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following conditions: MS/MS analysis with starting
collision-induced dissociation energy of 30 eV, negative
ionization mode.
2.5. A ntimicrobial assay
The antibacterial and antifungal activities were carried
out in the Microbial Department, National Research
center, using the disc diffusion method (7). A filter paper
sterilized disc saturated with measured quantity (20 µl) of
the sample (5mg/ml) (100µg/disc) is placed on a plate (9
cm diameter) containing a solid bacterial medium (nutrient
agar) or a fungal medium (potato dextrose agar) which has
been seeded with the spore suspension of the test
organism. Incubation was at 37ºC, 24 h for bacteria and at
28ºC, 72 h for fungi. All measurements were done in
methanol as a solvent which has zero inhibition activity.
The antimicrobial activity of the tested compounds were
examined with gram positive bacteria, Bacillus cereus and
Staphylococcus aureus ATCC 6538, and gram negative
bacteria Salmonella typhimurium ATCC 25566,
Escherichia coli NRRN 3008 and Pseudomonas aeruginosa
ATCC 10145 and fungus Candida albicans
EMCC105.The obtained results are compared with the
reference antibiotics 20 µl from 5mg/ml solution
Cephradine (Bristol Myers-Squibb) and Ketoconazole
(Janssen Pharmaceutica NV ), (100µg/disc). All these steps
were carried out under aseptic conditions. The test was
carried out in triplicates. The diameter of the clear zone of California, USA) at ? 595 nm and a reference wavelength
inhibition surrounding the sample is taken as a measure of
the inhibitory power of the sample against the particular
test organism according the following equation: %
inhibition = sample inhibition zone (cm) / plate diameter
(cm) x 100). The Minimum Inhibition Concentration
(MIC) was determined after incubation of the bacterial
strains at 37 º C for 24 h and the fungal strain at 28º C for to the formula: (1- (Reading of extract / Reading of
72 h in the presence of serial dilutions of the test
compounds. Each MIC experiment was repeated three
times. The MIC was defined as the lowest concentration at
which no visible growth was observed.
2.6. Statistical analysis
The results obtained in all analyses were expressed in
mean of three ±SE (standard error). Student?s t- test using
GraphPad QuickCalcs on line Software, Inc. CA, US A.
by comparing the inhibition zones of samples to those of
antibiotics Statistical significance was set at P <0.01.
2.7. In vitro assessment of cytotoxicity
Cell viability was assessed by the mitochondrial
dependent reduction of yellow MTT (3-(4, 5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to
purple formazan (8). All the following procedures were
done in a sterile area using a Laminar flow cabinet
biosafety class II level (Baker, SG403INT, Sanford, ME,
USA). Cells were suspended in RPMI 1640 medium for
MCF7 (Breast carcinoma cell line) HCT 116 (Colon
carcinoma cell line) and HEPG2 (liver cell carcinoma).The
media are supplemented with 1% antibiotic-antimycotic
260
Elsayed A. Aboutabl et al.
mixture (10,000U/ mL Potassium Penicillin, 10,000µg/ml
Streptomycin Sulphate and 25µg/mL Amphotericin B), 1%
L-glutamine and 10% fetal bovine serum and kept at 37 ºC
under 5% CO2.Cells were batch cultured for 10 days, then
seeded at concentration of 104 cells/well in fresh complete
growth medium in 96-well microliter plastic plates at 37
ºC for 24 hours under 5% CO2 using a water jacketed
Carbon dioxide incubator (Sheldon, TC2323, Cornelius,
OR, USA). Media was aspirated, (without serum) fresh
medium was added and cells were incubated either alone
(negative control) or with different concentrations of
sample (100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78
µg/mL), Doxorubicin was used as reference drug at
aforementioned concentration; triplicate being prepared for
each individual dose. After 48 hours of incubation,
medium was aspirated, 40µL MTT salt (2.5µg/mL) were
added to each well and incubated for further four hours at
37ºC under 5% CO2. To stop the reaction and dissolving
the formed crystals, 200 µL of 10% Sodium dodecyl
sulphate (SDS) in deionized water was added to each well
and incubated overnight at 37ºC. A positive control which
composed of 100µg/mL was used as a known cytotoxic
natural agent (A nnona cherimola leaves) who gives 100%
lethality under the same conditions (9). The absorbance
was then measured using a microplate multi-well
reader (Bio-Rad Laboratories Inc., model 3350, Hercules,
at ? 620 nm. A statistical significance was tested between
samples and negative control (cells with vehicle) using
independent t-test by SPSS 11 program. DMSO is the
vehicle used for dissolution of plant extracts and its final
concentration on the cells was less than 0.2%. The
percentage of change in viability was calculated according
negative control)) x 100.
3. RESULTS
3.1. UPLC of acetone extract
The UPLC profile for monitoring the separation
process is illustrated in (Figure 1) the compounds
tentatively identified from mass are compiled in (Table 1).
The elution order followed a sequence of decreasing
polarity, whereby flavonoid diglucosides eluted first,
followed by monoglucosides then free aglycones and
highly oxygenated triterpenoids.
3.2. A ntimicrobial effect of acetone extract
As shown in Table 2 the tested extract is clearly active
against gram negative bacteria represented by Escherichia
coli and Salmonella typhimurium reaching 100% the
activity of the reference antibiotic followed by the gram
negative bacterium Pseudomonas aeruginosa (90%). At the
same time our extract exhibited antimicrobial activity
against gram positive bacteria Bacillus cereus and
Staphylococcus aureus with inhibitory effect of two thirds
the reference antibiotic. With respect to Candida albicans
the tested extract exerts mild activity against this pathogen.
These results are of great value to biological applications
@Real Academia Nacional de Farmacia. Spain