Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina with antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA)

Article abstract of DOI:10.1016/j.phytochem.2014.07.028

Galloylated flavonol rhamnosides identified as kaempferol-3-O-(2″,3″,4″-tri-O-galloyl)-α-l-rhamnopyranoside, quercetin-3-O-(3″,4″-di-O-galloyl)-α-l-rhamnopyranoside, and quercetin-3-O-(2″,3″,4″-tri-O-galloyl)-α-l-rhamnopyranoside, together with five known

Full text of DOI:10.1016/j.phytochem.2014.07.028

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina
with antibacterial activity against methicillin-resistant Staphylococcus
aureus (MRSA)
a
b
b
a
a,
⇑
Elaine Wan Ling Chan , Alexander I. Gray , John O. Igoli , Sui Mae Lee , Joo Kheng Goh
a
School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor, Malaysia
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom
b
a r t i c l e i n f o
a b s t r a c t
0
0
00 00
Article history:
Galloylated flavonol rhamnosides identified as kaempferol-3-O-(2 ,3 ,4 -tri-O-galloyl)-
a
-L
-rhamnopyr-
Received 29 October 2013
Received in revised form 18 July 2014
Available online xxxx
00 00
00 00 00
anoside, quercetin-3-O-(3 ,4 -di-O-galloyl)-a-L-rhamnopyranoside, and quercetin-3-O-(2 ,3 ,4 -tri-O-
galloyl)-a-L-rhamnopyranoside, together with five known galloylated and non-galloylated flavonol
rhamnosides, were isolated from leaves of Calliandra tergemina (L.) Benth. Their structures were estab-
lished using spectroscopic methods and their antibacterial activities against methicillin-resistant Staph-
ylococcus aureus (MRSA) were evaluated by a microdilution method.
Keywords:
Calliandra tergemina
Leguminosae
Ó 2014 Elsevier Ltd. All rights reserved.
Flavonol rhamnosides
Antimicrobial
Methicillin-resistant Staphylococcus aureus
1
. Introduction
The genus Calliandra belongs to the family Leguminosae and
This paper describes the isolation, structure elucidation and the
structure–activity relationships of the isolated compounds.
subfamily Mimosaceae. The family has about 200 species of flow-
ering plants native to tropical America, Madagascar and India. Sev-
eral species are planted as ornamental plants (Mattagajasingh
et al., 2006). There is, however, presently no paper describing the
chemical constituents and bioactivity of Calliandra tergemina
although there are reports on several related species including Cal-
liandra portoricensis, Calliandra californica and Calliandra haemato-
cephala (Aguwa and Lawal, 1988; Encarnacion et al., 1994; Agunu
et al., 2005; Moharram et al., 2006; Orishadipe et al., 2010). Preli-
minary screening of the leaf extracts of C. tergemina displayed anti-
bacterial activity against Micrococcus luteus, Bacillus cereus,
methicillin-sensitive Staphylococcus aureus (MSSA) and methicil-
lin-resistant Staphylococcus aureus (MRSA), respectively (Chew
et al., 2011). The activity against MRSA is interesting as the latter
is a major cause of infections in hospitals and resistant to all
known beta-lactam antibiotics (Otsuka et al., 2008; Chew et al.,
2. Results and discussion
Five crude extracts were obtained from the leaves of C. terge-
mina of which the ethyl acetate extract had the highest anti-
MRSA activity (Table 1); and from his extract, eight flavonol gly-
cosides were isolated using a combination of silica gel column
and gel filtration chromatography. Three new compounds were
identified by their NMR ( H, C, HSQC, HMBC), UV and mass
spectra, optical rotations, and by comparison with previously
reported spectroscopic data. The five known galloylated and
1
13
0
0
non-galloylated flavonol rhamnosides were quercetin-3-O-(3 -O-
galloyl)- -rhamnopyranoside (4) (Moharram et al., 2006),
quercetin-3-O- -rhamnopyranoside (5) (Eldahshan, 2011),
kaempferol-3-O-(2 ,3 -di-O-galloyl)-
a-L
a-L
0
0
00
a
-
L
-rhamnopyranoside
(6)
0
0
2
011). Thus a bioassay-guided fractionation of C. tergemina leaf
(Lee et al., 2009), kaempferol-3-O-(3 -O-galloyl)-a-L-rhamnopyr-
anoside (7) (Mahmoud et al., 2001) and kaempferol-3-O-
extracts, coupled with chromatographic separation; was employed
to isolate the active constituents. This yielded three new galloylat-
ed flavonol rhamnosides and five known flavonol rhamnosides.
a-L-
rhamnopyranoside (8) (Eldahshan, 2011); respectively.
0
0
00 00
Kaempferol-3-O-(2 ,3 ,4 -tri-O-galloyl)-
a-
L
-rhamnopyranoside
-rhamnopyranoside (2)
and quercetin-3-O-(2 ,3 ,4 -tri-O-galloyl)- -rhamnopyranoside
0
0
00
(
1), quercetin-3-O-(3 ,4 -di-O-galloyl)-a-L
0
0
00 00
a-L
⇑
Corresponding author. Tel.: +60 3 5514 6108; fax: +60 3 5514 6364.
E-mail address: goh.joo.kheng@monash.edu (J.K. Goh).
(3) are new and their 1D and 2D NMR and HRESIMS spectra are
provided as Supporting information (Figs. S1–S3).
http://dx.doi.org/10.1016/j.phytochem.2014.07.028
031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Chan, E.W.L., et al. Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina with antibacterial activity
against methicillin-resistant Staphylococcus aureus (MRSA). Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.028

2
E.W.L. Chan et al. / Phytochemistry xxx (2014) xxx–xxx
0
0
Table 1
face of the sugar; i.e. H-1 did not have a 1,3-diaxial relationship
Antimicrobial activity of different solvent extracts of C. tergemina leaves.
00
00
00
with H-3 and H-5 . Therefore, H-1 must be equatorial and on
the opposite side of the sugar from H-3 and H-5 as in
0
0
00
Extract/control
Diameter of zone of inhibition (mm)
a-L-
rhamnosides. The NOESY experiment also exhibited correlations
MRSA ATCC
33591)
MRSA (Clinical
Isolate 1)
MRSA (Clinical
Isolate 2)
0
0
00
00
(
between the sugar protons (H-1 , 2 and 3 ) and the aromatic pro-
0
0
tons H-2 /6 giving further proof of the rhamnosyl attachment in C-
3. The absolute configuration of the rhamnose was determined to
Vancomycin
Hexane
Dichloromethane
Ethyl Acetate
Methanol
21.0 ± 0.0
20.2 ± 0.3
18.5 ± 0.5
*
*
*
nd
nd
nd
nd*
nd*
nd*
be L-based on the optical rotation after acid hydrolysis of com-
15.2 ± 0.3
13.0 ± 0.0
11.2 ± 0.3
pound 1. Thus, compound 1 was identified as kaempferol-3-O-
13.0 ± 0.0
11.0 ± 0.0
10.5 ± 0.5
00 00 00
nd*
nd*
nd*
(2 ,3 ,4 -tri-O-galloyl)-
a-
L
-rhamnopyranoside.
The
complete
Water
1
13
assignment of the H and C NMR resonances was made possible
Vancomycin (0.030 mg/disc); Plant extract (1 mg/100
means of three replicates ± SD.
ll per disc). Values are the
by analysis of its HMBC and HSQC spectra (S1).
00 00
Compound 2, quercetin-3-O-(3 ,4 -di-O-galloyl)-a-L-rhamno-
⁄
(
nd) non-detectable.
pyranoside, was also obtained as a yellow amorphous powder. Its
ꢀ
Q-TOFMS gave a molecular ion peak at m/z 751.1156 for [MꢀH]
1
attributed to a molecular formula of C35
28
H O
19. The H NMR spec-
troscopic data of compound 2 (Table 3 and S2) showed the pres-
2
.1. Structural elucidation of 1–3
ence of an ABX-type aromatic system, this being characteristic of
0
0
a 3 ,4 -disubstituted B-ring of a flavone with the ring protons at
0
0
00 00
0
0
Compound 1, kaempferol-3-O-(2 ,3 ,4 -tri-O-galloyl)-
a
-
L
-
H-2 (d
H
7.61, d, J = 2.1 Hz), H-6 (d
7.14, d, J = 8.3 Hz), respectively. Two doublets with meta
6.31 (1H, d, J = 2.1 Hz) and 6.52 (1H, d, J = 2.1 Hz)
H
7.50, dd, J = 8.3, 2.1 Hz) and
0
rhamnopyranoside, was obtained as a yellow amorphous powder.
H-5 (d
H
ꢀ
Its Q-TOFMS gave a molecular ion at m/z 887.1322 for [MꢀH] ,
couplings at d
H
1
supporting a molecular formula of C42
H
32
O
22. In the H NMR spec-
were observed for the H-6 and H-8 of the 5,7-dihydroxy substi-
tuted ring A of the flavonol moiety. The carbohydrate proton region
was similar to that of compound 1, and there were also resonances
trum of 1 (Table 3 and S1), two doublets with meta couplings were
observed at d 6.31 (H-6) and d 6.53 (H-8) for the 5,7-dihydroxy
A-ring, and two aromatic doublets characteristic of a 1,4-disubsti-
tuted benzene ring at d
also suggested the flavonol moiety was kaempferol. A proton sin-
glet due to an –OH group at C-5, chelated to the carbonyl at C-4,
was also observed at d
ter proton correlated with C-5, C-6 and C-10. In the carbohydrate
region, there were signals for a secondary methyl doublet at d
H
H
0
0
for a secondary methyl signal at d
H
0.88 (3H, d,H-6 ), an anomeric
0
0
0
0
00
H
7.22 (H-3 /H-5 ) and d
H
8.03 (H-2 /H-6 )
proton at d
H
5.78 (d, H-1 ) and four oxygen-bearing methines at d
4.62 (br dd, H-2 , whose position is shielded compared to that of H-
2 in 1), 5.46 (dd, H-3 ), 5.41 (t, H-4 ) and 3.62 (dq, H-5 ), respec-
tively. This indicated that compound 2 was a quercetin derivative.
The presence of two galloyl moieties in compound 2 was inferred
from the two-proton singlets observed in the aromatic region of
its H NMR spectrum due to H-2 and H-6 of the galloyl groups.
These resonances were similar to the H NMR spectroscopic data
for quercetin 3 -O-galloyl-rhamnopyranoside and quercetin
2 ,3 -di-O-galloyl-rhamnopyranoside (Moharram et al., 2006;
H
0
0
0
0
00
00
00
H
12.57 ppm. In the HMBC spectrum, this lat-
H
00
00
1
000
000
0
.99 (3H, d, H-6 ), an anomeric proton at d
H
5.97 (d, H-1 ) and four
00
00
1
oxygen-bearing methines at d 5.98 (dd, H-2 ), 5.70 (dd, H-3 ),
5
pound 1 was a kaempferol rhamnoside derivative. The H NMR
spectrum of 1 was also similar to that of kaempferol-3-O-
rhamnopyranoside (8) and kaempferol-3-O-(2 ,3 -di-O-galloyl)-
H
00
00
00
.42 (t, H-4 ), and 3.71 (dq, H-5 ). These data suggested that com-
1
00 00
a
-L
-
Eldahshan, 2011), except for the position of the galloyl groups.
00
00
The positions of the galloyl groups in compound 2 were deter-
0
0
00
a
-
L-rhamnopyranoside (6), (Lee et al., 2009; Eldahshan, 2011),
mined to be at C-3 and C-4 on the basis of the down-field shift
0
0
00
00
except for one additional galloyl moiety. The rhamnose H-1 pro-
of H-3 (d
H
= 5.46) and H-4 (d
H
= 5.41) compared to compound 5
ton of 1 showed a weak correlation to a carbon at 133.7 ppm (C-
with no galloyl groups (d
loyl group at C-3 (d = 5.27, 3.80), respectively. The C NMR spec-
H
H
= 3.51, 3.20) and compound 4 with a gal-
0
0
13
3
) in a similar fashion to that shown in the HMBC spectrum for
compound 8. The galloylation of the rhamnosyl group in 1 was evi-
trum was similar to that of compound 1; the carbonyl signals at
dent from the three sets of galloyl protons appearing as two-proton
165.0 and 165.5 were assigned to the two galloyl groups and the
methyl carbon for the rhamnosyl group (C-6 ) was observed at
0
0
singlets at d
H
6.98, 7.01 and 7.17 ppm. The positions of substitution
of the galloyl groups were determined by analysis of the down-
16.8 ppm. Similarly, the small J value (1.8 Hz) of the anomeric pro-
00
00
field shifts of the sugar protons: H-2 (d
H
5.98), H-3 (d
H
5.70)
ton at d
H
5.78 in compound 2 and correlations in its 2D NMR spec-
-configuration for the
00
and H-4 (d
H
5.42) compared to 8 with no galloyl groups (d
H
tra similar to 1 also indicated the a
00
3
3
5
.47, 3.97, 3.08), 7 with a galloyl group at C-3 (d
.79), and 6 with two galloyl groups at C-2 and C-3 (d
.50, 3.83), respectively. Similar down-field shifts of the rhamnosyl
H
4.56, 5.24,
rhamnosyl moiety. Compound 2 was thus identified as quercetin-
00
00
00 00
H
5.94,
3-O-(3 ,4 -di-O-galloyl)-a-L-rhamnopyranoside.
0
0
00 00
Compound 3, quercetin-3-O-(2 ,3 ,4 -tri-galloyl)-
pyranoside, was similarly obtained as a yellow amorphous powder.
Its molecular formula was determined to be C42 23, based on
its Q-TOFMS, which gave a molecular ion peak at m/z 903.1264
a-L-rhamno-
00
00 00
protons have been reported for kaempferol-3-O-(2 ,3 ,4 -tri-O-
acetyl-rhamnopyranoside (Usia et al., 2004). In its 13C NMR spec-
trum, three carbonyl carbons from the galloyl groups were
32
H O
ꢀ
1
13
observed at d
of the rhamnosyl moiety (C-6 ) being at d
ASANOESY spectrum, the methyl protons (H-6 , d
C
164.5, 164.9 and 165.2 ppm, with the methyl carbon
[MꢀH] . The H NMR, C NMR, COSY, HSQC and HMBC spectra
00
1
1
C
16.9 ppm. In the H– H
of compound 3 were similar to those of compound 2 except for
00
1
H
0.99) correlated
5.41). That the latter two protons
were axial was deduced by the large coupling constant (J = 10.0 Hz)
an additional galloyl group in compound 3. The H NMR spectrum
00
00
with H-5 (d
H
3.71) and H-4 (d
H
showed three aromatic singlets for three galloyl groups at 7.16,
7.03 and 6.98 ppm. The positions of attachment of the galloyl
groups in compound 3 were deduced as for compound 1 which
00
00
and H-3 had the same coupling constant with H-4 indicating that
00
00
00
it was also axial, while H-2 must be equatorial as the coupling
showed very similar downfield shifts of H-2 (d
H
5.99), H-3 (d
H
00
00
00
00
constant to H-3 was small (J = 3.1 Hz). H-3 and H-5 had strong
5.66) and H-4 (d
H
5.41). This was further confirmed by analysis
of its C NMR spectrum with three carbonyl carbons for the galloyl
groups at d 164.6, 164.9 and 165.3 ppm; the 2D NMR correlations,
1
3
NOE interactions with one another indicative of their 1,3-diaxial
00
orientation. The rhamnosyl anomeric proton (H-1 ,
d
H
5.97,
C
J = 1.8 Hz) did not have any detectable NOE interactions with H-
as well as chemical shifts for the flavonoid nucleus, were also
almost identical with that of 2 while those of the sugar mirrored
0
0
00
3
nor H-5 showing that these three protons were not on the same
Please cite this article in press as: Chan, E.W.L., et al. Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina with antibacterial activity
against methicillin-resistant Staphylococcus aureus (MRSA). Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.028

E.W.L. Chan et al. / Phytochemistry xxx (2014) xxx–xxx
3
Fig. 1. Structures of isolated compounds 1–8.
those of 1. Thus compound 3 was identified as quercetin-3-O-
the antioxidant activity of galloylated flavonol rhamnosides has a
direct correlation with the number of galloyl groups (Moharram
et al., 2006). The results obtained here, however, showed that
esterification of the rhamnoside moiety was more important in
conferring anti-MRSA activity than the corresponding flavonol
rhamnoside and there was no correlation between anti-MRSA
activity and the degree of esterification. Antibacterial activity for
the flavonol rhamnosides could be increased by multiple esterifica-
tion of the rhamnoside moiety. Although the anti-MRSA activity of
the isolated compounds was low, these flavonol rhamnosides
might be able to reverse the susceptibility of MRSA towards b-lac-
tam antibiotics when used in combination as flavonoids with a low
antibacterial activity have been found to intensify the susceptibil-
ity of MRSA towards b-lactam antibiotics at a concentration much
lower than the MIC (Sato et al., 2004).
0
0
00 00
(
2 ,3 ,4 -tri-O-galloyl)-a-L-rhamnopyranoside (Fig. 1).
2
2
.2. Biological evaluation
.2.1. Antibacterial activity against MRSA
Compounds 1, 2, 3 and 6 with multiple galloyl groups showed
weak antibacterial activity against three strains of MRSA at
56 g/mL (Table 2). There was no detectable anti-MRSA activity
2
l
for the compounds without galloyl groups or with a single galloyl
group. These results were consistent with those reported for
kaempferol and quercetin rhamnosides with para-coumaric acid
esters (Otsuka et al., 2008). Antibacterial activities were observed
for the acetylated rhamnosides, but not for the non-acylated
kaempferol or quercetin rhamnosides (Otsuka et al., 2008). How-
ever, flavonol with acylated coumaroyl groups seem to possess
more potent antibacterial activity against MRSA than flavonol with
acylated galloyl groups. In addition, the degree of esterification
was not directly correlated to the antibacterial activity as the com-
pounds with two and three galloyl groups had the same minimum
inhibitory concentration (MIC). This is in contrast to the report that
2.2.2. Effects of flavonol rhamnosides on MRSA cells
The morphological changes of MRSA cells after treatment with
C. tergemina leaf extracts and the flavonol rhamnosides were
observed under a scanning electron microscope (SEM) to deter-
mine the mode of action of the flavonol rhamnosides on MRSA
Table 2
Minimum inhibition concentrations (MICs) of flavonol rhamnosides from C. tergemina.
Compounds/Extracts
Number of galloyl groups
Minimum inhibition concentration (lg/mL)
MRSA ATCC (33591)
MRSA (Clinical Isolate 1)
MRSA (Clinical Isolate 2)
Vancomycin (control)
4
4
4
Ethyl acetate extract
512
512
512
1
2
3
4
5
6
7
8
3
2
3
1
0
2
1
0
256
256
256
>1000
>1000
256
>1000
>1000
256
256
256
>1000
>1000
256
>1000
>1000
256
256
256
>1000
>1000
256
>1000
>1000
Compound 1, 6, 7 and 8 were kaempferol rhamnoside derivatives while compound 2, 3, 4 and 5 were quercetin rhamnoside derivatives.
Please cite this article in press as: Chan, E.W.L., et al. Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina with antibacterial activity
against methicillin-resistant Staphylococcus aureus (MRSA). Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.028

4
E.W.L. Chan et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 2. Scanning electron microscope (SEM) photograph of MRSA treated with ethyl acetate leaf extracts and isolated active compounds of C. tergemina at 512
MICs). (A) Control; (B) treated with ethyl acetate leaf extracts; kaempferol derivatives: (C) treated with compound 1; (D) treated with compound 6; quercetin derivatives: (E)
treated with compound 2; (F) treated with compound 3.
lg/mL (2ꢁ
Table 3
1
H NMR spectroscopic data of 1–3 in (CD
3
)
2
CO at 400 MHz. d
H
in ppm and (J values in Hz).
Moiety
Position
1
2
3
6
8
2 /6
6.31 (d, 2.1)
6.53 (d, 2.1)
8.03 (d, 8.8)
–
7.22 (d, 8.8)
–
6.31 (d, 2.1)
6.52 (d, 2.1)
–
7.61 (d, 2.1)
–
7.14 (d, 8.4)
7.50 (dd, 8.4, 2.1)
12.68 (s)
5.78 (d, 1.8)
4.62 (br dd, 2.8, 1.8)
5.46 (dd, 10.1, 2.8)
5.41 (t, 10.1)
3.62 (dq, 10.1, 6.3)
0.88 (d, 6.3)
7.04 (s)
6.30 (d, 2.1)
6.52 (d, 2.1)
–
7.64 (d, 2.1)
–
7.18 (d, 8.4)
7.55 (dd, 8.4, 2.1)
12.58 (s)
5.97 (d, 1.9)
5.99 (dd, 3.2, 1.9)
5.66 (dd, 10.1, 3.2)
5.41 (t, 10.1)
3.67 (dq, 10.1, 6.2)
0.97 (d, 6.2)
7.16 (s)
Kaempferol/
Quercetin
0
0
0
0
0
0
0
2
3
5
6
/5
–
OH (C-5)
12.57 (s)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
00
00
Rhamnose
1
2
3
4
5
6
2
2
2
5.97 (d, 1.8)
5.98 (dd, 3.1, 1.8)
5.70 (dd, 10.0, 3.1)
5.42 (t, 10.0)
3.71 (dq, 10.0, 6.2)
0.99 (d, 6.2)
7.17 (s)
000
000
000
Galloyl A
Galloyl B
Galloyl C
/ 6
/ 6
/ 6
7.01 (s)
6.98 (s)
7.07 (s)
–
7.03 (s)
6.98 (s)
cells. In the control cells, the MRSA cells appeared as grape-like
clusters (Fig. 2A). After treating the MRSA cells with 512 g/mL
Phenolic compounds are reported to act specifically on the bacte-
rial membrane and cause bacterial cell leakage (Johnston et al.,
2003; Nohynek et al., 2006). Polyphenols in tea such as epicatechin
and gallocatechin gallate have been shown to cause leakage of cel-
lular contents in Lactobacillus plantarum and Neisseria meningitides
(Cho et al., 2010). This leakage can be clearly observed in Fig. 2C,
when MRSA cells were treated with compound 1. Phenolic com-
pounds, such as these flavonol rhamnosides, are able to cause
l
(
2
2ꢁ MICs) of the ethyl acetate extract and isolated compounds 1,
, 3 and 6, the grape-shaped cluster of MRSA cells were disrupted
and the remaining cells showed leakage of cellular contents
Fig. 2C–F). There were no differences in the antibacterial effects
(
on the MRSA cells between the quercetin and kaempferol rhamno-
sides. The compounds all caused cellular leakage of MRSA cells.
Please cite this article in press as: Chan, E.W.L., et al. Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina with antibacterial activity
against methicillin-resistant Staphylococcus aureus (MRSA). Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.028

E.W.L. Chan et al. / Phytochemistry xxx (2014) xxx–xxx
5
Table 4
rhamnosides showed their structure–activity relationship where
the esterification of the rhamnoside moiety was important in con-
ferring anti-MRSA activity compared to the corresponding flavonol
rhamnosides. There was no correlation between the degree of
esterification and the antibacterial activity of these flavonol
rhamnosides against MRSA. The lytic effect of the flavonol rhamno-
sides on MRSA were also confirmed by a scanning electron micro-
scope imaging. Overall, the structure–activity-relationship of
flavonol rhamnosides observed in this study could serve as a refer-
ence for the synthesis and design of new antimicrobial drugs.
1
3C NMR data of compounds 1–3.
Moiety
Position
1
2
3
2
3
4
5
6
7
8
9
1
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
157.2
133.7
178.0
162.4
98.9
157.7
134.2
178.3
162.3
99.0
157.9
133.0
178.0
162.4
98.9
Kaempferol/quercetin
164.3
93.9
164.2
93.8
164.3
94.0
157.7
104.9
121.7
131.0
116.0
159.8
116.0
131.0
98.0
157.2
104.9
122.2
116.1
145.2
148.0
115.8
121.9
100.8
68.2
157.2
104.9
122.0
116.1
145.3
148.1
115.9
122.1
97.8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4. Experimental
4.1. General
0
0
0
0
0
0
00
Rhamnose
69.3
69.5
69.1
69.7
Silica gel (Kieselgel 60, 70–230, and 230–400 mesh, Merck KGaA,
Darmstadt, Germany), and Sephadex LH-20 (Pharmacia Fine Chem-
icals AB, Uppsala, Sweden) were used for column chromatography
(CC). Optical rotations were measured on an Atago AP-300 auto-
matic polarimeter. Ultraviolet (UV) absorption spectra were mea-
sured in MeOH using a HPLC with a UV/VIS detector separated by a
72.1
70.3
68.4
16.8
70.4
70.3
68.7
16.9
68.6
16.9
Galloyl A
Galloyl B
120.0
109.3
145.2
138.4
164.5
119.9
109.2
145.0
138.3
164.9
120.3
109.2
144.98
138.1
165.0
120.5
109.2
145.0
138.1
165.5
119.9
109.3
145.25
138.5
164.6
119.85
109.3
145.0
138.32
164.9
00 000
00 000
00
/6
/5
C-18 reversed phase column (Assentis 25 cm ꢁ 4.9 mm ꢁ 5
lm).
C@O
The HPLC profiles were processed with Agilent software to obtain
0
0
0
0
000
00 000
00 000
000
1
2
3
4
the UV kmax. Nuclear magnetic resonance (NMR) spectra 1D and 2D
/6
/5
1
were acquired on a Bruker-AVANCE-III operating at 400 MHz ( H)
1
3
and 100 MHz ( C). Mass spectra were carried out on a Q-TOF Mass
Spectrometer (Agilent), in the negative ion mode of ionisation.
C@O
0
0
0
0
0000
Galloyl C
1
2
3
4
119.7
109.2
145.0
138.2
165.2
119.83
109.1
145.0
138.3
165.3
00 000
/6
00 000
/5
0000
4.2. Plant material
C@O
Leaves of C. tergemina were collected in May 2011 from Klang
Valley in Peninsular Malaysia and were identified by Anthonysamy
Savarimuthu of the Plant Taxonomy Unit of School of Science,
Monash University Sunway campus Malaysia where a voucher
specimen (MUM-LEGUM-001) is deposited in the herbarium.
3 2
Recorded in ((CD ) CO) at 100 MHz. Values in ppm (d).
cellular leakage because they may diffuse through the bacterial
cells’ cytoplasmic membrane and thus increase the cells’ perme-
ability. The cellular leakage could be proteins, nucleic acids, and
inorganic ions such as potassium or phosphates. Cell shrinkage
and cell lysis were also observed in MRSA cells after exposure to
the isolated compounds. This is because at a higher concentration,
phenolic compounds might inhibit permease causing denaturation
of the bacterial proteins. The disruption of cell membrane could
subsequently cause cell shrinkage and cell lysis that may eventu-
ally lead to cell death (Maris, 1995). Phenolic compounds are also
reported to inactivate intra-cytoplasmic enzymes by forming
unstable complexes and causing the lipophilic molecules to be
trapped by the membrane phospholipids. An increase in phenolic
content will disrupt the equilibrium of surface macromolecules.
This may affect the association of cell-surface polysaccharides
and result in changes of bacterial shape and size (Dauang et al.,
4.3. Extraction and Isolation
Freeze-dried C. tergemina leaves (1.5 kg) were extracted succes-
sively by adding 10 L each of hexane, CH
2 2
Cl , EtOAc, MeOH and
then H O at 25 °C for 3 days. The extracts were evaporated under
2
reduced pressure. The EtOAc extract (40.0 g) was subjected to silica
CC using an 80 mm column and eluted gradient-wise with mix-
tures of hexane–CHCl , CHCl –EtOAc and EtOAc–MeOH (2.5 L for
3 3
each solvent mixture) to obtain 15 fractions of 500 ml each. Frac-
tion 11 on evaporation to dryness yielded compound 8 (53.1 mg)
while fraction 14 on treatment with EtOAc followed by MeOH gave
compound 5 (35.2 mg) as a precipitate. 2.0 g of fraction 2 which
showed the highest anti-MRSA activity was re-applied to a
2
011). This explains the changes in the size and shape of the MRSA
4
0 mm silica column using 200 ml of the same solvent systems
as above to obtain five sub-fractions. Sub-fractions 2–2
311.0 mg), 2–3 (486.0 mg) and 2–4 (105.0 mg) also showed
cells after treatment with the compounds. The morphological fea-
tures of the MRSA cells observed under the SEM indicates that
compounds 1, 2, 3 and 6 had bactericidal effects on MRSA cells
rather than a bacteriostatic one.
(
anti-MRSA activity and were separately subjected to gel filtration
chromatography using Sephadex LH-20 eluting with MeOH.
Sub-fraction 2-2-5 yielded compound 6 (34.3 mg), sub-fraction
2-2-7 gave compound 4 (15.9 mg), sub-fraction 2-2-9 yielded com-
pound 7 (36.3 mg), sub-fraction 2-2-15 gave compound 2 (6.5 mg),
and sub-fraction 2-2-17 on evaporation yielded compound 1
(18.2 mg). Sub-fraction 2-3-8 and 2-3-10 also yielded compound
1 (9.3 mg) and compound 3 (4.8 mg), respectively, after evapora-
tion while sub-fraction 2–4-5 gave compound 3 (4.4 mg).
3
. Concluding remarks
Eight flavonol rhamnosides, including three new galloylated fla-
vonol rhamnosides, were isolated from the leaves of C. tergemina.
This is the first report on the phytochemical study of C. tergemina.
The anti-MRSA activity of the extracts and isolated flavonol
Please cite this article in press as: Chan, E.W.L., et al. Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina with antibacterial activity
against methicillin-resistant Staphylococcus aureus (MRSA). Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.028

6
E.W.L. Chan et al. / Phytochemistry xxx (2014) xxx–xxx
4
4
.4. Analytical data
.4.1. Kaempferol-3-O-(2 ,3 ,4 -tri-O-galloyl)-
tion (MIC) determinations were made in triplicate and vancomycin
was used as positive control for the three strains of methicillin-
resistant S. aureus, MRSA (ATCC: 33591), MRSA (Clinical Isolate 1)
and MRSA (Clinical Isolate 2). Compounds were dissolved in 5%
00
00 00
a-
L-rhamnopyranoside
(1)
Yellow amorphous powder; UV (MeOH) kmax (nm): 212, 236,
2
DMSO in H O. A serial two-fold dilution of the compounds was
mixed with MHB in 96-well sterile microtitre plates to give a final
2
5
1
2
(
75, 318, 340; [
a
]
D
+25.3° (c 0.1 in MeOH), for H (400 MHz,
1
3
ꢀ1
CD )
3 2
CO) and C (100 MHz, (CD
3
)
2
CO) NMR spectroscopic data,
concentration of 0.02–1.00 mg ml . Inocula of 50
l
l of the 24-h
ꢀ
5
see Tables 3 and 4; Q-TOF MS m/z 887.1322 [MꢀH] (calculated
culture of each bacterial strain, containing approximately 5 ꢁ 10
CFU/ml, was applied into MHB supplemented with the tested com-
pounds. MIC values were obtained after incubation at 37 °C for
24 h. The MICs were recorded as the lowest concentrations of
tested compounds which completely inhibited bacterial growth.
31
for C42H O22, 887.1307).
00
00
4
.4.2. Quercetin-3-O-(3 ,4 -di-O-galloyl)-
a-L-rhamnopyranoside (2)
Yellow amorphous powder; UV (MeOH) kmax (nm):212, 228,
2
5
1
2
(
68, 350 nm; [
a
]
D
+27.7° (c 0.1 in MeOH), for H (400 MHz,
1
3
CD )
3 2
CO) and C (100 MHz, (CD
)
3 2
CO)NMR spectroscopic data,
4.5.3. Scanning Electron Microscope (SEM) analysis
ꢀ
see Tables 3 and 4; Q-TOF MS m/z 751.1156 [MꢀH] (calculated
After the MIC determination with microdilution, the bacterial
27
for C35H O19, 751.1147).
cells from the 96 well plates with 2ꢁ MIC of the respective tested
compounds, positive and negative controls were transferred sepa-
rately into a centrifuge tube. The bacterial cells were spin down
and washed thrice with phosphate buffer saline (PBS). On a glass
00
00 00
4
.4.3. Quercetin-3-O-(2 ,3 ,4 -tri-O-galloyl)-
a-L-rhamnopyranoside
(3)
Yellow amorphous powder; UV (MeOH) kmax nm: 215, 260,
slide, 20
and extract treated cells were fixed with 2.5% glutaraldehyde for
5 min and were later washed with PBS. The specimens were then
dried with liq. N and freeze dried. Using a splutter coater, the
ll of bacterial suspension was left to dry. The control
2
5
1
3
50 nm; [
a
]
D
+27.3° (c 0.1 in MeOH), for H (400 MHz, (CD
3 2
) CO)
1
3
and C (100 MHz, (CD CO) NMR spectroscopic data, see Tables 3
and 4; Q-TOF MS m/z 903.1264 [MꢀH] (calculated for C42
3 2
)
4
ꢀ
31 23
H O ,
2
9
03.1256).
dried specimens were coated with a thin layer of platinum and
were examined by a Scanning Electron Microscope.
4
.4.4. Acid hydrolysis
2 4
Compound 1 (7 mg) was hydrolysed with 1 M H SO (2 ml) at
room temperature overnight. The reaction mixture was then parti-
tioned with EtOAc (3 mL). The aqueous layer was neutralised with
Acknowledgements
1
.0 M Ba(OH)
under pressure to dryness. After treatment with EtOAc, MeOH
and H O, -rhamnose was obtained as white crystalline powder
2
ꢂ8H
2
O (2 mL), filtered with glass wool and reduced
The authors are thankful to Monash University Malaysia for
financial support to EWLC for a visit to the Strathclyde Institute
of Pharmacy and Biomedical Sciences, University of Strathclyde
Glasgow for the use their NMR facilities and the University of
Malaya Kuala Lumpur for LCMS analysis. We are grateful to Mr.
Anthonysamy Savarimuthu (formerly of the University Putra
Malaysia) for identifying the plant material.
2
L
from the aqueous layer. The identity and purity of the sugar was
confirmed by direct comparison (HPLC and TLC) with an authentic
sample. The configuration of the rhamnose was determined to be L-
2
5
[a
]
D
ꢀ7.8° (c 0.6 in H
2
O) (Hudson and Yanovsky, 1917; Lee et al.,
2
5
2
4
4
009): [
a]
D
ꢀ7.2° (c 0.05 in H O).
2
Appendix A. Supplementary data
.5. Biological assay
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
.5.1. Test microorganisms and media
All strains of MRSA used were sub-cultured from our laboratory
0
7.028.
stock culture collection, including clinical isolates of MRSA. Muel-
ler–Hinton broth (MHB; Difco, MD, USA) were used for culturing
the bacteria.
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Please cite this article in press as: Chan, E.W.L., et al. Galloylated flavonol rhamnosides from the leaves of Calliandra tergemina with antibacterial activity
against methicillin-resistant Staphylococcus aureus (MRSA). Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.028