Flavonol glucuronides and C-glucosidic ellagitannins from Melaleuca squarrosa

Article abstract of DOI:10.1016/j.phytochem.2008.04.004

Two flavonoids and three ellagitannins, squarrosanins A, B, and C, were isolated from the leaves of Melaleuca squarrosa. The flavonoids were characterized structurally as kaempferol-3-O-(2″-O-galloyl)-glucuronide and herbacetin-3-O-glucuronide, while the ellagitannins were characterized as monomeric and dimeric C-glucosidic ellagitannins by application of spectroscopic and chemical methods. The antioxidant effect of the polyphenolic constituents of the M. squarrosa leaves was also examined in vitro, and C-glucosidic tannins including oligomers were shown to be more effective radical scavengers against 1,1-diphenyl-2-picrylhydrazyl (DPPH) than flavonoids and ordinary ellagitannins.

Full text of DOI:10.1016/j.phytochem.2008.04.004

Phytochemistry 69 (2008) 3062–3069
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Flavonol glucuronides and C-glucosidic ellagitannins from Melaleuca squarrosa
Morio Yoshimura a, Hideyuki Ito b, Kyoko Miyashita b, Tsutomu Hatano b, Shoko Taniguchi b,
Yoshiaki Amakura a, Takashi Yoshida a,
*
a College of Pharmaceutical Sciences, Matsuyama University, Bunkyo-cho, Matsuyama 790-8578, Japan
b Department of Pharmacognosy, Okayama University Graduate School of Medicine, Dentistry, Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Two flavonoids and three ellagitannins, squarrosanins A, B, and C, were isolated from the leaves of Mel-
aleuca squarrosa. The flavonoids were characterized structurally as kaempferol-3-O-(200-O-galloyl)-glucu-
ronide and herbacetin-3-O-glucuronide, while the ellagitannins were characterized as monomeric and
dimeric C-glucosidic ellagitannins by application of spectroscopic and chemical methods. The antioxidant
effect of the polyphenolic constituents of the M. squarrosa leaves was also examined in vitro, and C-glu-
cosidic tannins including oligomers were shown to be more effective radical scavengers against 1,1-
diphenyl-2-picrylhydrazyl (DPPH) than flavonoids and ordinary ellagitannins.
Received 25 February 2008
Received in revised form 2 April 2008
Available online 22 May 2008
Keywords:
Melaleuca squarrosa
Myrtaceae
Flavonol glucuronide
C-Glucosidic ellagitannin
Squarrosanin A
Squarrosanin B
Squarrosanin C
Antioxidant
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Melaleuca squarrosa Donn ex Sm. (family Myrtaceae) is a small
evergreen tree that grows in New Zealand and the southern and
southeastern parts of Australia. In our continuing study of natural
polyphenolic products from myrtaceous plants (Okuda et al.,
1982b; Yoshida et al., 1992, 1996; Lee et al., 1997; Ito et al.,
2000, 2004), we reported the isolation of four novel C-glucosidic
ellagitannin oligomers, melasquanins A–D, together with many
other hydrolyzable tannins from the water-soluble portion of an
aqueous acetone extract of M. squarrosa leaves (Yoshida et al., in
press). C-glucosidic ellagitannin oligomers of this plant, including
cowaniin (Ito et al., 2007; Yoshida et al., in press), a complex tannin
dimer, were found to be composed of two or three molecules of
casuarinin and/or stachyurin (Okuda et al., 1983; Nonaka et al.,
1990), which are linked to each other by a wide variety of C–C cou-
pling mode(s) (Yoshida et al., in press). Further examination of the
nonpolar and polar fractions of the plant has led to the isolation of
two new flavonoids (1 and 2) and three additional ellagitannins,
designated squarrosanins A (3), B (4), and C (5). This paper de-
scribes the elucidation of their structures and their antioxidant
activity.
The aqueous acetone homogenate of air-dried leaves of M.
squarrosa was successively extracted with ether, ethyl acetate,
and n-butanol. Each extract obtained after concentration in vacuo
was separately submitted to a combination of chromatographic
steps over silica gel, Diaion HP-20, Toyopearl HW-40, and MCI
GEL CHP-20P to yield compounds 1–5. Five known compounds,
grandis-3 (Crow et al., 1971), messagenic acid I (Macías et al.,
1998), oleanolic acid, betulinic acid, and angophorol (Wollenweber
et al., 2000) from the ether extract and six polyphenols, catechin,
ellagic acid, valoneic acid dilactone, strictinin, 1,2,3,6-tetragalloyl-
glucose, and casuarictin (Okuda et al., 1983) in addition to C-gluco-
sidic ellagitannin monomers (casuarinin (6) and stachyurin (7))
from the ethyl acetate extract, and dimers reported in the preced-
ing paper (Yoshida et al., in press) from the n-butanol and aqueous
extracts were also obtained. These were identified based on com-
parisons of the respective spectroscopic data with that of data re-
ported in the literature.
Compound 1 was obtained as a yellow amorphous powder.
High resolution electrospray ionization mass spectra (HRESIMS)
and 13C NMR spectra established the molecular formula of 1 as
C28H22O16. The UV spectrum showed maxima characteristic of a
flavonoid at 267 and 349 nm. The 1H NMR spectrum displayed
A2B2 signals at d 6.95 and 8.08 (2H each d, J = 9.0 Hz) and two
meta-coupled signals at d 6.22 and 6.44 (each J = 2.5 Hz), indicating
the presence of a kaempferol skeleton. The spectrum also implied
* Corresponding author. Tel.: +81 89 926 7128.
E-mail address: tyoshida@cc.matsuyama-u.ac.jp (T. Yoshida).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.04.004

M. Yoshimura et al. / Phytochemistry 69 (2008) 3062–3069
3063
the presence of a galloyl group and a glucuronyl moiety as sug-
gested from the 2H-singlet at d 7.22 and the five sequential spin-
system of the aliphatic proton region. These structural components
were confirmed by the production of kaempferol, gallic acid, glu-
curono-6,3-lactone, and glucuronic acid upon acid hydrolysis of
1. The HMBC experiment showed correlations from anomeric H-
100 (d 6.00) to C-3 (d 133.8) and from a downfield shifted H-200 (d
5.21) to ester carbonyl carbon (d 166.6), which in turn showed a
three-bond correlation with a galloyl proton signal, indicating the
location of the glucuronyl residue at O-3 and the galloyl group at
O-200. The structure of compound 1 was thus established as
kaempferol-3-O-(200-O-galloyl)-glucuronide (see Fig. 1).
Compound 2 was assigned the molecular formula C21H18O13
based on HRESIMS. The NMR features of 2 closely resembled those
of 1, except for a lack of galloyl protons and one of the A-ring pro-
tons. Acid hydrolysis of 2 yielded glucuronic acid, glucurono-6,
3-lactone, and an aglycone. The aglycone showed an HMBC
correlation between a chelated 5-OH proton at d 11.96 and a
hydrogen-bearing aromatic carbon at d 98.9, indicating it to be
8-hydroxykaempferol, which was confirmed by direct comparison
of the 1H NMR spectrum and HPLC co-chromatography with an
authentic sample (herbacetin) (Sikorska et al., 2004). The location
of the sugar residue was established by HMBC correlation between
H-100 and C-3. Therefore, compound 2 was assigned as herbacetin-
3-O-glucuronide.
(358 nm), indicated the presence of an ellagic acid moiety as found
in 3. Considering a Cotton effect at 234 nm with amplitude for one
HHDP group in the CD spectrum, the structure of squarrosanin B
was deduced as 4, which was substantiated by HMQC and HMBC
correlations (Fig. 2). The presence of a glucose core and an ellagic
acid moiety linked at C-1 in 4 was chemically verified by treatment
with acid in boiling water bath, giving a hydrolyzate (8), which was
identified with that obtained from casuarinin (6) of known struc-
tures in a similar manner. The pyranose structure of 8 was assigned
on the basis of a large coupling constant of the H-1 signal (d,
J = 10.0 Hz) at d 6.46 in the 1H NMR spectrum and of the molecular
formula C20H16O13 determined by HRESIMS. The derivative 8 was
also obtained from the C-1 epimer of 6, stachyurin (7), thereby
indicating that the pyranose formation occurred through a benzylic
cation during the reaction to have a stable b-configuration for the
anomeric center. Thus, although the reaction did not provide an ex-
pected evidence for the C-1 configuration in 4, the 1R-configura-
tion was tentatively assigned by the small coupling constant of
the H-1 signal (d 6.39, br d, J = 3.0 Hz) in 1H NMR and a clear
ROE correlation for H-1/H-3 in the ROESY (Fig. 3). Namely, among
possible rotations for C1–C2 bond, anti-conformation in which the
two bulky substituents (ellagic acid and C3–C6 chain) are as far
apart as possible (Fig. 3) would likely be adoptable. Thus, the most
plausible structure of squarrosanin B was assigned as 4.
Squarrosanin C (5) was indicated to be a dimeric congener of 4
Squarrosanins A (3), B (4), and C (5) were shown to be ellagitan-
nins by their spectroscopic features as well as via the production of
gallic and ellagic acids upon acid hydrolysis.
by ESIMS giving
a pseudomolecular ion peak at m/z: 1872
[M+NH4]+ corresponding to the molecular formula C82H54O51
,
characteristic UV absorption at 367 nm, and a proton signal at d
7.54 indicative of the presence of ellagic acid. The 1H and 13C
NMR spectra indicated that 5 is a dimer composed of 4 and
stachyurin (7), in which the C-1 of 7 and an HHDP group of 4 par-
ticipate in the formation of a C–C bond linking the monomers, as
indicated by an upfield shift of H-10 at d 4.69 (s) and four singlets
due to the HHDP protons. Note that one of the H-6 methylene pro-
ton signals of the glucose core shifted to a remarkably higher field
(d 2.97, d, J = 13.0 Hz) than that of 4 (d 4.03, d, J = 13.0 Hz), which is
a diagnostic shift for alienanin B-type C-glucosidic dimers (Yoshida
et al., in press). The proposed structure (5) was supported by good
correspondence of the glucose carbon resonances of 5 with those of
4 and the glucose-II (Glc-II) core in alienanin B (9) (Nonaka et al.,
1991) (Table 1). The positions of the acyl groups were secured by
HMBC correlations among aromatic and glucose protons through
three-bond coupling with carbonyl carbons as illustrated in
Fig. 2. The atropisomerism of the chiral HHDP groups in 5 was
determined to be all S-series by a strong positive Cotton effect at
231 nm in CD. The C-1 configuration was also tentatively assigned
to be R by the small coupling constant of H-1 (d 6.36, br s) similar
to that of 4, leading to the gross structure 5 for squarrosanin C.
Present and previous studies (Yoshida et al., in press) indicated
that M. squarrosa is a rich source of C-glucosidic ellagitannins
including oligomers. Although hydrolyzable tannins are well docu-
mented to exhibit antioxidative activity (Yoshida et al., 1989) as
well as diverse biological properties such as anti-tumor promotion
(Ito et al., 1999), host-mediated antiproliferative effects against
Sarcoma 180 in mice (Miyamoto et al., 1993), and antiviral effects
(Sakagami et al., 1999), the antioxidant properties of the M. squar-
rosa polyphenols have not been examined. Polyphenolic com-
pounds together with previously isolated ones from this plant
were thus evaluated for their radical scavenging effect against
1,1-diphenyl-2-picrylhydrazyl (DPPH). All of the tested polypheno-
lic constituents exhibited stronger activity than a positive control,
trolox (Table 2). The C-glucosidic ellagitannins, including complex
tannins and oligomers (EC50 1.6–3.6 lM), were found to be more
effective radical scavengers than flavonoids (EC50 14.8–30.4 lM)
and ordinary ellagitannins (EC50 5.5–8.0 lM) such as tellimagran-
dins I and II as shown in Table 2. The activity increased depending
Squarrosanin A (3), a pale brownish amorphous powder, was
shown to have the formula C55H32O33 from its HRESIMS and 13C
NMR spectroscopic data. Compound 3 showed 1H and 13C NMR
spectra characteristic of C-glucosidic ellagitannins such as casuar-
inin (6) and stachyurin (7). That is, a 2H-singlet (d 7.21) and three
1H-singlets (d 6.56, 6.59, 6.85) in the aromatic region of the 1H
NMR spectrum were accounted for by the presence of a galloyl
and two hexahydroxydiphenoyl (HHDP) groups among which
one HHDP group participates biogenetically in the formation of a
C–C bond arising from a phenol–aldehyde coupling (Okuda et al.,
1995). The presence of an ellagic acid moiety in the molecule
was also indicated by the 1H NMR (d 7.57, 1H, s) and 13C NMR
(see Section 3) spectra along with UV (kmax 367 nm) data. Open-
chain glucosyl signals in the 13C NMR spectrum of 3 were similar
to those of 7 except for a remarkable upfield shift (d 65.5 in
7 ? d 41.4 in 3) for C-1 resonance, suggesting that the hydroxyl
group at C-1 in 7 is replaced by ellagic acid through an additional
C–C bond in 3. The allocation of the ellagic acid moiety in 3 was
substantiated from the long-range correlations of the glucose H-1
and H-2 signals with ellagic acid carbons (C-4, C-5, and C-6) in
the HMBC spectrum. Other HMBC correlations illustrated in
Fig. 2 were consistent with the proposed structure. The H-1 signal
{d 5.79 (d, J = 1.0 Hz)} in 3 showed an unusual, larger downfield
shift than that of 7 {d 4.82 (d, J = 1.5 Hz)} in the 1H NMR spectrum,
despite the absence of an oxygen function. This may be explained
by a deshielding effect caused from the coplanar H-1 and ellagic
acid planes. The assignment of the (S)-configuration at the chiral
HHDP groups was determined from a strong positive Cotton effect
at 235 nm in the circular dichroism (CD) spectrum (Okuda et al.,
1982a). From the above data, the structure of squarrosanin A (3)
was established as depicted.
Squarrosanin B (4) was isolated as a pale brownish amorphous
powder, and its molecular formula was determined as C41H28O26
by HRESIMS. The 1H and 13C NMR spectra of 4 (Table 1) were sim-
ilar to those of 6 and 7 except for a remarkable downfield shift of
an aromatic proton and upfield shifts of the H-2 and H-3 signals
(d 4.04 and 4.15) of a glucose residue. The low-field aromatic pro-
ton signal at d 7.56, along with characteristic UV absorption

3064
M. Yoshimura et al. / Phytochemistry 69 (2008) 3062–3069
OHHO
HO
HO
OH
OH
OH
OH
OH
OH
HO
O
OH
HO
O
CO
CH2O
CO
O
HO
H
O
O
O
O
COOH
O
OH
O
COOH
1
O
O
CO
O
O
OC
O
O
OH
1"
O
OH
CO
OH
1"
CO
OH
OH
OH
OH
HO
HO
OH
HO
OH
OH
OH
O
OH
OH
OH HO
HO
2
1
3
OHHO
OHHO
HO
HO
OH
OH
HO
HO
OH
OH
CO
CH2O
CO
O
CO
CO
O
OH
CH2O
HO
O
O
HO
HO
O
1
1
1
H
R
O
O
HO
O
CO
O
HO
OC
OH
O
OH
OH
OC
R'
CO
O
O
OH
OH
OH
OH
HO
HO
HO
HO
O
HO
OH
OH
8
HO
HO
4
OH
OH
HO
OHHO
O
6: R =H , R '=OH
7: R =OH, R '=H
O
OHHO
OHHO
HO
OH
OH
HO
OH
OH
HO
OC
HO
CO
CO
O
CO
CO
O
CH2O
CH2O
1'
1
H
O
O
O
O
HO
OC
OH
O
OH
OH
OH
H
CO
CO
HO
HO
HO
O
HO
OH
HO
HO
OH
HO
OH
OH HO
HO
OH
O
5
O
OHHO
HO
HO
OH
OH
OHHO
HO
OH
OH
HO
CO
CH2O
CO
O
CO
CH2O
CO
O
Glc-II
Glc-I
1'
1
H
O
O
O
CO
O
CO
O
O
OC
OC
H
OH
OH
CO
CO
HO
OH
HO
OH
HO
HO
OH
HO
HO
OH HO
OH
OH
OH
HO
HO
OHHO
OH
OH
9
OHHO
OH
OHHO
HO
HO
OH
OH
OH
HO
CO
CO
OH
OH
O
CH2O
CO
CH2O
CO
O
O
1
L
O
O
CO
O
OC
1
H
CO
O
O
CO
O
OC
H
HO
HO
CO
HO
OH
HO
HO
HO
O H
OH
HO
OH
OH
OH HO
HO
OH
OH
O
OH
O
OH HO
HO
L=
OH
HOH C
OH OH
OH
11
OH
10
Fig. 1. Structures of compounds 1–11.

M. Yoshimura et al. / Phytochemistry 69 (2008) 3062–3069
3065
OHHO
OHHO
HO
HO
OH
H
HO
H
OH
H
OH
HO
OH
OH
H
CO
CO
O
CO
CO
O
2
4
HO
H
O
1
O
CH2O
CH2O
5
H
H
H
H
H
H
1
6
1
H
H
H
O
O
O
CO
O
HO
O
O
OH
OH
O
OC
OC
OH
H
H
CO
H
OH
OH
OH
HO
HO
O
H
HO
OH
OH
OH
HO
OH
HO
HO
HO
HO
OH HO
3
O
O
4
OHHO
OHHO
HO
OH
H
HO
HO
OH
HO
OH
OH
H
H
CO
CH2O
CO
O
CO
CH2O
CO
O
H
H
H
H
H
H
1
1'
H
O
H
H
O
O
CO
O
OC
HO
OC
OH
O
H
H
OH
H
CO
H
HO
HO
HO
OH
OH
OH
O
HO
OH
HO
HO
HO
OH
OH HO
HO
OH
O
O
5
Fig. 2. HMBC correlations of compounds 3–5.
on the molecular size (trimers > dimers > monomers), but the dif-
ference in potency was small. Interestingly, squarrosanins A (3),
B (4), and C (5), which have a C–C linked ellagic acid moiety at
C-1 of the open-chain glucose residue, showed less potency than
their congeners having an HHDP or catechin unit instead of the el-
lagic acid (e.g., stenophyllanin A (11) (complex tannins)), casuari-
HMBC experiments, the JCH value was set at 6 Hz. ESIMS spectra
were recorded on a Micromass Auto Spec OA-Tof mass spectrome-
ter (solvent: 50% aqueous MeOH + 0.1% NH4OAc for positive mode;
CH3CN for negative mode). Normal phase HPLC was conducted on a
YMC-Pack SIL A-003 (YMC Co., Ltd.) column (4.6 i.d. ꢀ 250 mm)
developed
with
n-hexane–MeOH–tetrahydrofuran–HCO2H
nin (6) (C-glucosidic tannin monomer), and alienanin
B
(9)
(55:33:11:1) containing oxalic acid (450 mg/1.0 L) (flow rate:
1.5 mL/min; 280 nm UV detection) at room temperature. Re-
versed-phase HPLC was performed on a YMC-Pack ODS-A A-302
(YMC Co., Ltd.) column (4.6 i.d. ꢀ 150 mm) developed with
10 mM H3PO4–10 mM KH2PO4–CH3CN (44:44:12) (solvent A) and
MeOH–H2O (4:1, v/v) (solvent B), with each at a flow rate of
1.0 mL/min; detection, 280 nm UV or diode array detector (DAD)
(HITACHI L-7445) 200–600 nm at 40 °C. Column chromatography
(dimer), respectively, suggesting that the ellagic acid unit has less
contribution toward the radical scavenging potency than the HHDP
unit.
2.1. Concluding remarks
These findings may provide the basis for further clarifying the
antioxidant-related actions of the M. squarrosa leaves, which are
rich in C-glucosidic tannins, and may contribute toward the devel-
opment of a new resource of agents beneficial to human health.
OH HO
HO
HO
OH
OH
3. Experimental
CO
CH2O
CO
O
H
3.1. General
H
H
H
H
H
C3
O
H
H
OH
UV spectra on a HITACHI U-2000-10 spectrophotometer and CD
spectra on a JASCO J-720W spectrometer were measured. Optical
rotations were recorded on a JASCO DIP-1000 polarimeter. Elemen-
tal analyses were recorded on a Yanaco CHN corder MT-5. NMR
spectra were recorded on a Varian VXR-500, Bruker AVANCE500
(500 MHz for 1H and 126 MHz for 13C) or Varian INOVA 600 instru-
ment (600 MHz for 1H and 150 MHz for 13C), and chemical shifts
are given in d (ppm) values relative to those of the solvent [ace-
tone-d6 (dH 2.04; dC 29.8)] on a tetramethylsilane scale. In the
HO
OH
OC
OH
O
Ellag
OH
OH
OH
OH
HO
HO
O
HO
HO
OH
Newman projection
O
O
Fig. 3. ROESY correlations of compound 4.

3066
M. Yoshimura et al. / Phytochemistry 69 (2008) 3062–3069
Table 1
1H, 13C NMR spectroscopic data for the glucose moieties of 4–7 and 9 in acetone-d6 + D2O
Position
NMR
4a
5a
6a
7a
9b
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
1
2
3
4
5
6
6.39 br d
(3.0)
4.15 dd
(2.0, 3.0)
4.04 br s
75.0
6.36 br s
74.6
5.58 d
(5.0)
4.63 dd
(2.5, 5.0)
5.42 t
67.6
4.82 d
(1.5)
4.90 t
(1.5)
5.00 dd
(1.5, 2.5)
5.69 dd
(2.5, 8.5)
5.30 dd
(3.0, 8.5)
4.01 d
65.5
5.62 d
(4.8)
4.61 dd
(1.8, 4.8)
5.37 t
67.4
74.4
71.8
73.3
71.7
64.8
4.14 br s
3.92 br s
74.6
71.2
73.8
70.2
65.9
76.7
69.8
74.2
71.2
64.6
81.0
72.0
73.3
70.9
64.5
77.0
69.2
74.6
69.4
65.6
(2.5)
(1.8)
5.35 dd
(3.0, 8.5)
5.68 dd
(3.0, 8.5)
4.03 d
5.26 dd
(3.0, 8.5)
5.68 br dd
(2.0, 8.5)
2.97 d
5.44 dd
(2.5, 8.5)
5.31 dd
(3.0, 8.5)
4.04 d
5.28 dd
(1.8, 9.0)
5.18 dd
(3.6, 9.0)
2.99 d
(13.0)
(13.0)
(13.0)
(13.0)
(12.6)
4.85 dd
(3.0, 13.0)
4.03 br dd
(13.0)
4.84 dd
(3.0, 13.0)
4.91 dd
(3.0, 13.0)
4.03 br d
(12.6)
10
20
30
40
50
60
4.69 br s
40.5
80.9
74.7
73.5
71.6
64.4
4.75 d
(1.8)
4.92 t
(1.8)
4.98 t
(1.8)
5.63 dd
(1.8, 9.0)
5.27 dd
(3.0, 9.0)
4.03 br d
(12.6)
40.3
80.8
74.6
73.3
71.4
64.3
4.92 br dd
(1.0, 2.0)
4.98 t
(2.0)
5.62 dd
(2.0, 8.5)
5.29 br dd
(3.0, 8.5)
4.03 d
(13.0)
4.78 dd
(3.0, 13.0)
4.79 dd
(3.0, 12.6)
J values (Hz) are in parenthesis.
a
1H NMR: 500 MHz and 13C NMR: 126 MHz.
1H NMR: 600 MHz and 13C NMR: 150 MHz.
b
was carried out on a Diaion HP-20, MCI GEL CHP-20P (Mitsubishi
Chemical Co.), and Toyopearl HW-40 (coarse grade; Tosoh Co.).
over MCI GEL CHP-20P (1.1 i.d. ꢀ 16 cm) eluted with H2O ? aque-
ous MeOH (10% ? 20% ? 30% ? 40% ? 50%) ? 100% MeOH to
yield strictinin (15.9 mg) and kaempferol-3-O-(200-O-galloyl)-glu-
curonide (1) (22.0 mg). The H2O–MeOH (4:6, v/v) eluate was di-
vided into three portions, and each portion was separately
subjected to column chromatography over MEGA BOND ELUT
C18 with H2O-MeOH (9:1, v/v) giving casuarinin (6) (327.6 mg),
1,2,3,6-tetragalloylglucose (12.2 mg), and valoneic acid dilactone
(4.3 mg). The MeOH eluate gave ellagic acid (16.4 mg), whereas
H2O–acetone (3:7, v/v) eluate was purified by column chromatog-
raphy over MCI GEL CHP-20 (1.1 i.d. ꢀ 17 cm) with aqueous MeOH
(30% ? 100% MeOH) ? H2O–acetone (3:7, v/v) as eluates to give
casuarictin (22.1 mg).
3.2. Plant material
The leaves of M. squarrosa were supplied from the herbal garden
of POLA Chemical Industries, Inc. A voucher specimen (OKP-
MY98005) is deposited at the Medicinal Herbal Garden of Okayama
University Graduate School of Medicine, Dentistry, Pharmaceutical
Sciences.
3.3. Extraction and isolation
Dried leaves of M. squarrosa (1.4 kg) were homogenized in H2O–
acetone (3:7, v/v, 11 L) at room temperature. The filtered homoge-
nate was concentrated in vacuo below 40 °C to 1 L and extracted
with Et2O (1 L ꢀ 3), EtOAc (1 L ꢀ 3), and n-BuOH (1 L ꢀ 3) succes-
sively to give Et2O (53.7 g), EtOAc (35.4 g), n-BuOH (82.9 g), and
H2O (135.8 g) extracts. Fractionations of the respective extracts
were achieved by monitoring either normal- or reversed-phase
HPLC. The Et2O extract (22.0 g) was applied to a silica gel column
(63–200 lm; Merck, 5.0 i.d. ꢀ 50 cm) with CHCl3 ? CHCl3–MeOH
(95:5 ? 9:1 ? 4:1 ? 1:1) as eluants. The CHCl3–MeOH (95:5, v/
v) eluate was further subjected to Toyopearl HW-40 (coarse grade,
2.2 i.d. ꢀ 60 cm) followed by preparative HPLC to yield grandis-3
(4.9 mg), angophorol (5.4 mg), betulinic acid (8.9 mg), messagenic
acid I (11.0 mg), and oleanolic acid (47.6 mg). A part (2.00 g) of the
EtOAc extract was subjected to Toyopearl HW-40 (coarse grade, 2.2
i.d. ꢀ 45 cm) with aqueous MeOH (30% ? 40% ? 50% ? 60% ?
100% MeOH) ? H2O–acetone (3:7, v/v) as eluants. The H2O–MeOH
(6:4, v/v) eluate afforded (+)-catechin (35.5 mg). The H2O–MeOH
(1:1, v/v) eluate was further subjected to column chromatography
The n-BuOH extract (82.9 g) was applied to a Diaion HP-20 (6.5
i.d. ꢀ 70 cm) column, eluated with H2O ? aqueous MeOH
(10% ? 20% ? 30% ? 50%) ? MeOH ? H2O–acetone (3:7, v/v). An
aliquot of the H2O–MeOH (8:2, v/v) eluate (3.00 g) was further sub-
jected to column chromatography over Toyopearl HW-40 (coarse
grade, 2.2 i.d. ꢀ 60 cm) eluted with aqueous MeOH (50% ?
60% ? 70%) ? 100% MeOH ? H2O–acetone (3:7, v/v) and MCI
GEL CHP-20P (1.1 i.d. ꢀ 40 cm) eluted with H2O ? aqueous MeOH
(10% ? 20% ? 30% ? 50%) ? 100% MeOH, to yield strictinin
(28.9 mg), pterocarinin
A
(10) (120.4 mg), stachyurin (7)
(35.3 mg), casuarinin (6) (482.6 mg), alienanin B (9) (96.8 mg),
pedunculagin (23.0 mg), and stenophyllanin A (11) (47.2 mg),
respectively. An aliquot of the H2O–MeOH (7:3, v/v) eluate
(3.00 g) was further subjected to column chromatography over
Toyopearl HW-40 (coarse grade, 2.2 i.d. ꢀ 60 cm) eluted with
aqueous MeOH (50% ? 60% ? 70%) ? 100% MeOH ? H2O–ace-
tone (3:7, v/v) and MCI GEL CHP-20P (1.1 i.d. ꢀ 40 cm) eluted with
H2O ? aqueous MeOH (10% ? 20% ? 30% ? 50%) ? MeOH, to
yield herbacetin-3-O-glucuronide (2) (62.5 mg), strictinin

M. Yoshimura et al. / Phytochemistry 69 (2008) 3062–3069
3067
and 178.3 (C-4). ESIMS m/z: 615 [M+H]+. HRESIMS m/z: 637.0787
Table 2
[M+Na]+ and calcd for C28H22O16 + Na, 637.0806.
Radical scavenging effect of polyphenols against DPPH
EC50 (lM)a
3.5. Herbacetin 3-O-glucuronide (2)
Reference compound
Trolox
36.5
A yellow amorphous powder, [a]D ꢁ20.0 (c = 1.0, MeOH). UV
kmax (MeOH) nm (log e): 213 (sh 4.55), 278 (4.27), and 328
(3.99). 1H NMR (600 MHz, DMSO-d6): d 3.25 (1H, t, J = 9.0 Hz, H-
200), 3.26 (1H, t, J = 9.0 Hz, H-300), 3.36 (1H, br dd, J = 9.0, 9.6 Hz,
H-400), 3.58 (1H, d, J = 9.6 Hz, H-500), 5.47 (1H, d, J = 9.0 Hz, H-100),
6.28 (1H, s, H-6), 6.88 (2 H, d, J = 9.0 Hz, H-30, 50), 8.12 (2H, d,
J = 9.0 Hz, H-20, 60), 8.67 (1H, s, 8-OH), 10.19 (1 H, s, 40-OH), 10.51
(1H, s, 7-OH), and 11.96 (1H, s, 5-OH). 13C NMR (150 MHz,
DMSO-d6): d 71.6 (C-400), 74.1 (C-200), 75.9 (C-300), 76.2 (C-500), 98.9
(C-6), 101.4 (C-100), 103.8 (C-4a), 115.3 (C-30, 50), 121.1 (C-10),
125.1 (C-8), 131.2 (C-20, 60), 132.9 (C-3), 145.0 (C-2), 152.9 (C-8a),
153.6 (C-5), 156.4 (C-7), 160.3 (C-40), 169.9 (C-600), 177.7 (C-4).
ESIMS m/z: 479 [M+H]+. HRESIMS m/z: 479.0898 [M+H]+ and calcd
for C21H18O13 + H, 479.0826.
Flavonoids
Kaempferol-3-O-(200-O-G)-
glucuronide (1)
14.8
30.4
Herbacetin-3-O-glucuronide (2)
Ellagitannins and related polyphenols
Ellagic acid
Gallic acid
Monomers
18.4
15.5
Composing unit
HHDP + 1 Galloyl
HHDP + 2 Galloyl
HHDP + 3 Galloyl
2 HHDP
Strictinin
5.9
8.0
7.6
5.5
Tellimagrandin I
Tellimagrandin II
Pedunculagin
C-glucosidic tannin monomers
Casuarinin (6)
Pterocarinin A (10)
Composing unit (monomer)
2 HHDP + Galloyl
Stachyurin (7) + Lyxose
Stachyurin (7) + Catechin
3.3
2.5
3.6
Stenophyllanin A (11)
C-glucosidic tannin oligomers
Alienanin B (9) (dimer)
Casuglaunin A (dimer)
Casuglaunin B (dimer)
Melasquanin A (dimer)
Melasquanin B (trimer)
Melasquanin C (trimer)
Melasquanin D (trimer)
3.6. Hydrolysis of (1) and (2)
2.5
2.6
2.2
2.5
1.8
2.3
1.6
Stachyurin (7) + 6
Stachyurin (7) + 6
Stachyurin (7) + Catechin + 6
Stachyurin (7) + 6
(Stachyurin (7)) ꢀ 2 + 6
(Stachyurin (7)) ꢀ 2 + 6
(Stachyurin (7)) ꢀ 2 + 6
(Related congener)
(10), (11)
(6)
Compound 1 (5 mg) was treated with 1% HCl (2 mL) in a boiling
water bath for 12 h to yield kaempferol, gallic acid, glucuronic acid,
and glucurono-6,3-lactone, which were identified by comparison
with authentic samples on HPLC (UV or RI). Compound 2 was sim-
ilarly treated, and the products were identified as herbacetin, glu-
curonic acid, and glucurono-6,3-lactone on HPLC.
C-glucosidic tannin with C1-ellagic acid
Squarrosanin A (3) (monomer)
Squarrosanin B (4) (monomer)
Squarrosanin C (5) (dimer)
4.2
6.0
3.2
(9)
3.7. Squarrosanin A (3)
a
Each value is a mean value of triplicate data.
A pale brownish amorphous powder, [a]D +30.3 (c = 1.0, MeOH).
UV kmax (MeOH) nm (log e): 217 (4.78), 272 (4.51), and 366 (3.80).
CD (MeOH) [h] (nm): +16.6 ꢀ 104 (235), ꢁ6.3 ꢀ 104 (261), and
+4.5 ꢀ 104 (284). 1H NMR (500 MHz, acetone-d6 + D2O): d 4.07
(1H, d, J = 13.0 Hz, H-6), 4.92 (1H, br t, H-2), 4.99 (1H, dd, J = 3.0,
13.0 Hz, H-6), 5.35 (1H, t, J = 2.0 Hz, H-3), 5.39 (1H, dd, J = 3.0,
8.5 Hz, H-5), 5.79 (1H, d, J = 1.0 Hz, H-1), 5.92 (1H, dd, J = 2.0,
8.5 Hz, H-4), 6.56, 6.59, 6.85 (each 1H, s, HHDP-H), 7.21 (2H, s, gal-
loyl-H), and 7.57 (1H, s, aromatic-H). 13C NMR (126 MHz, acetone-
d6 + D2O): d 41.1 (C-1), 64.5 (C-6), 71.1 (C-5), 72.8 (C-4), 75.1 (C-3),
82.0 (C-2), 106.0, 107.4, 108.7, 127.9 (each 1C, HHDP C-3), 110.0
(2C, galloyl C-2, 6), 106.9, 109.6, 111.7, 113.4, 114.6, 122.4, 137.2,
137.8, 139.5 (2C), 148.8 (2C), 159.9, 160.7 (ellagic acid moiety),
115.4, 115.5, 116.3, 116.7 (each 1C, HHDP C-1), 120.6 (galloyl C-
1), 123.0, 125.0, 126.7, 126.8 (each 1C, HHDP C-2), 135.1, 136.0,
136.4, 137.8 (each 1C, HHDP C-5), 139.4 (galloyl C-4), 142.8,
142.9, 144.27, 144.30 (2C), 145.1, 145.2, 145.7 (HHDP C-4, 6),
146.1 (2C, galloyl C-3, 5), 166.3, 167.7, 168.5, 169.0, 169.8 (each
1C, ester carbonyls). ESIMS m/z: 1221 [M+H]+, 1238 [M+NH4]+,
1243 [M+Na]+. HRESIMS m/z: 1243.0722 [M+Na]+ and calcd for
C55H32O33 + Na, 1243.0724.
(64.3 mg), casuarinin (6) (394.7 mg), stenophyllanin
A (11)
(66.6 mg), alienanin B (9) (121.7 mg), casuglaunin A (14.0 mg),
cowaniin (7.0 mg), melasquanin A (13.9 mg), and casuglaunin B
(14.8 mg).
The water-soluble portion (37.58 g) was subjected to column
chromatography over Diaion HP-20 (6.5 i.d. ꢀ 45 cm) with
H2O ? aqueous
MeOH
(10% ? 20% ? 30% ? 50%) ? 100%
MeOH ? H2O–acetone (3:7, v/v). The H2O–MeOH (1:1, v/v) eluate
was further applied to Toyopearl HW-40 (coarse grade, 2.2
i.d. ꢀ 60 cm) with 70% MeOH ? MeOH ? MeOH–H2O–acetone
(7:2:1) ? MeOH–H2O–acetone (7:1:2) ? H2O–acetone (3:7, v/v),
MCI GEL CHP-20P (1.1 i.d. ꢀ 40 cm) with aqueous MeOH
(10% ? 20% ? 30% ? 50%) ? MeOH ? H2O–acetone (3:7, v/v)
and YMC GEL ODS AQ12S50 (1.1 i.d. ꢀ 40 cm) with aqueous MeOH
(10% ? 20%
? 30% ? 50%) ? MeOH to give alienanin B (9)
(265.8 mg), casuglaunin B (236.1 mg), and squarrosanins A (3)
(20.0 mg), B (4) (43.3 mg), and C (5) (22.6 mg).
3.4. Kaempferol 3-O-(200-O-galloyl)-glucuronide (1)
A yellow amorphous powder, [a]D ꢁ102.6 (c = 1.0, MeOH). UV
kmax (MeOH) nm (log e): 218 (sh 4.63), 267 (4.41), 349 (4.14). 1H
NMR (500 MHz, acetone-d6 + D2O): d 3.80 (1H, t, J = 9.5 Hz, H-400),
3.89 (1H, t, J = 9.5 Hz, H-300), 3.94 (1H, t, J = 9.5 Hz, H-500), 5.21
(1H, dd, J = 8.0, 9.5 Hz, H-200), 6.00 (1H, d, J = 8.0 Hz, H-100), 6.22
(1H, d, J = 2.5 Hz, H-6), 6.44 (1H, d, J = 2.5 Hz, H-8), 6.95 (2H, d,
J = 9.0 Hz, H-30, 50), 7.22 (2H, s, galloyl-H), and 8.08 (2 H, d,
J = 9.0 Hz, H-20, 60). 13C NMR (126 MHz, acetone-d6 + D2O): d 72.8
(C-400), 74.8 (C-200), 74.9 (C-300), 76.5 (C-500), 94.5 (C-8), 99.5 (C-6),
99.7 (C-100), 105.2 (C-4a), 110.2 (galloyl C-2, 6), 116.1 (C-30, 50),
121.4, 122.0 (C-10, galloyl C-1), 131.9 (C-20, 60), 133.8 (C-3), 139.0
(galloyl C-4), 145.9 (galloyl C-3, 5), 157.7, 157.8, 161.1, 162.4 (C-
2, C-5, C-8a, C-40), 165.2 (C-7), 166.6 (galloyl C-7), 170.6 (C-600),
3.8. Squarrosanin B (4)
A pale brownish amorphous powder, [a]D ꢁ11.7 (c = 1.0, MeOH).
UV kmax (MeOH) nm (log e): 215 (4.66), 256 (4.49), and 358 (3.79).
CD (MeOH) [h] (nm): +7.8 ꢀ 104 (234), ꢁ3.1 ꢀ 104 (259), and
+1.5 ꢀ 104 (281). 1H NMR (500 MHz, acetone-d6 + D2O): d 6.52,
6.84 (each 1H, s, HHDP-H), 6.96 (2H, s, galloyl-H), and 7.56 (1H,
s, aromatic-H); for sugar protons, see Table 1; 13C NMR
(126 MHz, acetone-d6 + D2O): d 107.2, 108.9 (each 1C, HHDP C-3),
109.6 (2C, galloyl C-2, 5), 105.8, 109.4, 111.6, 113.0, 114.3, 125.2,
135.8, 136.8, 139.3, 140.3, 148.5, 149.1, 159.9, 160.3 (each 1C, ella-
gic acid moiety), 115.4, 116.0 (each 1C, HHDP C-1), 120.3 (galloyl
C-1), 125.8, 126.0 (each 1C, HHDP C-2), 136.2, 136.4 (each 1C,

3068
M. Yoshimura et al. / Phytochemistry 69 (2008) 3062–3069
HHDP C-5), 139.0 (galloyl C-4), 144.2, 144.3 (each 1C, HHDP C-6),
145.0, 145.1 (each 1C, HHDP C-4), 145.7 (2C, galloyl C-3, 5),
166.4, 168.0, 169.2 (each 1C, ester carbonyls); for sugar carbons,
see Table 1; ESIMS m/z: 954 [M+NH4]+, 959 [M+Na]+. HRESIMS
m/z: 959.0704 [M+Na]+ and calcd for C41H28O26 + Na, 959.0767.
3.13. Antioxidant activity
The antioxidant activity was evaluated by DPPH radical in the
usual way (Yoshida et al., 1989). Briefly, a 2 mL portion of extract
or trolox (12.5, 25, 50, 100, 200 lM) in MeOH was added to a
15 ꢀ 105 mm assay tube containing 0.5 mL of freshly prepared
DPPH solution (1 mM) in MeOH. The tubes were vigorously vortex
mixed, covered, and left in the dark at room temperature. After
30 min, the absorbance at 520 nm was measured on a spectropho-
tometer. The degree of discoloration of the solution indicates the
scavenging efficacy of the added substance. All experiments were
performed in triplicate.
3.9. Squarrosanin C (5)
A pale brownish amorphous powder, [a]D +10.4 (c = 1.0, MeOH).
UV kmax (MeOH) nm (log e): 214 (4.48), 272 (4.24), and 367 (3.41).
CD (MeOH) [h] (nm): +17.3 ꢀ 104 (231), ꢁ2.8 ꢀ 104 (257), and
+3.5 ꢀ 104 (282). 1H NMR (500 MHz, acetone-d6 + D2O): d 6.44,
6.54, 6.76, 6.83 (each 1H, s, HHDP-H), 7.04, 7.08 (each 2H, s, gal-
loyl-H), and 7.54 (1H, br s, aromatic-H); for sugar protons, see Ta-
ble 1; 13C NMR (126 MHz, acetone-d6 + D2O): d 106.1, 107.5, 108.7,
109.8, 114.4, 114.7, 115.5 (2C), 115.7, 116.1, 116.3, 116.6 (HHDP C-
1, 10, 3, 30), 109.9 (2C, galloyl C-2, 5), 110.1 (2C, galloyl C-20, 50),
105.8, 109.0, 111.9, 113.2, 125.3, 134.4, 135.7, 139.3 (2C), 148.6,
149.3, 159.7, 160.3 (ellagic acid moiety), 121.0, 121.6 (each 1C, gal-
loyl C-1, C-10), 125.1 (2C), 125.3, 126.7, 127.0, 127.1 (HHDP C-2, 20),
136.1, 136.5, 136.8, 136.9, 137.0, 137.1 (each 1C, HHDP C-5, 50),
138.8, 140.4, 143.5, 143.9, 144.1, 144.3, 144.4, 144.5, 145.1,
145.2, 145.3, 145.8 (each 1C, HHDP C-4, 6, 40, 6’), 145.6, 146.0 (each
2C, galloyl C-3, 5, 30, 50), 166.0, 166.1, 166.5, 168.1, 168.2, 168.5,
169.4, 169.5 (each 1C, ester carbonyls); for sugar carbons, see Table
1; ESIMS m/z: 1872 [M+NH4]+. Anal. Found: C, 45.11; H, 3.82.
C82H54O51ꢂ17.5 H2O requires: C, 47.37; H, 4.10%.
Antioxidant activity (after 30 min) was defined as the percent
discoloration of a DPPH solution and was calculated by the follow-
ing equation:
Antioxidant effect ð30Þ ¼ 100 ꢀ ðAsample=AcontrolÞ:
Acknowledgements
We thank Mr. Y. Kitada of the Central Research Laboratory of
the Pola Chemical Industries Inc., for kindly supplying the plant
material. The NMR experiments were performed at the SC-NMR
Laboratory of Okayama and Matsuyama Universities.
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3.10. Acid hydrolysis of casuarinin (6) and stachyurin (7)
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A yellow amorphous powder. 1H NMR (500 MHz, MeOH-d4): d
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br t, J = 10.0 Hz, H-2), 3.87 (1H, dd, J = 3.5, 12.0 Hz, H-6), 3.94
(1H, d, J = 12.0 Hz, H-6), 6.46 (1H, d, J = 10.0 Hz, H-1), 7.47 (1H, s,
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111.7, 113.7, 115.1, 124.2, 137.4, 137.6, 140.6, 141.6, 148.5,
149.5, 160.9, 161.2 (each 1C, ellagic acid moieties). ESIMS m/z:
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A solution of each compound (1–2 mg) in 0.5% H2SO4 (1.0 mL)
was heated in a boiling water bath for 8 h. After cooling, the reac-
tion mixture was adsorbed on an ODS Mega Bond Elut cartridge
(1 g). After washing with H2O, the product was eluted with MeOH,
and the concentrated solution was analyzed by reversed-phase
HPLC (solvent A) to detect ellagic (Rt 38.7 min) and gallic (Rt
2.6 min) acids, which were identified by co-chromatography with
authentic specimens.

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