Tyrosinase inhibitors from the leaves of Eucalyptus globulus

Article abstract of DOI:10.1016/j.fitote.2019.104418

A new isoiphionane sesquiterpene, named (3S, 5S, 7S, 10R)-3, 11-dihydroxyisoiphion-4-one (1), two new phloroglucinol glycosides, named eucalglobuside A (2) and eucalglobuside B (3), along with 15 known compounds were isolated from the leaves of Eucalyptus globulus. Their structures were elucidated based on extensive spectroscopic analysis and in comparison with literature data. The absolute configuration of compound 1 was determined by ECD calculation. All isolates were evaluated their inhibitory activities against the mushroom tyrosinase. As a result, three sesquiterpenoids, 1, 5β, 11-dihydroxy-iphionan-4-one (5), and (?)-globulol (8), exhibited the most potent activities with IC₅₀ values of 14.17 μM, 10.08 μM and 9.79 μM, respectively.

Full text of DOI:10.1016/j.fitote.2019.104418

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Tyrosinase inhibitors from the leaves of Eucalyptus globulus
Qiao-Mei Lin, Yong Wang, Jin-Hai Yu, Yun-Lai Liu, Xue Wu,
Xiao-Ru He, Zhi-Wang Zhou
PII:
S0367-326X(19)31955-0
DOI:
https://doi.org/10.1016/j.fitote.2019.104418
FITOTE 104418
Reference:
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
29 September 2019
31 October 2019
4 November 2019
Please cite this article as: Q.-M. Lin, Y. Wang, J.-H. Yu, et al., Tyrosinase inhibitors
from the leaves of Eucalyptus globulus, Fitoterapia (2018), https://doi.org/10.1016/
j.fitote.2019.104418
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2018 Published by Elsevier.

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Tyrosinase inhibitors from the leaves of Eucalyptus globulus
a
a
b
a
a
c
a,*
Qiao-Mei Lin , Yong Wang , Jin-Hai Yu , Yun-Lai Liu , Xue Wu , Xiao-Ru He , Zhi-Wang Zhou
aSchool of Pharmacy, Nanchang University, Nanchang 330006, PR China
bSchool of Biological Science and Technology, University of Jinan, Jinan 250022, PR China
cKey Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of TCM, Nanc
hang 330004, PR China
A new isoiphionane sesquiterpene, named (3S, 5S, 7S, 10R)-3, 11-dihydroxyisoiphion-4-one (1),
two new phloroglucinol glycosides, named eucalglobuside A (2) and eucalglobuside B (3), along
with 15 known compounds were isolated from the leaves of Eucalyptus globulus. Their structures
were elucidated based on extensive spectroscopic analysis and in comparison with literature data.
The absolute configuration of compound 1 was determined by ECD calculation. All isolates were
evaluated their inhibitory activities against the mushroom tyrosinase. As a result, three
sesquiterpenoids, 1, 5, 11-dihydroxy-iphionan-4-one (5), and ()-globulol (8), exhibited the most
potent activities with IC50 values of 14.17 M, 10.08 M and 9.79 M, respectively.
Key words Eucalyptus globulus; Eucalyptus; isoiphionane; phloroglucinol; tyrosinase inhibit
ors
1
. Introduction
Eucalyptus globulus Labill, an Australian native tall timber tree, is widely
distributed in the southwestern provinces of China. Its fruit is known as ‘Yi Kou
Zhong’ in China and used as a well-known folk medicine to treat flu, dysentery,
eczema, scald, and rheumatism [1]. Previous chemical investigations on this plant
afforded many structurally diverse secondary metabolites which comprise
phloroglucinol derivatives [2], terpenes [3], flavonoids [4], and sterols [5]. In recent
years, plants of the Eucalyptus genus have been a hot research topic of natural
products due to the increasing discovery of characteristic phloroglucinol derivatives
with diverse biological activities, such as antiviral [6], cytotoxic [7−12], antibacterial
[
[
13], anti-inflammatory [14], anti-HIV [15], antifungal [16, 17], immunosuppressive
18], as well as PTP1B inhibitory effects [14, 19].
Tyrosinase, a metal monooxygenase enzyme, plays a key role in the biosynthesis of
mammalian melanin which is responsible for the variation in the colors of eyes, skin,
and hair [20]. Therefore, tyrosinase inhibitors can be used in cosmetic and medicinal
industry to reduce skin hyperpigmentations, like age spots, melasma, and chloasma.
In addition, tyrosinase is responsible for the detrimental browning of many
plant-derived foods, which lead to the application of tyrosinase inhibitors in food
preservation by preventing these undesirable enzymatic browning reactions. As part
of our ongoing work to discover new tyrosinase inhibitors from plant source [21, 22],
an ehthanolic extract of the leaves of E. globulus was intensively investigated. This
effort led to the isolation of three new compounds, including one isoiphionane
*Corresponding author.
E-mail address: Zhouzw@ncu.edu.cn (Z.-W. Zhou).

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sesquiterpene (1) and two phloroglucinol glycosides (2 and 3) (Fig. 1), together with
1
(
4
5 known compounds, chromene glucoside (4) [23], 5, 11-dihydroxy-iphionan-4-one
5) [24], proximadiol (6) [25], ()--eudesmol (7) [26], ()-globulol (8) [27],
β,10α-aromadendranediol (9) [28], vomifoliol (10) [29], isololiolide (11) [30],
eucalyptin (12) [31], (+)-rhododendrol (13) [32],
-(4-hydroxy-3-methoxyphenyl)-2R-butanol (14) [33], ursolic acid lactone (15) [34],
-acetoxyurs-11-en-28, 13-olide (16) [35], pinoresinol (17) [36],
,5-dimethylhydroquinone (18) [37]. In this paper, we describe the isolation and
4
3
2
structural elucidation of the new compounds as well as the tyrosinase inhibitory
activity of all isolates.
Fig. 1 The chemical structures of compounds 14
2
. Results and discussion
2
D
5
Compound 1 ([]
10.73, c = 0.10, CH
3
OH) was obtained as a colorless oil and
deduced from its positive HRESIMS
shown to have the molecular formula C15
H
26
O
3
+
+
(
m/z 277.1770 [M+Na] , calcd for C15
H
26
O Na , 277.1774), requiring three degrees
3
1
13
of unsaturation. Its H- and C-NMR spectral data (Table 1), in combination with
HSQC spectrum, indicated a total of 15 carbon signals, comprising three tertiary
methyls [
one acetyl moiety [
.56 (1H, dd, J = 9.6, 7.8 Hz); 
H
1.22 (3H, s)/
2.17 (3H, s); 
76.8]. Moreover, the NMR data also showed five
C
27.1, 
H
1.22 (3H, s)/
C
27.2, and 
H
0.90 (3H, s)/ 21.9],
C
H
C
31.9, 218.3], and one oxygenated methine [
H
4
C
3
methylenes, one methine [
H
1.58 (1H, m); 
C
44.9], and three sp quaternary carbons
1
13
[

C
65.0, 43.6, 73.2]. All aforementioned H- and C-NMR data indicated 1 to be a
1
1
bicyclic sesquiterpene. The H- H COSY correlations and HSQC spectra revealed the
presence of two proton-bearing structural fragments as drawn with bold lines in Fig. 2.
In the HMBC spectrum, correlations from H-14 to C-1, C-5, C-9, and C-10, H-6 to
C-3, C-5, and C-10, and that from H-3 to C-4 could be observed. These HMBC
correlations assembled above two proton-bearing fragments into a bicyclic
hydrindane system and located the methyl of C-14 and the acetyl unit at C-10 and C-5,
respectively, considering the chemical shift of C-5 (
C
65.0) and C-10 ( 43.6). In
C
addition, HMBC correlations of H-12(13) with C-7 and C-11 connected the
isopropanol moiety with C-7. Thus, the planar structure of 1 was determined as shown
in Fig. 2 as identical to that of 3, 11-dihydroxyisoiphion-4-one, a known

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isoiphionane-type sesquiterpene [38], except for the downfield shift of H-3 in 1,
suggesting it is a stereoisomer of the latter. The relative stereochemistry of 1 was
established from the observed correlations of the NOESY experiment (Fig. 2). The
hydrindane skeleton of 1 was assigned as cis-fused configuration according to the
NOESY correlation of H-14/H-15, which was supported by the chemical shift of the
methyl of C-14 (
C
21.9) [39]. The -orientation of 3-OH and H-7 were similarly
established by the NOESY correlation of H-3/H-14 and H-12(13)/H-15, respectively.
1
1
Fig. 2 H- H COSY (
), key HMBC (HC), and NOE (H
H) correlations of compound 1
The absolute configuration of 1 was determined by ECD calculation employing the
time-dependent density functional theory Electronic Circular Dichroism
(TDDFT-ECD) method [40]. As shown in Fig. 3, the measured ECD spectrum of 1
displayed the opposite curve with the calculated 3R, 5R, 7R, 10S-1 enantiomer. On the
basis of above evidence, the structure of 1 was elucidated as shown and named (3S,
5
S, 7S, 10R)-3, 11-dihydroxyisoiphion-4-one. As far as we know, isoiphionane
sesquiterpenes have a very narrow distribution in plants and mainly have been
reported occurring in the family of Compositae [41−44] and Lauraceae [45, 46]. The
co-occurrence of the isoiphionane- and iphionane-type sesquiterpenes 1 and 5, and the
eudesmanes 6 and 7 in E. globulus reinforces the suggested biogenetic
inter-relationship between those rarely naturally occurring rearranged sesquiterpenes
and eudesmanes [42, 47]. Thus, compound 1, which might have been formed by
oxidative cleavage of C-3/C-4 double bond of an eudesmane precursor
(+)--eudesmol [48] leading to a 3, 4-diketo derivative, followed by aldol
condensation between C-3 and C-5, is thus being reported for the first time in the
Myrtaceae.
Fig. 3 Calculated and experimental CD spectra of 1
2
D
5
Compound 2 ([]
-6.40, c = 0.13, CH
3
OH), a yellow amorphous powder, gave a
molecular formula of C19
H
24
O
9
as determined by HRESIMS at m/z 419.1321
+
1
[
M+Na] (calcd 419.1318). The H-NMR spectrum (Table 1) of 2 displayed one

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aldehyde proton (
aromatic methyl (
m, 1H)], and one glucosyl moiety. The coupling constant of the anomeric proton [δ
.69 (d, J = 7.6 Hz, H-1)] suggested a -configuration for the glucose. In the
H
10.37, s, 1H), one singlet olefinic proton ( 6.54, s, 1H), one
H
H
2.44, s, 3H), one isopropyl unit [δ 1.35 (d, 6.8 Hz, 6H) and 3.08
H
(
H
4
1
3
C-NMR (Table 1) and HSQC spectra of 2, nineteen carbon signals were observed
including one conjugated aldehyde carbon ( 198.3), three methyls ( 9.1, 21.2, and
29.4), eight sp carbons together with a set of signals
arising from a glucose moiety [ 107.0 (C-1), 75.6 (C-2), 77.9 (C-3), 71.3 (C-4),
8.2 (C-5), and 62.5 (C-6)]. All aforementioned NMR data suggested compound 2 is
C
C
2
2
1.2), one methine carbon (
C
C
7
a phloroglucinol glycoside [30, 49], the characteristic metabolite type within the
genus Eucalyptus. The 1D-NMR data of 2 was closely related to those of chromene
glucoside (4), a co-metabolite previously isolated by Ito et. al [30] from the leaves of
E. cypellocarpa, except for the absence of one olefinic proton signal and the
appearance of one isopropyl group. These observations indicated 2 had a
2
-isopropyl-2H-furan moiety instead of a 2, 2-dimethyl-2H-pyran ring in 4.
1
1
Comprehensive analysis of the 2D-NMR spectra of 2, especially H- H COSY and
HMBC (Fig. 4), allowed the establishment of the planar structure. The isopropyl
1
1
fragment as drawn with bold lines in Fig. 4 was established on the basis of H- H
COSY spectrum. In the HMBC, the correlations of H-3/C-2, C-8, and C-9,
H
3
-13(14)/C-2 and H-12/C-2 established the 2-isopropyl-2H-furan segment fused at
C-8 and C-9. The HMBC correlations from H -11 to C-6, C-7, and C-8, and that from
3
H-10 to C-4 located the methyl of C-11 and the –CHO unit at C-7 and C-5,
respectively. Moreover, the HMBC correlation of H-1/C-6 allowed the attachment of
the glucose to C-6. Acid hydrolysis of 2 liberated a sugar that was identified as
D-glucose by TLC and optical rotation comparison with a standard sample. Therefore,
compound
-hydroxy-2-isopropyl-7-methylbenzofuran 6-O--D-glucopyranoside and named
eucalglobuside A.
2
was
determined
to
be
5-formyl-
4
Table.1 H- and 13C-NMR Data for Compounds 13 ( in ppm, J in Hz)
1
1a 2b
3b
No.
1

H

C

H

C

H

C
2.06, td, 12.6, 6.0
.32, ddd, 12.6, 9.6, 3.6
2.20, m (overlapped)
39.1
1
2
30.9
166.2
165.9
1
.79, dddd, 13.5, 12.6, 7.8, 3.6
4.56, dd, 9.6, 7.8
3
4
5
6
76.8
218.3
65.0
6.54, s
98.6
156.4
112.3
154.9
6.79, s
99.5
153.1
111.2
158.7
2.17, dd, 12.6, 3.0
27.9
1
.65, t, 12.6
7
8
1.58, tt, 12.6, 3.0
1.62, m
44.9
24.0
108.7
160.8
104.9
161.8
1
.18, m
9
1.44, td, 13.8, 3.6
40.2
115.6
113.5

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1
.34, dt, 13.8, 3.6
1
0
1
43.6
73.2
27.2
27.1
21.9
31.9
10.37, s
2.44, s
198.3
9.1
10.48, s
2.25, s
196.9
7.4
1
1
1
1
1
2
3
4
5
1.22, s
1.22, s
0.90, s
2.17, s
3.08, m
29.4
21.2
21.2
3.04, m
29.3
21.1
21.1
1.35, d, 6.8
1.35, d, 6.8
1.35, d, 6.8
1.35, d, 6.8
1






4.69, d, 7.6
3.56, m
3.43, m
3.42, m
3.17, m
107.0
75.6
77.9
71.3
78.2
4.97, d, 7.6
3.56, m
3.45, m
3.40, m
3.37, m
105.4
75.4
78.0
71.2
78.7
2
3
4
5
6
3.75, dd, 12.0, 2.0 62.5
.66, dd, 12.0, 4.8
. bRecorded at 400 MHz in methanol-d
.
4
3.89, dd, 12.0, 1.6 62.5
3.71, dd, 12.0, 5.2
3
aRecorded at 600 MHz in methanol-d
4
1
1
Fig. 4 H- H COSY (
), and key HMBC (HC) correlations of compounds 2 and 3
Compound 3 was obtained as a yellow amorphous powder and shared the same
molecular formula C19
combination with the H-NMR data (Table 1), compound 3 can be deduced as the C-4
glycosidation isomer of 2 due to their closely similar H-NMR spectra except for the
H
24
O with 2 according to their identical HRESIMS. In
9
1
1
up-field shift of H-11 and downfield shift of H-3, H-10, and H-1 in 3. The HMBC
correlation of H-1/C-4 corroborated above our deduction and located the -glucose
[
JH-1/H-2 = 7.6 Hz] unit at C-4 (Fig. 4). The planar structure of 3 was then fully
established based on more detailed HMBC correlations together with HSQC and
1
1
H- H COSY spectra (Fig. 4). Acid hydrolysis of 3 produced D-glucose, which was
identified by TLC and optical rotation comparison with a standard sample. Compound
3
was
therefore
determined
to
be
5
-formyl-6-hydroxy-2-isopropyl-7-methylbenzofuran
4-O--D-glucopyranoside,
namely, eucalglobuside B.
Compounds 118 were tested for their inhibitory activities against the mushroom
tyrosinase (Table 2). To our delight, compounds 1, 5 and 8 exhibited the most potent
activities with IC50 values of 14.17 M, 10.08 M and 9.79 M, respectively,
followed by 14 with an activity (IC50 = 21.75 M) comparable with that of the
positive control, kojic acid. Compounds 4, 12, and 13 displayed moderate activities
with IC50 values ranging from 33.43 to 49.16 M. Compounds 2, 3, and 17 showed
weak activities (50 M < IC50 < 100 M), while the remaining compounds were

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inactive (IC50 > 100 M). For those phloroglucinol glycosides (24), the
glycosidation position at C-6 is more favorable for the inhibitory activity than that of
C-4, but there is no remarkable difference between the 2-isopropyl-2H-furan moiety
and the 2, 2-dimethyl-2H-pyran ring in activity.
Table 2. Tyrosinase inhibitory activity of compounds 118
compound IC50 (μM)a compound IC50 (μM)a compound IC50 (μM)a
1
2
3
4
5
6
14.17±0.73
57.082.52
91.763.41
49.160.12
10.080.18
>100
7
8
>100
9.790.08
>100
13
14
15
16
17
18
42.630.43
21.750.33
>100
9
10
11
12
＞100
>100
>100
74.570.26
>100
33.430.14
Kojic acid 17.320.24
aEach value represents the mean ± SD of three determinations.
3
. Materials and methods
.1. General experimental procedures
3
NMR spectra were recorded on a Bruker AVANCE Ⅲ 600 or a Bruker Avance Ⅲ
4
00 NMR spectrometer with tetramethylsilane (TMS) as an internal reference. All
HRESIMS spectra were analyzed on a Waters UPLC-QTOF or an AB Sciex Triple
TOF 5600 system. Optical rotations were measured on an Anton Paar MCP-200
polarimeter. CD spectra were measured on a JASCO J-815 spectropolarimeter in
MeOH. Silica gel (300−400 mesh, Qingdao Marine Chemical Plant, Qingdao, P. R.
China), C18 reversed-phase silica gel (150−200 mesh, Merck), MCI gel (CHP20P,
7

5−150 μM, Mitsubishi Chemical Industries Ltd.), and Sephadex LH-20 gel (75−150
M, GE Healthcare) were used for column chromatography. Precoated silica gel
GF254 plates (Qingdao Marine Chemical Plant) were used for TLC. Semipreparative
HPLC was performed on an Agilent 1200 system equipped with a VWD G1314B
detector and a Zorbax SB-C18 column (250 × 10 mm, 5 μm). All solvents used were of
analytical grade (Xilong Chemical Reagent Co., Ltd., Guangdong, P. R. China). Kojic
acid was purchased from the Solarbio Science & Technology Co., Ltd (Beijing, P. R.
China). Both the mushroom tyrosinase and L-DOPA were purchased from the
Sigma-Aldrich Co. LLC (St. Louis, MO, USA).
3
.2. Plant material
The leaves of E. globulus (5.0 kg) was purchased from Quanzhou County, Guangxi
Province, P. R. China, in October 2016, and identified by Professor Yun Ling (School
of Pharmacy, Nanchang University, P. R. China). A voucher specimen (EG-201610)
has been deposited in School of Pharmacy, Nanchang University, P. R. China.
3
.3. Extraction and isolation
The dry and powdered leaves of E. globulus were extracted with 95% EtOH three
times at room temperature. The crude extract (210.0 g) was then suspended in water
and subsequently extracted with petroleum ether, EtOAc, and n-BuOH, successively.

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The EtOAc extract (60.5 g) was fractionated by MCI gel column chromatography
(CC) using a CH
3
OH/H O gradient system (30: 70 100: 0，v/v) to afford ten
2
fractions, Fr.1−Fr.10. Fr.2 (1.45 g) was separated over Sephadex LH-20 (EtOH) to
yield four subfractions, Fr.2A−Fr.2D. Fr.2A (135.0 mg) was purified by CC on silica
gel (petroleum ether/acetone 8: 12: 1，v/v), followed by semi-preparative HPLC
(
CH
min). Fr.2C (70.0 mg) was purified by semi-preparative HPLC (CH
.0 ml/min) to afford 9 (4.0 mg, 41.6 min). Fr.3 (1.90 g) was subjected to CC with
Sephadex LH-20 (EtOH) to give six subfractions, Fr.3A−Fr.3F. Fr.3B (153.2 mg) was
purified by semi-preparative HPLC (CH CN/H O, 30: 70, 3.0 ml/min) to give 2 (2.5
mg, 50.9 min) and 3 (3.0 mg, 56.0 min). Fr.3E (103.8 mg) was purified by
semi-preparative HPLC (CH CN/H O, 30: 70, 3.0 ml/min) to afford 4 (1.8 mg, 32.7
min), and 18 (4.2 mg, 21.3 min). Fr.3C (261.0 mg) was purified on an ODS column
30: 70 80: 20，v/v) to afford 6 (26.0 mg). Fr.4 (3.60 g) was separated by Sephadex
LH-20 (EtOH) to give six subfractions Fr.4A−Fr.4F. Fr.4C (343.7 mg) was purified
by CC on silica gel (CHCl /MeOH, 30: 1), followed by semi-preparative HPLC
CH CN/H O, 20: 80, 3.0 ml/min) to afford 10 (3.0 mg, 12.8 min) and 11 (4.8 mg,
7.0 min). Fr.5 (1.64 g) was separated over an ODS column chromatography eluted
with a MeOH/H O gradient (70: 30~90: 10, v/v) to give five subfractions
Fr.5A−Fr.5E. Fr.5C (69.2 mg) was then purified by semi-preparative HPLC
CH CN/H O, 35: 65, 4.0 ml/min) to afford 7 (4.2 mg, 30.7 min), 8 (10.0 mg, 33.5
3
CN/H
2
O, 25: 75, 3.0 ml/min) to afford 1 (1.0 mg, 36.4 min) and 5 (3.2 mg, 32.8
3
CN/H O, 25: 75,
2
3
3
2
3
2
(
3
(
1
3
2
2
(
3
2
min), and 12 (2.3 mg, 40.0 min). Fr.1 (1.6 g) was subjected to CC with Sephadex
LH-20 (EtOH) to give three subfractions, Fr.1A−Fr.1C. Fr.1B (740.8 mg) was
separated on silica gel CC (petroleum ether/acetone 10: 12: 1, v/v) to afford seven
subfractions (Fr.1B
1
−Fr.1B
7
). Fr.1B
4
(42.9 mg) was then purified by semi-preparative
HPLC (CH CN/H O, 15: 85, 3.0 ml/min) to afford 13 (1.1 mg, 37.8 min) and 14 (1.8
3
2
mg, 44.1 min). Fr.6 (1.2 g) was separated over Sephadex LH-20 (EtOH) to give three
subfractions, Fr.6A−Fr.6C. Fr.6A (103.4 mg) was further purified by
HPLC(CH
3
CN/H O, 60: 40, 4.0 ml/min) to afford 15 (4.3 mg, 30.2 min), 16 (3.4 mg,
2
4
5.0 min), and 17 (3.8 mg, 21.1 min).
2
D
5
(
0
[
3S, 5S, 7S, 10R)-3, 11-dihydroxyisoiphion-4-one (1): Colorless oil, []
10.73 (c =
1
13
.10, CH
3
OH), H- and C-NMR data: see Table 1; (+) HRESIMS m/z 277.1770
+
+
M+Na] (calcd for C15
H
26
O
3
Na , 277.1774).
2
D
5
eucalglobuside A (2): Yellow amorphous powder, []
-6.40 (c = 0.13, CH
3
OH), UV
1
13
(
DMSO), max (log ε) 254 (3.85), 285 (3.63) nm, H- and C-NMR data: see Table 1;
+
+
(+) HRESIMS m/z 419.1321 [M+Na] (calcd for C19
H
24
O Na , 419.1318).
9
2
D
5
eucalglobuside B (3): Yellow amorphous powder, []
-15.33 (c = 0.15, CH
3
OH), UV
1
13
(
DMSO), max (log ε) 254 (3.92), 285 (3.77) nm, H- and C-NMR data: see Table 1;
+
+
(+) HRESIMS m/z 419.1321 [M+Na] (calcd for C19
H
24
O Na , 419.1318).
9
3
.4. ECD calculation for compound 1
The preliminary conformation of compound 1 was established by the Chem3D-
_
Pro_14.1 software using the MM2 force field overlaid with key correlations

Journal Pre-proof
observed in the ROESY spectrum. Further conformational searches were carried out
by the Maestro 10.2 software using mixed torsional/Low-mode sampling method with
MMFFs force field in an energy window of 3.01 Kcal/mol. The re-optimization and
the following TD-DFT calculations of the re-optimized conformations were all
performed with Gaussian 09 program package at the B3LYP/6-311G(d, p) level. The
harmonic vibrational frequencies were also calculated to confirm the stability of the
re-optimized conformers. Finally, the SpecDis 1.64 software was used to obtain the
Boltzmann-averaged ECD spectra.
3
.5. Hydrolysis of compounds 2 and 3
Compounds 2 and 3 (each 1.6 mg) were treated with 2 M HCl solution (3 mL) at
9
0 °C for 2 h. After the completion of hydrolysis, the reaction mixture was extracted
with EtOAc (10 mL × 3), and both the organic and the water layers were evaporated
to dryness under reduced pressure. The carbohydrate obtained from the water layer
(
0.5 mg) was dissolved in 2 mL of distilled water and kept overnight before the
determination of optical rotation. TLC of the sugar from both 2 and 3 on a silica gel
plate with CHCl /MeOH/H O (9: 6: 1) showed that it had an identical R value (0.32)
O) values of +40.90 and +41.35,
3
2
f
2
D
5
to glucose. The isolated glucose had []
(c 0.025, H
2
respectively.
3
.6. Tyrosinase inhibitory assay
The tyrosinase assay was conducted as reported by us previously [21]. All testing
samples were dissolved in dimethyl sulfoxide (DMSO) and used at concentration of
00, 50, 25, 10, 5 μg/mL (or μM for pure compounds), respectively. The tyrosinase
1
inhibitory activity assay was performed in 96-well microplates with 200 μL total
testing solution. The assay mixtures consists 40 μL of sample solution, 40 μL L-DOPA
(2 mmol/L in 0.1 M phosphate buffer pH 6.8), and 40 μL of mushroom tyrosinase
solution (2 U/mL in 0.1 M phosphate buffer pH 6.8). The assay mixture was
incubated at room temperature for 30 min, and the absorbance at 475 nm was
measured in triplicate with a microplate reader (Thermo Electron Corporation, CA,
USA). Kojic acid, a known tyrosinase inhibitor, was used as positive control.
Conflict of Interest
The authors declare that there are no conflicts of interest with this work.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of
China (No. 21362023 and 30901855), the Natural Science Foundation of Jiangxi
Province, China (No. 20142BAB215021 and 20151BAB205083), and the Project of
Jiangxi Provincial Education Department (No. GJJ150843).
Appendix A. Supplementary data
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Highlights

Three new compounds, including one isoiphionane sesquiterpene (1) and two
phloroglucinol glycosides (2 and 3), together with 15 known compounds were
isolated from Eucalyptus globulus.


The absolute configuration of compound 1 was determined by ECD calculation.
Three sesquiterpenoids, 1, 5, 11-dihydroxy-iphionan-4-one (5), and ()-globulol
(8), exhibited the best activities against the mushroom tyrosinase with IC50 values
of 14.17 M, 10.08 M and 9.79 M, respectively, more potent than the positive
control kojic acid.

Figure 1

Figure 2

Figure 3

Figure 4