Research On Mechanism Of Citrus Leaves Against Bovine Mastitis

Objective: This study was designed on explore mechanism of Citrus leaves against bovine mastitis,including anti-inflammatory,anti-bacteria and anti-oxidation in vitro.Methods:(1)The toxic effect of Citrus leaves on BMECs and inflammatory cells was measured by MTT method.The inflammatory model was induced by culture of S.aureus.Control group,infection group and Citrus leaves group was established,in which the total epithelial cells’ RNA and protein were acquired.TNF-α,IL-1β,IL-6,SOD,ACACA,FASN,CSN2,CSN3 gene expression was administered by RT-PCR.TNF-α,NO,IL-6,IL-1β was detected by ELISA kit.(2)The minimal inhibitory concentrations(MIC)of Citrus leaves on S.aureus ATCC 29213 was detected by broth microdilution method.(3)The anti-oxidation of Citrus leaves was detected by the ability to eliminate free radicals--DPPH· and O2ˉ·.The HPLC chromatographic condition of Citrus leaves was established,through which to acquire spectrum of Citrus leaves and to develop cluster analysis.The relationship between chromatographic elements and anti-oxidation was explained by stepwise regression analysis.Results:(1)Big difference was really existed among Citrus leaves that act on inflammatory cells model.The anti-inflammatory effect of Citrus tangerine Tanaka was the strongest,which could inhibit the gene expression of main inflammatory factors,such as TNF-α 、 IL-6 、 IL-1β and significantly improve the level of SOD.The anti-inflammatory effect of other Citrus leaves had no consistent.The influence of some Citrus leaves to inflammatory genes was not statistically significant(P > 0.05).Citrus tangerine Tanaka could not promote milk ingredients or add the expression of gene--ACACA、FASN、CSN2、CSN3.Some hybrids and pomelo class could significantly promote the gene expression of CSN2 and CSN3(P < 0.05).(2)The anti-bacteria of Citrus.leaves to S.aureus ATCC 29213 had big difference.Citrus tangerine Tanaka had obvious effect that could completely inhibit the growth of S.aureus ATCC 29213 at29.6813mg/m L.The rest had weak antibacterial ability.(3)The clearance rate of Citrus leaves to DPPH· and O2ˉ· was 2.08% ~ 29.06% and 0.4% ~ 14.45%,respectively.HPLC chromatogram illustrated that the chemical elements of Citrus leaves were informative.There was high similarity of the same general type while difference was obvious between different classes.The stepwise regression method is adopted to screen chemical compositions,there were 6 ingredients respectively had the biggest contribution to eliminate DPPH· and O2ˉ·.Conclusion: The anti-inflammatory,anti-bacteria and anti-oxidation of Citrus leaves did not show obvious effectiveness,which explained Citrus leaves against bovine mastitis was not completely through those mechanisms.Among samples,anti-inflammatory and anti-bacteria of Citrus tangerine Tanaka was a little better than other varieties.Traditional varieties showed better than emerging varieties against mastitis.The varieties could be considered when choosing Citrus leaves as medicinal material resources.