Primary Culture Of Cells From Mythimna Separata And Cytoxicity Assay Of Several Natural Products Against The Cells

The hemocyte cell,fat body cell, embryo cell, midgut cell from Mythimna separata had been cultured. The cytoxicities of Celangulin V, fraxinellone and dictamnine against the midgut cell of Mythimna separata had been assayed. The cytoxicities of a series of derivatives of Celangulin against the SFE-HA-8212 cell had been assayed too.1.Cell cultureThe hemocyte cells from the larvas of Mythimna separata were primary cultured . The result showed that the cells grew well in M199 insect medium. The medium turned to black after thirty minutes when the cells were inoculated, the colour would be normal after changed the medium several times. The cells would grow a month later and the clones appeared after three months.The fat body cells from the pupas of Mythimna separata were primary cultured. We got tow types of cells:ovoid and spindle cells. The M199 insect medium did not fit the cells grow, but they grew well in Grace's insect medium and TNM-FH insect medium. The ovoid cells grew like ovoids and attached very closed. The cells was attachment inhibited. The spindle cells grew very slowly and they attached too close to pass a generation.The midgut cells from the larvas of Mythimna separata were cultured too. The result showed that the cells grew well in TNM-FH insect medium than M199 insect medium and Grace's insect medium. The cells grew steadily and looser after a month. A year later, the spindle cells appeared. but they grew very slowly and need three months to pass a generation.The embryo cells from the eggs of Mythimna separata were also cultured. TNM-FH was the best medium in the three. The ovoid and spindle cells were cultured. One month later the ovoid cells grew like a bunch of fibers and there were many ovoid cells around the fibers after tow months. The spindle cells appeared after six months, but the cells were smaller than the midgut cells. There were many small ovoid cells around the spindle cells with the times went on.2. The cytoxicity assayThe toxicities of Celangulin V, fraxinellone and dictamnine against the midgut cells from Mythimna separata were assayed with the methods of MTT, Neutral Red and Trypan Blue Stain. The curves of the different solvents to the formazane were measured in the method of MTT. The maximum absorption wavelength of acidate isopropyl alcohol was 558nm, 10%SDS was 568nm and DMSO was 497nm. These three phytochemicals had obvious toxicity to the cell. The LC50s of CelangulinⅤagainst the midgut cell by the methods of MTT, Neutral Red and Trypan Blue Stain were 9.08, 7.79 and 10.94 mg·L-1, those of fraxinellone were 27.85, 31.77 and 36.42 mg·L-1, and those of the dictamnine were 186.66, 164.00 and 189.43 mg·L-1.The toxicities of fifteen derivatives of Celangulin against the SFE-HA-8212 cells were assayed with the method of MTT. The results showed that there were six derivatives had obvious toxicity to the cells, and the LC50s of them were under 100 mg·L-1, the LC50 of the best one was 37.51 mg·L-1. There were also five derivatives had toxicity to the cell, and the LC50 of the worst one was 232.63 mg·L-1. The activities became worse with the groups got complexer in diafuran and acetone protected derivatives, It was indicated that there maybe existent Structure Activity Relationship in the derivatives of Celangulin.