JP2006131594A - Carcinogenisis preventing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a safe and effective carcinogenesis preventing agent particularly containing at least one kind of chalcogen compounds, coumarin compounds, flavanone compounds, and diacetylene compounds. <P>SOLUTION: This carcinogenesis preventing agent comprises at least one chalcogen compound selected from the group consisting of dorsmanin A, xanthoangerol I, and xanthoangerol J, at least one coumarin compound selected from the group consisting of xanthotoxin, isopimpinellin, and osthenol, at least one flavanone compound selected from the group consisting of isobavachin, mandeleaflavanone B, and 8-geranyl naringenin, and a diacetylene compound of (11S,16R)-dihydroxyoctadeca-9Z,17-dien-12,14-diyn-1-yl acetate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a carcinogenic preventive agent, and particularly to a carcinogenic prophylactic agent containing at least one of chalcone compounds, coumarin compounds, flavanone compounds and diacetylene compounds.

Conventionally, it is known that an extract from Ashitaba, which is a perennial periphyceae, has a carcinogenicity-preventing action (see Patent Document 1). Moreover, it is publicly known that there are chalcone compounds effective as carcinogenesis preventing agents (see Non-Patent Document 1).
JP 2003-155248 A okuyama et al., Plant Med. 57, 242, 1991

However, Patent Document 1 does not specifically disclose a specific component having a sufficient effect for preventing carcinogenesis. On the other hand, in view of the great need for carcinogenesis prevention, there is great expectation that a safe and effective carcinogenesis preventing agent will not be obtained.
An object of the present invention is to provide a safe and effective carcinogenic preventive agent.

  In order to solve the above-mentioned problems, the present invention provides a new cancer preventive agent comprising an Ashitaba component as an active ingredient. That is, the present invention has an important meaning in finding a carcinogenesis-preventing action in an Ashitaba component, and the carcinogenesis-preventing agent according to claim 1 of the present invention is dolsmannin A (hereinafter referred to as “compound 1” in the present invention). Xanthoangelol I (hereinafter also referred to as “Compound 2” in the present invention), Xanthoangelol J (hereinafter also referred to as “Compound 3” in the present invention). At least one chalcone compound selected from the group consisting of: xanthotoxin (hereinafter sometimes referred to as “compound 4” in the present invention), isopinepineline (hereinafter also referred to as “compound 5” in the present invention). ), At least one coumarin selected from the group consisting of ossenol (hereinafter also referred to as “compound 6” in the present invention). Product, isobabatine (hereinafter also referred to as “compound 7” in the present invention), mandelea flavanone B (hereinafter also referred to as “compound 8” in the present invention), 8-geranylnaringenin (hereinafter referred to as the present invention) (11S, 16R) -dihydroxyoctadeca-9Z, 17-diene, which is at least one flavanone compound selected from the group consisting of: and (11S, 16R) -dihydroxyoctadeca-9Z It is characterized by containing at least one compound out of -12,14-diin-1-yl acetate (hereinafter also referred to as “compound 10” in the present invention).

If it is a compound as mentioned above, it is excellent in the carcinogenesis prevention effect and is also highly safe.
In addition, the compound of the present invention described above includes medically used and pharmacologically acceptable salts of each compound. Examples of such salts include alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt and copper salt. Metal salts such as nickel salts and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine Salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, Tris (Hydroxyme And an amino amine salt such as glycine salt, lysine salt, arginine salt, ornithine salt and asparagine salt.

Moreover, when the compound of this invention of Claim 1 mentioned above forms a solvate (for example, hydrate), all these are also contained in this invention. For example, when the compound of the present invention is left in the air or recrystallized, it may absorb moisture, adsorbed water may adhere, or form a hydrate. Such solvates are also included in the present invention.
Furthermore, the compound of the present invention described in claim 1 has several asymmetric carbon atoms, and therefore various optical isomers exist. In the present invention, unless otherwise specified, all of these isomers including racemic compounds and mixtures of these isomers are included.
The carcinogenesis preventive agent according to claim 2 of the present invention is characterized in that, in claim 1, the compound is obtained by treating ashitaba.

  According to the present invention, a carcinogenic preventive agent that is safe and excellent in carcinogenic preventing effect is provided.

Next, the present invention will be described specifically by way of examples. That is, the isolation of the compounds 1 to 10 from the ascibatus exudate, the structural analysis and identification of the compounds 1 to 10, and the effect confirmation test of the carcinogenic preventive agent according to the present invention will be described.
[Isolation of Compounds 1-10 from Ashitaba Leachate]
First, Ashitaba leachate (leachate from stem cut surface, 300 g) was dried under reduced pressure to obtain a dried product (22.3 g). This was separated with n-hexane / methanol / water (95:95:10) to obtain an n-hexane fraction (0.504 g). The methanol / water fraction was further separated with ethyl acetate / water (1: 1) to obtain an ethyl acetate fraction (15.3 g). Further, the remaining water fraction was separated with n-butanol / water (1: 1) to obtain an n-butanol fraction (0.184 g) and a water fraction (5.79 g).

  The ethyl acetate fraction obtained as described above was fractionated by silica gel (500 g) column chromatography. As eluent, n-hexane / ethyl acetate [95: 5 (1.5 L), 8: 2 (2.0 L), 1: 1 (4.0 L), 0: 1 (5.0 L)], ethyl acetate / Methanol [4: 1 (2.0 L), 1: 1 (3.5 L), 0: 1 (0.5 L)] in order, and fractionated into 12 fractions (Fr. A to L: Fr. A (181.2 mg), Fr.B (819.3 mg), Fr.C (944.0 mg), Fr.D (7817.2 mg), Fr.E (3576.6 mg), Fr.F (529.0 mg) ), Fr.G (170.9 mg), Fr.H (179.0 mg), Fr.I (54.5 mg), Fr.J (79.8 mg), Fr.K (76.3 mg), Fr.L (33.1 mg)).

  Fr. D was further fractionated by silica gel (500 g) column chromatography. As an eluent, n-hexane / ethyl acetate [0: 1 (0.5 L), 9: 1 (1.5 L), 85:15 (5.0 L), 8: 2 (1.0 L), 75:25 (0.5L), 7: 3 (3.0L), 6: 4 (0.5L), 1: 1 (1.0L), 4: 6 (0.5L), 3: 7 (0.5L) 2: 8 (0.5 L), 1: 9 (0.5 L), 0: 1 (0.5 L)], ethyl acetate / methanol [7: 3 (1.5 L), 1: 1 (0.5 L) ), 3: 7 (0.5 L), 0: 1 (0.5 L)] in order, and fractions 13 (Fr. D1 to D13: Fr. D1 (14.9 mg), Fr. D2). (3.6 mg), Fr.D3 (24.2 mg), Fr.D4 (23.1 mg), Fr.D5 (138.4 mg), Fr.D6 (1251.0 mg) Fr.D7 (1533.1 mg), r.D8 (754.6 mg), Fr.D9 (679.4 mg), Fr.D10 (1663.0 mg), Fr.D11 (90.2 mg), Fr.D12 (119.7 mg), Fr.D13 (230 0.9 mg)).

Fr. D5 is preparative HPLC using an octadecyl silica (ODS) column (column: Hypersil® ODS (particle diameter 5 μm, length 25 cm × inner diameter 10 mm; eluent: methanol / water / acetic acid = 75: 25: 1; flow rate 3 Compound 8 (4.4 mg; retention time 15.2 minutes) was isolated.
Fr. D9 was fractionated by ODS (10 g) column chromatography. Fraction D9a to D9d: Fr. D9a (183) were fractionated using methanol / water [7: 3 (1.3 L), 1: 0 (0.4 L) in order of addition] as an eluent. .6 mg), Fr.D9b (393.4 mg), Fr.D9c (24.7 mg), Fr.D9d (34.1 mg)).

  This Fr. D9b is preparative HPLC using an ODS column (column: Pegasil ODS (manufactured by Senshu Scientific Co., Ltd., particle size 5 μm, length 25 cm × inner diameter 10 mm); eluent: methanol / water / acetic acid = 80: 20: 1; flow rate 2 0.02 mL / min), compound 2 (3.4 mg; retention time 18.4 min), and compound 3 (1.8 mg; retention time 20.0 min), compound 9 (3.6 mg; retention time 31. 0 min) was isolated.

  Fr. E was fractionated by ODS (120 g) column chromatography. Fractionation was performed using methanol / water [7: 3 (3.0 L), 8: 2 (0.9 L), 1: 0 (0.4 L)] in the order of addition) as an eluent, and fraction 9 (Fr. E1 to E9: Fr.E1 (110.8 mg), Fr.E2 (1008.1 mg), Fr.E3 (320.9 mg), Fr.E4 (125.5 mg), Fr.E5 (90.3 mg), Fr E6 (62.4 mg), Fr.E7 (52.2 mg), Fr.E8 (1417.5 mg), Fr.E9 (44.7 mg)).

  This Fr. E1 is preparative HPLC using an ODS column (column: ERC-ODS-2532 (manufactured by Yokohama Rika Co., Ltd., particle diameter 5 μm, length 25 cm × inner diameter 10 mm); eluent: methanol / water / acetic acid = 65: 35: 1; A flow rate of 2.0 mL / min was performed, and Compound 4 (2.7 mg; retention time 7.6 minutes) and Compound 6 (2.1 mg; retention time 14.8 minutes) were isolated.

  In addition, Fr. E4 is preparative HPLC using an ODS column (column: ERC-ODS-2532 (manufactured by Yokohama Rika Co., Ltd., particle size 5 μm, length 25 cm × inner diameter 10 mm); eluent: methanol / water / acetic acid = 65: 35: 1; A flow rate of 2.0 mL / min was performed, and Compound 5 (1.6 mg; retention time 10.8 minutes) and Compound 10 (22.0 mg; retention time 51.6 minutes) were isolated.

  Fr. F was fractionated by ODS (30 g) column chromatography. As eluent, methanol / water [6: 4 (0.4 L), 7: 3 (0.3 L), 8: 2 (0.8 L), 9: 1 (0.3 L), 1: 0 (in order of charging)] 0.4L)], ethyl acetate (0.4L), and fractionated into 10 fractions (Fr.F1 to F9: Fr.F1 (80.1 mg), Fr.F2 (54.1 mg), Fr. F3 (71.9 mg), Fr.F4 (31.5 mg), Fr.F5 (45.2 mg), Fr.F6 (17.9 mg), Fr.F7 (47.4 mg), Fr.F8 (32.3 mg) ), Fr.F9 (2.4 mg), Fr.F10 (10.2 mg)).

This Fr. F3 is preparative HPLC using an ODS column (column: Pegasil ODS (manufactured by Senshu Scientific Co., Ltd., particle size 5 μm, length 25 cm × inner diameter 10 mm); eluent: methanol / water / acetic acid = 65: 35: 1; flow rate 2 Compound 7 (8.9 mg; retention time 19.6 minutes) was isolated.
Fr. F4 is preparative HPLC using ODS column (column: Pegasil ODS (manufactured by Senshu Scientific Co., Ltd., particle size 5 μm, length 25 cm × inner diameter 10 mm); eluent: methanol / water / acetic acid = 80: 20: 1; flow rate 2 Compound 1 (2.4 mg; retention time 31.2 minutes) was isolated.

  As the solvent for preparing the leachate of Ashitaba, water, alcohols (methanol, ethanol, isopropyl alcohol, etc.), ketones (acetone, methyl ethyl ketone, etc.), ethers (diethyl ether, diisopropyl ether, dimethoxyethane, tetrahydrofuran) ), Fatty acid esters (ethyl acetate, butyl acetate, etc.), halogenated hydrocarbons (chloroform, methylene chloride, dichloroethane, etc.), aromatic hydrocarbons (benzene, toluene, etc.), aliphatic hydrocarbons (n- One kind of solvent such as hexane, cyclohexane, or a mixed solvent of two or more kinds can be used.

Further, the extraction method is not limited to the above method, and it may be directly extracted with a low polarity solvent such as hexane without going through a process of gradually extracting from a water-soluble or high polarity solvent to a low polarity solvent. .
Moreover, the part of Ashitaba immersed in a solvent is not specifically limited, Any parts, such as a stem, a leaf, and a root, may be sufficient.

[Structural analysis and identification of compounds 1 to 10]
Structural analysis and identification were performed on the above 10 types of compounds (compounds 1 to 10), and the results will be described.
Structural analysis of compounds 1 to 10 is MS, IR, UV, ¹ H-NMR, ¹³ C-NMR, two-dimensional NMR correlation spectroscopy ( ¹ H- ¹ HCOSY), heteronuclear multiquantum coherence method (HMQC), Heterogeneous nuclear remote multiquantum correlation (HMBC) and nuclear overhauser exchange spectroscopy (NOESY) were used. The structures of compounds 1 to 10 by this structural analysis are shown in FIG.

Compounds 2 and 3 were novel compounds not described in the literature so far. The ¹ H-NMR and ¹³ C-NMR data of the novel compounds 2 and 3 are shown in Tables 1 and 2 together with the HMBC data. Further, in FIG. 2, the nuclear overhauser effect (NOE) correlation obtained by the NOESY spectrum method of the novel compounds 2 and 3 is shown by both arrows together with the three-dimensional structural formula.

  The structures of other known compounds are described in literature values of various spectral data with the corresponding compounds (Compound 1: Ngadjui et al., Phytochemistry, 48, 349, 1998; Compound 4: Masuda et al., Phytochemistry, 47, 13 Compound 5: Furuya et al., Chem. Pharm. Bull., 15, 1362, 1967; Compound 6: Murray et al., Tetrahedron, 27, 1247, 1971; Compound 7: Komatsu et al., Chem.Pharm.Bull., 26, 3863, 1978; Compound 8: Rao et al., Phytochemistry, 46,1271; 1997; Compound 9: Shirataki et al., Chem. , Vol. 33, 444 pp., 1985; Compound 10: Liu et al., Phytochemistry, 49, pp. 211 pp., Was confirmed by comparison with 1998). However, the carcinogenic effect of these compounds is not known.

The properties and spectral data of the novel compounds 2 and 3 are shown below.
(1) Xanthoangelol I (2 ′, 3 ′-[2-methyl-2- (4-methyl-3-pentenyl) pyrano] -4,4-dihydroxychalcone) (Compound 2):
Yellow crystals. Melting point: 106-109 ° C. Specific rotation [α] ²⁵ _D : 8.00 (concentration c = 0.1%, solvent: methanol). UV (solvent: methanol) λ _max (unit: nm): 206, 350. IR v _max (unit: cm ⁻¹ ): 3267, 2971, 2930 (Ar—O), 1639 (C═O), 1588, 1512 (derived from aromatic ring), 1441, 1334, 1287, 1233, 1167, 1072, 832, 811.
EI-Ms m / z (%): 392 (70) [M ⁺ ] 1269 (85), 149 (100)
High resolution EI-MS (m / z value): 392.9888 (theoretical value C ₂₅ H ₂₈ O ₄ [M ⁺ ], 392.987)
The data of ¹³ C-NMR, ¹ H-NMR and HMBC of Compound 2 are shown in Table 1.
¹³ C-NMR (125 MHz), ¹ H-NMR (500 MHz), and HMBC spectrum data (solvent: methanol-d ₄ ) of Compound 2

The numerical value in parentheses is the coupling constant J value (Hertz).
(2) Xanthoangelol J (2 ′, 4,4′-trihydroxy-3 ′-(3-hydroxy-3,7-dimethyl-2-octaenyl) chalcone) (Compound 3):
Yellow crystals. Melting point: 120-123 ° C. Specific rotation [α] ²⁵ _D : 6.0 (concentration = 0.1%, solvent: methanol). UV (solvent: methanol) λ _max (unit: nm): 206, 368. IR v _max (unit: cm ⁻¹ ): 3369, 2969, 2928 (Ar—O), 1605 (C═O), 1562, 1512 (derived from aromatic ring), 1491, 1442, 1370, 1218, 1292, 1236, 1168, 1107, 832, 799.
EI-Msm / z (%): 410 (11), 3929 (42), 269 (50), 203 (36), 149 (100), 120 (34).
High resolution EI-MS (m / z values): 410.2096 (theoretical value _{C 25 H 30 O 5 [M} +], 410.2093).
The data of ¹³ C-NMR, ¹ H-NMR and HMBC of Compound 3 are shown in Table 2.
¹³ C-NMR (12, ⁵ MHz), ¹ H-NMR (500 MHz), and HMBC spectrum data (solvent: acetone-d ₆ ) of Compound 3

The numerical value in parentheses is the coupling constant J value (Hertz).
The structure determination of novel compounds 2 and 3 will be described below.
Compound 2 was shown to have the molecular formula C ₂₅ H ₂₈ O ₄ by high resolution EI-MS ([M ⁺ ] m / z: 392.987). The IR spectrum showed the presence of hydroxyl groups, carbonyl groups and aromatic rings. ¹ H-NMR spectrum (δ (ppm), CD ₃ OD) shows a signal of a benzene proton having a substituent at the para position from 6.80 (2H, d), 7.47 (2H, d), Signals attributed to two benzene protons located at ortho positions relative to each other were shown at 6.40 (1H, d) and 7.44 (1H, d). This suggests that the compound has a chalcone skeleton having a substituent at the 4, 2 ′, 3 ′ and 4 ′ positions. Further, 1.44 (s), 1.60 (s), 1, 67 (2H, m), 2.12 (2H, dd), and 5.03 (1H, t) indicate the presence of a dimethylallyl group. It was. In addition to the above data, a comparison of ¹ H-NMR and ¹³ C-NMR data with Dorsmanin A (Ngadiui et al., Phytochistry, 48, 349, 1998) shows that there is a pyran ring at the 2 ′ and 3 ′ positions. It was revealed. The correctness of this structure was confirmed by analysis of ¹ H- ¹ HCOSY, HMQC, HMBC, and NOESY spectra.

Compound 3 was shown to have the molecular formula C ₂₅ H ₃₀ O ₅ by high resolution EI-MS ([M ⁺ ] m / z: 410.2096). The IR spectrum showed the presence of hydroxyl groups, carbonyl groups and aromatic rings. ¹ H-NMR spectrum (δ (ppm), acetone-d ₆ ) shows a signal of a benzene proton having a substituent at the para position from 6.87 (2H, d) and 7.67 (2H, d), and 6.45 (1H, d) and 7.91 (1H, d) show signals attributed to two benzene protons located in the ortho positions relative to each other. This suggests that the compound has a chalcone skeleton having a substituent at the 4, 2 ′, 3 ′ and 4 ′ positions. Furthermore, 1.19 (s), 1.50 (2H, m), 1.57 (s), 1.61 (s), 1.65 (2H, m), 2.08 (2H, m), 2 .71 (2H, m), 5.10 (1H, tt) suggests the presence of a geranyl group having a hydroxyl group. In addition to the above data, a geranyl group is substituted at the 3 'position by comparison with xanthoangelol (Baba et al., Phytochemistry, 29, 3907, 1990) in ¹ H-NMR and ¹³ C-NMR data. It became clear. The correctness of this structure was confirmed by analysis of ¹ H- ¹ HCOSY, HMQC, HMBC, and NOESY spectra.

Compounds 1 to 10 are not limited to natural compounds extracted from Ashitaba as long as they are represented by the above chemical structural formulas, but may be obtained by modifying natural compounds or by chemical synthesis. May be.
The above-described compounds 1 to 10 of the present invention can be used alone or in combination.
Compounds 1 to 10 can be combined with a suitable pharmaceutical carrier or diluent to form a pharmaceutical, which can be formulated by any conventional method, and can be a solid, semi-solid or It can be formulated into a liquid dosage form. In prescribing, it is good also as a compounding agent with another pharmaceutical active ingredient.

  For example, various preparations described in the Japanese Pharmacopoeia, that is, solid preparations for internal use such as tablets, pills, capsules, granules, powders, dry extracts, troches, flow extracts, elixirs, spirits It can be formulated into liquid preparations such as syrups and limonades, external liquids such as tinctures, liniments and lotions, and external preparations such as plasters, ointments and poultices. If administration is possible, inhalants, aerosols, injections, eye drops, suppositories and the like may be formulated according to the intended use.

In oral administration, it is preferable to administer to adults in the range of 0.5 to 500 mg / kg body weight.
In addition, the compounds 1 to 10 according to the present invention are useful not only for use as a carcinogenic agent, but also for producing functional foods and drinks effective for preventing carcinogenesis by being added to various foods and drinks. . Examples of the form of such functional foods and drinks include granules, tablet confectionery, jelly, strawberries, beverages and the like. In addition, the compounds 1 to 10 according to the present invention can be used not only as foods eaten by humans but also added to feeds of animals other than humans such as pets, livestock, and sports animals.

[Confirmation test for carcinogenesis prevention effect]
Next, a test conducted for confirming the effect of the present invention will be described.
(Epstein-Barr virus (EBV) activation inhibition test)
The EBV activation suppression test is a test known as a primary screening method for searching for an anti-carcinogenic promoter, and is an excellent test in that it is rapid and excellent in quantitativeness, and in addition, a trace active ingredient can be detected.

First, the test procedure will be described with reference to the scheme of FIG. This procedure is based on the method of Tokuda et al. (Cancer Letters, 40, 309, 1988).
(1) To 1 × 10 ⁶ / mL Raji (radio) cells, as a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) at a concentration of 20 ng / mL was added 32 pmo1, and further TPA N-butyric acid was added which acts as a synergistic effect for activity expression.
(2) A predetermined amount of a test substance dissolved in water, ethanol, or dimethyl sulfoxide was added thereto, and cultured at 37 ° C. for 48 hours.
(3) After completion of the culture, expression of EBV early antigen was detected by an indirect fluorescent antibody method using serum from patients with nasopharyngeal cancer (including antibodies derived from cells activated by TPA).

After performing the test according to the above procedure, the expression rate of the EBV early antigen of the test substance additive was determined by setting the expression rate of the EBV early antigen of the group (control) to which only TPA was added as 100%. The expression inhibition rate (%) of the test substance EBV early antigen was calculated.
Expression inhibition rate of EBV early antigen = 100-Expression rate of EBV early antigen in test substance added group (%) Formula (1)

The test was performed using compounds 1 to 10 obtained from the Ashitaba leachate as described above as test substances, and preparing each test substance at various concentrations (molar ratio of test substance / TPA). The results of this test are shown in Table 3. In addition, curcumin was used as a test compound as a reference compound.
EBV expression inhibition rate (%) of 10 kinds of chalcones, coumarins, flavanones, and diacetylene compounds

  The numerical value in parentheses in the column where the concentration of the test substance is 1000 times the molar concentration indicates the survival rate (%) of the raji cells. It can be said that the higher this value, the smaller the adverse effect on normal cells, that is, the higher the safety. As shown in Table 3, the component of Ashitaba leachate has a compound with a high inhibition rate. Compared with curcumin (Cancer Letters, 159, 135, 2000), which is known to have a carcinogenic preventing effect, compounds 2, 3, 6-9 are 100 times as much as curcumin at 1000-fold molar concentration. % Value was 9 to 12% stronger than curcumin at 10-fold molar concentration. This confirmed that the anti-tumor promoter activity was excellent. Furthermore, since these showed high survival rate of raji cells in this assay, they can be expected as carcinogenic preventive agents with high safety.

Furthermore, since the inhibition test with respect to NO disorder | damage | failure was performed for compounds 1-10, and it showed activity, it demonstrates.
(Suppression test for NO damage)
(±)-(E) -4-methyl-2-[(E) -hydroxyimino] -5-nitro-6-methoxy-3-hexenamide (NOR1) treatment is a primary screening for carcinogenic initiator. It is a test known as law. NO is a gaseous free radical, and it is said that the risk of gastric cancer and colon cancer increases when its overproduction becomes chronic. From this, it can be said that suppressing excessive NO production is effective in preventing carcinogenesis. This test is excellent in that it can rapidly detect trace active ingredients.

The test method will be described. This procedure conforms to the method of Tokuda et al. (Cancer Letters 205, page 9, 2004).
(1) 5 × 10 ⁵ / mL human normal liver-derived cells obtained from Eagle's minimum required medium are cultured for 3 days.
(2) A test substance is added thereto, and after 1 minute, NOR, which is a NO donor, is added and cultured in a CO ₂ incubator.
(3) Measure the morphological change of the cells with an optical microscope (measure the number of cells that showed more than 250 morphological changes).

After performing the test according to the above procedure, the group to which no test substance was added was used as a control, and the inhibition rate against NO damage was calculated by the following formula.
Inhibition rate for NO damage = cells whose morphology was changed only by NOR1 (%) / cells whose morphology was changed when NOR1 and a test substance were added (%) (2)
Table 4 shows the results of the test conducted using compounds 1 to 10 obtained from the Ashitaba leachate as described above as test substances. As reference compounds, curcumin (Cancer Letters, 159, 135, 2000), which is known to have a carcinogenic effect, and 2- (4-carboxyphenyl) -4,4, which is a NO scavenger, are used. The results of using 5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) as a test substance are also shown.

  As shown in Table 4, compounds 2, 3, 6, 8, and 9 showed activity similar to or higher than that of the reference compound. This confirmed that it has anticarcinogenic initiator activity. These compounds can be expected as an agent for preventing carcinogenesis.

It is a figure which shows the chemical structural formula of the compounds 1-10 which concern on this invention. It is a figure explaining the nuclear overhauser effect (NOE) correlation obtained by the NOESY spectrum method of the compounds 2 and 3 which concern on this invention. It is a figure explaining the procedure of an EBV activation suppression test. It is a figure explaining the procedure of the suppression test with respect to NO trouble.

Claims (2)

  Drusmannin A, xanthoangelol I (IUPAC name: 2 ', 3'-[2-methyl-2- (4-methyl-3-pentenyl) pyrano] -4,4'-dihydroxychalcone), xanthoangele At least one chalcone selected from the group consisting of roll J (IUPAC name: 2 ', 4,4'-trihydroxy-3'-(3-hydroxy-3,7-dimethyl-2-octaenyl) chalcone) At least one coumarin compound selected from the group consisting of a compound, xanthotoxin, isopine pinerin, ossenol, at least one flavanone compound selected from the group consisting of isobabatin, mandelea flavanone B, 8-geranylnaringenin, And (11S, 16R) -dihydroxyoctadeca-9Z, which is a diacetylene compound. Carcinogenesis preventive agent characterized by containing at least one compound of the 7-diene -12,14- diyne-1 Iruasetato.   The carcinogenic preventive agent according to claim 1, wherein the compound is obtained by treating Ashitaba.

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