Phytomedicine 21 (2014) 1695–1701

Contents lists available at ScienceDirect

Phytomedicine
journal homepage: www.elsevier.de/phymed

Pharmacological insight into the anti-inflammatory activity of

sesquiterpene lactones from Neurolaena lobata (L.) R.Br. ex Cass
R. McKinnon a,∗,1 , M. Binder b,1 , I. Zupkó c,1 , T. Afonyushkin b , I. Lajter d , A. Vasas d ,
R. de Martin b , C. Unger e , H. Dolznig e , R. Diaz f , R. Frisch f , C.M. Passreiter g , G. Krupitza h ,
J. Hohmann d , B. Kopp a , V.N. Bochkov b
a
Department of Pharmacognosy, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria
b
Department of Vascular Biology and Thrombosis Research, Center of Physiology and Pharmacology, Medical University of Vienna, Schwarzspanierstrasse
17, A-1090 Vienna, Austria
c
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
d
Department of Pharmacognosy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
e
Institute of Medical Genetics, Medical University of Vienna, Währinger Strasse 10, A-1090 Vienna, Austria
f
Institute of Ethnobiology, Playa Diana, San José/Petén, Guatemala
g
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
h
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions
Received 4 June 2014 including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated
Received in revised form 23 July 2014 sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed
Accepted 29 July 2014
at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated
sesquiterpene lactones, as well as a structure–activity relationship and in vivo anti-inflammatory data.
Keywords:
Methods: The effect of the extract and its compounds on the generation of pro-inflammatory proteins
Neurolaena lobata
was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their
Natural products
Anti-inflammatory
potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo
Sesquiterpene lactones anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-
Chemokines induced paw edema model in rats.
Results: The compounds and extract inhibited LPS- and TNF-␣-induced upregulation of the pro-
inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced
elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the
extract inhibited the development of acute inflammation in rats.
Conclusions: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory
cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines.
Structure–activity analysis revealed the importance of the double bond at C-4–C-5 and C-2–C-3 and
the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises
further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases.

© 2014 Elsevier GmbH. All rights reserved.

Introduction

Neurolaena lobata (L.) R.Br. ex Cass. (Asteraceae) is a Cen-

Abbreviations: ECGS, endothelial cell growth supplement; ELISA, enzyme-linked
tral American plant used in traditional medicine throughout
immunosorbent assay; HUVEC, tert telomerase reverse transcriptase technol-
ogy immortalized human umbilical vein endothelial cells; IL, interleukin; NF-kB, the Caribbean for the treatment of inflammatory diseases, can-
nuclear factor-kB; qPCR, real-time PCR; PSF, penicillin–streptomycin–fungizone; cer, skin ailments, malaria, diabetes, pain, and as insect repellent
TNF, tumor necrosis factor. (Gracioso et al., 1998; Passreiter and Isman, 1997). Several research
∗ Corresponding author. Tel.: +43 1 4277 55262; fax: +43 1 4277 9552.
groups confirmed the in vitro anti-inflammatory, anti-neoplastic
E-mail addresses: ruxandra.mckinnon@univie.ac.at, ruxy13@hotmail.com
and anti-protozoal activity of the plant, and showed its anti-
(R. McKinnon).
1
These authors contributed equally to this work. feedant and anti-viral potential (Bedoya et al., 2008; Berger et al.,

http://dx.doi.org/10.1016/j.phymed.2014.07.019
0944-7113/© 2014 Elsevier GmbH. All rights reserved.
1696 R. McKinnon et al. / Phytomedicine 21 (2014) 1695–1701
2001; François et al., 1996; Passreiter and Isman, 1997; Unger
et al., 2013; Walshe-Roussel et al., 2013). In addition, organic
fractions of N. lobata hydroalcoholic extracts demonstrated
anti-ulcerogenic and anti-nociceptive effect in vivo (Gracioso
et al., 1998, 2000). The analgesic activity was suggested to be
due to the interference with the inflammatory process. Phar-
macological and phytochemical characterization of the plant
attributed the different therapeutic effects to sesquiterpene lac-
tones which were identified as main constituents. Up to date,
eleven sesquiterpene lactones of the germacranolide and fura-
noheliangolide type have been isolated from N. lobata, namely
neurolenin A–F, lobatin A–C, 9␣-hydroxy-8␤-isovaleryloxy-
calyculatolide, and 9␣-acetoxy-8␤-isovaleryloxy-calyculatolide
(Manchand and Blount, 1978; Passreiter, 1998; Passreiter et al.,
1995).
Sesquiterpene lactones are secondary metabolites that con-
fer potent anti-inflammatory properties to medicinal plants such
as Arnica montana, Artemisia absinthium, and Tanacetum parthe-
nium (Merfort, 2011; Wichtl, 2002). In contrast to nonsteroidal
anti-inflammatory drugs (NSAIDs) that inhibit the enzyme cyclo-
oxygenase and can cause unwanted side effects, the mode of
action underlying the anti-inflammatory effect of sesquiterpene
lactones has been explained by mechanisms such as inhibi-
tion of the nuclear factor-kB (NF-kB) and of the production of
inflammatory cytokines (Berges et al., 2009; Cho et al., 2000;
Lyss et al., 1997). This has raised interest in sesquiterpene
lactones as prospective therapeutics for the treatment of inflam-
mation.
A recent study reported anti-inflammatory activity of N. lobata
leaf extract and five isolated sesquiterpene lactones, i.e., neurolenin
B, a mixture of neurolenins C and D, lobatin B and 8␤-isovaleryloxy-
9␣-hydroxy-calyculatolide as illustrated by their inhibitory action
on TNF-␣ production in LPS-stimulated THP-1 cells (Walshe-
Roussel et al., 2013). These data raised our interest in fur-
ther pharmacological studies regarding the anti-inflammatory
activity of N. lobata and its sesquiterpene lactones. The
present work investigated the effect of eight sesquiterpene
lactones isolated from N. lobata on the generation of pro-
inflammatory proteins (interleukin-8 (IL-8) and E-selectin) in
vitro. Based on these data a structure-activity relationship
was established. The anti-inflammatory effect of the extract
and the most active compounds was further studied at the
mRNA level, as well as in carrageenan-induced rat paw edema
model.
Materials and methods
Plant material
N. lobata (L.) R.Br. ex Cass. was collected in February 2011 in
Guatemala, Departamento Petén, near the north-western shore
of Lago Petén Itzá, 0.5 km NNW of San José in the area of the
Chakmamantok-rock formation (16 59 16 N, 89 53 45 W) and
within the botanical garden of the Institute for Ethnobiology
(Unger et al., 2013). Voucher specimens (leg. R. Diaz, det. R. O.
Frisch 03. 02. 2011/a,b, Chakmamantok) were archived at the
herbarium of the Institute for Ethnobiology, San Jose, Guatemala.
Voucher specimen (No. 813) has also been deposited at the
Herbarium of the Department of Pharmacognosy, University of
Szeged, Szeged, Hungary. The fresh plant material (the aerial plant
parts, leaves, caulis and florescence) of N. lobata was air dried in
Guatemala, sent to Austria and stored deep-frozen until subse-
quent extraction. According to The Plant List data base, accepted
synonyms are N. lobata var. indivisa Donn.Sm. and N. lobata var.
lobata.
General procedures
Column chromatography (CC) was carried out on polyamide
(ICN, Eschwege, Germany); vacuum liquid chromatography (VLC)
on silica gel G (15 mm, Merck, Darmstadt, Germany); preparative
thin-layer chromatography (preparative TLC) on silica gel 60 F254
and RP-18 F254 plates (Merck, Darmstadt, Germany); and rotation
planar chromatography (RPC) on silica gel 60 GF254 with a Chro-
matotron instrument (Model 8924, Harrison Research, Palo Alto,
CA, USA). NMR spectra were recorded in CDCl3 on a Bruker Avance
DRX 500 spectrometer at 500 MHz (1 H) and 125 MHz (13 C), with
TMS as internal standard. Two-dimensional data were acquired and
processed with standard Bruker software. In the COSY, HSQC and
HMBC experiments, gradient-enhanced versions were used. Mass
spectrometry was carried out on API 2000 instrument (Framing-
ham, MA, USA) with an atmospheric pressure chemical ionization
(APCI) interface. The source temperature was 350 ◦ C. All solvents
used for the isolation of the compounds were purchased from Molar
Chemicals Ltd. (Budapest, Hungary).
Extraction and isolation
Dried and ground aerial parts of the plant (3.00 kg), which was
stored at −70 ◦ C before processing, was crushed in a blender and
then percolated with MeOH (50 L) at room temperature. The crude
extract was concentrated in vacuo and subjected to solvent–solvent
partition first with 5 × 1 l petroleum ether (A), then with 5 × 1 l of
CH2 Cl2 (B) and finally with 5 × 1 l of EtOAc (C). The CH2 Cl2 phase
(95.4 g) was chromatographed on a polyamide column (287 g) with
mixtures of MeOH and H2 O (1:4, 2:3, 3:2 and 4:1, 3 l of each) as
eluents. Fractions with similar composition were combined accord-
ing to TLC monitoring, affording fractions BI–BVII. The fraction BII
(33.6 g) obtained with MeOH–H2 O (1:4) was subjected to silica gel
VLC using a gradient system of cyclohexane–EtOAc–EtOH (from
30:10:0 to 30:30:6). The CC fractions were combined into nine
fractions (BII/1-BII/9) after TLC monitoring.
Fraction BII/4 obtained by CC on silica gel with
cyclohexane–EtOAc 1:1 was separated by RPC with a gradi-
ent system of CH2 Cl2 –acetone (from 100:0 to 9:1). The subfraction
eluted with CH2 Cl2 –acetone 97:3 was separated by RPC with a
gradient system of CH2 Cl2 –acetone (100:0, 99:1, 98:2, 95:5, 90:10,
70:30), and then further purified by preparative TLC on silica
gel with the mobile phase cyclohexane–EtOAc–EtOH (30:20:0.5),
affording the isolation of neurolenin A and neurolenin B.
Fraction BII/5 obtained with cyclohexane–EtOAc 1:1 was sep-
arated on VLC with CH2 Cl2 –acetone gradient system (from 99:1
to 4:1). The subfraction eluted with CH2 Cl2 –acetone 95:5 was fur-
ther fractionated with RPC using a mobile phase of CH2 Cl2 –aceton
(from 99:1 to 4:1), and finally separated by preparative RP-TLC,
using MeOH–H2 O 7:3, to yield neurolenin C and neurolenin D.
Fraction BII/6 was separated by CC on silica gel, using a
gradient system of CH2 Cl2 –acetone (from 99:1 to 4:1). The sub-
fraction eluted with CH2 Cl2 –acetone 97:3, was subjected to
preparative TLC, using a system of cyclohexane–EtOAc–EtOH
(60:60:1) to give 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide and
lobatin A. Moreover, the subfraction obtained with CH2 Cl2 –acetone
95:5 was further purified by preparative TLC on silica gel with
cyclohexane–EtOAc–EtOH (60:60:1), which led to the isolation
of lobatin B. Finally, the subfraction eluted with MeOH was
purified first by RP-TLC using MeOH–H2 O (7:3), and then by
preparative TLC using cyclohexane–EtOAc–EtOH (60:60:1), to yield
8␤-isovaleryloxy-9␣-hydroxy-calyculatolide. The isolated com-
pounds were identified by comparison of their APCI-MS, 1 H NMR
and 13 C NMR data with those published in the literature (Blair et al.,
2002; Herz and Kumar, 1980; Passreiter et al., 1995).
 R. McKinnon et al. / Phytomedicine 21 (2014) 1695–1701
Quantification of sesquiterpene lactones
Quantitative high-performance liquid chromatography (HPLC)
analyses were carried out according to the previously reported
method (Passreiter, 1998) using a Nucleosil 120-5 C18 column
(Macherey & Nagel GmbH & Co. KG, Düren, Germany, length
125 mm/diameter 4.6 mm), mobile phase MeOH–H2 O (45:55), and
flow 1.5 ml min−1 . The compounds were detected at 215 nm.
Cell culture
Telomerase reverse transcriptase immortalized human umbili-
cal vein endothelial cells (HUVECtert) were maintained in medium
199 (M199) supplemented with 20% fetal bovine serum, 1%
penicillin–streptomycin–fungizone (PSF), 1% l-glutamine, and 0.4%
endothelial cell growth supplement (ECGS). THP-1 cells were cul-
tured in RPMI 1640 medium. Cells were incubated at 37 ◦ C in a
humidified atmosphere of 5% CO2 . The media and serum were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA), trypsin and PSF
were from Lonza (Walkersville, MD, USA), ECGS from Technoclone
(Vienna, Austria), and l-glutamine from BioWhittaker® Reagents,
Lonza.
Interleukin-8 and E-selectin ELISA
HUVECtert or THP-1 cells were seeded in 96-well plates in M199
medium containing 5% serum and cultured over night. Then, cells
were pretreated with compounds or extract for 20 min prior to
stimulation with 100 ng/ml LPS (Sigma-Aldrich, Missouri, USA) or
30 ng/ml TNF-␣ (PeproTech, New Jersey, USA) for 6 h. Secreted
IL-8 was determined in the media of treated cells using human
CXCL8/IL-8 immunoassay kit (R&D Systems, Minneapolis, USA). For
the detection of E-selectin, cells were fixed with 0.1% glutaralde-
hyde in PBS and then incubated with 300 ng/ml CD62E mouse anti
human antibody (R&D Systems, Minneapolis, USA) and horseradish
peroxidase-linked sheep anti mouse IgG (GE Healthcare, Wauke-
sha, USA). The optical density was measured at 450 nm using
Synergy Reader H4 (BioTek, Vermont, USA).
Analysis of mRNA levels by qPCR
HUVECtert cells were seeded in 96-well plates in M199
medium containing 5% serum and cultured over night. Then,
cells were pretreated with compounds and extract for 20 min
prior to stimulation with 100 ng/ml LPS for 6 h. Following incu-
bation time, RNA was isolated using QIAzol lysis reagent (Qiagen,
Hilden, Germany) as previously described (Grienke et al., 2011).
900 ng total RNA were used for reverse transcription with a
MuLV-RT and Oligo d(T) primers (Applied Biosystems, Carls-
bad, CA). Real-time PCR (qPCR) was employed to determine
the relative expression of the studied genes. Design of primers
was facilitated by PRIMER3 software from the Whitehead Insti-
tute for Biomedical Research (Cambridge, MA) and reference
mRNA sequences of respective genes from the GeneBank
(www.ncbi.nlm.nih.gov). Primers 5 -ctcttggcagccttcctgatt-
30(forward) and 5 -tatgcactgacatctaagttctttagca-3 (reverse)
were used for IL-8, and 5 -ggtttggtgaggtctgctc-3 (forward) and
5 -tgatctgtcccggaactgc-3 (reverse) for E-selectin. Relative quantifi-
cation of the genes of interest was normalized to the expression
levels of the housekeeping gene ␤2-microglobulin using the
mathematical model applied by Pfaffl (Kadl et al., 2002).
Carrageenan-induced paw edema in rats
The anti-inflammatory action of the extract in vivo was
investigated by means of rat paw edema test as described
1697
Table 1
%-Content ± Stdev of sesquiterpene lactones in N. lobata leaves and extract. Neu-
rolenin A (Lob 1), neurolenin B (Lob 2), neurolenin C (Lob 3), neurolenin D (Lob 4),
lobatin A (Lob 5), lobatin B (Lob 6), 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide (Lob
7), 8␤-isovaleryloxy-9␣-hydroxy-calyculatolide (Lob 8).
Compound Content leaves (%) Content extract (%)
Lob 1 0.013 ± 0.005 0.10 ± 0.03
Lob 2 0.124 ± 0.023 1.89 ± 0.05
Lob 3 0.210 ± 0.031 4.74 ± 0.18
Lob 4 0.169 ± 0.019 7.14 ± 0.41
Lob 5 0.016 ± 0.008 0.75 ± 0.23
Lob 6 1.710 ± 0.209 42.41 ± 0.13
Lob 7 0.120 ± 0.02 3.28 ± 0.21
Lob 8 0.240 ± 0.03 14.10 ± 0.88
Total 2.602 74.41
before (Háznagy-Radnai et al., 2012). Animals were treated
in accordance with the European Communities Council Direc-
tives (86/609/ECC) and the Hungarian Act for the Protection
of Animals in Research (XXVIII.tv.32.§). Experiments involving
animal subjects were carried out with the approval of the
Hungarian Ethical Committee for Animal Research (registration
number: IV./01758-2/2008). Mature male Sprague-Dawley rats
(9 rats/group, 175–200 g) were treated intraperitoneally with
20 and 60 mg/kg extract dissolved in physiological saline con-
taining 20% dimethylsulfoxide. Control group was treated with
vehicle, while dexamethasone (0.5 mg/kg) was used as a refer-
ence anti-inflammatory agent. The local inflammatory response
was elicited 60 min later by a subplantar injection of 0.5 mg car-
rageenan (Marine Colloids, Springfiled, NJ, USA) dissolved in 0.1 ml
isotonic saline. The contralateral foot was injected with physio-
logical saline. The volume of the paws was determined 5 h later
with a plethysmometer (Hugo Sachs Elektronik, March-Hugstetten,
Germany). The edema volume is presented as the difference of
the volumes of the carrageenan-injected and the contralateral
paws.
Statistical analysis
For statistical analysis GraphPad PrismTM software version 4.03
(GraphPad Software Inc., La Jolla, CA) was used. Student’s t test
(Figs. 2–4), two-way ANOVA (Fig. 5) and one-way ANOVA (Fig. 6)
were employed for the comparison of two groups and p val-
ues < 0.05 were considered significant (*).
Results
Isolation of sesquiterpene lactones from N. lobata
Separation of the CH2 Cl2 phase of the MeOH leaf extract using
column, rotational planar, and preparative thin layer chromatogra-
phy resulted in purification of eight sesquiterpene lactones. Mass
spectrometry and nuclear magnetic resonance analysis showed
that five of the compounds were germacranolide sesquiterpene
lactones: neurolenin A (Lob 1), neurolenin B (Lob 2), neu-
rolenin C (Lob 3), neurolenin D (Lob 4) and lobatin A (Lob 5)
(Fig. 1). The remaining three were of furanoheliangolide type:
lobatin B (Lob 6), 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide
(Lob 7) and 8␤-isovaleryloxy-9␣-hydroxy-calyculatolide (Lob
8).
Quantification by HPLC indicated that the content of sesquiter-
pene lactones in the plant material varied from 0.01% (Lob 5) to
0.24% (Lob 8), while in the CH2 Cl2 phase it varied between 0.10%
(Lob 1) and 42.41% (Lob 6) (Table 1). The total amount of sesquiter-
pene lactones (2.60%) was found to be relatively high compared
to plant material investigated in 1998 (Passreiter, 1998; Passreiter
1698 R. McKinnon et al. / Phytomedicine 21 (2014) 1695–1701
Fig. 1. Chemical structures of sesquiterpene lactones isolated from the leaves
of N. lobata. The abbreviations are: neurolenin A (Lob 1), neurolenin B (Lob
2), neurolenin C (Lob 3), neurolenin D (Lob 4), lobatin A (Lob 5), lobatin B
(Lob 6), 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide (Lob 7), 8␤-isovaleryloxy-9␣-
hydroxy-calyculatolide (Lob 8).
and Aldana, 1998). The CH2 Cl2 phase also showed a high content
of total sesquiterpenes (74.41%).
Decreased secretion of IL-8 in LPS- and TNF-˛-stimulated
endothelial cells
To assess the effect of the eight sesquiterpene lactones present in
the extract (CH2 Cl2 phase of the MeOH extract) on the cytokine IL-
8, endothelial cells were pretreated with different concentrations
of compounds (1–10 ␮M) or extract (10, 30 ␮g/ml) and stimulated
with LPS and TNF-␣ for 6 h. Treatment with extract or any of the
eight sesquiterpene lactones decreased the LPS-induced secretion
of IL-8 in a dose-dependent manner (Fig. 2A). At the highest tested
concentration (10 ␮M) all compounds strongly down-regulated the
LPS-induced production of IL-8 protein, with Lob 2, Lob 5, Lob 6
and Lob 7 being the most effective. Lob 2, Lob 6, Lob 7 (10 ␮M)
and extract (10, 30 ␮g/ml) caused a significant decrease in IL-8
production also in TNF-␣-stimulated endothelial cells (Fig. 2B).
The WST-1 viability assay determined that concentrations of up
to 10 ␮M isolated compounds and 30 ␮g/ml extract were not toxic
to endothelial cells (Supplementary Material). Hence, the observed
anti-inflammatory activity was not due to a direct toxic effect.
Down-regulation of the expression of E-selectin on LPS- and
TNF-˛-stimulated endothelial cells
Similarly, the adhesion molecule E-selectin was monitored in
endothelial cells by ELISA as a second inflammation marker. The
extract (5 ␮g/ml) and isolated compounds (5 ␮M) significantly
down-regulated the expression of E-selectin on LPS- and TNF-␣-
stimulated endothelial cells (Fig. 3A and B). The highest activity
was indicated by Lob 6 and succeeded by Lob 7, Lob 2 and Lob 5.
Decreased secretion of IL-8 in LPS-stimulated THP-1 monocytic
cells
In order to test the effect of the compounds on a second cell
line, the production of IL-8 inflammatory protein induced by LPS
was assessed in THP-1 monocytes. The extract (5 ␮g/ml) and eight
sesquiterpene lactones (5 ␮M) significantly decreased the secretion
of IL-8 (Fig. 4). Lob 6 proved to be most effective, followed by Lob
7, Lob 2 and Lob 5.
Down-regulation of IL-8 and E-selectin mRNA expression in
endothelial cells
In order to test whether N. lobata components modulate expres-
sion of inflammatory genes at the mRNA level, endothelial cells
were treated with the three most active sesquiterpene lactones, Lob
6, Lob 7 and Lob 2, followed by analysis of LPS-induced expression of
mRNA encoding for IL-8 and E-selectin. Relative mRNA expression
of the IL-8 and E-selectin genes in endothelial cells was strongly
inhibited by the lactones when compared to activation with LPS
alone (Fig. 5A and B). Lob 2 exerted a significant effect only on
E-selectin.
Reduced carrageenan-induced edema in rats
Both applied doses of N. lobata extract (20 and 60 mg/kg)
exerted significant anti-inflammatory actions in the rat paw edema
test (Fig. 6). The suppression of local edema formation by the
higher dose was more that 50%. Treatment with 0.5 mg/kg dexa-
methasone, as expected, resulted in substantial inhibition of
inflammation.
Discussion
IL-8 is a pro-inflammatory cytokine with implications in innate
immunity and several inflammatory diseases (Campbell et al.,
2013). Another molecule with a role in inflammatory response is
the cell surface glycoprotein E-selectin, which mediates the adhe-
sion of leukocytes to endothelial cells (Ley, 2003; Ley et al., 2007).
In normal cells expression of IL-8 and E-selectin is highly regulated
and reduced. IL-8 and E-selectin can be induced by inflammation
signals such as the bacterial endotoxin LPS and the cytokine TNF-
␣. At the concentration of 10 ␮M all tested compounds strongly
decreased secretion of IL-8 in LPS-stimulated endothelial cells.
The most active sesquiterpene lactones, neurolenin B (Lob 2),
lobatin A (Lob 5), lobatin B (Lob 6) and 8␤-isovaleryloxy-9␣-
acetoxy-calyculatolide (Lob 7) also down-regulated the production
of IL-8 protein in TNF-␣-induced endothelial cells. The eight
compounds (5 ␮M) demonstrated significant effect on another
inflammation marker, the adhesion molecule E-selectin. After stim-
ulation with LPS and TNF-␣, all tested sesquiterpene lactones
down-regulated the expression of E-selectin on endothelial cells.
The anti-inflammatory activity of the eight compounds was also
observed in THP-1 monocytes where they significantly reduced the
production of IL-8 after stimulation with LPS. Lobatin B (Lob 6), the
dominant compound of the extract as indicated by HPLC quantifi-
cation, showed the most potent anti-inflammatory effect in both
cell lines. Lobatin B (Lob 6) was followed by 8␤-isovaleryloxy-9␣-
acetoxy-calyculatolide (Lob 7), neurolenin B (Lob 2) and lobatin
A (Lob 5). In HUVECtert cells, BAY 11-7082 (5 ␮M), a potent NF-
kB inhibitor often used as positive control for anti-inflammatory
activity, showed ∼90% inhibition of the LPS- and TNF-␣-induced
production of IL-8 and E-selectin when compared with the con-
trol (Vogl et al., 2013). Similar results were obtained in the present
study with lobatin B (Lob 6) in HUVECtert cells. Previously, lobatin
B (Lob 6) also showed potent effect in LPS-stimulated THP-1 cells,
 R. McKinnon et al. / Phytomedicine 21 (2014) 1695–1701 1699

Fig. 2. Dose-dependent effect of N. lobata extract and its components on the induction of IL-8 protein after stimulation with LPS (A) or TNF-␣ (B) in HUVECtert cells. Cells
were treated with compounds and extract 20 min prior to stimulation with LPS or TNF-␣. IL-8 release was measured by ELISA after 6 h. The abbreviations are: neurolenin
A (Lob 1), neurolenin B (Lob 2), neurolenin C (Lob 3), neurolenin D (Lob 4), lobatin A (Lob 5), lobatin B (Lob 6), 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide (Lob 7), 8␤-
isovaleryloxy-9␣-hydroxy-calyculatolide (Lob 8), CH2 Cl2 phase of the MeOH N. lobata extract (Extr). Values are mean ± S.E.M. of sextuplicate measurements. * p < 0.05, **
p < 0.01, *** p < 0.001 as compared to LPS or TNF-␣ activation (t test).

with stronger anti-inflammatory activity than that of the positive
control parthenolide (Walshe-Roussel et al., 2013). Hence, lobatin
B (Lob 6) exhibits an anti-inflammatory effect comparable to that
of the known NF-kB inhibitors BAY 11-7082 and parthenolide.
In the present study eight compounds comprising structurally
uniform series of germacranolide sesquiterpenes were investi-
gated for in vitro anti-inflammatory activity, which generated
information concerning their structure–activity relationship. The
structural variation within the set of compounds stems from
(i) changes in the substitution on C-8 and C-9, (ii) the num-
ber and positions of the double bonds, and (iii) the presence
or absence of an ether functionality between C-3 and C-10. The
highest activity was exerted by lobatin B (Lob 6); this com-
pound differs from 8␤-isovaleryloxy-9␣-hydroxy-calyculatolide
(Lob 8) only in the double bond at C4–C5, indicating the
importance of this olefin for the anti-inflammatory potency.
1700 R. McKinnon et al. / Phytomedicine 21 (2014) 1695–1701
Fig. 3. Effect of N. lobata extract and its components on the induction of E-selectin
protein after stimulation with LPS (A) or TNF-␣ (B) in HUVECtert cells. Cells were
treated with 5 ␮M compounds and 5 ␮g/ml extract 20 min prior to stimulation with
LPS or TNF-␣. E-selectin expression was measured by ELISA after 6 h. The abbre-
viations are: neurolenin A (Lob 1), neurolenin B (Lob 2), neurolenin C (Lob 3),
neurolenin D (Lob 4), lobatin A (Lob 5), lobatin B (Lob 6), 8␤-isovaleryloxy-9␣-
acetoxy-calyculatolide (Lob 7), 8␤-isovaleryloxy-9␣-hydroxy-calyculatolide (Lob
8), CH2 Cl2 phase of the MeOH N. extract (Extr). Values are mean ± S.E.M. of sex-
tuplicate measurements. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to LPS or
TNF-␣ activation (t test).
Comparison of 8␤-isovaleryloxy-9␣-hydroxy-calyculatolide (Lob
8) and 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide (Lob 7), differ-
ing solely in the C-9 substituent, demonstrates an increase in the
anti-inflammatory effect with acetylation at this position. This
Fig. 4. Influence of N. lobata extract and its components on the induction of IL-8
protein after stimulation with LPS in THP-1 cells. Cells were treated with 5 ␮M com-
pounds and 5 ␮g/ml extract 20 min prior to stimulation with LPS. IL-8 release was
measured by ELISA after 6 h. The abbreviations are: neurolenin A (Lob 1), neurolenin
B (Lob 2), neurolenin C (Lob 3), neurolenin D (Lob 4), lobatin A (Lob 5), lobatin B
(Lob 6), 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide (Lob 7), 8␤-isovaleryloxy-9␣-
hydroxy-calyculatolide (Lob 8), CH2 Cl2 phase of the MeOH N. lobata extract (Extr).
Values are mean ± S.E.M. of sextuplicate measurements. * p < 0.05, ** p < 0.01, ***
p < 0.001 as compared to LPS activation (t test).
Fig. 5. Effect of N. lobata extract and components on the relative mRNA expression
of IL-8 (A) and E-selectin (B) after stimulation with LPS in HUVECtert cells. Cells
were treated with 5 ␮M compounds and 5 ␮g/ml extract 20 min prior to stimulation
with LPS for 6 h. mRNA expression was determined by qPCR. Data are presented as
fold expression of mRNA in relation to untreated control. The abbreviations are:
neurolenin B (Lob 2), lobatin B (Lob 6), 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide
(Lob 7), CH2 Cl2 phase of the MeOH N. lobata extract (Extr). Values are mean ± S.E.M.
of six tuplicate measurements. * p < 0.05, ** p < 0.01 as compared to LPS activation
(ANOVA).
observation is supported by other structurally related pairs of
compounds. For example, neurolenin B (Lob 2) with a 9-acetoxy
group exhibited higher activity than neurolenin D (Lob 4) with a
9-hydroxy group.
Comparison of neurolenin B (Lob 2) and lobatin A (Lob 5), which
are different in regard to the position of an olefin group in the 10-
membered ring, revealed that C-2–C-3 double bond is preferred
regarding the anti-inflammatory effect. Furthermore, the higher
efficacy of neurolenin D (Lob 4) compared to that of neurolenin
Fig. 6. Paw edema volumes of rats treated with vehicle (5 ml/kg), CH2 Cl2 phase
of the MeOH N. lobata extract (Extr) (20 and 60 mg/kg) and dexamethasone (Dex)
(0.5 mg/kg) as reference agent. ** p < 0.01 as compared to the vehicle group (n = 9)
(ANOVA).
 R. McKinnon et al. / Phytomedicine 21 (2014) 1695–1701
C (Lob 3) demonstrated that the position of isovaleryloxy group at
C-8 is more favorable than on C-9.
Further in vitro testing of the three most active compounds,
lobatin B (Lob 6), 8␤-isovaleryloxy-9␣-acetoxy-calyculatolide (Lob
7), neurolenin B (Lob 2) showed that the structures significantly
down-regulated mRNA expression of the IL-8 (with the exception
of Lob 2) and E-selectin genes in HUVECtert cells stimulated with
LPS. Most probably, the missing 3,10 ether functionality accounts
for the lack of significant effect of Lob 2 on the expression of the
IL-8 gene.
The anti-inflammatory property of the extract was confirmed in
vivo by means of paw edema test in rats. Since the extract showed
high content of total sesquiterpene lactones with lobatin B (Lob
6) as major constituent, it is suggested that the sesquiterpene lac-
tones are responsible for the in vivo effect. Since the most common
mechanism of action associated with the activity of sesquiterpene
lactones is the inhibition of NF-kB (Merfort, 2011), future work
should address the effect of lobatin B and related compounds on
NF-kB and proteins involved in its regulation such as the inhibitor
of NF-kB (IkB) and the IkB kinase complex (IKK).
Taken together, the present study shows that sesquiterpene
lactones from N. lobata exert their anti-inflammatory activity by
preventing the induction of inflammatory cell adhesion molecules
and chemokines in endothelial and monocytic cells stimulated with
bacterial products and cytokines. Moreover, structure–activity
analysis of the compounds revealed the importance of the double
bond at C-4–C-5 and C-2–C-3 and the acetyl group at C-9 for the
anti-inflammatory activity. In vivo confirmation of the pharmaco-
logical effect raises further interest in the therapeutic potential of
lobatin B and related compounds.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors thank Dr. Peter Forgo and Dr. Nikoletta Jedlinszki
(Department of Pharmacognosy, University of Szeged, Hungary)
for the NMR and MS measurements, respectively. This publication
was supported by the European Union and co-funded by the Euro-
pean Social Fund (TÁMOP-4.2.2.A-11/1/KONV-2012-0035), as well
as a grant from the Austrian Science Foundation (FWF; grant NFN
S10713-B13 to Valery Bochkov). Financial support of the Hungarian
Scientific Research Fund (OTKA K109846) is gratefully acknowl-
edged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.phymed.
2014.07.019.
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