Antibacterial synergy between rutin and florfenicol enhances therapeutic spectrum against drug resistant Aeromonas hydrophila.

Emergence of antibiotic resistant bacteria has necessitated the drive to explore competent antimicrobial agents or to develop novel formulations to treat infections including Aeromonas hydrophila. The present study investigates the synergistic antibacterial effects of citrus flavonoid rutin and florfenicol (FF) against A. hydrophila in vitro and in vivo. Rutin is extracted and purified from Citrus sinensis peel through preparative HPLC and characterized through TLC, GC-MS and 1H and 13C NMR analyses. Though rutin did not display significant antibacterial activity, it modulated FF activity resulting in four-fold reduction in the MIC value for FF. The anti-biofilm potential of synergistic association of rutin and FF was validated by protein analysis, quantification of exopolysaccharide (EPS) and microscopy studies using sub-MIC doses. Besides antibacterial action, in vivo studies showed that Rutin/FF combination enhanced host immunity by improving blood cell count, anti-protease, and lysozyme activities as well as decreased the oxidative stress and the pathological changes of tilapia Oreochromis niloticus against A. hydrophila infection. No significant DNA damages or clastogenic effects were detected in tilapia challenged with A. hydrophila under Rutin/FF treatment. It is shown that an acute-phase Lipopolysaccharide binding protein (LBP) enhances the innate host defence against bacterial challenge. Semi quantitative RT-PCR and western blot results revealed the significant increase of LBP in the supernatant of tilapia monocytes/macrophages challenged with A. hydrophila upon treatment. The study findings substantiate that the combination of natural molecules with antibiotics may open up possibilities to treat MDR strains.

Dietary supplementation with rutin has pro-/anti-inflammatory effects in the liver of juvenile GIFT tilapia, Oreochromis niloticus.

Dietary supplementation with rutin may have some pharmacological qualities including anti-inflammatory effects. Kupffer cell activation resulted in increased transcription of pro- and anti-inflammatory cytokines. The main purpose of this study was to investigate the pro- and anti-inflammatory activities in juvenile freshwater tilapia, Oreochromis niloticus, in response to 0.1 or 0.3 g/kg dietary supplementation of rutin. Results showed that hepatic IgM, anti-inflammatory-cytokines, and pro-inflammatory cytokines were significantly decreased in groups treated with high doses of rutin. Hepatic IgM and anti-inflammatory cytokines (IL-10 and IFN-γ) transcripts were significantly decreased, whereas the transcripts of the pro-inflammatory cytokines, TNFα and IL-1ß were significantly decreased, whereas IL-8 was significantly increased. The number of Kupffer cells in rutin-treated groups was significantly decreased, and scanning electron micrographs showed that rutin enriched the number of gut microvilli and secretion pits. With the phenomena of cell apoptosis occurred in the rutin groups, the present study demonstrated that optimum levels of rutin may be beneficial but excessive level may cause liver impairment, which may be absorbed by the gut and then transported to the liver.

[Synthesis and identification of rutin complete antigen and analysis its immunogenicity].

OBJECTIVE: Synthesis and identification of complete antigen of rutin, the traditional Chinese medicine active ingredient, and develop rapid detection of rutin using enzyme-linked immunoassay method (ELISA). Immunogenicity of the complete antigen was also studied. METHOD: Prepare the complete antigen by sodium periodate solution and identified by UV scanning and SDS-PAGE test. Male New Zealand white rabbits were immunized by the antigen to obtain the antiserum. RESULT: The results of UV analysis showed that the coupling ratio of complete antigen is 13: 1. SDS-PAGE display of the artificial antigen was delayed compared with bovine serum protein. The titer of rutin antibody is 1:4 000. The sensitivity of IC50 was 5.37 mg x L(-1), the lowest detection limit was 1 mg x L(-1), the average recovery was 102%, the intra and interspecific RSD were less than 10%, cross-reactivity rate of antibodies and other analogs were less than 1%. CONCLUSION: Rutin complete antigen was synthesized successfully, and the rapid detection of rutin by ELISA method was successfully established.

Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion. The resulting food-borne peptides have recently been shown to be potential activators of microbial autolysis. The main question that needs to be answered in order to verify the hypothesis is whether oral proteases are able (and to what extent) to lyse/mediate lysis of intestinal microorganisms in situ. Methods based on up-to-date molecular biology techniques to allow investigation of the influence of exogenous proteases on microbial lysis processes in vivo (in the intestines) need to be developed. Research testing of this hypothesis may have an important impact in development of novel preparations for the systemic enzyme therapy.