Effect of Agrimonia eupatoria L. and Origanum vulgare L. Leaf, Flower, Stem, and Root Extracts on the Survival of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most antibiotic multi-resistant bacteria, causing chronic pulmonary disease and leading to respiratory failure and even mortality. Thus, there has been an ever-increasing search for novel and preferably natural antimicrobial compounds. Agrimonia eupatoria L. and Origanum vulgare L. shoots are commonly used as teas or alcoholic tinctures for their human health-promoting and antibacterial properties. Here, we explored the antimicrobial effects of all plant parts, i.e., leaf, flower, stem, and root extracts, prepared in water or in 60% ethanol, against P. aeruginosa. The impact of these extracts on bacterial survival was determined using a luminescent strain of P. aeruginosa, which emits light when alive. In addition, the antimicrobial effects were compared with the antioxidant properties and content of phenolic compounds of plant extracts. Ethanolic extracts of O. vulgare roots and flowers showed the highest antimicrobial activity, followed by A. eupatoria roots. In particular, chlorogenic acid, the ethanolic extract of O. vulgare roots contained high levels of protocatechuic acid, hesperidin, shikimic acid, rutin, quercetin, and morin. The synergistic effects of these phenolic compounds and flavonoids may play a key role in the antibacterial activity of teas and tinctures.

The effects of heavy metal ions, phthalates and ochratoxin A on oxidation of carcinogenic aristolochic acid I causing Balkan endemic nephropathy.

OBJECTIVES: Balkan endemic nephropathy (BEN) is a chronic progressive fibrosis associated with upper urothelial carcinoma (UUC). Aetiology of BEN is still not fully explained. Although carcinogenic aristolochic acid I (AAI) was proven as the major cause of BEN/UUC, this nephropathy is considered to be multifactorial. Hence, we investigated whether other factors considered as potential causes of BEN [a mycotoxin ochratoxin A (OTA), Cd, Pb, Se and As ions and organic compounds (i.e. phthalates) released from lignite deposits in BEN areas] can influence detoxication of AAI, whose concentrations are crucial for BEN development. METHODS: Oxidation of AAI to 8-hydroxyaristolochic acid I (AAIa) in the presence of Cd, Pb, Se, As ions, dibutylphthalate (DBP), butylbenzylphthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP) and OTA by rat liver microsomes was determined by HPLC. RESULTS: Only OTA, cadmium and selenium ions, and BBP inhibited AAI oxidation by rat liver microsomes. These compounds also inhibited activities of CYP1A1 and/or CYP2C6/11 catalysing AAI demethylation in rat livers. Therefore, these CYP inhibitions can be responsible for a decrease in AAIa formation. When the combined effects of these compounds were investigated, the most efficient inhibition was caused by OTA combined with BBP and selenium ions. CONCLUSION: The results show low effects of BBP, cadmium and selenium ions, and/or their combinations on AAI detoxication. No effects were produced by the other metal ions (Pb, As) and phthalates DBP and DEHP. This finding suggests that they do not influence AAI-mediated BEN development. In contrast, OTA might influence this process, by inhibition of AAI detoxication.

Impact of beta-naphthoflavone on genotoxicity of food-derived carcinogens.

OBJECTIVES: Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, which frequently occur in the human diet. Their metabolic activation to reactive species binding to DNA is mediated by cytochromes P450 (CYPs) 1A1 and 1A2. Thus, levels and activities of these CYPs are crucial for initiation of BaP- and PhPI-mediated carcinogenesis. Here, the effect of CYP1A1/2 induction due to their prototype flavonoid inducer, ß-naphthoflavone (BNF), on BaP- and PhPI-derived DNA adduct formation in rats was examined. METHODS: Male rats pretreated with BNF were treated with a single dose of either carcinogen by oral gavage. Nuclease P1 version of 32P-postlabeling assay and online column-switching liquid chromatography-electrospray ionization-tandem mass spectrometry were used to detect and quantify covalent DNA adducts formed by BaP and PhIP in-vivo, respectively. Expression of CYP1A1/2 enzymes was examined by Western blot. Enzymatic activities of CYP1A1/2 were assessed using their marker substrates (ethoxyresorufin and methoxyresorufin). RESULTS: Treatment of rats with a single dose of BNF produced an increase in levels CYP1A1/2 and CYP1A1 proteins in liver and small intestine, respectively. An increase in CYP1A1 protein expression found in both organs correlated well with specific activities of these CYPs. The CYP1A1 expression levels and its specific activity in small intestine decreased along the length of the organ, being highest in its proximal part and lowest in its distal part. The BNF induction of CYP1A1/2 resulted in a significant increase in the formation of BaP- and PhIP-DNA adducts in liver and in the distal part of the small intestine, respectively. Thus, pretreatment of rats with BNF did not prevent the PhIP and BaP activation, but vice versa, enhanced their genotoxicity. CONCLUSIONS: The results of this study demonstrate that the administration of only a single dose of CYP-inducing flavonoid prior to the intake of food carcinogens may increase the risk of a tumor formation.

Role of cytochromes P450 in metabolism of carcinogenic aristolochic acid I: evidence of their contribution to aristolochic acid I detoxication and activation in rat liver.

OBJECTIVE: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model. METHODS: DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC. RESULTS: Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed. CONCLUSION: Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.