Antimicrobial Activity of Single And Combined Extracts of Medicinal Plants From Cameroon

Selected Cameroonian plants have been investigated for their antifungal and antibacterial activity against five species: four bacteria, namely S. aureus, S. pyogenes, S. mutans, Pseudomonas aeruginosa and the yeast C. albicans. The solvents used for plant extraction were either ethanol or water. The in vitro antimicrobial activity was performed by disk diffusion and microdilution method. The aqueous extracts showed no activity whereas the ethanolic extracts showed a significant antibacterial activity, which may be associated to the high content in tannins shown by some of the extracts. In conclusion, this work adds to the accumulating evidences supporting the development of new alternatives to antibiotics based on the use of natural products

Molecular cloning and biochemical characterization of Xaa-Pro dipeptidyl-peptidase from Streptococcus mutans and its inhibition by anti-human DPP IV drugs

Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans, we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg-1, while the Vmax and Km were 7 μM min-1 and 89 ± 7 μM (n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent (Ki = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents

Fluorescence study on rat epithelial cells and liposomes exposed to aromatic nitroxides

This study was performed to evaluate the effects, if any, of aromatic nitroxides, namely, indolinic nitroxides, on membrane fluidity of rat epithelial cells using steady-state fluorescence. These nitroxides are being increasingly considered as new and versatile compounds to reduce oxidative stress in biological systems. Hence, the results obtained in this study will give more insights on the interaction of these compounds with biological structures which at present is lacking, especially in view of their possible application as antioxidant therapeutic agents. The probes DPH and Laurdan which give information on the hydrophobic and hydrophilic-hydrophobic regions of the membrane bilayer, respectively, showed that nitroxide 1 (1,2-dihydro-2-methyl-3H-indole-3-one-1-oxyl) significantly increases membrane fluidity, whereas the corresponding phenylimino nitroxide derivative 2 (1,2-dihydro-2-methyl-3H-indole-3-phenylimino-1-oxyl) leads to membrane rigidification. The aliphatic nitroxide TEMPO included in this study for comparison produced no modifications. Consequently, it appears that the structure of the heterocyclic rings (aromatic or aliphatic) and the substituents may affect membrane fluidity differently. (C) 2004 Elsevier Inc. All rights reserved

Structure-function relationships in bovine thymus 20S proteasome: a fluorimetric study

The structure-function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle. (C) 2001 Elsevier Science B.V. All rights reserved

Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects

Peroxynitrite is a potent reactive oxygen species that is believed to mediate deleterious protein modifications in a wide variety of neurodegenerative disorders. In this study, we have analysed the effects of oxidative damage induced by peroxynitrite on a cysteine-free mutant of dihydrofolate reductase (SE-DHFR), from a functional and a structural point of view. The peroxynitrite-mediated oxidation results in the inhibition, concentration-dependent, of the catalytic activity. This effect is strongly influenced by the HCO(3)(-)/CO(2) buffering system, that we observed to significantly affect the yield of protein oxidation by modulating the peroxynitrite-induced modification of aromatic residues. Because of this effect, in presence of bicarbonate system, we have observed a protection of enzymatic activity of SE-DHFR with regard to peroxynitrite. The thermodynamic stability of the oxidized protein has been studied in comparison with the non-oxidized protein by differential scanning calorimetry. The thermodynamic parameters obtained showed a decrease of stability of SE-DHFR upon oxidation, evaluated in terms of Gibbs free energy of about 1.25 kcal/mol at 25 degrees C, with respect to the non-oxidized protein. Together, these data indicate that structural and functional alterations induced by peroxynitrite may play a direct role in compromising DHFR function in multiple pathological conditions

Antioxidant and Ex Vivo Immune System Regulatory Properties of Boswellia serrata Extracts

Boswellia serrata (BS) is an important traditional medicinal plant that currently represents an interesting topic for pharmaceutical research since it possesses several pharmacological properties (e.g., anti-inflammatory, antimicrobial, and antitumour). The safety and versatility of this dietary supplement should allow for its use in numerous pathological conditions; however the quality of the extracts needs to be standardized to increase the clinical success rate resulting from its use. In the present study, different commercially available B. serrata extracts were employed to compare their AKBA content and in vitro antioxidant power. Furthermore, their ability to modulate the immune system regulatory properties was investigated. Our results showed that the AKBA content varied from 3.83 ± 0.10 to 0.03 ± 0.004%, with one sample in which it was not detectable. The highest antioxidant power and phenolic content were shown by the same extract, which also exhibited the highest AKBA concentration. Finally, the BS extracts showed the ability to influence the regulatory and effector T-cell compartments. Our results suggest that frankincense should be further investigated for its promising potentiality to modulate not only inflammation/oxidative stress but also immune dysregulation, but attention should be paid to the composition of the commercial extracts

Rapid Assay to Evaluate the Total Antioxidant Capacity in Donkey Milk and in more Common Animal Milk for Human Consumption

The milk antioxidants, by preventing lipid peroxidation, maintain milk quality, but they also exert a beneficial effect on the consumer’s health, in particular that of infants. Donkey Milk (DM), for its nutritional, functional and bioactive components, seems to be one of the best substitutes of breast milk when the latter is not available. However, there are few data about its antioxidant properties. In this study, the Total Antioxidant Capacity (TAC) of donkey milk was determined by means of an in micro-plate assay. DM samples were analyzed at the first, third and fifth month of the lactation period (n 6/period), comparing results to those obtained in milk of different dairy species (goat, ewes, cows) and in breast milk using the same assay. The lactation periods did not affect the TAC of DM, whereas significant different values (P<0.001) were observed between species. The breast milk showed the lowest TAC value, followed by its progressive increase in donkey, cow’s, goat’s and ewe’s milk. The rapid test here adopted can be successfully employed for a reliable monitoring of the TAC in DM and, thanks to the constant antioxidant supply, DM can also be sponsored as a valid alternative to infant milk nutrition

DNA binding and oxidative DNA damage induced by climacostol\u2013copper(II) complexes: Implications for anticancer properties

Climacostol is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids. Climacostol exerts a potent antimicrobial activity against a panel of bacterial and fungal pathogens. In addition it inhibits the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death via intrinsic pathway. In this work, we investigated the possibility that climacostol exerts a prooxidant effect, inducing plasmid DNA strand breakage and eukaryotic DNA damage in presence of Cu(II) ions. Inhibition of DNA breakage using SOD, catalase and neocuproine confirmed the involvement of reactive oxygen species and Cu(I) ions in the DNA damage. UV\u2013visible absorption changes and mass spectrometric analysis identified a product of reaction as a deprotonated form of climacostol. Study of the interaction with DNA, using fluorescence spectroscopic techniques, showed that climacostol binds with DNA. Given the structure\u2013activity relationship of this compound and the mechanism of its prooxidant effect, we propose that the Cu(II)-mediated oxidative DNA damage by climacostol could explain its antimicrobial and antiproliferative activity

Characterization and Biological Activities of In Vitro Digested Olive Pomace Polyphenols Evaluated on Ex Vivo Human Immune Blood Cells

Olive pomace (OP) represents one of the main by-products of olive oil production, which still contains high quantities of health-promoting bioactive compounds. In the present study, three batches of sun-dried OP were characterized for their profile in phenolic compounds (by HPLC-DAD) and in vitro antioxidant properties (ABTS, FRAP and DPPH assays) before (methanolic extracts) and after (aqueous extracts) their simulated in vitro digestion and dialysis. Phenolic profiles, and, accordingly, the antioxidant activities, showed significant differences among the three OP batches, and most compounds showed good bioaccessibility after simulated digestion. Based on these preliminary screenings, the best OP aqueous extract (OP-W) was further characterized for its peptide composition and subdivided into seven fractions (OP-F). The most promising OP-F (characterized for its metabolome) and OP-W samples were then assessed for their potential anti-inflammatory properties in ex vivo human peripheral mononuclear cells (PBMCs) triggered or not with lipopolysaccharide (LPS). The levels of 16 pro-and anti-inflammatory cytokines were measured in PBMC culture media by multiplex ELISA assay, whereas the gene expressions of interleukin-6 (IL-6), IL-10 and TNF-α were measured by real time RT-qPCR. Interestingly, OP-W and PO-F samples had a similar effect in reducing the expressions of IL-6 and TNF-α, but only OP-W was able to reduce the release of these inflammatory mediators, suggesting that the anti-inflammatory activity of OP-W is different from that of OP-F

Multi-Targeted Anticancer Activity of Imidazolate Phosphane Gold(I) Compounds by Inhibition of DHFR and TrxR in Breast Cancer Cells

A class of phosphane gold(I) compounds, made of azoles and phosphane ligands, was evaluated for a screening on the regards of Breast Cancer cell panels (BC). The compounds possess N-Au-P or Cl-Au-P bonds around the central metal, and they differ for the presence of aprotic or protic polar groups in the azoles and/or the phosphane moieties to tune their hydrophilicity. Among the six candidates, only the compounds having the P-Au-N environment and not displaying neither the hydroxyl nor carboxyl groups in the ligands were found active. The compounds were screened by MTT tests in SKBR3, A17, and MDA-MB231 cancer cells, and two compounds (namely the 4,5-dicyano-imidazolate-1yl-gold(I)-(triphenylphosphane, 5, and 4,5-dichloro-imidazolate-1yl-gold(I)-triphenylphosphane, 6) were found very cytotoxic, with the most active with an IC50 value of 3.46 μM in MDA-MB231 cells. By performing enzymatic assays in the treated cells lysates, the residual enzymatic activity of dihydrofolate reductase (DHFR) has been measured after cell treatment for 4 or 12 h in comparison with control cells. Upon 12 h of treatment, the activity of DHFR was significantly reduced in both SKBR3 and A17 cells by compounds 5 and 6, but not in human MDA-MB231 cells; interestingly, it was found remarkably high after 4 h of treatment, revealing a time dependence for the DHFR enzymatic assays. The DHFR inhibition data have been compared to those for the thioredoxin reductase (TrxR), the most recognized molecular target for gold compounds. For this latter, similar residual activities (i.e., 37 and 49% for the match of SKBR3 cells and compound 5 or 6, respectively) were found. Binding studies on the regards of ct-DNA (calf-thymus-DNA) and of plasma transporters proteins, such as BSA (bovine serum albumin) and ATF (apo transferrin), were performed. As expected for gold compounds, the data support strong binding to proteins (Ksv values range: 1.51 ÷ 2.46 × 104 M−1) and a weaker interaction with ct-DNA's minor groove (Ksv values range: 1.55 ÷ 6.12 × 103 M−1)