Li Chao-Pin, Xu Li-Fa, Liu Qun-Hong, Zhang Chao, Wang Jian, Zhu Yu-Xia
Department of Etiology and Immunology, Medical College, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China.
World J Gastroenterol. 2004 Feb 1;10(3):433-6. doi: 10.3748/wjg.v10.i3.433.
To explore an ideal method for extracting protoporphyrin disodium (PPN) from unanticoagulated animal blood, and to study the inhibitory effects of PPN on HBV-DNA duplication and its cytotoxicity to 2.2.15 cell strain.
Protoporphyrin methyl ester and other intermediate products were prepared with protoheme separated from protein hydrolysates of coagulated animal blood, which were finally made into PPN and detected quantitatively with an ultraviolet fluorescent analyzer. Ten microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml of PPN-aqueous solution were added into culture medium for 2.2.15 cells respectively. Eight days later, the drug concentration in supernatant from the culture medium was detected when inhibition rate of HBeAg, cell survival rate when inhibition rate of HBeAg was 50% (ID50), and when survival cells in experimental group were 50% of those in control group (CD50), and the therapeutic index (TI) was also detected. PPN with different concentration of 10 microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml was respectively mixed and cultivated with HepG2 2.2.15 cell suspension, and then the inhibition of PPN against HBV-DNA was judged by PCR.
The extract of henna crystal was identified to be PPN. When the concentrations of PPN were 160 microg/ml and 80 microg/ml, the inhibition rates of HBeAg were 89.8% and 82.4%, and the cell survival rates were 98.7% and 99.2%.
It is suggested that PPN can be extracted from unanticoagulated animal blood. PPN can inhibit HBV-DNA expression and duplication in vitro, and has no cytotoxicity to liver cells. Further study and application of PPN are warranted.
探索从未抗凝动物血液中提取原卟啉二钠（PPN）的理想方法，并研究PPN对乙肝病毒脱氧核糖核酸（HBV-DNA）复制的抑制作用及其对2.2.15细胞株的细胞毒性。
用从凝固动物血液的蛋白水解物中分离出的原卟啉制备原卟啉甲酯及其他中间产物，最终制成PPN并用紫外荧光分析仪进行定量检测。分别将10微克/毫升、20微克/毫升、40微克/毫升、80微克/毫升和160微克/毫升的PPN水溶液加入2.2.15细胞培养基中。8天后，检测培养基上清液中的药物浓度、HBeAg抑制率为50%时的细胞存活率（ID50）、实验组存活细胞为对照组50%时的浓度（CD50），并检测治疗指数（TI）。将10微克/毫升、20微克/毫升、40微克/毫升、80微克/毫升和160微克/毫升不同浓度的PPN分别与HepG2 2.2.15细胞悬液混合培养，然后通过聚合酶链反应（PCR）判断PPN对HBV-DNA的抑制作用。
鉴定出指甲花结晶提取物为PPN。当PPN浓度为160微克/毫升和80微克/毫升时，HBeAg抑制率分别为89.8%和82.4%，细胞存活率分别为98.7%和99.2%。
提示可从未抗凝动物血液中提取PPN。PPN在体外可抑制HBV-DNA表达和复制，对肝细胞无细胞毒性。PPN值得进一步研究和应用。
