Abstract
Chenopodium ambrosioides L. (C. ambrosioides) has been used as dietary condiments and as traditional medicine in South America. The oil of Chenopodium ambrosioides L. (C. ambrosioides) can be used as a natural antioxidant in food processing. It also has analgesic, sedating, and deworming effects, and can be used along with the whole plant for its medical effects: decongestion, as an insecticide, and to offer menstruation pain relief. This study was conducted to investigate the cytotoxicity and apoptosis effects of an essential oil from C. ambrosioides in vitro. The cytotoxicity evaluation of the essential oil from C. ambrosioides on human normal liver cell line L02 was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. AO/EB dual fluorescent staining assay and Annexin V-FITC were used for apoptosis analysis. The changes in mitochondrial membrane potential (MMP) were analyzed with 5,5,6,6′-tetrachloro-1,1,3,3,-tetraethyl-imidacarbocyanine iodide (JC-1) dye under a fluorescence microscope. The level of apoptosis related protein expression was quantified by Western blot. The L02 cells were treated with the essential oil from C. ambrosioides at 24, 48, and 72 h, and the IC50 values were 65.45, 58.03, and 35.47 μg/mL, respectively. The AO/EB staining showed that viable apoptotic cells, non-viable apoptotic cells, and non-viable non-apoptotic cells appeared among the L02 cells under the fluorescence microscope. Cell cycle arrest at the S phase and cell apoptosis increased through flow cytometry in the L02 cells treated with the essential oil. MMP decreased in a concentration-dependent manner, as seen through JC-1 staining under the fluorescence microscope. In the L02 cells as shown by Western blot and qPCR, the amount of the apoptosis-related proteins and the mRNA expression levels of cytochrome C, Bax, Caspase-9, and Caspase-3 increased, Bcl-2 decreased, and Caspase-12, which is expressed in the endoplasmic reticulum, showed no obvious changes in protein amount or mRNA expression level. The essential oil form C. ambrosioides had a cytotoxic effect on L02 cells. It could inhibit L02 cell proliferation, arrest the cell cycle at the S phase, and induce L02 cell apoptosis through the endogenous mitochondrial pathway.
Highlights
Chenopodium ambrosioides L. (C. ambrosioides) is an annual or perennial aromatic herb that belongs to the Chenopodiaceae subfamily of plants
The results showed the green fluorescence (JC-1 monomers, treated as depolarized mitochondria) of the L02 cells in the carbonylcyanide-p-chlorophenyl hydrazone (CCCP) positive control group (Figure 2Cd), indicating that the membrane potential (MMP) was lower in the L02 cells
We investigated the potential mechanism through which the essential oil from C. ambrosioides may induce apoptosis in L02 cells by Quantitative Polymerase Chain Reaction (qPCR)
Summary
Entire C. ambrosioides plants were collected from vacant land in the suburbs of Chengdu, Sichuan, China and verified as C. ambrosioides by Professor Danwei Ma, Sichuan Normal. The collected plants were dried in a cool place. C. ambrosioides essential oil was extracted through a steam distillation method, dried with anhydrous sodium and stored at −20 ◦ C. The cells were incubated at 37 ◦ C with 5% CO2 in a humiditysaturated incubator, which was used while the experiments were in the log growth phase
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