JPH0410447B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the formula () [In the formula, when R ₁ is a hydrogen atom, R ₂ is α-L-
Rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl group, when R ₁ is a hydroxyl group, R ₂
is β-D-xylopyranosyl-(1→2)-α-L
- represents an arabinopyranosyl group] This is an anticancer agent containing a triterpene glycoside represented by the following as an active ingredient. As a result of screening tests for the antitumor effects of various herbal medicines, the present inventors found that the root of Patriniascabriosae folia Fisch of the family Valerianaceae and the root of Akebia of the family Lardizabalaceae.
quinata (Thunb.) Decne] fruits have been found to have excellent anticancer effects. Focusing on this fact,
While separating its active ingredients, the inventors discovered that the triterpene glycoside represented by the formula () exhibits a strong anticancer effect, and based on this new knowledge, they completed the present invention. To obtain the triterpene glycoside represented by the formula (), which is the active ingredient of the drug of the present invention, for example, extract the root of Ominae or the fruit of Akebia with methanol and dissolve the extract obtained in water. After extracting with ether, the aqueous layer is further extracted with n-butanol or an acid/n-butanol mixture. After concentrating the n-butanol layer or the acetic acid:n-butanol mixture layer under reduced pressure, a methanol-acetone mixture is added to form a precipitate. The precipitate generated from the n-butanol layer was hydrolyzed by adding 0.5N KOH, then neutralized with 0.5N HCl, and n-butanol was added to this to remove non-hydrolyzed products with n-butanol. After extraction and concentration of the n-butanol layer under reduced pressure, it was subjected to silica gel column chromatography and eluted with chloroform-methanol-water (25:3:0.3) to obtain the formula ().
The triterpene glycoside shown is obtained. In addition, the precipitate generated from the n-butanol mixture layer is
After dissolving in n-butanol, it is subjected to silica gel column chromatography and eluted with chloroform-methanol-water (25:3:0.3) to obtain the triterpene glycoside represented by formula (). Methanol extraction of the above-mentioned raw material herbal medicines (root of Ominae and fruit of Akebia) can be carried out at room temperature or with heating. It is desirable to do so. A specific example of the production of the triterpene glycoside represented by formula (), which is the active ingredient of the drug of the present invention, is as follows. Specific example 1 After cutting the root of Ominae and drying it, take 10g and put it in a flask equipped with a reflux condenser.
ml of methanol was added and the mixture was heated and extracted on a 75°C water bath for 1 hour. After filtering the extract, add the residue again.
100 ml of methanol was added and the mixture was heated and extracted on a 75°C water bath for 1 hour. The extract was filtered, combined with the previous extract, 200 ml of water was added, and concentrated under reduced pressure to 50 ml. After extracting the resulting concentrate with 50 ml of ether, the aqueous layer was further saturated with water.
-Extracted three times with 50 ml of butanol. The n-butanol layers were combined, washed with 50 ml of water saturated with n-butanol, concentrated under reduced pressure to 50 ml, and added to this.
200 ml of methanol-acetone mixture was added, and the mixture was left to stand at room temperature for 3 hours, centrifuged, and the resulting precipitate was collected. The obtained precipitate was dissolved in 30 ml of 0.5N KOH solution and hydrolyzed, and then neutralized with 0.5N HCl.
This was extracted three times with 50 ml of n-butanol saturated with water, and the n-butanol layers were combined, washed with 50 ml of water saturated with n-butanol, and then concentrated under reduced pressure to 10 ml. This was subjected to silica gel column chromatography using silica gel 40, eluted with chloroform-methanol-water (25:3:0.3), and 10 ml of the eluate was collected using a fraction collector.
Aliquots were taken one by one. A portion of each fraction obtained was subjected to thin layer chromatography using silica gel G, and chloroform-methanol-water (25:
3:0.3), the fractions containing triterpene glycosides were combined, and 100 ml of water was added to concentrate to 30 ml. The powder obtained by freeze-drying this was dissolved in 5 ml of distilled water, 20 ml of methanol-acetone mixture was added thereto, left for 2 hours, the precipitated crystals were filtered out, and dried in a desiccator. The active ingredient of the drug of the present invention is represented by the formula (), where R ₁ is a hydrogen atom and R ₂ is α-L-
Rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl group, white needle-like oleanoic acid 3-O-α-L-rhamnopyranosyl-
25 mg of (1→2)-α-L-arabinopyranoside (hereinafter referred to as triterpene glycoside A) was obtained. Specific Example 2 After drying and pulverizing Akebia fruit, 1 kg was taken, 100 g of methanol was added, and the fruit was heated and extracted in an extractor for 1 hour, followed by centrifugation when cold to obtain an extract. 10 ml of methanol was added to the residue again and an extract was obtained in the same manner. This extract was combined with the previous extract and concentrated to a concentration of 10% using a falling membrane concentrator, and then further concentrated to a concentration of 5% by adding 10% of water. After extracting the obtained concentrate twice with ether (1), the aqueous layer was further extracted with acetic acid:n-butanol (2:1) mixture (5).
Extracted three times. Acetic acid: n-butanol (2:
1) Combine the mixed liquid layers and add acetic acid: n-butanol (2:
1) After washing the mixed solution with saturated water 5, it was concentrated under reduced pressure to give 5, and to this was added 20 methanol-
An acetone mixture was added, the mixture was left to stand at room temperature for 3 hours, and centrifuged, and the resulting precipitate was collected. After dissolving the obtained precipitate in 500 ml of n-butanol, 50 ml each was subjected to silica gel column chromatography using silica gel 40, eluted with chloroform-methanol-water (25:3:0.3), and the eluate was A fraction of 50 ml was collected using a fraction collector. A portion of each fraction obtained was subjected to thin layer chromatography using silica gel G and chloroform-
Developed with methanol-water (25:3:0.3) and combined the fractions containing triterpene glycosides.
Add 1 portion of water and concentrate to 500 ml. The powder obtained by freeze-drying this was dissolved in 100ml of distilled water,
500 ml of a methanol-acetone mixture was added to this and left to stand for ₂ hours, filtering out the precipitated crystals and drying in a desiccator. At the atom, R ₂ is β-D-xylopyranosyl-(1→
2) -α-L-arabinopyranosyl group, white needle-like hederagenin 3-O-β-D-xylopyranosyl-(1→2)-α-L-arabinopyranoside (hereinafter referred to as triterpene) (referred to as glycoside B)
3.2g was obtained. The properties of triterpene glycoside A and triterpene glycoside B obtained in Specific Examples 1 and 2 were as follows. Triterpene glycoside A Color/Property White needle crystals Melting point 220℃ (measured value) 220-224℃ (literature value) Specific optical rotation [α] ²⁰ _D = +10° (measured value) (c = 1.05,
MeOH) [α] ²⁰ _D = +10° (literature value) (c = 1.05,
MeOH) [Literature name: Chem.Pharm.Bull.27(10)
2388-2393 (1979)] Triterpene glycoside A Color/property White needle-like crystals Melting point 249℃ (measured value) 249.5-25035℃ (literature value) Specific optical rotation [α] ²⁰ _D = +37.5° (measured value ) (c=1.05,
MeOH) [α] ²⁰ _D = +37° (literature value) (c = 1.05,
MeOH) [Literature name: Chem.Pharm.Bull.24(9)
1935-1939 (1972)] Next, the anticancer effect of the drug of the present invention and its safety will be explained using experimental examples. Experimental Example 1 Ehrlichi's ascites carcinoma was transplanted into an ICR albino mouse, and one week later, ascites was collected, and 1×10 ⁷ Ehrrich's ascites carcinoma cells per mouse were transplanted into the peritoneal cavity of another mouse. There were 5 animals in each group, and each test drug (triterpene glycoside A and triterpene glycoside B) was intraperitoneally administered once a day for 5 days starting the day after cancer transplantation. Seven days after transplantation, the abdomen was opened to remove ascites fluid, which was centrifuged at 2000G for 5 minutes to separate cancer cells. Volume of this cancer cell (Packed Cell Volume)
The ratio of the difference in the volume of cancer cells between the control group and the administration group to the volume of cancer cells in the control group is taken as the cancer cell growth inhibition rate, and a dose-effect curve is drawn. Led the amount of inhibition by 50%. In addition, in the control group, physiological saline was administered intraperitoneally. The results are shown in Table 1.

[Table] Experimental Example 2 Effect of the drug of the present invention on Hela cultured cells MEM medium (manufactured by Eagle) to which 5% bovine serum was added was used as a maintenance medium. Place ⁴ Hela cells (1 x 10) and maintenance medium in a 35 mm plastic Petri dish,
Cultured in an incubator at 5% _CO2 . twenty four
After a period of time, when HeLa cells were observed to have implanted in approximately 50% of the bottom of the Petri dish, test drugs (triterpene glycoside A and triterpene glycoside B) adjusted to various concentrations were added and left in contact for 15 minutes. The maintenance medium was then removed and washed twice with Hanks solution. Maintenance medium was added again to this, and after culturing for 4 days at 37°C in a 5% CO ₂ incubator, the cells were stained with crystal violet, and 8 to 10 cell populations were counted as one colony under a microscope. . The control group was contacted only with physiological saline, and the HeLa cell colony formation inhibition rate is expressed as a percentage of the number of colonies in the control group relative to the difference in the number of colonies between the control group and the test drug group, and is 50%. Inhibitory concentration (IC ₅₀ ) values were calculated. The results are shown in Table 2.

[Table] As is clear from the results shown in Tables 1 and 2, this substance exhibited excellent antitumor activity both in vivo and in vitro. Experimental Example 3 Acute toxicity test of the drug of the present invention Using ddY mice (10 mice per group), the drug of the present invention was administered orally, intraperitoneally, and intravenously.The _LD50 value was the same as that of oral administration. case 3000
When administered intraperitoneally and intravenously, the dose was 120 mg/Kg or more. Considering the results of the above examples, the effective dosage of the drug of the present invention as an antitumor agent is determined by the age of the patient,
Although it varies depending on the body weight and severity of the disease, it is usually appropriate for adults to take 500 to 700 mg at a time, up to 2 to 3 times a day, or up to 700 mg a day by intravenous drip or intramuscular injection. Within this range, the drug of the present invention appears to exhibit high safety. The triterpene glycoside, which is the active ingredient of the drug of the present invention, can be used as an anticancer drug as it is, but it is necessary to add excipients, adjuvants, etc. that are used in conventional drugs to it, and to use it as it is as an anticancer drug. According to the information, it can also be used in preparations such as powders, granules, tablets, capsules, syrups, suppositories, enteric-coated preparations and injections. For oral administration, they can be formulated into tablets, pills, capsules, powders, granules, etc. using at least one excipient such as starch, lactose, sucrose, mannitrate, carboxymethyl cellulose, etc. In addition to the above-mentioned excipients, this type of preparation may contain, for example, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc, binders such as dextrin, crystalline cellulose, polyvinylpyrrolidone, gum arabic, corn starch, and gelatin. A disintegrating agent such as an agent, potato starch, carboxymethyl cellulose, etc. can be used. It can also be administered as a suspension, emulsion, syrup, or elixir, and these dosage forms may contain flavoring agents and coloring agents. As a parenteral preparation, it can be mixed with a suitable base to form a cream, ointment, poultice, or suppository. As a diluent, distilled water for injection, physiological saline, aqueous dextrose solution, vegetable oil for injection, propylene glycol, polyethylene glycol, etc. can generally be used. Furthermore, if necessary, an isotonizing agent, a solubilizing agent, a stabilizer, a preservative, a soothing agent, etc. may be added as appropriate. Moreover, in the case of this type of dosage form, it is desirable to dissolve it in a sterile injection medium. EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto in any way. Example 1 Triterpene glycoside obtained in specific example 1
200 g of A was mixed with 95 g of microcrystalline cellulose and 5 g of magnesium stearate, and the mixture was compressed using a single-shot tablet machine to produce tablets with a diameter of 7 mm and a weight of 300 mg. One tablet of the present invention contains 200 mg of triterpene glycoside A obtained in Example 1. Take 3 tablets at a time, 3 times a day, depending on your symptoms. Example 2 Triterpene glycoside obtained in specific example 2
100 g of B was mixed with 400 g of corn starch, water was added and kneaded, and the mixture was granulated using a screen having a mesh size of 1 mm x 1 mm and dried to obtain granules. 1 g of this granule contains 200 mg of triterpene glycoside B obtained in Example 2. This granule is taken at a dose of 3g three times a day depending on the symptoms. Example 3 Triterpene glycoside obtained in specific example 2
100 g of B was mixed with 90 g of lactose and 10 g of magnesium stearate, and 500 mg each was filled into hard capsules. One capsule of the present invention contains 250 mg of triterpene glycoside B obtained in Specific Example 2. This capsule formulation is taken at a time of 3 capsules twice a day depending on the symptoms. Example 4 Triterpene glycoside obtained in specific example 1
70 g of A was dissolved in 250 ml of water, and 3 ml of orange essence and simple syrup were added to make a syrup preparation with a total volume of 1000 ml. 1 ml of this syrup contains 70 mg of triterpene glycoside A obtained in Example 1. This syrup is 7 to 10 ml at a time, 3 times a day, depending on the symptoms.
Take multiple doses. Example 5 The tablet obtained in Example 1 was coated with a film of cellulose acetate phthalate (CAP) to form an enteric agent. One tablet of the present invention contains 200 mg of triterpene glycoside A obtained in Example 1. Take 3 tablets at a time, 2 to 3 times a day, depending on your symptoms. Example 6 Triterpene glycoside obtained in specific example 1
100 g of A was mixed with 90 g of lactose and 10 g of magnesium stearate, and 400 mg each was filled into enteric coated capsules. One capsule of the present enteric-coated capsule contains 200 mg of triterpene glycoside A obtained in Example 1. This capsule is taken at a time, 3 capsules, 3 times a day, depending on the symptoms. Example 7 Triterpene glycoside obtained in specific example 1
50 g of A was dissolved in 1 part of distilled water for injection heated to 60°C according to a conventional method for manufacturing injections, and after making the solution isotonic with sodium chloride, the solution was sealed in an ampoule. 1 ml of this injection contains 50 mg of triterpene glycoside A obtained in Example 1. Depending on the symptoms, up to 14ml of this injection can be injected intramuscularly per day, or intravenously administered at the same time as 200ml of Ringer's solution or other infusion.

Claims (1)

[Claims] 1 Formula () [In the formula, when R ₁ is a hydrogen atom, R ₂ is α-L-
Rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl group, when R ₁ is a hydroxyl group, R ₂
is β-D-xylopyranosyl-(1→2)-α-L
- represents an arabinopyranosyl group] An anticancer agent containing a triterpene glycoside represented by the following as an active ingredient.