JPS638088B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anticancer agent containing an ellagitannin compound. Tannin is a type of polyphenol derivative contained in plants, and is broadly classified into two types: hydrolyzable tannins and condensed tannins.Furthermore, among hydrolyzable tannins, ellagic acid is produced by hydrolysis. are collectively called ellagitannins. Since ancient times, tannins have been used as leather tanning agents and as oral medicines for gastrointestinal disorders, but the present inventors have discovered that some of the ellagitannins have mutated into certain carcinogens. Focusing on significantly suppressing the autogenicity,
As a result of intensive research on ellagitannins extracted from various plants, nobotanin A, nobotanin C, gemin A, lugosine D, lugosine E, isorgosine D, cornuciin A, corialiin A, corialiin C, and enotein B were found to be mutagenic carcinogens. It has been found that in addition to suppressive effects, it also has an extremely strong cancer cell suppressive effect. The present invention has been made based on this knowledge. Regarding ellagic acid, which is a common hydrolysis product of each ellagitannin, there is no known effect on the mutagenicity of benzo[α]pyrene-7,8-diol-9,10-epoxide, which is a carcinogen. However, the effect of suppressing cancer cells, that is, the anticancer effect, has not been clarified. Therefore, the anticancer activity of the ellagitannin compounds extracted from plants and confirmed according to the present invention was discovered for the first time by the present inventors. The anticancer agent of the present invention is at least one compound selected from the group of ellagitannin compounds consisting of nobotanin A, nobotanin C, gemin A, lugosine D, lugosine E, isorgosine D, cornuciin A, corialiin A, corialiin C, and enotein B. It is characterized by containing as an active ingredient. The structural formula and properties of each compound are shown below. Color/Property: Gray-white amorphous powder Specific optical rotation: [α] _D +51° (c=1.0, MeOH) Elemental analysis: Molecular formula C ₇₅ H ₅₂ O ₄₈・8H ₂ O Theoretical value (%) C, 48.22; H, 3.54 Actual value (%) C, 48.25; H, 3.80 Fast atom bombardment mass spectrum (FAB Mass
Spectrum): Parent peak undetectable UV absorption spectrum (UV Spectrum): λ ^MeOH _nax
nm (logε): 219 (5.07), 270 (4.77) Circular dichroism spectrum (CD Spectrum):
(MeOH), [θ] (nm): +242000 (227), +
197000 (235), -71000 (262), +34000 (282), -
19000 (303) Thin layer chromatography: (cellulose, 7% acetic acid): Rf0.47 (FeCl ₃ , gray-blue) Proton nuclear magnetic resonance spectrum ( ¹ H-NMR
Spectrum): δppm (400MHz, acetone- _d6 ):
7.19, 7.16, 6.99, 6.92, 6.75, 6.65, 6.53,
6.49, 6.47, 6.45, 6.44, 6.43, 6.41, 6.33,
6.32, 6.22 Color/Property: Gray-white amorphous powder Specific optical rotation: [α] _D +38° (c=1.0, MeOH) Elemental analysis: Molecular formula C ₁₁₆ H ₈₀ O ₇₄・22H ₂ O Theoretical value (%) C, 45.62; H, 4.09 Actual value (%) C, 45.64; H, 3.81 FAB-MS: Parent peak undetectable UV: λ ^MeOH _nax nm (logε): 223 (5.29), 270 (5.
00) CD: (MeOH), [θ] (nm): +457000 (227),
+356000 (235), -124000 (262), +45000 (283),
-45000 (312) Thin layer chromatography: (cellulose, 7% acetic acid): Rf0.41 (FeCl ₃ gray-blue) ¹ H-NMR: δppm (200MHz, acetone-d ₆ ):
7.30, 7.28, 7.19, 7.18, 7.14, 7.13, 7.04,
7.00, 6.99, 6.71, 6.68, 6.56, 6.53, 6.51,
6.50, 6.49, 6.48, 6.45, 6.40, 6.26, 6.22,
6.16, 6.15 Color/Property: Gray-white amorphous powder Specific optical rotation: [α] _D +156° (c=1.6, EtOH) Elemental analysis: Molecular formula C ₈₂ H ₅₆ O ₅₂・7H ₂ O Theoretical value (%) C, 49.3; H, 3.5 Actual value (%) C, 49.2; H, 3.5 FAB-MS: m/z1873 [(M+H) ⁺ ] UV: λ ^MeOH _nax nm (logε): 222 (5.26), 272 (4.
92) CD: (MeOH), [θ] (nm); +335000 (235),
-68000 (262), +65000 (283) Thin layer chromatography: (cellulose, 7% acetic acid): Rf0.31 ¹ H-NMR: δppm (200MHz, acetone- _d6 ):
7.35, 7.31, 7.05, 7.00, 6.88, 6.70, 6.67,
6.65, 6.56, 6.51, 6.48, 6.38, 6.17, 5.85,
5.59, 5.54, 5.38, 5.31, 5.24, 5.22, 5.19,
4.52, 3.79, 3.69 Color/Property: Gray-white amorphous powder Specific optical rotation: [α] _D +118° (c=1.0, acetone) Elemental analysis: Molecular formula C ₈₂ H ₅₈ O ₅₂・9H ₂ O Theoretical value (%) C, 48.34; H, 3.76 Actual value (%) C, 48.31; H, 3.64 FAB MS: m/z1898 [(M+Na) ⁺ ] UV: λ ^MeOH _nax nm (logε): 219 (5.17), 227 (4.
84) CD: (MeOH) [θ] (nm): +235000 (224), -
35000 (258), +78000 (280) Thin layer chromatography: (cellulose, 7% acetic acid): Rf0.29 ¹ H-NMR: δppm (200MHz, acetone- _d6 )
7.149, 7.032, 7.025, 7.02, 6.99, 7.146, 6.67,
6.49, 6.47, 6.26, 6.20, 6.15, 5.85, 5.81,
5.61, 5.54, 5.33, 5.16, 4.54, 4.48, 3.83,
3.79 Color/Property: Pale and amorphous powder Specific optical rotation: [α] _D +140° (c=1, acetone) Elemental analysis: Molecular formula C ₇₅ H ₅₄ O ₄₈・5H ₂ O Theoretical value (%) C, 49.58; H, 3.56 Actual value (%) C, 49.78; H, 3.27 FAB-MS: Parent peak undetectable UV: λ ^MeOH _nax nm (logε): 220 (5.19), 275 (4.
87) CD: (MeOH) [θ] (nm): +224000 (224), -
53000 (258), +96000 (282) Thin layer chromatography: (cellulose, 7% acetic acid): Rf0.34 ¹ H-NMR: δppm (200MHz, acetone- _d6 ):
7.16, 7.09, 7.08, 7.034, 7.028, 6.99, 6.67,
6.49, 6.47, 6.251, 6.245, 6.17 Color/Appearance: Pale and amorphous powder Specific optical rotation: [α] _D +75° (c=0.5, MeOH) Elemental analysis: Molecular formula C ₈₂ H ₅₈ O ₅₂・7H ₂ O Theoretical value (%) C, 49.20; H, 3.62 Actual value (%) C, 49.49; H, 3.77 FAB-MS: m/z1897 [(M+Na) ⁺ ] UV: λ ^MeOH _nax nm (logε): 218 (5.33) 277
(4.86) CD: (MeOH) [θ] (nm); +31100 (285), -
60200 (255), +24.95 (228) Thin layer chromatography: (cellulose, 7% acetic acid) Rf0.40 ¹ H-NMR: δppm (400MHz, acetone- _d6 );
7.24, 7.12, 7.07, 7.01, 6.99, 6.94, 6.69,
6.65, 6.51, 6.19, 6.17, 6.12, 5.82, 5.63,
5.56, 5.54, 5.30, 5.22, 5.21, 5.10, 4.51,
4.34, 3.87, 3.86 Color/Property: Gray-white amorphous powder Specific optical rotation: [α] _D +78° (c=1.0, MeOH) Elemental analysis: Molecular formula C ₆₈ H ₅₀ O ₄₄・11H ₂ O Theoretical value (%) C, 46.16; H, 4.10 Actual value (%) C, 46.21; H, 3.97 FAB-MS: m/z1593 [(M+Na) ⁺ ] UV: λ ^MeOH _nax nm (logε): 218 (5.14), 271 (4.
81) CD: (MeOH) [θ] (nm); +330000 (220), +
200000 (230), -130000 (260), +130000 (285) Thin layer chromatography; (cellulose, 7% acetic acid): Rf0.34 ¹ H-NMR: δppm (400MHz, acetone- _d6 ):
Aromatic protons 7.05-7.04 (2Hin total), 7.01-6.97 (2H), 6.90-6.81 (2H), 7.09-7.04 (1H, partially overlapped with galloyl group signal), 6.71-6.67 (1H), 6.65- 6.63 (1H), 6.49-6.46 (1H), 6.21-6.16 (1H) Example of peak separation due to anomeric mixture formation Example of aromatic proton (1 piece): 6.21 (3/14H), 6.18 (3/14H) ), 6.17 (6/14H), 6.16 (2/14H) Aromatic proton of galloyl group (1 piece): 6.90 (6/14H), 6.87 (4/14H), 6.85 (12/14H), 6.81 (6 /14H) 3rd position proton of α-anomer of glucose core (both): 5.83 (3/14H), 5.81 (2/14H), 5.72 (3/14H), 5.70 (6/14H) β-anomer of left glucose 1st proton; 4.61 (6/14H), 4.53 (3/14H) (The reason why the NMR spectrum is complicated is that
to form four types of anomers) Color/Property: Gray-white amorphous powder Specific optical rotation: [α] _D +91° (c=0.1, acetone) Elemental analysis: Molecular formula C ₈₂ H ₅₈ O ₅₂・12H ₂ O Theoretical value (%) C, 47.09; H, 3.95 Actual value (%) C, 47.02; H, 4.08 FAB-MS: m/z1898 [(M+Na) ⁺ ] UV: λ ^MeOH _nax nm (logε); 224 (5.20), 280 (4.
92) CD: (MeOH) [θ] (nm); +284000 (230), -
38000 (261), +60000 (282) Thin layer chromatography: (cellulose, 7% acetic acid); Rf0.27 ¹ H-NMR: δppm (200MHz, acetone-d ₆ );
7.27, 7.18, 6.70, 7.039, 7.036, 6.991, 6.985,
6.68, 6.65, 6.51, 6.12, 6.06, 5.83, 5.81,
5.61, 5.59, 5.37, 5.35, 5.26, 5.21, 4.51,
4.47 (In addition, the H-6′ signal of glucose
overlaps with HDO peak at 3.8ppm) Color/Property: Pale and amorphous powder Specific optical rotation: [α] _D +99° (c=0.1, MeOH) Elemental analysis: Molecular formula C ₈₉ H ₆₂ O ₅₇ ×14H ₂ O Theoretical value (%) C, 46.57; H, 3.95 Actual value (%) C, 46.63; H, 4.15 FAB-MS: m/z2065 [(M+Na) ⁺ ] UV: λ ^MeOH _nax nm (logε): 220 (5.24), 277 (4.
93) CD: (MeOH) [θ] (nm); +350000 (231), -
50000 (263), +69000 (285) Thin layer chromatography: (cellulose, n-
BuOH-n-PrOH- _H2O (2:1:3v/v)
upper layer) Rf・0.43 ¹ H−NMR: δppm (200MHz, acetone−d ₆ );
7.23, 7.17, 6.68, 7.006, 6.993, 6.968, 6.965,
7.15, 6.66, 6.46, 6.45, 6.36, 6.10, 6.01,
5.55, 5.51, 5.82, 5.76, 5.19, 5.10, 4.49,
4.42, 5.33, 5.25, 3.84, 3.69 Color/Property: Gray-white amorphous powder Specific optical rotation: [α] _D +220° (c=0.5, MeOH) Elemental analysis: Molecular formula C ₆₈ H ₅₀ O ₄₄・8H ₂ O Theoretical value (%) C, 47.62; H, 3.88 Actual value (%) C, 47.47; H, 3.61 FAB-MS: m/z1593 [(M+Na) ⁺ ] UV: λ ^MeOH _nax nm (logε); 218 (5.08), 268 (4.
77) CD: (MeOH) [θ] (nm); +379000 (218), -
43200 (258), +104000 (280) Thin layer chromatography: (cellulose, 7% acetic acid) Rf0.50 ¹ H-NMR: δppm (200MHz acetone-d ₆ );
7.25, 7.19, 7.00, 6.70, 6.66, 6.50, 6.31,
6.20, 6.11, 5.87, 5.60, 5.45, 5.25, 5.19,
5.00, 4.92, 4.59, 4.48, 4.16, 3.85 Each of the above compounds can be obtained by homogenizing fresh or dried raw material plants in water, methanol, ethanol, acetone, or a highly polar solvent mixture of each, and then filtering. , concentrate the liquid under reduced pressure at 40℃ or below, remove low polar solvent soluble substances from the obtained concentrated extract by extracting with chloroform or ether, etc., and extract from the highly polar solvent layer with acetic acid ethyl ester or BuOH. It can be obtained by isolating and purifying it by subjecting it to various types of adsorption or partition chromatography. As an isolation and purification method, for example, Cephadex LH20
(Pharmacia), Toyopearl HW-40 (Toyo Soda Kogyo), Amberlight XAD (Organo), Diaion HP-20 (Mitsubishi Kasei), droplet counter current chromatography (droplet)
counter current chromatography), centrifugal counter current chromatography
chromatography), and high-performance liquid chromatography using Deverosil 60-5 (Nomura Chemical). The plants used to obtain the medicament according to the invention and their parts used are shown in the table below.

[Table] The active ingredients of the anticancer agent of the present invention can be prepared into liquid preparations, powders, granules, tablets, and enteric-coated preparations according to conventional methods using appropriate solvents, excipients, adjuvants, etc. commonly used in preparations. The drug can be made into various dosage forms such as and capsules. Moreover, the anticancer agent of the present invention may contain other active ingredients. For oral administration, at least one excipient,
For example, using starch, lactose, sucrose, mannitrate, carboxymethyl cellulose, etc.
It can be formulated into capsules, powders, granules, 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. Disintegrants such as esters, barley starch, carboxymethylcellulose, etc. can be used. In addition, the drug of the present invention can be used in suspensions, emulsions,
It can also be administered as a syrup or elixir, and these various dosage forms may contain flavoring agents and coloring agents. When manufacturing injections, diluents generally include distilled water for injection, physiological saline, aqueous dextrose solution, vegetable oil for injection, propylene glycol,
Polyethylene glycol or the like can be used. Furthermore, an isotonizing agent, a stabilizer, a preservative, a non-permeable 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. Hereinafter, the present invention will be explained in more detail with reference to Examples. Production Example 1 600 g of dry powder of Coriaria japonica A.Gray leaves was homogenized three times with an acetone aqueous solution (acetone/water = 7/3 (v/v)), filtered, and the liquid was heated at 40°C. Concentrate under reduced pressure.
This concentrated extract is extracted six times with 600 ml of chloroform to remove chloroform-soluble components. After extracting the remaining aqueous layer six times with 600 ml of ethyl acetate, the solvent was distilled off from the combined organic layers to obtain 73.5 g of ethyl acetate extract. moreover,
After extracting the remaining aqueous layer seven times with 600 ml of n-butanol, the solvent of the combined organic layers was distilled off to obtain 55.8 g of n-butanol extract. Add 2.2 g of acetic acid ethyl ester extract and 3.2 g of n-butanol extract to Sephadex LH-20 (diameter 22 mm, length 4 cm) and Toyopearl HW-40 (diameter 22 mm, length 43 cm).
It was subjected to column chromatography using . Separation of ethyl acetate extract using Sephadex LH-20 uses ethanol, ethanol/
Using a methanol mixture and then methanol while gradually increasing the methanol ratio, collect fractions 873 to 5 ml each, which are eluted with 100% methanol.
877 was collected and dried under reduced pressure to obtain 33 mg of coryliin A, which is one of the active ingredients of the drug of the present invention. On the other hand, the separation of n-butanol extract using Toyopearl HW-40 was first performed using an ethanol aqueous solution (ethanol:water = 7:3 (v/v)).
Elute with 950ml then ethanol:acetone:
Water = 65:5:30 (v/v)), fractionated into 5 ml portions, collected fractions 361 to 439, and dried under reduced pressure to obtain 313 mg of coryliin A.
Therefore, in this example, a total of 346 mg of coryliin A was obtained, and the yield was about 1.4% based on the weight of the dry powder of the raw material. Production Example 2 Oenothera erythrosepala
6.5 kg of dry powder of leaves of Borbas was homogenized three times with an aqueous acetone solution (acetone:water = 7:3 (v/v)), filtered, and the liquid was concentrated under reduced pressure at below 40°C to 940 ml. . Ether 940 from concentrate
Ether soluble components were removed by extracting 10 times with ml. n-butanol 400% from the remaining aqueous layer
After extracting 5 times with 1.5 ml of water, the solvent was distilled off to obtain 32 g of n-butanol extract. 7 g of this n-butanol extract was subjected to column chromatography using Sephadex LH-20 (diameter 22 mm, length 43 cm) using ethanol, an ethanol/methanol mixture, then methanol as the eluent, gradually increasing the proportion of methanol. It was separated using 5ml
The eluates of fractions 350 to 425 were collected and dried under reduced pressure to obtain 538 mg of enotein B, which is one of the active ingredients of the drug of the present invention. The yield based on the weight of the dry powder of the raw material was 0.4%. The substance obtained in the above example is
centrifugal counter current chromatography
Alternatively, the purity can be increased by subjecting it to preparative high performance liquid chromatography using Deverosil 60-5. Formulation example 1 60g of the active ingredient of the present invention is made into a fine powder, and this is mixed with lactose.
220 g of magnesium stearate and 20 g of magnesium stearate, and this mixture was compressed using a single-shot slug tablet machine to make slug tablets with a diameter of 20 mm and a weight of approximately 2.3 g. This was crushed using an oscillator, sized, and sieved. Separately, good granules of 20 to 50 meshes were obtained. Each gram of this granule contains 200 mg of the active ingredient, but 0.5 to 2.5 g should be taken at a time depending on the symptoms. Formulation Example 2 90 g of microcrystalline cellulose and 10 g of magnesium stearate were added to 100 g of the active ingredient according to the present invention and mixed, and this mixture was compressed using a single-shot tablet machine to produce tablets with a diameter of 9 mm and a weight of 200 mg. Each tablet contains 100 mg of the active ingredient, but 1 to 5 tablets should be taken at a time, depending on the symptoms. Formulation example 3 100g of the active ingredient according to the present invention is made into a fine powder, and this
Capsules were obtained by filling 100 mg each into hard capsules. This capsule contains 100 active ingredients in one capsule.
mg, but take 1 to 5 capsules at a time depending on your symptoms. Formulation Example 4 20 g of the active ingredient according to the present invention was dissolved in distilled water for injection 1 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 20 mg of the active ingredient. This injection is administered at a dose of 25 mg/day depending on the symptoms.
ml can be administered by subcutaneous, intravenous or intramuscular injection. Next, the anticancer effect and safety of the drug of the present invention will be explained using the following test examples. Test Example 1 Effect on mice transplanted with Sarcoma 180 cells Sarcoma 180 cells were intraperitoneally transplanted into ddY mice, and one week later, ascites containing the expanded Sarcoma 180 cells was obtained. The above Sarcoma180 cells were transplanted intraperitoneally into 6-week-old female ddY mice at 1×10 ⁵ cells per mouse. A single group of 6 animals was administered the test substance 4 days before tumor cell transplantation, and the survival rate ^* and 60-day survival rate were examined. The results are shown in the table below. In addition,
The average survival time of the control group was 12.8±1.5 days. Furthermore, no tumor cell proliferation was observed in any of the mice that survived for 60 days.

【table】

[Table] Test Example 2 Sarcoma180 cells were transplanted into the peritoneal cavity of a ddY mouse, and one week later, ascites containing proliferated Sarcoma180 cells was obtained. The above Sarcoma180 cells were transplanted intraperitoneally into 6-week-old female ddY mice at 1×10 ⁵ cells per mouse. 6 animals per group, 1 after tumor cell transplantation
Intraperitoneal administration on day 4, day 7, survival rate ^*
and 60-day survival rate. The results are shown in the table below. The average survival time for the control group was 13.7 days.
It was ±1.2 days. Furthermore, no tumor cell proliferation was observed in any of the mice that survived for 60 days.

[Table] * represents the same meaning as in Example 1.
Test Example 3 Effect on MM2 mouse breast cancer transplanted mice MM2 mouse breast cancer cells were transplanted intraperitoneally into C3H/He mice and allowed to proliferate, and one week later, ascites fluid was collected. 5×10 ⁵ cells were implanted intraperitoneally into C3H/He mice. A single group of 6 mice was administered coryliin A intraperitoneally 4 days before cell transplantation, and the survival rate ^* and 60-day survival rate were examined. The results are shown in the table below. The average survival time of the control group was 20.2±0.4 days.

[Table] * represents the same meaning as Test Example 1.
Test Example 4 MM2 mouse mammary cancer cells were implanted intraperitoneally into C3H/He mice and allowed to proliferate, and the ascites fluid was collected one week later. Transplant 5 x ¹⁰ cells per animal. Each group consisted of 6 animals, and coryliin A was administered intraperitoneally on the 1st, 4th, and 7th day after cancer cell transplantation, and the survival rate ^* and 60-day survival rate were examined. The results are shown in the table below. The average survival time of the control group was 21.1±0.7.

[Table] * represents the same meaning as in Example 1.
Test Example 5 The 50% lethal dose (LD ₅₀ ) of coryliin A to ddY mice was determined by the probit method. The results are shown in the table below.

[Table] From the above results, the dosage of the active ingredient in the anticancer agent of the present invention varies depending on the patient's age, weight, and degree of disease, but is usually 100 to 500 ml per dose for adults.
mg, oral administration of 300 mg to 1500 mg per day is considered appropriate, and intravenous infusion of 500 mg or less per day, or
Intramuscular injection is considered appropriate.

Claims (1)

[Claims] 1 Containing at least one compound selected from the group of ellagitannin compounds consisting of nobotanin A, nobotanin C, gemin A, lugosin D, rugosin E, isorgosin D, cornuciin A, corialiin A, corialiin C, and enotein B. Characteristic anticancer drug.