5味中药醇提物对尼罗罗非鱼白细胞免疫活性的作用

将超微五倍子、白头翁、地榆、柯子、虎杖粉碎后过160目筛,按照料液比1∶10将药粉与60%的乙醇水溶液混合,再用超声波粉碎机超声1 h,用滤纸过滤收集滤液,置于多样品平行蒸发仪中浓缩后制得中药提取物。用吸取0.2 mL抗凝剂的无菌注射器自尼罗罗非鱼尾静脉采血0.5 mL,与L-15细胞培养基按体积比1∶9的比例混合成血浆,与中草药提取液单用、联用共孵育,利用氮蓝四唑还原法和流式细胞仪技术分别测定中草药提取液对尼罗罗非鱼外周血白细胞呼吸爆发活性和白细胞吞噬活性的影响。试验结果显示,五倍子、白头翁、地榆、柯子、虎杖单用均能显著提高白细胞氧呼吸爆发活性,呼吸爆发增加率分别为128.68%、123.96%、28.37%、44.44%和78.93%;试验组白细胞吞噬率分别较对照组增加44.04%、44.66%、95.00%、10.35%和64.93%。在两味中药双联用试验中,对白细胞氧呼吸爆发活性呈协同作用的组合为白头翁+地榆,呼吸爆发联用指数为1.28;对白细胞吞噬活性呈协同作用的组合为白头翁+虎杖、柯子+虎杖、五倍子+虎杖和白头翁+柯子,吞噬联用指数分别为11.45、6.20、3.03和1.06。

In Vitro Effect of Alcohol Extracts of 5 Traditional Chinese Medicines on Leukocyte Immune Activity in Peripheral Blood of Nile Tilapia

Chinese gall(Galla Chinensis), Chinese pulsaiilla root(Radix Pulsatillae), garden burnet root(Radix Sanguisorbae), fructus chebulae(Terminalia chebula), and giant knotweed rhizome(Rhizoma Polygoni Cuspidati) were powdered and sieved through a 160 mesh sieve, and then mixed with 60% aqueous solution of ethanol at the ratio of material to liquid 1∶10,finally ultrasonicated by an ultrasonic pulverizer for 1 hour. The filtrate was collected by filtration through a filter paper, and concentrated in a multi-sample parallel evaporator to obtain a Chinese medicine extract. About 0.5 mL of blood was collected from the caudal vein of Nile tilapia(Oreochromis niloticus) using a sterile syringe with 0.2 mL of anticoagulant, and mixed with L-15 cell medium at a ratio of 1∶9 by volume to form plasma. The Chinese herbal medicine extract was incubated with the Chinese herbal medicine extract alone and in combination and the effects of Chinese herbal medicine extract on the respiratory burst activity and leukocyte phagocytic activity in peripheral blood leukocytes of Nile tilapia were determined by nitrogen blue tetrazolium reduction method and flow cytometry. Single use of Chinese gall, Chinese pulsaiilla root, garden burnet root, fructus chebulae, and giant knotweed rhizome was shown to lead to significantly enhance the leukocyte oxygenation burst activity by 128.68%, 123.96%, 28.37%, 44.44%, and 78.93%, and the phagocytic rate elevate by 44.04%, 44.66%, 95.00%, 10.35% and 64.93%, respectively. In the double-use experiment of two traditional Chinese medicines, the combination showing synergistic effect on the leukocyte oxygen respiratory burst activity was Chinese pulsaiilla root + garden burnet root, with the combined respiratory burst index of 1.28. There were 4 combinations showing synergistic effects on phagocytic activity of leukocyte, including Chinese pulsaiilla root + giant knotweed rhizome, fructus chebulae + giant knotweed rhizome, Chinese gall + giant knotweed rhizome, and Chinese pulsaiilla root + fructus chebulae, with the phagocytic combined index of 11.45, 6.20, 3.03, and 1.06, respectively.