Preventive effects of chebulic acid isolated from Terminalia chebula ...

570 H.-S. Lee et al. / Journal of Ethnopharmacology 131 (2010) 567–574
Fig. 3. ROS scavenging activities of chebulic acid against glycer-AGEs. DCFH-DA-loaded HUVEC were exposed to BSA (A), 100 �g/ml of glycer-AGEs (B), 100 �g/ml of
glycer-AGEs + 5 �M chebulic acid (C), 100 �g/ml of glycer-AGEs + 10 �M chebulic acid (D), and 100 �g/ml of glycer-AGEs + 25 �M chebulic acid (E) for 24 h. Data (A–E) were
expressed as a histogram of the fluorescence peak channel by flow cytometry, and their fluorescence intensities were assayed with fluorescence spectrometry (F).
2.6. Cytotoxicity of glycer-AGEs and chebulic acid
The cytotoxicities of the glycer-AGEs and various concentrations
(1–100 �M) of chebulic acid were analyzed by MTT colorimetric
assays (Mosmann, 1983). The cells were incubated with chebulic
acid with/without 100 �g/ml of glycer-AGEs for 24 h. At the end of
the treatment time, all the wells were aspirated, refilled with MTT
solution, and incubated for 3 h (Roffey et al., 2007).
2.7. Detection of ROS in HUVEC
The production of intracellular ROS was determined by DCFH-
DA by fluorescence spectrophotometry (Shukla et al., 2006), and
flow cytometry. DCFH-DA was dissolved in dimethyl sulfoxide
(DMSO) as a 10 mM stock solution, and kept frozen at −20 ◦ C. To
load the cells, DCFH-DA from the stock solution was mixed with
M199 medium. Next, 20 �M DCFH-DA-loaded HUVEC were incubated
for 30 min, washed twice with M199, and exposed to various
concentrations of chebulic acid and/or 100 �g/ml of glycer-AGEs.
After 24 h, the cells were harvested by trypsinization, and washed
with cold phosphate buffered saline (PBS) twice. ROS were measured
by flow cytometry (FACS Calibur, Becton Dickinson, NJ, USA)
at a level of 10,000 events for each test. Fluorescence was obtained
by a histogram of the peak channel using the CellQuest program
(Becton Dickinson), and the fluorescence intensity was determined
with excitation of 485 nm and emission of 530 nm using a plate
reader (VICTOR3 TM , PerkinElmer, MA, USA).
2.8. Assessment of transendothelial electrical resistance (TER)
The effects of glycer-AGEs on TER were measured in HUVEC
according to the method of Kazakoff et al. (1995). The HUVEC were
seeded at 3 × 10 5 cells per inset on a type 1 rat tail collagen coated
membrane (pore size, 0.4 �m) contained in the apical chamber of
a 12-well trans-well system (Corning, NY, USA). After monolayer
confluence was achieved (>120 cm 2 ), the HUVEC were treated
with chebulic acid and glycer-AGEs. TER was measured every 4 h
over 24 h. The measurements were taken in triplicate, and the data
were expressed as relative percentages.
2.9. Adhesion assay of THP-1
The monocyte adhesion assay was performed as described previously
(Hiraoka et al., 2004). Briefly, THP-1 cells were prepared