Abstract:

Objective To evaluate the anti-Echinococcus multilocularis effect of total alkaloids of Sophora moorcroftiana (TA-SM) in water solution and tablet forms in vitro and in vivo. Methods The protoscolices of E. multilocularis were isolated from the Mongolian gerbils and cultured in a culture flask (10 000 protoscolices/flask). The culture flasks were randomly assigned into the culture medium control group, solvent control group, the albendazole (ABZ) positive control group, the albendazole sulfoxide (ABZ-SO) positive control group, and the low, medium and high concentration groups of TA-SM as water solution groups and as tablet form groups, in which the protoscolices were co-cultured with RPMI 1640 complete medium, or the final concentration of 0.1% dimethyl sulfoxide (DMSO), 10 μg/ml ABZ, 10.60 μg/ml ABZ-SO, or the final concentration of 5 μg/ml (low concentration), 10 μg/ml (medium concentration) and 20 μg/ml (high concentration) of TA-SM in water solution and tablet forms at 37 ℃ and 5% CO₂ for 7 days. The samples collected every day were stained with trypan blue and observed under inverted microscope to check the survival and calculate the survival rate. The relative expression level of alkaline phosphatase (ALP) in the culture supernatant and caspase 3 in protoscolices was detected on the fifth day of co-culture. Fifty-four female Kunming mice were injected with protoscolices of E. multilocularis (0.2 ml/mouse, 10⁴ protoscolices/ml) intraperitoneally and were randomly assigned into the normal saline group, tablet group and water solution group 4 months post-infection. In the water solution group and tablet group, mice were treated with 28 mg/(kg·d) of TA-SM in water solution or tablet forms, respectively, by oral gavage, once a day. In the normal saline group mice were given the same volume of normal saline only. On the 15th, 30th and 60th days of treatment, six mice in each group were randomly selected to dissect, the mass of cyst was weighed, and the rate of cyst inhibition was calculated. The liver tissue sections were prepared, and the pathological changes of the liver tissue were observed using HE staining, the ultrastructure of liver tissue was observed by scanning electron microscope (SEM). One-way ANOVA was used for inter-group comparison, and LSD-t test was used for pairwise comparison. Results On the 7th day of co-culture in vitro, the protoscolices in the medium and in the solvent control groups were oval in shape, and the protoscolices were intact and clear without damage, showing typical invagination. In the ABZ and ABZ-SO and positive control group, the protoscolices became longer, and the sucker was visible, showing the typical eversion type. In the low and medium concentration groups of TA-SM in water solution and tablet forms, the protoscolices were mainly invaginated, and occasionally everted. The morphology of the protoscolices in the high concentration groups of TA-SM in water solution and tablet forms was mostly eversion type. On the 7th day of co-culture in vitro, the survival rates of the protoscolices in the low, medium and high concentration groups of TA-SM in water solution and tablet forms were (34.7 ± 2.75)%, (31.5 ± 10.12)%, (27.6 ± 2.51)% and (30.8 ± 4.15)%, (29.8 ± 8.11)%, (25.2 ± 4.30)%, respectively, with no statistically significant difference between the TA-SM in water solution group and tablet form group (t = 1.36, 0.22, 0.83, all P > 0.05). The survival rates in the TA-SM in water solution group and tablet form group were all lower than that of the medium control group [(78.4 ± 9.68)%] (F = 32.30, 38.53; all P < 0.01) and the solvent control group [(82.7 ± 5.45)%] (F = 55.15, 67.50; all P < 0.01), but there were no significant differences with ABZ positive control group (23.85 ± 19.63)%] (F = 0.53, 0.29; all P > 0.05) or the ABZ-SO positive control group [(18.93 ± 3.08)%] (F =4.44, 3.17; all P > 0.05). On the fifth day of co-culture in vitro, the relative expression levels of ALP in the culture supernatant in the low, medium and high concentration groups of the TA-SM in water solution groups and the tablet form groups, the ABZ and ABZ-SO positive control groups, the medium and solvent control groups were 0.15 ± 0.01, 0.17 ± 0.03, 0.18 ± 0.03 and 0.21 ± 0.04, 0.29 ± 0.04, 0.32 ± 0.04; 0.17 ± 0.01 and 0.15 ± 0.01; 0.15 ± 0.01 and 0.13 ± 0.01, respectively, in which the TA-SM in tablet form groups were higher than medium and solvent control groups, ABZ and ABZ-SO positive control groups (F = 18.49, 22.34, 14.86, 18.06; P < 0.01) and the TA-SM in water solution groups (t = 6.86, 3.78, 5.19; P < 0.05). On the fifth day of co-culture in vitro, the relative expression levels of caspase 3 in protoscolices in the low, medium and high concentration groups of TA-SM in water solution groups and tablet form groups, ABZ and ABZ-SO positive control groups, medium and solvent control groups were 0.14 ± 0.01, 0.13 ± 0.01, 0.14 ± 0.01 and 0.21 ± 0.02, 0.36 ± 0.04, 0.42 ± 0.04; 0.10 ± 0.01 and 0.17 ± 0.01; 0.13 ± 0.01 and 0.10 ± 0.01, respectively, in which the TA-SM in tablet form groups were higher than the medium and solvent control groups, ABZ and ABZ-SO positive control groups (F = 58.97, 69.18, 71.81, 46.77; all P < 0.01) and the TA-SM in water solution group (t = 4.99, 10.24, 10.82; P < 0.01). The results of the in vivo experiment showed that after 60 days of treatment, the cyst inhibition rates of the tablet group and the water solution group were (24.4 ± 4.15)% and (17.2 ± 3.71)%, respectively, with a statistically significant difference between them (t = 3.15, P < 0.05). The HE staining results showed that after 60 days of treatment, the hepatocytes in the saline group were swollen, there were a large number of inflammatory cells infiltrating around, and there were a large number of vacuolar degeneration in the cytoplasm. The morphology of hepatocytes in the water solution group and the tablet group tended to be normal, the inflammatory cells were significantly reduced, and the vacuolar degeneration in the cytoplasm was reduced. SEM results showed that after 15 days of treatment, the typical vesicular structure appeared in the liver tissue lesions of mice in the saline group, while the typical vesicular structures in the liver tissue lesions of mice in the tablet group and the water solution group were significantly reduced. Conclusion TA-SM in water solution and tablet forms have certain suppressive effects on protoscolices of E. multilocularis in vitro and in vivo. The efficacy in vitro test is essentially the same as that of ABZ, and the efficacy of TA-SM in tablet form is better than that in water solution.

Key words: Echinococcus multilocularis, Sophora moorcroftiana, Total alkaloids, Dosage form