Tyrosinase inhibitors from the leaves of Eucalyptus globulus

Journal Pre-proof 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

Reference:

FITOTE 104418

To appear in:

Fitoterapia

Received date:

29 September 2019

Revised date:

31 October 2019

Accepted date:

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

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

Journal Pre-proof

Tyrosinase inhibitors from the leaves of Eucalyptus globulus Qiao-Mei Lina, Yong Wanga, Jin-Hai Yub, Yun-Lai Liua, Xue Wua, Xiao-Ru Hec, Zhi-Wang Zhoua,* 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

ro

of

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

re

-p

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

lP

1. Introduction

Jo ur

na

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: [email protected] (Z.-W. Zhou).

Journal Pre-proof

re

-p

ro

of

sesquiterpene (1) and two phloroglucinol glycosides (2 and 3) (Fig. 1), together with 15 known compounds, chromene glucoside (4) [23], 5, 11-dihydroxy-iphionan-4-one (5) [24], proximadiol (6) [25], ()--eudesmol (7) [26], ()-globulol (8) [27], 4β,10α-aromadendranediol (9) [28], vomifoliol (10) [29], isololiolide (11) [30], eucalyptin (12) [31], (+)-rhododendrol (13) [32], 4-(4-hydroxy-3-methoxyphenyl)-2R-butanol (14) [33], ursolic acid lactone (15) [34], 3-acetoxyurs-11-en-28, 13-olide (16) [35], pinoresinol (17) [36], 2,5-dimethylhydroquinone (18) [37]. In this paper, we describe the isolation and structural elucidation of the new compounds as well as the tyrosinase inhibitory activity of all isolates.

2. Results and discussion

lP

Fig. 1 The chemical structures of compounds 14

Jo ur

na

Compound 1 ([]25D 10.73, c = 0.10, CH3OH) was obtained as a colorless oil and shown to have the molecular formula C15H26O3 deduced from its positive HRESIMS (m/z 277.1770 [M+Na]+, calcd for C15H26O3Na+, 277.1774), requiring three degrees of unsaturation. Its 1H- and 13C-NMR spectral data (Table 1), in combination with HSQC spectrum, indicated a total of 15 carbon signals, comprising three tertiary methyls [H 1.22 (3H, s)/C 27.1, H 1.22 (3H, s)/C 27.2, and H 0.90 (3H, s)/C 21.9], one acetyl moiety [H 2.17 (3H, s); C 31.9, 218.3], and one oxygenated methine [H 4.56 (1H, dd, J = 9.6, 7.8 Hz); C 76.8]. Moreover, the NMR data also showed five methylenes, one methine [H 1.58 (1H, m); C 44.9], and three sp3 quaternary carbons [C 65.0, 43.6, 73.2]. All aforementioned 1H- and 13C-NMR data indicated 1 to be a bicyclic sesquiterpene. The 1H-1H 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 (C 43.6). In 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

Journal Pre-proof

Fig. 2 1H-1H COSY (

), key HMBC (HC), and NOE (H

of

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.

H) correlations of compound 1

Jo ur

na

lP

re

-p

ro

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, 5S, 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

Compound 2 ([] -6.40, c = 0.13, CH3OH), a yellow amorphous powder, gave a molecular formula of C19H24O9 as determined by HRESIMS at m/z 419.1321 [M+Na]+ (calcd 419.1318). The 1H-NMR spectrum (Table 1) of 2 displayed one 25 D

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

aldehyde proton (H 10.37, s, 1H), one singlet olefinic proton (H 6.54, s, 1H), one aromatic methyl (H 2.44, s, 3H), one isopropyl unit [δH 1.35 (d, 6.8 Hz, 6H) and 3.08 (m, 1H)], and one glucosyl moiety. The coupling constant of the anomeric proton [δH 4.69 (d, J = 7.6 Hz, H-1)] suggested a -configuration for the glucose. In the 13 C-NMR (Table 1) and HSQC spectra of 2, nineteen carbon signals were observed including one conjugated aldehyde carbon (C 198.3), three methyls (C 9.1, 21.2, and 21.2), one methine carbon (C 29.4), eight sp2 carbons together with a set of signals arising from a glucose moiety [C 107.0 (C-1), 75.6 (C-2), 77.9 (C-3), 71.3 (C-4), 78.2 (C-5), and 62.5 (C-6)]. All aforementioned NMR data suggested compound 2 is 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. Comprehensive analysis of the 2D-NMR spectra of 2, especially 1H-1H COSY and HMBC (Fig. 4), allowed the establishment of the planar structure. The isopropyl fragment as drawn with bold lines in Fig. 4 was established on the basis of 1H-1H COSY spectrum. In the HMBC, the correlations of H-3/C-2, C-8, and C-9, H3-13(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 H3-11 to C-6, C-7, and C-8, and that from 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 2 was determined to be 5-formyl4-hydroxy-2-isopropyl-7-methylbenzofuran 6-O--D-glucopyranoside and named eucalglobuside A. Table.1 1H- and 13C-NMR Data for Compounds 13 ( in ppm, J in Hz) 1a

No. 1

H

C

2.06, td, 12.6, 6.0

39.1

2b

H

3b

C

H

C

1.32, ddd, 12.6, 9.6, 3.6 2

2.20, m (overlapped)

30.9

166.2

165.9

1.79, dddd, 13.5, 12.6, 7.8, 3.6 3

4.56, dd, 9.6, 7.8

76.8

6.54, s

98.6

6.79, s

99.5

4

218.3

156.4

153.1

5

65.0

112.3

111.2

27.9

154.9

158.7

6

2.17, dd, 12.6, 3.0 1.65, t, 12.6

7

1.58, tt, 12.6, 3.0

44.9

108.7

104.9

8

1.62, m

24.0

160.8

161.8

40.2

115.6

113.5

1.18, m 9

1.44, td, 13.8, 3.6

Journal Pre-proof 1.34, dt, 13.8, 3.6 10

43.6

10.37, s

198.3

10.48, s

196.9

11

73.2

2.44, s

9.1

2.25, s

7.4

1.22, s

27.2

3.08, m

29.4

3.04, m

29.3

13

1.22, s

27.1

1.35, d, 6.8

21.2

1.35, d, 6.8

21.1

14

0.90, s

21.9

1.35, d, 6.8

21.2

1.35, d, 6.8

21.1

15

2.17, s

31.9

1

4.69, d, 7.6

107.0

4.97, d, 7.6

105.4

2

3.56, m

75.6

3.56, m

75.4

3

3.43, m

77.9

3.45, m

78.0

4

3.42, m

71.3

3.40, m

71.2

5

3.17, m

78.2

3.37, m

78.7

6

3.75, dd, 12.0, 2.0

62.5

3.89, dd, 12.0, 1.6

62.5

3.66, dd, 12.0, 4.8

3.71, dd, 12.0, 5.2

at 400 MHz in methanol-d4.

ro

at 600 MHz in methanol-d4.

bRecorded

Fig. 4 1H-1H COSY (

lP

re

-p

aRecorded

of

12

), and key HMBC (HC) correlations of compounds 2 and 3

Jo ur

na

Compound 3 was obtained as a yellow amorphous powder and shared the same molecular formula C19H24O9 with 2 according to their identical HRESIMS. In combination with the 1H-NMR data (Table 1), compound 3 can be deduced as the C-4 glycosidation isomer of 2 due to their closely similar 1H-NMR spectra except for the 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 H-1H 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

Journal Pre-proof 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 IC50 (μM)a

compound

IC50 (μM)a

compound

IC50 (μM)a

1

14.17±0.73

7

>100

13

42.630.43

2

57.082.52

8

9.790.08

14

21.750.33

3

91.763.41

9

>100

15

>100

4

49.160.12

10

＞100

16

>100

5

10.080.18

11

>100

17

74.570.26

6

>100

12

33.430.14

18

>100

Kojic acid

17.320.24

value represents the mean ± SD of three determinations.

ro

aEach

of

compound

3.1. General experimental procedures

-p

3. Materials and methods

Jo ur

na

lP

re

NMR spectra were recorded on a Bruker AVANCE Ⅲ 600 or a Bruker Avance Ⅲ 400 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, 75−150 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.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

The EtOAc extract (60.5 g) was fractionated by MCI gel column chromatography (CC) using a CH3OH/H2O gradient system (30: 70 100: 0，v/v) to afford ten 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 (CH3CN/H2O, 25: 75, 3.0 ml/min) to afford 1 (1.0 mg, 36.4 min) and 5 (3.2 mg, 32.8 min). Fr.2C (70.0 mg) was purified by semi-preparative HPLC (CH3CN/H2O, 25: 75, 3.0 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 (CH3CN/H2O, 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 (CH3CN/H2O, 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 (CHCl3/MeOH, 30: 1), followed by semi-preparative HPLC (CH3CN/H2O, 20: 80, 3.0 ml/min) to afford 10 (3.0 mg, 12.8 min) and 11 (4.8 mg, 17.0 min). Fr.5 (1.64 g) was separated over an ODS column chromatography eluted with a MeOH/H2O 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 (CH3CN/H2O, 35: 65, 4.0 ml/min) to afford 7 (4.2 mg, 30.7 min), 8 (10.0 mg, 33.5 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.1B1−Fr.1B7). Fr.1B4 (42.9 mg) was then purified by semi-preparative HPLC (CH3CN/H2O, 15: 85, 3.0 ml/min) to afford 13 (1.1 mg, 37.8 min) and 14 (1.8 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(CH3CN/H2O, 60: 40, 4.0 ml/min) to afford 15 (4.3 mg, 30.2 min), 16 (3.4 mg, 45.0 min), and 17 (3.8 mg, 21.1 min). (3S, 5S, 7S, 10R)-3, 11-dihydroxyisoiphion-4-one (1): Colorless oil, []25D 10.73 (c = 0.10, CH3OH), 1H- and 13C-NMR data: see Table 1; (+) HRESIMS m/z 277.1770 [M+Na]+ (calcd for C15H26O3Na+, 277.1774). eucalglobuside A (2): Yellow amorphous powder, []25D -6.40 (c = 0.13, CH3OH), UV (DMSO), max (log ε) 254 (3.85), 285 (3.63) nm, 1H- and 13C-NMR data: see Table 1; (+) HRESIMS m/z 419.1321 [M+Na]+ (calcd for C19H24O9Na+, 419.1318). eucalglobuside B (3): Yellow amorphous powder, []25D -15.33 (c = 0.15, CH3OH), UV (DMSO), max (log ε) 254 (3.92), 285 (3.77) nm, 1H- and 13C-NMR data: see Table 1; (+) HRESIMS m/z 419.1321 [M+Na]+ (calcd for C19H24O9Na+, 419.1318). 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

3.6. Tyrosinase inhibitory assay

re

-p

ro

of

Compounds 2 and 3 (each 1.6 mg) were treated with 2 M HCl solution (3 mL) at 90 °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 CHCl3/MeOH/H2O (9: 6: 1) showed that it had an identical Rf value (0.32) to glucose. The isolated glucose had []25D (c 0.025, H2O) values of +40.90 and +41.35, respectively.

Jo ur

na

lP

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 100, 50, 25, 10, 5 μg/mL (or μM for pure compounds), respectively. The tyrosinase 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 References

Journal Pre-proof [1] J. F. Zeng, S. Q. Liu, In Chinese Flora (Zhongguo Zhiwu Zhi); Science Press: Beijing, 1984; Vol. 53, Chapter 1, pp 31−52. [2] K. Osawa, H. Yasuda, H. Morita, K. Takeya, H. Itokawa, Eucalyptone from Eucalyptus globulus, Phytochemistry 40 (1995) 183−184. [3] G. G. Santos, J. C. N. Alves, J. M. L. Rodilla, A. P. Duarte, A. M. Lithgow, J. G. Urones, Terpenoids and other constituents of Eucalyptus globulus, Phytochemistry 44 (1997) 1309−1312. [4] B. S. Yun, I. K. Lee, J. P. Kim, S. H. Chung, G. S. Shim, I. D. Yoo, Lipid peroxidation inhibitory activity of some constituents isolated from the stem bark of Eucalyptus globulus, Arch. Pharm. Res. 23 (2000) 147−150. [5] P. S. Vankar, J. Srivastava, K. Molcanov, B. Kojic-Prodic, Withanolide A series steroidal lactones from Eucalyptus globulus bark, Phytochem Lett. 2 (2009) 67−71. [6] M. Takasaki, T. Konoshima, M. Kozuka, K. Yoneyama, S. Yoshida, H. Tokuda, H. Nishino, A.

of

Iwashima, Inhibitors of skin-tumor promotion. ⅩⅢ. Inhibitory effects of Euglobals and their tumors, Biol. Pharm. Bull. 18 (1995) 288−294.

ro

related compounds on epstein-barr virus activation and on two-stage carcinogenesis of mouse skin [7] J. Wang, W. Z. Zhai, Y. K. Zou, J. J. Zhu, J. Xiong, Y. Zhao, G. X. Yang, H. Fan, M. T. Hamann,

-p

G. Xia, J. F. Hu, Eucalyptals D and E, new cytotoxic phloroglucinols from the fruits of Eucalyptus globulus and assignment of absolute configuration, Tetrahedron Lett. 53 (2012) 2654−2658.

re

[8] S. Yin, J. J. Xue, C. Q. Fan, Z. H. Miao, J. Ding, J. M. Yue, Eucalyptals A−C with a new skeleton isolated from Eucalyptus globulus, Org. Lett. 9 (2007) 5549−5552. [9] X. J. Qin, L. Y. Jin, Q. Yu, H. Liu, A. Khan, H. Yan, X. J. Hao, L. K. An, H. Y. Liu,

lP

Eucalypglobulusals A−J, formyl-phloroglucinol−terpene meroterpenoids from Eucalyptus globulus fruits, J. Nat. Prod. 81 (2018) 2638−2646. [10] T. A. Pham, I. S. Mohammad, V. T. Vu, X. L. Hu, C. Birendra, A. Ulah, C. Guo, X. Y. Lu, W. C.

na

Ye, H. Wang, Phloroglucinol derivatives from the fruits of Eucalyptus globulus and their cytotoxic activities, Chem. Biodivers. 15 (2018) e1800052.

Jo ur

[11] H. Liu, M. Y. Feng, Q. Yu, H. Yan, Y. Zeng, X. J. Qin, L. He, H. Y. Liu, Formyl phloroglucinol meroterpenoids from Eucalyptus tereticornis and their bioactivities, Tetrahedron 74 (2018) 1540−1545.

[12] Z. C. Shang, M. H. Yang, K. L. Jian, X. B. Wang, L. Y. Kong, 1H NMR-guided isolation of formyl-phloroglucinol meroterpenoids from the leaves of Eucalyptus robusta, Chem. Eur. J. 22 (2016) 11778−11784.

[13] K. Osawa, H. Yasuda, H. Morita, K. Takeya, H. Itokawa, Macrocarpals H, I, and J from the leaves of Eucalyptus globulus, J. Nat. Prod. 59 (1996) 823–827. [14] X. J. Qin, M. Y. Feng, H. Liu, W. Ni, T. Rauwolf, J. A. Jr. Porco, H. Yan, L. He, H. Y. Liu, Eucalyptusdimers A−C, dimeric phloroglucinol−phellandrene meroterpenoids from Eucalyptus robusta, Org. Lett. 20 (2018) 5066−5070. [15] M. Nishizawa, M. Emura, Y. Kan, H. Yamada, K. Ogawa, N. Hamanaka, Macrocarpals: HIV-RTase inhibitors of Eucalyptus globulus, Tetrahedron Lett. 33 (1992) 2983−2986. [16] Z. C. Shang, M. H. Yang, R. H. Liu, X. B. Wang, L. Y. Kong, New formyl phloroglucinol meroterpenoids from the leaves of Eucalyptus robusta, Sci. Rep. 6 (2016) 39815. [17] Z. C. Shang, C. Han, J. L. Xu, R. H. Liu, Y. Yin, X. B. Wang, M. H. Yang, L. Y. Kong, Twelve formyl phloroglucinol meroterpenoids from the leaves of Eucalyptus robusta, Phytochemistry 163 (2019) 111–117.

Journal Pre-proof [18] T. A. Pham, X. L. Hu, X. J. Huang, M. X. Ma, J. H. Feng, J. Y. Li, J. Q. Hou, P. L. Zhang,

V. H.

Nguyen, M. T. Nguyen, F. Xiong, C. L. Fan, X. Q. Zhang, W. C. Ye, H. Wang, Phloroglucinols with immunosuppressive activities from the fruits of Eucalyptus globulus, J. Nat. Prod. 82 (2019) 859−869. [19] Y. Yu, L. S. Gan, S. P. Yang, L. Sheng, Q. F. Liu, S. N. Chen, J. Li, J. M. Yue, Eucarobustols A-I, conjugates of sesquiterpenoids and acylphloroglucinols from Eucalyptus robusta, J. Nat. Prod. 79 (2016) 1365−1372. [20] J. Y. Lin, D. E. Fisher, Melanocyte biology and skin pigmentation, Nature 445 (2007) 843−850. [21] Y. Wang, L. Y. Xu, X. Liu, X. R. He, G. Ren, L. H. Feng, Z. W. Zhou, Artopithecins A–D, prenylated 2-arylbenzofurans from the twigs of Artocarpus pithecogallus and their tyrosinase inhibitory activities, Chem. Pharm. Bull. 66 (2018) 1199–1202. [22] L. Y. Xu, X. Liu, Q. M. Lin, Y. Wang, X. R. He, Z. W. Zhou, Prenylflavanones and lignans from

of

the twigs of Artocarpus pithecogallus, Magn. Reson. Chem. 57 (2019) 506–511. [23] H. Ito, M. Koreishi, H. Tokuda, H. Nishino, T. Yoshida, Cypellocarpins A−C, phenol glycosides

ro

esterified with oleuropeic acid, from Eucalyptus cypellocarpa, J. Nat. Prod. 63 (2000) 1253−1257.

-p

[24] X. L. Gao, Z. M. Xiong, G. Zhou, Y. L. Li, First enantioselective total syntheses and absolute configurations of 4,5-dioxo-seco--eudesmol and 5,11-dihydroxyiphionan-4-one, two aglycones

re

of naturally occurring sesquiterpenes with new skeletons, Synthesis 1 (2001) 37–39. [25] F. E. Evans, D. W. Miller, T. Cairns, G. V. Baddeley, E. Wenkert, Structure analysis of proximadiol cryptomeridiol by carbon 13C nmr spectroscopy, Phytochemistry 21 (1982) 937–938.

lP

[26] M. Tebbaa, A. E. Hakmaoui, A. Benharref, M. Akssira, Short and efficient hemisynthesis of α-eudesmol and cryptomeridiol, Tetrahedron Lett. 52 (2011) 3769–3771. [27] M. Miyazawa, T. Uemura, H. Kameoka, Biotransformation of sesquiterpenoids, (-)-globulol and

na

(+)-ledol by Glomerella cingulata, Phytochemistry 37 (1994) 1027–1030. [28] A. S. R. Anjaneyulu, K. S. Sagar, M. J. R. V. Venugopal, Terpenoid and steroid constituents of the

Jo ur

Indian ocean soft coral Sinularia maxima, Tetrahedron 51 (1995) 10997–11010. [29] T. T. Jong, M. Y. Jean, Constituents of Houttuyniae cordata and the crystal structure of vomifoliol, J. Chin. Chem. Soc. 40 (1993) 399–402. [30] J. Kimura, N. Maki, New loliolide derivatives from the brown alga Undaria pinnatifida, J. Nat. Prod. 65 (2002) 57–58.

[31] F. Huq, L. N. Misra, An alkenol and C-methylated flavones from Callistemon lanceolatus leaves, Planta Medica 63 (1997) 369−370. [32] B. Das, S. P. Rao, K. V. N. S. Srinivas, J. S. Yadav, Stereospecific synthesis and absolute configuration of (+)-rhododendrol, Phytochemistry 33 (1993) 1529−1530. [33] B. Das, M. Takhi, K. V. N. S. Srinivas, J. S. Yadav, Phenolics from needles of himalayan Taxus baccata, Phytochemistry 33 (1993) 1489−1491. [34] H. Wang, Y. Fujimoto, Triterpene esters from Eucalyptus tereticornis, Phytochemistry 33 (1993) 151−153. [35] M. Katai, T. Terai, H. Meguri, Triterpenoids of the bark of Pieris japonica D. Don (Japanese name: asebi). Ⅱ. 13C nuclear magnetic resonance of the -lactones of ursane- and oleanane-type triterpenes, Chem. Phurm. Bull. 31 (1983) 1567−1571. [36] L. H. Liu, J. X. Pu, J. F. Zhao, S. X. Mei, X. D. Yang, Y. B. Wang, H. B. Zhang, L. Li, A new lignan from Boschniakia himalaica, Chin. Chem. Lett. 15 (2004) 43−45.

Journal Pre-proof [37] A. Patel, F. Liebner, T. Netscher, K. Mereiter, T. Rosenau, Vitamin E chemistry. nitration of non--tocopherols: products and mechanistic considerations, J.Org. Chem. 72 (2007) 6504−6512. [38] A. A. Ahmed, J. Jakupovic, Sesqui- and monoterpenes from Jasonia montana, Phytochemistry 29 (1990) 3658−3661. [39] M. N. Todorova, E. T. Tsankova, New sesquiterpenoids from Achillea clypeolata, Phytochemistry 52 (1999) 1515–1518. [40] G. Pescitelli, T. Bruhn, Good computational practice in the assignment of absolute configurations by TDDFT calculations of ECD spectra, Chirality 28 (2016) 466–474. [41] P. Ceccherelli, M. Curini, M. C. Marcotullio, A. Menghini, Structure of cyperanic acid, a new sesquiterpene from Dittrichia Viscosa. J. Nat. Prod. 51 (1988) 1006–1009. [42] M. N. Todorova, E. T. Tsankova, New sesquiterpenoids from Achillea clypeolata. Phytochemistry 52 (1999) 1515–1518

of

[43] M. G. El-Ghazouly, N. A. El-Sebakhy, A. A.Seif El-Din, C. Zderoa, F. Bohlmann, Sesquiterpene xylosides from Iphiona scabra. Phytochemistry 26 (1987) 439–443.

ro

[44] A. A. Ahmed, J. Jakupovic, Sesqui- and monoterpenes from Jasonia montana. Phytochemistry 29 (1990) 3658–3661.

-p

[45] F. R. Garcez, W. S. Garcez, L. Hamerski, A. C. M. Miranda, Eudesmane and rearranged eudesmane sesquiterpenes from Nectandra cissiflora. Quim. Nova. 33 (2010) 1739–1742.

re

[46] J. S. Yu, J. Baek, H. B. Park, E. Moon, S. Y. Kim, S. U. Choi, K. H. Kim, A new rearranged eudesmane sesquiterpene and bioactive sesquiterpenes from the twigs of Lindera glauca (Sieb. et Zucc.) Blume. Arch. Pharm. Res. 39 (2016) 1628–1634.

lP

[47] H. Hikino, T. Kohama, T. Takemoto, Rearrangement of 4, 5-epoxy-eudesmanes with boron trifluoride. Tetrahedron 25 (1969) 1037–1045.

[48] M. Toyota, Y. Yonehara, I. Horibe, K. Minagawa, Y. Asakawa. A revision of the positive sign of

na

the optical rotation and its maximum value of -eudesmol. Phytochemistry 52 (1999) 689–694. [49] L. W. Tian, Y. J. Zhang, C. Qu, Y. F. Wang, C. R. Yang, Phloroglucinol glycosides from the fresh

Jo ur

fruits of Eucalyptus maideni, J. Nat. Prod. 73 (2010) 160–163.

Journal Pre-proof

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.

Jo ur

na

lP

re

-p

ro

of



Figure 1

Figure 2

Figure 3

Figure 4