Phloroglucinol diglycosides accompanying hydrolyzable tannins from Kunzea ambigua

Article abstract of DOI:10.1016/j.phytochem.2008.03.003

Six phloroglucinol diglucosides-kunzeaphlogins A-F (1-6) and a hydrolyzable tannin, kunzeatannin A (7)-were isolated along with 10 known polyphenols from the leaf extract of Kunzea ambigua. Structural elucidation of these compounds was based on spectroscopic analyses and chemical properties.

Full text of DOI:10.1016/j.phytochem.2008.03.003

Phytochemistry 69 (2008) 3080–3086
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phloroglucinol diglycosides accompanying hydrolyzable tannins from Kunzea
ambigua
a,
c
Naoki Kasajima b, Hideyuki Ito a, , Tsutomu Hatano , Takashi Yoshida
*
*
a Department of Pharmacognosy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan
b School of Pharmacy, Shujitsu University, Nishigawara, Okayama 703-8516, Japan
c Matsuyama University, Bunkyo-cho, Matsuyama 790-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Six phloroglucinol diglucosides—kunzeaphlogins A–F (1–6) and a hydrolyzable tannin, kunzeatannin A
(7)—were isolated along with 10 known polyphenols from the leaf extract of Kunzea ambigua. Structural
elucidation of these compounds was based on spectroscopic analyses and chemical properties.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 17 January 2008
Received in revised form 20 February 2008
Available online 23 April 2008
Keywords:
Kunzea ambigua
Myrtaceae
Phloroglucinol
Tannin
Kunzeaphlogins
Kunzeatannin
1. Introduction
(Toyopearl HW-40, Diaion HP-20, MCI GEL CHP-20P, YMC-GEL
ODS-AQ 120-S50 and/or Sephadex LH-20 columns) to afford three
Kunzea ambigua (SM.) Druce belonging to the Myrtaceae is na-
tive to Australia (Tasmania, New South Wales, and Victoria). Its
essential oil containing monoterpenes and sesquiterpenes has
been used in New Zealand as a remedy for the treatment of diar-
rhoea, cold, inflammation, and wounds (David, 1997; Khambay
et al., 2002). Besides the reported phloroglucinol derivatives, we
previously isolated six galloylated chromone C-glucosides (kunz-
eachromones A–F) and a dimeric flavonol glycoside (kunzeagin
A) (Ito et al., 2004b), as well as acylated phloroglucinol derivatives
(kunzeanones A–C) (Ito et al., 2004a), from the leaf extract of this
plant. Further investigation on phenolic constituents has led to iso-
lation of 17 polyphenols including seven new compounds (six
phloroglucinol glucosides and a hydrolyzable tannin). We describe
herein the structural elucidation of these compounds.
new compounds [kunzeaphlogins C–E (3–5)] and three known
compounds [3-O-p-coumaroylquinic acid (Haslam et al., 1961),
40,60-dihydroxyisobutrophenone
20-O-b-glucoside
(Vanc-
raenenbroeck et al., 1965) and pedunculagin (8) (Hatano et al.,
1988)] from the ethyl acetate extract, and five new compounds
[kunzeaphlogins A (1), B (2), D (4), F (6) and kunzeatannin A (7)]
along with eight known compounds [4-O-b-glucopyranosyl-cis-
coumaric acid (Cui et al., 1990), 3-O-p-coumaroylquinic acid, 4-
O-p-coumaroylquinic acid, ellagic acid, 3,30-di-O-methylellagic
acid (Duc Do et al., 1990), 3,30-di-O-methylellagic acid 4-O-b-glu-
coside (Pakulski and Budzianowski, 1996), casuarinin (Okuda
et al., 1983) and praecoxin A (Hatano et al., 1991)] from the n-buta-
nol extract.
Kunzeaphlogin A (1) was obtained as a pale yellow amorphous
powder. Its molecular formula of C23H34O14 was determined by
high-resolution electrospray ionization mass spectroscopy (HRE-
SIMS) [m/z 535.1991].
2. Results and discussion
The 1H NMR spectrum of 1 showed signals due to an isovaleroyl
group {two secondary methyl groups [dH 0.91, 0.87 (3H each, d,
J = 7.0 Hz)], a methylene signal [dH 3.15 (1H, dd, J = 6.5, 16.5 Hz),
2.88 (1H, dd, J = 7.5, 16.5 Hz)] and a methine signal [dH 2.16 (1H,
m)]}, and an aromatic proton [dH 6.28 (1H, s)]. The presence of
two glucose residues was also suggested by two anomeric proton
resonances [dH 5.04 (d, J = 7.5 Hz, H-10) and 4.80 (d, J = 9.5 Hz, H-
100)] and other aliphatic proton signals characteristic of 4C1
2.1. Structure elucidation of new compounds
The ethyl acetate and n-butanol extracts of an aqueous acetone
homogenate of the dried leaves of K. ambigua were purified by a
combination of various column chromatographic techniques
* Corresponding authors. Tel./fax: +81 86 251 7937.
E-mail address: hito@cc.okayama-u.ac.jp (H. Ito).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.03.003

N. Kasajima et al. / Phytochemistry 69 (2008) 3080–3086
3081
glucopyranose residue, which were assigned by 1H–1H COSY. The
13C NMR spectrum of 1 supported the presence of a phloroglucinol
moiety (dC 165.5, 164.1, 161.0, 106.4, 105.9, 95.4), an isovaleroyl
group (dC 206.8, 53.5, 25.4, 23.1, 22.6) and two glucose moieties
(Table 1). In the HMQC spectrum of 1, the anomeric proton signals
at dH 5.04 and 4.80 correlated with carbon resonances at dC 101.3
and 74.7, respectively, suggesting that kunzeaphlogin A shares C-
and O-glucosidic characters (see Fig. 1).
The HMBC spectrum of 1 showed long-range correlations be-
tween H-10 (dH 5.04) and C-2 (dC 161.0) and between the H-100 sig-
nal (dH 4.80) and carbon resonances at dC 164.1 (C-4), 106.4 (C-5)
and 165.5 (C-6) (Fig. 2). The aromatic proton at dH 6.28 correlated
with the carbons at dC 105.9 (C-1), 161.0 (C-2), 164.1 (C-4) and
106.4 (C-5). Structure 1 thus assigned to kunzeaphlogin A was ver-
ified by the NOESY measurement, which showed NOEs between
the H-10 and the aromatic proton at dH 6.28. The O-glycosylated su-
Kunzeaphlogins B (2) and C (3) were shown to have molecular
formulas of C37H42O22 and C44H46O26, respectively, by HRESIMS.
Their UV spectral patterns (2; kmax 219, 280 and 322sh, 3; kmax
219, 280 and 321sh) were similar to that of 1. The 1H and 13C
NMR spectroscopic data of 2 and 3 were also similar to those of
compound 1, except for the appearance of galloyl signals [dH
7.07, 7.06 (2H each, s) in 2; dH 7.17, 7.115, 7.109 in 3]. The location
of the two galloyl groups at O-60 and O-600 of the glucose residues in
2 was determined by comparison of the 1H NMR spectrum of 2
with that of 1, which showed a remarkable downfield shift (D
0.67–0.87 ppm) of H-60and H-600 in 2. Three galloyl groups in com-
pound 3 were similarly allocated at O-60, O-600 and O-400 by a large
downfield shift of H-400 (D 1.82 ppm) of 3 on comparing the 1H
NMR spectroscopic data with those of 2. The structural relationship
between 2 and 3 was confirmed by enzymatic hydrolysis of 3 with
tannase, which afforded 2 and 1, along with gallic acid. Conse-
quently, the structures of kunzeaphlogins B and C are represented
by formulas 2 and 3, respectively.
Kunzeaphlogin D (4), a pale yellow amorphous powder, had the
molecular formula of C25H38O14 as indicated by HRESIMS. A phlor-
oglucinol skeleton of 4 was deduced from analysis of the UV (kmax
217, 271 and 321sh) and NMR spectra. The 1H and 13C NMR spectra
of 4 exhibited the presence of an isovaleroyl group [dC 210.4, 53.8,
26.0, 22.9, 22.7; dH 3.16 (1H, dd, J = 7.0, 15.5 Hz), 2.93 (1H, dd,
J = 7.0, 15.5 Hz), 2.08 (1H, m), 0.84, 0.83 (3H each, d, J = 7.0 Hz)].
The spectra also showed signals due to two aromatic methyl
groups [dC 10.9, 9.8; dH 2.27, 2.14 (3H each, s)] and a phloroglucinol
moiety [dC 159.4, 157.3, 153.4, 118.5, 117.3, 116.8] as well as two
glucose entities (Table 2). The HMBC spectrum of 4 showed corre-
lations of H-10 to C-2; H-100 to C-4; 3-Me to C-2, C-3 and C-4; 5-Me
to C-4, C-5 and C-6; H-8 to C-7, C-9, C-10 and C-11; H-9 to C-8, C-
10, C-11; H-10, 11 to C-9 (Fig. 2). The NOESY spectrum of 4 showed
correlations of 3-Me with both anomeric proton signals H-10 and
H-100, and a correlation between 5-Me and H-100. Acid hydrolysis
of 4 yielded glucose and aglycone (4a). A liberated sugar from 4
gar obtained from acid hydrolysis of 1 was identified as D-glucose
by co-chromatography with an authentic sample on HPLC
equipped with an optical rotation detector and also by positive
reaction to D-glucose oxidase (Miwa et al., 1972). Although the C-
glycosidic sugar in 1 lacks direct evidence, its assignment is likely
based on the analogy of the 1H and 13C NMR spectroscopic data to
those of the values reported in the literature (Agrawal, 1989).
Based on these findings, the structure of kunzeaphlogin A was
determined as formula 1.
Table 1
13C NMR spectroscopic data for kunzeaphlogins A (1)–C (3) (126 MHz, acetone-d6
+
D2O)
Carbon
1
2
3
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
105.9
161.0
95.4
164.1
106.4
165.5
206.8
53.5
106.1
160.8
95.6
164.4
106.4
165.4
206.7
53.5
106.18
160.9
95.7
164.5
106.19
165.7
206.9
53.4
was identified as D-glucose by reversed-phase HPLC with an optical
rotation detector and positive reaction to D-glucose oxidase. The
structure of 4a was consistent with its 1H NMR spectroscopic data.
Based on these data, the structure of kunzeaphlogin D was estab-
lished as 4.
25.4
23.1
22.6
25.5
23.0
22.6
25.5
22.9
22.5
The molecular formula of kunzeaphlogin E (5) was determined
to be C32H42O18 by HRESIMS. The 1H and 13C NMR spectra of 5 as-
signed from COSY, TOCSY, HMQC and HMBC were very similar to
those of 4 except for the presence of an extra signal [dH 7.12 (2H,
s)] due to a galloyl group and downfield shifts of H-600 [dH 4.57
(1H, d, J = 2.0, 12.0 Hz), 4.28 (d, J = 4.0, 12.0 Hz)]. The downfield-
shifted signals (H-600) were assigned by correlation with the
anomeric proton at dH 4.78 (H-100) in the TOCSY spectrum of 5.
Enzymatic hydrolysis of 5 with tannase afforded 4 and gallic acid.
Consequently, kunzeaphlogin E (5) was characterized as a monog-
allate of kunzeaphlogin D (4).
O-Glucose
C-1’
C-2’
C-3’
C-4’
101.3
74.1
101.0
74.0
101.0
74.0
77.7a
70.8b
77.6a
62.1c
79.1d
70.9e
78.9d
63.7f
77.3g
70.3
C-5’
C-6’
71.7g
63.8h
C-Glucose
C-1’’
C-2’’
C-3’’
C-4’’
74.7
72.2
79.3
70.7b
81.6
61.9c
74.7
72.3
77.3d
70.2e
75.1
64.8f
74.7
72.2
77.0g
72.2
C-5’’
C-6’’
75.1g
64.2h
Kunzeaphlogin F (6) showed a pseudomolecular ion peak
(M + H)+ at m/z 549, which is 14 mass units (CH2) less than that
of 4, and its molecular formula (C24H36O14) was confirmed by HRE-
SIMS. The UV, 1H and 13C NMR spectra of 6 were similar to those of
4. A distinguishing feature from 4 in the NMR spectra was a lack of
methylene signals, suggesting that the isovaleroyl group in 4 is re-
placed by an isobutyryl group {two methyl signals [dC 19.8, 17.5; dH
1.12, 0.92 (3H each, d, J = 6.0 Hz)], methine signal [dC 48.8; dH 3.90
(1H, m)], carbonyl carbon signal (dC 211.8)} in 6. The HMQC, HMBC
and NOESY spectroscopic data were fully consistent with structure
6 for kunzeaphlogin F.
Galloyl
C-1”’
120.92
120.85
120.93
120.86
120.8
C-2”’, 6”’
C-3’”, 5”’
109.8 (2C)
109.7 (2C)
145.78 (2C)
145.77 (2C)
110.1 (4C)
109.9 (2C)
145.8 (2C)
145.7 (2C)
145.69 (2C)
139.12
138.91 (2C)
167.3
167.2
C-4”’
C-7”’
138.91
138.87
167.4
Kunzeatannin A (7) showed the (M + NH4)+ ion peak at m/z 1586
167.2
in ESI-MS, corresponding to the molecular formula of C68H48O44
.
166.8
Methylation of 7 with diazomethane followed by methanolysis
gave methyl tri-O-methylgallate (10), dimethyl hexamethoxydi-
a–h
Interchangeable.

3082
N. Kasajima et al. / Phytochemistry 69 (2008) 3080–3086
OH
CH2OR
OH
O
HO
OH
HO
HO
HO
OH
O
1"
1'
3
1'
2
HO
O
7
O
CH2OR
4
3
2
O
O
7
CH2OH
4
5
H
H
H
OH
ROH2C
R'O
O
11
9
5
H
8
1
R'
6
1"
1
6
10
OH
O
OH
O
OH
OH
R
R'
R
R'
8
9
10, 11
H
G
-CH2CH(CH3)2
-CH2CH(CH3)2
H
G
G
H
H
G
4
5
1
2
3
8
9, 10
-CH(CH3)2
6
H
3 OH
HO
OH
O
2
6
7
C
1
4
OH
G =
5
O
OH
OH
4a
Fig. 1. Chemical structures of compounds 1–6.
OH
O
CH2OH
OH
HO
OH
HO
HO
HO
OH
CH2OH
O
H
1''
1'
1'
3
3
HO
O
CH2OH
O
H
O
O
O
H
H
OH
5
5
HOH2C
HO
O
1''
1
1
HMBC
NOE
HMBC
NOE
H
OH
OH
O
OH
OH
4
1
Fig. 2. HMBC and NOESY correlations of kunzeaphlogin A (1) and kuzneaphlogin D (4).
Table 2
13C NMR spectroscopic data for kunzeaphlogins D (4)–F (6) (126 MHz, acetone-d6
D2O)
+
phenate (11), and trimethyl octa-O-methyltergallate (12) (Fig. 2),
indicating that 7 is a dimeric hydrolyzable tannin with a tergalloyl
group as the connecting unit of monomers. The 1H NMR spectrum
of 7 was complicated owing to a formation of multiple signals for
each proton, suggesting that this compound exists as an equilib-
rium mixture of a- and b-anomers at sugar portion(s). Neverthe-
less, the presence of a galloyl, two HHDP and a tergalloyl group
was demonstrated by aromatic proton signals composed of a 2H
singlet and seven 1H singlets, most of which were paired by doubly
duplicated (four lines) signals.
Carbon
4
5
6
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
116.8
157.3
118.5
159.4
117.3
153.4
210.4
53.8
116.8
158.1
118.4
159.4
117.4
153.6
210.3
53.8
116.4
151.2
117.5
157.5
117.1
154.6
211.8
48.8
C-9
26.0
26.0
19.8
The atropisomerism of chiral HHDP and tergalloyl groups in 7
was shown to be in the (S)-series by a strong positive Cotton ef-
fect at 240 nm in the CD spectrum (Okuda et al., 1982). The sugar
carbon resonances in the 13C NMR spectrum of 7 showed close
correspondence with those of pedunculagin (8) and tellimagran-
din I (9) (Wilkins and Bohm, 1976) (Table 3), which indicated that
7 is an ellagitannin dimer biogenetically produced through C–O
oxidative coupling forming a tergalloyl group between a hexa-
hydroxydiphenoyl group of 8 and a galloyl group of 9. Structure
8 was presumed to be the most plausible one for kunzeatannin
A, taking into consideration the most frequently encountered cou-
pling mode in biogenesis of ellagitannin oligomers. The tergalloyl
group is known to easily isomerize into an isomeric valoneoyl
group by Smiles rearrangement (Yoshida et al., 1993). To verify
the proposed structure, 7 was treated with phosphate buffer
(pH 7.2) to yield an isomerized product, which was identified with
the known hydrolyzable tannin, camelliin A (13) (Yoshida et al.,
C-10
C-11
3-CH3
5-CH3
22.9
22.7
10.9
9.8
23.1
22.9
10.8
9.9
17.5
10.5
10.0
O-Glucose
C-1’, 1’’
C-2’, 2’’
C-3’, 3’’
C-4’, 4’’
C-5’, 5’’
C-6’, 6’’
104.9, 104.8
75.04, 74.97
77.3, 77.1a
71.0, 70.8
105.3, 104.8
75.5, 75.1
77.7, 77.5b
71.8, 70.7
77.3, 75.6b
63.2, 62.9
104.3 (2C)
74.34, 74.25
77.0 (2C)c
70.4, 70.1
76.6, 76.3c
61.6, 61.3
77.1, 77.0a
62.3, 62.1
Galloyl
C-1000
121.8
C-2000, 6000
C-3000, 5000
C-4000
110.1 (2C)
146.0 (2C)
138.8
C-7000
166.7
a–c
Interchangeable.

N. Kasajima et al. / Phytochemistry 69 (2008) 3080–3086
3083
Table 3
addition to previously reported kunzeachromones A–F may be
characteristic constituents of this plant.
13C-NMR spectroscopic data for the sugar moieties of kunzeatannin (7), pedunculagin
(8) and tellimagrandin I (9) (126 MHz, acetone-d6 + D2O)
Carbon
7
8
9
3. Experimental
a
b
a
b
a
b
C-1
C-2
C-3
C-4
C-5
C-6
C-1’
C-2’
C-3’
C-4’
C-5’
C-6’
91.2
75.3
75.5
69.8
66.7
63.83b
90.6
73.2
71.3a
70.9a
66.9
63.4b
94.9
77.9
77.3
69.4
71.6
63.79b
96.0
74.5
73.6
91.8
75.6
75.8
69.9
67.4
63.6
95.4
78.3
77.6
69.6
72.5
3.1. General
1H and 13C NMR spectra were recorded on a Varian VXR-500
instrument (500 MHz for 1H and 126 MHz for 13C), and the chem-
ical shifts are given in d (ppm) values relative to that of the solvent
[acetone-d6 (dH 2.04; dC 29.8)] and tetramethylsilane. The standard
pulse sequences that were programmed into the instrument (VXR-
500) were used for each two-dimensional measurement. The JCH
value was set at 6 Hz in the HMBC spectra. Optical rotations were
measured with a Jasco DIP-1000 polarimeter. UV spectra were
measured with a Hitachi U-2000 spectrophotometer. ESIMS was
recorded on a Micromass Auto Spec OA-TOF mass spectrometer
(solvent: MeOH:H2O (1:1, v/v) containing 0.1% NH4OAc; flow rate:
0.02 mL/min). Normal-phase HPLC was conducted on a YMC-Pack
SIL A-003 column (4.6 mm i.d. ꢀ 250 mm; YMC Co., Japan), and
developed at room temperature with a solution of n-hexane/
MeOH/tetrahydrofuran/HCO2H (55:33:11:1 containing 450 mg/L
oxalic acid) (solvent A) (detection: UV 280 nm). Reversed-phase
HPLC was performed with a YMC-Pack ODS-A A-302 column
(4.6 mm i.d. ꢀ 150 mm), and developed at 40 °C with 10 mM
H3PO4/10 mM KH2PO4/MeCN (42.5:42.5:15, solvent B), 10 mM
H3PO4/10 mM KH2PO4/AcOEt/EtOH (45:45:8:2, solvent C) and
MeOH/H2O (60:40, solvent D). Column chromatography was per-
formed on Diaion HP-20, MCI GEL CHP-20P (Mitsubishi Kasei Co.,
Japan), Toyopearl HW-40 (coarse grade; Tosoh Co., Japan), YMC-
GEL ODS-AQ 120-50S (YMC Co. Ltd.) or Mega Bond Elut C18 (Varian
Inc.).
91.2
72.9
71.1
71.1
67.2
63.5
96.7
74.1
73.5
71.9
72.0
a,b
Interchangeable.
1990) (Fig. 3). Based on these data, kunzeatannin A is represented
by the structure 7.
2.2. Concluding remarks
In this study, K. ambigua was demonstrated to contain novel
diglucosidic phloroglucinols, kunzeaphlogins. Although only a
phloroglucinol diglucoside having both O- and C-glucosidic link-
ages in the molecule has been isolated from Dryopteris crassirhi-
zome (Dryopteridaceae) (Chang et al., 2006), kunzeaphlogins A
(1), B (2) and C (3) are the second examples of this type of phloro-
glucinol. In addition, kunzeaphlogins B (2), C (3) and E (5) were
also the first isolated products of the galloylated phloroglucinol
diglucosides from natural sources. These galloylated glucosides in
OH
OH
O
HO
HO
O
C
O
C
O
HO
HO
HO
HO
O
O
O
O
C
O
OH
OH
OH
OH
C
O
OH
OH
O
O
O
O
CO
OH
CO
OH
HO
HO
OC
OH
OC
OH
OH
OH
HO
OH
buffer (pH 7.2)
HO
OH
9
OH
O
O
HO
HO
O
C
O
C
O
O
HO
HO
O
O
O
O
HO
O
O
C
O
OH
O
C
O
OH
O
8
CO
CO
CO
HO
CO
HO
OH
OH
HO
HO
OH
OH
HO
HO
OH
OH
OH HO
OCH3
OH HO
7
13
H3COOC
H3CO
H3CO
COOCH3
OCH3
H3COOC
COOCH3
OCH3
OCH3
H3CO
H3CO
COOCH3
OCH3
OCH3
1) CH2N2
2) NaOMe
H3CO
H3CO
COOCH3
O
OCH3 OCH3
OCH3 OCH3
H3CO
10
11
12
Fig. 3. Isomerization of kunzeatannin A (7) to camelliin A (13) by smiles rearrangement.

3084
N. Kasajima et al. / Phytochemistry 69 (2008) 3080–3086
3.2. Plant materials
mixture was passed through a Mega Bond Elut C18 cartridge col-
umn and washed with H2O. The aqueous solution was concen-
trated and analyzed by reversed-phase HPLC with an optical
rotation detector (Shodex OR-2; Showa Denko Co.) [column, TSK-
gel Amide-80 (4.6 mm i.d. ꢀ 250 mm) (Tosoh Co.); solvent,
MeCN/H2O (75:25); column temp., 35 °C; flow rate, 1.0 mL/min]
K. ambigua (SM.) Druce was collected in April 1998 from the
herbal garden of Pola Co. Ltd. (Japan). A voucher specimen has been
deposited in the Medicinal Herbal Garden of Okayama University
(specimen No. OKP-MY98003).
to detect D-glucose (Rt; 9.2 min, Rt; 9.8 min) as positive peaks iden-
3.3. Extraction and isolation
tical with those of authentic glucose. Positive reaction to
oxidase was also confirmed.
D-glucose
Dried leaves (700 g) of K. ambigua were homogenized in ace-
tone–H2O (7:3, v/v) (10 L), and the homogenate was filtered and
concentrated. The concentrated solution was extracted succes-
sively with ether, EtOAc and water-saturated n-BuOH. A portion
(2.0 g) of the EtOAc extract (11.0 g) was applied to a Toyopearl
HW-40 column (2.2 cm i.d. ꢀ 40 cm) and eluted with H2O contain-
ing increasing amounts of MeOH in a stepwise gradient. The
MeOH–H2O (1:4, v/v) eluate was subjected to column chromatog-
raphy over an MCI GEL CHP-20P (1.1 cm i.d. ꢀ 25 cm) with
aqueous MeOH, to yield kunzeaphlogin D (4) (23.7 mg). The
MeOH–H2O (3:7, v/v) eluate was further purified by column chro-
matography over an MCI GEL CHP-20P column (1.1 cm
i.d. ꢀ 25 cm), to yield 3-O-p-coumaroylquinic acid (8.9 mg), 40,60-
dihydroxyisobutrophenone 20-O-b-glucoside (11.0 mg) and kun-
zeaphlogin E (5) (37.4 mg). The MeOH–H2O (6:4, v/v) eluate was
similarly subjected to chromatographic purification over an YMC-
gel ODS-AQ 120-S50 (1.1 cm i.d. ꢀ 44 cm) with aqueous MeOH as
eluant to yield kunzeaphlogin C (3) (11.0 mg). The other portion
(3.3 g) of the EtOAc extract was subjected to column chromatogra-
phy over Toyopearl HW-40 (2.2 cm i.d. ꢀ 42 cm) with aqueous
MeOH in a stepwise gradient. The MeOH–H2O (6:4, v/v) eluate
was similarly applied to a YMC-GEL ODS-AQ 120-S50 (1.1 cm
i.d. ꢀ 44 cm) column eluted with aqueous MeOH, yielding peduncu-
lagin (8) (156.8 mg) and 3 (21.2 mg). A 10.1-g portion of the n-BuOH
extract (24.6 g) was fractionated by column chromatography over
Diaion HP-20 (5.0 cm i.d. ꢀ 50 cm), and developed with H2O and
increasing amounts of MeOH in a stepwise gradient. The MeOH–
H2O (3:7, v/v) eluate was purified by column chromatography over
Toyopearl HW-40 (2.2 cm i.d. ꢀ 40 cm) and/or a Mega Bond Elut C18
cartridge with aqueous MeOH to afford 3-O-p-coumaroylquinic acid
(4.9 mg), 4-O-p-coumaroylquinic acid (3.1 mg), casuarinin (4.9 mg),
4-O-b-glucopyranosyl-cis-coumaric acid (20.2 mg), ellagic acid
(4.0 mg), kunzeaphlogin A (1) (3.1 mg) and praecoxin A (28.4 mg).
The MeOH–H2O (1:1, v/v) eluate was further purified by chromatog-
raphy on Toyopearl HW-40 (2.2 cm i.d. ꢀ 40 cm) and YMC-GEL
ODS-AQ 120-S50 column (1.1 cm i.d. ꢀ 22 cm) with aqueous MeOH,
or a Sephadex LH-20 (1.1 cm i.d. ꢀ 28 cm) with EtOH–MeOH to
yield 1 (10.2 mg), 2 (5.7 mg) and 6 (4.2 mg).
3.6. Kunzeaphlogin B (2)
23
Pale yellow amorphous powder; ½aꢁD ꢂ 45 (c 1, MeOH); UV
(MeOH) kmax (loge) 219 (4.67), 280 (4.31), 322sh (3.98) nm; 1H
NMR (acetone-d6 + D2O, 500 MHz) dY; 7.07, 7.06 (2H each, s, gal-
loyl-H, H0), 6.26 (1H, s, H-3), 5.17 (1H, d, J = 6.5 Hz, glc-10), 4.88
(1H, d, J = 9.5 Hz, glc-100), 4.58 (1H, br d, J = 10.5 Hz, glc-60), 4.52
(1H, br d, J = 10.5 Hz, glc-600), 4.41 (1H, dd, J = 5.0, 12.0 Hz, glc-600),
4.32 (1H, dd, J = 4.0, 12.0 Hz, glc-60), 4.00 (1H, t, J = 9.5 Hz, glc-200),
3.5–3.9 (overlapping DHO, glc-20–50, 300–500), 3.09 (1H, dd, J = 6.01,
15.5 Hz, H-8), 2.84 (1H, dd, J = 7.5, 15.5 Hz, H-8), 2.13 (1H, m, H-
9), 0.85, 0.81 (3H each, d, J = 6.5 Hz, H-10, 11); for 13C NMR spectro-
scopic data, see Table 1; ESIMS m/z: 677 [M-galloyl group]+, 839
[M + H]+, 861 [M + Na]+; HRESIMS m/z 839.2347 [M + H]+
(C37H42O22 + H, 839.2246).
3.7. Kunzeaphlogin C (3)
23
Pale yellow amorphous powder; ½aꢁD ꢂ 41 (c 1, MeOH); UV
(MeOH) kmax (loge) 219 (4.88), 280 (4.88), 321sh (3.98) nm; 1H
NMR (acetone-d6 + D2O, 500 MHz) dY; 7.17, 7.115, 7.109 (2H each,
s, galloyl-H, H0, H00), 6.31 (1H, s, H-3), 5.34 (1 H, t, J = 9.5 Hz, glc-400),
5.20 (1H, d, J = 7.0 Hz, glc-10), 5.07 (1H, d, J = 9.5 Hz, glc-100), 4.66
(1H, dd, J = 2.5, 12.0 Hz, glc-60), 4.46 (1H, br d, J = 11.0 Hz, glc-600),
4.36 (1H, dd, J = 5.0, 12.0 Hz, glc-60), 4.29 (1H, dd, J = 5.0, 11.0 Hz,
glc-600), 4.11 (1H, m, glc-500), 4.05 (1H, t, J = 9.5 Hz, glc-200), 3.93
(1H, t, J = 9.5, glc-300), 3.87 (1H, m, glc-50), 3.6–3.7 (3H, m, glc-20–
40), 3.16 (1H, dd, J = 6.0, 15.5 Hz, H-8), 2.96 (1H, dd, J = 6.0,
15.5 Hz, H-8), 2.23 (1H, hept, J = 6.0 Hz, H-9), 0.92, 0.89 (3H each,
d, J = 6.0 Hz, H-10, 11); for 13C NMR spectroscopic data, see Table
1; ESIMS m/z 991 [M + H]+, m/z 1013 [M + Na]+; HRESIMS m/z
991.2350 [M + H]+ (C44H46O26 + H, 991.2356).
3.8. Kunzeaphlogin D (4)
23
Pale yellow amorphous powder; ½aꢁD þ 38 (c 1, MeOH); UV
(MeOH) kmax (loge) 217 (4.34), 271 (4.11), 321sh (3.83) nm; 1H
NMR (acetone-d6 + D2O, 500 MHz) dY; 11.6 (1H, s, 6-OH), 4.70
(1H, d, J = 7.5, glc-100), 4.50 (1H, d, J = 7.5, glc-10), 3.85 (2H, m, glc-
60, 600), 3.64 (1H, dd, J = 2.5, 10.0 Hz, glc-600), 3.62 (1H, dd, J = 5.5,
10.0 Hz, glc-60), 3.46–3.55 (overlapping DHO, glc-20, 30, 200, 300),
3.40 (1H, br t, J = 10.0 Hz, glc-400), 3.35 (1H, br t, J = 10.0 Hz, glc-
40), 3.20 (1H, ddd, J = 2.5, 5.5, 10.0 Hz, glc-500), 3.11 (1H, ddd,
J = 2.5, 5.5, 10.0 Hz, glc-50), 3.16 (1H, dd, J = 7.0, 15.5 Hz, H-8),
2.93 (1H, dd, J = 7.0, 15.5 Hz, H-8), 2.27 (3H, s, 3-Me), 2.14 (3H, s,
5-Me), 2.08 (1H, m, H-9), 0.84, 0.83 (3H each, d, J = 7.0 Hz); for
13C NMR spectroscopic data, see Table 2; ESIMS m/z 563 [M + H]+,
m/z 580 [M + NH4]+; HRESIMS m/z 580.2567 [M + NH4]+
(C25H38O14 + H, 580.2605).
3.4. Kunzeaphlogin A (1)
23
Pale yellow amorphous powder; ½aꢁD ꢂ 19 (c 1, MeOH); UV
(MeOH) kmax (loge) 226 (4.15), 287 (4.02), 321sh (3.55) nm; 1H
NMR (acetone-d6 + D2O, 500 MHz) dY; 6.28 (1H, s, H-3), 5.04 (1H,
d, J = 7.5 Hz, glc-10), 4.80 (1H, d, J = 9.5 Hz, glc-100), 4.00 (1H, t,
J = 9.5 Hz, glc-200), 3.91 (1H, dd, J = 2.5, 11.5 Hz, glc-60), 3.52 (1H, t,
J = 7.5 Hz, glc-400), 3.48 (1H, t, J = 9.5 Hz, glc-300), 3.3–3.8 (overlap-
ping DHO, H-2, 6, glc-20–60 glc-500–600), 3.15 (1H, dd, J = 6.5,
16.5 Hz, H-8), 2.88 (1H, dd, J = 7.5, 16.5 Hz, H-8), 2.16 (1H, m, H-
9), 0.91, 0.87 (3H each, d, J = 7.0 Hz, H-10, 11); for 13C NMR spectro-
scopic data, see Table 1; ESIMS m/z 535 [M + H]+, 552 [M + NH4]+;
HRESIMS m/z 535.1991 [M + H]+ (C23H34O14 + H, 535.2027).
3.9. Acid hydrolysis of 4
3.5. Acid hydrolysis of 1
A solution of 4 (5.1 mg) in 1 M HCl (2.0 mL) was heated at
100 °C for 1 h in a boiling water bath. After cooling, reaction mix-
tures were partitioned between AcOEt and water to yield 4a from
the AcOEt soluble portion and glucose from the water-soluble
A solution of 1 (0.5 mg) in 1 M HCl (1.0 mL) was heated at
100 °C for 1 h in a boiling water bath. After cooling, the reaction

N. Kasajima et al. / Phytochemistry 69 (2008) 3080–3086
3085
portion.
D
-glucose was identified by HPLC with an optical rotation
-glucose oxidase.
(7H in total, each s, HHDP-H and tergalloyl-H), 5.86 (t,
J = 10.5 Hz, H-30a), 5.64 (t, J = 9.5 Hz, H-30b), 5.61 (d, J = 4.0 Hz, H-
10a), 5.45 (dd, J = 3.5, 10.0 Hz, H-3a), 5.42 (d, J = 3.0 Hz, H-1a),
5.29 (dd, J = 8.0, 9.5 Hz, H-20b), 5.22 (m, H-3b, 60a), 5.21 (m, H-
60b), 5.19 (m, H-6b, 20a), 5.18 (m, H-6a), 5.16 (m, H-40b), 5.10 (m,
H-40a), 5.09 (m, H-4a), 5.08 (m, H-4b), 5.03 (m, H-2a), 5.024 (d,
J = 8.5 Hz, H-1b), 5.018 (d, J = 8.0 Hz, H-10b), 4.83 (t, J = 8.5 Hz, H-
2b), 4.65 (br dd, J = 8.0, 10.0 Hz, H-50a), 4.59 (m, H-5a), 4.24 (br
dd, J = 3.5, 10.0 Hz, H-50b), 4.18 (m, H-5b); for 13C NMR spectro-
scopic data, see Table 3; ESIMS m/z 1586 [M + NH4]+.
detector and a positive reaction to
D
3.10. Kunzeaphlogin D aglycone (4a)
Brown oil: 1H NMR (acetone-d6, 500 MHz) dY; 2.98 (2H, d,
J = 7.5, H-8), 2.23 (1H, m, H-9), 2.08 (6H, s, 3-Me, 5-Me), 0.93
(6H, d, J = 7.0, H-10, 11).
3.11. Kunzeaphlogin E (5)
23
Pale yellow amorphous powder; ½aꢁD þ 31 (c 1, MeOH); UV
3.15. Methylation of 7 followed by methanolysis
(MeOH) kmax (loge) 216 (4.71), 274 (4.40), 337sh (3.90) nm; 1H
NMR (acetone-d6 + D2O, 500 MHz) dY; 7.12 (2H, s, galloyl-H),
4.78 (1H, d, J = 7.0 Hz, glc-100), 4.57 (1H, dd, J = 2.0, 12.0 Hz, glc-
600), 4.53 (1H, d, J = 7.5 Hz, glc-10), 4.28 (1H, d, J = 4.0, 12.0 Hz, glc-
600), 3.35–3.60 (overlapping DHO, glc-20–40, 60, 200–500), 3.15 (1H,
m, H-8), 3.14 (1H, m, glc-50), 2.95 (1H, dd, J = 6.5, 16.5 Hz, H-8),
2.22 (3H, s, 3-Me), 2.14 (3H, s, 5-Me), 2.12 (1H, m, H-9), 0.88,
0.86 (3H each, d, J = 7.0 Hz, H-10, 11); for the 13C NMR data, see Ta-
ble 2; ESIMS m/z 715 [M + H]+, m/z 732 [M + NH4]+, m/z 737
[M + Na]+; HRESIMS m/z 732.2590 [M + NH4]+ (C32H42O18 + H,
732.2715).
Compound 7 (1.0 mg) in EtOH was methylated with CH2N2–
Et2O at room temperature for 8 h. The residue obtained after re-
moval of the solvent was directly methanolyzed with 1% NaOMe
in MeOH (1.0 mL) at room temperature for 4 h to give methyl tri-
O-methylgallate (10), dimethyl hexamethoxydiphenate (11) and
trimethyl octa-O-methyltergallate (12), which were identified by
co-chromatography on the reversed-phase HPLC (solvent D).
3.16. Isomerization of 7 to camelliin A by smiles rearrangement
A solution of 7 (0.2 mg) in a phosphate buffer (pH 7.2) (0.5 mL)
was treated at room temperature for 1 h. The reactant was identi-
fied as camelliin A (13) by co-chromatography on normal phase
HPLC (Rt 15.2 min, solvent A) and reversed-phase HPLC (Rt 5.4,
6.0 min, solvent C).
3.12. Partial hydrolysis of 2, 3 and 5 with tannase
A solution of each compound (0.2–1.0 mg) in H2O (0.2–0.5 mL)
was treated at 37 °C for an appropriate time with two drops of tan-
nase, which was obtained from Aspergillus niger. After addition of
EtOH, the reaction mixture was evaporated to dryness. The residue
was analyzed by normal and reversed-phase HPLC. The products
besides gallic acid from each compound were as follows and iden-
tified by co-chromatography with authentic specimens,
respectively:
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific
Research (No. 10557207) from the Ministry of Education, Science,
Sports and Culture, Japan. Thanks are due to Mr. Y. Kitada of the
Central Research Laboratory of the Pola Corporation for the kind
supply of the plant materials. The NMR experiments were per-
formed at the SC-NMR Laboratory of Okayama University.
Production of 1 and 2 from 3: reaction time 20 h; normal and
reversed-phase HPLC (solvents A, B).
Production of 4 from 5: reaction time 48 h; normal and
reversed-phase HPLC (solvents A, B).
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23
Pale yellow amorphous powder; ½aꢁD þ 30 (c 1, MeOH); UV
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Pale brown amorphous powder; ½aꢁD þ 54 (c 1, MeOH); UV
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