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UPLC/ Tandem mass and antimicrobial activity of Khaya senegalensis (A.Juss.)
thus they encouraged us to study the minimal inhibitory
concentration (MIC) of the extract against each microbial
organism (Table 2). The obtained results revealed that
MIC of the tested extract was about 5 µ L (25µg) per disc
against gram negative bacteria Escherichia coli,
Pseudomonas aeruginosa and Salmonella typhimurium,
in addition to the yeast pathogen Candida albicans.
Meanwhile, MIC against gram positive bacteria Bacillus
cereus and Staphylococcus aureus ranged 5 µ L (25µg)
=10 µL (50 µg) per disc. These results confirm the
stronger inhibitory effect of the extract against gram
negative bacteria as the smaller the MIC the stronger
antimicrobial effect.
3.3. Cytotoxicity of acetone extract
The acetone extract was tested in-vitro on human
carcinoma cell line (HepG2 – MCF7 – HCT 116). The
concentration of extract that is required for 50% inhibition senegalensis flowers Table (1); amongst them, some were
in cell viability (IC50) against MCF7 was IC50 =88.1
(µg/mL), compared to Doxorubicin IC50 = 26.1 (µg/mL),
but neither HEPG2 nor HCT 116 cell lines were
inhibited at tested concentrations.
4. DISCUSSION
Coupling of liquid chromatography (LC) with
ultraviolet (UV) and electrospray ionization (ESI) mass
spectrometric has been demonstrated as a powerful tool for
the identification of compounds in plant extracts (10). The
method described in our study allowed identification of
seventeen compounds from Khaya senegalensis flowers.
Flavonoids
UV absorption of flavonoids shows the two
characteristic maxima in the ranges ?max240-285 nm (band
II) and 300-370 (band I) (11).
Five flavonoid glycosides (di and mono-sugar) were
identified. Quercetin glycosides were isolated before from
Khaya senegalensis (A. Juss.) leaves (12), while this is the
first report of luteolin and isorhamnetin glycosides in this effect of 200 mg/ml Khaya Senegalensis ethanol stem bark
plant. The neohesperidoside sugar moiety
(rhamnoglucoside 1 2)) in compound 1 is concluded
from fragment at m/z 505 (M-H-120)- characteristic for the
neohesperidoside sugar rather than rutinoside
(rhamnoglucoside (1 6)) (13). UV absorption of
compound 1 at ?max 370 nm (band I) indicates presence of
free 3-OH suggesting the attachment of sugar moiety at 7-
O-. On the other hand, compounds 2, 3 and 5 showed band
I UV absorption at ?max range 350-355 nm indicating
substituted 3- OH flavonol. So they were identified as
isorhamnetin-3-O neohesperidoside, quercetin-3-O-
rutinoside and quercetin-3-O- rhamnoside, respectively.
Peaks at m/z 479 and m/z 463 for isorhamnetin
neohesperidoside and rutin, respectively, were due to
sequential loss of terminal sugar moiety (rhamnose) before
loss of the other moiety (glucose) and give base peak at
m/z 315 and m/z 301 representing the two aglycones
respectively.
Limonoids
Furthermore, 12 limonoids were tentatively identified,
@Real Academia Nacional de Farmacia. Spain
viz: mexicanolides, phragmalins (as khayanolides) and
angolensate, based on exact masses, fragmentation
pathways, elemental constituents and phytochemical
dictionary of natural products database. The UV
absorptions at ?max 250-290 nm are due to the transition
interaction between two chromophores of the unsaturated
lactone and the furan ring in limonoid compounds (5). A
common feature was observed, in the mass fragmentation
of limonoids, which are characterized by the loss of a
major fragment m/z 44 to give a base peak referred to (M-
H-CO2)- resulting from ring cleavages of cyclic anhydride,
other common fragments were m/z (M-H-60)- and (M-H-
88)- resulting from loss of C2O2H4 and C3O3H4,
respectively. Mexicanolide type limonoid had
characteristic fragment m/z (M-H-28)-, due to the loss of
intramolecular carbonyl group.
Compounds were identified for the first time in K.
previously reported from other organs of K. senegalensis
such as pentahydroxy-oxo-tricyclomeliacate and
khayanone (mexicanolide type) have been reported before
from stem bark (14,15), while the acetyl derivative of the
former in addition to khayanolides and khayseneganins
(phragmalin type), have been previously isolated from
stem bark, leaves and twigs (5,16), furthermore, 2-
hydroxymexicanolide and destigloylswietenin have been
isolated from K. senegalensis seeds (17). On the other
hand, 6, 8-dihydroxycarapin have been isolated before
from K. anthotheca (18).
Acetone extract of K. senegalensis flowers showed
moderate activity against MCF7 (Breast carcinoma cell
line) compared to doxorubicin, with no activity on HCT
116 (Colon carcinoma cell line) and HEPG2 (liver cell
carcinoma). The results implied a selective cytotoxicity of
K. senegalensis (A.Juss.) flowers acetone extract , which is
in accordance with that reported for limonoids from Citrus
reticulata (19). However , Khaya senegalensis Bark
Extract was reported to inhibit growth of HT-29, HCT-15
and HCA-7 Colorectal cancer cells with IC50 (1.00 , 0.30 ,
0.22 µg/µl ) respectively (20). Moreover, K. senegalensis
flowers oil displayed cytotoxic activity against HepG-2,
MCF-7 and HCT-116 cell lines with LC50 (61.1, 79.7, 61
ppm) respectively (21).
Adebayo and Osman, (22) tested the antimicrobial
extract against Escherichia coli and Salmonella typhi, they
found the zone of inhibition = 20.10 and 17.10 mm
respectively. Preceding study reported the MIC values
were ranged from 390.63 to 1560 µg/mL for Khaya
senegalensis bark aqueous acetone extract (23). In fact, a
deferential cytotoxic and antimicrobial effect of flavonoids
and limonoids depend on several factors: Their chemical
structure, concentration, and also on the type of cancer cell
lines or the microorganisms revealing somewhat different
sensitivity toward different compounds (1,24- 26).
5. CONCLUSION
This is the first report of flavonoids and limonoids
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