cabrochik, shihamachi, hoteichiku, chinmeihachiku, shimahoteichiku and kechiki, as well as genus Narihidake, moss moss, moss moss, citrus moss , And porpoise genus porphyra, porphyra cinerea, phoenix vulgaris, crocodile and cypress, and the genus Physcomitrium spp.
Taimenchiku, Ryukyuchiku, Medake, Hagariwamedake, Hazelnut mushroom, Azmanezasa, Himesimatake, Kenezasa, Oshiromachiku, Akebonosasa, Kamuroza and Iyosudare, and selected from the group of the genus Lactarius, Kamaza, Yadachi, and Ryakugaku, Rakugachi The antitumor agent composition according to claim 1.