Flavonoids and Other Compounds from Ouratea ferruginea (Ochnaceae) as Anticancer and Chemopreventive Agents

The chemical study of the extracts from leaves and stems of Ouratea ferruginea allowed the identification of a new isoflavone, 5-hydroxy-7,3′4′5′-tetramethoxyisoflavone, and twenty two known compounds, including friedelin, 3β-friedelinol, lupeone, a mixture of sitosterol, stigmasterol and campesterol, sitosteryl- and stigmasteryl-3-O-β-D-glucopyranosides, 5,4′-dihydroxy-7,5′,3′-trimethoxyisoflavone, 5,4′-dihydroxy-7,3′-di-methoxyisoflavone (7,3′-di-O-methylorobol), 5,7,4′-trihydroxy-3′,5′-dimethoxyisoflavone (piscigenin), 2R,3R-epicatechin, syringic acid, 2,6-dimethoxybenzoquinone, 2,6-dimethoxyhydroquinone, syringic and ferulic aldehyde, a mixture of vanillic acid, 1-hydroxy-2-methoxy-4-(1E-3-hydroxy-1-propenyl)-benzene and 3,5-dimethoxy-4-hydroxy-dihydrocinamaldehyde, besides amenthoflavone and 7-O-methylamenthoflavone (sequoiaflavone) which are considered as chemotaxonomic markers of Ouratea. The structures were identified by IR, 1H- and 13C-NMR and GC-MS, HPLC-MS, besides comparison with literature data. The inhibitory effects of 5,4′-dihydroxy-7,5′,3′-trimethoxyisoflavone, 7,3′-di-O-methylorobol, piscigenin and 7-O-methylamenthoflavone on cytochrome P450-dependent 7-ethoxycoumarin O-deethylase (ECOD) and glutathione S-transferase (GST) were evaluated in vitro. The 5,4′-dihydroxy-7,5′,3′-trimethoxy-isoflavone was the best inhibitor, inhibiting almost 75% of GST activity. Sequoiaflavone was the most potent inhibitor, inhibiting ECOD assay in 75%. These activities allow us to consider both these flavonoids as potential anticancer and chemopreventive agents.


Introduction
Ouratea Alb. genus is the largest within the Ochnaceae family with approximately 120 species [1]. Plants in this family are known to be rich in flavonoids and biflavonoids. Biflavonoids can be used as chemotaxonomic markers of the genus [2]. Some biflavonoids, as well as extracts of Ouratea species showed important biological activities such as DNA topoisomerase inhibition, cytotoxic, antitumoral activities and other pharmacological activities [3].
The cancer protective effects of flavonoids have been attributed to a wide variety of mechanisms, including free radical scavenging, modifying enzymes that activate or detoxify carcinogens, and inhibiting the induction of the transcription factor activator protein-1 (AP-1) activity by tumor promoters [4]. Among the enzymes, which are likely to play important roles in carcinogenesis that interact with flavonoids, cytochromes P450 (CYPs) and glutathione S-transferases (GST), are the most extensively studied.
Cytochrome P450s (monooxygenase enzymes) are enzymes that catalyze a variety of chemical reactions such as O-dealkylation, N-dealkylation, O-hydroxylation and N-hydroxylation and thus play an important role primarily in phase I of xenobiotic metabolism. They help their excretion and activate the endogenous compounds (estradiol, testosterone, 11-deoxycortisol, arachidonic acid and vitamin D) to modify the products that are involved in the regulation of physiological and cellular processes (homeostasis, endocrine control, inflammation and cellular proliferation) [5]. Many naturally occurring flavonoids have been shown to modulate the CYP450 system, including the induction of specific CYP isoenzymes, and the activation or inhibition of these enzymes.
Glutathione S-transferases (GSTs, EC 2.5.1.18) catalyze the binding of a large variety of electrophiles to the sulfydryl group of glutathione, that are involved in the detoxification of (oxygen) radicals, and have a main function in the binding and transport of a wide variety of harmful compounds [6]. The major carcinogenic agents are exogenous or metabolically generated reactive oxygen species (ROS) and electrophiles arising from the environment and from in vivo normal oxidative processes. Enhanced ROS levels are involved in initiation and promotion of tumor and may ultimately lead to carcinogenesis [7].
The present paper reports the first phytochemical study of extracts from stems and leaves of Ouratea ferruginea Engl, describing the isolation and identification of twenty four compounds, including five isoflavones and two biflavonoids. Four isolated compounds were evaluated on ECOD (CYP1A1) and GST in vitro inhibition.

Biological Activity
The tested flavonoids revealed important anti-cancer properties. These properties may be attributed to their capacity of inhibiting phase I enzymes or activating phase II enzymes. These phase I and phase II metabolizing enzymes are the key to carcinogen metabolism. Thus inhibition or enhancement of their activities are a significantly chemopreventative cancer effect. The potential of compounds listed in Table 1 to modulate the activity of drug-metabolizing enzymes, i.e., phase I, cytochromes P450 (Cyp) enzymes and phase II detoxifying enzymes (gluthation S-transferase) was investigated. ECOD assay was selected as a phase I marker enzyme. ECOD is catalyzed by many CYP enzymes, including CYP1A1, CYP2A6, CYP2B6, and CYP2E1, and also CYP3A to a lesser extent. The CYP 1 family consists of 1A1, 1A2, and 1B1 members that are capable of activating procarcinogens and high CYP1A1 activity is also associated with colorectal cancer [19]. CYP2A6 is a hepatic enzyme predominantly with some expression in specialized extrahepatic cell types. Studies about the CYP have revealed that CYP2A6 is the high-affinity metabolizer of nicotine and several procarcinogens present in cigarette smoke [20]. CYP2B is a large subfamily that encodes versatile catalysts of xenobiotics and steroid hydroxylation. CYP2B6 plays a role in the metabolism of endogenous substrates including testosterone steroid [21]. CYP2E1, an ethanol-inducible enzyme, is important in the field of toxicology and carcinogenesis, and it also has a role in drug metabolism [22]. CYP3A is the largest subfamily of CYP enzymes expressed in the liver and gastrointestinal tract. It is involved in the metabolism of 50% of therapeutic agents as well as in the activation of toxic and carcinogenic substances. As indicated in Table 1, all four compounds inhibited ECOD activity, using rat hepatic microsomes as an enzyme source. Sequoiaflavone (15) was the most potent inhibitor, inhibiting ECOD assay in 75.2%, by comparing the enzyme activity 24.8% with control 100%.
Flavonoids have been reported in literature to display significant chemopreventative effects. Some classes of flavonoids, like flavones (apigenin, chrysin), flavonols (fisetin, kaempferol, morin, myrisetin, quercetin), flavanones (hesperetin and naringenin) and flavan [(±)-catechin] were studied in 7-ethoxycoumarin O-deethylase (ECOD) in liver microsomes from rats treated with 3-methylcholanthrene (MC). All the flavonoids studied inhibited microsomal ECOD activity in the following order: flavones > flavonols > flavanones, except (±)-catechin that had no effect. The double bond between C2 and C3 of the C ring, the keto group as well as the hydroxyl groups in the C5 and C7 positions of A ring in the flavones and the hydroxyl group in the C3 position of C ring in the flavonol classes, respectively, were important factors for the inhibition of the enzyme [23].
Sequoiaflavone is a flavone dimer and has a double bond between C2 and C3 of the C ring and hydroxyl groups in the C5 and C7 positions of a ring. Inhibition of CYPs enzymatic activity by biflavonoids has not been cited in the literature. Most studies report the inhibition of CYP1-catalyzed by 7-ethoxyresorufin O-deethylation (EROD) activity [24].
Flavonoids with multiple hydroxyl groups have been documented as effective CYP1 inhibitors, whereas little is known regarding the inhibitory activities of polymethoxylated flavonoids on CYP1 enzymes. Recent studies suggest that CYP1A1 and CYP1B1 enzymes show substrate specificity for flavonoids with multiple methoxy groups [25].
Inhibition of CYP1 enzymatic activity has been reported for genistein, a prominent isoflavone with tumor suppressing activity, found primarily in nuts and soy-based products. Genistein demonstrated effective inhibition of CYP1 enzyme activity by EROD and the DMBA-induced DNA damage assay [26]. Biochanin A, the 4′-methoxylated derivative of genistein, was a more effective inhibitor of CYP1A1 and CYP1B1 enzymatic activities, as it was earlier noted for the flavone acacetin [26]. Substitution of a methoxy group at the 4′-position enhances the inhibition of the isoflavones towards the CYP1A1 and CYP1B1 enzymes [27]. 5,7-Dimethoxyflavone (DMF) isolated from a Malaysian Piper species, reduced CYP1A1 EROD activity of the HepG2 cell virtually down to zero [28]. It is remarkable that two so seemingly similar compounds as DMF and chysin can have such different effects, i.e., the 5,7-dimethoxy compound is a potent inhibitor of CYP1A1 protein whereas the 5,7-dihydroxy compound is a potent inducer [28]. Thus, methylation of flavonoids seems to be an important feature determining enzyme inhibitory properties [25].
Natural products have been in use since ancient times as medicines and spices, and the use of herbal remedies and dietary supplements is ever increasing. It is now well established that plant chemicals can affect or modulate drug-metabolizing enzymes [29]. With this resurgence of interest in natural products, focus on the interaction of the latter with xenobiotic-metabolising enzymes has received increased attention. Many flavonoids are reported to interact with phase II-enzymes like GST, increasing or reducing its activity. Flavonoids such as daidzein, genistein, morin and silymarim have been report to possess GST activity inductor effects [4]. Other flavonoids isolated from plant species (Croton sp., Curcuma sp., Eurycoma sp., Orthosiphon sp. and Cinnamomum sp.) have inhibitory effects on glutathione S-transferases in blood platelets as well as in cancer cell lines [30]. During treatment of many cancers, there is often development of drug resistance in a tumour that was originally sensitive to treatment resulting in a phenomenon known as multidrug resistance (MDR). Many mechanisms are involved in MDR and these include alterations in drug transport resulting in impaired entry or enhanced efflux of the drug from the tumour cell, enhanced DNA repair, alterations in target proteins and alterations in drug metabolism [31]. One of the most important detoxifying enzymes that is involved in MDR belongs to GST family.
In this report, the effect of Ouratea ferruginea flavonoids toward in vitro GST activity was evaluated in the hepatic cytosolic fraction of rat. Our study has revealed that the compounds inhibited rat GST activity in vitro. Piscigenin (11) and 5,4′-dihydroxy-7,3′,5′-trimethoxyisoflavone (9) showed the best inhibitory effects, inhibiting almost 70 and 75% of GST activity, respectively.
The inhibitory effect of 7,3′-di-O-methylorobol (10) on GST activity was not observed. GSTs are promising therapeutic targets because specific isozymes are overexpressed in a wide variety of tumours [32].
Considering the structure activities relationships of the tested isoflavones, the observed effect of 9, 10, and 11 (Table 1), allow us to propose that the ring B of 4′-hydroxy-3′,5′-dimethoxyisoflavone made a difference in the GST enzyme inhibition. It may also be observed that the presence of a hydroxyl or methoxyl group in position C-7 causes a similar effect in the inhibition of GST enzyme as can be seen in Table 1 comparing isoflavones 9 and 11. However, the absence of a methoxy group at C-5′ has a significant effect for the GST as observed with compounds 9 and 10.

Plant Material
The leaves and stems of Ouratea ferruginea Engl. (Ochnaceae) were collected in the campus of Embrapa in Belém, Pará State, Brazil. The species was identified by one of the authors (S. T. Rodrigues), a voucher specimen (Nº IAN-183954) was deposited at the herbarium of Embrapa Amazônia Oriental, Belém, Pará, Brazil.

Extraction and Isolation
The dried and powdered leaves (968.57 g) and stems (3645.97 g) were extracted initially with 4 L n-hexane (leaves) and 12 L dichloromethane (stems), and both with methanol (4 L leaves and 12 L stem), five times at room temperature by three days each extraction. Part of the OFLMFA fraction (2.50 g) was chromatographed on a silica gel column eluted with CHCl3 and increasing the polarity with MeOH. Twenty four fractions were collected and analyzed by TLC. Fraction 10-11 (630 mg) was chromatographed by the same method described above and twelve subfractions were collected. Subfraction 6-9 (169.0 mg) was filtered on Sephadex LH-20 and yielded 2R,3R-epicatechin (13, 13 mg) which absolute configuration was defined by circular dichroism spectrum analysis (500-200 nm; 22 °C). Substance 13 (2.0 mg) was treated with Ac 2 O/pyridine (1:1) to yield the peracetyl derivative 13a.
The structures of the compounds were determined by IR, 1 H-and 13 C-NMR (1D an 2D techniques), mass spectrometry including GC-MS and HPLC-MS analysis of natural compounds and some derivatives.

Preparation of Liver Fractions
Male Wistar rats (130 ± 150 g weight) were obtained from Universidade da Zona Oeste (UEZO). The animals were kept under light-and temperature-controlled conditions and were provided with rodent laboratory chow and water ad libitum. The rats were euthanized prior to the preparation of cytosolic and microsomal fractions. The livers were removed, placed in ice-cold 0.1 M phosphate buffer (pH 7.4) and then homogenized. Fractions were prepared by differential centrifugation [33] and protein content was determined by Peterson's methods [34].

CYP Inhibition Assays
Ethoxycoumarin O-desalkylation by rat liver microsomes was used as a probe to determine CYP1A activity. Ethoxycumarin (1 mM), NADPH (1 mM), magnesium chloride (10 mM) and rat liver microsomes (5.5 μg) were incubated in phosphate buffer (0.1 M), pH 7.8 (0.5 mL final volume). Incubations were at 37 °C for 5 min. The reaction was terminated with trichloroacetic acid (TCA) 2.5% followed by centrifugation (1000 g, 10 min). Immediately before fluorescence determination, 2 mL of glycine-NaOH buffer were added and fluorescence of cumarin was determined (excitation: 390 nm and emission 440 nm). CYP activity was calculated as amount of produced cumarin per minute per protein mg. Assays containing phytochemicals were compared with control with DMSO (dimethylsulfoxide).

GST Inhibition Assays
The conjugation of glutathione to 1-chloro-2,4,-dinitrobenzene (CDNB) was measured as a non-specific substrate for GST activity [35]. To a quartz cuvette reduced cytosolic fraction (10 μg), CDNB (2 mM) and 100 mM potassium phosphate buffer (pH 6.1) were added in a final volume of 1 mL. The cuvette was placed in a spectrophotometer and the reaction initiated by the addition of glutathione (5 mM). The increase in absorbance at 340 nm over a 3 min period was measured at room temperature. An extinction coefficient of 9.6 cm/mM was used to determine activity from the initial slope of the reaction. GST activity was calculated as amount of produced conjugated per minute per protein mg. Assays containing phytochemicals were compared with control with DMSO (dimethylsulfoxide).

Conclusions
The results obtained in this work allowed us to expand the knowledge about the chemical composition of the genus Ouratea and the Ochnaceae family in particular. This is first chemical study of the species Ouratea ferruginea. In this work 24 substances were identified in the extracts of stems and leaves. The substance 7,3′-dimethylorobol (10) is being registered for the first time in Ochnaceae. No record was found in the literature of the substance 5-hydroxy-7,3′,4′,5′-tetramethoxyisoflavone (12). The compounds 5,4′-dihydroxy-7,3′,5′-trimethoxyisoflavone (9) and piscigenin (11), have not been previously identified in Ouratea. The presence of biflavonoids such as amentoflavone (14) in the extracts of leaves confirms the botanical classification, since they are quimiossistematic markers of the genus. The stems of Ouratea ferruginea is a source of methoxyl isoflavones derivatives.
In this report, we also describe the ECOD and GST inhibitory assays. All the tested substances in inhibition assays showed inhibitory effects against at least in one of the enzymes used (ECOD and GST). The moderate and strong inhibitory effects are relevant, because little is known about the enzymatic inhibition effects of methoxyisoflavones and biflavonoids.