Pyronane monoterpenoids from the fruit of Gardenia jasminoides

Article abstract of DOI:10.1021/np800002z

Five new pyronane-type monocyclic monoterpenoids, jasminodiol (1), jasminoside H (6), 6′-O-sinapoyljasminoside A (7), 6′-O- sinapoyljasminoside C (8), and jasminoside I (9), together with four known analogues, were isolated from the fruit of Gardenia jasminoides. The structures of the new metabolites were characterized using spectroscopic data, and the absolute configurations of 1, 6, and 7 were established using circular dichroism (CD) analysis. Compound 1 showed tyrosinase inhibitory activity (IC ₅₀ 2.2 mM).

Full text of DOI:10.1021/np800002z

J. Nat. Prod. 2008, 71, 995–999
995
Pyronane Monoterpenoids from the Fruit of Gardenia jasminoides
Quan Cheng Chen,† UiJoung Youn,† Byung-Sun Min,‡ and KiHwan Bae*,†
College of Pharmacy, Chungnam National UniVersity, Daejeon 305-764, Korea, and College of Pharmacy, Catholic UniVersity of Daegu,
Gyeongsan 712-702, Korea
ReceiVed January 2, 2008
Five new pyronane-type monocyclic monoterpenoids, jasminodiol (1), jasminoside H (6), 6′-O-sinapoyljasminoside A
(7), 6′-O-sinapoyljasminoside C (8), and jasminoside I (9), together with four known analogues, were isolated from the
fruit of Gardenia jasminoides. The structures of the new metabolites were characterized using spectroscopic data, and
the absolute configurations of 1, 6, and 7 were established using circular dichroism (CD) analysis. Compound 1 showed
tyrosinase inhibitory activity (IC50 2.2 mM).
The fruit of Gardenia jasminoides Ellis (Rubiaceae) is widely
used as traditional medicine in many Asian countries for its
cholagogue, sedative, diuretic, antiphlogistic, homeostatic, and
antipyretic effects.1 Previous investigations of G. jasminoides
revealed the presence of a variety of terpenoids, including mono-
cyclic monoterpenoids and their glycosides,2,3 iridoids and their
glycosides,4–6 triterpenoids, crocetin and its glycosides,7–9 vanillic
acid glycosides, and quinic acid.10 The present study describes the
structural elucidation of five new pyronane monoterpenoids from
the EtOAc- and n-BuOH-soluble fractions, partitioned from the
MeOH extract of the fruit of G. jasminoides, as well as their relative
tyrosinase inhibitory activities.
group adjacent to the R,ꢀ-unsaturated ketone, two oxygenated
methylene groups, and a gem-dimethyl attached to a quaternary
carbon. This combined data indicated that 1 was a monocyclic
monoterpenoid. The locations of two oxygenated methylene groups
were determined by the oxygenated methylene protons at δ 3.84
(2H, br d, J ) 4.2 Hz, H-7), which showed HMBC correlations
with C-1 (δ 36.5), C-5 (δ 168.6), and C-6 (δ 50.7), and the
oxygenated methylene protons at δ 4.40 (1H, dd, J ) 17.4, 1.2
Hz, H-10a)/4.20 (1H, dd, J ) 17.4, 1.2 Hz, H-10b), correlating
with C-4, C-5, and C-6. The absolute configuration at C-6 of 1
was established as S, based on the negative Cotton effects at 328.0
nm (∆ꢀ -0.35), 249.0 nm (∆ꢀ -1.17), and 211.0 nm (∆ꢀ -1.39)
in the CD spectrum, compared with those of jasminoside B (2).2
Therefore, the structure of 1 was established as a monocyclic
monoterpenoid and was named jasminodiol.
Results and Discussion
The MeOH extract of the fruit of G. jasminoides was partitioned
into n-hexane-, EtOAc-, and n-BuOH-soluble fractions. From the
EtOAc- and n-BuOH-soluble fractions, chromatography produced
nine compounds (1-9). Four of these compounds were identified
as jasminoside B (2), crocusatin-C (3), epijasminoside A (4), and
jasminoside A (5).2,11
Compound 6 was isolated as an optically active, white, amor-
phous powder, [R]22 -70.0 (c 0.22, MeOH). Its HRFABMS
D
exhibited a molecular ion at m/z 493.2253 [M + H]+, in agreement
with a molecular formula of C22H36O12. The spectroscopic data of
6 were similar to those of epijasminoside A (4), except for the
obvious differences due to the presence of additional glucosyl
signals in the 1H and 13C NMR spectra (Table 2). After enzymatic
hydrolysis of 6, the sugar was identified as D-glucose by TLC and
GC analysis.12 Two anomeric proton resonances at δ 4.22 (1H, d,
1
J ) 7.8 Hz, H-1′) and 4.38 (1H, d, J ) 7.8 Hz, H-1′′) in the H
NMR spectrum of 6 indicated ꢀ-glycosidic linkages. The deshielded
methylene carbon signal at δ 70.3 (C-6′) in the 13C NMR spectrum
of 6 suggested that two sugar moieties formed a ꢀ-gentiobiosyl
moiety by a 1′′f6′ connection, which was further confirmed by
observation of the correlation between H-1′′ and C-6′ in the HMBC
spectrum. In addition, H-1′ showed long-range HMBC correlations
with the oxygenated methylene carbon C-7 (δ 69.4), indicating that
the ꢀ-gentiobiose was linked to the 7-OH of the aglycone (Figure
1). The CD spectrum of 6 showed negative Cotton effects at 329.0
nm (∆ꢀ -0.25), 240.0 nm (∆ꢀ -1.58), and 205.0 nm (∆ꢀ -2.21),
suggesting the S absolute configuration at C-6.2 Accordingly, the
structure of 6 was elucidated to be a monoterpenoid diglucoside,
named jasminoside H.
Compound 1 was obtained as an optically active, colorless oil,
[R]22D -54.5 (c 0.22, MeOH). Its HREIMS exhibited a molecular
ion at m/z 184.1098, corresponding to a molecular formula of
C10H16O3, with three degrees of unsaturation. The IR spectrum
indicated the presence of hydroxy (3390 cm-1) and ketone (1650
cm-1) groups. The 1H and 13C NMR data of 1 were similar to those
of jaminoside B (2),2 except for the absence of the sugar moiety
Compound 7 was obtained as an optically active, pale yellow,
amorphous powder, [R]22 +50.0 (c 0.40, MeOH). Its molecular
D
formula, C27H36O11, was established by HRFABMS, which showed
a quasi-molecular ion at m/z 537.2275 [M + H]+. The presence of
hydroxy groups (3360 cm-1), an aromatic ring (1608, 1520 cm-1),
and conjugated unsaturated carbonyl groups (1710, 1680, 1615
cm-1) was indicated by the IR spectrum. Two overlapping proton
singlets at δ 6.92 (2H, s, H-5′′, 9′′) and 3.88 (6H, s, 6′′, 8′′-OCH3)
observed in the 1H NMR spectrum of 7, together with an
overlapping methine carbon signal at δ 107.1 (C-5′′, 9′′), an
overlapping quaternary carbon signal at δ 148.7 (C-6′′, 8′′), and
1
(Table 1). Combined analysis of H and 13C NMR spectra of 1
revealed the presence of an R,ꢀ-unsaturated ketone [δ 6.17 (1H, s,
H-4), δ 123.9 (C-4), 168.6 (C-5), and 202.9 (C-3)], a methylene
* To whom correspondence should be addressed. Tel: +82-42-821-5925.
Fax: +82-42-823-6566. E-mail: baekh@cnu.ac.kr.
† Chungnam National University.
‡ Catholic University of Daegu.
10.1021/np800002z CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/28/2008

996 Journal of Natural Products, 2008, Vol. 71, No. 6
Chen et al.
Table 1. NMR Spectroscopic Data of Compounds 1 and 2
1
2
position
δC, mult.
δH (J in Hz)
HMBC
δC, mult.
δH (J in Hz)
1
2
36.5, qC
50.1, CH2
36.5, qC
50.1, CH2
2.74, d (17.1)
2.03, d (17.1)
1, 3, 4, 6, 8, 9
1, 3, 4, 6, 8, 9
2.75, d (17.1)
2.03, d (17.1)
3
4
5
6
7
8
9
10
202.9, qC
123.9, CH
168.6, qC
50.7, CH
62.5, CH2
27.5, CH3
29.4, CH3
65.1, CH2
202.9, qC
125.4, CH
164.4, qC
50.8, CH
62.3, CH2
27.5, CH3
29.4, CH3
71.7, CH2
6.17, s
2, 6, 10
6.28 br s
2.08, t (4.2)
3.84 br d (4.2)
1.15, s
1.03, s
4.40, dd (17.4, 1.2)
4.20, dd (17.4, 1.2)
1, 2, 4, 5, 7, 9, 10
1, 5, 6
1, 2, 6, 9
1, 2, 6, 8
4, 5, 6
2.14, t (3.9)
3.87, m
1.15, s
1.04, s
4.70, dd (16.5, 1.5)
4.32, dd (16.5, 1.5)
4.35, d (7.5)
3.25, m
3.34, m
3.28, m
4, 5, 6
1′
2′
3′
4′
5′
6′
104.5, CH
75.2, CH
78.2, CH
71.8, CH
78.3, CH
62.9, CH2
3.37, m
3.87, m
3.65, dd (12.0, 4.8)
an overlapping O-methyl carbon signal at δ 57.0 (6′′, 8′′-OCH3),
observed in the 13C NMR and DEPT spectra, suggested the presence
of a symmetrical 1,3,4,5-tetrasubstituted benzene ring (Table 2).
Additionally, the 1H and 13C NMR spectra of 7 showed trans
double-bond signals [δ 6.42 (1H, d, J ) 15.9 Hz, H-2′′), 115.8
(C-2′′), 7.62 (1H, d, J ) 15.9 Hz, H-3′′), 147.4 (C-3′′)], of which
H-3′′ showed HMBC correlations with C-4′′ (δ 126.7), C-5′′ (δ
107.1), and C-6′′ (δ 148.7). Additionally, H-3′′ showed HMBC
correlation with the C-1′′ carbonyl carbon (δ 169.1) (Figure 1).
This data revealed the presence of a sinapoyl group. In addition,
the 1H and 13C NMR spectra of 7 showed typical glucosyl signals.
The configuration of the glycosidic linkage unit was determined
as ꢀ, on the basis of the coupling constant observed for the anomeric
δ 4.67 (1H, dd, J ) 15.3, 1.5 Hz, H-10a)/4.53 (1H, dd, J ) 15.3,
1.5 Hz, H-10b) and the anomeric carbon (C-1′) of glucose, as well
as correlations between the sugar protons at δ 4.49 (1H, dd, J )
12.0, 2.4 Hz, H-6′a)/4.36 (1H, dd, J ) 12.0, 6.0 Hz, H-6′b) and
the C-1′′ carbonyl carbon (δ 169.1) of the sinapoyl group. Therefore,
compound 8 was determined to be 6′-O-sinapoyljasminoside C.
Compound 9 was obtained as a colorless, amorphous powder.
The molecular formula of 9, C22H36O12, was deduced from its
HRFABMS, which showed a quasi-molecular ion at m/z 493.2214
[M + H]+. The NMR data of 9 were similar to those of jasminoside
E,2 except for the presence of additional glucosyl signals in the 1H
and 13C NMR spectra (Table 2). The ꢀ-linkages of the glucosidic
units were consistent with the coupling constants observed for the
anomeric protons at δ 4.32 (1H, d, J ) 7.5 Hz, H-1′) and 4.42
(1H, d, J ) 7.8 Hz, H-1′′). The downfield shift of the methylene
carbon at δ 70.3 (C-6′) in the 13C NMR spectrum of 9 suggested
that two sugar moieties formed a ꢀ-gentiobiosyl moiety via a 1′′f6′
connection, which was confirmed by the HMBC correlation between
H-1′′ and C-6′. Additionally, the anomeric proton (H-1′) showed a
long-range HMBC correlation with the oxygenated methylene
carbon at δ 67.0 (C-7), indicating that the ꢀ-gentiobiose was linked
to the 7-OH of aglycone. Thus, the structure of 9 was characterized
as a monoterpenoid diglucoside, named jasminoside I.
All isolated compounds are pyronane monoterpenoids. Aside
from the compounds isolated previously from G. jasminoides, many
pyronane monoterpenoids are found in Crocus satiVus L.2,6,11,13–18
Some of these pyronane monoterpenoids showed significant tyro-
sinase inhibitory activity.11,15 Thus, our isolated compounds were
evaluated for their antityrosinase activity. The antityrosinase activity
of 1 (IC50 2.2 mM) was lower than that of kojic acid (IC50 0.2
mM); however, 1 showed more significant activity than that of
hydroquinone (IC50 4.5 mM), a known commercial whitening agent
in cosmetics.19 The other compounds showed no or very weak
activities (data not shown). The inhibition kinetics of 1, with three
concentrations (1.0, 2.0, and 3.0 mM), were measured every minute
for a total of 50 min, following a preincubation of tyrosinase for
10 min (Figure 2). The results showed a dose-dependent correlation
for the tyrosinase inhibitory activity of 1.
1
proton H-1′ (δ 4.27, d, J ) 7.8 Hz). The remaining H and 13C
NMR signals were very similar to those of crocusatin-C (3), which
was initially isolated from the pollen of Crocus satiVus L.11 The
chemical shifts of the six carbons at δ 104.3 (C-1′), 75.1 (C-2′),
78.3 (C-3′), 71.8 (C-4′), 75.6 (C-5′), and 64.6 (C-6′) in the 13C
NMR spectrum of 7 indicated that the sugar was a 1′,6′-disubstituted
glucose. In the HMBC spectrum of 7, the anomeric proton (H-1′)
showed long-range correlations with the oxygenated methylene
carbon at δ 69.2 (C-7), suggesting that the glucosyl was linked to
the 7-OH of crocusatin-C with the same pattern as jasminoside A.
Furthermore, the oxygenated methylene protons at δ 4.48 (1H, dd,
J ) 12.0, 2.4 Hz, H-6′a)/4.38 (1H, dd, J ) 12.0, 6.0 Hz, H-6′b)
showed long-range HMBC correlations with the carbonyl carbon
at δ 169.1 (C-1′′) of the sinapoyl group, indicating that the sinapoyl
group was connected to the 6′-OH of the glucosyl moiety (Figure
1). The CD spectrum of 7 showed positive Cotton effects at 253.0
nm (∆ꢀ +0.65) and 200.0 nm (∆ꢀ +1.51), suggesting the 6R
absolute configuration.2 Thus, the structure of 7 was assigned as
6′-O-sinapoyljasminoside A.
Compound 8 was purified as a pale yellow, amorphous powder.
Its molecular formula, C27H34O11, was established by HRFABMS,
with a mass of [M + H]+ (m/z 535.2184, calcd 535.2179). The 1H
and 13C NMR spectral data of 8 were similar to those of jasminoside
C together with those of sinapoyl moiety in 7.2 The ꢀ-linkage of
the glucosidic unit was consistent with the coupling constant of
the anomeric proton at δ 4.39 (1H, d, J ) 7.5 Hz, H-1′). The
chemical shifts of the six carbons at δ 104.2 (C-1′), 75.2 (C-2′),
78.1 (C-3′), 71.8 (C-4′), 75.7 (C-5′), and 64.7 (C-6′) in the 13C
NMR indicated that the sugar was a 1′,6′-disubstituted glucose. The
above data suggested that the structure of 8 was jasminoside C
with a sinapoyl moiety connected to 6′-OH of the glucosyl moiety.
The linkage among glucose and aglycones was deduced by the
HMBC correlations between the oxygenated methylene protons at
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a JASCO DIP-370 polarimeter. UV spectra were obtained using a
Beckman Du-650 recording spectrophotometer. CD spectra were
measured on a JASCO J-715 CD/ORD spectropolarimeter. FT-IR
spectra were obtained using a JASCO Report-100 infrared spectrometer.
FT-NMR spectra were recorded on a Bruker DRX-300 spectrometer

Pyronane Monoterpenoids from Gardenia jasminoides
Journal of Natural Products, 2008, Vol. 71, No. 6 997

998 Journal of Natural Products, 2008, Vol. 71, No. 6
Chen et al.
was applied to an RP HPLC [Gilson TRILUTION LC system; YMC-
pack Pro C18 (250 × 20 mm, i.d.) column; MeOH-H2O (2:8, v/v);
UV detection, 254 nm; flow rate 6 mL/min], producing 1 (25.9 mg), 2
(145.0 mg), 3 (22.0 mg), 4 (21.0 mg), 5 (16.5 mg), 6 (182.0 mg), and
9 (6.0 mg). The EtOAc-soluble fraction (70.0 g) was chromatographed
on a Si gel column (200-300 mesh, 3.6 × 50 cm) and eluted with a
gradient of acetone in CHCl3, yielding a crude fraction containing 7
and 8. This was further purified by chromatography on a Sephadex
LH-20 column (2.5 × 50 cm) with CH2Cl2-MeOH (1:1, v/v) and then
by HPLC over a YMC-pack Pro C18 (250 × 10 mm, i.d.) column
using MeOH-H2O (6:4, v/v) as the mobile phase, followed by UV
240 nm for detection, to give 7 (7.5 mg) and 8 (7.0 mg).
Jasminodiol (1): colorless oil; [R]22 -54.5 (c 0.22, MeOH); UV
D
(MeOH) λmax (log ε) 248 (3.91) nm; CD (MeOH) 211.0 (∆ε -1.39),
249.0 (∆ε -1.17), 328.0 (∆ε -0.35) nm; IR (KBr) νmax 3390, 2900,
1
1650, 1045 cm-1; H NMR (300 MHz, CD3OD) and 13C NMR (75
Figure 1. Selected HMBC (f) correlations of compounds 6 and 7.
MHz, CD3OD), see Table 1; HREIMS m/z 184.1098 [M]+ (calcd for
C10H16O3, 184.1099).
Jasminoside H (6): white, amorphous powder; [R]22D -70.0 (c 0.22,
MeOH); UV (MeOH) λmax (log ε) 246 (4.06) nm; CD (MeOH) 205.0
(∆ε -2.21), 240.0 (∆ε -1.58), 329.0 (∆ε -0.25) nm; IR (KBr) νmax
3400, 2925, 1680, 1070 cm-1; H NMR (300 MHz, CD3OD) and 13C
1
NMR (75 MHz, CD3OD), see Table 2; HRFABMS m/z 493.2253 [M
+ H]+ (calcd for C22H37O12, 493.2285).
6′-O-Sinapoyljasminoside A (7): white, amorphous powder; [R]22
D
+50.0 (c 0.40, MeOH); UV (MeOH) λmax (log ε) 216 (4.45), 244 (4.42),
337 (4.14) nm; CD (MeOH) 200.0 (∆ε +1.51), 253.0 (∆ε +0.65), 318.0
(∆ε +0.40), 339.0 (∆ε +0.38) nm; IR (KBr) νmax 3360, 2920, 1710,
1680, 1615, 1608, 1520, 1440, 1110 cm-1 1H NMR (300 MHz,
;
CD3OD) and 13C NMR (75 MHz, CD3OD), see Table 2; HRFABMS
m/z 537.2275 [M + H]+ (calcd for C27H37O11, 537.2336).
6′-O-Sinapoyljasminoside C (8): white, amorphous powder; [R]22
D
-12.5 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 213 (4.50), 235 (4.37),
284 (4.15), 339 (4.18) nm; IR (KBr) νmax 3360, 2920, 1700, 1640, 1440,
1
1300, 1080 cm-1; H NMR (300 MHz, CD3OD) and 13C NMR (75
MHz, CD3OD), see Table 2; HRFABMS m/z 535.2184 [M + H]+ (calcd
for C27H35O11, 535.2179).
Jasminoside I (9): white, amorphous powder; [R]22D -7.5 (c 0.40,
MeOH); UV (MeOH) λmax (log ε) 254 (4.07) nm; IR (KBr) νmax 3400,
Figure 2. Inhibitory effect of jasminodiol (1) on tyrosinase in a
dose-dependent manner. Mushroom tyrosinase and L-Dopa were
incubated in the absence (control) and presence of 1 (1, 2, and 3
mM).
1
2900, 1670, 1440, 1070 cm-1; H NMR (300 MHz, CD3OD) and 13C
NMR (75 MHz, CD3OD), see Table 2; HRESIMS m/z 493.2214 [M +
H]+ (calcd for C22H37O12, 493.2285).
(1H NMR, 300 MHz; 13C NMR, 75 MHz) using CD3OD as the solvent
and tetramethylsilane (TMS) as an internal standard. Chemical shifts
(δ) were expressed in ppm with reference to the TMS signals. Two-
dimensional (2D) NMR (HMQC, HMBC) experiments were performed
on a Bruker Avance 500 spectrometer. HRMS was measured on a JMS-
700 Mstation mass spectrometer. Semipreparative HPLC were con-
ducted on a TRILUTION LC with a UV/vis-151 detector, a 321 pump,
a 402 syringe pump, and a GX-271 liquid handler (Gilson, Inc.), using
a YMC-pack Pro C18 (250 × 20 mm, i.d.) column. Column chroma-
tography was performed using Si (Kieselgel 60, 70-230 mesh and
230-400 mesh, Merck). TLC was performed on Merck precoated silica
gel 60 F254 and/or RP-18 F254s plates (0.25 mm), and compounds were
observed under UV 254 and 365 nm, or visualized by spraying the
dried plates with 10% H2SO4, followed by heating at 180 °C. Optical
density (OD) values in the tyrosinase inhibitory activity assays were
read on an Emax Precision microplate reader.
Enzymatic Hydrolysis of 6. Naringinase (100.0 mg) was added to
a suspension of 6 (50.0 mg) in 50 mM acetate buffer (pH, 5.5), and
the mixture was stirred at 37 °C for 3 h. The reaction mixture was
extracted with EtOAc (20 mL × 3 times) and evaporated to dryness.
The residue was dissolved in MeOH and applied to RP HPLC [Gilson
trilution system; YMC-pack Pro C18 (250 × 20 mm, i.d.) column;
MeOH-H2O (2:8, v/v); UV detection, 240 nm], yielding 3 (5.0 mg)
and 4 (4.5 mg), which were identified by comparison of the spectro-
scopic data with literature values. The H2O layer was concentrated and
passed through a Sep-Pak C18 cartridge (Waters, Milford, MA). The
sugar was identified as D-glucose by TLC in EtOAc-MeOH-AcOH
(13:4:3:3) with an authentic D-(+)-glucose (Sigma) (Rf 0.35), in
conjunction with the following GC analysis. The remaining eluate was
concentrated to dryness, and the residue was stirred with D-cysteine
methyl ester hydrochloride, hexamethyldisilazane, and trimethylsilyl
chloride in pyridine using the same procedures as in previous reports.12
After the reactions, the supernatant was analyzed by GC [column: GL
capillary column TC-1 (GL Science, Inc.) 0.25 mm × 30 m, detector,
FID; detector temp, 270 °C; injector temp, 270 °C; carrier gas, N2;
column temperature, 230 °C]. A peak corresponding to D-glucose
appeared at a tR of 21.6 min.
Plant Material. The dried, ripe fruit of G. jasminoides was purchased
from a pharmacy store in Daejeon, Korea, in July 2006, and the fruit
was identified by one of the authors (K.B.). A voucher specimen (CNU
1516-3) was deposited at the herbarium in the College of Pharmacy,
Chungnam National University.
Extraction and Isolation. A dried fruit slice of G. jasminoides (4.5
kg) was extracted with hot MeOH (5 L × 3 times) for 2 days. The
MeOH extracts were filtered, combined, and concentrated in Vacuo,
resulting in a residue (1.0 kg). The residue was suspended into H2O
and then fractionated successively with n-hexane, EtOAc, and n-BuOH,
producing an n-hexane-soluble fraction (120.0 g), EtOAc-soluble
fraction (80.0 g), and n-BuOH-soluble fraction (700.0 g), respectively.
The n-BuOH-soluble fraction was dissolved in a minimum amount of
50% MeOH and then applied to a Diaion HP 20 column (100 × 20
cm, i.d.) with H2O and MeOH gradient eluent to give fractions GjB1-5.
GjB1 was subjected to an ODS column (50 × 2.5 cm, i.d.), using
MeOH-H2O (1:2, v/v) as eluent. The third subfraction from this column
Tyrosinase Inhibitory Activity Assay. The mushroom tyrosinase
and L-Dopa used for the bioassay were purchased from Sigma Chemical
Co. Antityrosinase activity was measured by spectrophotometry,
according to the method of Mason and Peterson with minor modifica-
tions.20 The test substance was dissolved in 0.1 mL of 10% DMSO in
aqueous solution and incubated with 0.1 mL of 135 U/mL mushroom
tyrosinase in phosphate buffer solution (PBS, pH 6.8) at 25 °C for 10
min, and then 0.1 mL of L-Dopa (0.5 mM, PBS pH 6.8) was added.
The reaction mixture was incubated for 5 min. The amount of
dopachrome in the mixture was determined by the optical density (OD)
at 475 nm using an Emax Precision microplate reader. Kojic acid and
hydroquinone (Sigma Chemical Co.) were used as positive control

Pyronane Monoterpenoids from Gardenia jasminoides
Journal of Natural Products, 2008, Vol. 71, No. 6 999
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follows: % inhibition ) {[(A - B) - (C - D)]/(A - B)} × 100 (A,
OD at 475 nm without test substance; B, OD at 475 nm without test
substance and tyrosinase; C, OD at 475 nm with test substance; D,
OD at 475 nm with test substance, but without tyrosinase).
Acknowledgment. This research was supported by the Korea Food
and Drug Administration (07092 Crude Drugs 326). We are grateful
to Korean Basic Science Institute (KBSI) for measuring the NMR
spectra.
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