Dimeric Flavonol Glycoside and Galloylated C-Glucosylchromones from Kunzea ambigua

Article abstract of DOI:10.1021/np030367s

A novel dimeric flavonol glycoside linked through a methylene group, kunzeagin A (1), and six new chromone C-glucosides, kunzeachromones A-F (2-7), were isolated along with seven known compounds from the leaf extract of Kunzea ambigua. The structures of these compounds were elucidated on the basis of spectroscopic analyses and chemical properties. Kunzeachromones A-F provided additional examples of galloylated C-glucosidic chromones occurring in the Myrtaceae. Kunzeagin A (1) and major constituents of this plant (6-C- and 8-C-glucosylchromones and their monogallates) exhibited potent inhibitory effects on activation of Epstein-Barr virus early antigen induced by 12-O-tetradecanoylphorbol 13-acetate in Raji cells.

Full text of DOI:10.1021/np030367s

J . Nat. Prod. 2004, 67, 411-415
411
Dim er ic F la von ol Glycosid e a n d Ga lloyla ted C-Glu cosylch r om on es fr om Ku n zea
a m bigu a
Hideyuki Ito,† Naoki Kasajima,†,§ Harukuni Tokuda,‡ Hoyoku Nishino,‡ and Takashi Yoshida*,†
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, J apan, and
Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, J apan
Received August 8, 2003
A novel dimeric flavonol glycoside linked through a methylene group, kunzeagin A (1), and six new
chromone C-glucosides, kunzeachromones A-F (2-7), were isolated along with seven known compounds
from the leaf extract of Kunzea ambigua. The structures of these compounds were elucidated on the
basis of spectroscopic analyses and chemical properties. Kunzeachromones A-F provided additional
examples of galloylated C-glucosidic chromones occurring in the Myrtaceae. Kunzeagin A (1) and major
constituents of this plant (6-C- and 8-C-glucosylchromones and their monogallates) exhibited potent
inhibitory effects on activation of Epstein-Barr virus early antigen induced by 12-O-tetradecanoylphorbol
13-acetate in Raji cells.
Ta ble 1. 1H and 13C NMR Spectral Data for Kunzeagin A (1)
Kunzea ambigua (SM.) Druce, which is a plant of the
(δH, 500 MHz; δC, 126 MHz, in DMSO-d6)
Myrtaceae family, is a bushy shrub that is endemic to
eastern Australia (Tasmania, New South Wales, and
Victoria). Its essential oil, which contains R-pinene, R-ter-
pinene, and 1,8-cineole, has been used as an insecticide and
antiseptic and as a remedy for the treatment of wounds.1
Although myrtaceous plants are known to be rich in both
polyphenols and terpenoids,2,3 there have been no reports
on the polyphenol constituents of K. ambigua. As part of
our studies on the polyphenolics of the Myrtaceae family,
we have investigated the constituents of this plant and
isolated 14 polyphenols, including seven newly discovered
phenolic compounds. In this report, we describe the eluci-
dation of the structures of these novel compounds. Some
of the major constituents of the plant were also evaluated
for an in vitro inhibitory effect on activation of Epstein-
Barr virus early antigen (EBV-EA) induced by tumor
promoter.
position
δH
δC
2
157.14, 157.08
134.2, 130.0
3
4
5
178.2, 177.9
158.7(U), 156.3(L)
5-OH
13.17 (1H, s, U),a
13.02 (1H, s, L)
6
110.0(U), 106.3(L)
164.0(U), 160.7(L)
93.3(U), 105.0(L)
154.6(U), 152.1(L)
103.8(2C)
7
8
6.35 (1H, s, U)
9
10
11
4.05 (2H, br s, U),
1.98 (3H, s, L)
16.3(U), 8.1(L)
1′, 6′
2′, 5′
7.37, 7.23 (each 1H,
dd, 2.0, 8.5, H-6′)b
7.49, 7.27 (each 1H,
d, 2.0, H-2′)
121.5, 121.0, 121.3(2C)
116.2, 115.8, 115.62,
115.56
6.86, 6.83 (each 1H,
d, 8.5, H-5′)
3′
4′
145.4, 145.3
148.54, 148.50
Resu lts a n d Discu ssion
rhamnose
1′′
A concentrated aqueous acetone homogenate of dried
leaves was extracted successively with ether, ethyl acetate,
and n-butanol, thereby producing an extract and a water-
soluble portion at each step. The ethyl acetate and n-
butanol extracts were each subjected to a series of column
chromatography steps, which involved Diaion HP-20, Toyo-
pearl HW-40, MCI GEL CHP-20P, and Sephadex LH-20
columns, resulting in the purification of a new flavonoid
named kunzeagin A (1), six new chromones, kunzeachro-
mones A-F (2-7), and seven known compounds. The
known compounds were identified as quercitrin, pinoquer-
cetin (6-methylquercetin),4 myricetin 3-O-â-galactopyra-
noside,5,6 6-â-C-glucopyranosyl-5,7-dihydroxy-2-isopropyl-
chromone (8),6 6-â-C-(2′-O-galloylglucopyranosyl)-5,7-dihy-
droxy-2-isopropylchromone (9),7 8-â-C-glucopyranosyl-5,7-
dihydroxy-2-isopropylchromone (10),8 and 8-â-C-(2′-O-gal-
loylglucopyranosyl)-5,7-dihydroxy-2-isopropylchromone (11).7
5.25, 5.23 (each 1H, br s) 102.1, 102.0
3.1-3.6 71.43, 71.35, 70.8(2C),
70.6, 70.5, 70.3, 70.2
2′′-5′′
6′′
0.83, 0.80 (each 3H, d, 6) 17.69, 17.67
a
b
U: upper unit, L: lower unit. J values (Hz) are in paren-
theses.
(M + H)+]. Its UV spectrum (λmax 271 and 352 nm) was
indicative of its flavonol skeleton. The H NMR spectrum
1
of 1 in DMSO-d6 contained six hydrogen-bonded hydroxyl
proton signals at δ 13.17, 13.02, 9.66, 9.64, 9.31, and 9.28;
the first two signals were characteristic of a 5-OH group.
The spectrum exhibited two ABX system sets [δ 7.49/7.27
(each d, J ) 2.0 Hz); 6.86/6.83 (each d, J ) 8.5 Hz); 7.37/
7.23 (each dd, J ) 2.0, 8.5 Hz)] due to B-ring protons, and
only one A-ring proton appeared as a singlet at δ 6.35. The
presence of two rhamnose residues was also suggested by
two secondary methyl signals (δ 0.83/0.80, each d, J ) 6.0
Hz) and two anomeric proton signals [δ 5.23/5.25 (each br
s)]. These spectral features, together with that of an
isolated methylene (δ 4.05, br s) and a methyl signal (δ
1.98, s), imply that 1 is a dimer of quercitrin, in which each
glycoside is linked through a methylene bridge between the
A-rings, with one of the A-rings containing a methyl group
at C-6 or C-8. This assumption was consistent with the 13C
NMR data (Table 1). The HMBC measurement indicated
Kunzeagin A (1) was obtained as yellow needles, and its
molecular formula of C44H42O22 was established by high-
resolution electrospray ionization (HRESI) MS [m/z 923.2233
* To whom correspondence should be addressed. Tel & Fax: +81-86-
251-7936. E-mail: yoshida@pharm.okayama-u.ac.jp.
† Okayama University.
§ Present address: School of Pharmacy, Shujitsu University, Nishi-
gawara, Okayama 700-8516, J apan.
‡ Kyoto Prefectural University of Medicine.
10.1021/np030367s CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/20/2004

412 J ournal of Natural Products, 2004, Vol. 67, No. 3
Ito et al.
Ta ble 2. 1H NMR Spectral Data for Kunzeachromones A-F (2-7) (500 MHz, DMSO-d6)
proton
OH-5
2
3
4
5
6
7
13.49
12.98
(1H, s)
6.21
13.41
(1H, s)
6.10
13.51
12.92
(1H, s)
6.08
12.94
(1H, s)a
6.07
(1H, s)
6.11
(1H, s)
6.11
H-3
(1H, s)
(1H, s)
6.10
(1H, s)
(1H, s)
(1H, s)
6.20
(1H, s)
6.24
H-6
(1H, s)
(1H, s)
(1H, s)
H-8
6.29
6.26
6.28
(1H, s)
2.81
(1H, hept, 7)
1.18
(1H, s)
2.28
(3H, s)
(1H, s)
2.28
(3H, s)
H-11
H-12, 13
3.02
(1H, hept, 7)
1.44, 1.39
2.40
(3H, s)
2.47
(3H, s)
(6H, d, 7)
(each 3H, d, 7)
glucose
C-1′
5.03
5.06
4.85
5.02
4.86
5.04
(1H, d, 10)
5.97
(1H, br t, 10)
5.25
(1H, t, 10)
3.55
(1H, m)
3.43
(1H, d, 10)
5.88
(1H, t, 10)
5.34
(1H, t, 10)
3.63
(1H, t, 10)
3.51
(1H, d, 10)
5.67
(1H, m)
3.42-3.46
(1H, d, 10)
5.97
(1H, m)
5.25
(1H, t, 10)
3.42-3.54
(1H, d, 10)
5.56
(1H, t, 10)
3.52
(1H, m)
3.52
(1H, m)
3.25
(1H, d, 10)
5.81
(1H, t, 10)
5.32
(1H, t, 10)
3.65
(1H, t, 10)
3.46
C-2′
C-3′
C-4′
C-5′
C-6′
3.25
(1H, m)
3.25
(1H, m)
3.73
3.42-3.54
(1H, m)
3.75
(1H, m)
3.78
(1H, m)
3.72
(1H, m)
3.75
3.75
(1H, br d, 11.5)
3.50
(1H, dd, 6.5, 11.5)
(1H, br d, 11)
3.51
(1H, m)
(1H, br d, 11.5)
3.42-3.46
(1H, br d, 11)
3.42-3.54
(1H, br d, 11.5)
3.46
(1H, m)
(1H, br d, 11)
3.55
(1H, dd, 5, 11)
galloyl
C-2′′, 6′′
6.87, 6.69
6.84, 6.63
6.77
6.87, 6.64
6.75
6.88, 6.63
(each 2H, s)
(each 2H, s)
(2H, s)
(each 2H, s)
(2H, s)
(each 2H, s)
a
J values (Hz) are in parentheses.
that each 5-OH proton correlated through two- and three-
bond couplings with C-5, C-6, and C-10 of each A-ring. The
signals assignable to each C-6 (δ 110.0 and 106.3) were also
correlated with the methylene and methyl protons, respec-
tively, which establishes the location of those alkyl sub-
stituents. The location of the rhamnosyl residues was
deduced by the correlation of each anomeric proton with
the C-3 signals of upper and lower units, respectively. The
other long-range correlations are illustrated in the formula.
have been reported to date. To the best of our knowledge,
kunzeagin A constitutes a new class of naturally occurring
bisflavonol glycoside that is bonded through a methylene
bridge. The existence of this class of flavonol in the
Myrtaceae is not surprising, because many formylated
phloroglucinol derivatives and methylated flavonol mono-
mers have been found among the plants of this family.8,9
Kunzeachromone A (2), which is a pale yellow amor-
phous powder, exhibited an [M + H]+ ion peak at m/z
687.1359 in the HRESIMS, corresponding to the molecular
formula C32H30O17. The 1H NMR spectrum of 2 revealed
the presence of a hydrogen-bonded OH at δ 13.49, two 2H
singlets due to galloyl groups at δ 6.87 and 6.69, two 1H
singlets at δ 6.29 and 6.07, a methine proton signal at δ
2.81 (1H, hept, J ) 7 Hz) coupled with two methyl signals
at δ 1.18 (6H, d, J ) 7 Hz), and aliphatic proton signals
arising from a â-glucopyranosyl moiety (Table 2). These
data were similar to those for 6-â-C-(2′-O-galloylglucopy-
ranosyl)-5,7-dihydroxy-2-isopropylchromone (9),7 except for
the presence of the extra galloyl group. In the 13C NMR
spectrum of 2, resonances that were ascribable to two
galloyl, isopropyl, chromone, and glucose moieties were also
observed (Table 3). Treatment of 2 with tannase furnished
6-â-C-glucopyranosyl-5,7-dihydroxy-2-isopropylchromone (8)
and 9, as well as gallic acid. The location of the extra galloyl
group at C-3′ was determined by comparison of the 1H
NMR spectrum of 2 with that of 9, which revealed a
remarkable downfield shift (ca. ∆ 1.8 ppm) of H-3′ in 2.
On the basis of these findings, the structure of kunzeach-
romone A was determined as 2.
1
Acid hydrolysis of 1 yielded an aglycone whose H NMR
and HRESIMS spectra were coincident with the proposed
structure. A liberated sugar from 1 was identified as
L-rhamnose by co-chromatography with an authentic sample
on an HPLC system that was equipped with an optical
rotation detector. The L-series of rhamnose was also
confirmed by enzymatic hydrolysis of 1 with naringinase.
On the basis of these data, structure 1 was assigned to
kunzeagin A.
Kunzeachromone B (3) has the molecular formula
C
32H30O17, which is the same as that of 2, as revealed by
HRESIMS, which showed an [M + H]+ ion peak at m/z
687.1360. The H and 13C NMR spectral data for 3 (Tables
1
2 and 3) were similar to those for 8-â-C-(2′-O-galloylglu-
copyranosyl)-5,7-dihydroxy-2-isopropylchromone (11), ex-
cept for resonances due to the extra galloyl group, which
suggests that compound 3 is 8-C-digalloylglucosyl-5,7-
Numerous flavonoids, including prenylated flavonoids,
biflavonoids, and flavan-3-ol oligomers (condensed tannins),

C-Glucosylchromones from Kunzea ambigua
J ournal of Natural Products, 2004, Vol. 67, No. 3 413
Ta ble 3. 13C NMR Spectral Data for Kunzeachromones A-F (2-7) (126 MHz, DMSO-d6)
carbon
2
3
4
5
6
7
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
glucose
C-1′
C-2′
C-3′
C-4′
C-5′
174.9
105.3
182.2
160.9
106.4
163.2
93.4
157.0
103.1
32.4
174.5
105.6
182.4
161.0
97.9
162.6
102.1
157.2
104.0
33.1
167.8
167.7
106.3
182.0
161.1
108.1
163.1
93.4
157.1
103.0
19.9
167.6
167.7
108.0
182.2
161.0
98.0
162.4
103.9
157.1
102.0
20.1
107.0
182.0
160.4
108.1
162.4
93.9
157.0
102.9
20.0
107.8
182.2
160.8
97.6
162.2
103.9
157.1
102.8
20.0
19.78
19.75
20.3
20.1
70.7
69.7
77.6
68.6
81.7
61.2
70.8
70.2
77.0
68.9
81.9
61.3
70.8
72.0
76.7
70.8
82.0
61.6
70.7
69.7
77.6
68.7
81.8
61.2
70.6
72.5
76.1
70.8
81.7
61.5
70.7
70.1
77.0
68.5
81.5
61.0
C-6′
galloyl
C-1′′
C-2′′, 6′′
C-3′′, 5′′
C-4′′
C-7′′
119.7, 119.0
119.6, 118.7
119.9
119.7, 119.1
119.7
119.6, 118.7
108.9 (2C), 108.7 (2C)
145.4 (2C), 145.3 (2C)
138.4, 138.3
108.9 (2C), 108.7 (2C)
145.5 (2C), 145.4 (2C)
138.7, 138.6
108.9 (2C)
145.4 (2C)
138.2
108.9 (2C), 108.7 (2C)
145.5 (2C), 145.3 (2C)
138.5, 138.4
108.8 (2C)
145.5 (2C)
138.8
108.9 (2C), 108.7 (2C)
145.6 (2C), 145.5 (2C)
138.7, 138.5
165.4, 164.4
165.4, 164.8
164.8
165.3, 164.6
165.0
165.5, 164.8
Kunzeachromones E (6) and F (7) were isomers of 4 and
5 at the position of the C-glucosyl residue, on the basis of
their respective molecular formulas of C23H22O13 and
C
30H26O17 and their NMR spectra, which were similar,
except for the chemical shifts of H-6 to H-8 and C-6 to C-8,
respectively. Therefore, compounds 6 and 7 were assumed
to be the mono- and digallate forms of 8-â-C-glucopyrano-
syl-5,7-dihydroxy-2-methylchromone (13; isobiflorin).11 Treat-
ment of 6 and 7 with tannase afforded isobiflorin (13) and
gallic acid. The locations of the galloyl groups in each
compound were evident from the 1H NMR spectra that
were assigned by COSY (Table 2). Consequently, the
structures of kunzeachromones E and F were represented
by 6 and 7.
Galloylated C-glucosylchromones have been identified
only in Baeckea frutescens7 and Syzygium aromaticum11
in Myrtaceae. Isolation of kunzeachromones from the genus
Kuzea would thus be of chemotaxonomical significance. It
is also noteworthy that kunzeachromones A (2), B (3), D
(5), and F (7) are the first examples of C-glucosylchromone
digalloyl esters.
dihydroxy-2-isopropylchromone. The galloylation at C-3′ of
the glucosyl residue of 3 was substantiated by a notable
downfield shift of H-3′ (ca. ∆ 1.8 ppm) relative to 11.
Enzymatic hydrolysis of 3 with tannase afforded 8-â-C-
glucopyranosyl-5,7-dihydroxy-2-isopropylchromone (10), 11,
and gallic acid. On the basis of these data, the structure of
kunzeachromone B was established to be 3.
Polyphenols possessing galloyl unit(s) represented by
(-)-epigallocatechin gallate (EGCG) from green tea and
many hydrolyzable tannins have been reported to be
possible chemopreventive agents of cancer on the basis of
their remarkable suppression of tumor promotion in mul-
tistep mechanisms of chemical carcinogenesis.12-14 To
develop a beneficial function of the K. ambigua leaves, some
major constituents obtained in the present study were
assessed for their inhibitory effects on the activation of
EBV-EA using Raji cells, which has been frequently used
as an in vitro preliminary screening test for searching
possible anti-tumor-promoting agents in nature.15 Tested
samples were 6-C- and 8-C-glucosidic chromones (8, 10) and
their monogallates (9, 11) in addition to bisflavonoid (1).
The results are shown in Table 4. All of them significantly
inhibited (67-76%) EBV-EA activation induced by 12-O-
tetradecanoylphorbol 13-acetate (TPA) at a concentration
of 500 mol ratio/TPA without exhibiting cytotoxicity. These
activities were more potent or equivalent to that (65.1%
inhibition) of EGCG. It is noteworthy that 6-C-glucosyl-
chromones were more potent than 8-C congeners, and
Kunzeachromones C (4) and D (5) had molecular formu-
las C23H22O13 and C30H26O17, respectively, by HRESIMS.
The 1H and 13C NMR spectra of 5 (Tables 2 and 3) were
very similar to those of 2, except for the presence of a
methyl signal (δH 2.28 and δC 19.9) in place of the isopropyl
group at C-2 in 2, which indicates that 5 is the 2′,3′-
digallate of 6-â-C-glucopyranosyl-5,7-dihydroxy-2-methyl-
chromone (12; biflorin).10 The 1H NMR spectrum of 4
exhibited a 2H singlet due to one galloyl group (δ 6.67) and
an upfield shift in H-3′ compared with that of 5 (δ 5.25 in
5 f δ 3.42-3.46 in 4); the remaining signals were similar
to those of 5. The structural relationship between 4 and 5
was confirmed chemically by enzymatic hydrolysis of 5 with
tannase, which afforded 4 and biflorin (12), along with
gallic acid. The 13C NMR spectra of 4 and 5 were also
consistent with the proposed structures.

414 J ournal of Natural Products, 2004, Vol. 67, No. 3
Ito et al.
Ta ble 4. Relative Ratio of EBV-EA Activationa with Respect to
Positive Control (100%) in the Compounds from K. ambigua
chromatography over an MCI GEL CHP-20P column (1.1 cm
i.d. × 25 cm) to yield 6-â-(2′-O-galloylglucopyranosyl)-5,7-
dihydroxy-2-isopropylchromone (9) (39 mg) and 8-â-(2′-O-
galloylglucopyranosyl)-5,7-dihydroxy-2-isopropylchromone (11)
(31 mg). The 50% MeOH eluate was similarly chromato-
graphed over an MCI GEL CHP-20P column (1.1 cm i.d. × 25
cm) with aqueous MeOH, and a Sephadex LH-20 column (1.1
cm i.d. × 28 cm) with EtOH/MeOH, to afford kunzeagin A (1)
(44 mg) and pinoquercetin (4 mg). The other portion (3.3 g) of
the EtOAc extract was subjected to column chromatography
over a Toyopearl HW-40 column (2.2 cm i.d. × 42 cm) with
aqueous MeOH in a stepwise gradient. The 30% and 40%
MeOH eluates were purified by column chromatography over
a YMC-GEL ODS-A 120-S50 column (1.1 cm i.d. × 44 cm) with
aqueous MeOH to afford 9 (44 mg), 11 (71 mg), and myricetin
3-O-â-galactopyranoside (10 mg), respectively. The 50% and
60% MeOH eluates were separately rechromatographed over
a YMC-GEL ODS-A 120-S50 column (1.1 cm i.d. × 44 cm) with
aqueous MeOH, yielding kunzeachromones A (2) (23 mg), B
(3) (17 mg), D (5) (31 mg), and F (7) (25 mg). A 10.1 g portion
of the 24.6 g n-BuOH extract was fractionated by column
chromatography over a Diaion HP-20 column (5.0 cm i.d. ×
50 cm) and developed with H2O and increasing amounts of
MeOH in a stepwise gradient. The 30% MeOH eluate was
purified by column chromatography over a Toyopearl HW-40
column (2.2 cm i.d. × 40 cm) and/or a Mega Bond Elut C18
cartridge with aqueous MeOH to afford myricetin 3-O-â-
galactopyranoside (3 mg). The 100% MeOH eluate was further
purified on a Toyopearl HW-40 column (2.2 cm i.d. × 35 cm)
and an MCI GEL CHP-20P column (1.1 cm i.d. × 23 cm) with
aqueous MeOH and a Sephadex LH-20 column (1.1 cm i.d. ×
21 cm) with EtOH-MeOH to yield 1 (11 mg) and 8 (14 mg).
The remainder of the n-BuOH extract was pooled and further
purified by column chromatography over Diaion HP-20, MCI
GEL CHP-20P, and YMC-GEL ODS AQ 120-50S columns and
a Mega Bond Elut cartridge with aqueous MeOH, and/or a
Sephadex LH-20 column with EtOH-MeOH, to afford kun-
zeachromones C (4) (24 mg) and E (6) (19 mg).
concentration (mol ratio/TPA)b
1000
500
100
10
1
8
9
10
4.3 (70)c
4.6 (70)
5.9 (70)
9.1 (70)
11.5 (70)
6.4 (70)
32.5
23.7
26.8
29.6
31.6
34.9
66.1
65.0
70.4
72.5
74.7
68.1
90.5
91.0
94.8
100
100
87.7
11
EGCGd
a
Values represent percentages relative to the positive control
b
value (100%). TPA concentration was 20 ng (32 pmol)/mL.
c Values in parentheses are the viability percentage of Raji cells;
unless otherwise stated, the viability percentage of Raji cells was
d
more than 80%. Reference compound.
galloylation on these skeletones resulted in reducing the
inhibitory effect on EBV-EA activation. Further in vivo
anti-tumor-promoting effects and other biological activity
of the compounds in K. ambigua are under investigation.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
measured on a Yanaco micro-melting point apparatus and are
1
uncorrected. The H and 13C NMR spectra were recorded on a
Varian VXR-500 instrument (500 MHz for 1H, and 126 MHz
for 13C), and the chemical shifts are given in δ (ppm) values
relative to that of the solvent [DMSO-d6 (δH 2.49; δC 39.7),
acetone-d6 (δH 2.04; δC 29.8)] and tetramethylsilane. The
standard pulse sequences that were programmed into the
instrument (VXR-500) were used for each two-dimensional
measurement. The J CH value was set at 6 Hz in the HMBC
spectra. Optical rotations were measured with a J asco DIP-
1000 polarimeter. UV spectra were measured with a Hitachi
U-2000 spectrophotometer. ESIMS was recorded on a Micro-
mass Auto Spec OA-TOF mass spectrometer (solvent: 50%
aqueous MeOH 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 i.d. × 250 mm; YMC Co. Ltd.) and developed
at room temperature with a solution of n-hexane/MeOH/
tetrahydrofuran/formic acid (55:33:11:1) that contained 450
mg/L oxalic acid (flow rate: 1.5 mL/min; 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/EtOH/EtOAc (45:
45:8:2, solvent A) or 10 mM H3PO4/10 mM KH2PO4/MeCN
(42.5:42.5:15, solvent B; 40:40:20, solvent C). Column chro-
matography was carried out on Diaion HP-20, MCI GEL CHP-
20P (Mitsubishi Kasei Co.), Toyopearl HW-40 (coarse grade;
Tosoh Co.), YMC-GEL ODS AQ 120-50S (YMC Co., Ltd.),
Sephadex LH-20 (Pharmacia Fine Chemicals Co., Ltd.), and
Mega Bond Elut C18 (Varian Inc.) columns.
Ku n zea gin A (1): yellow needles, mp 243-245 °C (from
MeOH); [R]23D -282° (c 1, MeOH); UV (MeOH) λmax (log ꢀ) 271
(4.67), 352 (4.53) nm; for the 1H and 13C NMR spectral data,
see Table 1; ESIMS m/z 923 [M + H]+, 940 [M + NH4]+;
HRESIMS m/z 923.2233 [M + H]+ (C44H42O22+H, 923.2246).
Hyd r olysis of 1. A 10 mg solution of 1 in 1 M HCl (5 mL)
was heated for 1 h in a boiling water bath. After cooling, the
reaction mixture was separated using a Mega Bond Elut C18
cartridge column to yield the aglycone of 1 (5 mg) from the
MeOH eluate.
To a 1 mL solution of 1 (0.6 mg) in phosphate buffer (pH
3.8) was added 1.0 mg of naringinase (N-1385, Sigma), and
the mixture was incubated at 40 °C for 48 h. The HPLC
analysis revealed the completion of hydrolytic cleavage of the
glycosidic linkage.
Aglycon e of 1: yellow powder; 1H NMR (500 MHz, acetone-
d6+D2O) δ 8.03, 7.73 (each 1H, d, J ) 2.0 Hz, H-2′), 7.89, 7.62
(each 1H, dd, J ) 2.0, 8.5 Hz, H-6′), 6.98, 6.94 (each 1H, d, J
) 8.5 Hz, H-5′), 6.71 (1H, s, U-H-8), 4.15 (2H, s, U-H-11), 2.01
(3H, s, L-H-11); ESIMS m/z 631 [M + H]+; HRESIMS m/z
631.1313 [M + H]+ (C32H22O14+H, 631.1089). The water eluate
was neutralized and analyzed by reversed-phase HPLC with
an optical rotation detector (Shodex OR-2; Showa Denko Co.,
Ltd.) [column, TSK-gel Amide-80 (4.6 mm i.d. × 250 mm)
(Tosoh Co.); solvent, MeCN/H2O (75:25); column temp, 35 °C],
to detect L-rhamnose (tR 6.5 and 8.6 min) as the negative peaks
identical with that of an authentic specimen.
P la n t Ma ter ia ls. K. ambigua (SM.) Druce was collected
in April 1998 from the herbal garden of Pola Co. Ltd., J apan.
The voucher specimen has been deposited in the herbarium
of Pola Co. Ltd and in the Medicinal Herbal Garden of
Okayama University (specimen no. OKP-MY98003).
Extr a ction a n d Isola tion . Dry leaves (700 g) of K. am-
bigua were homogenized in 70% aqueous acetone (10 L), and
the homogenate was filtered and concentrated. The concen-
trated solution was extracted successively with ether, EtOAc,
and water-saturated n-BuOH. A 2.0 g portion of the 11.0 g
EtOAc extract was chromatographed over a Toyopearl HW-
40 column (2.2 cm i.d. × 42 cm) with H2O containing
increasing amounts of MeOH in a stepwise gradient. The 20%
and 30% MeOH eluates were subjected separately to column
chromatography over an MCI GEL CHP-20P column (1.1 cm
i.d. × 25 cm) with aqueous MeOH, to yield 6-â-C-glucopyra-
nosyl-5,7-dihydroxy-2-isopropylchromone (8) (13 mg) and 8-â-
C-glucopyranosyl-5,7-dihydroxy-2-isopropylchromone (10) (21
mg). The 40% MeOH eluate was further purified by column
Ku n zea ch r om on e A (2): pale yellow amorphous powder;
[R]23 +30° (c 1, MeOH); UV (MeOH) λmax (log ꢀ) 216 (4.83),
D
258 (4.50), 277 (4.41) nm; for 1H and 13C NMR spectral data,
see Tables 2 and 3; ESIMS m/z 687 [M + H]+; HRESIMS m/z
687.1359 [M + H]+ (C32H30O17+H, 687.1361).
Ku n zea ch r om on e B (3): pale yellow amorphous powder;
[R]23 -23° (c 1, MeOH); UV (MeOH) λmax (log ꢀ) 217 (4.84),
D
258 (4.48), 277 (4.42) nm; for 1H and 13C NMR spectral data,
see Tables 2 and 3; ESIMS m/z 687 [M + H]+; HRESIMS m/z
687.1360 [M + H]+ (C32H30O17+H, 687.1361).

C-Glucosylchromones from Kunzea ambigua
J ournal of Natural Products, 2004, Vol. 67, No. 3 415
Ku n zea ch r om on e C (4): colorless fine needles, mp 212-
EBV-EA Assa y. The inhibition of EBV-EA activation was
assayed using Raji cells (virus nonproducer), the EBV genome-
carrying lymphoblastoid cells derived from Burkitt’s lym-
phoma, which were cultured in 10% fetal bovine serum (FBS)
in RPMI-1640 medium (Nissui). The indicator cells (Raji, 1 ×
106/mL) were incubated at 37 °C for 48 h in the medium (1
mL) containing n-butyric acid (4 mM), TPA [20 ng (32 pM) in
DMSO 2 µL] as inducer, and a known amount of test compound
in 5 µL of DMSO. Smears were made from the cell suspension,
and the activated cells that were stained by EBV-EA positive
serum were detected by a conventional indirect immunofluo-
rescens technique. In each assay, at least 500 cells were
counted, and the number of stained cells was recorded.
Triplicate assays were carried our for each compound. The
average EBV-EA induction of the test compound was expressed
as a relative ratio to the positive control experiment (100%)
with n-butyric acid plus TPA in which EA induction was
ordinarily 35%.
214 °C (from MeOH); [R]23 -87° (c 1, MeOH); UV (MeOH)
D
λmax (log ꢀ) 210 (4.73), 258 (4.45), 274 (4.23) nm; for 1H and
13C NMR spectral data, see Tables 2 and 3; ESIMS m/z 507
[M + H]+, 529 [M + Na]+; HRESIMS m/z 507.1193 [M + H]+
(C23H22O13+H, 507.1139).
Ku n zea ch r om on e D (5): pale yellow amorphous powder,
[R]23 -23° (c 1, MeOH); UV (MeOH) λmax (log ꢀ) 215 (4.74),
D
259 (4.33), 277 (4.32) nm; for 1H and 13C NMR spectral data,
see Tables 2 and 3; ESIMS m/z 659 [M + H]+; HRESIMS m/z
659.1238 [M + H]+ (C30H26O17+H, 659.1248).
Ku n zea ch r om on e E (6): colorless needles, mp 204-205
°C (from MeOH); [R]23 -135° (c 1, MeOH); UV (MeOH) λmax
D
(log ꢀ) 210 (4.72), 257 (4.40), 281 (4.25) nm; for 1H and 13C
NMR spectral data, see Tables 2 and 3; ESIMS m/z 507 [M +
H]+, 529 [M + Na]+; HRESIMS m/z 507.1085 [M + H]+
(C23H22O13+H, 507.1139).
Ku n zea ch r om on e F (7): pale yellow amorphous powder,
[R]23D -4° (c 1, MeOH); UV λmax (MeOH) nm (log ꢀ) 216 (4.78),
257 (4.39), 277 (4.34); for 1H and 13C NMR spectral data, see
Tables 2 and 3; ESIMS m/z 659 [M + H]+; HRESIMS m/z
659.1263 [M + H]+ (C30H26O17+H, 659.1248).
Ack n ow led gm en t. 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, J apan.
Thanks are due to Mr. Y. Kitada of the Central Research
Laboratory of the Pola Corporation for kindly supplying plant
material. The NMR experiments were performed at the SC-
NMR Laboratory of Okayama University.
P a r tia l Hyd r olyses of 2-7 w ith Ta n n a se. A solution of
each compound (0.4-0.5 mg) in H2O (0.4 mL) was treated at
37 °C for an appropriate time with two drops of tannase, which
was obtained from Aspergillus niger, as described previously.16
After the addition of EtOH, the reaction mixture was evapo-
rated to dryness. The residue was analyzed by normal- and
reversed-phase HPLC. The liberated compounds in addition
to gallic acid from each compound were as follows and
identified respectively by co-chromatography with authentic
specimens: Biflorin (12) was obtained by hydrolysis of 4 (5
mg) with tannase and identified by comparison of the spectral
data with those reported in the literature.10 12: pale yellow
amorphous powder; 1H NMR (500 MHz, DMSO-d6) δ 13.39
(1H, s, 6-OH), 6.37 (1H, s, H-8), 6.16 (1H, s, H-3), 4.55 (1H, d,
J ) 10.0 Hz, Glc-H-1′), 3.0-4.1 (6H, m, Glu-H-2′-6′), 2.34 (3H,
s, CH3); ESIMS m/z 335 [M + H]+. Isobiflorin (13), which was
previously isolated from Eucalyptus cypellocarpa,17 was used
as standard sample upon comparison on HPLC.
2: reaction time 8 h; normal- and reversed-phase HPLC
(solvent C) resulted in peaks for 6-â-C-glucopyranosyl-5,7-
dihydroxy-2-isopropylchromone (8) and 6-â-C-(2′-galloylglu-
copyranosyl)-5,7-dihydroxy-2-isopropylchromone (9).
3: reaction time 24 h; normal- and reversed-phase HPLC
(solvent C) resulted in peaks for 8-â-C-glucopyranosyl-5,7-
dihydroxy-2-isopropylchromone (10) and 8-â-C-(2′-galloylglu-
copyranosyl)-5,7-dihydroxy-2-isopropylchromone (11).
4: reaction time 48 h; normal- and reversed-phase HPLC
(solvent B) resulted in peaks for biflorin (12).
Refer en ces a n d Notes
(1) David, H. J . J . PCT Int. Appl. 1997, 14 pp.
(2) Yazaki, Y. Phytochemistry 1977, 16, 138-139.
(3) Okuda, T.; Yoshida, T.; Hatano, T.; Yazaki, K.; Ashida, M. Phy-
tochemistry 1980, 21, 2871-2874.
(4) Mani, R.; Ramanathan, V.; Venkataraman, K. J . Sci. Ind. Res. 1956,
15B, 490-495.
(5) Foo, L. Y.; Lu, Y.; Molan, A. L.; Woodfield, D. R.; McNabb, W. C.
Phytochemistry 2000, 54, 539-548.
(6) Markham, K. R.; Ternai, B.; Stanley, R.; Geiger, H.; Mabry, T.
Tetrahedron 1978, 34, 1389-1397.
(7) Satake, T.; Kamiya, K.; Saiki, Y.; Hama, T.; Fujimoto, Y.; Endang,
H.; Umar, M. Phytochemistry 1999, 50, 303-306.
(8) Wollenweber, E.; Wehdea, R.; Dorra, M.; Langb, G.; Stevens, J . F.
Phytochemistry 2000, 55, 965-970.
(9) Ghisalberti, E. L. Phytochemistry 1996, 41, 7-22.
(10) Ghosal, S.; Kumar, Y.; Singh, S.; Ahad, K. Phytochemistry 1983, 22,
2591-2593.
(11) Tanaka, T.; Orii, Y.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull.
1993, 41, 1232-1237.
(12) Yoshizawa, S.; Horiuchi, H.; Fujiki, H.; Yoshida, T.; Okuda, T.;
Sugimura, T. Phytother. Res. 1987, 1, 44-47.
(13) Okabe, S.; Suganuma, M.; Imayoshi, Y.; Taniguchi, S.; Yoshida, T.;
Fujiki, H. Biol. Pharm. Bull. 2001, 24, 1145-1148.
(14) Ito, H.; Miyake, M.; Nishitani, E.; Mori, K.; Hatano, T.; Okuda, T.;
Konoshima, T.; Takasaki, M.; Kozuka, M.; Mukainaka, T.; Tokuda,
H.; Nishino, H.; Yoshida, T. Cancer Lett. 1999, 143, 5-13.
(15) Ito, Y.; Kawanishi, M.; Harayama, T.; Takabayashi, S. Cancer Lett.
1981, 12, 175-180.
(16) Yoshida, T.; Tanaka, K.; Chen, X.-M.; Okuda, T. Chem. Pharm. Bull.
1989, 37, 920-924.
(17) Ito, H.; Koreishi, M.; Tokuda, H.; Nishino, H.; Yoshida, T. J . Nat.
Prod. 2000, 63, 1253-1257.
5: reaction time 48 h; normal- and reversed-phase HPLC
(solvent B) resulted in peaks for biflorin (12) and kunzeach-
romone C (4).
6: reaction time 150 h; normal- and reversed-phase HPLC
(solvent B) resulted in peaks for isobiflorin (13).
7: reaction time 48 h; normal- and reversed-phase HPLC
(solvent B) resulted in peaks for isobiflorin (13) and kunzeach-
romone E (6).
NP030367S