Iridoid and phenylpropanoid glycosides from the roots of Lantana montevidensis

Article abstract of DOI:10.1007/s00044-017-1817-x

A new iridoid glycoside; 6-O-β-D-xylopyranoside-shanzhiside methyl ester (1) along with six known compounds; shanzhiside methyl ester (2), lamalbid (3), geniposidic acid (4), theveside (5), verbascoside (6) and arenarioside (7) were isolated from the roots of Lantana montevidensis. The structures of the compounds were determined through 1D and 2D NMR spectroscopic data analysis, HRESIMS, electronic circular dichorism and UPLC-UV/MS method. The total extract, chloroformic (F1) and aqueous (F2) fractions together with the isolated compounds were tested for their antimicrobial, antiprotozoal, antiplasmodial, anti-inflammatory, monoamine oxidase inhibition and cell viability activities in addition to free radical scavenging activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The phenylpropanoid compounds (6 and 7) resulted in a potent antioxidant activity. Total methanolic extract together with the aqueous fraction (F2) showed decrease in reactive oxidative stress with 57 and 66%, respectively, while the chloroformic fraction (F1), together with the total methanolic extract, showed a decrease in iNOS with IC₅₀ values 5 and 30 μg/mL, respectively. Compounds 1, 2, 3, 6, and 7 showed inhibition in the reactive oxidative stress with values 50, 60, 57, 63, and 52%, respectively. Both F1 and F2 fractions demonstrated measurable inhibition of MCF-7 breast cancer cell growth, with IC₅₀ value 0.3 mg/mL. Compounds 2 and 7 showed mild monoamine oxidase inhibition. None of the tested compounds showed antimicrobial, antiplasmodial or antiprotozoal activity.

Full text of DOI:10.1007/s00044-017-1817-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1817-x
ORIGINAL RESEARCH
Iridoid and phenylpropanoid glycosides from the roots of Lantana
montevidensis
Nesma M. Mohamed1,2 Makboul A. Makboul2 Salwa F. Farag2
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Amer H. Tarawneh3,4 Shabana I. Khan1,3 Tracy A. Brooks3 Yan-Hong Wang1
Samir A. Ross1,3
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Received: 19 October 2016 / Accepted: 16 February 2017
© Springer Science+Business Media New York 2017
Abstract A new iridoid glycoside; 6-O-β-D-xylopyrano-
side-shanzhiside methyl ester (1) along with six known
compounds; shanzhiside methyl ester (2), lamalbid (3),
geniposidic acid (4), theveside (5), verbascoside (6) and
arenarioside (7) were isolated from the roots of Lantana
montevidensis. The structures of the compounds were
determined through 1D and 2D NMR spectroscopic data
analysis, HRESIMS, electronic circular dichorism and
UPLC-UV/MS method. The total extract, chloroformic (F1)
and aqueous (F2) fractions together with the isolated
compounds were tested for their antimicrobial, anti-
protozoal, antiplasmodial, anti-inflammatory, monoamine
oxidase inhibition and cell viability activities in addition to
free radical scavenging activity using the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay. The phenylpropanoid com-
pounds (6 and 7) resulted in a potent antioxidant activity.
Total methanolic extract together with the aqueous fraction
(F2) showed decrease in reactive oxidative stress with 57
and 66%, respectively, while the chloroformic fraction (F1),
together with the total methanolic extract, showed a
decrease in iNOS with IC50 values 5 and 30 μg/mL,
respectively. Compounds 1, 2, 3, 6, and 7 showed inhibition
in the reactive oxidative stress with values 50, 60, 57, 63,
and 52%, respectively. Both F1 and F2 fractions demon-
strated measurable inhibition of MCF-7 breast cancer cell
growth, with IC50 value 0.3 mg/mL. Compounds 2 and 7
showed mild monoamine oxidase inhibition. None of the
tested compounds showed antimicrobial, antiplasmodial or
antiprotozoal activity.
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Keywords Lantana Iridoids Phenylpropanoids Anti-
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inflammatory Anticancer Reactive oxidative stress
Introduction
The Verbenaceae family is comparatively a large family of
mainly tropical flowering plants. It comprises about 100
genera and 2000 species distributed in tropics and sub-
tropics, mainly in temperate zone of Southern Hemisphere
(Lawrence 1951; Datta 1970 and Core 1955). The Verbe-
naceae plants are well known for their uses in the traditional
medicinal systems of various countries. Quite a number of
the plants have been reported to contain bio-active phyto-
chemicals with important pharmacological effects. Among
these plants Lantana montevidensis (Spreng.) Briq, a plant
that is popularly known as “chumbinho”. It is a fast
spreading, creeping, perennial herb that is native to Brazil
and Uruguay. It was introduced as an ornamental plant and
is considered to be invasive species in many parts of the
world. The plant has various potential medicinal uses
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1817-x) contains supplementary material,
which is available to authorized users.
* Samir A. Ross
sross@olemiss.edu
1
National Center for Natural Products Research, Research Institute
of Pharmaceutical Sciences, School of Pharmacy, The University
of Mississippi, University, Oxford, MS 38677, USA
2
Pharmacognosy Department, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt
3
Department of BioMolecular Sciences, School of Pharmacy,
University of Mississippi, University, Oxford, MS 38677, USA
4
Department of Chemistry, Tafila Technical University, P.O. Box
179, Tafila, Jordan

Med Chem Res
including treatment of fever, influenza, asthma, and bron-
chitis (Munir 1996; Nagao et al. 2002; Barreto et al. 2010;
Montanari et al. 2011; Sousa et al. 2011). In South America,
it is employed in native medicine for the treatment of
broncho-pulmonary diseases, headaches and sunstroke
(Munir 1996; Nagao et al. 2002; Barreto et al. 2010;
Montanari et al. 2011; Sousa et al. 2011). The plant also
was subjected to a number of phytochemical studies that
included investigation of the volatile oils of the leaves and
flowers (Makboul et al. 2013a), and isolation of pentacyclic
triterpenoids and flavonoids together with the assessment of
different leaf and root extracts and isolated compounds for
their biological activities such as free radical scavenging,
antimicrobial, antiprotozoal, anti-inflammatory, analgesic,
and antipyretic activities (Makboul et al. 2013a, b; Makboul
et al. 2014; Mohamed et al. 2016). In the current study, we
report herein the isolation of one new iridoid glycoside; 6-
O-β-D-xylopyranoside-shanzhiside methyl ester (1), along
with six other known compounds: shanzhiside methyl ester
(2), lamalbid (3), geniposidic acid (4), theveside (5), ver-
bascoside (6), and arenarioside (7) from the methanolic
extract of L. montevidensis. The antimicrobial, anti-
protozoal, antiplasmodial, anti-inflammatory, anticancer,
monoamine oxidase inhibition, and free radical scavenging
activities were assessed. Mentioned in the Results and dis-
cussion section
(PDA) detector, and single quadrupole detectors (SQD).
Column chromatography was carried on Silica gel G60
(60–120 mesh, Merck, Darmstadt, Germany), Sephadex
LH-20 (Mitsubishi Kagaku, Tokyo, Japan). Analytical TLC
was conducted on precoated aluminum sheets silica 60
F254, 0.25 mm (E-Merck, Darmstadt, Germany).
Plant material
The roots of Lantana montevidensis (Spreng.) Briq. were
collected during the flowering stage in the period of
February–November 2013 from the gardens of Assiut
University, Assiut, Egypt. The plant was authenticated and
identified by the late Prof. N. E. Keltawy, Professor of
Ornamental, Horticulture, and Floriculture, Faculty of
Agriculture, Assiut University, Assiut, Egypt. A voucher
sample (No. 2009 LM) has been deposited at the Herbarium
of Pharmacognosy Department, Faculty of Pharmacy,
Assiut University, Assiut, Egypt.
Extraction and isolation of constituents
The air-dried powdered roots of L. montevidensis (1.5 Kg)
were exhaustively extracted using 70% MeOH (5L × 4) by
maceration at room temperature. The methanolic extract
was concentrated under reduced pressure to afford a dark
brown viscous residue (112 g) which was suspended in
distilled water (200 mL) and partitioned using CHCl3
(1L × 4) to yield two major fractions, the chloroformic
fraction F1 (40 g) and aqueous fraction F2 (70 g). About
40 g of the aqueous fraction was subjected to Diaion HP-20
CC which was eluted with H2O then MeOH to yield two
fractions F-2-1 (13.2 g) and F-2-2 (24.8 g), respectively.
The methanolic subfraction (F-2-2) was subjected to VLC
on silica gel (900 g) which was eluted using CHCl3−MeOH
mixture in the order of increasing polarities to yield three
fractions FA-FC. FA (7.3 g) was subjected to silica gel (200
g) CC using EtOAc−MeOH as eluent in gradient elution.
Fractions eluted with EtOAc−MeOH (8:2) were collected
and concentrated to yield fraction FA-1 (1.2 g). Fraction
FA-1 was furthermore purified using Sephadex LH-20 (100
g) CC using MeOH as eluent to yield two main fractions
FA-1-1 which was collected and concentrated to yield
compound 2 (50 mg) and fraction FA-1-2 (750 mg). Frac-
tion FA-1-2 was subjected to silica gel (30 g) CC using
CHCl3−MeOH as eluent in gradient elution. The fractions
eluted with CHCl3−MeOH (8.5:1.5) were collected and
concentrated to yield compound 6 (100 mg), while the
fractions eluted with CHCl3−MeOH (7.5:2.5) were col-
lected and concentrated to yield compound 7 (33 mg). FB
(6.3 g) was subjected to silica gel (200 g) CC using
CHCl3−MeOH as eluent in gradient elution. Fractions
eluted with CHCl3−MeOH (9:1) were collected and
Experimental
General procedures
Specific rotations were measured using a Rudolph Research
AutoPol IV polarimeter at room temperature; UV spectra
were recorded by a Hewlett-Packard 8452 A UV−Vis
spectrometer; IR spectra were acquired using Bruker Tensor
27 and MIRacle ATR FT−IR spectrometers (Bruker
Optics) and reported in wave number cm−1. All NMR
experiments were carried out on a Varian—Mercury AS
400 MHz NMR spectrometer (Varian Inc., USA). The
chemical shifts values are reported in ppm (δ) and the
coupling constants (J) are presented in Hertz. The spin
multiplicities are given as singlet (s), doublet (d), triplet (t),
q (quartet), and multiplet (m). High-resolution mass spectra
were obtained using an HRESITOFMS spectrometer with
the Analyst QS software for data acquisition and processing
(Agilent Series 1100 SL, ESI source model #G1969A,
Agilent Technologies, Palo Alto, CA, USA). The UPLC
used for UPLC-UV/MS method was an Acquiry UPLCTM
BEH C18 column (100 mm × 2.1 mm I.D., 1.7 μM) and
was used on Waters Acquity UPLCTM system (Waters
Corp.) that includes a binary solvent manager, sample
manager, heated column compartment, photodiode array

Med Chem Res
concentrated to yield compound 4 (9 mg), while the frac-
tions eluted with CHCl3-MeOH (8.5:1.5) collected and
concentrated to yield compound 3 and the fractions eluted
with CHCl3-MeOH (7.5:2.5) collected and concentrated to
yield compound 1 (5 mg). About 1 g of FC was purified
using sephadex LH 20 (100 g) CC using MeOH as eluent to
afford compound 5 (7 mg).
Determination of sugar configuration
The sugar composition and its configuration were deter-
mined according to the reported method by Wang and co-
workers (Wang et al. 2012). In brief, 1 mg of compound 1
was hydrolyzed using 200 μL of 2 M HCl at 90 °C for 2 h.
After hydrolysis, the reaction mixture was neutralized with
200 μL of 9M NH4OH and dried with high purity N2 gas.
Monosaccharide standards, including D-(+)-xylose, L-
(-)-xylose, L-rhamnose, D-(+)-glucose, and L-(-)-glucose
together with the hydrolysis products of compound 1 were
derivatized, respectively. The final products of each sample
were analyzed by UHPLC-UV-MS. The mobile phase
consisted of water with 0.05% formic acid (A) and acet-
onitrile/methanol/isopropanol (50:25:25, v/v) with 0.05%
formic acid (B). Analysis was performed using gradient
elution at a flow rate 0.3 mL/min: 14% B to 16.5% B in 22
min, and increasing B to 100% B in the following 0.5 min.
The PDA detection wavelength was 254 nm and ESI source
of SQD was used in positive mode. The configuration of the
sugar unit was determined by comparing with reference
standards, retention time and mass spectra.
6-O-β-D-xylopyranoside-shanzhiside methyl ester (1)
20
Oily colourless residue; [α]D −17.996 (c0.05, MeOH);
UV (MeOH) λmax 232 nm; IR(film) νmax 3343, 2926, 1700,
1
1639, 1460 cm−1; H NMR (CD3OD, 400 MHz,): Agly-
cone: δ = 7.51 (1H, d, J = 1.2 Hz, H-3), 5.77 (1H, d, J =
2.4 Hz, H-1), 4.53 (1H, t, J = 4.4 & 2.0 Hz, H-6), 3.89 (3H,
s, OCH3), 3.43 (1H, m, H-5), 2.80 (1H, dd, J = 8.8 & 2.4
Hz, H-9), 2.10 (1H, d, J = 14.8 Hz, H-7α), 1.91(1H, dd, J
= 14.8 & 4.8 Hz, H-7β), 1.40 (3H, s, CH3-10), Glucose: δ
= 4.72 (1H, d, J = 7.6 Hz, H-1’), 3.34–3.46 (4H, m, H-2”-
H-5”), 4.01 (1H, d, J = 2.4 Hz, H-6’a), 3.75 (1H, m, H-6’b),
Xylose: δ = 4.45 (1H, d, J = 7.6 Hz, H-1”), 3.32–3.46 (3H,
m, H-2”-H-5”), 3.98 (1H, d, J = 2.4 Hz, H-5”a), 3.31(1H,
dd, J = 4 & 1.6 Hz, H-5”b); 13C NMR (CD3OD, 100 MHz,):
δ = 168.8 (C-11), 153.7 (C-3), 109.7 (C-4), 103.0 (C-1”),
99.9 (C-1’), 95.0 (C-1), 83.4 (C-6), 79.6 (C-8), 78.3 (C-3’),
78.0 (C-5’), 77.7 (C-3”), 74.9 (C-2”), 74.6 (C-2’), 71.6 (C-
4”), 71.2 (C-4’), 62.8 (C-6’), 67.0 (C-5”), 52.6 (C-9), 51.7
(OCH3), 46.0 (C-7), 40.5 (C-5), 25.6 (C-10) HRESIMS
m/z 573.1554 [M+Cl]- (calcd. for C22H34O15Cl, 573.1573).
Biological activities
Free radical scavenging assay
The free radical scavenging activity of compounds 1–7 was
measured by spectrophotometric method (Qureshi et al.
2010). One milli litre of ethanolic solutions of the test
compound (50 and 100 μg/mL) was mixed with 1 mL of the
ethanolic solution of DPPH (200 μM) (SigmaAldrich, St.
Louis, MO, USA). Similarly, 1 mL of ethanolic of ascorbic
acid (50 and 100 μg/mL) was mixed with 1 mL DPPH
solution and used as a positive control. One mL of ethanol
alone was used as a blank. After mixing, all the solutions
were incubated in dark for 20 min and absorbance was
measured at 517 nm (Qureshi et al. 2010). The experiments
were performed in triplicate and average percent scavenging
activity was calculated as follows: Scavenging % =
(Absorbance of control − Absorbance of test compound) /
Absorbance of control
Conformational analysis and ECD calculation
Conformational analysis of possible stereoisomer of com-
pound 1 based on NMR data was performed with Schrö-
dinger Macromodel 9.9 (Schrödinger, LLC, New York)
employing the OPLS2005 (optimized potential for liquid
simulations) force field in MeOH (Macro Model 9.9,
Schrodinger LLC, 2012). Two conformers within a
2 kcal/mol energy window from the global minimum were
selected for each stereoisomer, based on Boltzmann dis-
tribution as calculated from Schrödinger software. The
output files for these conformers were prepared by Avo-
gadro software for Gaussian 09 calculation, geometrical
optimization, and energy calculation was applied on these
conformers at B3LYP/6-31G (Frisch et al. 2010). Vibra-
tional evaluation was done at the same level to confirm
minimal excitation energy (denoted by wavelength in nm),
rotatory strength dipole, and dipole length that were cal-
culated in MeOH by TD-DFT/B3LYP/6-31G performed by
the Gaussian 09 software package (Frisch et al. 2010). ECD
curves were obtained in the SpecDis 1.62 program (Bruhn
et al. 2014).
Reporter gene assay for the inhibition of NF-κB activity
Human chondrosarcoma (SW1353) cells were cultured in
DMEM/F12 medium supplemented with 10% FBS,
100 U/mL penicillin G sodium, and 100 μg/mL streptomy-
cin at 37 °C in an atmosphere of 5% CO2 and 95%
humidity. The cells were transfected with NF-κB luciferase
plasmid construct were plated in 96-well plates at a density
of 1.25 × 105 cells/well. After 24 h, cells were treated with
the test compounds, and after incubating for 30 min,

Med Chem Res
phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) (70
ng/mL) was added and further incubated for 6–8 h. Luci-
ferase activity was measured using a Luciferase Assay Kit
(Promega, Madison, WI, USA). Percent decrease in luci-
ferase activity was calculated relative to the vehicle control.
Parthenolide (Sigma-Aldrich, St. Louis, MO, USA) was
used as a positive control. (Ma et al. 2007)
Antimicrobial screening
The extracts and the isolated compounds were tested for
antibacterial activity against Staphylococcus aureus (ATCC
29213), methicillin resistant S. aureus (ATCC 33591)
(MRSA), Escherichia coli (ATCC 35218), Pseudomonas
aeruginosa (ATCC 27853), Mycobacterium intracellulare
(ATCC 23068) and antifungal activity against Candida
albicans (ATCC 90028), C. glabrata (ATCC 90030), C.
krusei (ATCC 6258), Cryptococcus neoformans (ATCC
90113), Aspergillus fumigatus (ATCC 204305). Cipro-
floxacin and Amphotericin-B (ICN Biomedicals, Aurora,
Ohio) were used as positive control for bacteria and fungi,
respectively (Bharate et al. 2007).
Assay for the inhibition of iNOS activity
Mouse macrophages (RAW264.7) were cultured in phenol
red-free RPMI medium supplemented with 10% bovine calf
serum, 100 U/mL penicillin G sodium, and 100 μg/mL
streptomycin. Cells were seeded in 96-well plates (1 × 105
cells/well) and incubated for 24 h for a confluency of ≥75%.
The cells were treated with the test compounds, and after
30 min, lipopolysaccharide (LPS) (Sigma-Aldrich) (5 μg/
mL) was added and further incubated for 24 h. The activity
of iNOS was determined in terms of the concentration of
NO by measuring the level of nitrite in the cell culture
supernatant using Griess reagent (Sigma-Aldrich, St. Louis,
MO, USA). Percent inhibition of nitrite production by the
test compound was calculated in comparison to the vehicle
control. IC50 values were obtained from dose−response
curves. Parthenolide (Sigma-Aldrich, St. Louis, MO, USA)
was used as the positive control (Zaki et al. 2013).
Antiprotozoal screening
Leishmania donovani promastigote, L. donovani amasti-
gote, L. donovani amastigote/THP1 cells and Trypanosoma
brucei brucei strains were used for antiprotozoal screening.
The in vitro anti-leishmanial and anti-trypanosomal assays
were performed on cell cultures of L. donovani promasti-
gotes, axenic amastigotes, THP1-amastigotes, and Trypa-
nosoma brucei trypomastigotes by Alamar Blue assays, as
described earlier (Makler and Hinrichs 1993). Difluor-
omethylornithine (DFMO, ICN Biomedicals, Aurora, Ohio)
was used as a positive control.
Assay for the inhibition of cellular oxidative stress
Antiplasmodial screening
The cellular antioxidant activity was measured in HepG2
cells according to a method previously described (Wolfe
and Rui 2007). The method measures the ability of test
compounds to prevent intracellular generation of peroxyl
radicals in response to 2,2′-azobis(2-amidinopropane)
dihydrochloride (ABAP, Sigma-Aldrich), a generator of
peroxyl radicals. The HepG2 cells were seeded in the wells
of a 96-well plate at a density of 6 × 104 cells/well and
incubated for 24 h. The medium was then removed and cells
were washed with PBS before treating with the test com-
pounds diluted in serum-free medium containing 25 μM
2′,7′ dichlorofluorescin diacetate (DCFH-DA, Invitrogen,
Carlsbad, CA, USA) for 1 h. After removing the medium,
the cells were treated with 600 μM ABAP and the plate was
immediately placed on a SpectraMax plate reader for kinetic
measurement every 5 min for 1 h (37 °C, excitation at 485
nm and emission at 538 nm). Quercetin (SigmaAldrich, St.
Louis, MO, USA) was included as the positive control. The
area under the curve (AUC) of fluorescence vs. time was
used to calculate cellular antioxidant activity (CAA) units
from the following equation: CAA unit = 100−[(AUC
sample/AUC control) × 100].
All isolates were tested against chloroquine-sensitive (D6,
Sierra Leone) and resistant (W2, Indo China) strains of P.
falciparum by measuring plasmodial LDH activity, as
described earlier (Makler and Hinrichs 1993), in addition to
the VERO mammalian cell line as an indicator of general
cytotoxicity. The selectivity indices (SI), and the ratio of
VERO IC50 to either D6 or W2 IC50, were calculated. IC50
values were calculated using the XLfit software. Chlor-
oquine (ICN Biomedicals, Aurora, Ohio) was used as a
positive control.
Monoamine oxidase inhibition (MAOI) assay
Recombinant human MAO-A and MAO-B were used to
investigate the effect of compounds using the kynuramine
deamination assay adapted for 96-well plates (Samoylenko
et al. 2010). The IC50 values were determined using range
of concentrations (0.001 to 100 μM). All experiments were
carried out in duplicate. Phenelzine, cloryline, and deprenyl
(Sigma Chemical, St Louis, MO, USA) were used as
positive controls.

Med Chem Res
Cell viability assay
two anomeric signals, one for the common glucose moiety
attached to C-1 of the iridoidal skeleton at δH 4.72, d, J =
7.6 Hz (H-1′)/δC 99.9 (C-1′) and the other at δH 4.45, d, J =
7.6 (H-1′′)/δC 103.0 (C-1′′) was for the additional sugar unit,
in which the sugar signals was found to be in agreement
with those of xyloside (Breitmaier and Voelter 1990). The
downfield shift of oxygenated methine proton H-6 to δH
4.53 and carbon C-6 to δC 83.4 highly recommends the
placement of the xylose sugar moiety at C-6. This was
further confirmed by the HMBC correlations (Fig. 2) which
showed the presence of correlations between H-6 (δH 4.53)
with C-1′′ (δC 103.0), C-4 (δC 109.7), C-5 (δC 40.5), C-7 (δC
MCF-7 breast cancer cells were maintained in exponential
growth conditions in DMEM media supplemented with
10% FBS and 1% penicillin/streptomycin at 37 °C in a
humid environment with 5% CO2 until used for experi-
ments. To measure compound effects on cell viability, 5 ×
103 cells were plated in 90 μL of media in 96-well plates.
Cells were allowed to attach overnight. The following day,
10 μL of a 10× compound stock as added to the cells in
doses covering a 5–6 log range (0–100 μM for pure com-
pounds and 0–1 mg/mL for whole extracts) and plates were
incubated at 37 °C for 72 h. Twenty micro litre of an
3-(4,5-dimethylthiazol-2-yl)-5(3carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (2 mg/
mL in PBS; Promega, Madison, WI) supplemented with 5%
Phenazine methosulphate was added and incubated for
2–4 hrs before absorbance was measured at 490 nm. Back-
ground was subtracted from the absorbance (compounds at
each concentration plus media and MTS without cells),
which were further normalized to % of untreated control.
Cells were treated in triplicate. As applicable, IC50 values
were determined by non-linear regression with GraphPad
Prism software (La Jolla, CA).
1
46.0), C-8 (δC 79.6) and C-9 (δC 52.6) and H-1H COSY
correlations (Fig. 2) which showed correlations between H-
6 (δH 4.53) with H-5 (δH 3.43, m), H-5 with H-9 (δH 2.80)
and H-9 with H-1 (δH 5.77). The ROESY spectrum showed
correlations between H-9 (δH 2.80, dd, J = 8.8 & 2.4 Hz)
with H-5 (δH 3.43, m) and H-7β (δH 1.91, dd, J = 14.8 &
4.8 Hz) indicated that H-5 and H-9 are both β-oriented while
correlations between CH3-10 (δH 1.40, s) with H-7α (δH 2.10,
d, J = 14.8 Hz), H-6 (δH 4.53, t, J = 4.4 & 2 Hz) and H-1 (δH
5.77, d, J = 2.4 Hz) supported that H-1, H-6, and CH3-10 are
all α-oriented. The stereochemistry of the compound was
further more confirmed using the electronic circular dichro-
ism in which the experimental ECD spectrum showed a peak
of negative cotton effect at 250 nm and positive at 227 nm.
The ECD spectrum (Fig. 3) of the possible stereoisomer of
compound 1 was calculated to match with the experimental
one. Also, the identity of the present sugar moieties as well as
their absolute configurations was determined using UHPLC-
UV/MS method. The hydrolysate of the compound 1 was
treated following the reported procedures (Wang et al. 2012).
From the chromatograms and mass spectra of the derivative
of compound 1, D-glucose and D-xylose was easily identi-
fied. By the comparison of the spectral data of compound 1
(Table 1) with the reported analogues (Dinda et al. 2007),
compound 1 was identified as 6-O-β-D-xylopyranoside-
shanzhiside methyl ester.
The known compounds were identified by analyzing the
spectroscopic data (1D and 2D NMR) and comparing their
data with those in the literature to be shanzhiside methyl ester
(2) (Yue et al. 2013), lamalbid (3) (Kobayashi et al. 1986),
geniposidic acid (4) (Güvenalp et al. 2006), theveside (Ford
and Bendall 1980), verbascoside (6) (Akademir et al. 2004),
and arenarioside (7) (Andary and Private 1985), respectively.
The phenolic constitute is considered one of the major
group of compounds acting as primary antioxidants or free
radical terminators and this activity was assessed for the
isolated compounds (1–7) using the DPPH method. The
DPPH reacts with an antioxidant compound, which can
donate hydrogen, and reduce DPPH changing its color from
deep violet into yellow color which was measured using UV
spectrophotometer (Padmanabhan and Jangle 2012). All the
Results and discussion
The aqueous fraction F2 was subjected to a series of chro-
matographic separation steps to yield one new iridoidal
glucoside (1) and six known compounds (2–7) (Fig. 1).
Compound 1 was obtained as a colorless oily residue. Its
molecular formula was established as C22H34O15 which is
consistent with the molecular ion [M+Cl]- at m/z 573.1554
(calcd.: 573.1573) found in the negative mode HRESIMS
spectrum and this was furthermore confirmed by spectral
data. It was found to have a great similarity to compound 2
which was identified as shanzhiside methyl ester, except for
the presence of an additional pentose sugar moiety. The
structure of the compound and the placement of the two
sugars were established using the available 1D (1H- & 13C-
NMR) and 2D (HMQC, HMBC, H-1HCOSY & ROESY)
1
NMR spectral data, in which all the data are given in the
supplementary material Appendix 1(Figs. A1–A7). The 1H-
and 13C-NMR spectra displayed signals characteristic for
the iridoidal nucleus presented by an enol ether unit [δH
7.51, d, J = 1.2 Hz (H-3)/ δC 153.7 (C-3) & 109.7 (C-4)]
with a carbomethoxy group [δH 3.80, s (OCH3)/δC 51.7
(OCH3) & δC 168.8 (C-11)], a dioxygenated methine [δH
5.77, d, J = 2.4 Hz (H-1)/δC 95.0 (C-1)], two aliphatic
methines [δH 2.80, dd, J = 8.8 & 2.4 Hz (H-9)/δC 52.6 (C-9)
and δH 3.43, m (H-5) / 40.5 (C-5)] and a methyl group [δH
1.40, s (H-10)/δC 25.6 (C-10)]. Also the spectra displayed

Med Chem Res
Fig. 1 List of isolated
compounds
Fig. 2 a Important HMBC and
1H-1H COSY correlations of
compound 1. b Important
ROESY correlations of
compound 1
isolated compounds were tested for radical-scavenging
activity against DPPH organic free radicals. The results
were exhibited in the form of percent of inhibition
(Table 2), in which compounds 6 and 7 showed radical
scavenging activity with value 100%, while the iridoid
glycosides compounds 1–5 showed radical scavenging
activity with values 73.80, 63.79, 66.56, 57.99, and 72.53%
in concentration 100 μg/mL.
Inflammation is considered as a risk factor for several
types of cancer, as well as a contributing factor in obesity
and metabolic disorders. The activation of NF-κB in
response to pro-inflammatory signals is associated with
many diseases caused by unregulated inflammation. Since
NF-κB is highly activated at the sites of inflammation in
diverse diseases, the compounds that can suppress NF-κB
activation have potential as anti-inflammatory agents (Tak
and Firestein 2001). Excessive generation of nitric oxide
(NO) and reactive oxygen species (ROS) also contributes
significantly to the progress of inflammation and subsequent
development of metabolic syndrome, characterized by
obesity, diabetes, and cardiovascular disease. Inhibition of
inducible nitric oxide synthase (iNOS) can reduce the
intracellular NO production. Hence, NF-κB, iNOS, and
ROS have been considered as important targets for
inflammation (Ko and Auyeung 2013; Zhao et al. 2014).
The total methanolic extract and the aqueous fraction F2
showed a decrease in the oxidative stress ROS with values
57 and 66% respectively, while the chloroformic fraction

Med Chem Res
Fig. 3 The experimental and calculated ECD spectra of compound 1
Table 1 1H- and 13C-NMR spectral data for compound 1 (400 and
100 MHz) in CD3OD
Table 2 Free radical scavenging activity of the isolated metabolites
1–7
No.
δC
Type
δH
Compounds
Antioxidant %
100 μg
50 μg
1
2
3
4
5
6
7
95.0
CH
–
5.77, d, J = 2.4 Hz
–
153.7
109.7
40.5
–
1
73.80%
63.69%
66.56%
57.99%
72.53
54.50%
45.42%
50.69%
37.45%
46.87%
100%
CH
C
7.51, d, J = 1.2 Hz
2
–
3
CH
CH
CH2
3.43, m
4
83.4
4.53, t, J = 4.4 & 2 Hz
5
46.0
1.91, dd, J = 14.8 and 4.8 Hz, H-7β
6
100%
2.10, d, J = 14.8 Hz, H-7α
7
100%
100%
8
79.6
52.6
25.6
168.8
99.9
74.6
78.3
71.2
78.0
62.3
C
–
Ascorbic acid
100%
100%
9
CH
CH3
C
2.80, dd, J = 8.8 & 2.4 Hz
1.40, s
10
11
1’
2’
3’
4’
5’
6’
–
CH
CH
CH
CH
CH
CH2
4.72, d, J = 7.6 Hz
3.45, m
F1 didn’t show any activity. Also, compounds 1, 2, 3, 6, and
7 decreased cellular oxidative stress by inhibiting ROS
generation values 50, 60, 57, 63, and 52%, respectively,
with comparison to the positive control quercetin which
showed decrease in the oxidative stress with value 72%.
Compounds 1–7, as well as the aqueous fraction F2 from
which these compounds were isolated, did not show any
inhibition of iNOS as they did not affect cellular nitric oxide
levels in LPS treated macrophages. Furthermore, the
increase in transcriptional activity of NF-κB in PMA-treated
cells was also not suppressed by any of the tested com-
pounds (1–7). Hence, these results indicated that their anti-
inflammatory effect is not mediated by inhibition of iNOS
or NF-κB activity. However, the total methanolic extract
and the chloroformic fraction F1 showed inhibition to the
3.46, m
3.40, m
3.34, m
4.01, d, J = 2.4 Hz
3.75, m
1”
2”
3”
4”
5”
103.0
74.9
77.7
71.6
67.0
CH
CH
CH
CH
CH2
4.45, d, J = 7.6 Hz
3.32, m
3.46, m
3.41, m
3.98, d, J = 2.4 Hz
3.31, dd, J = 4 and 1.6 Hz
3.80, s
OCH3
51.7
CH3

Med Chem Res
iNOS with IC50 values 30 and 5 μg/mL, respectively, using
Parthenolide as a positive control with IC50 value 0.26 μg/
mL. This may be attributable to the high content of olea-
nolic acid, oleanonic acid, and 25-hydroxy oleanolic acid
which were isolated before from the chloroformic fraction
(F1) of the methanolic extract of Lantana montevidensis
roots (Mohamed et al. 2016) and showed in this study
inhibition in iNOS with IC50 values 6.9, 7.4, and 9.0 μg/mL
in comparison to parthenolide which showed IC50 value
0.26 μg/mL. Also, oleanonic acid and 25-hydroxy oleanolic
acid showed inhibition toward NF-κB with IC50 values 49
and 10, in comparison to parthenolide which showed IC50
value 1.0 μg/mL. All the potential anti-inflammatory activ-
ities of the extracts, compounds 1–7, oleanolic acid, olea-
nonic acid, and 25-hydroxy oleanolic acid are listed in
Table 3.
Both the chloroformic and aqueous fractions, F1 and F2
respectively, demonstrated measurable inhibition of MCF-7
breast cancer cell growth, with IC50 value 0.3 mg/mL
(Fig. 4). None of the pure compounds inhibited cell growth
Table 3 Potential anti-
inflammatory activity of the
extracts and compounds 1−7
Compounds
Stock conc.
(μg/mL)
inhibition of
iNOS
inhibition of
NF-κB
IC50 (μg/mL)
inhibition of ROS
generation*
IC50 (μg/mL)
%
1
5
5
5
5
5
5
5
5
5
5
NA
NA
NA
NT
NT
NA
NA
6.9
7.4
9
NA
NA
NA
NT
NT
NA
NA
NA
49
50
60
57
–
2
3
4
5
–
6
52
63
NA
NA
NA
7
Oleanolic acid
Oleanonic acid
25-Hydroxy oleanolic
acid
10
Total methanolic
extract
20
30
NA
57
Chloroform Fr. (F1)
Aqueous Fr. (F2)
Parthenolide
20
20
–
NA
5
NA
NA
1.0
NT
66
NA
NT
72
0.26
NT
Quercetin
–
NA no activity, NT not tested
*at 125 µg/mL for pure and 500 µg/mL for extract
Fig. 4 Effect of pure compounds 1–7 a and the chloroformic (F1) and aqueous (F2) fractions b on the viability of MCF-7 breast cancer cells

Med Chem Res
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by 50% or more, and consequently IC50 values could not be
determined.
Compounds 2 & 7 showed mild inhibition for mono-
amine oxidase. Compound 2 showed inhibition of MAO-A
and B with IC50 values 39.48 and 27.77 µM, while com-
pound 7 inhibited monoamine oxidase MAO-B with weak
potency with IC50 value 65.02 µM using phenelzine as a
reference with IC50 values 0.27 and 0.14 µM in inhibition of
MAO-A and MAO-B, respectively. However compounds 3,
5, and 6 showed no inhibition to monoamine oxidase and
compounds 1 and 4 were not tested due to lack of
amount. More information is available in the supportive
material appenddix 1 (Table A1).
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Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson
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Acknowledgements The authors are grateful to the Egyptian Gov-
ernment and the National Center for Natural Products Research, The
University of Mississippi, School of Pharmacy, Mississippi, USA for
their financial support. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the
aforementioned institutions. We are also thankful to Dr. Baharthi
Avula for providing the HRESIMS, Dr. Babu L. Tekwani and Dr.
Narayan D. Chaurasiya for carrying out the MAOI assay and to Dr.
Melissa Jacob for performing the antimicrobial assay.
Ma G, Khan S, Benavides G, Schuhly W, Fischer N, Khan IA, Pasco
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Compliance with ethical standards
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Conflict of interest The authors declare that they have no competing
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Investigation of essential oil and biological activities of Lantana
montevidensis (Spreng.) Briq.cultivated in Egypt. J Nat Pharm
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