Malek Besbes Hlila, Habib Mosbah, Kaouther Majouli et al.148
Stock solutions of these compounds were prepared in HPLC-grade methanol,
wrapped in aluminum foil then stored at 4°C. Ribavirin (Sigma) was used as
the positive control in the anti-CVB3 test.
2.2. Samples
Scabiosa arenaria was collected in May 2010 from the area of Monastir
(Tunisia) at the flowering stage. The plant material was kindly identified by
Pr. Fethia Harzallah Skhiri, High Institute of Biotechnology of Monastir,
Tunisia. A voucher specimen (S. arenaria) (Sa 110) was deposited in the
Laboratory of Bioorganic Chemistry and Natural Products at the Faculty of
Sciences of Monastir, Tunisia. The flowers, the fruits, the (stems and leaves)
and the roots were air-dried at room temperature for two weeks and reduced to
coarse powder. The powdered samples (250 g) were separately, extracted by
maceration with MeOH–H 2 O 80:20 (v/v) for 72 hours at room temperature. A
repetitive extraction of the same material three times with solvent was carried
out. After filtration, a portion of obtained extracts was dried completely for
further biological activities and the residual aqueous layer was further
subjected to a successive extraction with ethyl acetate (EtOAc) and butanol (n-
BuOH) to yield dried fractions for each organ, knowing that, the extracts was
evaporated in a rotary evaporator and dried by vacuum pump. Sixteen extracts
were obtained and maintained at 4°C before analysis.
2.3. Cell Line
The Vero cells were derived from a kidney of normal, adult, African green
monkey (Cercopithecus) by Yasumura and Kawakita at Japan Chiba
University in 1962. This cell line has been extensively used for virus
replication studies and plaque assays. Vero cells (kindly provided by Pr. Bruno
Pozzetto, Laboratory of Bacteriology-Virology, Saint-Etienne, France) were
used for culturing enterovirus strains. Vero cells were maintained in RPMI
1640 (Roswell Park Memorial Institute medium) supplemented with 10% fetal
bovine serum (FBS), L-Glutamin (2 mM), penicillin (100 U/mL), and
streptomycin (100 μg/mL). Cells were incubated at 37°C in a 5% CO 2
humidified atmosphere.