ORIGINAL ARTICLES
a Finnigan LTQ FT Ultra mass spectrometer (Thermo Fisher Scientific, Bremen,
the crude extract and higher activities of the isolated compound 14,
4.56 0.82 μg/ml. These values are lower than that of the positive
control Trolox which had an ED50 of 27.0 13.41 μg/ml.
Germany) equipped with a Nanomate ESI interface (Advion Biosystems, USA). An
electrospray voltage of 1.7 kV (+/−) and a transfer capillary temperature of 200 °C
were applied. Collision induced dissociation (CID) was performed in the ion trap using
a normalized collision energy of 35 %, activation time of 30 ms, 0.25 activation Q and
a precursor ion isolation width of 2 amu. High resolution product ions were detected in
the Fourier transform ion cyclotron resonance (FTICR) cell of the mass spectrometer.
ORAC measurements were performed on a FLUOstar Omega Microplate Reader -
BMG LABTECH. UVrecording was made on a Shimadzu UV–Visible-1601 spectro-
photometer and optical rotation was obtained on a Kruess optronic Polarimeter. Paper
chromatographic analysis (PC) was carried out on Whatman No. 1 paper, using solvent
systems: (1) H2O; (2) 6% HOAc; (3) BAW (n-BuOH–HOAc–H2O, 4:1:5, upper layer)
2.3.2. Cytotoxicity against tumor cell lines, NRU assay
NRU assay was used to assess the cytotoxicity of the Melaleuca
extract and the new isolate 14 against three different solid tumor
cell lines. The IC50 values for cytotoxicity of the extract and the
isolated compound 14 are given in Table 2. The vehicle in which
the test samples were dissolved had no influence on measured
parameters. Using etoposide (positive control for cytotoxicity)
viability of the three tested cell lines was reduced to 40 to 60%.
The extract and the novel compound showed the highest cyto-
toxic activity against colon cancer cell line HCT 116 with IC50 of
83.08 0.71μg/mL and 24.08 1.57 μg/mL respectively.
3.2. Plant material
Leaves of Melaleuca leucadendra were collected from the Zoo garden at Cairo, on
September, 2016 and identified by Prof. Salwa Kawashty at the Department of Phyto-
chemistry and Plant Systematic, National Research Centre (NRC), Cairo, Egypt.
A voucher specimen (M 1732) has been deposited at the herbarium of the NRC.
Table 2: Cytotoxicity (IC50 μg/ml) of the extract of Melaleuca leuca-
dendra and the new isolate 14
3.3. Preparation of extract
Fresh leaf material (2 kg) was extracted with hot EtOH/H2O (3:1, 3 times, each with 3
l, for 8 h, under reflux). The solvent was evaporated under vacuum. The resulting dry
sticky material thus left (200 g), dissolved in 200 ml H2O, was applied to a Polyamide
6S chromatographic column (Riedel-de Haen, Seelze, Hannover, Germany) and
eluted with H2O, followed by H2O/MeOH mixtures of decreasing polarity to yield
ten major fractions (I – X). Following removal of the solvents the received dried ten
fractions were as follow: fraction I eluted with H2O; II with 10 %, III with 20 %,
IV with 30 %, V with 40 %, VI with 50 %, VII with 60 %, VIII with 70 %, IX with
80 % and X with MeOH). The fractions individually collected were subjected to two
dimensional paper chromatography (TDPC).
Liver carcinoma
cell line (Huh-7)
Breast cancer
MCF-7
Colon cancer
HCT 116
104 3.55 102.61 5.91 83.08 0.71 Extract
35.76 2.51 31.08 3.83 24.08 1.57 Myricetin 3-O--β-4C1-ga-
lacturonide
3.4. Isolation and identification of phenolics
Compound 1 (37 mg) was purely isolated from fraction II (2.5 g, eluted with H2O) by
repeated column fractionation over MCI-gel, using H2O for elution followed by prep.
PC for subfraction 3, using BAW (n-BuOH–HOAc–H2O, 4:1:5, upper layer) as solvent.
Compound 2 (108 mg) was separated pure from fraction 3 (2.6 g eluted with 20 % aqueous
MeOH) by Sephadex LH-20 column fractionation, using n-BuOH water saturated with
H2O for elution, followed by crystallization from H2O. Compound 3 was isolated from
fraction IV (8.17 g, eluted with 40 % aqueous MeOH) by MCI gel column fractionation,
using H2O as solvent, followed Prep. PC, using BAW as solvent. Compound 4 (29 mg)
was purely isolated from 1.3 g of fractionV (eluted by 40 % aqueous MeOH) by extraction
by EtOAc, the residue dissolved in ethanol, filtered and the dried filtrate was applied
on MCI gel column eluted with 50 % aqueous MeOH and the eluate was then purified
over Sephadex LH-20 using 50 % aqueous MeOH as solvent to afforded pure sample of
compound 5 (24 mg). FractionVI (1.56 g, eluted with 60 % aqueous MeOH) was fraction-
ated into H2O and MeOH sub-fractions by being applied to MCI gel column. The MeOH
sub-fraction was dried and extracted with ether. The ether contained pure compound
6 (23 mg). The residue was subjected to prep. PC using 6 % aqueous acetic acid as solvent,
whereby, compounds 7 (18 mg), 8 (21 mg), 9 (17 mg), 10 (30 mg) were individually sepa-
rated. Fraction 7 (1.56 g, eluted with 70 % aqueous MeOH) was applied to a Sephadex
LH-20 column, eluted with n-BuOH saturated with H2O and the eluted dark purple band
(under UV) was dried under vacuum. Prep. PC of the received dried material using 6 %
aqueous acetic acid afforded a pure sample of compound 11 (19 mg). Compound 12
(20 mg) was isolated pure from fraction 8 (1.5 g, eluted with 80 % aqueous MeOH) by
prep. PC and elution with 6 % aqeous acetic acid. Prep. PC of fraction IX (1.8 g, eluted with
90 % aqueous MeOH) using 30 % aqueous acetic acid as solvent yielded another amount
of pure sample of compound 12 (34 mg) beside pure sample of 13 (22 mg). Fraction X
(1.5 g, eluted with MeOH) was exhaustively extracted with ether, the ether was filtered on
and removed under vacuum. The ether insoluble portion was subjected to repeated prep.
PC (3 times), using 6 % aq, acetic acid to yield a chromatographically pure sample of
14 (30 mg). Application of the received ether extract on a silica gel column and elution
with n-hexane:EtOAC (1:1) afforded pure samples of 15 (16 mg) and 16 (12 mg).
Myricetin 3-O-β-4C1-galactopyranuronoide (14): A faint yellow amorphous powder; [α]
D25 -0.79 (c, 0.15 in MeOH); Rf-values: 0.50 (H2O), 0.26 (6 % HOAc), 0.24 (BAW). UV
λmax nm in MeOH: 266, 267, 295, 36; NaOAc: 235, 274; NaOAc–H3BO3: 260, 302, 386;
AlCl3: 270, 303, 393; Complete acid hydrolysis of 14 (11 mg in 5 ml, 2 N aq. HCl, at 100 °C
for 2 h) yielded myricetin and galacturonic acid; Controlled acid hydrolysis (8 mg, 10%
aq. AcOH, 30 min, 100 °C) yielded only myricetin at the end; HRESI-FTMS (negative
ions) of 14: [M – 1]- : m/z = 493.2628 corresponding to a molecular mass of 494 and a
molecular formula of C21 H17 O14, as has been established by HRESIMS (calc.: 493.3513)
in negative mode. 1-D and 2-D 1H and 13C NMR data: Table 1.
2.3.3. Determination of phagocytic activity
Heparinized (10 IU/ml) rat blood grouped samples were centrifuged
at 3000 rpm for 10 min, then the buffy coat was aspirated and care-
fully layered on histopaque neutrophil isolation medium (Biowest),
pH 7.3 by a ratio 1:3 in a siliconized centrifuge tube and the gradient
was centrifuged at 990 rpm for 25 min at 4 °C. The pellet containing
neutrophils and RBCs was transferred into 50 ml sterile falcon tube.
A total of 20 ml sterile, cold distilled water was added and let stand
for, exactly 45 s to lyse RBCs. Isotonicity was restored by the addi-
tion of 10 ml of sterilized normal saline, and then the content was
centrifuged at 3000 rpm for 10 min. The pellet was washed 3 times
by Hanks balanced salt solution (HBSS) and re-suspended in 1 ml
and kept at 4 ºC until used (Hogan et al. 1990).
Twenty four hours tryptic soy broth culture of Staphylococcus
aureus was adjusted to 52.5% transmission as each 1 ml contained
5x107 to 108 CFU. The broth containing the bacteria was centri-
fuged at 3000 rpm for 10 min and the pellet was washed twice with
HBSS. The bacteria were opsonized with 1 ml of 10% homologous
serum (collected from 5 different rat sera) for 30 min at 37 ºC with
gentle shaking. After opsonization, the bacteria were centrifuged,
washed once and suspended in 1ml HBSS (Silva et al. 1988).
Ten-microliter volumes of bacterial suspensions were added to neutro-
phil phagocytic cells in a 10:1 ratio obtained from different concen-
tration plant extract groups in 1 ml of HBSS and incubated for 2 h at
37 °C under rotation. Subsequently, the cells were centrifuged for
4 min at 110 x g and the supernatant was removed. The pellet was
streaked on clean glass slides, and stained by the addition of 200 μl
of acridine orange (15 mg/liter) for 1 min. The slide preparations
were examined under Axio Imager Z2 fluorescence microscope using
transmitted light. Photographs were taken using AxioCam MRc3
S/N 4299 film with the shutter set for 2 min (Nagl et al. 2002). These
results proved that Melaleuca leucadendra extract led to a significant
increase in phagocytic index of neutrophils by increasing the dose.
These findings suggest an immunostimulant behavior.
3.5. Biological assays
3.5.1. Radical scavenging effect, DPPH assay
3. Experimental
The estimation was done according to the method of Brand-Williams and Cuvelier
(1995). DPPH, a stable radical, is reduced after reaction with an antioxidant compound
and its absorbance at 517 nm is than reduced. The reaction mixture contained 500 l of
test extract, 375 μl ethanol and 125 μl of a 1 mM freshly prepared DPPH solution in
ethanol. Different concentrations of test samples were prepared while the final concen-
tration of DPPH in the reaction mixture was 0.125 mM. After incubation of the mixture
at 37 °C for 30 min in the dark, the absorbance was measured at 517 nm. Blank samples
contained the same amount of methanol and DPPH solution. All experiments were
3.1. General
NMR spectra were acquired in DMSO-d6 on a Bruker Avance 400 NMR spectrometer,
at 400 MHz. Standard pulse sequence and parameters were used to obtain 1D- 1H and
13C APT, and 2D- HSQC and HMBC spectra. 1H chemical shifts (δ) were measured in
ppm, relative to TMS and 13C NMR chemical shifts to DMSO-d6 and were converted
to TMS scale by adding 39.49. High resolution ESI mass spectra were measured using
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