ABSTRACT
OBJECTIVE: To compare the differences of macrophages phagocytic activities between Dioscorea opposita Thunb. cv. Tiegun(abbreviated as TG) and Dioscorea opposita Thunb. but not Tiegun(abbreviated as NTG). METHODS: CCK-8 assay was used to test the cytotoxicity. On the basis of non-toxic dose, the high content screening(HCS) cell imaging analysis was applied to test the phagocytic ability of RAW264.7 cells engulfing GFP-E. coli in vitro, and the phagocytic rates between different kinds of samples at various concentrations were calculated. Furthermore, the biological potency was measured according to the qualitative response parallel method to better evaluate the difference of TG and NTG, by translating phagocytic rates into biological potency. RESULTS: At the range of 0.695 - 5.56 mg(crude drug) •mL-1, all 11 batches samples have no toxicity. The results of HCS displayed that every sample could promote cell phagocytic activity in varying degrees at a certain concentration. RAW264.7 cells could engulf the GFP-E. coli, and the images of HCS reflected the situation of phagocytosis clearly. In addition, the results of biopotency showed that the biopotency value of different samples was between 39.56 to 100, and the average biopotency value of TG was significantly higher than NTG (P < 0.05). CONCLUSION: In vitro experiments showed that all the samples could promote the phagocytic activity of RAW264.7 cells at different degrees. And the average biopotency value of TG was significantly higher than NTG, which is consistent with the saying that "TG has a better quality".
ABSTRACT
Objective: To establish a high content screening (HCS) method by testing the phagocytic function of RAW 264.7, and to evaluate the effect of Dioscorea opposita on cell proliferation and phagocytosis in vitro. Methods: CCK-8 was used to measure the proliferation of RAW 264.7, and HCS was helpful to determine the ability of RAW 264.7 to engulf GFP-E.coli. Results: The best method was established by examining the time of administration, the amount of bacteria added, and the time of bacterial stimulation: administration time of 12 h, 50 times of bacteria, and stimulation time of 1.5 h. Then, the repeatability of this method was tested, and the RSD value was 2.31%. Compared with the group without drugs, at the concentration of 0.156-1.25 mg/mL, the water extract of D. opposita could promote the proliferation, and improve the average phagocytosis rate of every single RAW264.7 cell. And the samples had the strongest effect on proliferation and phagocytosis when the concentration was 1.25 mg/mL. Conclusion: An HCS method is established and firstly used in D. opposita. HCS has the advantage of accurate, intuitive, and high throughput. D. opposita can not only promote cell proliferation, but also improve the phagocytic ability of each single RAW264.7 in vitro. A new method is provided for further study on immune activity and on the mechanism of enhancing the immune activity that some tonic herbs may have, such as D. opposita.