Alkenylresorcinols and cytotoxic activity of the constituents isolated from Labisia pumila

Article abstract of DOI:10.1016/j.phytochem.2012.04.008

Phytochemical investigation on the leaves of Labisia pumila (Myrsinaceae), an important medicinal herb in Malaysia, has led to the isolation of 1-O-methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol (1), labisiaquinone A (2) and labisiaquinone B (3). Along with these, 16 known compounds including 1-O-methyl-6-acetoxy-5-pentadecylresorcinol (4), 5-(pentadec-10Z-enyl)resorcinol (5), 5-(pentadecyl)resorcinol (6), (-)-loliolide (7), stigmasterol (8), 4-hydroxyphenylethylamine (9), 3,4,5-trihydroxybenzoic acid (10), 3,4-dihydroxybenzoic acid (11), (+)-catechin (12), (-)-epicatechin (13), kaempferol-3-O-α-rhamnopyranosyl-7-O-β-glycopyranoside (14), kaempferol-4′-O-β-glycopyranoside (15), quercetin-3-O-α- rhamnopyranoside (16), kaempferol-3-O-α-rhamnopyranoside (17), (9Z,12Z)-octadeca-9,12-dienoic acid (18) and stigmasterol-3-O-β- glycopyranoside (19) were also isolated. The structures of these compounds were established on the basis of 1D and 2D NMR spectroscopy techniques ( ¹H, ¹³C, COSY, HSQC, NOESY and HMBC experiments), mass spectrometry and chemical derivatization. Among the constituents tested 1 and 4 exhibited strongest cytotoxic activity against the PC3, HCT116 and MCF-7 cell lines (IC₅₀ values ≤10 μM), and they showed selectivity towards the first two-cell lines relative to the last one.

Full text of DOI:10.1016/j.phytochem.2012.04.008

Phytochemistry 80 (2012) 42–49
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Alkenylresorcinols and cytotoxic activity of the constituents isolated
from Labisia pumila
Nabil Ali Al-Mekhlafi a, Khozirah Shaari a,b, Faridah Abas a,c, Ralf Kneer d, Ethel Jeyaseela Jeyaraj e,
Johnson Stanslas e, Naoshi Yamamoto f, Toshio Honda f, Nordin H. Lajis a,b,
⇑
a Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia
b Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia
c Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia
d Borneo Plant Technology Sdn. Bhd., Kuching, Sarawak, Malaysia
e Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia
f Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41, Shinagawa, Tokyo 142-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 April 2011
Received in revised form 20 February 2012
Available online 26 May 2012
Phytochemical investigation on the leaves of Labisia pumila (Myrsinaceae), an important medicinal herb
in Malaysia, has led to the isolation of 1-O-methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol (1), labis-
iaquinone A (2) and labisiaquinone B (3). Along with these, 16 known compounds including 1-O-methyl-
6-acetoxy-5-pentadecylresorcinol (4), 5-(pentadec-10Z-enyl)resorcinol (5), 5-(pentadecyl)resorcinol (6),
(ꢀ)-loliolide (7), stigmasterol (8), 4-hydroxyphenylethylamine (9), 3,4,5-trihydroxybenzoic acid (10),
Keywords:
Labisia pumila
Myrsinaceae
Cytotoxicity
3,4-dihydroxybenzoic acid (11), (+)-catechin (12), (ꢀ)-epicatechin (13), kaempferol-3-O-
anosyl-7-O-b-glycopyranoside (14), kaempferol-40-O-b-glycopyranoside (15), quercetin-3-O-
pyranoside (16), kaempferol-3-O- -rhamnopyranoside (17), (9Z,12Z)-octadeca-9,12-dienoic acid (18)
a
-rhamnopyr-
a
-rhamno-
a
and stigmasterol-3-O-b-glycopyranoside (19) were also isolated. The structures of these compounds were
established on the basis of 1D and 2D NMR spectroscopy techniques (1H, 13C, COSY, HSQC, NOESY and
HMBC experiments), mass spectrometry and chemical derivatization. Among the constituents tested 1
and 4 exhibited strongest cytotoxic activity against the PC3, HCT116 and MCF-7 cell lines (IC50 values
1,4-Benzoquinone derivatives
Alkylresorcinol
610
l
M), and they showed selectivity towards the first two-cell lines relative to the last one.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Despite its long use in traditional medicine, it is only recently
that the herb was exploited commercially as health supplements.
Nowadays, products in the forms of tablets, capsules, health drinks
and tonics, derived from the plant extracts have become common
in pharmacy and retail outlets (Latiff, 1997; Singh et al., 2009).
The traditional uses of L. pumila are associated mainly with
estrogenic properties. This has been supported by several studies
on its biological effects (Jamal et al., 1998). The species also showed
antibacterial (Karimi et al., 2011), anti-inflammatory (Ibrahim et al.,
1996; Rasadah et al., 2001) and antioxidant (Mohamad et al., 2009)
properties. It has also been reported to reduce the risk of cardiovas-
cular diseases (Mohamud and Nazaimoon, 2009) and protect skin
cells from photo aging caused by UVB irradiation (Choi et al.,
2010). Plants of the Myrsinaceae family have been known to exhibit
Labisia pumila is a small herbaceous plant, belonging to the fam-
ily Myrsinaceae. It is widely distributed in the lowland and hilly
rain forests of Malaysia, Thailand, Indochina, the Philippines and
New Guinea (Jamia and Houghton, 2000; Ong, 2004; Stone, 1988;
Wiart and Wong, 2002). Three varieties of L. pumila are available
in Malaysia, classified as L. pumila var. alata, L. pumila var. pumila
and L. pumila var. Lanceolata (Stone, 1988). All of these are collec-
tively known as ‘‘Kacip Fatimah’’ among the local population.
L. pumila var. alata, has long been used in traditional medicine by
the indigenous communities in the Malay archipelago for the treat-
ing pre- and post-partum complications, menstrual disorders, in
addition to being used as a health tonic to regain vigor. Other tra-
ditional uses include for the treatment of dysentery, rheumatism
and anti-flatulence (Jaganath, 2000).
a
number of interesting pharmacological properties such as
´
anti-inflammatory, antiviral, anti-tumor (Kobayashi and De Mejia,
2005), cytotoxic (Chang et al., 2009), and antileishmanial
(Jimenez-Romero et al., 2007) activities. Investigations on a few
species of the Myrsinaceae family have revealed the presence of
various types of compounds including saponins, coumarins,
⇑
Corresponding author. Current address: Scientific Chairs Unit, College of
Medicine, Taibah University, P.O. Box 30001, Al-Madinah Al-Munawarah, Saudi
Arabia. Tel.: +60 3 89468082; fax: +60 3 89468080.
´
quinines (Kobayashi and De Mejia, 2005), triterpenoid saponins
E-mail address: nordinlajis@gmail.com (N.H. Lajis).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.04.008

N.A. Al-Mekhlafi et al. / Phytochemistry 80 (2012) 42–49
43
(Tang et al., 2009), hydroxyalkenylresorcinols (Jimenez-Romero
et al., 2007), alkenylphenols (Horgen et al., 1997), isoflavones (Kiku-
chi et al., 2009), dimeric lactones (Dat et al., 2007) and dimeric
benzoquinone derivatives (Liu et al., 2009). However, very little is
known about the phytochemistry of L. pumila. The medicinal impor-
tance of the species and the lack of information with regards to its
chemical constituents prompted this investigation on the plant.
Herein, identification of three new alkenylresorcinols, the occur-
rence of 16 other known compounds, and evaluation of the cyto-
toxic activity of some the isolated compounds are reported.
6.24) with C-6 (dC 131.7), C-1 (dC 151.8) and C-3 (dC 154.3), and be-
tween H-4 (dH 6.18) with C-6 (dC 131.7), C-2 (dC 98.4) and C-3 (dC
154.3), suggesting that the methoxyl and the hydroxyl groups are
located at C-1 and C-3, respectively. The connectivity of the alkenyl
chain to C-5 was established based on the key correlations ob-
served between H-10 (dH 2.39) and C-6 (dC 131.7), C-5 (dC 136.7),
as well as C-4 (dC 107.9).
The assignment of the double bond at C-100 was based on the
fragment ion peaks observed at m/z 348 [MꢀC3H7ꢀH]+ and 292
[MꢀC7H13ꢀH]+ resulting from the allylic cleavages (Suzuki et al.,
1996) (Scheme. 1). This was further confirmed by ozonolysis (Barr
et al., 1989) of the acetylated analogue (1c) which gave an alde-
hyde adduct (1d) exhibiting the protonated molecular ion peak
[M+H]+ at m/z 379.2119 (Scheme. 1). The configuration of the dou-
ble bond was assigned as Z based on the 13C NMR diagnostic chem-
ical shift value of the allylic carbons (dC 27.2), which differed
significantly from the reported chemical shift values of dC = 33
for the corresponding allylic carbons of E isomers (De Haan and
Van de Ven, 1973; Liu et al., 2009). On the basis of these consider-
ations, compound 1 was assigned as 1-O-methyl-6-acetoxy-5-
(pentadec-10Z-enyl)resorcinol. Recently, the 8(Z) isomer of this
compound was reported from Ardisia brevicaulis, also a member
of the Myrsinaceae family (Bao et al., 2010).
2. Results and discussion
The aqueous methanolic extract from the leaves of Labisia was
sequentially partitioned from water into CHCl3, EtOAc and n-BuOH
fractions. Subsequent chromatographic purification of each frac-
tion led to the isolation of three new compounds including an alkyl
resorcinol (1) and two dimeric 1,4-benzoquinone derivatives (2
and 3), in addition to 16 other known compounds. The structures
of these compounds were established mainly on the basis of anal-
ysis of NMR and mass spectroscopic data.
Compound 1 was obtained as colorless oil. The IR spectrum dis-
played absorptions at 3437 and at 1747 cmꢀ1 indicating the pres-
ence of hydroxyl and ester carbonyl functionalities, respectively.
The HRESI–MS of compound 1 exhibited a protonated molecular
ion [M+H]+ peak at m/z 391.2852 (calcd. for C24H38O4+H,
391.2843), which corresponded to the molecular formula
Compounds 2 and 3 were isolated as red semi-solids, both hav-
ing very similar UV, IR and NMR spectra. The UV and IR spectra
were indicative of the presence of a phenol and 1,4-benzoquinone
moieties, similar to those of belamcandaquinone found in Ardisia
species (Liu et al., 2009).
C24H38O4 and six degrees of unsaturation. Analysis of the NMR
The molecular formula of 2 was assigned C43H66O5 as deduced
from HR-ESI-MS, which exhibited a protonated molecular ion
[M+H]+ at m/z 663.4999 (calcd. for C43H66O5+H, 663.4989), thus
indicating 11 degrees of unsaturation. In the IR spectrum, absorp-
tion bands for hydroxyl was observed at 3283 cmꢀ1, for 1,4-qui-
none at 1679 and 1638 cmꢀ1, and for alkenyl at 1619 and
1601 cmꢀ1. The 1HNMR spectrum exhibited resonances for two
alkenyl side chains [dH 5.32 (4H, m), 2.00 (8H, m), 2.33 (1H, m),
2.26 (2H, m), 2.16 (1H, m), 1.41–1.20 (38H, m) and 0.89 (6H, t,
J = 6.5)], a methoxyl (3H, dH 3.83, s) and three aromatic [dH 6.21
(1H, s), 6.14 (1H, s) and 5.97 (1H, s)] protons. The 13CNMR spectrum
of 2 (Table 2), which displayed two signals for carbonyl groups (dC
188.8, 182.4) and 10 resonances in the aromatic region (dC 101.3–
159.3) supported the presence of 1,4-benzoquinone and phenyl
rings. Furthermore, the spectrum also exhibited signals for a meth-
oxyl (dC 56.4), olefinic (dC 130.1) and allylic (dC 27.4) carbons.
Inspection on key HMBC correlations (Fig. 2) as well as comparison
with literature data suggested the substitution pattern on the two
rings, and a C4-C10 linkage between the two rings (Fukuyama et al.,
1993; Liu et al., 2009). Meanwhile, the HMBC correlations observed
between H-7ax (dH 2.33) and C-2 (dC 182.2), C-3 (dC 147.3) and C-4
(dC 141.2), established the connectivity of one alkenyl chain at C-3,
whereas correlations observed between H-70 (dH 2.26) and C-10 (dC
112.3), C-20 (dC 143.1) and C-30 (dC 108.6), placed the other alkenyl
chain on C-20. The length of the two alkenyl chains was as a C15 unit
based on the ESI-MS (positive-ion mode) fragmentation pattern,
where two fragment ion peaks corresponding to 1,4-benzoquinone
(A) and phenyl (B) moieties at m/z 345 and 317, respectively, were
observed.
spectroscopic data of 1 indicated the presence of a tetra-substi-
tuted aromatic ring with substituents being an alkenyl chain, a hy-
droxyl, a methoxyl and an acetyl groups. The 1H NMR spectrum
showed a pair of aromatic meta-coupled protons at d 6.24 (H-2)
and 6.18 (H-4), as well as proton signals belonging to a methoxyl
group at d 3.71, olefinic methines at d 5.35 (overlapped, H-100, H-
110), two allylic methylenes at d 2.01 (overlapped, H-90, H-120),
methylene at 1.50 (m, H-20), eight other methylenes at d 1.32–
1.27 (overlapped), and a terminal methyl at d 0.89 (t, H-150). Anal-
ysis of 13C NMR spectrum supported these deductions by the sig-
nals assignable to the tetra-substituted benzene ring carbons and
to the C15 alkenyl chain (Table 1). Resonances for the methyl and
carbonyl carbons of acetyl group was also observed at dC 20.7 (dH
2.33, 3H, s) and 170.3, respectively. The substitution pattern on
the ring was further deduced based on HMBC correlations
(Fig. 1). Correlations between methoxyl protons at (dH 3.71) with
C-1 (dC 151.8) were observed, in addition to those of H-2 (dH
Table 1
1H and 13C NMR spectroscopic data for compound 1 measured in CDCl3.
Position
dH (J in Hz)
dC
1
2
3
4
151.8
98.4
6.24 (d, J = 2.5 Hz)
6.18 (d, J = 2.5 Hz)
154.3
107.9
136.7
131.7
32.2
30.3
29.6–30.2
27.2
130.0
130.0
27.2
22.6
14.3
5
6
10
2.39 (t, J = 7.5 Hz)
1.50 (m)
1.32–1.27 (m)
2.01 (m)
5.35 (m)
5.35 (m)
20
30–80
90
As in the case of 1, assignments of the double bonds in the alke-
nyl chains was accomplished by ozonolysis of the acetylated ana-
logue (2b), which gave an aldehyde adduct (2c) having the
sodiated molecular ion peak [M+Na]+ at m/z 661.3328 in its HRE-
SIMS (Scheme 2). On the basis of these considerations, the posi-
tions of the double bond were assigned to C-16, C-17 and C-160,
C-170 carbons. The configurations of the double bonds on the alke-
nyl chains were deduced as Z based on chemical shift values of the
allylic C-15 and C-18 carbons, which appeared as an overlapped
100
110
120
2.01 (m)
130–140
1.32–1.27 (m)
0.89 (t, J = 7.0 Hz)
3.71 (s)
150
2-OCH3
56.1
2.32 (s)
170.3
1-OCOCH3
1-OCOCH3
–
20.7

44
N.A. Al-Mekhlafi et al. / Phytochemistry 80 (2012) 42–49
O
O
CH3
C
O
CH3
O
10'
11'
15'
H3CO
H3CO
H
6
6
3
1
6
5
H
1
1a
OH
OH
Fig. 1. Structure of compound 1 and its key HMBC correlations (1a).
O
154 M- Ac-C14H27-H
H3C
O
348 M- C3H7-H
292 M- C7H13-H
H3CO
6
1b
OH
OAc
OAc
OH
OAc
OAc
I. O3/CH2Cl2
H3CO
H3CO
CHO
H3CO
Ac2O
Py, rt
-78 ºC
8
8
8
3
3
II. Me2S
OAc
1c
m/z 432
1
1d
m/z 390
m/z 378
Scheme 1. Key fragmentation (1B) and aldehyde adduct formation by ozonolysis of an acetylated analogue of 1.
Table 2
C
43H68O5+H, 665.5140), and consistent with 10 degrees of unsatu-
1H and 13C NMR spectroscopic data of compounds 2 and 3 measured in CDCl3.
ration. The 1H NMR spectrum of this compound was identical to 2,
except for the integration values of the olefinic and allylic protons,
which were equivalent for only two and four protons, respectively
(see Fig. 3). The position of the double bond on the alkenyl side-
chain was confirmed at C-160 and C-170 based on ozonolysis of
the acetylated analogue (3b) of 3, which gave aldehyde adduct
(3c) having the sodiated molecular ion peak [M+Na]+ at m/z
717.4303 in its HRESIMS (Scheme 3). In a similar manner, as in
the previous compounds, the configuration of the double bond
was assigned as Z based on the chemical shift value of the allylic
carbons (dC 27.4).
Position
Compound 2
dH (J in Hz)
Compound 3
dH (J in Hz)
dC
dC
1
2
3
4
5
6
–
–
–
–
159.3
182.4
147.3
141.2
188.8
107.6
27.1
–
–
–
–
159.2
182.4
147.2
141.1
188.0
107.6
27.1
–
–
5.97 (s)
2.33 (m)
2.16 (m)
1.20–1.41(m)
1.41 (m)
2.00 (m)
5.32 (m)
5.32 (m)
2.00 (m)
1.20–1.41 (m)
5.98 (s)
7eq
7ax
8–13
14
15
16
17
18
19
20
21
1-OCH3
10
2.33 (m)
2.16 (m)
1.15–1.41 (m)
1.41 (m)
2.01 (m)
5.35 (m)
5.35 (m)
2.01 (m)
1.26 (m)
1.26(m)
0.88 (t, J = 6.5 Hz)
3.84 (s)
–
–
28.4–30.1
28.4
27.4
130.1
130.1
27.4
32.2
22.6
14.3
56.4
112.3
143.1
108.6
156.7
101.3
153.9
33.8
28.4–30.1
28.4
27.4
130.1
130.1
27.4
32.2
22.6
28.4–30.3
28.4
27.4
130.1
130.1
27.4
32.2
22.6
14.4
56.5
112.6
143.4
108.6
156.8
101.2
153.8
33.7
28.4–30.3
28.4–30.3
28.4–30.3
28.4–30.3
28.4–30.3
28.4–30.3
32.2
The attachment of the alkenyl moiety was established to be at
the lower phenyl ring (ring-B) based on the results of ESI/MS/MS
experiment conducted on compound 3. Exertion of collision energy
on the protonated molecular ion at m/z 665 led to the fragmenta-
tion, of which the prominent fragments at m/z 317 and 347 were
assignable to the alkenylresorcinol 3d and alkylbenzoquinone 3e
cations, respectively. On account of these arguments, 2 and 3 were
therefore assigned labisiaquinone A and B, respectively. It is worth
mentioning that labisiaquinone A has recently been reported (Ali
and Khan, 2011) after our submission of this report for publication.
Sixteen other compounds were also isolated from this species
including1-O-methyl-6-acetoxy-5-pentadecylresorcinol (4) (Chang
et al., 2009), 5-(pentadec-10Z-enyl)resorcinol (5) (Occolowitz and
Wright, 1962), 5-(pentadecyl)resorcinol (6) (Suzuki et al., 1996),
(ꢀ)-loliolide (7) (Kimura and Maki, 2001), stigmasterol (8) (Misra
et al., 1984), hydroxyphenylethylamine (9) (Elgorashi et al., 2001),
3,4,5-trihydroxybenzoic acid (10) (Gottlieb et al., 1991), 3,4-dihy-
droxybenzoic acid (11) (Ban et al., 2007), (+)-catechin (12)
(Nahrstedt et al., 1987), (ꢀ)-epicatechin (13) (Ban et al., 2006),
0.89 (t, J = 6.5 Hz)
3.83 (s)
–
–
–
20
–
30
6.21 (s)
–
6.14 (s)
–
6.28 (s)
–
6.16 (s)
–
40
50
60
70
2.26 (m)
1.20–1.41 (m)
1.20 (m)
2.00 (m)
5.32 (m)
5.32 (m)
2,00 (m)
1.20 (m)
1.41 (m)
0.89 (t, J = 6.5 Hz)
2.23 (m)
1.15–141 (m)
1.15–141 (m)
1.15–141 (m)
1.15–141 (m)
1.15–141(m)
1.15–141 (m)
1.15–141 (m)
1.15–141 (m)
0.88 (t, J = 6.5 Hz)
80–130
140
150
160
170
180
190
200
210
22.6
14.4
kaempferol-3-O-
a-rhamnopyranosyl-7-O-b-glycopyranoside (14)
14.3
(Chen et al., 2007), kaempferol-40-O-b-glycopyranoside (15)
(Nørbæk and Kondo, 1999), quercetin-3-O-
(16) (Zhong et al., 1997), kaempferol-3-O-
a
a
-rhamnopyranoside
-rhamnopyranoside
signal at dC 27.4 ppm (De Haan and Van de Ven, 1973; Liu et al.,
2009).
(17) (Mizuno et al., 1990), (9Z,12Z)-octadeca-9,12-dienoic acid
(18) (Liu et al., 2004) and stigmasterol 3-O-b-glycopyranoside (19)
(Leitão et al., 1994). The identity of these compounds was confirmed
The molecular formula of 3 was determined as C43H68O5 by
HRESI-MS, which gave an [M+H]+at m/z 665.5136 (calcd. for

N.A. Al-Mekhlafi et al. / Phytochemistry 80 (2012) 42–49
45
OCH3
OCH3
H
O
O
1
A 2
17
6
CH321
16
21
5
16 17
4
7
5
O
HO
O
1'
B
4'
2'
OH
6'
'
7'
5
CH3
16' 17'
16'
17'
21'
5'
21'
3'
H
H
2
2a
OH
OH
Fig. 2. Structure of compound 2 and its key HMBC correlations (2a).
OCH3
OCH3
O
OCH3
O
O
CHO
CHO
I. O3/CH2Cl2
Ac2O
Py, rt
O
OH
O
AcO
9
9
O
AcO
9
9
9
9
-78 ºC
II. Me2S
OH
OAc
OAc
2
2c
2b
m/z 664
m/z 746
m/z 638
Scheme 2. Aldehyde adduct formation by ozonolysis of an acetylated analogue of 2.
OCH3
OCH3
O
H
O
1
A 2
6
5
O
21
21
4
7
5
O
HO
CH3
1'
2'
OH 6'
7'
CH3
5
16' 17'
21'
16'
17'
B
21'
5'
3'
H
H
4'
OH
3
3a
OH
Fig. 3. Structure of compound 3 and its key HMBC correlations (3a).
OCH3
OCH3
O
OCH3
O
O
I. O3/CH2Cl2
-78 ºC
II. Me2S
Ac2O
Py, rt
O
OH
O
AcO
9
9
9
9
O
AcO
14
9
CHO
OH
OAc
3b
OAc
3
3c
m/z 666
m/z 748
m/z 694
OCH3
HO
O
O
OH
3d
3e
m/z 347
m/z 317
Scheme 3. Alkenyl resorcinol (3d), alkyl benzoquinone (3e) cation fragments, and aldehyde adduct formation by ozonolysis of an acetylated analogue of 3.
by analysis of their spectroscopic data as well as by comparison of
these and their physical properties, with those of the literature.
Twelve of the isolated compounds (1–6, 9, 11, 13, 14, 16 and 17)
activity in the preliminary screening and hence, the compounds
were tested against PC-3, HCT-116 and MCF-7 cells to obtain the
growth inhibitory parameters (Table 3). The benefit of testing
against the three cell lines is it provides information on the poten-
tial selectivity of a compound. Although a limited number of cell
lines were used for the assessment of cytotoxic selectivity, it gives
an initial indication on the therapeutic value of a compound. It has
were screened for cytotoxic activity at 10 lM against MCF-7 breast
cancer cells. Only compounds displaying >50% cell death were se-
lected for dose–response curves to obtain the three parameters
(GI50, TGI and LC50). Compounds 1 and 4 displayed the desired

46
N.A. Al-Mekhlafi et al. / Phytochemistry 80 (2012) 42–49
Table 3
In vitro cytotoxic activity of compounds 1 and 4 towards three cancer cell lines.
Compound
O
Growth inhibitory parameters (lM)
Cell line
GI50
TGI
1.2 0.0
1.0 0.1
15.7 2.1
LC50
8.0 1.7
7.7 6.4
41.3 2.3
PC-3
HCT-116
MCF-7
0.3 0.0
0.3 0.0
0.4 0.1
CH3
C
O
H3CO
6
1
OH
PC-3
HCT-116
MCF-7
0.4 0.1
0.3 0.0
0.5 0.1
1.6 0.4
1.6 0.3
15.0 1.7
10.0 4.4
12.0 8.7
40.0 0.0
O
CH3
C
O
H3CO
6
4
OH
Doxorubicina
PC-3
HCT-116
MCF-7
0.7 0.1
0.6 0.1
0.6 0.1
4.0 0.2
2.0 0.2
1.9 0.2
20.0 0.5
5.0 0.5
5.5 0.5
The cell lines used were: human prostate (PC-3), colon (HCT-116) and breast (MCF-7) cancer cells. The cells were treated for 96 h with at least four different concentrations of
compounds ranging from 0.1 to 100 M. MTT assay (Mosmann, 1983) was used to calculate GI50, TGI and LC50 values (expressed in M). Values are mean of three
l
l
independent experiments and errors represent the SD values.
a
Doxorubicin was used as a positive control.
been well established that compounds with cancer-type selectivity
are beneficial in cancer chemotherapy. The GI50 values of 1 and 4
were similar in all the cell lines. However, when the TGI values
were taken into consideration, the MCF-7 cells were less sensitive
approximately by 15-fold when compared with PC-3 and HCT-116
cells. A similar pattern was observed at the LC50 level, in which the
compounds were approximately 5-fold more active against PC-3
and HCT-116 cells as compared with MCF-7 cells. Therefore,
although the selectivity was not discerned at the GI50 level, the
compounds displayed selectivity for PC-3 and HCT-116 cells at
the TGI and LC50 levels. The clinically used anticancer agent
doxorubicin, which was used as a positive control, exhibited
sub-micromolar to low micromolar growth inhibitory activities.
It is of interest to note that 1 and 4 were more potent than doxo-
rubicin at the GI50 level.
Structurally, 1 and 4 are closely related. While the alkyl chain in
the former contains a double bond, the alkyl chain of the latter
does not. The results therefore suggested that the occurrence of
double bond moiety in the alkyl chain does not cause any differ-
ence in cytotoxic activity. Similar results have been observed on
the relative phytotoxicity of lipid benzoquinones with various lev-
els of unsaturation (Kagan et al., 2003). Cytotoxic activity of 4 and
(8Z)-pentadecenyl isomer of 1 isolated from Ardisia virens and
A. brevicaulis, respectively has been reported recently (Bao et al.,
2010; Chang et al., 2009). These compounds also showed similar
cytotoxic activity pattern based on their IC50 values towards
A459, MCF-7, PANC-1, NCI-H460 and SF-268 cancer cell lines,
same Myrsinaceae family. Compounds 1 and 4 had the strongest
cytotoxic activity at equipotent submicromolar growth inhibition
(GI50) in the tested cancer cell lines. More importantly, they exhib-
ited cancer-type selectivity against PC-3 and HCT-116 cells at the
TGI and LC50 levels.
4. Experimental
4.1. General experimental procedures
Melting points were recorded on a Koffler hot-stage apparatus
and were uncorrected. The UV and IR spectra were recorded on a
Varian UV–VIS 50 and Perkin–Elmer 1650 FTIR spectrophotome-
ters, respectively. 1D NMR and 2D NMR spectra were acquired
on a Varian Unity 500 MHz spectrometer either in CDCl3, ace-
tone-d6, or CD3OD. ESI–MS were determined using JEOL JMS-T
100LP spectrometer and HRESI–MS were determined using a Finn-
iganLTQ-Orbitrap Discovery mass spectrometer. The chemical
shifts (d) were determined from residual solvent peaks. Column
chromatography was performed using silica gel Merck 7734 (70–
230 mesh ASTM), Merck 9385 (230–400 mesh ASTM)and reversed
phase silica gel Merck 10167. Sephadex LH-20 (Fluka) was em-
ployed to purify the isolated compounds or remove chlorophyll.
TLC analyses were carried out on Merck silica gel DC-plastikfolien
60 F254 plastic sheets with detection accomplished by spraying
with 10% H2SO4 followed by heating at 100 °C, or by visualizing
with a UV lamp at 254 and 366 nm. Semi-preparative HPLC system
consisting of a JASCO pump PU-2089 plus and JASCO absorbance
detector model UV-2077 plus 4-k intelligent UV/VIS detector
linked by JASCO ChromNAV version 1.09.03 software. Semi-pre-
although their growth inhibitory parameters (GI50, TGI and LC50
were not measured.
)
parative HPLC column used was XterraC18 OBD 5
l
m (19ꢁ
3. Concluding remarks
150 mm), with the solvent system used was acetonitrile-H2O
(70:30–100:0, over 45 min) or (0:100–100:0) at flow rate 5 ml/
min.
A comprehensive phytochemical investigation on the leaves of
L. pumila led to the isolation of nineteen compounds (1–19), three
of which (1–3) were new. Their structures were established on the
basis of spectroscopic data, and the assignments of the double
bond in the alkyl chain were confirmed by ozonolysis followed
by ESI-MS analysis. In chemotaxonomic sense, the constituents of
L. pumila are closely related to Ardisia species, a genus from the
4.2. Plant material
Fresh leaves of L. pumila were obtained from Pahang which was
supplier from F. A. Herbs Sdn. Bhd., Shah Alam, Malaysia in March

N.A. Al-Mekhlafi et al. / Phytochemistry 80 (2012) 42–49
47
2008. They were cleaned, chopped into small pieces (3–5 mm
thickness), and dried in the shade. voucher specimen
(ACP0084/08) was identified by Dr. Shamsul Khamis (A Resident
Botanist) and deposited at the Herbarium of the Laboratory of Nat-
ural Products, Institute of Bioscience, Universiti Putra Malaysia.
CHCl3–MeOH to give fractions E2/1 (95 mg; from the 10% MeOH
eluate), E2/2 (312 mg; 35% MeOH) and E2/3 (180 mg; 100% MeOH).
Subfraction E2/2 (312 mg) was fractionated by a Sephadex LH-20
column and eluted with MeOH to afford four fractions (E2/2/1–E2/
2/4). Subfraction E2/2/2 was subjected to semi-preparative RP HPLC
to afford two pure compounds, 3,4,5-trihydroxybenzoic acid (10;
4 mg; tR 17.9) and 3,4-dihydroxybenzoic acid (11; 3 mg; tR 22.0).
Fraction E4 (1.31 g) was applied to normal-phase silica gel CC
and eluting with a gradient polarity with MeOH (0–100%) in CHCl3
to give three subfractions (E4/1–E4/3). The second subfraction E4/2
(325 mg) was separated by Sephadex LH-20 column chromatogra-
A
4.3. Extraction and isolation
The dried leaves (1.5 kg) were ground and extracted with
MeOH–H2O (80:20), three times at room temperature. The crude
extract (about 214 g) was obtained after evaporating the solvent
in vacuo. The crude extract was then fractionated by sequential
liquid–liquid partitioning of H2O and with n-hexane, CHCl3, EtOAc
and n-BuOH to give 57.7, 5.4, 25.1 and 44.2 g of the respective
fractions.
phy using MeOH as mobile phase to afford five subfractions (E4/2/1
–
E4/2/5), the subfractionE4/2/5 (90 mg) was further separated by
semi-preparative HPLC to give two pure compounds, (+)-catechin
(12; 3 mg; tR 24.75) and (ꢀ)-epicatechin (13; 8 mg; tR 25.63).
Fraction E5 (1.45 g) was applied to a silica gel column using
CHCl3–MeOH (100–0, 0–100, v/v) for elution, which afforded six
fractions (E5/1–E5/6). Fraction E5/4 (235 mg) was passed through a
Sephadex LH-20 column with MeOH as eluant to give three frac-
tions (E5/4/1–E5/4/3). Subfraction E5/4/2 (120 mg) was purified by
The n-hexane fraction (15 g) was subjected to flash column chro-
matography (CC) on reversed phase C18 column, eluted with
MeOH–H2O (8:2–1:0), to give 23 fractions. Fractions with similar
TLC pattern were combined to yield five major combined fractions
(H1–H5). Fraction H2 (136 mg) was reapplied to a silica gel column
eluted with n-hexane–EtOAc (1:0–0:1, v/v) to afford (9Z,12Z)-
octadeca-9,12-dienoic acid (18, 19 mg). Further separation of frac-
tion H3 (3.5 g) by silica gel CC was performed with n-hexane–EtOAc
(1:0–0:1, v/v) as eluent to afford four subfractions (H3/1–H3/4).
Subfraction H3/1 (1.515 g) was then subjected to semi-preparative
reversed phase HPLC to yield four compounds, 5-(pentadec-
10Z-enyl)resorcinol (5; 25.3 mg; tR 21.5), 1-O-methyl-6-
acetoxy-5-(pentadec-10Z-enyl)resorcinol (1; 45 mg; tR 23.6),
5-(pentadecyl)resorcinol (6; 10 mg; tR 25.5) and 1-O-methyl-6-
acetoxy-5-pentadecylresorcinol (4; 39 mg; tR 27.5). Fraction H5
(0.525 mg) was purified by Sephadex LH-20 eluted with MeOH to
afford three subfractions (H5/1–H5/3). Subfraction H5/2 (395 mg)
was then applied to semi-preparative reversed phase HPLC to yield
labisiaquinone A (2; 120 mg; tR 46.2) and labisiaquinone B (3;
42 mg; tR 63.0). Subfraction H5/3 (100 mg) was subjected to silica
gel CC, eluted with n-hexane–EtOAc (4:1–0:1) to give stigmas-
terol-3-O-b-glycopyranoside (19, 22 mg).
The crude CHCl3 fraction of L. pumila leaves (4.5 g) was fraction-
ated by Sephadex LH-20 column, eluting with CHCl3–MeOH (70:30
v/v) to give 22 subfractions, and all fractions with similar chro-
matograms were combined (based on TLC pattern) and evaporated
to dryness under reduced pressure to afford four major fractions
(C1–C4). Fraction C1 (2.8 g) was separated by silica gel CC and
eluted with CHCl3–acetone (1:0–0:1) to give seven fractions (C1/
1–C1/7). Fraction C1/3 (47 mg) was further purified by silica gel CC,
eluted with n-hexane–acetone (4:1–3:2) to give (ꢀ)-loliolide (7;
5 mg). Fraction C3 (164 mg) was subjected to silica gel CC and
eluted with CHCl3–acetone (1:0–0:1), to give four subfractions
(C3/1–C3/4). Fraction C3/3 (36 mg) was further applied to a silica
semi-preparative HPLC to yield kaempferol-3-O-a-rhamnopyrano-
syl-7-O-b-glycopyranoside (14; 23 mg; tR 25.81). Fraction E7
(1.75 g) was separated over a Sephadex LH-20 column and eluted
with MeOH to give four subfractions (E7/1–E7/4). Subfraction E7/4
(531 mg) was subjected to silica gel CC (CHCl3–MeOH gradient elu-
tion), yielding four fractions (E7/4/1–E7/4/4). Subfraction E7/4/3
(276 mg) was further separated by semi-preparative HPLC to
afford three pure compounds, kaempferol-40-O-b-glycopyranoside
(15; 3 mg; tR 30.02), quercetin-3-O-
a-rhamnopyranoside (16;
8 mg; tR 30.35) and kaempferol-3-O-
a-rhamnopyranoside (17;
120 mg, tR 32.80).
4.4. Ozonolysis
Compound 1 (7.1 mg, 0.020 mmol) was dissolved in pyridine
(0.5 ml) and Ac2O (0.5 ml) and the resulting mixture was allowed
to stand for 12 h at room temperature. The mixture was concen-
trated under reduced pressure to afford 1c quantitatively. A stream
of O3 was bubbled into a solution of 1c in CH2Cl2 (2.0 ml) for
0.5 min at -78 °C and excess ozone was removed by a stream of
Ar. The reaction mixture was treated with Me2S (0.1 ml) at
ꢀ78 °C and the mixture was warmed to room temperature during
the period of 3 h, and stirred for further 3 h at room temperature.
The mixture was concentrated under reduced pressure and
purified by PLC (EtOAc/n-hexane; 1:2) to afford 1d (1 mg,
0.0026 mmol) as brown oil. The same reaction conditions of ozon-
olysis of 1 were applied for the ozonolysis of compounds 2 and 3 to
give acetylated analogue (2b, 3b) and an aldehyde adduct (2c, 3c).
gel column to give kaempferol-3-O-a-rhamnopyranoside (17;
4.4.1. 1-O-Methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol (1)
19 mg) after elution with CHCl3–MeOH (4:1 v/v). Fraction C4
(175 mg) was treated with MeOH to give stigmasterol (8; 5 mg)
as a residue and the MeOH soluble was subjected to silica gel CC
with CHCl3–MeOH (4:1 v/v) as eluant to afford quercetin-3-O-
Colorlessoil; UV (MeOH) kmax nm (loge): 229 (3.34), 274 (3.27);
IR (thin film) vmax cmꢀ1: 3437, 2926, 2857, 1747, 1609, 1191; neg-
ative ESI–MS: m/z 390 [MꢀH]ꢀ, 347, 153, 292; positive HRESI–MS
m/z 391.2852 [M+H]+, (calcd. for C24H38O4+H, 391.2843); for
1HNMR (500 MHz, CDCl3) and 13CNMR (125 MHz, CDCl3) spectro-
scopic data, see Table 1.
a-rhamnopyranoside (16; 3 mg).
A portion of the EtOAc fraction (13 g) was subjected to re-
versed-phase silica gel CC, eluted with H2O–MeOH (100:0–0:100,
v/v) to yield 45 fractions. Each fraction (200 ml) was examined
by TLC, and fractions with similar TLC patterns were combined to
yield ten major fractions (E1–E10). Fraction E1 (628 mg) was sepa-
rated into four subfractions (E1/1–E1/4) by chromatography over
Sephadex LH-20 with MeOH as eluent. The E1/4 subfraction
(198 mg) was subjected to silica gel CC eluted with CHCl3–MeOH
(100–0, 0–100, v/v) to give 4-hydroxyphenylethylamine (9;
15 mg). Fraction E2 (600 mg) was fractionated using a solid-phase
extraction (SPE) cartridge over normal-phase silica and eluted with
4.4.2. Labisiaquinone A (2)
Red semisolid; UV (MeOH) kmax nm (loge): 268 (4.03), 223
(4.01); IR (KBr, disc) mmax cmꢀ1: 3283, 2925, 2854, 2683, 1679,
1638, 1619, 1600, 1507, 1457, 1339, 1228, 1148, 1053, 847, 722;
negative ESI–MS: m/z 661 [MꢀH]ꢀ, 437, 345, 317; positive HR–
ESI–MS m/z 663.4999 [M+H]+, (calcd. for C43H66O5+H, 663.4989);
for 1H NMR (500 MHz, CDCl3) and 13CNMR (125 MHz, CDCl3) spec-
troscopic data, see Table 2.

48
N.A. Al-Mekhlafi et al. / Phytochemistry 80 (2012) 42–49
4.4.3. Labisiaquinone B (3)
day 4, AC = absorbance of control cells at day 4, A0 = absorbance
on day 0.
Red semisolid; UV (MeOH) kmax nm (loge): 271 (4.01), 220
(3.97); IR (KBr, disc) mmax cmꢀ1: 3283, 2925, 2854, 2683, 1679,
1638, 1619, 1600, 1507, 1457, 1339, 1228, 1148, 1053, 847, 722;
positive ESI–MS: m/z 665 [M+H]+, 633, 582, 469, 439, 317; positive
HR–ESI–MS m/z 665.5136 [M+H]+, (calcd. for C43H68O5+H,
665.5140); for 1HNMR (500 MHz, CDCl3) and 13CNMR (125 MHz,
CDCl3) spectroscopic data, see Table 2.
Acknowledgements
The authors wish to thank Universiti Putra Malaysia for provid-
ing the financial under the Research University Grant
Scheme (RUGS) No. 91178. The author (NHL) also wishes to thank
the Scientific Chairs Unit, Taiban University for support.
4.5. Cytotoxic assay
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