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Briefly, 5 mL of Tris-HCl buffer (0.05 M, pH = 8.2) was mixed with 4 mL of VCAF at different concentrations (5-100 [micro]g/mL, diluted in 50% ethanol) and VC at the same concentrations after a preheating period of 20 min at 25[degrees]C.
Cytoprotective Effects of VCAF. HepG2 cells were seeded at a density of 5 x [10.sup.3] cells/well into a 96-well plate and at a density of 5 x [10.sup.4] cells/well into 6-well plates.
Our results indicated that the total flavonoid and phenolic contents of VCAF were 312.00 [+ or -] 10.00 mg/g rutin equivalent and 161.60 [+ or -] 24.33 mg/g gallic acid equivalent, respectively.
Moreover, three antioxidant assays demonstrated the strong antioxidant activity of VCAF compared with VC (Figure 1).
Cytotoxicity and Cytoprotective Effects of VCAF. No evidence of toxicity of VCAF (20-180 [micro]g/mL) was found in HL-7702 cells after treating for 24 and 48 h (Figure 2(a)).
As shown in Figure 4(c), VCAF pretreatment suppressed the increases in caspase-3 and caspase-8 compared with t-BHP treatment, but not caspase-9, which suggested that VCAF inhibited apoptosis through the downregulation of caspase-3 and caspase-8.
Previous research demonstrated that VCAF and its iridoid glycoside fraction exerted hepatoprotective activity against carbon tetrachloride- and acetaminophen-induced liver injury in mice, respectively .
VCAF suppressed the increase of early apoptosis in t-BHP-treated cells.
We found that VCAF markedly reduced the activities of ALT and AST and elevated the activity of AChE; thus, VCAF was shown to improve hepatic function.
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