Antiparasitic antioxidant phenylpropanoids and iridoid glycosides from Tecoma mollis

Article abstract of DOI:10.1016/j.fitote.2011.12.025

A radical scavenging guided phytochemical study on the stem bark of Tecoma mollis afforded seven active phenylpropanoid glycosides (1-7), including a new one (4), and one iridoid (8). The structures of the isolated compounds were elucidated on the basis of spectroscopic evidences and correlated with known compounds. Compounds (1-7) displayed promising antioxidant activity (DPPH assay) in relation to ascorbic acid (positive control). The antimicrobial activity for compounds (1-8) was evaluated against five bacterial and five fungal strains. The isolated compounds exhibited nonselective weak to moderate antimicrobial activity. The highest antileishmanial activity against Leishmania donovani was observed for compound (7) with an IC₅₀ value of 6.71 μg/ml, using pentamidine and amphotericin B as drug controls. Compound (5) exhibited moderate antimalarial activity (45% inhibition) against chloroquine sensitive (D6) clones of Plasmodium falciparum.

Full text of DOI:10.1016/j.fitote.2011.12.025

Fitoterapia 83 (2012) 500–507
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Antiparasitic antioxidant phenylpropanoids and iridoid glycosides from
Tecoma mollis
a
a
Wael M. Abdel-Mageed a, , Enaam Y. Backheet , Azza A. Khalifa ,
⁎
Zedan Z. Ibraheim a, Samir A. Ross b,c,⁎⁎
a
Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt
National Center for Natural Products Research, University of Mississippi, MS 38677, USA
Department of Pharmacognosy, School of Pharmacy, University of Mississippi, MS 38677, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A radical scavenging guided phytochemical study on the stem bark of Tecoma mollis afforded
seven active phenylpropanoid glycosides (1–7), including a new one (4), and one iridoid
(8). The structures of the isolated compounds were elucidated on the basis of spectroscopic
evidences and correlated with known compounds. Compounds (1–7) displayed promising
antioxidant activity (DPPH assay) in relation to ascorbic acid (positive control). The antimicro-
bial activity for compounds (1–8) was evaluated against five bacterial and five fungal strains.
The isolated compounds exhibited nonselective weak to moderate antimicrobial activity. The
highest antileishmanial activity against Leishmania donovani was observed for compound (7)
with an IC50 value of 6.71 μg/ml, using pentamidine and amphotericin B as drug controls.
Compound (5) exhibited moderate antimalarial activity (45% inhibition) against chloroquine
sensitive (D6) clones of Plasmodium falciparum.
Received 22 September 2011
Accepted in revised form 17 December 2011
Available online 8 January 2012
Keywords:
Tecoma mollis
Phenylpropanoid glycosides
Iridoid
Radical scavenging activity
Antileishmanial activity
Antimalarial activity
Antimicrobial activity
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
cultivated ornamentals [2,3]. The wood is widely used for tim-
bering, construction and in building of ships and for export due
Tecoma mollis Humb. and Bonpl., Family Bignoniaceae is an
upright shrub to a large tree attaining 7 m in height. The plant
is native to South United States, Mexico to Venezuela and
Argentina and is known by the following synonyms: Tecoma
velutina, Lindl.; and Tecoma stans var. velutina, Hort. [1]. The
plant has been introduced to Egypt in 1982 as an ornamental
plant for its timber and showy bell-shaped flowers and culti-
vated in the experimental station of Faculty of Agriculture,
Assiut Univ., Egypt. T. mollis is a member of genus Tecoma
which is of economic importance both for lumber and as
to their hardness and resistance for environmental conditions
[4–6]. To the best of our knowledge, little studies were focusing
on the phytochemistry and biological activity of T. mollis [7],
and this is the first study describing in details the chemistry
of the polar constituents as well as the potential biological
activities of T. mollis. The only reported phytochemical study
of this plant exhibited the isolation of triterpenoid as well as
flavonoid glycosides [7]. The reported biological studies of the
leaves of T. mollis include analgesic, antipyretic and CNS
depressant activities [7]. Also, the leaves of T. mollis have been
used in traditional medicine as hypoglycaemic due to its alka-
loidal contents [8,9]. In general, the genus Tecoma is rich in
phytoconstituents and previous studies revealed the isolation
and identification of many constituents of different chemical
classes as phenylpropanoids [10,11], iridoids [11–16], flavo-
noids [7, 17–21], alkaloids [22–24], triterpenoids [7], phenolics
[25] and indolic compounds [26]. Many biological activities
were also reported for this genus such as anti-inflammatory,
⁎
Correspondence to: W.M. Abdel-Mageed, Pharmacognosy Department,
Faculty of Pharmacy, Assiut University, Assiut, Egypt. Tel.: +20 146020352;
fax: +20 088 2332776.
⁎⁎ Correspondence to: S.A. Ross, Department of Pharmacognosy, School of
Pharmacy, University of Mississippi, MS 38677, USA. Tel.: +20 146020352;
fax: +20 088 2332776.
E-mail addresses: Waelabdelmageed@yahoo.com (W.M. Abdel-Mageed),
sross@olemiss.edu (S.A. Ross).
0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2011.12.025

W.M. Abdel-Mageed et al. / Fitoterapia 83 (2012) 500–507
501
hypoglycaemic, spasmolytic, diuretic, antimicrobial and for
protozoal diseases [27–35]. Other reported biological activities
include antioxidant, anti-proliferative, insecticidal and as as-
tringent for the treatment of diarrhea, dysentery and enteritis
[36–40].
residue (185 g, 18.5%) was mixed with 500 mL of distilled
H2O, and defatted with CHCl3. The aqueous layer was sub-
jected to DIAION HP20 column (25×1.5 cm) and eluted with
H2O, MeOH and Me2CO, successively. The MeOH eluant (31 g)
was subjected to medium pressure liquid chromatography
(MPLC) on silica gel column using CHCl3–MeOH mixtures in a
manner of increasing polarities. Hundred and five fractions
(100 mL each) were collected and monitored on TLC (silica
gel) using CHCl3–MeOH–H2O (80:20:0.5), (70:30:3) and
(55:40:5), as well as on RP-C18 using H2O–MeOH (70:30),
(50:50) and (30:70) as solvent systems and Anisaldehyde/
H2SO4 as a spraying reagent. Similar fractions were combined
to yield five groups.
In the course of our ongoing research activities toward the
isolation of biologically active compounds from plants growing
in Egypt either wild or cultivated, in particular the species of
diverse chemical constituents with various reported biological
activities, and as a part of our collaboration with the National
Center for Natural Products Research, School of Pharmacy,
University of Mississippi, USA, for differential antimicrobial
and antiparasitic screening, we had the opportunity to continue
the work on the stem bark of the cultivated T. mollis to investi-
gate its chemical constituents and potential biological activities.
In this study, we report the isolation of seven phenylpro-
panoid glycosides and one iridoid from the stem bark of
T. mollis, one of which (4) is a new phenylpropanoid glycoside.
All structures were established by spectroscopic analyses, as
well as using accurate mass measurement.
Group 1 (fractions 1–22, eluent: CHCl3–MeOH, 8:2) (3.6 g)
was re-chromatographed on RP-C18 column using
eluent MeOH–H2O (70:30) to provide seven sub-
fractions. Part (40 mg) of sub-fraction 1–3 (900 mg)
was purified by HPLC (Luna 5 μ C18 100 Å, 10 μm×
10 mm×250 mm) using
a gradient of 0–100%
CH3CN–H2O over 40 min to give compound (2)
(9.7 mg, yellow powder).
2. Materials and methods
Group 2 (fractions 23–60, eluent: CHCl3–MeOH, 7:3) (8.3 g)
was re-chromatographed on silica gel column using
EtOAc–MeOH in a manner of increasing polarities to
provide five sub-fractions. Part (65 mg) of sub-
fraction 2–4 (3.9 g) was purified by RP HPLC using a
gradient of 0–100% CH3CN–H2O over 40 min to afford
compound (1) (13.6 mg, yellow powder) and com-
pound (6) (19.2 mg, yellow powder).
Group 3 (fractions 61–78, eluent: CHCl3–MeOH, 7:3) (6.9 g)
was subjected to gel filtration chromatography
using a Sephadex LH-20 column with CHCl3–MeOH
(3:2) to provide nine fractions. Part (80 mg) of sub-
fraction 3–6 (3.4 g) was purified by RP HPLC using a
gradient of 0–100% CH3CN–H2O over 40 min to give
compound (5) (14.1 mg, yellow powder) and (4)
(16.4 mg, yellow powder).
Group 4 (fractions 79–96, eluent: CHCl3–MeOH, 6:4) (5.1 g)
was re-chromatographed on RP-C18 column using
5-70% aq. MeOH as eluent to provide six sub-
fractions. Part (60 mg) of sub-fractions 2–3 (1.8 g)
were purified by RP HPLC using a gradient of
0–100% CH3CN–H2O over 40 min to give compound
(3) (7.1 mg, yellow powder) and (7) (13.1 mg,
yellow powder).
2.1. General experimental procedures
IR spectra were measured on an Ati Mattson Genesis Series
FTIR machine. UV absorption was done on a UV–vis spectrom-
eter (Perkin-Elmer Instruments, Lambda 25).The 1D and 2D-
NMR experiments were recorded on Varian Unity INOVA
400 MHz NMR systems. Low resolution ESI-MS data were
obtained using a Perseptive Biosystems Mariner LC-MS, and
high-resolution ESI-MS data were obtained on a Finnigan
MAT 900 XLT system. HPLC separations were carried out
using a Phenomenex RP column (C18, 250×10 mm, 5 μm)
and an Agilent 1200 series gradient pump monitored using a
DAD G1315B variable-wavelength UV detector. Column
chromatography (CC) was performed using a silica gel
(Kieselgel 60 Å, 40–63 μM mesh size, Fluorochem, UK), size
exclusion chromatography (Sephadex LH-20, 25–100 mm
mesh size, SIGMA, Germany) and using TLC pre-coated silica-
gel 60 F254 (0.25 mm, ALUGRAM® SIL G/UV254, Macherey-
Nagel, Germany) and RP-18 F254S plates (0.25 mm, Merck,
Germany). All flash chromatography was performed on Biotage
cartridge flash columns (Si 40 M and C18 HS 40 M) using a
Biotage Flash system, Charlottesville, USA.
Group 5 (fractions 97–105, eluent: CHCl3–MeOH, 6:4)
(3.4 g) was re-chromatographed on Sephadex
LH-20 column with CHCl3–MeOH (1:1) to provide
seven sub-fractions. Part (50 mg) of sub-fractions
4–6 (826 mg) was purified by RP HPLC using a
gradient of 0–80% CH3CN–H2O over 40 min to give
compound (8) (16.8 mg, yellow powder).
2.2. Plant materials
The stem barks were collected in June 2005 from the trees
cultivated in the experimental station of Faculty of Agriculture,
Assiut Univ., Egypt. The plant was authenticated by Dr. Gamal
Taha, Dept. of Horticulture, Faculty of Agriculture, Assiut
Univ., Egypt. A voucher specimen (no. 2011TM) was deposited
at the Pharmacognosy Dept., Faculty of Pharmacy, Assiut Univ.,
Egypt.
2.4. Acid hydrolysis of isolated glycosides [41]
2.3. Extraction and isolation
Isolated glycosides (5 mg, each) were dissolved in 0.2 N
H2SO4 (5 mL) and heated at 95 °C for 1 h. After cooling, the
reaction mixture was extracted with Et2O (2 mL×3). The
aqueous layer was neutralized with NaHCO3, evaporated to
dryness, and extracted with pyridine. The pyridine extract
One kg of the air-dried powdered stem barks of T. mollis
were extracted by maceration in 70% EtOH (2 L×3). The
alcoholic extract was concentrated and the solvent free

502
W.M. Abdel-Mageed et al. / Fitoterapia 83 (2012) 500–507
was then analyzed on silica gel TLC (EtOAc–MeOH–H2O–
AcOH 13:3:3:4). Rhamnose (Rf 0.52), Apiose (Rf 0.48) and
glucose (Rf 0.32) were detected by comparison with authen-
tic samples.
was seen. The MFC/MBC is defined as the lowest test con-
centration that kills the organism (allows no growth on
agar).
2.6.2. In vitro antileishmanial assay
2.5. DPPH radical-scavenging assay
The in vitro antileishmanial activity was evaluated against
a culture of L. donovani promastigotes grown in RPMI 1640
medium supplemented with 10% GIBCO fetal calf serum at
26 °C [48,49]. A three-day-old culture was diluted to 5×105
promastigotes/ml. Drug dilutions (50–3.1 μg/ml) were pre-
pared directly in cell suspension in a 96-well plate, followed
by incubation (26 °C, 48 h). Growth of leishmanial promasti-
gotes was determined by the Alamar Blue assay (BioSource
International, Camarillo, CA) [50–52].
Firstly, radical scavenging activity of the isolated glyco-
sides against stable DPPH• was performed with a rapid TLC
screening method using 0.2% DPPH in MeOH. 30 min after
spraying, the active compounds appear as yellow spots
against purple background [42,43]. In a second experiment,
spectrophotometric assay was carried out according to the
method of Yen and Chen [44]. Briefly, 2.0 mL of a wide
range of concentrations (2.5–120 μM) of test sample (in
MeOH) was added to 2.0 mL of 100 μM of methanolic solu-
tion of DPPH. The mixture was vortexed for 1 min and then
left to stand at room temperature for 30 min in dark. When
DPPH• reacts with an antioxidant compound, it changes its
color from deep-violet to light-yellow which is measured at
517 nm on a UV/visible light spectrophotometer. Absorption
of blank sample containing the same amount of MeOH and
DPPH solution was prepared and measured daily. The exper-
iment was carried out in triplicate, using ascorbic acid as a
positive control. The percentage reduction of the DPPH, Q,
referred to “inhibition or quenching” was calculated by the
following formula [45]:
Standard fluorescence was measured by a Fluostar Galaxy
plate reader (excitation wavelength, 544 nm; emission
wavelength, 590 nm). Pentamidine and amphotericin
B
were used as the drug controls. Percent growth was calculat-
ed and plotted against the tested concentrations in order to
determine the IC50 and IC90 values.
2.6.3. In vitro antimalarial activity
The assay for antimalarial activity was based on the deter-
mination of plasmodial LDH activity. For the assay, a suspen-
sion of red blood cells infected with D6 strain of Plasmodium
falciparum (200 μL, with 2% parasitemia and 2% hematocrit in
RPMI 1640 medium supplemented with 10% human serum
and 60 μg/mL amikacin) was added to the wells of a 96-
well plate containing 10 μL of serially diluted test samples.
The plate was flushed with a gas mixture of 90% N2, 5% O2,
and 5% CO2 and incubated at 37 °C, for 72 h in a modular in-
cubation chamber (Billups-Rothenberg, CA). Parasitic LDH
activity was determined according to the procedure of Mak-
ler and Hinrichs [53]. Briefly, 20 μL of the incubation mixture
was mixed with 100 μL of the Malstat™ reagent (Flow Inc.,
Portland, OR) and incubated at room temperature for
30 min. Twenty microliters of a 1:1 mixture of NBT/PES
(Sigma, St. Louis, MO) was then added and the plate is further
incubated in the dark for 1 h. The reaction was then stopped
by the addition of 100 μL of a 5% acetic acid solution. The
plate was read at 650 nm. Artemisinin and chloroquine
were included in each assay as antimalarial drug controls.
IC50 values were computed from the dose response curves.
To determine the selectivity index of antimalarial activity of
compounds their in vitro cytotoxicity to mammalian cells
was also determined. The assay was performed in 96-well tis-
sue culture-treated plates. Vero cells (monkey kidney fibro-
blasts) were seeded to the wells at a density of 25,000 cells/
well and incubated for 24 h. Diluted samples were added
and plates were again incubated for 48 h. The number of via-
ble cells was determined by Neutral Red assay as described
earlier [54]. IC50 values were obtained from dose response
curves. Doxorubicin was used as a positive control for
cytotoxicity.
Qð% inhibitionÞ ¼ ½ðAB−AAÞ=ABꢀ ꢁ 100
where: AB—absorption of blank sample (t=0 min);
AA—absorption of tested extract solution (t=30 min).
2.6. Biological assays
2.6.1. Antimicrobial assay
All organisms are obtained from the American Type
Culture Collection (Manassas, VA). They include the fungi
Candida albicans ATCC 90028, Candida glabrata ATCC 90030,
Candida krusei ATCC 6258, Cryptococcus neoformans ATCC
90113, and Aspergillus fumigatus ATCC 90906 and the bacteria
Staphylococcus aureus ATCC 29213, methicillin-resistant S.
aureus ATCC 43300 (MRSA), Pseudomonas aeruginosa
ATCC 27853, Mycobacterium intracellulare ATCC 23068 and
Escherichia coli (ATCC 35218). Susceptibility testing was
performed for all organisms, except M. intracellulare, using
modified versions of the CLSI/NCCLS methods [46]. M.
intracellulare susceptibility was tested using a modified
Franzblau-method [47]. Samples dissolved in DMSO were
serially diluted in DMSO/saline (20%/0.9%) and transferred
in duplicate to 96-well flat bottom microplates. Microbial
inocula were prepared by correcting the OD630 of microbe
suspensions in incubation broth to afford final target inocu-
la. Controls [fungi: amphotericin B; bacteria: ciprofloxacin
(ICN Biomedicals, OH)] were included in each assay. All
organisms were read at 630 or 544(ex)/590(em)nm (M.
intracellulare and A. fumigatus) prior to and after incuba-
tion. Percent growth was plotted versus test concentration
to afford the IC50. Minimum fungicidal or bactericidal con-
centrations were determined by removing 5 μL from each
clear well, transferring to agar, and incubating until growth
Verpectoside
B (Crassoside) (3): yellow amorphous
powder (7.1 mg). [α]23 +133 (c 0.08, MeOH); UV: λmax
D
(MeOH): 218, 301, 348 nm; IR (KBr) vmax 3450, 1693, 1617,
1513 cm−1; HRESIMS m/z 809.24707 ([M+Na]+, calcd for
C
35H46NaO20,
809.24801, Δ=−1.0 ppm); 1H (400 MHz,
DMSO-d6) and 13C (100 MHz, DMSO-d6) NMR data: see
Table 1.

W.M. Abdel-Mageed et al. / Fitoterapia 83 (2012) 500–507
503
Table 1
1H (400 MHz) and 13C-NMR (100 MHz) data of 3 and 4 in DMSO-d6.
No.
3
3
4
4
δ1H/ppm, mult, J/Hz
δ13C/ppm, mult.
δ1H/ppm, mult, J/Hz
δ13C/ppm, mult.
Aglycone
1
2
3
4
5
6
7
8
–
129.1, s
116.3, d
145.0, s
143.5, s
115.5, d
119.6, d
35.0, t
–
129.5, s
116.3, d
145.0, s
143.5, s
115.5, d
119.5, d
35.0, t
6.62 (1H, brs)
–
6.61 (1H, d, 1.8)
–
–
–
6.62 (1H, d, 8.0)
6.49 (1H, d, 8.0)
2.68 (2H, t, 7.8)
a. 3.88 (1H, m)
b.3.62 (1H, m)
6.62 (1H, d, 8.0)
6.49 (1H, dd, 1.8, 8.0)
2.69 (2H, m)
a. 3.82 (1H, m)
b.3.63 (1H, m)
70.3, t
70.3, t
Sugar 1
Glucose
Glucose
101.3, d
81.1, d
77.7, d
69.2, d
74.3, d
60.8, t
Glucose
Glucose
101.2, d
80.6, d
77.2, d
68.8, d
70.7, d
62.5, t
1′
2′
3′
4′
5′
6′
4.57 (1H, d, 7.8)
3.56 (1H, m)
3.86 (1H, m)
4.77 (1H, m)
3.50 (1H, m)
a. 3.38 (1H, m)
b. 3.34 (1H, m)
–
4.65 (1H, d, 7.2)
3.62 (1H, m)
3.91 (1H, m)
4.90 (1H, m)
3.81 (1H, m)
a. 4.03 (1H, dd, 5.0, 12.0)
b. 3.95 (1H, m)
–
C_O
CH3
–
–
170.1, s
20.6, q
–
1.96 (3H, s)
Caffeoyl moiety
1″
2″
3″
4″
5″
6″
7″
8″
–
125.5, s
114.7, d
145.5, s
148.5, s
115.8, d
121.4, d
145.6, d
113.6, d
165.7, s
–
125.9, s
114.8, d
145.6, s
148.5, s
115.8, d
121.5, d
145.8, d
113.2, d
165.6, s
7.00 (1H, brs)
–
7.02 (1H, brs)
-
-
6.76 (1H, d, 8.1)
6.98 (1H, d, 8.1)
7.46 (1H, d, 15.9)
6.19 (1H, d, 15.9)
–
-
6.75 (1H, d, 8.0)
6.98 (1H, d, 8.0)
7.46 (1H, d, 16.0)
6.20 (1H, d, 16.0)
–
C_O
Sugar2
1″′
2″′
3″′
4″′
Rhamnose
Rhamnose
101.3, d
70.4, d
70.4, d
71.8, d
Rhamnose
Rhamnose
101.1, d
70.4, d
70.2, d
71.7, d
5.03 (1H, brs)
3.81 (1H, m)
3.27 (1H, m)
3.11 (1H, m)
3.34 (1H, m)
0.96 (3H, d, 6.2)
5.02 (1H, brs)
3.81 (1H, m)
3.27 (1H, m)
3.11 (1H, m)
3.34 (1H, m)
0.98 (3H, d, 6.2)
5″′
6″′
68.9, d
18.2, q
69.0, d
18.2, q
Sugar 3
1″″
2″″
3″″
4″″
Glucose
Glucose
101.8, d
74.3, d
77.1, d
69.7, d
77.1, d
61.0, t
Glucose
Glucose
101.8, d
74.3, d
77.1, d
69.7, d
77.1, d
60.9, t
4.54 (1H, d, 7.5)
2.99 (1H, t, 7.6)
3.12 (1H, m)
3.11 (1H, m)
3.16 (1H, m)
a. 3.68 (1H, m)
b. 3.52 (1H, m)
4.53 (1H, d, 7.5)
2.99 (1H, t, 7.6)
3.12 (1H, m)
3.12 (1H, m)
3.16 (1H, m)
a. 3.68 (1H, m)
b. 3.51 (1H, m)
5″″
6″″
Tocomoloside (6′-O-acetyl verpectoside B) (4): yellow
Their structures were elucidated by extensive 1D, 2D NMR
analysis, accurate mass measurements and by comparing
with the reported data.
Six known phenylpropanoids (1–3, 5–7) were identified as
acteoside (Verbascoside) (1) [55,56], 6′-O-acetylverbascoside
(2) [57], verpectoside B (Crassoside) (3) [58], luteoside A (5)
[41,59], isoacteoside (isoverbascoside) (6) [57,60], luteoside B
(7) [41,61] and one iridoid, ixoside (8) [61–63]. All physical
and spectral data of these compounds were in agreement
with the respective published data.
amorphous powder (16.4 mg). [α]23 +116.3 (c 0.2,
D
MeOH
MeOH); UV:λ
: 220, 291, 345 nm ; IR (KBr) vmax 3433,
max
1730, 1688, 1624, 1523 cm−1; HRESIMS m/z 851.25623 ([M+
Na]+, calcd for C37H48NaO21, 851.25858, Δ=−3.0 ppm); 1H
(400 MHz, DMSO-d6) 13C (100 MHz, DMSO-d6) NMR data: see
Table 1.
3. Results and discussion
A combination of flash or medium-pressure liquid chro-
matography (MPLC), gel filtration and HPLC of the MeOH
fraction obtained from the stem bark of T. mollis afforded
seven phenylpropanoids (1–7) and one iridoid (8) (Fig. 1).
Compound 4 was obtained as a yellow amorphous powder
([α]23D +116.3 (c 0.2, MeOH)). HRESIMS showed pseudomo-
lecular ion peaks at m/z 829.27435 [M+H]+ and 851.25623
[M+Na]+ consistent with a molecular weight of 828 amu.

504
W.M. Abdel-Mageed et al. / Fitoterapia 83 (2012) 500–507
Fig. 1. Chemical structures of isolated compounds 1–8 (Glc = Glucose, Api= Apiose).

W.M. Abdel-Mageed et al. / Fitoterapia 83 (2012) 500–507
505
Table 2
Phenylpropanoid glycosides are water soluble natural
products widely distributed in the plant kingdom. Their signif-
icant biological activities such as enzyme inhibition, immuno-
modulatory, antibacterial and cytotoxic activities have been
reported [64]. Recently, phenylpropanoid glycosides were
demonstrated in vitro assays to have antioxidative and free rad-
ical scavenging activities [65–70]. The antioxidant activity of
the isolated phenylpropanoids (1–7) was determined by
using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical
scavenging system. The IC50s ranged from 9.8 to 16.8 μM
(Table 2). The antioxidative effect of these compounds was re-
lated to the number of free phenolic hydroxyl groups in the
form of 3,4-dihydroxy (catechol) moiety in their structures
which explain the close similarity of their antioxidant activity.
Absence or blocking of the hydroxyl groups by methyl group
leads to dramatic decrease of the antioxidant activity [71].
The sequence of the strength of the antioxidant activity was
shown to be 1>3>2>6>5>4>7. The iridoid glycoside
(ixoside) (8) exhibited very weak antioxidant activity with
IC50=109.2 μM. The free radical scavenging effects of the phe-
nylpropanoids proven here may play an important role in the
actions of the Tecoma species, and partly explain the mecha-
nisms of the activities of phenylpropanoids against neoplasma
[66,72] and inflammation [72,73] in which free radical are seri-
ously involved.
The antifungal and antibacterial activity for the isolated
compounds was evaluated against Gram positive, Gram neg-
ative bacteria and fungi. The percentage of inhibition (% inh.)
of each strain was determined and compared with those for
commercially available antibiotics (Table 3). In general, weak
activity was observed for isolated compounds against fungal
strains particularly A. fumigatus ATCC 90906. The highest activ-
ity was observed for compounds (6) and (7) with inhibition
of 30% and 40% respectively. Similar was observed against
Gram +ve and −ve bacteria with highest inhibition occurred
for Mycobacterium intracellulare by compound (7).
DPPH radical scavenging activity of fractions and isolated compounds.
Sample
IC50 in μg/ml (μM)
Total ext.
99.3
>200
52.8
39.6
18.7
28.8
14.1
128.6
6.1 (9.8)
7.5 (11.2)
8.6 (10.9)
12.8 (15.5)
11.7 (14.6)
8.5 (13.7)
12.7 (16.8)
42.4 (109.2)
2.1 (11.9)
Chloroform fraction
Hydro methanolic fraction
Group 1
Group 2
Group 3
Group 4
Group 5
Verbascoside (1)
6′-O-acetylverbascoside (2)
Verpectoside B (3)
Tocomoloside (4)
Luteoside A (5)
Isoverbascoside (6)
Luteoside B (7)
Ixoside (8)
Ascorbic acid
The molecular formula was established as C37H48O21, thus
implying 14 degrees of unsaturation. The UV:λ
MeOH
: 220, 291,
max
345 nm, while IR spectrum showed strong absorption bands
attributed to the hydroxyl and ester groups at 3433 and 1730,
1688 cm−1, respectively. The 1D and 2D NMR spectra in
DMSO-d6 (Table 1) of 4 revealed the phenylpropanoid glyco-
sides pattern and were very similar to those of 3[58]. The 1H
NMR spectral data showed the presence of two sets of ABX
system for β-(3,4-dihydroxyphenyl) ethoxy moiety and
caffeoyl moiety, as well as two trans-olefinic protons, three
anomeric protons for two β-glucopyranosyl and one α-
rhamnopyranosyl units, and an acetyl signal. The 1H and 13C
NMR spectra were very similar to those of 3 except for the ad-
ditional signal of acetyl group (δH 1.96 for COCH3), (δC 20.6 for
CH3) and (δC 170.1 for CO). Comparison of the 13C NMR spectral
data of 4 with those of 3 (Table 1) revealed downfield shift in
the position of C-6′ (+1.7 ppm) and upfield shift of C-5′
(3.6 ppm) of the glucosyl moiety indicating that the acetyl
group is at C-6′ of the glucosyl moiety. Furthermore, the
HMBC spectrum showed the three bond correlation between
H-6′ (δH 3.95 and 4.03) of the glucosyl moiety and carbonyl
carbon (δC 170.1) of the acetyl group. Acid hydrolysis of 4
yielded sugars rhamnose and glucose. Therefore, the structure
of compound 4 was elucidated as 6′-O-acetyl-verpectoside B.
Compound 4 was identified as a new natural product for
which we propose the name tocomoloside.
Compounds (1–8) were tested against the protozoan para-
site L. donovani, using pentamidine and amphotericin B as drug
controls (Table 4). The highest in vitro antileishmanial activity
was observed for compounds 1, 5, and 7 with percentage of
inhibition [83% (IC50 30.08), 92% (IC50 15.07) and 92% (IC50
6.71), respectively] (Table 4). Compounds 2 and 6 showed rel-
atively moderate activity with percentage of inhibition [62%
(IC50 >40), and 78% (IC50 >40), respectively], while the least
active compounds are 3, 4 and 8 with percentage of inhibition
(19%, 21% and 13%, respectively). It is obvious from Table 4
Table 3
Antimicrobial activity of isolated compounds (1–8) against different bacteria and fungi.
Compound Compound S. aureus MRS
E. coli P. aeruginosa M. intracellulare C. Albicans C. glabrata C. krusei A. fumigatus C. neoformans
number
conc.
% inh.
% inh. % inh. % inh.
% inh.
% inh.
% inh.
% inh.
% inh.
% inh.
(μg/ml)
1
2
3
4
5
6
7
8
13
13
13
13
13
13
13
13
7
11
9
5
0
5
8
0
8
10
1
6
14
10
2
5
3
2
6
0
6
3
4
0
4
8
6
0
4
16
6
16
9
21
7
11
2
14
6
12
5
8
5
1
9
0
11
1
0
0
1
4
0
8
0
14
3
6
12
4
11
6
8
11
10
9
8
9
11
14
17
30
41
0
7
1
33
6
8
10

506
W.M. Abdel-Mageed et al. / Fitoterapia 83 (2012) 500–507
Table 4
of compounds to use as antileishmanial drugs. Moderate anti-
malarial activity was observed with compound 5 against chlo-
roquine sensitive (D6) clones of P. falciparum.
In vitro antileishmanial activity of isolated compounds (1–8).
Compound
Number
Compound
conc.
L. donovani
% inh.
IC50
(μg/ml)
IC90
(μg/ml)
(μg/ml)
Acknowledgments
1
2
3
4
5
6
7
8
80
80
80
80
80
80
80
80
80
80
83
62
19
21
92
78
92
13
100
100
30.08
>40
ND
>40
>40
ND
The authors are thankful to Dr. Melissa Jacobs and Ms.
Marsha Wright for the antimicrobial testing and to Dr. Babu
Tekwani for the antileishmanial assay, and Dr. Shabana
khan for the antimalarial assay.
ND
ND
15.07
>40
6.71
ND
0.34
1.37
>40
>40
22.28
ND
0.43
1.62
Amphotericin Ba
Pentamidinea
Appendix A. Supplementary data
IC50
:
concentration causing 50% growth inhibition. IC90
:
concentration
Supplementary data to this article can be found online at
doi:10.1016/j.fitote.2011.12.025.
causing 90% growth inhibition.
ND: not determined. a: control.
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