RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

Transcriptomic and biochemical analyses revealed antifungal mechanism of trans-anethole on Aspergillus flavus growth.

Plant volatile compounds have great potential for preventing and controlling fungal spoilage in post-harvest grains. Recently, we have reported the antifungal effects of trans-anethole, the main volatile constituent of the Illicium verum fruit, on Aspergillus flavus. In this study, the inhibitory mechanisms of trans-anethole against the growth of A. flavus mycelia were investigated using transcriptomic and biochemical analyses. Biochemical and transcriptomic changes in A. flavus mycelia were evaluated after exposure to 0.2 µL/mL trans-anethole. Scanning electron microscopy showed that trans-anethole treatment resulted in the surface wrinkling of A. flavus mycelia, and calcofluor white staining confirmed that trans-anethole treatment disrupted the mycelial cell wall structure. Annexin V-fluorescein isothiocyanate/propidium iodide double staining suggested that trans-anethole induced apoptosis in A. flavus mycelia. Reduced mitochondrial membrane potential and DNA damage were observed in trans-anethole-treated A. flavus mycelia using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine and 4',6-diamidino-2-phenylindole staining, respectively. 2',7'- Dichloro-dihydro-fluorescein diacetate staining and biochemical assays demonstrated that trans-anethole treatment cause the accumulation of reactive oxygen species in the A. flavus mycelia. Transcriptome results showed that 1673 genes were differentially expressed in A. flavus mycelia exposed to trans-anethole, which were mainly associated with multidrug transport, oxidative phosphorylation, citric acid cycle, ribosomes, and cyclic adenosine monophosphate signaling. We propose that trans-anethole can inhibit the growth of A. flavus mycelia by disrupting the cell wall structure, blocking the multidrug transport process, disturbing the citric acid cycle, and inducing apoptosis. This study provides new insights into the inhibitory mechanism of trans-anethole on A. flavus mycelia and will be helpful for the development of natural fungicides. KEY POINTS: • Biochemical analyses of A. flavus mycelia exposed to trans-anethole were performed • Transcriptomic changes in trans-anethole-treated A. flavus mycelia were analyzed • An inhibitory mechanism of trans-anethole on the growth of A. flavus mycelia was proposed.

Isolation, Characterization and Antibacterial Activity of 4-Allylbenzene-1,2-diol from Piper austrosinense.

Isolation for antibacterial compounds from natural plants is a promising approach to develop new pesticides. In this study, two compounds were obtained from the Chinese endemic plant Piper austrosinense using bioassay-guided fractionation. Based on analyses of 1H-NMR, 13C-NMR, and mass spectral data, the isolated compounds were identified as 4-allylbenzene-1,2-diol and (S)-4-allyl-5-(1-(3,4-dihydroxyphenyl)allyl)benzene-1,2-diol. 4-Allylbenzene-1,2-diol was shown to have strong antibacterial activity against four plant pathogens, including Xanthomonas oryzae pathovar oryzae (Xoo), X. axonopodis pv. citri (Xac), X. oryzae pv. oryzicola (Xoc) and X. campestris pv. mangiferaeindicae (Xcm). Further bioassay results exhibited that 4-allylbenzene-1,2-diol had a broad antibacterial spectrum, including Xoo, Xac, Xoc, Xcm, X. fragariae (Xf), X. campestris pv. campestris (Xcc), Pectobacterium carotovorum subspecies brasiliense (Pcb) and P. carotovorum subsp. carotovorum (Pcc), with minimum inhibitory concentration (MIC) values ranging from 333.75 to 1335 µmol/L. The pot experiment showed that 4-allylbenzene-1,2-diol exerted an excellent protective effect against Xoo, with a controlled efficacy reaching 72.73% at 4 MIC, which was superior to the positive control kasugamycin (53.03%) at 4 MIC. Further results demonstrated that the 4-allylbenzene-1,2-diol damaged the integrity of the cell membrane and increased cell membrane permeability. In addition, 4-allylbenzene-1,2-diol also prevented the pathogenicity-related biofilm formation in Xoo, thus limiting the movement of Xoo and reducing the production of extracellular polysaccharides (EPS) in Xoo. These findings suggest the value of 4-allylbenzene-1,2-diol and P. austrosinense could be as promising resources for developing novel antibacterial agents.

Dissecting dietary and semisynthetic volatile phenylpropenes: A compile of their distribution, food properties, health effects, metabolism and toxicities.

Phenylpropenes represent a major subclass of plant volatiles, including eugenol, and (E)-anethole. They contribute to the flavor and aroma of many chief herbs and spices, to exert distinct notes in food, i.e., spicy anise- and clove-like to fruit. Asides from their culinary use, they appear to exert general health effects, whereas some effects are specific, e.g., eugenol being a natural local anesthetic. This review represents the most comprehensive overview of phenylpropenes with respect to their chemical structures, different health effects, and their food applications as flavor and food preservatives. Side effects and toxicities of these compounds represent the second main part of this review, as some were reported for certain metabolites generated inside the body. Several metabolic reactions mediating for phenylpropenes metabolism in rodents via cytochrome P450 (CYP450) and sulfotransferase (SULT) enzymes are presented being involved in their toxicities. Such effects can be lessened by influencing their pharmacokinetics through a matrix-derived combination effect via administration of herbal extracts containing SULT inhibitors, i.e., nevadensin in sweet basil. Moreover, structural modification of phenylpropanes appears to improve their effects and broaden their applications. Hence, such review capitalizing on phenylpropenes can help optimize their applications in nutraceuticals, cosmeceuticals, and food applications.

Laccase-Catalyzed Oxidation of Allylbenzene Derivatives: Towards a Green Equivalent of Ozonolysis.

Laccase-based biocatalytic reactions have been tested with and without mediators and optimized in the oxidation of allylbenzene derivatives, such as methyl eugenol taken as a model substrate. The reaction primarily consisted in the hydroxylation of the propenyl side chain, either upon isomerization of the double bond or not. Two pathways were then observed; oxidation of both allylic alcohol intermediates could either lead to the corresponding α,ß-unsaturated carbonyl compound, or the corresponding benzaldehyde derivative by oxidative cleavage. Such a process constitutes a green equivalent of ozonolysis or other dangerous or waste-generating oxidation reactions. The conversion rate was sensitive to the substitution patterns of the benzenic ring and subsequent electronic effects.

Modeling the hydroxylation of estragole via human liver cytochrome P450.

Natural compounds derived from plants are generally regarded safe and devoid of adverse effects. However, there are individual ingredients that possess toxic, genotoxic, and carcinogenic activities. These compounds when exposed at specific level become hazardous to health. Estragole (1-allyl-4-methoxybenzene) is a common component of spice plants. Its toxicity gets activated with the hydroxylation at benzylic carbon (C1') position by P450 enzymes present in the human liver. The present study grounds to explore the reaction mechanism of conversion of estragole to hydroxylated metabolite using computational methodology. Density functional theory (DFT)-based calculations were employed to explore the cytochrome P450-catalyzed mechanism at C1 position aliphatic hydroxylation of estragole. Overall reaction energy profile, electronic configuration, and 3D structure of all intermediates, transition states, and product complexes formed during the reaction along with their free energies were tried to be investigated.

Bioactivation of estragole and anethole leads to common adducts in DNA and hemoglobin.

Estragole and anethole are secondary metabolites occurring in a variety of commonly used herbs like fennel, basil, and anise. Estragole is genotoxic and carcinogenic in rodents, which depends on the formation of 1'-sulfoxyestragole after hydroxylation and subsequent sulfoconjugation catalyzed by CYP and SULT, respectively. It was hypothesized recently that anethole may be bioactivated via the same metabolic pathways. Incubating estragole with hepatic S9-fractions from rats and humans, specific adducts with hemoglobin (N-(isoestragole-3-yl)-valine, IES-Val) and DNA (isoestragole-2'-deoxyguanosine and isoestragole-2'-deoxyadenosine) were formed. An isotope-dilution technique was developed for the quantification of IES-Val after cleavage with fluorescein isothiocyanate (FITC) according to a modified Edman degradation. The same adducts, albeit at lower levels, were also detected in reactions with anethole, indicating the formation of 3'-hydroxyanethole and the reactive 3'-sulfoxyanethole. Finally, we conducted a pilot investigation in which IES-Val levels in human blood were determined during and after the consumption of an estragole- and anethole-rich fennel tea for four weeks. A significant increase of IES-Val levels was observed during the consumption phase and followed by a continuous decrease during the washout period. IES-Val may be used to monitor the internal exposure to the common reactive genotoxic metabolites of estragole and anethole, 1'-sulfoxyestragole and 3'-sulfoxyanethole, respectively.