Fig 1.
Kae exhibits greater antitumor effects than its glycosides. Cells (3 x 103/well) were added to wells in a 96-well plate and incubated with various concentrations of kae, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut for 72 h. The inhibitory rate of kae, kae-7-O-glu, kae-3-O-rha and kae-7-O-rut on cell proliferation was determined by the MTT assay.
(A) HepG2 cells. (B) CT26 cells. (C) B16F1 cells. (D) IC50 values of kae, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut. *P < 0.05, **P < 0.01. The data are shown as the mean ± SEM of three independent experiments.
Fig 2.
Kae induces liver cancer cell apoptosis, inhibits AKT phosphorylation, and induces caspase-dependent apoptosis in liver cancer cells.
(A) HepG2 cells were added to a 96 -well plate and incubated with kae, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut (0, 10 or 100 μM) for 24 h. HepG2 cell apoptosis was determined by annexin V/PI staining. (B) HepG2 cells were treated with kae, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut (10, 30 or 100 μM) for 24 h. Cleaved caspase-3, 7, and 9, cleaved PARP, and p-AKT protein levels were determined by Western blotting. (C) HepG2 cell apoptosis of three independent experiments were shown in the bar graph. (D)-(H) ImageJ software was used to analyze the levels of p-AKT, cleaved caspase-3, 7, 9 and cleaved PARP with GAPDH as the reference. *P < 0.05, **P < 0.01 The data are shown as the mean ± SEM of three independent experiments.
Fig 3.
Increased antioxidant and anti-inflammatory effects of kae compared to kae glycosides.
(A) The DPPH radical scavenging assay. (B) The ABTS radical scavenging assay. (C) IC50 values for DPPH and ABTS radical scavenging. (D) The T cells (1 x 106/well) were added to a 96-well plate with ConA (2.5 g/ml). Various concentrations of kae, kae-7-O-glu, kae-3-O-rha and kae-3-O-rut (0, 10, 30 or 100 μM) co-cultured with activated T cells were incubated for 24 h, 48 h and 72 h. The MTT assay was used to detect ConA-activated T cell proliferation. (E) RAW 264.7 cells (2 x 105 cells/ml) were added to a 96-well plate (100 μl/well). After 24 h, the cells were treated with LPS (500 ng/ml) and kae, kae-7-O-glu, kae-3-O-rha or kae-3-O-rut at various concentrations. Each concentration was tested twice in triplicate with the culture medium containing DMSO as the control. Each sample (100 μl) was added to an enzyme-labeled plate and the mixed Griess reagent (100 μl) was added. After 10 min, the absorbance was measured at 540 nm. The inhibitory effect of NO release on RAW 264.7 cells was calculated. (F) Steady state ROS concentration in the culturing medium was determined using the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, China). (G) IC50 values for the inhibition of T cells. *P < 0.05, **P < 0.01. The data are shown as the mean ± SEM of three independent experiments.
Fig 4.
Hydrolysis of kae glycosides by α-L-rhamnoside and/or β-glycosidase.
Effects of pH value, temperature, enzyme concentration and time on the hydrolysis rate of kae-3-O-rut by α-L-rhamnoside (A) and kae-3-O-glu by β-glucosidase (B). (C) Hydrolysis rates of kae-3-O-rut and kae-7-O-glu by α-L-rhamnoside and/or β-glucosidase under the optimal conditions. Each value represents the mean of three independent measurements.