The Pharmacological Study Of Galla Chinensis And The Effective Element Galic Acid On Spinibarbus Sinensis And Silurus Meridionalis

Chinese Traditional Medicine and the effective elements showed good effects on aquatic animals and gained more and more fishery workers close notice, so they had become the hotspot of aquatic drugs. They have many merits, such as natural sources, slight toxic and side effects, no drug residues and resistance, no harm on health, highly efficiency, and so on. They not only resolved the problems derived from antibiotics such as resistance in phytopathogens, overstandard residues, pollution on cultural surroundings, microecological unbalance, meet the requirements on the nutrition of aquatic animals and food security of human, and totally were in accordance with the disease prevention and cure principle on nuisanceless aquaculture and green aquatic products. So they have important economical and social benefits. Galla chinensis（GC） was one kind of Chinese Traditional Medicine often used and it is often used as an external drug for aquatic animals’ bacteriosis, such as white head-mouth disease, myxobacteria disease, Aeromonas hydrophila disease, Pseudomoniasis, Albo dermatosis, red-skin disease, furunculosis, Saprolegniasis, and so on. Gallic acid（GA） is one of the primary effective elements and at the same time itself is a kind of feed additives often used. The pharmacological study of GC and GA on aquatic animals in vivo and vitro were reported in the present study, the results are as follows:（1） The effective element GA from GC was taken as target drug to study the pharmacokinetics of GC. Spinibarbus sinensis were treated orally via stomach tube at a single dosage of 20 mg GA per kg fish weight fish weight at water temperature（25.0 ± 0.5） oC. High performance liquid chromatography（HPLC） method was used to determine the drug concentration in all tested tissues（serum, liver and kidney）. The mobile phase was CH3OH: H2O: H3PO4=20: 76: 4（V/V/V）, the flow rate was 0.5 ml/min, the wave was 273 nm. Under these conditions, the mean recovery was 92%-115% and the inter-day and intra-day coeffient of variability was 0.2- 0.76, that showed the method meet the requirement of pharmacokinetics methodology. Both compartment and non-compartment model analysis were utilized to obtain the pharmacokinetic parameters. The serum concentration-time data were fitted to a two-compartment open model with 1st order absorption. The absorption half-life（t1/2Kα） was 0.523 h and the distribution half-life（t1/2α） was 1.054 h and the elimination half-life（t1/2β） was 10.501 h in the serum. And all tesetd tissues of serum, liver and kidney reached to the maximum concentration（Cmax） at the time point of 1 h and the values were 9.166, 76.214 and 85.860 μg/ml or μg/g, respectively. The area under the concentration-time curve（AUC） and mean residence times（MRT） were 77.397 μgh/ml and 19.309 h in the serum, 402.106 μgh/g and 25.935 h in the liver, and 258.047 μg h/g and 6.480 h in the kidney, respectively.（2） S. sinesis were orally single-dosing administered with GA of 20 mg / kg fish weight at water temperature（25 ± 0.5） oC to study the effects of GC on the physiological and biochemical indices in the fish species. The blood samples were collected at 1 h, 12 h, 24 h and 48 h after administration and drug-free samples as controls. The physiological indices consist of RBC, WBC, HCV and Hb. The biochemical indices consist of Na+, Ca2+, K+, Mg2+, Cl-, GLU, TP, GLB, ALB, ALT, AST, ALP, TG, UREA, TC, HDL-C and LDL-C. The physiological indices: RBC, HCV and Hb level decreased while WBC increased. The biochemical indices: the content of GLU, UREA, ALT and AST changed sharply, they all fell afer rise and to the maximum at 12 h, TP（GLB and ALB） and TG level declined, other indices showed no obvious changes（p > 0.05）.（3） The oxidation and morphology of red blood cells（RBCs） from Silurus meridionalis in vitro exposure to GA were reported. RBCs were incubated with 0 to 80 mg/L GA for 4 h and 24 h at（27 ± 0.5） °C and at the same time the oxidative indices of Norfloxacin（NFLX） were measured, too. Drug evaluation was made based on these results. The results showed as follows:（a） Enzymatic antioxidants activities showed biphasic concentration-dependant curves with differing degrees were on the enzymatic antioxidants superoxide dismutase（SOD）, glutathione peroxidase（GPx） and catalase（CAT） activities with an induction or no significant effect at lower concentrations and inhibition at higher concentrations both in 4 and 24 h groups, the maximal induction occurred at 20 mg/L. Lipid peroxidation was on the contrary to the enzymatic antioxidants the malondialdehyde（MDA） contents decreased first then increased, the minimum occurred at 20 and 40 mg/L in 4 h and at 20 mg/L in 24 h. No significant differences were observed in RBC hemolysis for 4 h at all tested concentrations. Significant increase in 5 and 10 mg/L and decrease in 40 and 80 mg/L were found in RBC hemolysis for 24 h. The surfaces of minority RBC had crimples and deformation at the 80 mg/L and no significance were found at 5 to 40 mg/L compared to the controls in 4 h group. No significances at 5 to 20 mg/L and obvious morphological damage at 40 and 80 mg/L were found in 24 h group.（b） The oxidative effects and morphological changes of red blood cells（RBCs） during exposure to NFLX were measured. RBCs were incubated with 5 to 80 mg/L NFLX for 4 h at（27 ± 0.5） °C, drug-free samples served as controls. No significant differences were observed in RBC hemolysis and no obvious cytotoxicity at all tested concentrations. The results demonstrated that lower concentrations（< 20 mg/L） of NFLX do not cause oxidative damage and higher concentrations（≥ 20 mg/L） of NFLX can differentially inhibit enzymes SOD, GPx and CAT activities and promote lipid oxidation（LPO）. Furthermore, 20, 40 and 80 mg/L caused obvious morphological changes in RBCs.（4） The protection mechanism of GA to H₂O₂-induced oxidative damage on the RBCs from S. meridionalis at（27 ± 0.5） °C was further probed. The concentrations 10, 25 and 50 mg/L were chosen by hemolysis experiment, no drug no H₂O₂ as blank group and no drug only H₂O₂ as control group. ROS decreased in 10, 25 and 50 mg/L GA and the lowest in 25 mg/L. The activities of SOD, CAT and GPx in all concentration groups increased compared with controls. SOD activity has no statistics discrepancy compared with blanks. CAT activity of 10 mg/L has no statistics discrepancy and 25 and 50 mg/L increased markedly compared with blanks. GPx activity of 25 mg/L increased markedly compared with blanks and other concentration groups. MDA in the control group were increased evidently compared with the other groups. For DNA levels, middlely and severely damaged cell only exist in the control group and no damaged cell in 10- 50 mg/L groups were higher than blank and control groups and OTM in 10- 50 mg/L group were lower than blank and control groups.The main conclusions are as follows:（1） GC can be absorbed preferably and distributed extensively in S. sinensis, it can be kept in vivo for enough time to get curative effects. At the same time, GC eliminated rapidly and no drug were cumulated in vivo.（2） GC has some influences on the physiological and biochemical indices and eliminated gradually after 48 h contrasted to the drug-free samples in S. sinesis. That also said the high administration concentration is not suitable for fish.（3） The anti-oxidant and morphological effects of GA on RBCs from S. meridionalis were biphasic concentration- and time-dependant curves, which meant GA had both anti-oxidant and pro-oxidant property. And in the same concentration range NFLX did not show obvious anti-oxidant property.（4） The conception of RBC oxidative damage was introduced for the drug evaluation. Based on the effect of anti-oxidant and pharmacokinetics and pharmacodynamics experiments, NFLX can inhibit but GA can promote the anti-oxidative system. So GA is a more compatible and promising drug in fish compared to NFLX.（5） The fish often were hard to cure when the diseases were found. No significant differences in RBC hemolysis were observed after 4 h of exposure to all tesetd concentrations in the present study, however, the anti-oxidative indices and morphology were altered obviously. So this method is propitious for detecting fish diseases at an early stage.（6） GA can protect and repair the reactive oxygen species（ROS）, anti-oxidative enzymes, lipid oxidation and DNA level on H₂O₂-induced oxidative damage on the RBCs from S. meridionalis and the effects were concentration-dependant.