(+)-kunstlerone, a new antioxidant neolignan from the leaves of beilschmiedia kunstleri gamble

Article abstract of DOI:10.3390/molecules16086582

A new neolignan, 3,4-dimethoxy-3',4'-methylenedioxy-2,9-epoxy-6,7-cyclo- 1,8-neolign-11-en-5(5H)-one, which has been named (+)-kunstlerone (1), together with six known alkaloids: (+)-norboldine (2), (+)-N-methylisococlaurine (3), (+)-cassythicine (4), (+)

Full text of DOI:10.3390/molecules16086582

Molecules 2011, 16, 6582-6590; doi:10.3390/molecules16086582
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
(
+)-Kunstlerone, a New Antioxidant Neolignan from the Leaves
of Beilschmiedia kunstleri Gamble
1
1
1
2
Abbas Mollataghi , A Hamid A Hadi , Khalijah Awang , Jamaludin Mohamad , Marc
3
1,
Litaudon and Mat Ropi Mukhtar *
1
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia; E-Mails: abbas@siswa.um.edu.my (A.M.); ahamid@um.edu.my (A.H.A.H.);
khalijah@um.edu.my (K.A.)
2
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia; E-Mail: jamal@um.edu.my
3
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, 1, Avenue de la
Terrasse, 91198 Gif-sur-Yvette Cedex, France; E-Mail: marc.litaudon@icsn.cnrs-gif.fr
*
Author to whom correspondence should be addressed; E-Mail: matropi@um.edu.my;
Tel.: +603-79674048; Fax: +603-79674193.
Received: 31 May 2011; in revised form: 4 July 2011 / Accepted: 6 July 2011 /
Published: 4 August 2011
Abstract: A new neolignan, 3,4-dimethoxy-3′,4′-methylenedioxy-2,9-epoxy-6,7-cyclo-
1
,8-neolign-11-en-5(5H)-one, which has been named (+)-kunstlerone (1), together with six
known alkaloids: (+)-norboldine (2), (+)-N-methylisococlaurine (3), (+)-cassythicine (4),
+)-laurotetanine (5), (+)-boldine (6) and (-)-pallidine (7), were isolated from the leaves of
(
Beilschmiedia kunstleri. The structures were established through various spectroscopic
methods notably 1D- and 2D-NMR, UV, IR and LCMS-IT-TOF. (+)-Kunstlerone (1)
showed a strong antioxidant activity, with an SC50 of 20.0 µg/mL.
Keywords: Beilschmiedia kunstleri; alkaloid; neolignan; lauraceae; antioxidant
1
. Introduction
The Lauraceae family normally, with 30 genera and over 2,000 species, occurs throughout
Southeast Asia and tropical America [1,2]. In Malaysia, its contribution is about 213 species, from 16

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Molecules 2011, 16
genera [2]. Beilschmiedia species are known to produce many types of phytochemicals [3-12] with
varied biological activities such as O,O-dimethylcoclaurine isolated from Malaysian B. brevipes,
which exhibited significant cytotoxicity against P-388 murine leukemia cells with an IC value of
5
0
6
.5 μg/mL [13]. Besides the alkaloids, epoxyfuranoid lignans were also reported in the leaves of B.
tsangii [10]. Neolignans, whose precursors are the di- and trioxygenated cinnamic acids, have never
been reported so far in the species of Beilschmiedia; however they were detected in parts of other species
of Lauraceae such as in the fruits of Aniba riparia and in the leaves of Ocotea catharinensis [14-16].
In continuation of our search for new bioactive compounds from Malaysian flora [13,17], we have
performed a phytochemical study on the leaves of a Malaysian Lauraceae, Beilschmiedia kunstleri,
which has led to the isolation of a new neolignan; (+)-kunstlerone (1). In addition, six known
isoquinoline alkaloids, namely (+)-norboldine (2) [18], (+)-N-methylisococlaurine (3) [19],
(
+)-cassythicine (4) [20], (+)-laurotetanine (5) [21], (+)-boldine (6) [17] and (-)-pallidine (7) [22] were
also isolated (Figure 1). This paper describes the structural elucidation and the DPPH radical
scavenging activity of (+)-kunstlerone (1).
Figure 1. Chemical structures of compounds 1-7.
12
11
O
9
10
OCH3
H
H
2
3
4
8
1
6
5
7
1'
OCH3
2'
3'
O
O
6'
5'
4'
O
1
H CO
3
O
O
HO
NH
NCH3
NCH3
HO
H CO
3
H
H
H
H CO
3
H CO
3
HO
OH
OH
3
2
4
OH
H CO
3
H CO
HO
CO
3
NH
NCH
3
H CO
3
H
3
H
H
NCH3
H
H CO
3
H CO
3
H CO
3
O
OH
OH
6
7
5

6
584
Molecules 2011, 16
. Results and Discussion
2
+)-Kunstlerone, ꢀꢁꢂ ꢄꢃ ꢅ = +163.63° was obtained as a white amorphous solid. The LCMS-IT-TOF
(
+
revealed a pseudomolecular ion peak at m/z 371.1490 [M+H] , thus suggesting a molecular formula of
−
1
C H O (calc. 371.1495). The IR spectrum revealed absorption bands at 1,615 and 1,725 cm due to
2
1
23
6
1
the C=C and C=O stretching vibrations, respectively [14]. The H-NMR spectrum (see Table 1)
established the presence of three ABX type phenyl protons at δ 6.76 (1H, d, J = 8.1 Hz, H-5′), 6.71
(1H, br d, J = 8.1 Hz, H-6′), and 6.80 (1H, br s, H-2′)], two methoxyl groups at δ 4.08 and 3.69, a
methylenedioxy (δ 5.91, s, 2H), and protons of a propenyl group; H-11( δ 5.66, m), H -12 (δ 5.11, br
tran
d, 16.5 Hz) and Hcis -12 (δ 5.13, br d,11.0 Hz), respectively. The proton at C-7 of a cyclobutane moiety
showed significantly as a dd at δ 3.21 with coupling constants of 7.8 and 8.5 Hz, which indicated that
it is trans to H-8 (δ 2.83 m) and H-6 (δ 2.90 br d, 8.5 Hz) [23,24].
1
13
Table 1. H-NMR and C-NMR spectral data of (+)-kunstlerone (1) in CDCl (δ in ppm, J in Hz).
3
1
13
Position
H
C
-
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-1
C-2
C-3
C-4
C-5
C-6
C-7
132.12
107.25
147.97
146.39
108.32
119.69
46.01
6.80, br s
-
-
6.76, d, (8.1)
6.71, br d, (8.1)
-
4.32, s
79.11
-
159.47
138.46
193.10
50.16
-
-
2.90, d, (8.5)
3.21, dd,
44.58
(7.8, 8.5)
C-8
C-9
2.83, m
3.72, dd,
49.63
70.84
(
9.5, 3.9)
.90, d, (9.5)
2.38, m
3
C-10
C-11
C-12
41.76
136.51
119.41
5.66, m
5.13, d, (Jcis = 11.0),
5
.11, d, (Jtrans = 16.5)
5.91, s
OCH
2
O
101.08
58.46
60.57
3-OCH
3
4.08, s
3.69, s
4-OCH
3
1
3
The C-NMR spectrum (Table 1) indicated the presence of 21 carbon signals; two methyls, four
2
3
3
methylenes, four sp methines, four sp methines, six quaternary carbons and one carbonyl. The sp
quaternary carbon, C-1 bearing a propenyl group gave a signal at δ 46.0, while C-5 carbonyl peak was

6
585
Molecules 2011, 16
observed at δ 193.1. The correlations of H-7/C-1, H-9/C-1 and H-11/C-1 in the HMBC spectrum
further confirmed the position of propenyl fragment at C-1. In addition, the cross-peaks in the COSY
spectrum were observed between H-5′/H-6′, H-7/H-6, H-7/H-8, H-8/H-9, H-10/H-11and H-11/H-12 as
shown in Figure 2. Finally, thorough analysis of the COSY, HMQC and HMBC spectra allowed the
complete assignments of all protons and carbons of compound 1 (Table 1).
Figure 2. Selected HMBC and COSY correlations of (+)-kunstlerone (1).
12
O
9
1
0
OCH3
3
H
H
H
1
7
5
H
OCH3
1
'
3
'
O
O
5'
O
HMBC
COSY
The relative configuration of the five stereogenic centers of compound 1 was determined by the
correlations between H-6 (δ 2.90)/H-8 (δ 2.83), H-2/3-OCH (δ 4.08), H-6/H-10(δ 2.38) and H-7/H-
(δ 3.90) in the NOESY spectrum. Therefore, the relative configurations were assigned as 7R, 8R, 1R,
S and 2R, as depicted in Figure 3 [24].
3
9
6
Figure 3. Selected NOESY correlation of (+)-kunstlerone (1).
Antioxidant activity
(
+)-Kunstlerone (1) was tested for antioxidant activity using a DPPH radical scavenging activity
assay. The results have shown that it possesses a potent antioxidant activity, with scavenging capacity
SC50 = 20.0 µg/mL) comparable to that of ascorbic acid (SC50 = 14.0 µg/mL, Figure 4). The
antioxidant activities of neolignan glycosides from Verbascum salviifolium Boiss and neolignan
(

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Molecules 2011, 16
phenols from Nectandra grandiflora have been reported previously [25]. The mechanism of action for
the strong scavenging capacity of compound 1 is under investigation.
Figure 4. DPPH radical scavenging activity of the isolated new neolignan; (+)-kunstlerone
(
1) with an SC = 20.0 µg/mL.
50
3
. Experimental
3.1. General
The optical rotations were recorded on a JASCO (Japan) P1020 Polarimeter equipped with a
tungsten lamp; MeOH as solvent. The mass spectra were obtained from LCMS-IT-TOF, Shimadzu.
The ultraviolet spectra were obtained in MeOH on a Shimadzu UV-310 ultraviolet-visible
3
spectrometer. The Fourier Transform Infrared (FTIR) spectra were obtained with CHCl (NaCl
1
13
window technique) on a Perkin Elmer 2000 instrument. The H-NMR and C-NMR spectra were
recorded in deuterated chloroform on a JEOL 400 MHz spectrometer; chemical shifts are reported in
ppm on δ scale, and the coupling constants are given in Hz. Mayer’s reagent was used for alkaloid
screening. Aluminum TLC sheets and PTLC (20 × 20 cm Silica gel 60 F ) were used in the TLC
2
54
analysis. The TLC and PTLC spots were visualized under UV light (254 and 366 nm) followed by
spraying with Dragendorff’s reagent for an alkaloid detection. All solvents, except those used for bulk
extraction, were AR grade.
3.2. Plant materials
The leaves of Beilschmiedia kunstleri (Lauraceae) was collected from Hutan Simpan Sungai
Tekam, Jerantut, Pahang, Malaysia. The plant was identified by Mr. Teo Leong Eng. A voucher
specimen (KL5627) was deposited at the Herbarium of the Department of Chemistry, University of
Malaya, Kuala Lumpur, Malaysia and at the Herbarium of the Forest Research Institute, Kepong,
Malaysia.

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Molecules 2011, 16
.3. Extraction and isolation of neolignan and the alkaloids
The air-dried leaves (2.50 kg) of Beilschmiedia kunstleri Gamble were extracted exhaustively with
3
hexane (10.0 L) for 72 hours. The residual plant material was dried and left for 4 h after moistening
with 10% NH OH. It was then macerated with CH Cl (12.0 L) for four days. After filtration, the
4
2
2
supernatant was concentrated to 500 mL at room temperature (30 °C) followed by acidic extraction
with 5% HCl until a negative Mayer’s test result was obtained. The aqueous solution was made
alkaline to pH 11 with NH OH and re-extracted with CH Cl . This was followed by washing with
4
2
2
distilled H
The crude alkaloid (4.00 g) was subjected to exhaustive column chromatography over silica gel
column diameter = 2 cm, length =75 cm, silica gel 60, 70–230 mesh ASTM; Merck 7734) using
CH Cl gradually enriched with methanol (1% → 80% MeOH; 450 mL portions of eluent were used
2
O, dried over anhydrous sodium sulphate, and evaporation to give a crude alkaloid (4.50 g).
(
2
2
for each percentage) to yield 12 fractions. Purification of combined fractions 3–5 (0.4 g) using
preparative TLC (Merck KGaA silica gel 60 F ) afforded a neolignan identified as kunstlerone
2
54
(
1, 0.02%), pallidine (7, 0.08%, CH
2
Cl
2 2 2
-MeOH; 98:2) and norboldine (2, 0.07%, CH Cl -MeOH;
9
1
5:5). Fraction 9 afforded N-methylisococlaurine (3, 0.05%, CH Cl -MeOH; 98:2), and fractions 10–
2
2
1 (0.3 g) produced cassythicine (4, 0.03%; CH
Cl -MeOH; 98:2).
+)-Kunstlerone (1, Figure 1). ꢀꢁꢂ ꢄꢃ ꢅ = +163.63° (C = 2.37 × 10 M, MeOH), was obtained as a
white amorphous solid; UV max (MeOH): 404 (1.36) and 332 (4.00) nm; IR bands (KBr): 1,725,
2 2 2 2
Cl -MeOH; 97:3), laurotetanine (5, 0.01%, CH Cl -
MeOH; 98:2) and boldine (6, 0.04%, CH
2
2
−3
(
−
1
1
13
1
,654, 1,615, 1,491, 1,231, and 1,038 cm ; H-NMR (400 MHz, CDCl
3
) and C-NMR (100 MHz,
).
+
CDCl
3
): see Table 1; LCMS-IT-TOF, m/z: 371.1490 [M+H] (calc. 371.1495 for C21
H
23
O
6
3.4. Antioxidant assay
The free radical scavenging activity was determined using DPPH as described by Shimada et al.
26]. The DPPH radical scavenging activity assay is a decolorization assay that determines the activity
[
of antioxidants to directly react with DPPH stable free radical by observing its absorbance at 517 nm
with a spectrophotometer. A purple colored 1,1-diphenyl-2-picryl hydrazyl (DPPH), a stable free
radical which is reduced to α,α-diphenyl-β-picryl hydrazine and give yellow color when reacts with
antioxidant. The decolorization of purple color indicates the potential of antioxidants of the samples in
which increased decolorization shows the higher scavenging activity of the samples.
Briefly, 0.1 mM DPPH (1 mL) dissolved in ethanol was added to an ethanol solution (3 mL) of the
tested compound at different concentrations (25, 50, 100, 150, 200 µg/mL). An equal volume of
ethanol was added in the control test. The mixture was shaken vigorously and allowed to stand at room
temperature for 30 min. Then the absorbance at 517 nm was measured with a UV–VIS
spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical scavenging
activity. The percentage of scavenging of DPPH was calculated using the following equation:
ꢆ°ꢇꢆꢈ
DPPH scavenging effect (%) =
ꢉ 100
ꢆ°
o
where A is the absorbance of the control reaction and A1 is the absorbance in the presence of
the sample.

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Molecules 2011, 16
. Conclusions
This is the first communication on neolignans from the Beilschmiedia Kunstleri. To the knowledge
4
of the authors, kunstlerone (1) is the first neolignan reported in the family of Lauraceae bearing a
propenyl group at C-1 and the ether ring that is attached to C-2 and C-9. (+)-Kunstlerone (1) showed a
strong antioxidant activity with an SC of 20.0 µg/mL. This compound was also detected in neutral
5
0
fractions, showing that it could be isolated using an acid and base extraction. A proposed plausible
biogenetic pathway for 1 as shown in Scheme 1. Apparently, it results from the oxidative coupling
between the two precursors coniferyl alcohol and allyl-3,4,5-trihydroxybenzene. The primary hydroxyl
would give a Michael-type addition to α,β-carbonyl function and then, an enol addition to the quinone
methide (attack of C-6 to C-7) would give the cyclobutane ring. Finally, the dimer was O-methylated
at C-3 and C-4 and the methylenedioxy group was formed between the hydroxyl and methoxyl groups
of the coniferyl alcohol moiety. Alkaloids 2-7, which were identified in this study, belong to the
aporphine, benzylisoquinoline and morphinandienone type of alkaloids.
Scheme 1. Biogenetic pathway for kunstlerone (1).
Acknowledgements
The authors gratefully acknowledge the financial support provided by University of Malaya
Research Grant (UMRG RG045/11BIO), Centre of Natural Products and Drugs Development
(CENAR), Postgraduate Research Grant of University of Malaya (PS366/2010B) and CNRS Grant.

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Molecules 2011, 16
We also acknowledge the support given by Din Mohd Nor, Hasri Abdullah and Rafly Syamsir from
the Herbarium Group, University of Malaya. This work was carried out within the framework of an
official agreement between the CNRS and the University of Malaya (Malaysia).
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2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
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