Metabolic fate of orally administered phyllodulcin in rats

Article abstract of DOI:10.1021/np0400353

Naturally occurring phyllodulcin (1) was orally administered to rats to investigate its metabolic fate. Urinary metabolites were analyzed by three-dimensional HPLC. Phyllodulcin-3′-O-sulfate (2), phyllodulcin-3′-O-β-glucuronide (3), 2-[2-(3,4-dihydroxyphe

Full text of DOI:10.1021/np0400353

1604
J . Nat. Prod. 2004, 67, 1604-1607
Meta bolic F a te of Or a lly Ad m in ister ed P h yllod u lcin in Ra ts
Takaaki Yasuda, Saiko Kayaba, Kana Takahashi, Takahiro Nakazawa, and Keisuke Ohsawa*
Department of Phytochemistry, Tohoku Pharmaceutical University,
4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, J apan
Received February 12, 2004
Naturally occurring phyllodulcin (1) was orally administered to rats to investigate its metabolic fate.
Urinary metabolites were analyzed by three-dimensional HPLC. Phyllodulcin-3′-O-sulfate (2), phyllodulcin-
3′-O-â-glucuronide (3), 2-[2-(3,4-dihydroxyphenyl)ethyl]-6-hydroxybenzoic acid (4), and one novel bibenzyl
derivative, 2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-6-hydroxybenzoic acid (5), together with thunberginol
G (6) and hydrangenol (7) were isolated from the phyllodulcin-treated urine. 1 was extensively metabolized
to 4-6 by a rat fecal suspension after incubation for 24 h. Urinary excretion of 4-6 in rats administered
phyllodulcin orally was substantially reduced when the rats were treated with antibiotics to suppress
their intestinal flora. On the other hand, the incubation of 1 with rat liver S-9 mix showed the presence
of 7 together with 4 and 5.
Hydrangeae folium (Amacha in J apanese) is made from
the fermented and dried leaves of Hydrangea macrophylla
Seringe var. thunbergii Makino (Saxifragaceae) grown in
J apan. The plant contains a sweetening compound (1),
which is a dihydroisocoumarin derivative. Phyllodulcin is
well known in J apan as an oral refrigerant and as a
sweetener, since it is 600-800 times sweeter than sucrose.
It is also used to make tea served during the Hanamatsuri
(birth of Buddha) celebration. Several biological activities
have been observed for phyllodulcin, including antiallergic
effects on the Schultz-Dale reaction,1 inhibition of mi-
crosomal lipid peroxidation induced by NADPH and the
Fenton-type reaction,2 and inhibition of ACTH- and for-
skolin-induced steroidogenesis in the presence of Ca2+ in
the incubation media.3 Although some biochemical proper-
ties of 1 have been described, the metabolic fate of this
compound has yet to be reported. In our continuing study
on the metabolism of naturally occurring compounds, we
investigated the metabolic fate of 1 in rats.
one sulfate group at C-3′. On the basis of these data, 2 was
identified as phyllodulcin-3′-O-sulfate.
Here we describe the identification and structural elu-
cidation of urinary metabolites from rats administered 1
orally. In addition, we investigated the biotransformation
of 1 using antibiotic-treated rats, rat fecal suspension, and
rat liver S-9 mix, to study the site of metabolism.
Two distinct HPLC peaks, 2 and 3, together with
unchanged 1 were detected in the â-glucuronidase/aryl-
sulfatase nontreated urine of rats administered 1 orally.
When the urine was treated with â-glucuronidase/arylsul-
fatase, four peaks were detected: 4, 5, 6, and 7. Compounds
2-7 were isolated from the urine sample by chromatog-
raphy as described in the Experimental Section. Metabolite
2 was obtained as a white powder. Enzyme hydrolysis of 2
with arylsulfatase yielded 1, confirmed by tR agreement
using HPLC. The intense absorption at 1051 cm-1 in the
Metabolite 3 was obtained as a white powder. Enzyme
hydrolysis of 3 with â-glucuronidase yielded 1. The 1H
NMR spectrum showed one anomeric proton at δ 4.91 (d,
J ) 7.2 Hz), and negative-ion FABMS showed a molecular
ion peak at m/z 461 (M - H)- corresponding to a mono-
glucuronide. A comparison of the 13C NMR spectrum of 3
with 1 indicated that the C-3′ signal of 3 had shifted 0.13
ppm upfield, accompanied by downfield shifts of C-2′ (0.45
ppm) and C-4′ (1.0 ppm). These shifts indicated a glucu-
ronide group at C-3′. Heteronuclear multiple-bond correla-
tion spectroscopy (HMBC) data revealed a correlation
between the anomeric proton and C-3′. Thus, 3 was
identified as phyllodulcin-3′-O-â-glucuronide.
2-
IR spectrum and SO4 formation on carbonization sug-
gested a sulfate-conjugated structure for 2. Negative-ion
FABMS of 2 showed a base ion peak corresponding to (M
- H - SO3)- at m/z 285 along with fragment ion peaks at
m/z 387 (M - H + Na)- and 365 (M - H)-, thus indicating
one sulfate group in 2. A comparison of the 13C NMR
spectrum of 2 with 1 showed that the C-3′ signal of 2 had
shifted 3.9 ppm upfield, accompanied by downfield shifts
of C-2′ (7.5 ppm) and C-4′ (3.0 ppm). These shifts indicated
Metabolite 4 was identified as 2-[2-(3,4-dihydroxy-
phenyl)ethyl]-6-hydroxybenzoic acid, by comparison with
reference data.4 Furthermore, direct comparison of 4 with
an authentic sample prepared from phyllodulcin by EIMS
1
and H NMR spectra supported this observation.
Metabolites 6 and 7 were identified as thunberginol G
and hydrangenol, respectively, by direct comparison of
* To whom correspondence should be addressed. Tel: +81-22-234-4181.
Fax: +81-22-275-2013. E-mail: ohsawa@tohoku-pharm.ac.jp.
1
EIMS and H NMR spectral data with authentic samples.5
10.1021/np0400353 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/04/2004

Notes
Metabolite 5 was obtained as a white powder. The 1H
J ournal of Natural Products, 2004, Vol. 67, No. 9 1605
tively. This observation may be explained by the differences
in activities of bacterial enzymes in the demethylation and
reductive cleavage reaction pathways. This hypothesis was
confirmed by examination of the incubations of 5 and 6
with rat fecal suspensions. In fact, the incubations of 5 and
6 were shown to be almost completely converted to 4 in
rat fecal suspension under anaerobic conditions for 12 and
6 h, respectively. The metabolic pathways deduced from
the present study demonstrate that 1 was demethylated
to produce 6, reductively cleaved to produce 5, and a
combination of both or either process to produce 4. On the
other hand, 1 appeared to be converted to 7 via direct
demethylation and dehydroxylation in the liver. The iden-
tified phyllodulcin metabolites were all products of de-
methylation, dehydroxylation, and reductive cleavage reac-
tions. Since aromatic compounds such as flavonoids and
coumarins are partly metabolized to phenolic acids by
intestinal bacteria as well as by plants and aerobes,6-8 it
appears that ring fission in oxygen-containing heterocyclic
compounds through reductive or oxidative cleavage is a
common reaction of living organisms. In general, orally
administered phenolic compounds, especially those with a
low polarity, undergo hydroxylation and/or glucuronide and
sulfate conjugation primarily by the intestinal microflora
and secondarily in the liver and other tissues. On the other
hand, there are reports on the dehydroxylation and dem-
ethylation in rat liver for naturally occurring compounds,
such as the 7â-hydroxy-C27 plant sterol,9 nobiletin,10 and
tangeretin.11 Therefore, it is not surprising that phyllo-
dulcin could be directly converted to 7 by the liver and
associated enzymes. Furthermore, the conversion of 1 to 7
by rat liver S-9 fraction was not significantly inhibited by
SKF 525A (data not shown), suggesting that a rat liver
enzyme other than cytochrome P450 was involved in the
conversion.
and 13C NMR spectral data of 5 were similar to those of 4,
except that one of the three hydroxyl group signals in 4
had been replaced by a single methoxy group [δ 3.73 (1H,
s, -OCH3)]. It appeared that 5 possessed the same oxy-
genation pattern as 4. A NOE was observed between the
methoxy proton and H-5, indicating that the methoxyl
group was attached to C-4. Thus, 5 was concluded to be
2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-6-hydroxybenzo-
ic acid. Confirmation of the structure of 5 was made by
direct comparison with an authentic sample, which was
prepared from phyllodulcin by Pd-C treatment, using
1
EIMS and H NMR spectral data. Since the yields of 2, 3,
6, and 7 were very low, the stereochemistry at C-3 could
not be clarified.
To determine the contribution to the formation of 4-7
made by the intestinal microflora, we examined changes
in urinary metabolite excretion when rats were treated
with antibiotics. The urinary excretion of 4-6 significantly
decreased over 24 h after administration of 1 in rats treated
with antibiotics (4 and 6, p < 0.01; 5, p < 0.05), while the
excretion of 7 significantly increased (p < 0.05) when
compared with the nontreated rats. This result indicated
the importance of the gut flora in generating the urinary
metabolites 4-6. On the other hand, the main site of
formation of 7 was assumed to be the liver enzymes. These
findings prompted us to investigate the biotransformation
of 1 by the intestinal microflora and rat liver S-9 fraction.
During the metabolite formation and anaerobic incubation
of 1 with rat fecal microflora, 1 was almost completely
consumed after 36 h of incubation, and metabolites 4-6
were produced. Metabolite 4 increased progressively and
reached a maximum concentration at 24 h, then decreased
slightly on prolonged incubation. On the other hand,
metabolite 6 was produced, reached its maximum at 6 h,
and then decreased appreciably on prolonged incubation.
During the incubation period, only traces of 5 were
detected. These results suggested that 5 and 6 are meta-
bolic intermediates. As expected, after incubation under
anaerobic conditions for 12 and 6 h, compounds 5 and 6,
respectively, were almost completely converted to 4 in rat
fecal suspension.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Phyllodulcin was
isolated from Hydrangeae dulcis folium, the fermented and
dried leaves of Hydrangea macrophylla Seringe var. thunbergii
Makino. Authentic phyllodulcin as well as kanamycin sulfate,
tetracycline hydrochloride, and bacitracin were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, J apan). Ph-
thalylsulfathiazole was purchased from Tokyo Chemical In-
dustry Co., Ltd. (Tokyo, J apan). Arylsulfatase (type H-1),
â-glucuronidase (type H-2), glucose-6-phosphate, and proadifen
were purchased from Sigma (St. Louis, MO). Glucose-6-
phosphate dehydrogenase was from Oriental Yeast Co., Ltd.
(Tokyo, J apan). For column chromatography, Sephadex LH-
20 (Pharmacia Biotech, Uppsala, Sweden) and Wakosil C-200
(Wako Pure Chemical) were used. All other reagents were of
the highest purity commercially available. Uncorrected melting
points were determined on a Yanagimoto micro melting point
apparatus. Infrared (IR) spectra were measured with a Perkin-
Elmer FT-IR1725X spectrometer. NMR spectra were recorded
on J EOL J NM-EX 270 (1H, 270; 13C, 67.8 MHz), J NM-EX 400
(1H, 400; 13C, 100 MHz), and J NM-LA 600 (1H, 600; 13C, 150
MHz) spectrometers. Chemical shifts are given in δ values
(ppm) downfield relative to tetramethylsilane. Electron impact
(EI) and fast atom bombardment (FAB) MS were measured
on a J EOL J MS-DX 303 mass spectrometer. The HPLC system
was comprised of a CCMP-II pump, CO-8020 column oven
(Tosoh, Tokyo, J apan), and model MCPD-3600 photodiode
array detector (Otsuka, Osaka, J apan). HPLC conditions for
analysis of metabolites were as follows: column, CAPCELL-
PAK C18 (5 µm, 3.0 mm i.d. × 250 mm, Shiseido, Tokyo,
J apan); column temperature, 40 °C; flow rate, 0.5 mL/min;
detection, 200-400 nm; mobile phase, a linear gradient system
with 0.1% trifluoroacetic acid in H2O (A) and CH3CN (B), A/B
) 95/5 (0 min) f 40/60 (50 min).
During metabolite formation of 1 by rat liver S-9 fraction,
1 decomposed in a time-dependent manner and after 2 h
treatment was partly converted to 7, which at its maximum
accounted for 1.9% of the total 1. It was interesting to note
that 1 was also partly converted to 4 and 5, accounting for
maxima of 5.0% at 4 h and 3.0% at 6 h, respectively. This
conversion by rat liver S-9 was not substantially inhibited
by SKF 525A, the nonspecific cytochrome P450 inhibitor
(data not shown).
In the present study of in vivo metabolism of 1, six
urinary metabolites including sulfate and glucuronide
conjugates of 1 were detected in rat urine by HPLC. To
our knowledge, metabolite 5 is a novel bibenzyl derivative.
Furthermore, it was demonstrated that 4 and 5 were
produced by the intestinal microflora and rat liver S-9
fraction, while 6 or 7 were produced only by the intestinal
microflora or in the intact liver. These data were derived
from the use of antibiotic-treated rats and incubation of 1
with rat fecal microflora and rat liver S-9 fraction, respec-
tively. With HPLC analysis of rat fecal incubation mix-
tures, a substantial amount of 4 was found, while a small
amount of 6 and only a trace of 5 were detected. This
suggested that intermediates 5 and 6 were easily and
immediately converted to 4 by intestinal bacteria through
reductive cleavage and demethylation reactions, respec-

1606 J ournal of Natural Products, 2004, Vol. 67, No. 9
Notes
An im al Exper im en ts. Male Sprague-Dawley rats (6 weeks,
140-150 g) were purchased from J apan SLC, Inc. These
animals were housed at 22 ( 2 °C, humidity 55 ( 10%, in a
light (9:00-21:00)-controlled room with free access to water
and commercial rodent chow (CE-2, Clea J apan Inc., Tokyo,
J apan). After 3 days of feeding, food was withheld for 18 h,
and thereafter phyllodulcin (100 mg/kg body weight) uniformly
dispersed in 0.5% Tween 80 (ICN Pharmaceuticals, Ltd.) was
administered orally by direct stomach intubation. The animals
had free access to water and sugar during the experiments.
P r ep a r a tion of Ur in e Sa m p les. The urine samples were
collected over 24 h using a metabolic cage. The urine was
filtered through a 0.45 µm membrane filter, and then 20 µL
of the sample was injected into the HPLC.
En zym atic Hydr olysis of Ur in e Sam ples. A urine sample
(20 mL) was transferred to a test tube to which was added 5.0
mL of citrate buffer (pH 5.2) and 150 µL of â-glucuronidase
solution followed by incubation at 37 °C for 24 h. The incubated
solution was extracted three times with AcOEt (40 mL). The
organic layer was dried with anhydrous Na2SO4 overnight and
dried at 40 °C. The residue was dissolved in MeOH, and a 20
µL aliquot was injected into the HPLC.
Isola tion of Meta bolites. The urine sample (80 mL)
obtained from rats after oral administration of 1 (260 mg) that
had been incubated with â-glucuronidase/arylsulfatase was
extracted three times with AcOEt. The organic layer was dried
with anhydrous Na2SO4 for 24 h and evaporated to dryness
at 40 °C. The residue was dissolved in a small amount of
MeOH and chromatographed on Sephadex LH-20 with MeOH.
The fractions containing metabolites 3-6 were subjected to
preparative HPLC. The HPLC conditions were as follows:
column, Wakosil-II 5C18 (5 µm, 7.5 mm i.d. × 300 mm, Wako
Pure Chemicals Industries Ltd., Osaka, J apan); mobile phase,
H2O (A) and MeOH (B); linear gradient system, A/B ) 20/80
(0 min) f 80/20 (120 min); flow rate, 1.0 mL/min; detective
wavelength, 310 nm. Each metabolite fraction was evaporated
to dryness at 40 °C in vacuo to afford 4 (5 mg), 5 (0.5 mg), 6
(0.3 mg), and 7 (0.8 mg), respectively.
(COOH); FABMS m/z 287 (M - H)-; HREIMS m/z 288.0998
(calcd for C15H16O5, 288.0996).
Dem eth yla tion of 1. BBr3 (0.7 g, 0.27 mL) was added
dropwise to a solution of 1 (80 mg) in CH2Cl2 (5 mL) at -78
°C, then stirred at room temperature under a N2 atmosphere
for 24 h. The reaction mixture was evaporated in vacuo, and
the residue was dissolved in 10% KOH. The aqueous layer was
acidified with 10% H2SO4, extracted with AcOEt, and then
dried over anhydrous Na2SO4 and evaporated in vacuo. The
residue was dissolved in MeOH and chromatographed on
Sephadex LH-20 using MeOH as the eluant, and then chro-
matographed on Wakosil C-200 using n-hexane-AcOEt as
eluant. The fraction containing 6 was subjected to preparative
HPLC to give 6 (32 mg, 42% yield). HPLC conditions: column,
Wakosil-II 5C18 (5 µm, 7.5 mm i.d. × 300 mm, Wako Pure
Chemicals, Osaka, J apan); mobile phase, H2O (A) and MeOH
(B), linear gradient system, A/B ) 95/5 (0 min) f 40/60 (100
min); detection wavelength, 220 nm; flow rate, 1.0 mL/min at
room temperature.
Hyd r ogen a tion of 1 a n d 6. Phyllodulcin (1, 20 mg) and 6
(15 mg) were each dissolved in MeOH (14 mL) and hydroge-
nated over Pd-C (10%, 18 mg). The reaction mixtures were
stirred at room temperature under a H2 atmosphere for 1 h.
The reaction mixtures were filtered through
a 0.45 µm
membrane filter and the filtrates concentrated in vacuo to give
5 (20 mg, 100% yield) and 4 (16 mg, 100% yield), respectively.
An tibiotic Tr ea tm en t of An im a ls. Gut sterilization was
done according to the method of Goodwin et al.11 with minor
modifications. Rats were given a mixture of kanamycin sulfate
(45 mg), tetracycline hydrochloride (20 mg), bacitracin (1 mg),
and phthalylsulfathiazole (0.5 mg) orally once daily for 4 days.
After 1 h of the last dose on the fourth day, 1 (100 mg/kg) was
administered. Urine samples were collected for 24 h, incubated
with â-glucuronidase for 24 h, and then extracted three times
with AcOEt. The organic layer was dried over anhydrous Na2-
SO4 and evaporated to dryness at 40 °C. The residue was
dissolved in a small amount of MeOH and injected into the
HPLC. Statistical analysis was carried out by means of the
Student’s t-test (n ) 4). P values less than 0.05 were considered
to indicate statistical significance.
Com p ou n d 2: white powder; mp 118-119 °C; IR (KBr) νmax
3469, 1674, 1618, 1516, 1231, 1051 cm-1; 1H NMR (270 MHz,
DMSO-d6) δ 3.15 (1H, dd, J ) 3.4, 16.6 Hz, H-4 cis), 3.35 (1H,
J ) 3.4, 11.9 Hz, H-4 trans), 3.76 (3H, s, OCH3), 5.69 (1H, dd,
J ) 3.4, 11.9 Hz, H-3), 6.90 (2H, d, J ) 8.4 Hz, H-5, -7), 7.01
(1H, d, J ) 8.4 Hz, H-5′), 7.16 (1H, dd, J ) 2.1, 8.4 Hz, H-6′),
7.52 (1H, dd, J ) 8.4, 8.4 Hz, H-6), 7.61 (1H, d, J ) 2.1 Hz,
H-2′), 10.92 (1H, s, 8-OH); 13C NMR (67.8 MHz, DMSO-d6) δ
33.2 (C-4), 55.2 (OCH3), 79.6 (C-3), 108.0 (C-8a), 111.9 (C-5′),
114.9 (C-5 or -7), 117.9 (C-5 or -7), 119.0 (C-2′), 121.1 (C-6′),
129.3 (C-1′), 135.7 (C-6), 139.9 (C-4a), 142.1 (C-3′), 150.4 (C-
4′), 160.4 (C-8), 168.8 (C-1); FABMS m/z 387 (M - H + Na)-,
365 (M - H)-, 285 (M - H - SO3)-.
In cu ba tion of P h yllod u lcin w ith Ra t F eca l Su sp en -
sion . Fresh feces (1.0 g) obtained from male SD rats was
homogenized in 0.1 M phosphate buffer saline (PBS) (pH 7.4,
25 mL) by bubbling with CO2 gas to eliminate air, and
sediments were removed by filtration through gauze. The
filtrate was used as a fecal suspension in this experiment.
Each tube containing 1 (0.5 mg), 5 (0.1 mg), and 6 (0.1 mg)
in dimethyl sulfoxide (10 µL) and fecal suspension (3 mL) was
incubated at intervals at 37 °C in an anaerobic jar in which
air was replaced with oxygen-free CO2. The resulting mixture
was adjusted to pH ca. 3 with 0.05% trichloroacetic acid and
extracted three times with AcOEt (10 mL). The AcOEt layer
was concentrated to dryness in vacuo, and the residue was
dissolved in MeOH (1 mL). A 20 µL sample of the solution
was analyzed by HPLC.
In cu ba tion of P h yllod u lcin w ith Ra t Liver 9000g
Su p er n a ta n t. The rats were killed by decapitation and the
livers immediately removed. They were perfused in situ with
1.15% KCl solution, weighed, cut into small pieces, and then
homogenized in three volumes (w/v) of 0.1 M PBS (pH 7.4).
All subsequent procedures were performed at 0-4 °C. Nuclear
and celluar debris and mitochondoria were sedimented by
centrifugation at 9000g for 20 min at 4 °C. The supernatant
(S-9 fraction) was used for metabolism of phyllodulcin accord-
ing to the method described by Cooper and Brodie,13 with the
following modifications. The incubation mixture contained the
S-9 fraction (10 mL), 1 mM phyllodulcin dissolved in 100 µL
of MeOH, the NADPH generating system (1.3 mM NADP+,
40 units of glucose-6-phosphate dehydrogenase, 0.3 mM
glucose-6-phosphate), 3.3 mM MgCl2, and 0.1 M PBS (pH 7.4),
Com p ou n d 3: white powder; mp 173-180 °C; IR (KBr) νmax
1
3422, 1672, 1519, 1616, 1231 cm-1; H NMR (600 MHz, CD3-
OD) δ 3.21 (1H, dd, J ) 3.0, 16.5 Hz, H-4 cis), 3.37 (1H, dd, J
) 3.0,12.6 Hz, H-4 trans), 3.88 (3H, s, OCH3), 4.91 (1H, d, J )
7.2, anomeric proton), 5.63 (1H, dd, J ) 3.0, 15.0 Hz, H-3),
6.86 (2H, m, H-5, -7), 7.05 (1H, d, J ) 8.4 Hz, H-5′), 7.20 (1H,
dd, J ) 1.8, 8.4 Hz, H-6′), 7.37 (1H, d, J ) 1.8 Hz, H-2′), 7.47
(1H, dd, J ) 7.2, 8.4 Hz, H-6); 13C NMR (150 MHz, CD3OD) δ
35.5 (C-4), 56.8 (OCH3), 73.6 (C-4′′), 74.9 (C-2′′), 76.6 (C-5′′),
77.7 (C-3′′), 82.2 (C-3), 103.3 (C-1′′), 109.6 (C-8a), 113.7 (C-5′),
116.8 (C-5 or -7), 118.0 (C-2′), 119.5 (C-5 or -7), 122.3 (C-6′),
132.7 (C-1′), 137.6 (C-6), 141.8 (C-4a), 148.0 (C-3′), 151.5 (C-
4′), 163.3 (C-8), 171.6 (C-1), 180.5 (C-6′′); negative FABMS m/z
483 (M - H + Na)-, 461 (M - H)-, 285 (M - GlcUA)-.
Com p ou n d 5: white powder; mp 142-145 °C; IR (KBr) νmax
1
3400, 1666, 1610, 1510 cm-1; H NMR (400 MHz, CD3OD) δ
2.73 (2H, t, J ) 8.0 Hz, H-â), 3.16 (2H, t, J ) 8.0 Hz, H-R),
3.74 (3H, s, OCH3), 6.62 (1H, dd, J ) 2.2, 8.4 Hz, H-6′), 6.67-
6.68 (2H, m, H-3 or -5, -2′), 6.74 (1H, d, J ) 8.4 Hz H-3 or -5),
6.80 (1H, d, J ) 8.4 Hz, H-5′), 7.23 (1H, dd, J ) 8.4, 8.4 Hz,
H-4); 13C NMR (100 MHz, CD3OD) δ 39.0 (C-R), 39.5 (C-â),
56.6 (OCH3), 112.9 (C-3), 114.6 (C-1), 116.1 (C-5′), 116.6 (C-
2′), 120.6 (C-6′), 123.2 (C-5), 134.2 (C-4), 136.6 (C-1′), 146.0
(C-6), 147.3 (C-3′ or -4′), 147.4 (C-3′ or -4′), 162.6 (C-2), 174.4
in
a final volume of 12 mL. Protein concentration was
determined by the method of Lowry et al. with bovine serum
albumin as the standard protein. The amount of protein used
was 350 µg/mL, and the incubation was carried out at 37 °C
for 24 h. The mixture was extracted twice with AcOEt, and

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 9 1607
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the organic layer was then evaporated to dryness at 40 °C.
The residue was dissolved in MeOH, the mixture was filtered
through a 0.45 µm membrane filter, and then 20 µL of the
sample was injected into the HPLC.
Qu a n tita tive An a lysis of Meta bolites. A calibration
graph was prepared from peak areas obtained by injecting 20
µL of the sample solution for HPLC over the concentration
range 5-500 µg/mL for 1, 4-1580 µg/mL for 4, 5-50 µg/mL
for 5, 50-1500 µg/mL for 6, and 10-150 µg/mL for 7. The
resulting calibration graphs were linear, and each quantitative
value represents the mean of three experiments. The recover-
ies of standards added to each blank sample were 88.8-96.2%,
and the relative standard deviations were 1.24-3.19%.
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