Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC50 = 2.5 ± 0.6 µM, Ki = 2.4 ± 1.0 µM, kinact = 0.07 ± 0.01 min−1, kinact/Ki = 0.03 min−1 µM−1. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.

THE EFFECT OF PSEUDOCEDRELA KOTSCHYI (SCHWEINF.) HARMS EXTRACTS AGAINST STAPHYLOCOCCUS AUREUS GROWTH

Staphylococcus aureus is a ubiquitous bacteria that causes a serious health problem because of its multi-resistance to antibiotics. The aimed of this study was to evaluate the antibacterial activity of P. kotschyi root against S. aureus ATCC 29213 and S. aureus, a clinical strain. The phytochemical compound was sought in the extracts by standard staining tests and extractions were carried out by fractionation using solvents depletion method with increasing polarity. The method of dilution in liquid medium was used for the antibacterial tests. The results showed that the aqueous extract of the bark was effective against both S. aureus with MICs of 0.39 mg.mL-1. The MICs of ethanolic extract were 0.39 and 0.78 mg.mL-1, respectively on S. aureus ATCC 29213 and S. aureus. The steles of P. kotschyi roots were less effective. The ethanolic and aqueous extracts at concentrations = 2MICs, have been respectively bacteriostatic and bactericidal effects on tested germs. The kinetics of inhibition showed that the aqueous extract of the bark at 0.78 mg.mL-1, completely destroyed the two germs respectively in 4 h and 5 h. The action of ethanolic extract at 0.78 and 1.56 mg.mL-1 occurs late against both S. aureus at 6 h and 7 h. Phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, anthracenes, glycosides, saponosides and sterols, some of that may be responsible for the observed antimicrobial activity. This study proved the antibacterial activity of P. kotschyi roots that can be exploited as an antibiotic in the treatment of S. aureus infections.

In vitro antibacterial activity and in vivo efficacy of sulbactam-durlobactam against pathogenic Burkholderia species

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, B. mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen β-lactamase, which confers resistance to β-lactams. The β-lactam-β-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. Herein, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured minimal inhibitory concentrations (MICs) of SUL-DUR against BCC and B. gladioli (N = 150), B. mallei (N = 30) and B. pseudomallei (N = 28); studied the kinetics of inhibition of the PenA1 β-lactamase from B. multivorans and the PenI β-lactamase from B. pseudomallei by durlobactam; tested for blaPenA1 induction by SUL-DUR; and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 μg/mL. Durlobactam potently inhibited PenA1 and PenI with k2/K values of 3.9 x 106 M−1s−1 and 2.6 x 103 M−1s−1 and Ki app of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest SUL-DUR may be useful as a treatment for Burkholderia infections.

Anti-pathogenic efficacy of biogenic silver nanoparticles through adherence and biofilm inhibition in multidrug resistant ESKAPE pathogens

Background The current study aimed to produce AgNPs through a biogenic approach and assessed for their significant anti-pathogenic activities against multi-drug resistant ESKAPE pathogens. The biogenic AgNPs were synthesized through non-toxic manner and characterized by using UV-vis, XRD, DLS, TGA-DTA, FTIR, SEM along with EDX and UHRTEM were used to determine the absorption spectra, shape, size, thermal behaviour, functional groups, morphology, elemental constituents and defined particle size distribution profile, respectively. The AgNPs were evaluated for their anti-pathogenic effects against eleven strains of multi drug resistant ESKAPE pathogens by growth inhibition, biofilm adhesion, growth kinetics and Live/dead assays.Results The inhibitory range of AgNPs concentration was investigated as higher zones at escalating concentration (50 to 200 µg/ml). The growth kinetics of inhibition of all tested pathogens occurred after 4 hrs of treatment with AgNPs. Adherence assay exhibited highest inhibition in E. faecium (MCC 2763), P. aeruginosa (MTCC 1688) and E. species (MCC 2296) at 100µg/ml of AgNPs. The exposure of AgNPs increased the dead cell and consequently reduced cells density with AgNPs comparable with the effect of commercial antibiotics. The selected pathogens were found more sensitive to AgNPs than Cefotaxime/AgNO3 with the statistically significant (P < 0.05).Conclusion The emergence of drug resistance in ESKAPE pathogens are the extending reason for nosocomial infections, limiting the choice of antibiotics. Nanomaterials have been considered potential agents to prevent infections. Therefore, present study showed the broad spectrum potential and anti-pathogenic potency of biogenic AgNPs as an alternative to conventional antimicrobial agents.

Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4–7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (4.6, 2.83, 2.46 × 10−5 mM−1·s−1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC50 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Studies on Antimicrobial Activity and Kinetics of Inhibition by Plant Products in India (1990–2016)

The antimicrobial activity of herbal extracts or plant isolates has usually been evaluated in India using different antimicrobial susceptibility testing methods generally based on diffusion and dilution. There are different analytical approaches for the reliable evaluation of antimicrobial activity ascribed to medicinal plants against selected pathogenic microorganisms. Obtained results may provide scientific bases for the selective use of these natural plants as healing drugs, crop-protecting pesticides, or shelf-life-extending solutions. In general, antimicrobial susceptibility methodologies involve in vivo and in vitro studies; at present, the in vitro evaluation of antibacterial activity appears more popular. Diffusion methods have some limitations, although they are extensively used to determine the susceptibility of organisms isolated from specimen samples to applied antimicrobials and vice versa. Dilution methods are preferred in the case of more precise antimicrobial activity estimation, in terms of minimum inhibitory concentration. With regard to the inherent antimicrobial nature of herbal compositions, herbs, and herbal extracts, Indian researchers have evaluated the reliability of these antimicrobial agents against selected pathogens and have shown them to be effective. Researchers have also tried to establish linear regression correlation analyses on the basis of available inhibition results. This research is still evolving, and interesting results may be expected in the future.