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Abstract
Purpose:
This study examined the antioxidant and cancer cell growth inhibitory activities of an ethanol extract and different solvent fractions of Mesembryanthemum crystallinum L. (ice plant).
Methods:
The ice plant was freeze-dried, extracted with 99.9% ethanol, and then fractionated with hexane, ethyl acetate, butanol, and water. The total polyphenol content (TPC), total carotenoid content (TCC), 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity (RSA), and ferric reducing antioxidant power (FRAP) were measured. Assays using 2',7'-dichlorofluorescin-diacetate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were performed to measure the intracellular reactive oxygen species (ROS) and cell growth, respectively. Annexin V/propidium iodide staining and cell cycle analysis were performed for the detection of apoptosis and cell cycle arrest.
Results:
TPC, TCC, RSA, and FRAP of the ethanol extract (EE) were 3.7 mg gallic acid equivalent/g, 13.2 μg/g, 21.0% (at a concentration of 5 mg/mL), and 21.0% (at a concentration of 5 mg/mL), respectively. Among the different solvent fractions, the butanol fraction (BF) showed the highest TPC (5.4 mg gallic acid equivalent/g), TCC (86.6 μg/g), RSA (34.9% at 5 mg/mL), and FRAP (80.8% at 5 mg/mL). Treatment of HCT116 human colon cancer cells with EE and BF at concentrations of 250 and 500 μg/mL reduced the levels of intracellular ROS. Concomitantly, EE and BF resulted in the dose-dependent inhibition of cell growth (at the concentrations of 125, 250, and 500 μg/mL for 24~48 h) and the induction of apoptosis (at the concentrations of 250 and 500 μg/mL for 48 h) in HCT116 cells. An increased G2/M cell population was also found in the BF-treated cells.
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Fig. 1
Total polyphenol levels of ethanol extract and different solvent fraction prepared from Mesembryanthemum crystallinum L. GAE: gallic acid equivalent. All values are presented as mean ± SEM of ≥ 3 determinations. Different letters (a-c) mean significant difference among different samples by Duncan's multiple range test (p < 0.05).
Fig. 2
Total carotenoid levels of ethanol extract and different solvent fraction prepared from Mesembryanthemum crystallinum L. All values are presented as mean ± SEM of ≥ 3 determinations. Different letters (a-c) mean significant difference among different samples by Duncan's multiple range test (p < 0.05).
Fig. 3
DPPH radical scavenging activity of ethanol extract and different solvent fraction prepared from Mesembryanthemum crystallinum L. All values are presented as % of the control in the mean ± SEM of 3 determinations. Different letters (a-c) mean significant difference among different samples by Duncan's multiple range test (p < 0.05).
Fig. 4
Ferric reducing antioxidant power of ethanol extract and different solvent fraction prepared from Mesembryanthemum crystallinum L. All values are presented as % of the control in the mean ± SEM of 3 determinations. Different letters (a-d) mean significant difference among different samples by Duncan's multiple range test (p < 0.05).
Fig. 5
Intracellular ROS scavenging activity of ethanol extract and butanol fraction prepared from Mesembryanthemum crystallinum L. in HCT116 colon cancer cells. HCT116 cells were treated with ethanol extract and butanol fraction at the concentrations of 125, 250, and 500 μg/mL for 48 h. All values are presented as mean ± SEM of ≥ 3 determinations. Different letters (a-c) mean significant difference among different concentrations by Duncan's multiple range test (p < 0.05). Asterisks mean statistical difference between two different sample at a respective concentration by two-tailed student t-test (∗∗∗ p < 0.001).
Fig. 6
Growth inhibitory activities of ethanol extract and butanol fraction prepared from Mesembryanthemum crystallinum L. in HCT116 colon cancer cells. HCT116 cells were treated with ethanol extract and butanol fraction at the concentrations of 125, 250, and 500 μg/mL for 24 h (A) and 48 h (B). All values are presented as mean ± SEM of ≥ 3 determinations. Different letters (a-c) mean significant difference among different concentrations by Duncan's multiple range test (p < 0.05). Asterisks mean statistical difference between two different sample at a respective concentration by two-tailed student t-test (∗∗∗ p < 0.001).
Fig. 7
Correlation between intracellular ROS scavenging activity and growth inhibitory activities of the butanol fraction prepared from Mesembryanthemum crystallinum L. in HCT116 colon cancer cells. Pearson's correlation analysis was performed.
Fig. 8
Apoptosis-inducing activity of ethanol extract and butanol fraction prepared from Mesembryanthemum crystallinum L. in HCT116 colon cancer cells. HCT116 cells were treated with ethanol extract and butanol fraction at the concentrations of 250 and 500 μg/mL for 48 h. Representative histogram (A) and bar chart (B) of annexin/PI double staining assay. All values are presented as mean ± SEM of ≥ 3 determinations. Different letters (a-c) mean significant difference among different concentrations at a respective sample by Duncan's multiple range test (p < 0.05). Asterisks mean statistical difference between two different samples at a respective concentration by two-tailed student t-test (∗∗∗ p < 0.001).
Fig. 9
Representative cell cycle of HCT116 colon cancer cells after the treatments of ethanol extract and butanol fraction prepared from Mesembryanthemum crystallinum L. HCT116 cells (3 × 105 cells/well) were treated with ethanol extract or butanol fraction at the concentrations of 500 μg/mL for 48h. M1: G0/G1 phase; M2: G2/M phase; M3: S phase; M4: sub-G1 phase.
Table 1.
Cell cycle distribution of HCT116 colon cancer cells after the treatment of ethanol extract and butanol fraction prepared from Mesembryanthemum crystallinum L.
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