Antimutagenic activity of flavonoids from the heartwood of Rhus verniciflua

Journal of Ethnopharmacology 90 (2004) 73–79

Antimutagenic activity of flavonoids from the heartwood of Rhus verniciflua Kun-Young Park a , Geun-Ok Jung a , Kyung-Tae Lee b , Jongwon Choi c , Moo-Young Choi d , Gab-Tae Kim e , Hyun-Ju Jung e , Hee-Juhn Park e,∗ a

e

Department of Food and Nutrition, Pusan National University, Pusan, South Korea b College of Pharmacy, Kyung-Hee University, Seoul, South Korea c College of Pharmacy, Kyungsung University, Pusan, South Korea d Department of Food and Nutrition, Sangji University, Wonju, South Korea Division of Applied Plant Sciences, Sangji University, Wonju 220 702, South Korea

Received 29 August 2001; received in revised form 17 June 2003; accepted 22 September 2003

Abstract Pretreatment of the methanolic extract of the heartwood of Rhus verniciflua (Anacardiaceae) to rats prevented the activation of hepatic microsomal cytochrome P450 enzymes, inhibition of hepatic glutathione S-transferase by bromobenzene treatment, respectively, and therefore significantly decreased malondialdehyde content in the rat. The Ames test showed that the addition of 1.0 mg/plate of the methanolic extract or the EtOAc fraction of the Rhus verniciflua heartwood extract potentially inhibited the mutagenicity by aflatoxin B1 . Column chromatography of the EtOAc fraction yielded four flavonoids, garbanzol (1), sulfuretin (2), fisetin (3), fustin (4), mollisacasidin (5). When these components were subjected to the Ames test, it was found that sulfuretin might effectively prevent the metabolic activation or scavenge electrophilic intermediates capable of causing mutation. In contrast, fustin showed a dose-independent antimutagenic activity and it has mutagenic/antimutagenic activity. However, a mixture of sulfuretin and fustin (1:1) exhibited dose-dependent antimutagenicity indicating that sulfuretin inhibited the mutagenicity of fustin. These results suggest that the extract of Rhus verniciflua heartwood containing flavonoid complex could be a potent anticarcinogen. © 2003 Published by Elsevier Ireland Ltd. Keywords: Rhus verniciflua; Anacardiaceae; Ames test; Anticarcinogenic; Antimutagenic; Sulfuretin; Microsomal cytochrome P450

1. Introduction The exudate that can be obtained from the stem bark of Rhus verniciflua Stokes has been used mainly as a material for traditional paint, and lacquer in East Asian countries and is primarily composed of urushiol polymer (Kim et al., 1998). The stem bark of Rhus verniciflua contains a high level of urushiols, which are polymerized formation of a lacquer film by the radical-chain reaction (Hirota et al., 1998). The exudate was previously found to have anti-AIDS, a strong antioxidant and immune-enhancing activities (Miller et al., 1996). However, its therapeutic use is undermined by the strong shown by some of the population in East Asia, especially to western. In contrast, the heartwood of Rhus verniciflua does not cause this type of allergenic action, which implies that it does not contain urushiols. Moreover, this part of the plant has been used as a kind of tonic, for cancer ∗ Corresponding

author. Tel.: +82-33-730-0564; fax: +82-33-730-0564. E-mail address: [email protected] (H.-J. Park).

0378-8741/$ – see front matter © 2003 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2003.09.043

prevention and for removing the intoxication of smoking or lingering. Many xenobiotic substances activated hepatic microsomal enzymes and may damage DNA strands and cause lipid peroxidation (Park et al., 1996). In our studies upon the elucidation of anticarcinogenic principles, the anti-lipid peroxidation and antimutagenic effects of the methanolic extract of the heartwood of Rhus verniciflua were observed. During the isolation of active principles, we isolated the following species from the most active EtOAc fraction: garbanzol (1), sulfuretin (2), fisetin (3), fustin (4), and mollisacasidin (5).

2. Materials and methods 2.1. Plant material The heartwood of Rhus verniciflua stokes was collected in September 1999, on Chiak Mountain, Kangwon Province, Korea, and was identified by Professor G.T. Kim (Division

74

K.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 73–79

of Applied Plant Sciences, Sangji University, Wonju, South Korea). A voucher specimen (# natchem-18) of which is deposited in the herbarium of Applied Plant Sciences, Sangji University, Wonju, South Korea. 2.2. Extraction and isolation Dried heartwood (2 kg) of the stem of Rhus verniciflua was cut and extracted three times with MeOH under reflux and evaporated to give a viscous mass (280 g). This material was successively suspended in 3 l of H2 O and then partitioned with; 3 l of CHCl3 , EtOAc and n-BuOH in this order. These extracts were dried in vacuo to yield a CHCl3 fraction (68 g), EtOAc fraction (95 g) and n-BuOH fraction (70 g). A portion of the EtOAc fraction (20 g) was chromatographed on silica gel (600 g, 7 cm × 70 cm; Merck, Art 7734, Germany) with CHCl3 –MeOH–H2 O (73:27:10, lower phase; 5 l) as eluent to give five fractions (fractions 1–5), for further isolation. Column eluate was collected in 80 ml aliquots to afford 70 fractions and each was examined under UV (254 and 365 nm). The fractions was showing similar patterns on TLC were grouped and evaporated on a rotary evaporator to yield five fractions (fractions 1–5). Fraction 1 (560–640 ml) was found to be sterol glucoside (Rf 0.70, 30 mg) on co-TLC, but not fully analyzed. Fraction 2 (720–880 ml) afforded a white powder from MeOH (1, Rf 0.67, 30 mg). Repeated column chromatography of fraction 3 (1040–1200 ml) was carried out over silica gel with CHCl3 –MeOH (10:1) as eluent and yielded orange-yellow needles (2, Rf 0.65, 74 mg) on recrystallizing from MeOH. Compound 3 (Rf 0.59, 35 mg) was isolated by purifying fraction 4 (1280–1360 ml) by ODS column chromatography [diameter 3 cm, eluent: CHCl3 –MeOH (1:1)]. Fraction 5 (1360–1600 ml) containing impurities was subjected to ODS and Sephadex LH-20 column chromatography to give compound 4 (Rf 0.53, 190 mg). The compounds 1–5 were identified as garbanzol, sulfuretin, fisetin, fustin and mollisacasidin, respectively, from their physicochemical and spectroscopic data. 1 (garbanzol): White powder (35 mg) from MeOH, mp 207–208 ◦ C; EI-MS (70 eV) m/z: 272.3 (M+ , [C15 H12 O5 ]+ ) (Harborne, 1994a). 2 (sulfuretin): Orange-yellow prisms (160 mg) from MeOH, mp 280–285 ◦ C (dec.); EI-MS (70 eV) m/z: 270.3 (M+ , [C15 H10 O5 ]+ ) (Harborne, 1994b). 3 (fisetin): Yellowish needles (25 mg) from MeOH, mp > 300 ◦ C, EI-MS (70 eV) m/z: 286.3 (M+ , [C15 H10 O6 ]+ ) (Harborne, 1994c). 4 (fustin): White needles (250 mg) from MeOH, mp 228–229 ◦ C, [␣]D +28.3 (c, 0.9 in 50% aqueous acetone); EI-MS (70 eV) m/z: 288.3 (M+ , [C15 H12 O6 ]+ ) (Harborne, 1994d). 5 (mollisacasidin): White powder (40 mg) from MeOH, mp 127–128 ◦ C, [␣]D +12.6 (c, 1 in MeOH); EI-MS (70 eV) m/z: 290.3 (M+ , [C15 H14 O6 ]+ ) (Harborne, 1994e).

2.3. Animals Four-week-old Sprague–Dawley male rats were purchased from Korean Experimental Animal Co. and adapted under constant conditions (temperature: 20 ± 2 ◦ C, humidity: 40–60%, illumination: 12-h light:12-h dark cycle) for at least 2 weeks. Animals were fed with commercial standard rat diet and water ad libitum. 2.4. Administration of samples and bromobenzene Animals were orally administered daily with 100, 250 and 500 mg/kg of several extracts of Rhus verniciflua for 7 days. The control group was given 0.2 ml of 1% Tween-80 solution per 200 g. Then, bromobenzene (480 mg/kg) was intraperitoneally injected twice a day for 2 days. Animals were starved before sacrifice in order to prevent severe hepatic metabolism variations. 2.5. Preparation of enzyme source Animals were sacrificed by exsanguination by severing the abdominal aorta under anesthesia with CO2 gas. The liver was exhaustively perfused with ice-cold normal saline through the portal vein until uniformly pale and was then immediately removed and weighed. After being trimmed and minced, a piece of liver was homogenized with 4:vol. of ice cold 0.1 M potassium phosphate buffer (pH 7.5) solution. The homogenate was centrifuged at 600 × g for 10 min. The pellet was discarded and the supernatant was recentrifuged at 10,000 × g for 20 min. The supernatant was further centrifuged at 105,000×g for 60 min. The resulting supernatant, and cytosolic fraction, were used as an enzyme sources of glutathione S-transferase, and the microsomal fraction was used as an enzyme sources of cytochrome P450 , aniline hydroxylase and epoxide hydrolase. 2.6. Analytical methods Thiobarbituric acid (TBA) levels in the liver were measured as a marker of lipid peroxidation by the method of Ohkawa et al. (1979). Cytochrome P450 activity was determined according to the method of Omura and Sato (1964). Aniline hydroxylase activity (Bidlack and Lowery, 1982) was assayed by determining p-aminophenol formation from aniline hydrochloride. Epoxide hydrolase activity (Habig et al., 1974) was measured spectrophotometrically by monitoring the rate reduction of trans-stilbene oxide (TSO) at 229 nm as described previously. Glutathione S-transferase activity (Habig et al., 1974) was assayed by conjugated glutathione 2,4-dinitrobenzene formation from 1-chloro-2,4-dinitrobenzene. To determine glutathione content, the reaction mixture consisted of 0.5 ml homogenate and 0.5 ml of 4% sulfosalicylic acid. This mixture was centrifuged at 1000 × g for 10 min and 0.3 ml of the supernatant was added to 2.7 ml of the disulfide reagent. After 20 min at

K.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 73–79

room temperature, absorbance was read at 412 nm against a water blank. 2.7. Mutagens Aflatoxin B1 (AFB1 ) was purchased from the Sigma Chemical Co., St. Louis, MO, USA and dissolved in DMSO. N-Methyl-N -nitro-N-nitrosoguanidine (MNNG) was obtained from the Aldrich Chemical Co., Milwaukee, WI, USA and dissolved in distilled water. 2.8. Bacterial strains The Salmonalla typhimurium TA100 bacterial strain, a histidine-requiring mutant, was provided by Dr. B.N. Ames, University of California, Berkeley, CA, USA, and maintained as described by Maron and Ames (1983). The genotypes of the test strains were checked routinely for their histidine requirement, deep rough (rfa) character, UV sensitivity (uvr B mutation) and for the presence of R factor. 2.9. S9 fraction and S9 mix According to the method described by Maron and Ames (1983), male Sprague–Dawley rats were injected intraperitoneally with Aroclor 1254 dissolved in corn oil (500 mg/kg body weight). Five days after the injection, the rats were sacrificed, their livers were removed and minced in 0.15 M KCl, and then homogenized with using a Potter-Elvehjem apparatus. The homogenate was centrifuged at 9000 × g for 10 min in a refrigerated centrifuge, and the supernatant S9 fraction was distributed in 1.8–2.0 ml portions in plastic Nunc tubes, frozen quickly in a bed of crushed dry iced, and stored immediately at −80 ◦ C until use. The S9 required for the preparation of the S9 mix was thawed at room temperature and placed in a container of crushed ice. The S9 mix was prepared as soon as the S9 had thawed. The components of S9 mix were 8 mM MgCl2 , 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, and 100 mM sodium phosphate, pH 7.4, and S9 at a concentration of 0.04 ml/ml of mix. The S9 mix was prepared freshly for each mutagenicity assay. 2.10. Antimutagenicity test A modified plate incorporation procedure (Matsushima et al., 1980) was employed to determine the effect of the isolates (garbanzol, sulfuretin, fisetin, and fustin) on AFB1 -induced mutagenicity. In brief, 0.5 ml of S9 mixture (0.5 ml of phosphate buffer containing the direct mutagen, MNNG) was distributed in sterilized capped tubes in an ice bath, and then 0.1 ml of test bacterial suspension from an overnight culture (1 × 109 to 2 × 109 cells/ml) and 0.1 ml of test compounds (50 ␮l of mutagens and/or 50 ␮l of test compounds) were added. After vortexing gently and preincubating at 37 ◦ C for 30 min, 2 ml of the top agar

75

supplemented with l-histidine and d-biotin kept at 45 ◦ C was added to each tube and vortexed for 3 s. The resulting entire mixture was overlaid on the minimal agar plate. The plates were incubated at 37 ◦ C for 48 h and then the revertant bacterial colonies on each plate were counted. Toxicity tests were also carried out upon the samples on the different bacterial cells, and the sample concentrations employed for the antimutagenic test were found to be non-toxic. 2.11. Statistics The antimutagenicity assay results were analyzed using Student’s t-test.

3. Results and discussion Xenobiotic substances such as aflatoxin B1 , benzopyrene, and bromobenzene undergo epoxidation of their aromatic ring and glutathione conjugation in the liver. These epoxides or their intermediates induce lipid peroxidation in cell membranes as an electrophilic metabolite. Thus, a variety of aromatics including many natural aromatics often can be potential carcinogens. In addition, they can react with DNA strands to cause mutagenicity. Therefore, many xenobiotic substances could act as carcinogens (Choi et al., 1997). As an anticarcinogenic experiment, we investigated the in vivo anti-lipid peroxidation of an extract of Rhus verniciflua heartwood and its biochemical activity in bromobenzene-treated rats. The methanolic extract was found to inhibit lipid peroxidation as determined by the decreased malondialdehyde levels (Table 1). The activity level of hepatic microsomal enzymes such as cytochrome P450 and aniline hydroxylase was also considerably reduced. The activity level of epoxide hydrolase that metabolizes bromobenzene 3,4-oxide was significantly increased as was the activity level of glutathione S-transferase. In contrast, we found glutathione depletion, suggesting that it was consumed for the detoxification of bromobenzene. These results indicate that the methanolic extract effectively inhibited hepatic microsomal enzymes responsible for activating xenobiotic substances and resulted in the inhibition of lipid peroxidation. Therefore, the methanolic extract was found to have a potent inhibitory effect not only on the activation of xenobiotics but also on lipid peroxidation. In the Ames test, the methanolic extract inhibited strongly both aflatoxin B1 - and MNNG-induced mutagenicity (Table 2). Of the three fractions of the methanolic extract, EtOAc showed the more potent antimutagenicity in both experimental systems. In a search for active components, the EtOAc fraction was chromatographed over silica gel, ODS and Sephadex LH-20 to yield four compounds. These compounds were identified to be garbanzol (3,4 ,7-trihydroxyflavanone, 1), sulfuretin (3 ,4 ,6-trihydroxyaurone, 2), fisetin (3,3 ,4 ,7-tetrahydroxyflavone, 3), fustin (3,3 ,4 ,7-tetrahydroxy flavanone, 4), mollisacasidin (3␤,4␣,5,7,3 ,4 -pentahydroxyflavan, 5) by

Ascorbic acid

100 250 500 100

Abbreviations: MDA: malondialdehyde; Cyto-P450: cytochrome P450 ; GST: glutathione S-transferase. The values in the parentheses of the columns of MDA, Cyto-P450, p-aminophenol, and TSO represent percent inhibition rate, but the values in the parentheses of the columns of GST and glutathione represent percent activation rate. Data represent means ± S.D. (n = 10). The significance was tested by Student t-test (∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. control group). a Units represent nmol/g of tissue. b Units represent 1,2-dichlorobenzene nmol/mg protein/min. c Units represent ␮mol/g of tissue.

1.01 59.2 65.7 48.0 57.5 51.5 ± ± ± ± ± ± 203.6 177.4 193.2 189.3 190.7 198.4 11.4 ± 2.59∗∗∗ (100) 4.30 ± 0.70 (0) 7.32 ± 0.98∗∗ (43) 7.97 ± 1.33∗∗∗ (52) 8.43 ± 0.79∗∗∗ (56) 10.20 ± 0.95∗∗∗ (83) 0.19∗∗∗ (100) 0.316 (0) 0.41 (33) 0.19∗ (46) 0.35∗∗ (69) 0.32∗∗∗ (81) ± ± ± ± ± ± 0.54 1.34 1.08 0.97 0.79 0.69 0.228∗∗∗ (100) 0.262 (0) 0.149 (41) 0.126∗ (71) 0.114∗∗ (80) 0.136∗∗∗ (90) ± ± ± ± ± ± 0.65 1.16 0.95 0.80 0.75 0.70 2.33∗∗∗ (100) 1.67 (0) 1.23∗∗ (27) 0.63∗∗ (46) 1.67∗∗∗ (65) 1.36∗∗∗ (91) ± ± ± ± ± ± 23.7 46.9 40.6 36.2 31.9 25.8 Untreated Control MeOH extract

TSO (nmol)a p-Aminophenol (nmol)a Cyto-P450 (nmol)a MDA (nmol)a Dose (mg/kg) Group

Table 1 Effect of the MeOH extract of Rhus verniciflua heartwood on lipid peroxidation and its biochemical parameters in bromobenzene-treated male rats

GST (nmol)b

(100) (0) (60) (45) (51) (80)

2.15 1.89 1.78 1.98 1.90 2.03

± ± ± ± ± ±

1.01∗ (100) 0.63 (0) 0.66 (−42) 0.76 (35) 0.60 (4) 0.57 (54)

K.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 73–79 Glutathione (␮mol)c

76

spectroscopic methods (data not shown) as shown in Fig. 1. The physicochemical data of 1–5, melting point and [␣]D , were in agreement with the reported ones (Harborne, 1994a,b,c,d,e). The isolates were found to be aglycones, 5-deoxyflavonoids with much less molecular size, but not flavonoid glycosides, which are commonly found in natural source. In general, the 5-hydroxy-4-keto system inhibits metallic oxidation-reduction reaction by chelating action to metal ions (Lee et al., 1999). Based on the occurrence of 5-deoxyflavonoids in this plant, it appears that flavonoids without 5-OH may avoid the inhibition of laccase activity with cupric cation as cofactor. In case of injury of this plant, these flavonoids could be used as substrates for radical-dependent reactions in the presence of laccase to form various complex of antimicrobial substances (Eggert, 1997). Antimutagenic compounds with low molecular weights are more common than those with higher weights, based on our experience. The former may more readily penetrate cells and show rapid activity. The inhibitory effect of the EtOAc fraction on AFB1 induced mutagenicity was attributed to two flavonoids, sulfuretin and fisetin, (Table 3). The antimutagenicity of fisetin has been previously reported (Choi et al., 1994). A mutagen, AFB1 , undergoes an epoxidation process by a hepatic microsomal cytochrome P450 and successive epoxide hydrolysis and glutathione conjugation. The epoxide and other intermediates substitute for the nitrogens of nucleic acid in DNA strands and cleave DNA strands to cause mutagenicity. Moreover, several metabolic intermediates and reactive oxygen species (ROS) formed during the process of microsomal enzyme activation are also capable of breaking DNA strands. In contrast, MNNG requires no activation by hepatic microsomal enzymes to damage DNA and induce mutagenicity. The EtOAc fraction exhibited significant antimutagenicity in the MNNG-induced test as well as in the AFB1 -induced one (Table 2). This suggests that the EtOAc fraction inhibits microsomal enzyme activation or that the extract may directly protect DNA strands from the electrophilic metabolite of the mutagen. In the Ames test, the activity of sulfuretin was comparable to that of the EtOAc fraction (Tables 2 and 3). In the case of natural products, it is commonly found that isolated compounds have less activity than the corresponding extract. The reason may be attributable to the complex mix of components, several of which may be capable of defending the life-form in a variety of ways. Based on the dose-dependent behavior of sulfuretin, we are confident that it has an antimutagenic action. Many flavonoids can be carcinogenic or prooxidants to DNA at certain concentrations (Johnson and Loo, 2000). As the Ames test results of quercetin are shown in Table 3, it has a dose-independent effect, which indicates that it is not a functional antimutagen though it is known to have mutagenicity/antimutagenicity (Yoshino et al., 1999). Naringenin also did not show a satisfactory dose-dependency in the MNNG-induced test (Table 4). Fustin may have mutagenicity and antimutagenicity because it also showed a

K.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 73–79

77

Table 2 Effect of each extract from Rhus verniciflua on the mutagenicity induced by aflatoxin B1 (0.5 ␮g/plate) and MNNG (0.4 ␮g/plate) in Salmonella typhymurium TA100 Treatment (mg/plate)

Revertants/plate (AFB1 ) 0.25

Mutagen + MeOH extract Mutagen + CHCl3 extract Mutagen + EtOAc extract Mutagen + BuOH extract Mutagen (control) Spontaneous

542 487 211 943 953 130

Revertants/plate (MNNG)

0.5 ± ± ± ± ± ±

24a ,∗∗∗

(50)b

12∗∗∗ (57) 15∗∗∗ (90) 30 (1) 14 6∗∗∗

1.0

389 285 161 855 953 130

± ± ± ± ± ±

18∗∗∗

(69) 26∗∗∗ (81) 5∗∗∗ (96) 9∗∗ (12) 14 6∗∗∗

0.25

194 165 148 770 953 130

The significance was tested by Student t-test (∗ P < 0.05, ∗∗ P < 0.01 and a Values represent mean ± S.D. based on three experiments. b The values in parenthesis are the inhibition rates (%).

± ± ± ± ± ±

9∗∗∗

0.5

(92) 757 ± 5∗∗∗ (96) 739 ± 1∗∗∗ (98) 716 ± 42∗∗ (22) 637 ± 14 1014 ± 6∗∗∗ 143 ±

∗∗∗ P

1.0

19∗∗∗

(30) 659 ± 19∗∗∗ (32) 601 ± 13∗∗∗ (34) 440 ± 32∗∗∗ (43) 484 ± 26 1014 ± 3∗∗∗ 143 ±

19∗∗∗

(41) 461 ± 16∗∗∗ (47) 296 ± 28∗∗∗ (66) 225 ± 10∗∗∗ (61) 343 ± 26 1014 ± 3∗∗∗ 143 ±

< 0.001 vs. control group).

HO

OH

OH

OH HO

O

19∗∗∗ (63) 32∗∗∗ (82) 1∗∗∗ (91) 25∗∗∗ (77) 26 3∗∗∗

OH HO

HO

O

O

OH

OH O

O

O

3

2

1 OH

OH

OH

OH HO

O O

HO OH

OH

O OH

4

5

Fig. 1. Structure of flavonoids (1–5) isolated from Rhus veniciflua.

Table 3 Effect of the flavonoids from Rhus verniciflua on the mutagenicity induced by aflatoxin B1 (0.5 ␮g/plate) and MNNG (0.4 ␮g/plate) in Salmonella typhymurium TA100 Treatment (mg/plate)

Revertants/plate (AFB1 ) 0.125

Mutagen + 1 (garbanzol) Mutagen + 2 (sulfuretin) Mutagen + 3 (fisetin) Mutagen + 4 (fustin) Mutagen + 5 (mollisacasidin) Mutagen + quercetin Mutagen + naringenin Mutagen (control) Spontaneous

1267 859 837 723 741 500 372 953 130

Revertants/plate (MNNG) 0.250

± ± ± ± ± ± ± ± ±

16a

(–)b

24∗∗ (11) 36∗∗ (14) 20∗∗∗ (28) 38∗∗ (26) 27∗∗∗ (55) 13∗∗∗ (71) 14 6∗∗∗

1167 474 525 796 1066 857 334 953 130

The significance was tested by Student t-test (∗ P < 0.05, ∗∗ P < 0.01 and a Values represent mean ± S.D. based on three experiments. b The values in parenthesis are the inhibition rates (%).

0.125 ± ± ± ± ± ± ± ± ±

∗∗∗ P

16 (–) 8∗∗∗ (58) 26∗∗∗ (52) 22∗∗ (19) 22 (–) 21∗∗ (12) 7∗∗∗ (75) 14 6∗∗∗

683 936 848 1021 969 808 722 1014 143

< 0.001 vs. control group).

0.250 ± ± ± ± ± ± ± ± ±

12∗∗∗

(38) 28∗ (9) 13∗∗∗ (14) 23 (–) 11 (5) 18∗∗∗ (24) 21∗∗∗ (34) 26 3∗∗∗

947 909 762 1016 951 739 817 1014 143

± ± ± ± ± ± ± ± ±

23∗ (8) 20∗∗ (12) 20∗∗∗ (29) 21 (–) 22∗ (7) 30∗∗∗ (32) 15∗∗∗ (23) 26 3∗∗∗

78

K.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 73–79

Table 4 Effect of EtOAc fraction, its isolated flavonoids (2, 4) and the mixture (2 + 4) from Rhus verniciflua on the mutagenicity induced by MNNG (0.4 ␮g/plate) in Salmonella typhymurium TA100 Treatment (mg/plate)

Revertants/plate 0.125

MNNG + EtOAc fraction MNNG + 2 (sulfuretin) MNNG + 4 (fustin) MNNG + (2 + 4) (1:1) MNNG (control) Spontaneous

682 857 982 961 942 85

0.25

± ± ± ± ± ±

15a ,∗∗∗

(30)b

529 847 942 920 942 85

36∗ (10) 7 (–) 28 (–) 35 2∗∗∗

The significance was tested by Student t-test (∗ P < 0.05, ∗∗ P < 0.01 and a Values represent mean ± S.D. based on three experiments. b The values in parenthesis are the inhibition rates (%).

0.5 ± ± ± ± ± ±

∗∗∗ P

16∗∗∗

(48) 42∗ (11) 14 (–) 13 (3) 35 2∗∗∗

434 675 878 868 942 85

1.0 ± ± ± ± ± ±

29∗∗∗

(59) 30∗∗∗ (31) 17∗ (7) 23∗ (9) 35 2

231 448 822 725 942 85

± ± ± ± ± ±

23∗∗∗ (83) 9∗∗∗ (58) 11∗∗ (14) 10∗∗∗ (25) 35 2

< 0.001 vs. control group).

Table 5 Effect of EtOAc fraction, its isolated flavonoids (2, 4) and the mixture (2 + 4) from Rhus verniciflua on the mutagenicity induced by aflatoxin B1 (0.5 ␮g/ml) in Salmonella typhymurium TA100 Treatment (mg/plate)

Revertants/plate 0.125

AFB1 + EtOAc fraction AFB1 + 2 (sulfuretin) AFB1 + 4 (fustin) AFB1 + (2 + 4) (1:1) AFB1 (control) Spontaneous

841 1187 860 1242 1186 144

0.25 ± ± ± ± ± ±

24a ,∗∗

(33)b

60 (–) 33∗∗ (31) 23 (–) 37 7∗∗∗

0.5

360 906 974 958 1186 144

The significance was tested by Student t-test (∗ P < 0.05, ∗∗ P < 0.01 and a Values represent mean ± S.D. based on three experiments. b The values in parenthesis are the inhibition rates (%).

dose-independent action. To find the inhibitory effect of sulfuretin on the mutagenic property of fustin, an artificial mixture (1:1) between sulfuretin and fustin was subjected to the Ames test at different concentrations (Tables 4 and 5). The mixture showed dose-dependent antimutagenicity, though the activity did not reach that of sulfuretin alone. This suggests that sulfuretin protected against the mutagenicity of fustin. In summary, Rhus verniciflua extract and its component, sulfuretin, could be suitable anticarcinogenic agents. Acknowledgements This work was supported by the Basic Research Program of the Korea Science & Engineering Foundation (2000-2-20900-012-3). References Bidlack, W.R., Lowery, G.L., 1982. Multiple drug metabolism: p-nitroanisole reversal of acetone enhanced aniline hydroxylation. Biochemical Pharmacology 31, 311–317. Choi, J.S., Park, K.Y., Moon, S.H., Rhee, S.H., Young, H.S., 1994. Antimutagenic effect of plant flavonoids in the Salmonella assay system. Archives of Pharmaceutical Research 17, 71–75.

± ± ± ± ± ±

20∗∗∗

(79) (27) 45∗ (20) 18∗ (22) 37 7∗∗∗ 52∗

∗∗∗ P

296 623 1157 820 1186 144

1.0 ± ± ± ± ± ±

12∗∗∗

26∗∗∗

(85) (54)

19 (3) 3∗∗ (35) 37 7∗∗∗

219 279 1492 466 1186 144

± ± ± ± ± ±

30∗∗∗ (93) 21∗∗∗ (87) 39 (–) 27∗∗∗ (69) 37 7∗∗∗

< 0.001 vs. control group).

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