The article was published by Academy of Chemistry of Globe Publications
www.acgpubs.org/RNP © Published 05/01/2014 EISSN:
Rec. Nat. Prod. 8:3 (2014) 286-289
New Inhibitors of the DENV-NS5 RdRp from Carpolepis laurifolia
as Potential Antiviral Drugs for Dengue Treatment
, Alexandre Maciuk
, Cécilia Eydoux
, Edouard Hnawia
, Bruno Figadère
, Jean-Claude Guillemot
Laboratoire Insulaire du Vivant et de l’Environnement, Université de la Nouvelle-Calédonie, BP R4,
98851 Nouméa Cedex, Nouvelle-Calédonie
Laboratoire BioCIS, UMR CNRS 8076 Université Paris-Sud XI, 5 rue J.-B. Clément, 92296
Laboratoire AFMB, UMR 6098, Université Aix-Marseille, Campus de Luminy, 13288
Marseille cedex 09, France
(Received May 31, 2013; Revised November 6, 2013; Accepted December 14, 2013)
Abstract: Since a few decades the dengue virus became a major public health concern and no treatment is
available yet. In order to propose potential antidengue compounds for chemotherapy we focused on DENV RNA
polymerase (DENV-NS5 RdRp) which is specific and essential for the virus replication. Carpolepis laurifolia
belongs to the Myrtaceae and is used as febrifuge in traditional kanak medicine. Leaf extract of this plant has
been identified as a hit against the DENV-NS5 RdRp. Here we present a bioguided fractionation of the leaf
extract of C. laurifolia which is also the first phytochemical evaluation of this plant. Five flavonoids, namely
quercetin (1), 6-methylapigenin-7-methylether (2), avicularin (3), quercitrin (4) and hyperoside (5), together with
betulinic acid (6), were isolated from the leaf extract of C. laurifolia. All isolated compounds were tested
individually against the DENV-NS5 RdRp and compared with four other commercial flavonoids: isoquercitrin
(7), spiraeoside (8), quercetin-3,4’-di-O-glucoside (9) and rutin (10). Compounds 3, 4, 6, 8 and 10 displayed IC
ranging from 1.7 to 2.1 µM, and were the most active against the DENV-NS5 RdRp.
Keywords: Myrtaceae; Carpolepis laurifolia; Dengue Virus; Flavonoids; Triterpenes.
© 2014 ACG Publications. All rights reserved.
1. Plant Source
The Myrtaceae family is represented by 235 species in New Caledonia (NC), and 234 of them
are endemic to this island. Thus, all three species of Carpolepis are endemic to NC and has never been
investigated for their chemical content.
The leaves of Carpolepis laurifolia J.W.Dawson. were harvested in December 2009 at 480
meters high on the plateau de Tango (20°59'04'' S; 165°05'05' E ), in the North Province of NC
(harvesting authorization n° 609122852-2009). A voucher specimen (De Rus 27) was deposited at
Herbarium of the IRD center of Noumea (NOU) and identified by Dr. Jérome Munzinger (IRD).
Corresponding author: E- Mail: firstname.lastname@example.org; Phone: 00687 290252.
Antoine et al., Rec. Nat. Prod. (2014) 8:3 286-289
2. Previous Studies
Since a few decades the dengue virus (DENV) became the most prevalent and widespread
arthropod borne virus affecting human and no treatment is available yet. In order to find new agents
for chemotherapy, the viral DENV-NS5 RNA polymerase (DENV-NS5 RdRp) has been identified as a
target of choice for antidengue drug research . Previously, few natural compounds were described
for their inhibitory activity on the DENV-NS5 RdRp [2,3]. Among those compounds, biflavonoids
were the most active .
3. Present Study
During the screening of medicinal plants for antidengue activity, the crude extract obtained
from the leaves of Carpolepis laurifolia, an endemic species of Myrtaceae from NC, was found to be
particularly active on the DENV polymerase (91% inhibition at 10 µg/mL) and did not show any
cytotoxicity on MRC5 cells at 10 µg/mL. This plant was selected for further investigations. A
phytochemical screening led on the leaf powder underlined the abundance of flavonoids in leaves of
C. laurifolia which could be responsible of the antidengue potential of the plant.
340 g of dry leaf powder obtained from C. laurifolia were successively extracted at room
temperature with petroleum ether (3 × 1 L, 3h each) to obtain the fraction A (8.5 g) and with MeOH
80% (3 × 1 L, 3h each) to obtain fraction B (23.5 g). Methanolic extract (B) was reduced to 1 L and
extracted by EtOAc (3 × 1 L) to obtain B.2 (20 g). The petroleum ether extract (A) exhibited low
inhibitory activity on DENV-NS5 RdRp while methanol extract (B) and ethyle acetate part of
methanol extract (B.2) conserved the antidengue potential measured during the screening (data not
shown). The bioguided fractionation of B.2 allowed us to isolate five flavonoids (1-5) namely
quercetin (1), 6-methyl-7-methoxyapigenin (2), avicularin (3), quercitrin (4), hyperoside (5) together
with the triterpene betulinic acid (6). Purification was achieved by successive fractionations on silica
gel columns, Sephadex LH-20 and preparative HPLC. The purity of all tested compounds (> 95 %)
was assessed by HPLC-UV/MS. Spectroscopic data (MS,
C NMR) of all compounds were
consistent with published data [5-7]. Copies of the original spectra are obtainable from the
The biological activity on the DENV-NS5 RdRp of all compounds (Figure 1) was evaluated
individually and compared with four other commercial flavonoids: Isoquercitrine (7), spiraeoside (8),
quercetin-3,4’-di-O-glucoside (9) and rutin (10), purchased from Extrasynthèse. The DENV
polymerase activity was assayed by monitoring the incorporation of radiolabeled guanosine into a
homopolymeric cytosine RNA template, as previously described . The d-GTP was used as positive
Among tested compounds, the active flavonoids (1, 3, 4, 8 and 10) exhibited IC
DENV-NS5 RdRp ranging from 1.7 to 3.6 µM. The avicularine (3) was the most active flavonoid
tested here (IC
= 1.7 µM). According to the results obtained in this study (see Figure 1), the structure
of the genin significantly modulates the inhibitory activity of the DENV-NS5 RdRp and even small
modification of it can influence the activity measured on the DENV polymerase. Thus we can observe
that quercetin (1) was significantly more active than the C-methylapigenin derivative (2). Also, the
structure and position of the substituants on the quercetin modulated the inhibitory activity of the
DENV-NS5 RdRp. Indeed the avicularin (3) and quercitrin (4) with a rhamnose and arabinose
substituant in C3 respectively, demonstrated high inhibitory activity of DENV polymerase while the
activity dramatically decreased for hyperoside (5) and isoquercitrin (7) with a galactose and glucose in
the same position (Figure 1). Also, the spiraeoside (8) substituted by a glucose in C4’, strongly
inhibited the DENV-NS5 RdRp (IC
= 1.9 µM) whereas the additional glucose induced a loss of
activity for quercetin-3,4’-di-O-glucoside (9).
New inhibitors of the DENV-NS5 RdRp from Carpolepis laurifolia
1 (3.6 µM) 2 (> 30 µM) 3 (1.7 µM) 4 (2.1 µM)
5 (> 20 µM) 6 (1.7 µM) 7 (9.5 µM) 8 (1.9 µM)
9 (> 80 µM) 10 (2.1 µM)
Figure 1. Inhibition of the DENV-NS5 RdRp (IC
in µM) by compounds from C. laurifolia
(1-6) and other related commercial flavonoids monomers (7-10).
The flavonoids tested here have been previously described for various biological activities
including antiviral  but were tested for the first time for antidengue potential. We should note that
other flavonoids were previously described for antidengue activity [3, 10, 11]. Interestingly, betulinic
acid (6) isolated from C. laurifolia, was one of the stronger inhibitor of the DENV-NS5 RrRp among
tested compounds (IC
= 1.7 µM). This compound was previously described for several biological
activities including antiviral activity against HIV  but it is the first report for antidengue potential
of a triterpene. It will be interesting in the future to evaluate the activity of other triterpene derivatives
on the DENV-NS5 RdRp.
To the most of our knowledge, this study represents the first phytochemical study of a
Carpolepis species, an endemic genus in New-Caledonia, from the Myrtaceae family. This plant
appeared to be rich in quercetin derivatives (such as 1, 3, 4 and 5). This result is consistent with the
literature since those quercetin derivatives are frequently observed in the Myrtaceae species. The
presence of C-methyl apigenin derivatives (such as 2), that are less common in plant kingdom,
confirms that Carpolepis genus belong to the clade of Metrosideroideæ, in the phylogeny of
Myrtaceae. Indeed, those compounds have been isolated in the leaves of several species of this group,
including Metrosideros spp. . The leaf crude extract of this plant strongly inhibited the DENV-
NS5 RdRp and was not cytotoxic. Five flavonoids (1-5) and betulinic acid (6) were isolated from the
active fractions of this plant. The activity of all isolated compounds was determined individually on
the DENV-NS5 RdRp and compared with the activity of other four commercial flavonoids (7-10).
This study allowed us to identify five flavonoids (1, 3, 4, 8 and 10) and a triterpene (6), with high
potential for antidengue drug development.
This work was supported by a grant from the fond Pacifique. Authors are grateful to Karine
Leblanc for preparative HPLC. We also thank Aleksandra Grudniewska for the corrections and
commentaries of this article. The authors do not declare any conflict of interest with the presented data
from this article.
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