| Riemerella anatipestifer disease is an infectious disease that threats to the duck industry seriously,which is caused by Riemerella anatipestifer. There are21serotypes for Riemerella anatipestifer, andno powerful drug for RA desease except for the antibiotic therapy. But RA become resistant toantibiotics easily. Coptidis rhizoma Decoction is made of3kinds of Chinese herbs named Coptischinensis, Phellodendron amurense and Glycyrrhiza uralensis, and extracted form these three herbs.Coptidis rhizoma Decoction has a very high protection to ducks challenged by RA and naturallyinfected ducks. Drug compatibility and preparation method has been confered the national inventionpatent（ZL201010550373.8）. Our previous study found that RA lipopolysaccharide was one of theimportant pathogenic factor. In order to understand the mechanism of Coptidis rhizoma Decoction forRA desease, extraction,,purification of Riemerella anatipestifer lipopolysaccharide and effect onRiemerella anatipestifer LPS of Coptidis rhizoma Decoction in vivo and in vitro has been carried out inthis paper.The LPS is extracted by hot phenol water method depend on the serotype2Riemerellaanatipestifer, and some closely related substance are detected by phenol-sulphuric acid method,Coomassie Brilliant Blue method, agarose gel electrophoresis method. The result showed that theextraction ratio of LPS is1.36%, sugar content is1.48%, protein content is2.08%, and there is someother small DNA fragments, and the relative activity is75.6EU/mg. Relative to domestic endotoxinstandard materials (E. coli0111), the LPS has fewer impurities, low biological activity,.The Coptidis rhizoma Decoction is obtained according to3kinds of herbs of Coptis chinensisPhellodendron amurense and Glycyrrhiza uralensis with water extraction and alcohol precipitationmethod. The result showed that the drug concentration is122.7g/L, and there are some differentconcentrations of Coptidis rhizoma Decoction with the actual needs.The different concentrations of Coptidis rhizoma Decoction acted on RA(3×109CFU/mL),breaked RA(3×109CFU/mL) and LPS(10mg/mL) in external in12h. The effect on LPS of Coptidisrhizoma Decoction is researched by the chromogenic substrate limulus reagent method. The resultshowed that when drug effects in RA, the LPS concentration first by1.15EU/mg increased to2.68EU/mg then drops to0.43EU/mg with the increase of drug concentration; and when drugs effects toultrasonic RA and LPS, the LPS concentration decreased from,2.49EU/mg to0.21EU/mg and2.89EU/mg to0.26EU/mg, showing successively declining trend with the increase in drug concentration.This is because that when drug effects in RA, first the bacterial cell structure is damaged, resulting inRA adventitia LPS released in large quantities, low concentrations of the drug is insufficient to clear the large number of LPS, LPS concentration increased slightly. With the increase in drug concentration, alarge number of drugs can eliminate redundant LPS therefore performance for LPS concentrationincreased at first, then decreased this phenomenon. When drugs effects to ultrasonic RA and LPS, thislink does not exis that the broken bacteria release LPS, the amount of LPS is more stable, LPSconcentrations show a downward trend with the increase of drug concentration. In short, theexperiments show that: the Coptidis rhizoma Decoction has a significant role in the elimination of LPSin vitro.Ducklings were randomly divided into5groups,which is A, B, C, D, E. Then A, B group receivedintraperitoneal injection of RA (0.5mL1×107CFU/ml), C and D group received intraperitoneal injectionof LPS(10mg/ml), E group is vontrol group that received intraperitoneal injection of physiologicalsaline. Each duck fed30g/L Coptidis rhizoma Decoction5mL every6h with B group and D group,others drink water free. LPS concentration was measured in duck plasma every6h. Tests showed thatthe increase of the frequency of administration, the clinical and pathological manifestations of B, Dgroup significantly reduced, such as the sick duck necking reduce, started on feed, organ congestionrelief, typical lesions as cellulose inflammation disappear; the same time, the concentration of LPS from56.3EU/mg to1.7EU/mg and69.3EU/mg to2.3EU/mg; group A and group C clinical symptomssimilar, but group A is more serious, to pull Xifen, conjunctivitis and neurological symptoms; bodytemperature was significantly higher in group C, and did not discovered conjunctivitis; E group wasasymptomatic. Experiments confirmed that LPS is an important virulence factor of the RA, it alsoshows that: the Coptidis rhizoma Decoction has a significant role in the elimination of LPS in vivo.In summary, the study showed that: the Coptidis rhizoma Decoction play a significant role in theelimination of LPS in vivo and in vitro. The Coptidis rhizoma Decoction of elimination process to theRA LPS is one of the most important reasons for prevention and treatment of RA disease. |
