49
antibodies of of Anti-mouse p53 or Bcl-2 monoclonal antibodies (1:1000 dilution) for 1 h at room temperature and
washed in TBST (3 x 5 min). The blots were further incubated with the secondary antibody, horseradish
peroxidase-conjugated goat anti-mouse Ab (HRP) (1:2000 dilution) for 1 h and then washed in TBST (3 x 5 min).
Tetramethylbenzidine (TMB) substrate was added (in dark) for forming the HRP–substrate TMB precipitation
(blue precipitate). The developed bands were photographed. Actin protein was used as protein amount control in
SDS-PAGE by the blots were strip in stripping buffer containing 0.2 M glycine (pH 2.2), 150 mM NaCl and 0.1%
(v/v) Tween 20 for 30 min at 50°C, blocked with 0.1% (v/v) Tween-20 in Tris-buffered saline (TBST) containing
5% (w/v) nonfat dry milk for 20 min and washed with TBST (3 x 5 min). The blots were incubated with
antibodies, anti-actin, HRP and TMB solution as the above protocol. The blots were then photographed.
Results and discussion
The total phenolic contents were determined from regression equation of calibration curve using gallic
acid as a standard and expressed as gallic acid equivalents (GAE). The pomace extract contained total phenolic
compounds of 1,743.504 ± 0.003 ôg GAE/g (p<0.05). the yield of phenolic compounds in grape alcoholic extracts
was hightly increased with prolong extraction (Vladimir
et al
., 2005), as our pomace was extracted in 70%
ethanol. Yi
et al
. (1997) reported that the red wine Calzin and Shiraz grapes had the highest phenol content. This
data agree with Kanner
et al
. (1994) who reported that the red wine Cabernet Sauvignon and Shiraz grapes contain
higher concentrations of phenolics than the black seedless grapes. Our data indicated that Shiraz red grape was a
good source of phenolic compounds. The amount and types of phenolic compounds could be the main constituents
of plant extracts that affect their biological properties on other organisms.
MCF-7 cells were treated with pomace extract at various concentrations (10-1000 ôg/mL) for 6-24 h at
37
°
C then the percentage of cell viability was calculated
.
Significantly, the percentage of cell viability of cancer
cells was higher 50% after exposure to 200 (at 6 h) and 100 ôg/mL (at 12 and 24 h) of pomace extract with IC
50
values following incubation times (389.55, 129.74 and 62.06 ôg/mL at 6, 12 and 24 h, respectively, p<0.05). The
result of anti-proliferation was showed that the extracted pomace possessed good cytotoxicity against the
proliferated cancer cells all the treated times. Recent studies evaluated the abilities of phenolic compounds in
wines and grape extracts to reduce cell proliferation of various cancer cell types (Alkhalaf, 2007). Mertens-Talcott
et al
. (2008) confirmed that the differences in polyphenolic composition and antioxidant properties were expected
to result in significant differences in the anti-cancer cell proliferation of extracts.
The Shiraz pomace extract was applied onto MCF-7 cell culture at the range of 10-1000 ôg/mL for 6, 12
and 24 h. It appeared that nuclear morphology was changed after 5 hours of treatment. The nuclei of treated cells
were fragmented with blebbing appearance. While the nuclei of the untreated control cells were look normal. The
percentage of apoptotic cells was 50% higher after exposure to 500 and 200 ôg/mL of pomace extract at 12 and 24
h, respectively. Percentage of apoptosis of cells treated with pomace extract at 6 h and 12 h were lower compared
to 24 h at the same concentration tested.
