Antimutagenicity of hops (Humulus lupulus L.): bioassay-directed fractionation and isolation of xanthohumol.
The female inflorescences (hops) of the hop plant, Humulus lupulus L., are mainly used in the brewing industry to add bitterness and flavor to beer. Hops are known not only in beer brewing processes but also as a tranquilizer in folk medicines. Hops constituents have recently gained considerable attention due to its various biological effects. One of the most important finding has been the potential cancer chemopreventive properties of xanthohumol, a prenylated chalcone only present in the hop plant. It exhibits a broad spectrum of inhibition mechanisms at all stages of cancer development (Gerhauser, 2005; Stevens and Page, 2004). Given the health promoting potential of xanthohumol, it could find new applications, either as a phytopharmaceutical product or as a food additive.
We used bioassay-directed fractionation with Salmonella/microsomal assay against the food borne mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to identify antimutagenic components of hops. As the major antimutagenic compound we isolated pure xanthohumol. The antigenotoxic potential of the isolated xanthohumol was confirmed in mammalian test system with metabolically competent human hepatoma HepG2 cells.
2. Materials and methods
2.1. Preparation and fractionation of extract from hop pellets
Hop pellets of cultivar Golding were obtained from Pivovarna Lasko brewery (Lasko, Slovenia).
The pellets were ground and extracted with diethylether to give crude extract (sample DE). DE was fractionated by column liquid chromatography over Silica gel 60 (Merck, Darmstadt, Germany) using dichloromethane/methanol (93:7 (v/v)) as a mobile phase. 5 ml fractions were collected, analyzed by TLC and combined according to the spot pattern to yield samples E1-E6. Sample E4 was separated by semipreparative HPLC using Eurospher (120 x 16mm, C18, 5 [micro]m) column (Bia, Ljubljana, Slovenia) with 70% methanol in water at flow rate of 6ml/min. The eluate was detected at 280 nm and fractions collected every 30 s. Fractions were combined according to the chromatogram to yield samples E4a-E4e. Sample E4b was separated by semi-preparative HPLC on Eurospher column using gradient elution with acetonitrile (solvent A) and water (solvent B) at flow rate of 6ml/min and a gradient profile of 20% A from 0-15min, 20-30% A from 15-40 min, 30-70% A from 40-45 min, 70% A from 45-50 min, 70-20% A from 50-60 min, and 20% A from 60-70min to get samples E4[b.sub.I]-E4[b.sub.VIII].
2.2. Chemical analyses
1H NMR and 13C NMR spectra of the sample E4d were obtained with a Bruker AVANCE DP[X.sub.300] spectrometer, in DMSO-d6 containing tetramethylsilane as the internal standard. A mass spectrum was recorded with an ESI-MS mass spectrometer (AutoSpecE) using electron ionization (EI). The UV spectrum was measured with Beckman DU 530 Life Science UV/Vis Spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The spectroscopic data were identical to those already reported for xanthohumol by Hansel and Schulz (1988).
2.3. Determination of mutagenicity and antimutagenicity with the Salmonella typhimurium/microsomal (Ames) assay
For the bioassays the dry extract and fractions of the extract were dissolved in 1% DMSO (Sigma, Saint Louis, MO, USA). Mutagenicity and antimutagenicity of the samples were tested using the plate incorporation assay with S. typhimurium TA98 (Maron and Ames, 1983). To test antimutagenic activity of the samples, 10 [micro]g/ml 4-nitroquinoline (4-NQNO) (final concentration 1 [micro]g/plate) and 10 or 50nmol/ml IQ (final concentration 1 or 5nmol/plate) respectively, were added to the overnight grown bacterial suspension just prior to the experiment. 100 [micro]l of bacterial suspension (with or without the mutagen), 100 [micro]l of appropriately diluted sample and 500 [micro]l of PBS or S9 mix (containing 10% S9--Arachlor induced rat liver microsomal fraction (Moltox, Boone, NC, USA)) were added to 2 ml of molten soft agar containing a limited amount of histidine/biotin, mixed gently and poured onto minimal agar plates. [His.sup.+] revertants were counted after 48 h incubation at 37 [degrees]C. Three plates were used per experimental point. The antimutagenic potency was expressed as an inhibition factor (IF) calculated as IF = (number of mutagen induced revertants in the presence of the sample)/(number of mutagen induced revertants).
2.4. Determination of genotoxicity and antigenotoxicity with human hepatoma HepG2 cells
The HepG2 cells (the gift from Dr Firouz Darroudi, Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Netherlands) were grown in William's medium E (Sigma, St. Louis, MO, USA) containing 15% foetal bovine serum, 2mM L-glutamine and 100 U/ml penicillin/streptomycin at 37[degrees]C in 5% C[O.sub.2].
2.5. Determination of cytotoxicity
Cytotoxicity of E4d fraction was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) according to Mosmann (1983), with minor modifications. HepG2 cells seeded onto 96-well microtiter plates were exposed to different concentrations of the samples for 21 h, MTT was then added to a final concentration of 0.5 [micro]g/ml and the cells were incubated for another 3 h. The medium was removed and the formazan crystals were dissolved in DMSO. The absorbance ([A.sub.570]) was measured (reference filter 690 nm) using an ELISA microplate reader. Survival was determined by comparing the [A.sub.570/690] of the wells containing treated cells to those of non-treated cells.
2.6. Comet assay
The cells were treated with 0.1, 1 and 10[micro]g/ml of the E4d fraction alone or in combination with IQ (1 mM) for 24 h. The cells were then harvested by trypsinization and comet assay performed as described (Zegura and Filipic, 2004). The slides were stained with ethidium bromide (5 [micro]g/ml) and analyzed using a fluorescence microscope (Nikon, Eclipse 800). Images of 50 randomly selected cells per experimental point from each of the four cultures (experiment was performed twice, each time with two separate cultures of HepG2 cells) were analyzed with image analysis software (VisCOMET, TillPhotonics, Germany). The % of tail DNA was used to measure DNA strand breaks.
3. Results and discussion
3.1. Salmonella/microsomal assay directed fractionation of hops pellets
The bioassay directed fractionation of hop pellets is summarized in Fig. 1. The diethylether extract (DE) was not mutagenic for Salmonella typhimurium TA98 (Table 1). It showed a dose dependent inhibition of IQ induced mutations but no inhibition of 4-NQNO induced mutations (Table 1). Based on microscopic examination of the background lawn, no signs of toxicity were observed in bacteria treated with the extract alone or in combination with IQ, while the combination of 4-NQNO and the extract was toxic for bacteria. Thus the extract contains components that are active against indirect acting mutagen and therefore inhibition of IQ induced mutations was used as an assay to direct further fractionation of the extracts.
Separation of diethylether extract (DE) yielded six fractions (E1-E6), of which only fraction E4 significantly inhibited IQ-induced mutations (Table 2). Separation of the fraction E4 gave five fractions (E4a-E4e). Fraction E4b inhibited IQ-induced mutations by 50% without cytotoxic effect, while fractions E4d and E4e were cytotoxic (Table 2). Fractionation of E4b yielded seven (E4[b.sub.I]-E4[b.sub.VII]) fractions, and fraction E4[b.sub.VII] inhibited IQ-induced mutations (Table 2). Fractionation of E4[b.sub.VII] yielded six fractions, however none of them was antimutagenic (data not shown).
[FIGURE 1 OMITTED]
Fraction E4d was identified to be xanthohumol. At non-toxic concentrations (1-100 [micro]g/plate) this fraction exhibited a dose-related inhibition of IQ-induced mutations (Table 3). The result is in line with those of Miranda et al. (2000), who showed that xanthohumol inhibited mutagenicity of IQ, activated by rat liver S9 or c-DNA expressed human CYP1A2 in S. typhimurium TA98.
3.2. Antigenotoxicity of fraction E4d (xanthohumol) in human hepatoma HepG2 cells
Bacterial test systems have been used extensively for screening purposes as well as for the bioassay directed fractionation of antimutagens from different sources. However, several recent studies have shown that certain compounds that were protective in bacterial assay were not protective in human derived cells or were even genotoxic (Knasmuller et al., 2002). Human hepatoma HepG2 cells retain the activities of many drug-metabolizing enzymes in an inducible form (Knasmuller et al., 1998) and they have been shown to be appropriate indicator cells for the investigation of dietary antimu-tages as they enable detection of protective mechanisms that are not presented in other in vitro models (Knasmuller et al., 2002; Mersch-Sundermann et al., 2004).
In HepG2 cells we investigated the effect of fraction E4d on IQ induced DNA strand breaks, measured with the single-cell gel electrophoresis (comet) assay (Tice et al., 2000). Fraction E4d did not affect the viability of HepG2 cells and did not induce DNA damage (Fig. 2A), while it inhibited IQ induced DNA damage in a dose dependent manner (Fig. 2B).
The genotoxicity of IQ depends on its metabolic activation to reactive intermediates by a two-step process involving N-hydroxylation by CYP1A2, followed by esterification of N-hydroxylamine by N-acetyltransferase or sulfotransferase to reactive ester derivatives that covalently modify DNA (Schut and Snyderwine, 1999). Previous studies indicated that xanthohumol can inhibit activity of CYP1A enzymes (Henderson et al., 2000; Gerhauser et al., 2002) and CYPIA mediated formation of DNA reactive metabolites of IQ (Miranda et al., 2000). Therefore, it is likely that the protective effect of xanthohumol against IQ induced DNA damage in mammalian cells can also be attributed to inhibition of its activation. In conclusion, our study showed that xanthohumol is not genotoxic and that it exerts very strong protective effect against the genotoxicity of the food borne carcinogen IQ in bacteria and in mammalian cells. Our results provide additional evidence for cancer preventive potential of xanthohumol.
This study was supported by Slovenian Research Agency, program # PI-0245, by the EU grant HEPD-NA. QLRT-2001-02863 and by Lasko brewery (Lasko. Slovenia). We thank Prof. Roger Pain for critical reading of the manuscript.
[FIGURE 2 OMITTED]
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Javor Kac (a), Janja Plazar (b), Ales Mlinaric (a), Bojana Zegura (b), Tamara T. Lah (b), Metka Filipic (b,*)
(a) Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
(b) Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia
*Corresponding author. Tel.: +386 1 257 38 48; fax: + 386 1 257 38 47.
E-mail address: email@example.com (M. Filipic).
Table 1. Effect of diethylether extract (DE) on 4-NQNO- and IQ-induced mutations in S. typhimurium TA98 Number of revertants/plate [+ or -] SD Treatment S9 mix 0[micro]g/plate 10[micro]g/plate DE - 27 [+ or -] 1.0 26 [+ or -] 2.6 DE + 20 [+ or -] 1.0 21 [+ or -] 5.7 4-NQNO (a) + DE - 317 [+ or -] 19.4 299 [+ or -] 7.5 IQ (b) + DE + 613 [+ or -] 59 615 [+ or -] 70 (1.0) Number of revertants/plate [+ or -] SD Treatment 100[micro]g/plate 1000 [micro]g/plate DE 25 [+ or -] 3.6 25 [+ or -] 3.8 DE 19 [+ or -] 2.0 18 [+ or -] 4.7 4-NQNO (a) + DE 269 [+ or -] 9.6* Toxic IQ (b) + DE 436 [+ or -] 45 (0.71) 267 [+ or -] 65 (0.44) (a) 4-NQNO: 1 [micro]g/plate. (b) IQ: 5ng/plate; * toxic effect (reduced background lawn); inhibition factor (IF) is shown in parentheses. Table 2. Inhibition of IQ-induced mutations in S. typhimurium TA98 by DE derived fractions E1-E6; E4 derived fractions E4a-E4d and E4b derived fractions E4[b.sub.I]-E4[b.sub.VII] Sample [micro]g/plate Revertants/plate[+ or -]SD IF Control 46 [+ or -] 7 IQ (1 nmol/plate) 191 [+ or -] 13 1.00 E1 200 192 [+ or -] 45 1.01 (a) E2 200 153 [+ or -] 11 0.80 E3 200 142 [+ or -] 23 0.74 E4# 200# 104 [+ or -] 11# 0.54# E5 200 139 [+ or -] 29 0.73 E6 200 185 [+ or -] 42 0.97 Control 39 [+ or -] 0.7 IQ (1 nmol/plate) 257 [+ or -] 14 1.00 E4a 200 214 [+ or -] 60 0.83 E4b# 200# 132 [+ or -] 24# 0.51# E4c 200 156 [+ or -] 76 0.61 E4d# 200# 31# (a) E4e 200 145 [+ or -] 30 0.56 (a) Control 40 [+ or -] 1.7 IQ (1 nmol/plate) 219 [+ or -] 48 1.00 E4[b.sub.I] 40 229 [+ or -] 93 1.04 E4[b.sub.II] 60 218 [+ or -] 81 1.00 E4[b.sub.III] 60 156 [+ or -] 51 0.72 E4[b.sub.IV] 120 257 [+ or -] 85 1.17 E4[b.sub.V] 90 114 [+ or -] 25 0.52 (a) E4[b.sub.VI] 80 303 [+ or -] 106 1.38 E4[b.sub.VII]# 40# 50 [+ or -] 13# 0.23# (a) Toxic effect (reduced background lawn); active fractions are marked in bold. Note: Toxic effect (reduced background lawn); active fractions are marked indicated with #. Table 3. Dose dependent inhibition of IQ-induced mutations in S. typhimurium TA98 by fraction E4d (xanthohumol) Sample [micro]g/plate Revertants/plate [+ or -] SD IF Control 40 [+ or -] 5.1 IQ (1 nmol/plate) 188 [+ or -] 26.5 1.00 E4d 0.1 185 [+ or -] 8.3 0.99 E4d 1 159 [+ or -] 32.3 0.85 E4d 10 125 [+ or -] 27.3 0.67 E4d 100 78 [+ or -] 9.2 0.41
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|Author:||Kac, Javor; Plazar, Janja; Mlinaric, Ales; Zegura, Bojana; Lah, Tamara T.; Filipic, Metka|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Mar 1, 2008|
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