Antioxidant action and cytotoxicity on HeLa and NIH-3T3 cells of new quercetin derivatives

Quercetin is a natural polyphenol with proven health beneficial activities. In this study 15 new quercetin derivatives were prepared with the aim to enhance their bioavailability. Modification of their physicochemical properties could herewith improve the action in cells. The prepared compounds were tested for their antioxidant and cytotoxic activity. The ability to scavenge free radicals as well as ferric reducing antioxidant power of the new derivatives was not better than that of unmodified quercetin. But for acetylated esters a better cytotoxic activity was found on human cervical cancer cells HeLa than for the initial molecule. The best effect revealed chloronaphtoquinone quercetin (IC50=13.2 µM). For this compound comparable cytotoxic action on non-cancer murine fibroblast cells was detected (IC50=16.5 µM). The obtained results indicate that appropriate lipophilization of the quercetin molecule could improve its cytotoxic action in cells, probably due to its enhanced bioavailability.


Introduction
Cancer diseases represent with their contribution of 20 to 25% the second leading cause of death in the world. In 2007, 28 131 new cancer cases were registered in Slovakia. The incidence and mortality of cancer in recent years in the Slovak population is displaying a raising trend. For this rapid increase and large occurrence mainly prostate and colorectal cancers are responsible in males. In females the dramatic increase in cancer cases was caused by breast, female genital organs and lung cancers (Diba et al., 2012). It is therefore important to deal with the study of new potential anticancer drugs that are effective in selective inhibition of cancer cell growth.
Flavonoids represent such a group of natural, low toxic compounds. They are polyphenolics abundant in the plant kingdom. The average human diet contains a considerable amount of flavonoids and the major dietary sources Among flavonoids, quercetin is considered an excellent free radical scavenging antioxidant. Apart from this quercetin also exerts a direct pro-apoptotic effect on tumor cells and can indeed block the growth of several cancer cell lines at different phases of the cell cycle. Both these effects have been documented in a wide variety of cellular models as well as in animal models (Gibellini et al., 2011). Several studies demonstrated a significant role of quercetin in growth inhibition of breast, colon, prostate, ovary, endometrium and lung cancer cells (Baghel et al., 2012). But its effect is highly dependent on the ability to penetrate the lipophilic bilayer of the cell membranes.
Literature documents that the bioavailability of quercetin is not sufficient (Viskupičová et al., 2008). Authors tried to overcome this disadvantage via modification of the quercetin structure with groups of various polarities. According to published data, it can be concluded that improvement of anticancer properties was seen mainly upon flavonoid structure lipophilization (Cárdenas et al., 2006;Salem et al., 2011).
In this study we tested bioactivities of 15 novel quercetin derivatives with various acyl donors. We assessed their in vitro antioxidant and cytotoxic activity. The relationship between the observed action and polarity of molecules as well as the determined activities were also evaluated.

Quercetin derivatives
Quercetin structure was modified using simple condensation reactions or selective protection procedures and subsequent acylation with acylchlorides (Veverka et al., 2013). The structure of the compounds tested is outlined in Table 1. The purity of all the samples tested as determined by HPLC was approximately 99.8%.

Cell lines
The human tumor cell line HeLa and murine fibroblast NIH-3T3 cells were obtained from the ATCC (Rockville, MD, USA). The cells grown at 37 °C in humidified 5%-CO 2 and 95%-air atmosphere were in Dulbecco's modified eagle medium supplemented with 10% (vol/vol) inactivated bovine serum, penicillin G and streptomycin (100 mg/l). Before a uniform monolayer of cells was formed, cells were freed from the surface of the culture flask by a 0.25% solution of trypsin and were subcultivated two to three times a week. The cells were plated on the cultivation flask (surface 25 cm 2 ) at a density of 4×10 4 HeLa cells and 6×10 4 NIH-3T3 cells per ml medium and incubated for 24 h prior to the experiments.

Scavenging of DPPH radical
We used the simple spectrophotometric method according to Yen and Chen (1995) to determine the ability to scavenge free radicals. Binding of DPPH˙ radical by compounds causes its decolorization. The compounds tested were diluted in methanol to the final concentration 200 μM to 12.5 μM. Trolox served as standard antioxidant control. Reactions of samples with DPPH˙ solution were performed in 96-well plates and lasted for 10 min at room temperature in the dark. After that, the absorbance changes were measured spectrophotometrically at 520 nm (Multiskan FC, Thermo Scientific, DE). Each value is the mean of 5 wells with standard deviation. Inhibitory concentrations (IC 50 ) were expressed as the concentration of the derivative tested to react with one half of the DPPH˙ and were calculated by SigmaPlot.

Ferric reducing antioxidant power (FRAP)
The antioxidant reducing power of the derivatives tested was performed using the FRAP method according to Benzie and Strain (1996). Ferric to ferrous ion reduction at low pH causes the formation of a colored ferroustripyridyltriazine complex. Samples diluted in methanol (200 μM to 12.5 μM) and FRAP reagent reacted for 10 min at 37 °C in 96-well microplates. At the end, absorbance changes were measured spectrophotometrically at 593 nm (Multiskan FC, Thermo Scientific, DE). Each value is the mean of 5 wells with standard deviation. Inhibitory concentrations (IC 50 ) were expressed as the concentration of the derivative tested reducing one half of ferric complexes and were calculated by SigmaPlot.

MTT assay
The cytotoxic effect of the compounds prepared on cells was detected in vitro using the mitochondrial cytotoxic test according to Jantová et al. (2010) with modifications. Cell viability was evaluated using thiazolyl blue tetrazolium bromide (MTT), which indicates the metabolic activity of cells. The experiment was performed in 96-well microplates. The cells were seeded at a density of 3.5×10 3 HeLa or NIH-3T3 cells per well. Samples were dissolved in DMSO (stock solution 10 mM) and subsequently diluted in medium to the final concentration of 5 μM to 100 μM (concentration of DMSO 0.5%) and after 24 h they were added to the cells. Microplates were cultivated for 72 h in thermostat at 37 °C and 5% CO 2 atmosphere. After incubation thiazolyl blue tetrazolium bromide (3.33 mg/ml phosphate buffered saline, pH=7.4) was pipetted to each well and left to incubate for further two hours. Then the medium with MTT solution was removed. Formazan crystals in viable cells were dissolved in the lysis solution (4 mM HCl and 0.1% Nonidet P40 in ethanol). Microplates were shaken 15 min at 1500 rpm. Absorbance was measured at 540 nm and reference wavelength at 740 nm (Multiskan FC, Thermo Scientific, DE). Each value is

Results
We chose two simple spectrophotometric methods to investigate antioxidant properties of the prepared quercetin derivatives. As reference standard synthetic analogue of vitamin E (trolox) was used. The 50% effective doses of samples (IC 50 ) are shown in Table 2.
The cytotoxic action of the effective chloronaphtoquinone quercetin 5 was further observed also by light microscopy after 72 h of HeLa and NIH-3T3 cells cultivation. Microscopic observations (Figure 2) showed that control cells grew on the surface of the cultivation vessels and during 72 h of incubation a monolayer was formed. On the other hand, a significant decrease in cell number was found in cells treated with both concentrations studied (25 μM and 50 μM). Derivative 5 induced morphologic changes of HeLa and NIH-3T3 cells, which resulted within 72 h in cell rounding and cell separation from the cultivation surface.

Discussion
Many health beneficial effects of flavonoids are attributed to their ability to act as antioxidants. Experiments showed that flavonoids as single electron donors can stabilize and eliminate many radical forms, which in conditions of oxidative stress may initiate mutagenesis or carcinogenesis. The chemical structure of flavonoids allows them to chelate transition metals (Miller, 1996). The activity of antioxidants such as flavonoids may prevent damage of DNA, proteins, lipids and other important biomolecules. Quercetin is an effective antioxidant among natural compounds (Baghel et al., 2012). Our testing showed that in binding free radicals and in reducing metals it exerts better activities than the standard antioxidant trolox. Novel acylated and conjugated quercetin derivatives displayed lowered antioxidant capacity in comparison with unmodified quercetin (except monochloropivaloyl quercetin 2 in the FRAP method) ( Table 2). Of the quercetin derivatives screened the most active in both tests were monochloropivaloyl quercetin 2 and chloronaphtoquinone quercetin 5.
Investigations of lowered antioxidant capacity of compounds upon quercetin structure modification are in accordance with the literature because free hydroxyl groups in the structure are necessary to scavenge free radicals or chelate metals. Salem et al. (2011) found that acylation of isorhamnetin-3-O-glucoside decreased its radical scavenging activity. Lue et al. (2010) reported lowered ability of rutin laurate and rutin palmitate to bind metals. Tert-butylhydroxylation of quercetin lowered its ability to scavenge free radicals (Lebeau et al., 2000). Yet it seems that antioxidant activity in lipophilic media was improved in many cases after structure lipophilization (Filipe et al., 2009;Viskupicova et al., 2010). Since quercetin derivatives are metabolized to quercetin in the human body (Materska, 2008), reduced antioxidant capacity in the case of derivatives should not mean a serious problem.
In the last decade, there has been growing interest in searching for cancer prophylactic and therapeutic effects of natural compounds. Many in vitro and some in vivo studies confirmed that flavonoids displayed a range of anticancer actions. They are able to inhibit carcinogen activation, cancer cell proliferation, angiogenesis and metastasis. These compounds also induce apoptosis, have antioxidant and anti-inflammatory effects and reverse multidrug resistance (Genoux et al., 2011).
We monitored prepared quercetin derivatives for their cytotoxic action on human cervical cancer cells HeLa in the concentration range from 5 μM to 100 μM. For comparison non-cancer murine fibroblast cells NIH-3T3 were also evaluated. The MTT test and light microscopy were used for cytotoxicity measurements.
The cytotoxic screening of quercetin derivatives on HeLa and NIH-3T3 cells demonstrated that chloronaphtoquinone quercetin 5, tri(diacetylcaffeoyl)quercetin 11, di(tetraacetylquinoyl) quercetin 9 and pentaacetyl quercetin 6 were more effective than quercetin. The comparison of the structures of quercetin derivatives and their cytotoxic effect showed that acetylated esters were the most effective derivatives.
Other authors published similar results. Acylation of isorhamnetin-3-O-glucoside with ethyl laurate and ethyl butyrate increased their antiproliferative activity against Caco-2 cells (Salem et al., 2011). Cárdenas et al. (2006) found that naphtoflavons were slightly better in antiproliferation than initial flavone molecules. Acylation and alkylation of epicatechin resulted in enhanced inhibitory activity on cancer cells (Park et al., 2004). Introduction of various acyl and alkyl moieties to the flavonoid skeleton caused improvement of its cytotoxic properties. The most effective were dimethoxy flavonoids (Ou et al., 2011). In our experiments, acetyl moieties probably helped

Cell line
HeLa