| The immune mechanism research of aquatic animal disease have received tremendous attention in recent years and provided the basis of the effective prevention and treatment of the animal diseases. Traditionally, antibiotics were used for the prevention and treatment of aquatic animal disease as commonly used drugs, but their adverse reactions were obviously. However, more and more traditional Chinese medicines were found have antibacterial activity and improve the immunity of the aquatic animal with few side effects. Coptis chinensis Franch was familiar with its antibacterial activities and the major ingredient were the berberine, coptisine, palmatine, epiberberine and jatrorrhizine and the contents of berberine possess 6%-10%. Coptis chinensis was used as immunopotentiator to improve fish immunity have been reported, Hence, in order to clarify the material basis of Coptis chinensis Franch of improvement of the carps immunity, 4 alkaloids isolated from Coptis chinensis Franch and examined the level of complement C3 on the fish and explored what effects of berberine on grass carp complement C3, this research provided the basis of the effective treatment on aquatic animal disease of Rhizoma Coptidis.The main contents and results of this study:1. The extraction, isolation, identification of alkaloids from Coptis chinensis Franch.Using high-speed counter-current chromatography(HSCCC) to separated 4 al kaloids from the crude extract of the total alkaloids of Coptis chinensis Franch with chloroform-methanol-0.2 mol/L hydrochloric acid as a solvent system. 4 co mpounds were respectively confirmed as berberine, palmatine, epiberberine and c optisine by comparison 1H NMR and 13 C NMR data with its reported literature. Compared with the traditional separation methods, the high-speed counter-current chromatography(HSCCC) have advantage in separation of the Chinese herbal me dicine ingredient due to short separation cycle, less solvent consumption and sim ple operation.2. Study the effects of berberine hydrochloride on survival rate of grass car p infected with aeromonas hydrophila.Chosen 150 healthy and evenly weight grass carps randomly divided into 5 groups, the normal control group(NC), the model group(M), and 0.06 %, 0.12%, 0.24% of the berberine hydrochloride groups. The fishes of the NC and M groups were fed with the normal fodder and the medicated groups were fed with the fodder which was added different concentration of berberine hydrochloride.T ake10 g dose of fodder to each fish for 14 days, after then, the carps of the m odel and the medicated groups were injected with 0.2 ml A. hydrophila(108 cfu/ml) in enterocoelia and the control group with equivalent of sterile saline. Recor ded the carps of death data with three times and take the average of the data. T he result showed that the carps survival rates of medicated groups were 50 %, 70 %, 73.3 % and the model group(M) survival rate was only 10 %, along with statistical analysis, the carps survival rates of the medicated groups were signifi cantly increased(P<0.01). Indicated that the berberine hydrochloride can improve the immunity of grass carp with dose-related.3. Study the effects of berberine hydrochloride on the non-specific immunity of grass carp.Chosen 80 healthy and evenly weight grass carps randomly divided into 4 g roups, the normal control group(NC) and the medicated groups. The NC group carps were fed with the normal fodder, the medicated groups carps were fed wit h10 g dose of the fodder which were added different concentration of berberine hydrochloride, then observed the activity and appetite of the carps, collected the blood from the caudal vein of grass carps to determine the level of complement C3 and the activity of serum lysozyme and respiratory burst. Using SPSS 16.0 statistical software for analysis the experimental data, single factor variance analy sis and LSD multiple comparison were carried out on the samples. The results s howed that the level of complement C3 of the carps from the 12% and 24% be rberine hydrochloride groups was significantly increased(p < 0.05) compared wit h the control group and the lysozyme and NBT activity were increased, but they all have no significant difference(p > 0.05) on 7th day, on the 14 day, the lev el of complement C3 of carps from each medicated groups were significantly inc reased(p < 0.05) and the lysozyme activity increased, but no significant differen ce(p> 0.05), the respiratory burst activity of carps from 0.12%, 0.24% berberine hydrochloride groups were significantly increased(p < 0.05) and the respiratory burst activity of 0.06% medicated group was increased, but no significant differ ence(p > 0.05). The results indicated that the berberine hydrochloride could imp rove the non-specific immunity of grass carp.î—'î—'4. Study the effects of berberine hydrochloride on the complement C3 mRN A expression of grass carp.Chosen 80 healthy and evenly weight grass carps randomly divided into 4 g roups, the normal control group(NC) and 0.06 %, 0.12 %, 0.24% of the berberi ne hydrochloride groups. The NC group carps were fed with the normal fodder, the medicated groups carps were fed with the fodder which was added different concentration of berberine hydrochloride and fed with10 g dose of fodder to eac h fish for 14 days, observed the activity and appetite of fish, on the 14 th day, to ok the liver of the carps after narcotized and extracted the complement C3 mRN A from the liver of grass carp. Using Primer 5 software designed the primers an d the c DNA was synthesized by reverse transcription, the RNA concentration w as determined by nucleic acid quantitative instrument. Using SPSS 16.0 statistical software for analysis the experimental data, single factor variance analysis and LSD multiple comparison were carried out on the samples. The results showed t hat the expression of Complement C3 mRNA of carps from the medicated groups were raised and respectively escalated to 20.57 %, 27.35 %, 26.49 % compare d with the control group data and have significant difference(p < 0.05), indicate d that the berberine hydrochloride could raised the expression of the complement C3 mRNA on grass carp.5. Consumption of complement assayJoin the berberine hydrochloride in the blank serum of grass carps to respec tively prepared various concentrations of serum solution 2 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L to test complement consumption. The results showed that the concentration of berberine hydrochloride serum at 5 mg/L, there were few effects of activate complement C3, when the concentration of berberine hydrochloride serum up to 10 mg/L, the activation was obvious(P<0.05), which suggested that high-concentrations of berberine hydrochloride serum c ould changed the conformation of the complement C3 by combination with the c omplement C3 to activate C3 and presented a dose-response relationship.6. Study the pharmacokinetics of berberine on grass carps in vivo.Chosen 72 healthy grass carps(500 ± 10 g) and fed with berberine(5mg/m L) by 48mg/kg dose. Respectively collected the tail vein blood at 0.5h, 1h, 1.5h,2H, 3h, 4h, 5h, 6h, 8h, 12 h, 24 h, 36 h and repeat 6 times for each point, at th e same time, the methodology was taken by used blank plasma. The results indi cated this methodology performed good stability and repeatability, and the concen tration-time curves showed the plasma concentration reached its peak level(0.243mg/L) at 1.5h later by intragastric administration for the single dose(48mg/kg),which indicated that the peak concentration(Cmax) under intragastric administrati on for that doses was lower than the requirements of concentration that could ac tivates complement C3(5 mg/L). |
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