| PURPOSE: To evaluate the cytotoxicity effect of 9 natural products isolated; analyze the structure-activity relationship and free radicals-related mechanisms. To illustrate the new bioactivity features of compounds synthesized through linkage of benzoic nitrogen mustard and sesquiterpenes.METHOD: Human hepatoma cell line SMMC-7721 and HepG2, human cervical cancer cell line HeLa, human acute myeloid leukemia cell line HL-60 and human normal liver cell line L02 were used as models in this study. SRB method and trypan blue staining method were applied to evaluate cytoxicity, DCFH-DA fluorescence spectrophotometry was used to determine ROS generation, ammonium molybdate colorimetric assay was used to determine catalase activity.Tested compounds were as follows:(1)7 diterpenoids (No.1 to 7); (2) 2 benzofuran compounds (No. BF-1 and BF-2); (3) 5 species of benzoic acid nitrogen mustard sesquiterpene (No. BNMS-1-BNMS-5), benzoic nitrogen mustard, BNM) and a sesquiterpenes (1-on [N, N-2 (2-chloroethyl) amino] benzoyl-5α-hydroxy-4 (15),10 (14),11 (13)-triene-6a,12-Eucalyptus alkyl lactone) (S).RESULT:(1) The IC50 values of diterpenoids (1) and (6) on SMMC-7721, HeLa and HL-60 cells were less than 4μg/mL, showed potent cytotoxicity; IC50 values of compounds (2), (3) and (4) were between 2-33μg/mL; the IC50 value of compounds (5)and (7) were between 26-126μg/ mL.(2) The IC50 values of benzofuran compounds BF-1 and BF-2 on the normal liver cell line L02 were 171.2±3.3μg/mL and 79.0±4.1μg/mL, higher than that on hepatoma cell line HepG2,84.2±6.5μg/mL and 65.2±1.9μg/mL, which indicated that tumor cells (HepG2) are more sensitive to the compound than the normal cells (L02). Cells were treated with BF-1 and BF-2 under their IC50 for 48 h, and the reactive oxygen species level measured were 1.6 and 3.2 folds than control in HepG2 cells,1.2 and 1.8 folds than control in L02 cells; The catalase activity were decreased in HepG2 cells from the value 17.2±1.7 U/mgPr. of control to 12.8±0.4 and 6.4±0.1 U/mgPr. after treated with Compound BF-1 and BF-2. the catalase activity in L02 cells were decreased from the value of control group,17.7±1.2 U/mgPr. to 14.3±1.5 and 8.6±0.5 U /mgPr. in BF-1 and BF-2 treated samples. The catalase activity decreasing rate in HepG2 were higher than in L02.(3) The IC50 value of benzoic acid nitrogen mustard sesquiterpene BNMS-1 on SMMC-7721, HeLa, HL-60, and L02 cells were between 99.2-161.2μg/mL, the IC50 values of compounds BNMS-2, BNMS-3, and BNMS-4 were between 6.6-29.7μg/mL.The cytotoxiciy of these 4 compounds were in the order below: BNMS-3> BNMS-2≈BNMS-4> BNMS-1. BNMS-3 with two double bond in C3 to C4 and C11 to C13 (α-methylene-γ-butyrolactone) exhibited the most potent cytotoxicity; When the C3 to C4 double bond in BNMS-3 changed to C4 to C15, and the C11 to C13 double band changed to single band (a-methylene-y-butyrolactone was destroyed), that is BNMS-1, the cytotoxicity decreased to the lowest; Compound BNMS-2 can be synthesized from BNMS-3 with changing the double band from C3= C4 to C4= C15, and compound BNMS-4 can be originated from BNMS-3 through changing the double band between C11= C13 to single band, both of them had a same decreasing cytotoxicity compared to BNMS-3.(4) Human hepatoma cell line HepG2 and human normal liver cell line L02 were used to detect the cytotoxicity of BNMS-5 and its originated sesquiterpene (4 (15),10 (14),11 (13)-triene-6α,12-eudesmane sesquiterpene lactone) (S). The IC50 values of BNMS-5 and S on cells less than 100μg/mL, compound S on the hepatoma cell line HepG2 selective, and reactive oxygen species concentration with S increased; and BNMS-5 on HepG2 cell non-selective, and also unnecessary on the ROS induced concentration-dependent trend, its cytotoxicity has nothing to do with the reactive oxygen species mechanism.CONCLUSION:(1) The structure-activity analysis indicated that a-methylene cyclopentanone in the molecule structures was cytotoxicity center, and hydroxyl group seemed to exhibit a negative effect on the cytotoxicity, the more hydroxyls the less cytotoxicity. However, the position of hydroxyl group has very little effect on cytotoxicity.(2) According to the more potent cytotoxicity, the more ROS generation increasing in HepG2 than L02, and the more decreasing of catalase activity in HepG2 cells, we supposed that the mechanism of the cytotoxicity for these two benzofuran deravatives may relate to ROS theory.(3) The structure-activity analysis of BNMS-1, BNMS-2, BNMS-3 and BNMS-4 indicated that the double bond in C3 to C4 andα-methylene-γ-butyrolactone (double band in C11 to C13) were most important for the cytotoxicity of these compounds. (4) Results indicated that sesquiterpene S exhibited a more potent cytotoxicity on HepG2 cell than L02, and ROS generation were increased with concentration treatment increasing. While no selective effect of BNMS-5 were found, and no evident supported its cytotoxicity was related with ROS generation, although more potent cytotoxicity found in BNMS-5 than S.
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