Original Papers
icity than costunolide and 16. These results provided the first SAR
profile of sesquiterpene lactones and indicated that the α-methy-
lene-γ-lactone moiety plays a crucial role in TNF-α inhibition. Addi-
tionally, the epoxide derivative 6a might represent a lead compound
for further anti-TNF-α therapies, owing to its potent activity and re-
duced toxicity.
To isolate the active components, the hexane and EtOAc ex-
tracts were subjected to silica gel and Sephadex LH-20 column
chromatography followed by C-18 reversed-phase HPLC to obtain
10 sesquiterpenes (1–10) (▶ Fig. 1), together with one triterpene,
betulinic acid. The major metabolite (1), obtained on a gram
scale, was identified as costunolide from its spectroscopic data
and by comparison with data reported in the literature. Similarly,
the structures of compounds 2–10 were characterized by spectro-
scopic techniques and confirmed by comparing their 1D NMR
data with those reported previously [11–17].
To examine whether the isolated sesquiterpenes affected the
production of TNF-α in LPS-stimulated macrophage RAW264.7
cells, the cells were treated with the compounds at different con-
centrations in the presence of LPS and the levels of TNF-α in the
culture supernatant were measured by ELISA kit. TNF-α level sig-
nificantly increased in cells treated with LPS in comparison with
that in untreated control cells, as shown in ▶ Table 1. Among the
tested compounds, costunolide (1) and dehydrocostus lactone (4)
exhibited the most potent activity, with IC50 values of 2.05 and
2.06 µM, respectively. The activity of α- and β-cyclocostunolide
(6 and 7) was lower than that of 1 and 4, with IC50 values of 5.35
and 15.34 µM, respectively. The remaining compounds showed
IC50 values of over 20 µM, and were therefore deemed inactive.
Based on these results, the existence of an α-methylene-γ-lactone
moiety in the structure was considered crucial for activity, as de-
scribed previously [18,19]. Additionally, although costunolide (1)
showed strong inhibitory activity against TNF-α production, cell
viability, analyzed by MTT assay, markedly decreased (by up to
50% at 10 µM), as shown in ▶ Fig. 2.
In order to improve the activity and reduce the toxicity of the
compounds, some structural modification was performed and the
relationship between the activity and structure was studied. Since
several previous studies have revealed that α-methylene-γ-lac-
tone is a crucial building block of many natural products exhibit-
ing diverse biological activities including anti-inflammatory activ-
ity [20,21], it was considered important to retain this structural
moiety. To verify whether an α-methylene-γ-lactone moiety was
required for inhibition of TNF-α production, three derivatives, 11,
12, and 13, were prepared by methoxylation, basic hydrolysis, and
epoxidation of costunolide (1) [22], respectively (▶ Fig. 3), and
their activity was determined. As shown in ▶ Table 2, both 11
and 12, which had no α-methylene-γ-lactone moiety in their
structure, did not show any detectable activity even at the highest
concentration tested (50 µM), whereas compound 13 showed ac-
tive TNF-α inhibition. This result confirmed that the α-methylene-
γ-lactone moiety was essential for the anti-inflammatory effect on
TNF-α secretion in activated macrophages.
Introduction
Tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cyto-
kine, is an important mediator of inflammatory responses [1]. It
is mainly released by activated immune cells, such as macro-
phages and monocytes [2]. The excessive secretion of TNF-α has
been implicated in a diverse range of infectious and inflammatory
diseases, particularly Crohnʼs disease and rheumatoid arthritis [3].
The inhibition of this cytokine is thus considered an essential ap-
proach in the development of therapeutic agents. At present,
three drugs licensed as TNF-α blocking agents–infliximab, adali-
mumab, and etanercept–are available for treatment of rheuma-
toid arthritis and other inflammatory diseases [4]. Although effi-
cacious TNF-α inhibitors have already been developed, the chal-
lenge remains for researchers to identify more effective TNF-α in-
hibitors with reduced toxicity to treat various acute and chronic
inflammatory diseases.
The root of Saussurea lappa Clarke (Compositae), commonly
known as costus or kuth root [5], is used in various Indian Ayurve-
dic and Chinese traditional formulations for the treatment of ab-
dominal pain, distention, vomiting, allergy, and cancer [6, 7]. In
Southeast Asia, it has also been used to reduce fever and headache
and to treat diarrhea. In addition, the root extract has been used to
relieve syphilis in Japan [8]. This plant was found to be rich in ses-
quiterpenes, particularly sesquiterpene lactones, of which costu-
nolide is the main constituent. As the most abundant and
bioactive component of S. lappa root, costunolide is reported to
possess a broad range of biological activities including anti-cancer,
anti-ulcer, anti-bacterial, anti-hepatitis B virus, and anti-inflamma-
tory activities [9, 10]. However, few reports have examined the in-
hibition of TNF-α-induced inflammation by costunolide and other
sesquiterpene lactones isolated from S. lappa. Therefore, this
study was designed to investigate the anti-inflammatory activity
of isolated secondary metabolites from S. lappa root against TNF-
α inhibition. A number of semi-synthetic derivatives of sesquiter-
penes were also prepared and their activity was evaluated to clarify
the structure-activity relationship (SAR) of these compounds.
Results and Discussion
The anti-inflammatory effects of hexane, EtOAc, and MeOH ex-
tracts of S. lappa root were evaluated by measuring their potential
to inhibit TNF-α production in lipopolysaccharide (LPS)-stimulated
RAW264.7 macrophage cells. Both the hexane and EtOAc extracts
exhibited promising activity with IC50 values of 0.5 and 1.0 µg/mL,
respectively, while the MeOH extract did not show any significant
activity. Moreover, an MTT assay indicated that cell viability was
not affected by treatment with these extracts (Fig. 1S, Supporting
Information). These results indicate that the hexane and EtOAc
extracts of S. lappa root potently inhibited LPS-induced TNF-α re-
lease without affecting the viability of RAW264.7 cells.
As the main component, costunolide (1), a germacranolide-
type sesquiterpene, was used as the starting material for synthesis.
The 10-membered ring of this type of compound has been shown
to be highly prone to cyclization to a fused 6,6-bicyclic ring under
Choodej S et al. Inhibition of TNF-α-Induced… Planta Med