【作者】 郭海燕；

【导师】 张耀光；

【作者基本信息】 西南大学 ， 水产养殖， 2016， 博士

【摘要】 中药及其有效成分在水产动物的疾病防治等方面显示出明显的作用与效益,越来越受到水产工作者的重视,逐渐成为水产药物研究的热点。它们以取自天然、毒副作用小、无药物残留和抗药性、对健康无害、高效安全等优点,不但可以解决抗生素等化学合成药物引发的病原菌抗药性、残留超标、养殖环境污染、微生态平衡的破坏等问题,同时满足人们对食品安全和环境安全的需求,完全符合目前发展无公害水产养殖和生产绿色水产品的疾病防治原则,具有重要的经济和社会效益。五倍子（Galla chinensis,GC）是我国水产养殖常用的中药,具有显著的抑菌杀菌作用,主要用于防治水产动物的水霉病、白头白嘴病、赤皮病、气单胞菌病、假单胞菌病、粘细菌病、白皮病以及疖疮病等疾病。没食子酸（Gallic acid,GA）是五倍子的主要有效成分之一,具有较为肯定的抗氧化、抗病毒、抑菌等多种生物学活性,也常被用作饲料添加剂。本论文根据五倍子及没食子酸的作用和结构特点,以两种生活习性和食性完全不同的名特优养殖鱼类——中华倒刺鲃（Spinibarbus sinensis）和南方鲇（Silurus meridionalis）为对象,分别用在体和离体的方法开展了它们对两种鱼的药理学研究。具体研究结果如下:（1）在（25±0.5）℃水温条件下以GC的有效成分GA 20 mg/kg鱼体质量单剂量口灌GC醇提液后,以GA为目标药物研究GC在中华倒刺鲃体内的药物代谢动力学特征。药物浓度采用反相高效液相色谱法检测,流动相为甲醇:水:磷酸=20:76:4（v/v/v）,流速为0.8 ml/min,检测波长为273 nm,此条件下GA的平均回收率为92%–115%,日内精密度和日间精密度范围为0.2–6.71,符合药动学方法学的要求。药动学数据用房室模型和非房室模型法同时分析,血液中的药动学数据可以用一级吸收二室开放模型描述,药物在中华倒刺鲃体内血液、肝脏和肾脏均在1 h时达到最大药物峰,最大药物浓度（Cmax）值分别是:9.1656,76.2143和85.8602μg/ml或μg/g,其吸收、分布和消除半衰期分别是:0.523,1.054和10.501 h,药时曲线下面积（AUC）分别是77.3967,402.1059和258.0476μgh/ml或者μgh/g;平均驻留时间（MRT）分别是19.3091,25.935和6.4803h。（2）在（25±0.5）°c水温条件下以gc的有效成分ga20mg/kg鱼体质量单剂量口灌gc醇提液后,考察中华倒刺鲃体内的各生理生化指标的变化情况。取样时间分别是给药后1h、12h、24h和48h,分别称为1h组、12h组、24h组和48h组,另取未给药实验鱼为对照（称为对照组）,其红细胞数、白细胞数、血红蛋白质量浓度和红细胞压积等血液生理指标,钠（na+）、钾（k+）、钙（ca2+）、镁（mg2+）、氯（cl-）、甘油三酯（tg）、总胆固醇（tc）、高密度脂蛋白胆固醇（hdl-c）、低密度脂蛋白胆固醇（ldl-c）、血糖（glu）、总蛋白（tp）、球蛋白（glb）、白蛋白（alb）、尿素（urea）、尿酸（ua）、谷丙转氨酶（alt）、谷草转氨酶（ast）、碱性磷酸酶（akp）等血液生化指标的变化情况。随着给药后时间的延长,生理指标方面:红细胞数、红细胞压积和血红蛋白质量浓度降低,白细胞数升高;生化指标方面:血糖、尿酸、谷丙转氨酶和谷草转氨酶均先升后降,蛋白质（包括总蛋白和球蛋白）和甘油三酯水平略降,其他生化指标相对变化不具有统计学意义。（3）（27±0.5）oc时用南方鲇亚成体血液制备1%红细胞悬液,体外研究0,5,10,20,40和80mg/lga在不同时间对南方鲇红细胞抗氧化系统和表面形貌的影响,检测氧化指标超氧化物歧化酶（sod）、过氧化氢酶（cat）、谷胱甘肽酶（gpx）和戊二醛（mda,脂类过氧化产物）且观察红细胞相应的溶血和表面形貌的变化,同时还检测了诺氟沙星（norfloxacin,nflx）相应指标以做对比,并以此对两种药物进行药物评价。实验分为两部分,第一部分研究了ga对南方鲇红细胞抗氧化系统和表面形貌的影响与药物作用的时间和浓度的关系。结果显示:ga在0–80mg/l浓度范围内,与4h组相比,同一浓度24h组的溶血od值变大,sod,cat和gpx等抗氧化酶略有降低,mda值略有增大;而除4h组溶血不具有统计学意义差异,24h组溶血od先降后升外,无论是4h组还是24h组三种抗氧化酶均先升高再降低的趋势,mda先下降再升高的趋势,且24h浓度组的变化趋势更明显,反映在表面形貌上4h组除80mg/l浓度组可以看到少数红细胞表面略有皱缩变形,其它组与对照组无显著差异。24h组5,10,20mg/l浓度组与对照组相比红细胞均无显著变化,40和80mg/l浓度组红细胞表面损伤逐渐明显。第二部分选择ga和nflx对红细胞溶血均没有显著差异的4h时进行两种药物对南方鲇红细胞抗氧化系统和表面形貌影响的对比实验。结果显示:nflx对南方鲇红细胞的影响,与对照组相比,sod和gpx活性5mg/l时不具有统计学意义差异,20-80mg/l时显著降低（p<0.05）;cat活性5和20mg/l时显著升高,40mg/l时cat不具有统计学意义变化,80mg/l显著降低（p<0.05）。mda水平除5mg/l与对照组无差异,其它随着浓度的增加逐渐增大（p<0.05）。除5mg/l时没有明显损伤外,随着浓度增大,红细胞表面开始皱缩,边缘开始有缺口,80mg/l时损伤最为严重。（4）（27±0.5）°c时用200μmol/l过氧化氢（h2o2）诱导南方鲇幼鱼红细胞的过氧化损伤模型来进一步探讨ga的保护作用机制。根据溶血实验的结果选择10,25和50mg/lga浓度孵育24 h来研究GA对H₂O₂诱导的南方鲇红细胞氧化损伤的保护作用。以不加H₂O₂和GA的溶液为空白组,只加H₂O₂不加GA液为对照组,各GA浓度组的活性氧（ROS）均有所下降,25 mg/L时ROS为最低;各浓度的SOD、CAT和GPx活性比对照组有显著升高（p<0.05）,各给药组SOD活性与空白组之间不具有统计学意义差异;10 mg/L浓度组CAT活性与空白组不具有统计学意义差异,25和50 mg/L浓度组CAT活性显著大于空白组;25 mg/L浓度组GPx活性显著大于其它各浓度组和空白组（p<0.05）。对照组MDA含量比其它各组显著增大（p<0.05）,10 mg/L浓度组与空白组不具有统计学意义差异,25和50 mg/L组比空白组显著变小（p<0.05）。就DNA水平而言,只有在对照组中才有中度损伤和重度损伤的细胞存在,其它各组细胞均属无损伤细胞和轻度损伤细胞,10-50 mg/L浓度组的无损伤细胞高于空白组,更高于对照组,且10-50 mg/L浓度组的OTM明显的要小于对照组和空白组。主要研究结论:（1）单次口灌GC醇提液在中华倒刺鲃体内吸收迅速,分布广泛,在血液、肝脏和肾脏中浓度较高,能很好的发挥药效,并且药物在体内消除很快,不会在体内蓄积造成不良影响。（2）单次口灌GC醇提液对中华倒刺鲃幼鱼血液生理生化指标有一定的影响,随着药物的代谢,体内药物质量浓度逐渐降低,影响亦逐渐消除。也可因此说单次给药剂量不能过大。（3）一定浓度范围内（0–80 mg/L）,GA对健康南方鲇红细胞的抗氧化系统和表面形貌的影响是双向的,与浓度和作用时间相关。也就是说GA既有抗氧化性又有促氧化性。同样温度、浓度范围内NFLX对健康南方鲇红细胞的抗氧化系统和表面形貌没有明显的抗氧化性。（4）引入了红细胞氧化损伤的概念进行药物评价。根据红细胞抗氧化系统和表面形貌的变化可进行药物评价。依据GA和NFLX的抗氧化对比实验并结合之前两种药物对水产动物的药动学和药效学特点的研究,推荐GA在合适的给药剂量是一种更为安全有效的水产动物药物。（5）鱼病被发现时往往已经非常严重难以救治,本实验中药物作用4 h时红细胞溶血尚未有显著,但是其抗氧化指标和表面形貌已有一定的变化趋势,因此红细胞氧化损伤概念的引入还有利于鱼病的提前检出。（6）GA对低浓度H₂O₂诱导南方鲇红细胞的过氧化损伤有保护作用,可以从活性氧（ROS）、抗氧化酶、脂类氧化和DNA等方面修复,其保护作用的效果也与GA浓度相关。更多还原

【Abstract】 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.更多还原