Antibacterial, antifungal and antioxidant activities of whole plant chemical constituents of Rumex abyssinicus.

BACKGROUND: Antibiotic resistance has contributed to the burden of infectious diseases both in the hospital and community setting, and represents a great threat to public health. Previous studies have revealed the role of reactive oxygen species as intermediate mediators of tissue damage, following antibiotherapies, indicating the need of associating antioxidants to these treatments. Therefore, the present work was designed to study the antibacterial, antifungal and antioxidant activities of extracts and compounds from Rumex abyssinicus Jacq. (Polygonaceae), as well as to investigate the antibacterial mechanisms of action of the most effective agents. METHODS: The plant extracts were prepared by maceration in organic solvents followed by column chromatography of the EtOAc fraction and purification of different fractions which led to the isolation and characterization of pure compounds. The antimicrobial activities of the extracts/compounds and their combinations with ciprofloxacin and fluconazole were evaluated using the broth microdilution method by determining the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC). The effects of the extracts on the bacterial cell membrane and microbial respiratory chain dehydrogenase enzyme activity were determined by spectrophotometric methods. Antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and gallic acid equivalent antioxidant capacity (GAEAC) assays. RESULTS: Chrysophanol (1), physcion (2), Ergosta-6,22-diene-3,5,8-triol (3), emodin (4), 6-hydroxyemodin (citreorosein) (5), chrysophanein (6) and physcionin (7) were isolated from EtOAc fraction of R. abyssinicus and displayed different degrees of antimicrobial activities (MIC = 8-256 µg/mL). The MeOH extract and compounds 2 and 4 exhibited synergistic effects with ciprofloxacin and fluconazole. Compounds 1, 2 and the combined mixture of 6 + 7 displayed the highest antioxidant activity (GAEAC = 83.38-106.03 µg/mL). CONCLUSION: R. abyssinicus is a potential source of antibacterial, antifungal and antioxidant agents. The antibacterial mechanisms of action of the MeOH extract and compound 2 are due to disruption of the cytoplasmic membrane and inhibition of the microbial respiratory chain dehydrogenase enzyme activity. To the best of our knowledge, this is the first report of test samples and ciprofloxacin / fluconazole association against MDR strains. The observed activity of the isolated compounds against bacteria and fungi including MDR strains deserves further exploration.

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa.

BACKGROUND: Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa. METHODS: In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval. RESULTS: Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (ß = 0.490), both IMDM (ß = 0.256) and DMEM (ß = 0.198) media and the protein supplements NCS (ß = 0.052) and FBS (ß = 0.022); while for L. loa L3, in addition to feeder cells (ß = 0.259) and both IMDM (ß = 0.401) and DMEM (ß = 0.385) media, the protein supplements BSA (ß = 0.029) were found important in maintaining the worm motility. CONCLUSIONS: The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.