大黄酸在刺参、南美白对虾体内的代谢及大黄对刺参的免疫影响
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摘要
本研究分为三个部分,第一部分以刺参为实验对象,研究了单次体腔注射大黄酸后药物在刺参肌肉、呼吸树、体腔液和体壁中的代谢规律,探讨刺参对该药物的代谢特征,为生产中的实际应用提供理论指导。第二部分,研究南美白对虾单次肌肉注射大黄酸后药物在其肌肉、血淋巴、肝胰脏和鳃中的药物代谢动力学,为大黄及大黄制剂在治疗对虾烂鳃病方面提供组方依据。第三部分,在刺参空白饲料(鼠尾藻粉和海泥)中添加不同剂量的大黄粉,研究大黄对刺参免疫方面的影响,从免疫方面指导大黄的使用。主要结果如下:
一、大黄酸在刺参体内的药动学研究:在水温14±1℃条件下,给健康刺参体腔注射5.33mg/kg剂量的大黄酸,利用高效液相色谱(HPLC)法,测定大黄酸在其体腔液、呼吸树、肌肉、体壁中的药物浓度时间变化,数据分析采用3p87药代学软件。结果表明:除体壁中的大黄酸代谢规律符合一级吸收一室模型外,大黄酸在其它三种组织中均符合一级吸收二室模型。大黄酸在上述四种组织中的达峰时间(Tmax)分别为0.26h、0.67h、0.54h和0.88h;消除半衰期(T1/2β)分别为6.24h、26.1h、71.48h和8.93h;药时曲线下总面积(AUC)分别为69.29mg/L?h、105.6mg/L?h、132.38mg/L?h、20.99mg/L?h。以上研究表明,体腔注射大黄酸后,药物在刺参各组织中能迅速达到峰值,具有吸收快、消除半衰期长、代谢慢的特点,主要经呼吸树排出体外。
二、大黄酸在南美白对虾体内的研究:采用循环水槽养殖南美白对虾,水温控制在26~27℃。利用高效液相色谱法对南美白对虾单次肌肉注射5mg/kg大黄酸后的药物浓度进行检测,研究大黄酸在南美白对虾体内的药代动力学。测定南美白对虾单次肌注大黄酸后,不同时间点药物在其血淋巴、鳃、肌肉和肝胰脏中的浓度变化,利用3p97对药物-时间浓度进行拟合。实验结果表明大黄酸在南美白对虾四种组织中的药动学模型均符合一级吸收一室模型,主要药动学参数如下:大黄酸在南美白对虾血淋巴、鳃、肌肉、肝胰脏中的达峰时间(Tmax)分别为0.17h、0.23h、0.44h和2.73h;最大药物Cmax分别为1.81mg/L、2.06mg/L、0.44mg/L和2.31mg/L;表观分布容积(V)分别为2.62L/kg、2.33L/kg、10.35L/kg和0.96L/kg;药时曲线下总面积(AUC)分别为5.85mg/L?h、11.54mg/L?h、2.18mg/L?h和17.56mg/L?h;消除半衰期(T1/2β)分别为2.13h、3.72h、3.12h和2.36h。大黄酸在南美白对虾各组织中能较快达到药物浓度峰值,具有吸收快、分布广泛,清除能力强、消除速度快等特点。大黄酸在南美白对虾鳃部的含量较高,为养殖生产中使用大黄制剂治疗南美白对虾烂鳃病的合理性提供了理论依据。
三、大黄对刺参非特异性免疫的影响:刺参分为四组,一个空白对照组(投喂鼠尾藻和海泥),和三个分别含2%、5%、10%(质量分数)大黄粉的剂量实验组。各组连续投喂相应饲料28天,之后投喂空白饵料2周。在不同时间取样,测定刺参体腔液中的溶菌酶(LSZ)、超氧化物歧化酶(SOD)、碱性磷酸酶(AKP)、和酸性磷酸酶(ACP)的活性。结果显示,10%剂量组第七天时的LSZ活力比空白组提高了一倍,在第二十一天时2%剂量组和5%剂量组相对于空白组酶活分别提高了92.86%和96.43%;三个剂量组的SOD活力均在第七天达到最高,分别比空白组提高了4.36%、8.87%和9.28%,第二十八天时各剂量组酶活均低于空白组,且差异性显著(P<0.05);2%和5%剂量组的AKP酶活低于空白组,10%剂量组的AKP酶活在第二十一天时达到最高值,与空白组间的差异极显著,为空白组酶活的6.99倍;相对于空白各剂量组ACP的最大酶活分别为3.55倍、3.79倍和8.61倍。研究结果表明,大黄能提高刺参的非特异性免疫,建议投喂含2%和5%大黄的饲料三周,含10%大黄的饲料两周,可提高刺参的非特异性免疫,持续投喂时间不宜过长,各免疫指标在停止投喂药饵两周左右可恢复至平常水平。
This study is divided into three parts. In the first part Apostichopus japonicus is used as a test object. Pharmacokinetics of rhein was studied on coelomic fluid, respiratory trees, muscle and integument of Apostichopus japonicus after single dose injection, providing a theoretical guidance for the production of practical application. The second part is about pharmacokinetics of rhein in Penaeus vannamei after a single intramuscular injection. The concentrations of rhein in muscle, hemolymph, hepatopancreas and gill were detected, which can provide a basis for rhubarb and its preparation in the treatment of shrimp gill-rot disease. The third part is studied the effects of rhubarb on non-specific immune in Apostichopus japonicus, guiding the use of rhubarb in the aspect of immunization on Apostichopus japonicus. The main results are as follows:
Part one: Studies were conducted on the pharmacokinetics of rhein in Apostichopus japonicus. Coelomic fluid, respiratory trees, muscle and integument were sampled after coelomic injecting with a single dose of rhein 5.33mg/kg. The contents of rhein were determined by high performance liquid chromatography. The results showed that the peak time (Tmax), elimination half-time (T1/2β) and the area under the concentration-time curve (AUC) in Coelomic fluid, respiratory trees, muscle and integument were as follows: Tmax 0.26h, 0.67h, 0.54h and 0.88h ; T1/2β6.24h, 26.1h, 71.48h and 8.93h; AUC 69.29mg/L?h, 105.6mg/L?h, 132.38mg/L?h and 20.99mg/L?h. The concentration-time course of rhein in coelomic fluid, respiratory trees and muscle could be described by a two-compartment model, except for the course in integument being described by a one-compartment model. These results are suggesting that rhein can reach the peak time with a high speed absorption in Apostichopus japonicus after injection, while it is slowly metabolized with a long elimination half-time. Rhein is mainly excreted from respiratory trees.
Part two: Pharmacokinetics of rhein in Penaeus vannamei was studied. The concentrations of rhein in hemolymph, gill, muscle and hepatopancreas were determined by high performance liquid chromatography after intramuscular with a single dose of rhein 5mg/kg. The results showed that the peak time (Tmax), maximum concentration (Cmax), apparent volume of distribution (V), elimination half-time (T1/2β) and the area under the concentration-time curve (AUC) in hemolymph, gill, muscle and hepatopancreas were as follows: Tmax 0.17h、0.23h、0.44h and 2.73h; Cmax 1.81mg/L、2.06mg/L、0.44mg/L and 2.31mg/L; V 2.62L/kg、2.33L/kg、10.35L/kg and 0.96L/kg; T1/2β2.13h、3.72h、3.12h and 2.36h; AUC 5.85mg/L?h、11.54mg/L?h、2.18mg/L?h and 17.56mg/L?h. The concentration-time course of rhein in the four tissues could be described by a one-compartment model. These results are suggesting that rhein can reach the peak time with a high speed absorption in Penaeus vannamei after injection, and it is quickly metabolized with a short elimination half-time. Rhein mainly exists in the gill, which provides a theoretical basis on the Rhubarb treatment gill-rot disease in aquaculture production of Penaeus vannamei.
The activities of lysozyme (LSZ), superoxide dismutase (SOD), alkaline phosphatase (AKP), and acid phosphatase (ACP) were monitored in the coelomic fluid of Apostichopus japonicus at different days after feeding Rhubarb diet. The contents of rhubarb in diet were 2%、5% and 10%(by mass), besides a control group was set up. The time of feeding Rhubarb was 28 days, after that blank diet was fed. The results showed that the LSZ activity of 10% dose group has doubled the activity of the control group on the seventh day, while 2% and 5% dose group relative to the activity of the control group increased by 92.86% and 96.43% on the twenty-one day; SOD activity of all of the three dosage groups were highest in the seventh day, respectively increased 4.36%, 8.87% and 9.28% than that of the control group, but all of them were lower than the blank Group on the twenty-eighth days with a significant (P <0.05) difference; AKP activities of 2% and 5% dose group were lower than that of the control group, while the AKP activity of the 10% dose group reached the highest value on the twenty-day, which was 6.99 times than that of the blank one, displaying with a significant difference; relative to the control group, the maximum activity of ACP in each group were 3.55 times, 3.79 times and 8.61 times. The results show that rhubarb can increase non-specific immunity of Apostichopus japonicus. We recommend feeding with 2% and 5% of rhubarb for three weeks, containing 10% rhubarb diet two weeks can improve non-specific immunity of Apostichopus japonicus. Stop feeding about two weeks can restore to the normal level of immune parameters.
一、大黄酸在刺参体内的药动学研究:在水温14±1℃条件下,给健康刺参体腔注射5.33mg/kg剂量的大黄酸,利用高效液相色谱(HPLC)法,测定大黄酸在其体腔液、呼吸树、肌肉、体壁中的药物浓度时间变化,数据分析采用3p87药代学软件。结果表明:除体壁中的大黄酸代谢规律符合一级吸收一室模型外,大黄酸在其它三种组织中均符合一级吸收二室模型。大黄酸在上述四种组织中的达峰时间(Tmax)分别为0.26h、0.67h、0.54h和0.88h;消除半衰期(T1/2β)分别为6.24h、26.1h、71.48h和8.93h;药时曲线下总面积(AUC)分别为69.29mg/L?h、105.6mg/L?h、132.38mg/L?h、20.99mg/L?h。以上研究表明,体腔注射大黄酸后,药物在刺参各组织中能迅速达到峰值,具有吸收快、消除半衰期长、代谢慢的特点,主要经呼吸树排出体外。
二、大黄酸在南美白对虾体内的研究:采用循环水槽养殖南美白对虾,水温控制在26~27℃。利用高效液相色谱法对南美白对虾单次肌肉注射5mg/kg大黄酸后的药物浓度进行检测,研究大黄酸在南美白对虾体内的药代动力学。测定南美白对虾单次肌注大黄酸后,不同时间点药物在其血淋巴、鳃、肌肉和肝胰脏中的浓度变化,利用3p97对药物-时间浓度进行拟合。实验结果表明大黄酸在南美白对虾四种组织中的药动学模型均符合一级吸收一室模型,主要药动学参数如下:大黄酸在南美白对虾血淋巴、鳃、肌肉、肝胰脏中的达峰时间(Tmax)分别为0.17h、0.23h、0.44h和2.73h;最大药物Cmax分别为1.81mg/L、2.06mg/L、0.44mg/L和2.31mg/L;表观分布容积(V)分别为2.62L/kg、2.33L/kg、10.35L/kg和0.96L/kg;药时曲线下总面积(AUC)分别为5.85mg/L?h、11.54mg/L?h、2.18mg/L?h和17.56mg/L?h;消除半衰期(T1/2β)分别为2.13h、3.72h、3.12h和2.36h。大黄酸在南美白对虾各组织中能较快达到药物浓度峰值,具有吸收快、分布广泛,清除能力强、消除速度快等特点。大黄酸在南美白对虾鳃部的含量较高,为养殖生产中使用大黄制剂治疗南美白对虾烂鳃病的合理性提供了理论依据。
三、大黄对刺参非特异性免疫的影响:刺参分为四组,一个空白对照组(投喂鼠尾藻和海泥),和三个分别含2%、5%、10%(质量分数)大黄粉的剂量实验组。各组连续投喂相应饲料28天,之后投喂空白饵料2周。在不同时间取样,测定刺参体腔液中的溶菌酶(LSZ)、超氧化物歧化酶(SOD)、碱性磷酸酶(AKP)、和酸性磷酸酶(ACP)的活性。结果显示,10%剂量组第七天时的LSZ活力比空白组提高了一倍,在第二十一天时2%剂量组和5%剂量组相对于空白组酶活分别提高了92.86%和96.43%;三个剂量组的SOD活力均在第七天达到最高,分别比空白组提高了4.36%、8.87%和9.28%,第二十八天时各剂量组酶活均低于空白组,且差异性显著(P<0.05);2%和5%剂量组的AKP酶活低于空白组,10%剂量组的AKP酶活在第二十一天时达到最高值,与空白组间的差异极显著,为空白组酶活的6.99倍;相对于空白各剂量组ACP的最大酶活分别为3.55倍、3.79倍和8.61倍。研究结果表明,大黄能提高刺参的非特异性免疫,建议投喂含2%和5%大黄的饲料三周,含10%大黄的饲料两周,可提高刺参的非特异性免疫,持续投喂时间不宜过长,各免疫指标在停止投喂药饵两周左右可恢复至平常水平。
This study is divided into three parts. In the first part Apostichopus japonicus is used as a test object. Pharmacokinetics of rhein was studied on coelomic fluid, respiratory trees, muscle and integument of Apostichopus japonicus after single dose injection, providing a theoretical guidance for the production of practical application. The second part is about pharmacokinetics of rhein in Penaeus vannamei after a single intramuscular injection. The concentrations of rhein in muscle, hemolymph, hepatopancreas and gill were detected, which can provide a basis for rhubarb and its preparation in the treatment of shrimp gill-rot disease. The third part is studied the effects of rhubarb on non-specific immune in Apostichopus japonicus, guiding the use of rhubarb in the aspect of immunization on Apostichopus japonicus. The main results are as follows:
Part one: Studies were conducted on the pharmacokinetics of rhein in Apostichopus japonicus. Coelomic fluid, respiratory trees, muscle and integument were sampled after coelomic injecting with a single dose of rhein 5.33mg/kg. The contents of rhein were determined by high performance liquid chromatography. The results showed that the peak time (Tmax), elimination half-time (T1/2β) and the area under the concentration-time curve (AUC) in Coelomic fluid, respiratory trees, muscle and integument were as follows: Tmax 0.26h, 0.67h, 0.54h and 0.88h ; T1/2β6.24h, 26.1h, 71.48h and 8.93h; AUC 69.29mg/L?h, 105.6mg/L?h, 132.38mg/L?h and 20.99mg/L?h. The concentration-time course of rhein in coelomic fluid, respiratory trees and muscle could be described by a two-compartment model, except for the course in integument being described by a one-compartment model. These results are suggesting that rhein can reach the peak time with a high speed absorption in Apostichopus japonicus after injection, while it is slowly metabolized with a long elimination half-time. Rhein is mainly excreted from respiratory trees.
Part two: Pharmacokinetics of rhein in Penaeus vannamei was studied. The concentrations of rhein in hemolymph, gill, muscle and hepatopancreas were determined by high performance liquid chromatography after intramuscular with a single dose of rhein 5mg/kg. The results showed that the peak time (Tmax), maximum concentration (Cmax), apparent volume of distribution (V), elimination half-time (T1/2β) and the area under the concentration-time curve (AUC) in hemolymph, gill, muscle and hepatopancreas were as follows: Tmax 0.17h、0.23h、0.44h and 2.73h; Cmax 1.81mg/L、2.06mg/L、0.44mg/L and 2.31mg/L; V 2.62L/kg、2.33L/kg、10.35L/kg and 0.96L/kg; T1/2β2.13h、3.72h、3.12h and 2.36h; AUC 5.85mg/L?h、11.54mg/L?h、2.18mg/L?h and 17.56mg/L?h. The concentration-time course of rhein in the four tissues could be described by a one-compartment model. These results are suggesting that rhein can reach the peak time with a high speed absorption in Penaeus vannamei after injection, and it is quickly metabolized with a short elimination half-time. Rhein mainly exists in the gill, which provides a theoretical basis on the Rhubarb treatment gill-rot disease in aquaculture production of Penaeus vannamei.
The activities of lysozyme (LSZ), superoxide dismutase (SOD), alkaline phosphatase (AKP), and acid phosphatase (ACP) were monitored in the coelomic fluid of Apostichopus japonicus at different days after feeding Rhubarb diet. The contents of rhubarb in diet were 2%、5% and 10%(by mass), besides a control group was set up. The time of feeding Rhubarb was 28 days, after that blank diet was fed. The results showed that the LSZ activity of 10% dose group has doubled the activity of the control group on the seventh day, while 2% and 5% dose group relative to the activity of the control group increased by 92.86% and 96.43% on the twenty-one day; SOD activity of all of the three dosage groups were highest in the seventh day, respectively increased 4.36%, 8.87% and 9.28% than that of the control group, but all of them were lower than the blank Group on the twenty-eighth days with a significant (P <0.05) difference; AKP activities of 2% and 5% dose group were lower than that of the control group, while the AKP activity of the 10% dose group reached the highest value on the twenty-day, which was 6.99 times than that of the blank one, displaying with a significant difference; relative to the control group, the maximum activity of ACP in each group were 3.55 times, 3.79 times and 8.61 times. The results show that rhubarb can increase non-specific immunity of Apostichopus japonicus. We recommend feeding with 2% and 5% of rhubarb for three weeks, containing 10% rhubarb diet two weeks can improve non-specific immunity of Apostichopus japonicus. Stop feeding about two weeks can restore to the normal level of immune parameters.
引文
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