关键词: 细粒棘球蚴, 抗寄生虫中药, 水提物, 粗提物, 液相色谱与串联质谱联用分析

Abstract:

Objective To evaluate the in vitro effects of water extracts and organic solvent crude extracts of antiparasitic Chinese herb medicine against Echinococcus granulosus protoscolices Methods The protoscolices were isolated from the liver cyst of sheep infected with E. granulosus and cultured for 24 hours in vitro to collect protoscolices with vitality greater than 95%, which were transferred into 96 well plate (100 μl with 100 protoscolices). In the culture wells, the water extracts of 11 antiparasitic Chinese herb medicines, including Brucea javanica, Melia toosendan, Polygonum hydropiper, Pericarpium granati, Quisqualis linn, Areca catechu, Cyrtomium fortunei, Derris trifoliata, Semen iridis, Galla chinensis and Agrimonia pilosa, were applied at final concentrations of 0.8 mg/ml (low concentration group), 1.6 mg/ml (medium concentration group), and 3.2 mg/ml (high concentration group), respectively. After 72 hours of treatment, the protoscolices vitality was detected by eosin staining, and morphology was observed under inverted microscope to calculate the mortality rate. In addition, an albendazole group with the same concentration and a blank control group were designated. The crude herb extracts from flavonoids, polysaccharides, saponins and alkaloids showing apparent anti-protoscolices effect were cultured with protoscolices at a final concentration of 1.00 mg/ml for 72 hours. The morphology of protoscolices was observed to calculate the mortality rate. At the same time, blank control group was set up. Liquid chromatography and tandem mass spectrometry analysis were performed for those crude extracts showing significant anti-protoscolices effects. Single variant ANOVA analysis was used for inter-group comparisons, and LSD-t-test was used for pairwise comparison. Results After 72 hours of treatment with the traditional Chinese medicine water extracts, the protoscolices edges appeared blurred, the structure was loose, and the matrix was dissolved in the high-concentration groups with B. javanica, M. toosendan and P. hydropiper water extracts. The high concentration group of the other 8 Chinese herb medicine water extracts showed normal protoscolices morphology and clear structures. The albendazole group showed mild damage to the germinal layer of the original scalp. The blank control group had normal protoscolices morphology. After 72 hours of in vitro culturing, the protoscolices mortality rates in B. javanica, M. toosendan and P. hydropiper water extracts high concentration groups were (99.63 ± 0.57)%, (90.89 ± 1.10)% and (51.93 ± 0.60)%; (85.97 ± 1.50)%, (81.14 ± 1.19)% and (42.46 ± 0.56)% in the medium concentration groups, and (78.34 ± 1.35)%, (77.27 ± 0.92)% and (36.66 ± 0.60)% in the low concentration groups, respectively. The difference was statistically significant compared with the blank control group [(0.62 ± 0.51)%] (F = 180 678.22, 41 488.99, 44 346.38; 19 543.86, 27 887.32, 34 590.79; 20 059.467, 41 953.68, 17 993.77; P < 0.01 for all comparations) and compared with the albendazole group [(30.03 ± 2.02)%] (F = 6 585.72, 4 210.84, 646.46; 2 956.80, 2 849.68, 210.83; 2 365.92, 2 712.28, 58.12; P < 0.01 for all comparetions). There were no statistically significant differences in the mortality rates of the protoscolices between the control group and the high-concentration groups, the medium-concentration groups and the low-concentration groups of the other 8 Chinese herb medicine water extracts (all P > 0.05). The protoscolices in the groups of B. javanica flavonoids, M. toosendan flavonoids, B. javanica saponins, P. hydropipe saponins and P. hydropiper alkaloid showed wrinkling, loose structure, and matrix dissolution， and the mortality rates of the protoscolices in the groups were (98.33 ± 2.89)%, (96.67 ± 5.77)%, 100%, (99.33 ± 1.15)% and （56.67 ± 2.11)%, respectively. The differences were statistically significant compared with the blank control group (10.33 ± 2.51)% (F = 1 584.00, 563.71, 3 808.47, 3 099.52, 14.65; P < 0.01 for all comparisons). There was no statistically significant difference in the mortality rate of the protoscolices between the high-concentration group of other crude extracts of traditional Chinese herb medicine and the control group (P > 0.05 for all comparisons). The liquid chromatography combined with tandem mass spectrometry analysis showed that 741 and 398 metabolites were identified in the positive and negative ion modes, respectively. Among them, four flavonoids with significant differences were identified in the flavonoids crude extracts of B. javanica in the positive ion mode, namely lutein, 5-methyl-7-methoxyisoflavone, catechins, and sweet orange flavonoids. Eight species were identified in negative ion mode, namely proanthocyanidin B2 and quercetin B3 β-D-glucoside, citrin, hesperetin, quercetin, luteolin, apigenin, and daidzein. Conclusion The water extracts of B. javanica, M. toosendan, P. multiflorum, as well as B. javanica flavonoids, M. toosendan flavonoids, B. javanica saponins and P. hydropipe saponins reveal significant anti-echinococcus granulosus protoscolices in vitro, and their effects are superior to those of albendazole.

Key words: Echinococcus granulosus, Anti-parasitic Chinese herb medicine, Water extract, Crude extract, Liquid chromatography combined with tandem mass spectrometry analysis