Botanicals and phytochemicals from the bark of Hypericum roeperianum (Hypericaceae) had strong antibacterial activity and showed synergistic effects with antibiotics against multidrug-resistant bacteria expressing active efflux pumps.

ETHNOPHARMACOLOGICAL RELEVANCE: Infections due to multidrug-resistant (MDR) bacteria constitute a real problem in the public health worldwide. Hypericum roeperianum Schimp. ex A. Rich (Hypericaceae) is used traditionally for treatment of various ailments such as abdominal pains, constipation, diarrhea, indigestion, nausea, and bacterial diseases. AIM OF THE STUDY: This study was aimed at investigating the antibacterial and antibiotic-modifying activity of the crude methanol extracts (HRB), ethyl-acetate soluble fraction (HRBa), residual material (HRBb), and 11 compounds from the bark of Hypericum roeperianum against multi-drug resistant (MDR) bacteria expressing active efflux pumps. MATERIALS AND METHODS: The antibacterial activity, the efflux pump effect using the efflux pump inhibitor (EPI), phenylalanine-arginine-ß-naphthylamide (PAßN), as well as the antibiotic-modifying activity of samples were determined using the broth micro-dilution method. Spectrophotometric methods were used to evaluate the effects of HRB and 8,8-bis(dihydroconiferyl) diferulate (11) on bacterial growth, and bacterial membrane damage, whereas follow-up of the acidification of the bacterial culture was used to study their effects on bacteria proton-ATPase pumps. RESULTS: The crude extract (HRB), HRBa, and HRBb had selective antibacterial activity with MICs ranging from 16 to 512 µg/mL. Phytochemical 11 displayed the best antibacterial activity (0.5 ≤ MIC ≤ 2 µg/mL). The activity of HRB and 11 in the presence of EPI significantly increased on the tested bacteria strains (up to 32-fold). The activity of cloxacillin (CLO), doxycycline (DOX), and tetracycline (TET), was considerably improved (up to 64-fold) towards the multidrug-resistant Enterobacter aerogenes EA-CM64 strain. The crude extract (HRB) and 11 induced the leakage of bacterial intracellular components and inhibited the proton-ATPase pumps. CONCLUSIONS: The crude extract (HRB) and 8,8-bis(dihydroconiferyl)diferulate from the bark of Hypericum roeperianum are good antibacterial candidates that deserve further investigations to achieve antibacterial drugs to fight infections involving MDR bacteria.

8,8-bis-(Dihydroconiferyl)-diferulate displayed impressive cytotoxicity towards a panel of human and animal cancer cells.

BACKGROUND: Recalcitrant cancers appear as a major obstacle to chemotherapy, prompting scientists to intensify the search for novel drugs to tackle the cell lines expressing multi-drug resistant (MDR) phenotypes. PURPOSE: The purpose of this study was to evaluate the antiproliferative potential of a ferrulic acid derivative, 8,8-bis-(dihydroconiferyl)-diferulate (DHCF2) on a panel of 18 cancer cell lines, including various sensitive and drug-resistant phenotypes, belonging to human and animals. The mode of induction of cell death by this compound was further studied. METHODS: The antiproliferative activity, autophagy, ferroptotic and necroptotic cell death were evaluated by the resazurin reduction assay (RRA). CCRF-CEM leukemia cells were used for all mechanistic studies. A caspase-Glo assay was applied to evaluate the activity of caspases. Cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H2DCFH-DA) were assessed by flow cytometry. RESULTS: DHCF2 demonstrated impressive cytotoxic effects towards the 18 cancer cell lines tested, with IC50 values all below 6.5 µM. The obtained IC50 values were in the range of 1.17 µM (towards CCRF-CEM leukemia cells) to 6.34 µM (towards drug-resistant HCT116 p53-/- human colon adenocarcinoma cells) for DHCF2 and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against multidrug-resistant CEM/ADR5000 leukemia cells) for the reference drug, doxorubicin. DHCF2 had IC50 values lower than those of doxorubicin, against CEM/ADR5000 cells and on some melanoma cell lines, such as MaMel-80a cells, Mel-2a cells, MV3 cells and SKMel-505 cells. DHCF2 induced autophagy as well as apoptosis in CCRF-CEM cells though caspases activation, MMP alteration and increase of ROS production. CONCLUSION: The studied diferulic acid, DHCF2, is a promising antiproliferative compound. It deserves further indepth investigations with the ultimate aim to develop a novel drug to fight cancer drug resistance.

Arabinose Conjugates Diagnostic of Ferulate-Ferulate and Ferulate-Monolignol Cross-Coupling Are Released by Mild Acidolysis of Grass Cell Walls.

Ferulate (FA) units esterified to grass arabinoxylans are involved in cross-linking cell wall polymers. In this work, this contention is strengthened by the identification of FA homo- and heterodimers esterified to methyl arabinofuranoside (MeAra) units after their release from the xylan by mild acidolysis in dioxane/methanol/HCl. Acidolysis of poorly lignified maize bran cell walls provided diferulate (DFA) isomers, including those from 8-5, 8-O-4, and 5-5 interunit bonding, esterified to one or two MeAra units. Acidolysis of lignified grass samples released crossed dimers esterified to one MeAra unit and derived from the ß-O-4 coupling of coniferyl alcohol to FA esters. The evaluation of these heterodimeric esters by LC-UV of their aglycones revealed that the parent structures occur in significant amounts in lignified cell walls (0.5-1 mg/g expressed as FA equivalents). The present results position mild acidolysis as an efficient strategy to obtain improved details regarding the FA-mediated cross-linking of grass cell walls.

Metagenomic discovery of feruloyl esterases from rumen microflora.

Feruloyl esterases (FAEs) are a key group of enzymes that hydrolyze ferulic acids ester-linked to plant polysaccharides. The cow's rumen is a highly evolved ecosystem of complex microbial microflora capable of converting fibrous substances to energy. From direct cloning of the rumen microbial metagenome, we identified seven active phagemids conferring feruloyl esterase activity. The genomic inserts ranged from 1633 to 4143 bp, and the ORFs from 681 to 1359 bp. BLAST search reveals sequence homology to feruloyl esterases and esterases/lipases identified in anaerobes. The seven genes were expressed in Escherichia coli, and the proteins were purified to homogeneity. The FAEs were found to cover types B, C, and D in the feruloyl esterase classification system using model hydroxycinnamic acid esters. The release of ferulic acid (FA) catalyzed by these enzymes was established using natural substrates corn fiber (CF) and wheat insoluble arabinoxylan (WIA). Three of the enzymes were demonstrated to cleave diferulates and hence the capability to break down Araf-FA-FA-Araf cross-links. The wide variation in the sequence, activity, and substrate specificity observed in the FAEs discovered in this study is a confirming evidence that combined actions of a full range of FAE enzymes contribute to the high-efficiency fiber digestion in the rumen microbial ecosystem.

Diferulic acids in the cell wall may contribute to the suppression of shoot growth in the first phase of salt stress in maize.

In the first phase of salt stress the elongation growth of maize shoots is severely affected. The fixation of shape at the end of the elongation phase in Poaceae leaves has frequently been attributed to the formation of phenolic cross-links in the cell wall. In the present work it was investigated whether this process is accelerated under salt stress in different maize hybrids. Plants were grown in nutrient solution in a growth chamber. Reduction of shoot fresh mass was 50% for two hybrids which have recently been developed for improved salt resistance (SR 03, SR 12) and 60% for their parental genotype (Pioneer 3906). For SR 12 and Pioneer 3906, the upper three leaves were divided into elongated and elongating tissue and cell walls were isolated from which phenolic substances and neutral sugars were determined. Furthermore, for the newly developed hybrids the activity of phenolic peroxidase in the cell wall was analysed in apoplastic washing fluids and after sequential extraction of cell-wall material with CaCl2 and LiCl. The concentration of ferulic acid, the predominant phenolic cross-linker in the grass cell wall, was about 5mgg(-1) dry cell wall in elongating and in elongated tissue. The concentration of diferulic acids (DFA) was 2-3mgg(-1) dry cell wall in both tissues. Salt stress increased the concentration of ferulic acid (FA) and DFA in the parental genotype Pioneer 3906, but not in SR 12. Both genotypes showed an increase in arabinose, which is the molecule at which FA and DFA are coupled to interlocking arabinoxylan polymers. In SR 12, the activity of phenolic peroxidase was not influenced by salt stress. However, in SR 03 salt stress clearly increased the phenolic peroxidase activity. Results are consistent with the hypothesis that accelerated oxidative fixation of shape contributes to growth suppression in the first phase of salt stress in a genotype-specific manner.