α-Glucosidic hydroquinone derivatives from Viburnum erosum

Article abstract of DOI:10.1016/j.phytochem.2021.112782

Six undescribed compounds (1–6) were isolated from the leaves of Viburnum erosum along with four known compounds 7–10. The structures were determined by NMR and MS spectroscopic analyses, and their absolute configurations were established by chemical and

Full text of DOI:10.1016/j.phytochem.2021.112782

Phytochemistry 187 (2021) 112782
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
α
-Glucosidic hydroquinone derivatives from Viburnum erosum
Jinyoung Parka, Jiho Leea, Hyeon Seok Janga, Birang Jeonga, Seong Yeon Choia, Juyeol Kima,
Yong Soo Kwona, Heejung Yanga
,b,*
a College of Pharmacy, Kangwon National University, Chuncheon, 24341, Republic of Korea
b Bionsight, Inc., Chuncheon, 24341, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Six undescribed compounds (1–6) were isolated from the leaves of Viburnum erosum along with four known
compounds 7–10. The structures were determined by NMR and MS spectroscopic analyses, and their absolute
Viburnum erosum
Tyrosinase inhibition
β-arbutin
configurations were established by chemical and spectroscopic methods. Compounds 1–6 were
α-glucosidic
hydroquinone derivatives with different linear monoterpenoid structures. Compounds 1–10 were also evaluated
for their tyrosinase inhibitory activities, and 10 showed potent inhibition of tyrosinase enzyme with IC50 value of
37.9
μM compared to 47.6
μ
M of the positive control (β-arbutin).
1. Introduction
Krauze-Baranowska, 2015). β-arbutin ((2R,3S,4S,5R,6S)-2-(hydrox-
ymethyl)-6-(4-hydroxyphenoxy)oxane-3,4,5-triol)), a glycosylated hy-
droquinone, is found in many species of the Ericaceae and
The plants of genus Viburnum (Caprifoliaceae) are widely distributed
worldwide and contain terpenes, iridoids, flavonoids, lignans, phenols,
coumarins, lactones, and alkaloids (Wang et al., 2010). Several species
exhibit a wide range of biological activities including antitumor, anti-
oxidant, antimicrobial, antihyperglycemic, and hepatoprotective activ-
ities (Wang et al., 2010). Among the Viburnum species, Viburnum erosum
Thunb. (Caprifoliaceae) is a deciduous tree native to the East Asian
countries including Korea, China, and Japan. Neuroprotective and
anti-inflammatory lignans from the stems of V. erosum have been re-
ported, but their constituents have not been extensively investigated (In
et al., 2015).
Caprifoliaceae families, and is
a natural product with potent
anti-tyrosinase activity (Avonto et al., 2014; Migas and
Krauze-Baranowska, 2015). In the present study, potent tyrosinase
inhibitory constituents from the leaves of V. erosum are identified.
2. Results and discussion
In the pursuit of bioactive compounds with tyrosinase inhibitory
activities, compounds 1–10 were isolated from the EtOAc fraction of
V. erosum. The EtOAc sub-fractions, VEE1–8, showed different bioactive
profiles (Fig. 1).
Tyrosinase plays a key role in the browning of fruits, fungi, and
vegetables as well as melanogenesis in human skin (Zolghadri et al.,
2019). Tyrosinase is involved in melanin production, which causes skin
pigmentation and protects it from UV-induced damage. Tyrosinase in-
hibitors can be developed as skin-whitening agents in the cosmetic in-
dustry, and are considered as the most prominent targets for inhibiting
hyperpigmentation in skin cancer (Kim and Uyama, 2005). Many
plant-based natural products are used for the development of tyrosinase
inhibitors for cosmetic products as medical agents, but only a few
compounds are known to show inhibitory activities against tyrosinase
(Zolghadri et al., 2019).
VEE1 and VEE2 showed potent tyrosinase inhibitory activities with
weak cytotoxic activities against the B16F10 and HEp-2 cells, but frac-
tions VEE4–7 showed contrasting results. Therefore, VEE2 fraction with
sufficient volume (compared to VEE1) was subjected to further separa-
tion to isolate tyrosinase inhibitory compounds with weak cytotoxic
activities. Consequently, six undescribed
α-glucosidic hydroquinones
with linear monoterpenoids (1–6) with one known derivative (phlebo-
trichin, 7) (Takido et al., 1983), one monoterpenoid ((R)-menthiafolic
acid; 8) (Arslanian et al., 1990), one biflavonoid (amentoflavone; 9) (Li
et al., 2003), and one lignan ((ꢀ )-epipinoresinol; 10) (Shao et al., 2018)
were isolated (Fig. 2).
Phenolic compounds, hydroquinones, resveratrol, and tannins act as
substrates for the production of reactive quinones (Migas and
Compound 1 was obtained as a white powder and had the molecular
* Corresponding author. College of Pharmacy, Kangwon National University, Chuncheon, 24341, Republic of Korea.
E-mail address: heejyang@kangwon.ac.kr (H. Yang).
https://doi.org/10.1016/j.phytochem.2021.112782
Received 10 March 2021; Received in revised form 11 April 2021; Accepted 12 April 2021
Available online 27 April 2021
0031-9422/© 2021 Elsevier Ltd. All rights reserved.

J. Park et al.
Phytochemistry 187 (2021) 112782
d, J = 14.3 Hz, H-8′′)/113.9 (C-8′′) and 5.03 (2H, d, J = 16.2 Hz,
H-10′′)/116.9 (C-10′′)), two methylenes (2.43 (2H, dd, J = 7.3 and 14.5
Hz, H-4′′)/28.4 (C-4′′) and 2.37 (2H, dd, J = 6.8 and 7.9 Hz, H-5′′)/31.1
(C-5′′)), and one methyl (1.84 (3H, s, H-9′′)/12.6 (C-9′′)). The 13C NMR
signals at δC 169.4 (C-1′′), 146.8 (C-6′′), and 128.9 (C-2′′) suggested the
presence of one carboxylic and two olefinic quaternary carbons,
respectively. The COSY NMR data showed correlations between
H-3′′/H-4′′, H-4′′/H-5′′ and H-7′′/H-8′′, and the HMBC correlations from
H-3′′ to C-1′′ and C-9′′, H-4′′ to C-2′′, C-3′′, and C-6′′, H-5′′ to C-3′′, C-6′′,
C-7′′, and C-10′′, H-7′′ to C-5′′, C-6′′, and C-10′′, H-8′′ to C-6′′ and C-7′′,
H-9′′ to C-1′′, C-2′′, and C-3′′, and H-10′′ to C-5′′ and C-7′′ suggested the
presence of 2-methyl-6-methylene-2,7-octadienoate. Finally, the struc-
ture of compound 1 was completed based on the HMBC correlation
between H-6 of the β-arbutin moiety and C-1′′ of the 2-methyl-6-methy-
lene-2,7-octadienoyl group. Consequently, the structure of compound 1
was identified as 6-O-(2-methyl-6-methylene-2,7-octadienoyl)-arbutin.
Compound 2 was obtained as a white powder, and its molecular
formula was determined to be C22H30O9 by HRESIMS analysis (m/z
437.1834 [M-H]- calcd for 437.1812). The 1D and 2D NMR data of 2
showed similar patterns to those of 1, except for the signals of the linear
monoterpenoid moiety that was attached to C-6 of the glucopyranosyl
group. The 1D and HSQC NMR data showed the presence of two olefinic
groups (δH/δC 6.79 (1H, td, J = 1.4 and 7.3 Hz, H-3′′)/143.7 (C-3′′) and
5.39 (1H, t, J = 6.1 Hz, H-7′′)/125.7 (C-7′′)), one oxymethylene (4.08
(2H, d, J = 6.7 Hz, H-8′′)/59.4 (C-8′′)), two methylenes (2.36 (2H, dd, J
= 7.4 and 14.9 Hz, H-4′′)/28.0 (C-4′′) and 2.16 (2H, t, J = 7.6 and 7.6 Hz,
H-5′′)/39.1 (C-5′′)), and two methyl groups (1.85 (3H, s, H-9′′)/12.5 (C-
9′′) and 1.70 (3H, s, H-10′′)/16.2 (C-10′′)). The 13C NMR signals at δ
C
169.4 (C-1′′), 138.5 (C-6′′), and 128.9 (C-2′′) indicated the presence of
one carboxylic and two olefinic quaternary carbons, respectively. The
COSY NMR correlations between H-3′′/H-4′′, H-4′′/H-5′′, and H-7′′/H-
8′′, and the HMBC correlations from H-3′′ to C-1′′ and C-9′′, H-4′′ to C-2′′
and C-6′′, H-5′′ to C-3′′, C-6′′, C-7′′, and C-10′′, H-7′′ to C-5′′ and C-10′′, H-
8′′ to C-6′′ and C-7′′, H-9′′ to C-1′′, C-2′′, and C-3′′, and H-10′′ to C-6′′ and
C-7′′ suggested the presence of a 2,6-dimethyl-8-hydroxy-2,6-octadie-
noyl group. The location of this group was confirmed by the HMBC
correlation from H-6 to C-1′′, and 2 was identified as 6-O-(2,6-dimethyl-
8-hydroxy-2,6-octadienoyl)-arbutin.
Compound 3 was obtained as a white powder with a molecular for-
mula of C23H32O9, determined based on the HRESIMS data (m/z
451.1975 [M-H]- calcd for 451.1968). The 1D and 2D NMR data showed
similar patterns to those of 2, except for the presence of a methoxy group
at C-8′′ instead of a hydroxyl group. Significant changes in the chemical
shifts of C-6′′ (δC 140.8, +2.3 ppm), C-7′′ (δC 122.6, ꢀ 3.1 ppm), and C-8′′
(δ 69.7, +10.3 ppm) were observed compared to those of 2. The posi-
C
Fig. 1. Cytotoxic (A) and tyrosinase inhibition activities (B) of eight EtOAc sub-
fractions; VEE1–8 (100 mg/ml).
tion of the methoxy group at C-8′′ was confirmed by the HMBC corre-
lation between δ 3.28 (3H, s, H-11′′) and δ 69.7 (C-8′′). As a result, the
substituent attacHhed to the aglycone was deCtermined to be 2,6-dimethyl-
8-methoxy-2,6-octadienoate. Finally, the structure of compound 3 was
identified as 6-O-(2,6-dimethyl-8-methoxy-2,6-octadienoyl)-arbutin.
Compound 4 was isolated as a white powder and had the molecular
formula of C23H32O9 (HRESIMS; m/z 451.1975 [M-H]- calcd for
451.1968). Compound 4 was identified as a derivative of 7 with the
substitution of a methoxy group at C-6′′, which was confirmed by 1D and
formula, C22H28O8, based on HRESIMS analysis (m/z 419.1690 [M-H]-
calcd for 419.1706). The 1D and HSQC NMR data of 1 exhibited char-
acteristic signals at δH/δC 4.71 (1H, d, J = 7.4 Hz, H-1)/103.6 (C-1), 4.51
(1H, dd, J = 2.1 and 11.8 Hz, H-6)/65.1 (C-6), 4.24 (1H, dd, J = 7.2 and
11.8 Hz, H-6)/65.1 (C-6), 3.62 (1H, ddd, J = 2.1, 7.2 and 9.5 Hz, H-5)/
75.4 (C-5), 3.43 (H, m, H-3)/77.9 (C-3), 3.43 (H, m, H-2)/74.9 (C-2),
and 3.36 (1H, dd, J = 6.5 and 11.7 Hz, H-4)/71.9 (C-4) for the β-D-
glucosyl moiety, as well as 153.9 (C-4′), 152.3 (C-1′), 6.93 (2H, d, J =
8.9 Hz, H-2′ and 6′)/119.5 (C-2′, 6′) and 6.66 (2H, d, J = 8.9 Hz, H-3′ and
5′)/116.6 (C-3′ and 5′) for the hydroquinone group, with two hydroxyl
groups at the para position. The HMBC NMR data showed a correlation
between δH 4.71 (1H, d, J = 7.4 Hz, H-1) and δC 152.3 (C-1′), estab-
lishing the linkage of β-D-glucosyl and hydroquinone groups, and
confirmed that the compound 1 was a derivative of β-arbutin (Paw-
lowska et al., 2006). The 1D and HSQC NMR data also exhibited the
presence of two olefinic groups (δH/δC 6.82 (1H, td, J = 1.4 and 7.3 Hz,
H-3′′)/143.7 (C-3′′) and 6.40 (1H, dd, J = 10.9 and 17.7 Hz, H-7′′)/139.7
(C-7′′)), two exomethylenes (5.28 (1H, d, J = 14.3 Hz, H-8′′)/5.09 (1H,
2D NMR data. The chemical shifts of C-5′′ (δ 39.2, ꢀ 2.5 ppm), C-6′′ (δ
C
78.5, +4.8 ppm), C-7′′ (δC 144.3, ꢀ 1.6 ppm),CC-8′′ (δC 115.8, +3.3 ppm),
and C-10′′ (δ 21.9, ꢀ 6.0 ppm) confirmed the modification of C-6′′. The
C
HMBC correlation between δH 3.17 (3H, s, H-11′′) and δC 78.5 (C-6′′)
verified the location of the methoxy group. The absolute configuration
of C-6′′ in compound 4 was confirmed by the acid hydrolysis of 4 and 7.
The hydrolytic products of 4 and 7 had the same Rf values in the thin
layer chromatography (TLC) analysis (Fig. S37). The substituent
attached to the aglycone, β-arbutin, was determined to be (6S)-2,6-
dimethyl-6-methoxy-2,7-octadienoate. Consequently, 4 was identified
as 6-O-[(6S)-2,6-dimethyl-6-methoxy-2,7-octadienoyl]-arbutin.
Compound 5 was obtained as a white powder with a molecular
2

J. Park et al.
Phytochemistry 187 (2021) 112782
formula of C22H32O11, based on HRESIMS analysis (m/z 471.1868 [M-
H]- calcd for 471.1866). The 1D and 2D NMR data showed similar
patterns to those of 7, except for the signals of the linear monoterpenoid
moiety attached to C-6 of the glucopyranosyl group. The 1D and HSQC
NMR data indicated the presence of one olefinic group (δH/δC 6.83 (1H,
td, J = 1.1, and 7.4 Hz, H-3′′)/144.7 (C-3′′)), one oxymethine (3.50 (H,
dd, J = 3.2, and 7.9 Hz, H-7′′)/78.2 (C-7′′)), one oxymethylene (3.80 (H,
m, H-8′′) and 3.55 (H, m, H-8′′)/64.0 (C-8′′)), two methylenes (2.34 (2H,
m, H-4′′)/23.7 (C-4′′) and 1.70 (H, m, H-5′′)/1.54 (H, m, H-5′′)/38.6 (C-
5′′)), and two methyl groups (1.87 (3H, s, H-9′′)/12.5 (C-9′′) and 1.16
(3H, s, H-10′′)/22.1 (C-10′′)). The 13C NMR signals at δC 169.5 (C-1′′),
128.5 (C-2′′), and 74.6 (C-6′′) suggested the presence of one carboxylic
and two olefinic quaternary carbons, respectively. The HMBC correla-
tions from H-7′′ to C-6′′, C-8′′, and C-10′′ and H-10′′ to C-5′′, C-6′′, and C-
7′′ suggested the presence of the 2,6-methyl-6,7,8-trihydroxy-2-octae-
noyl group. The location of the derivative was confirmed by the
HMBC correlation from H-6 to C-1′′. Subsequently, compound 5 was
identified as 6-O-(2,6-methyl-6,7,8-trihydroxy-2-octaenoyl)-arbutin.
Compound 6 had the same molecular formula as 5 (C22H32O11 (m/z
471.1868 [M-H]- calcd for 471.1866)) as well as similar 1D and 2D NMR
data, except for the splitting pattern of H-4′′, H-5′′, and H-8′′ of the linear
8-trihydroxy-2-octaenoyl]-arbutin and 6-O-[(6S, 7S)-2,6-methyl-6,7,
8-trihydroxy-2-octaenoyl]-arbutin, respectively.
β-arbutin is the potent phenolic compound having the inhibitory
activity against tyrosinase enzyme and formulated as the anti-
melanogenic activity in the cosmetic industry (Migas and
Krauze-Baranowska, 2015). In the present study, compounds 1–7, de-
rivatives of β-arbutin, were expected to show tyrosinase inhibitory ac-
tivity owing to the presence of a glycosylated hydroquinone moiety, but
these did not show potent inhibition of tyrosinase (Fig. 4). It was spec-
ulated that the long chain of the monoterpenoid moiety could hinder the
binding of tyrosinase enzymes to the compounds. Interestingly, com-
pound 10, a lignan that was unambiguously identified as (ꢀ )-epipinor-
esinol based on the chemical shifts of the furofuran ring and negative
optical rotation, was isolated from the VEE2-3 fraction that showed the
highest tyrosinase inhibition activity (Shao et al., 2018). These results
are consistent with those reported in prior literature (Azhar-ul-Haq
et al., 2006).
3. Experimental
3.1. General experimental procedures
monoterpenoid moiety. Small changes in the chemical shifts of C-4′′ (δ
C
24.0, +0.3 ppm), C-5′′ (δC 37.9, ꢀ 0.7 ppm), and C-7′′ (δC 78.5, +0.3
ppm) in the 13C NMR data as well as H-5′′ (δH 1.57, +0.03 ppm), H-5′′
(δH 1.67, ꢀ 0.03 ppm), and H-8′′ (δH 3.75, ꢀ 0.05 ppm) in the 1H NMR
data suggested that 5 and 6 were stereoisomers with different stereo-
chemical configurations of the two hydroxyl groups at C-7 and C-8.
The absolute configuration of the hydroxyl moiety at C-7 of com-
pounds 5 and 6 was determined by the modified Mosher ester method
1D and 2D NMR data were obtained at 600 MHz using a Bruker
Avance Neo 600 (Brucker, Germany) spectrometer located at the Central
Laboratory of the Kangwon National University (Chuncheon, Korea).
Normal-phase silica gel Kieselgel 60 (40–60
μ
m, 230–400 mesh, Merck,
m, GE Healthcare, Swe-
Germany) and Sephadex LH-20 gel (18–111
μ
den) were used for column chromatography (CC). Fractions were
monitored by TLC using precoated silica gel plates (Kieselgel 60 F254
(Hoye et al., 2007). The ΔδSR (δ ꢀ δ ) values of H-4, H-5, and H-7 in 5
Merck, Germany) and reversed phase (RP)-C18 plates (Kieselgel 60 F254s,
S
and 6 showed similar values, indicatiRng that the hydroxyl moiety at C-7
in both compounds had the S-configuration (Fig. 3). The absolute
configuration of the hydroxyl moiety at C-6 was determined based on
the helicity rule using the electronic circular dichroism (ECD) data after
derivatization with [Mo2(OAc)4] because of the difference in the bulk-
iness of the two substituents around the hydroxyl moiety at C-6 (Snatzke
et al., 1981). The ECD measurements after complexation with
[Mo2(OAc)4] showed that compound 5 contained an erythro-2,3-diol
based on the negative Cotton effect in the band II region (418 nm,
ꢀ 0.35 mdeg), but 6 did not show significant changes (Fig. 3). These
results suggested that the absolute configurations of the hydroxyl moiety
at C-6 in 5 and 6 were opposite to those of 6-O-[(6S, 7R)-2,6-methyl-6,7,
Merck, Germany). The TLC spots were detected using 254- and 356-nm
UV light or H2SO4–EtOH (v/v) spray followed by heating. Preparative
high-performance liquid chromatography (HPLC) was performed using
an Empower Pro system equipped with a Waters 996 photodiode array
detector (Waters, Germany) using a HECTOR C18 column (5
μm, 21.2 ×
250 mm) and YMC-Actus Triart C18 column (s-5
μm, 12 nm, 20 × 250
mm) at a flow rate of 50 mL/min with monitoring at 210 nm. High-
purity solvents for the extraction, fractionation, and separation were
purchased from DaeJung (Sicheung, Korea).
Fig. 2. Chemical structures of compounds 1-10.
3

J. Park et al.
Phytochemistry 187 (2021) 112782
Fig. 3. ΔδSR (δS ꢀ δR) values (ppm, CDCl3) of the MTPA esters of 5 and 6 (A), and comparison of the ECD spectra of 5, 6, and their molybdenum derivatives in
DMSO (B).
National University, and identified by Prof. Yong Soo Kwon at the Col-
lege of Pharmacy, Kangwon National University.
3.3. Extraction and isolation
V. erosum leaves (2 kg) were extracted three times using 80% MeOH
with ultrasonic extraction for 3 h. The crude extract (365 g) was dis-
solved in H2O and partitioned successively using n-hexane (0.5 g),
EtOAc (66 g), and n-BuOH (123 g). A portion of EtOAc fraction (20 g)
was subjected to RP C18 medium-pressure liquid chromatography
(MPLC) using MeOH:H2O (1:1 to 1:0) solvent system to afford eight sub-
fractions (VEE1–VEE8). The fraction VEE2 (9.13 g) was separated by
silica gel MPLC using CH2Cl2:MeOH (9:1 to 3:7) to yield nine fractions.
Sub-fraction VEE2-2 (268 mg) was separated into seven sub-fractions
(VEE2-2-1–VEE2-2-7) by RP C18 MPLC using MeOH:H2O (1:1 to 1:0).
Compound 10 (10.3 mg) was purified from VEE2-2-5 (42 mg) by pre-
parative HPLC using a gradient solvent system of MeOH:H2O with 0.1%
formic acid (FA) (1:1, 5.0 mL/min). Fraction VEE2-4 (836 mg) was
further separated into four sub-fractions (VEE2-4-1–VEE2-6-4) by
Fig. 4. Tyrosinase inhibitory activities of compounds 1–10, isolated from
Sephadex LH-20 CC using MeOH. Compound 8 (153 mg) was purified
V. erosum. β-arbutin is used as the positive control.
from VEE2-4-1 (600 mg) with RP C18 MPLC using a gradient solvent
system of MeOH:H2O (1:1 to 1:0). Fraction VEE2-6 (2.82 g) was further
3.2. Plant material
separated into 14 sub-fractions (VEE2-6-1–VEE2-6-14) via Sephadex
LH-20 CC using MeOH. VEE2-6-4 (1.36 g) was subjected to RP C18 MPLC
Viburnum erosum Thunb. (Caprifoliaceae) leaves were collected from
using MeOH:H2O (1:1 to 1:0) to afford 12 sub-fractions (VEE2-6-4-
the Medicinal Plant Garden, Seoul National University, Korea, in
1–VEE2-6-4-12). Compounds 5 (6.3 mg) and 6 (5.2 mg) were purified
September 2004. (N37◦71′23.2′′, E126◦81′88.8′′) The herbarium
from VEE2-6-4-2 (19.4 mg) by preparative HPLC using a gradient sol-
(KNUVE-01) was deposited at the College of Pharmacy, Kangwon
vent system of MeOH:H2O with 0.1% FA (35:65, 5.0 mL/min).
4

J. Park et al.
Phytochemistry 187 (2021) 112782
Compound 7 (114 mg) was purified from VEE2-6-4-4 (340.6 mg) by
preparative HPLC using a gradient solvent system of MeOH:H2O with
0.1% FA (60:40, 5.0 mL/min). Compounds 1 (11.7 mg), 3 (19.4 mg), and
4 (11.1 mg) were purified from VEE2-6-4-7 (267 mg) by preparative
HPLC using a gradient solvent system of MeOH:H2O with 0.1% FA
(65:35, 10.0 mL/min). Sub-fraction VEE2-6-4-9 (89.9 mg) was further
separated with silica gel CC using CH2Cl2:MeOH (9:1 to 1:1). Compound
2 (0.9 mg) was purified from fraction VEE2-6-4-9-5 (3.2 mg) by pre-
parative HPLC using MeOH–H2O with 0.1% FA (72:28, 5.0 mL/min).
Compound 9 (198 mg) was recrystallized from VEE3.
Table 1
NMR spectroscopic data (methanol-d4, 150 MHz for 13C and 600 MHz for 1H
NMR) for compounds 1–6.
position
δC,
δH (J in
δC,
δH (J in
δC,
δH (J in
type
Hz)
type
Hz)
type
Hz)
1
2
3
1
2
3
4
103.6,
CH
4.71
103.6,
CH
4.71
103.5,
CH
4.71
d (7.4)
3.43 m
d (7.4)
3.43 m
d (7.4)
3.42 m
74.9,
CH
74.9,
CH
74.9,
CH
77.9,
CH
3.43 m
77.9,
CH
3.43 m
77.9,
CH
3.42 m
3.4. Structural characterization of undescribed compounds
71.9,
CH
3.36 dd
71.9,
CH
3.36 dd
(6.5,
71.9,
CH
3.35 t
(8.7)
(6.5, 11.7)
11.7)
3.4.1. 6-O-(2-methyl-6-methylene-2,7-octadienoyl)-arbutin (1)
25
D ꢀ 5.5 (c 0.001 MeOH); 1H and 13
C
5
6
75.4,
CH
3.62 ddd
(2.1, 7.2,
9.5)
75.4,
CH
3.62 m
75.4,
CH
3.62 ddd
(2.1, 5.8,
7.3)
white, amorphous powder; [
α]
NMR data, see Table 1; HRESIMS m/z 419.1690 [M ꢀ H]ꢀ (calcd for
C22H28O8 419.1706).
65.1,
CH2
4.24 dd
(7.2, 11.8),
65.0,
CH2
4.23 dd
(7.2,
65.1,
CH2
4.22 dd
(7.3,
11.8)
11.8),
4.51 dd
(2.2,
3.4.2. 6-O-(2,6-dimethyl-8-hydroxy-2,6-octadienoyl)-arbutin (2)
25
D ꢀ 14.3 (c 0.001 MeOH); 1H and 13
C
4.51 dd
4.51 dd
(1.9,
white, amorphous powder; [
α
]
(2.1, 11.8)
NMR data, see Table 1; HRESIMS m/z 437.1834 [M ꢀ H]ꢀ (calcd for
11.8)
11.8)
1′
152.3,
C
152.2,
C
152.2,
C
C
22H30O9 437.1812).
2′
119.5,
CH
6.93
119.5,
CH
6.93
119.5,
CH
6.93
3.4.3. 6-O-(2,6-dimethyl-8-methoxy-2,6-octadienoyl)-arbutin (3)
d (8.9)
6.66
d (8.9)
6.66
d (8.9)
6.66
25
white, amorphous powder; [
α]
D ꢀ 6.4 (c 0.001 MeOH); 1H and 13
C
3′
116.6,
CH
116.6,
CH
116.6,
CH
NMR, see Table 1; HRESIMS m/z 451.1975 [M ꢀ H]ꢀ (calcd for
d (8.9)
d (8.9)
d (8.9)
4′
153.9,
C
153.9,
C
153.9,
C
C
23H32O9 451.1968).
5′
116.6,
CH
6.66
116.6,
CH
6.66
116.6,
CH
6.66
3.4.4. 6-O-[(6S)-2,6-dimethyl-6-methoxy-2,7-octadienoyl]-arbutin (4)
d (8.9)
6.93
d (8.9)
6.93
d (8.9)
6.93
25
white, amorphous powder; [
α
]
D ꢀ 13.0 (c 0.001 MeOH); 1H and 13
C
6′
119.5,
CH
119.5,
CH
119.5,
CH
NMR, see Table 1; HRESIMS m/z 451.1975 [M ꢀ H]ꢀ (calcd for
d (8.9)
d (8.9)
d (8.9)
1′′
2′′
3′′
4′′
169.4,
C
169.4,
C
169.3,
C
C
23H32O9 451.1968).
128.9,
C
128.9,
C
128.8,
C
3.4.5. 6-O-[(6S, 7R)-2,6-methyl-6,7,8-trihydroxy-2-octaenoyl]-arbutin
(5)
143.7,
CH
6.82 td
143.7,
CH
6.79 td
(1.2, 7.2)
2.36 dd
(7.4,
143.6,
CH
6.78 td
(1.3, 7.3)
2.37 m
25
white, amorphous powder; [
α
]
D ꢀ 17.8 (c 0.001 MeOH); 1H and 13
C
(1.4, 7.3)
2.43 dd
NMR, see Table 1; HRESIMS m/z 471.1868 [M ꢀ H]ꢀ (calcd for
28.4,
CH2
28.0,
CH2
27.9,
CH2
(7.3, 14.5)
C
22H32O11 471.1866).
14.9)
5′′
31.1,
CH2
2.37 dd
39.1,
CH2
2.16 t
(7.6)
39.1,
CH2
2.21 dt
(7.4,
3.4.6. 6-O-[(6S, 7S)-2,6-methyl-6,7,8-trihydroxy-2-octaenoyl]-arbutin
(6.8, 7.9)
(6)
16.8)
6′′
7′′
146.8,
C
138.5,
C
140.8,
C
25
white, amorphous powder; [
α]
D ꢀ 9.4 (c 0.001 MeOH); 1H and 13
C
NMR, see Table 1; HRESIMS m/z 471.1868 [M ꢀ H]ꢀ (calcd for
139.7,
CH
6.4 dd
(10.9,
17.7)
125.7,
CH
5.39 t
(6.1)
122.6,
CH
5.34 m
C
22H32O11 471.1866).
8′′
113.9,
CH2
5.09
59.4,
CH2
4.08
69.7,
CH2
3.94
3.5. Tyrosinase inhibition assay
d (14.3),
5.28
d (6.7)
d (6.7)
Tyrosinase inhibitory activities of the fractions of V. erosum and
compounds 1–10 were determined using previously described methods
d (14.3)
1.84 s
9′′
12.6,
CH3
12.5,
CH3
16.2,
CH3
1.85 s
1.70 s
12.5,
CH3
16.4,
CH3
57.8,
CH3
1.85 s
1.71 s
3.28 s
with slight modifications (Zhai et al., 2020). First, 220 μL of phosphate
10′′
11′′
116.9,
CH2
5.03
buffer (0.1 M, pH 6.5), 20
(dissolved in ethanol), and 20
U/mL) were placed in the wells of a 96-well microplate. After incubation
for 10 min at 37 ◦C, 40
L of L-tyrosine (1.5 mM) was added to the re-
action mixture (300 L). Immediately, the absorbance was measured at a
μ
L of inhibitors with different concentrations
d (16.2)
μ
L of mushroom tyrosinase solution (2000
μ
4
5
6
μ
1
2
3
4
103.5,
CH
4.71
103.5,
CH
4.71
103.5,
CH
4.71
wavelength of 490 nm using a microplate reader, and after reacting for
17 min at 37 ◦C, the absorbance was measured again. The blank group, i.
e., the same volume of ethanol without the inhibitor was used, and
β-arbutin was used as the positive control. All experiments were per-
formed in triplicate for each compound. The relative tyrosinase activity
was calculated using the following expression: tyrosinase activity (%):
[(A1-A0)/(B1–B0)] × 100, where A0 is the absorbance of the sample
before the reaction and A1 is the absorbance of the sample after the
reaction. B0 and B1 are the absorbances before and after the reaction of
the blank group, respectively. The inhibitory effects of these compounds
on mushroom tyrosinase were expressed by the activity of the remaining
d (7.4)
3.43 m
d (7.4)
3.43 m
d (7.4)
3.43 m
74.9,
CH
74.9,
CH
74.9,
CH
77.9,
CH
3.43 m
3.34 m
77.9,
CH
3.43 m
77.9,
CH
3.43 m
3.36 m
71.9,
CH
71.9,
CH
3.35 dd
(6.5,
71.9,
CH
11.8)
5
75.4,
CH
3.62 ddd
(2.1, 7.3,
9.6)
75.4,
CH
3.63 ddd
(1.9, 7.5,
9.6)
75.4,
CH
3.63 ddd
(continued on next page)
5

J. Park et al.
Phytochemistry 187 (2021) 112782
3.7. Acid hydrolysis
Table 1 (continued)
position
δC,
δH (J in
δC,
δH (J in
δC,
δH (J in
The absolute configuration of the monoterpenoid moiety in 4 was
determined using a slightly modified literature procedure. Compounds 4
and 7 (1.0 mg each) were dissolved in 1 mL of 2 M HCl (H2O/1,4-
dioxane, 1:1, v/v), and each solution was heated at 90 ◦C for 4 h. After
cooling the solution, it was evaporated under N2, re-dissolved in water,
and extracted with n-hexane. The monoterpenoids from which the
monosaccharides were removed were extracted in n-hexane, and chro-
matographed on TLC plate.
type
Hz)
type
Hz)
type
Hz)
6
65.1,
CH2
4.22 dd
65.1,
CH2
4.21 dd
(7.4,
65.1,
CH2
4.22 dd
(7.4,
(7.3, 11.8),
11.8)
11.8)
4.51 dd
4.52 dd
(2.1,
4.52 dd
(1.1,
(2.0, 11.8)
11.8)
11.8)
1′
152.3,
C
152.3,
C
152.3,
C
2′
119.5,
CH
6.93
119.5,
CH
6.93
119.5,
CH
6.93
d (8.9)
6.66
d (8.9)
6.68
d (8.9)
6.68
3′
116.6,
CH
116.6,
CH
116.6,
CH
3.8. Preparation of MTPA esters of compounds 5 and 6
d (8.9)
d (8.9)
d (8.9)
4′
153.9,
C
153.9,
C
153.9,
C
(R)-(ꢀ )-
(3.3 L) and dried pyridine (3.3
(0.5 mg) in anhydrous CDCl3 (100
α
-methoxy-
α
-trifluoromethylphenylacetyl chloride (MTPACl)
L) were dissolved in a solution of 5 (or 6)
L). The mixtures were sealed and kept
μ
μ
5′
116.6,
CH
6.66
116.6,
CH
6.68
116.6,
CH
6.68
μ
d (8.9)
6.93
d (8.9)
6.93
d (8.9)
6.93
6′
119.5,
CH
119.5,
CH
119.5,
CH
overnight at room temperature. A capillary column was used to purify the
product with n-hexane:EtOAc (50:50, v/v) solution to obtain the pure (S)-
MTPA ester of 5 (or 6). (R)-MTPA esters were prepared in a similar manner
using (S)-MTPACl.
d (8.9)
d (8.9)
d (8.9)
1′′
2′′
3′′
4′′
169.4,
C
169.5,
C
169.5,
C
128.5,
C
128.5,
C
128.5,
C
143.5,
CH
6.80 td
(1.3, 7.6)
2.22 dt
(10.4,
144.7,
CH
6.83 td
(1.1, 7.4)
2.34 m
144.5,
CH
6.82 td
(1.3, 7.4)
2.33 dd
(7.9,
3.9. Preparation of Mo2-complexes of compounds 5 and 6
24.2,
CH2
23.7,
CH2
24.0,
CH2
The ECD spectra were recorded at room temperature in DMSO with
1.0 nm/step scans using a 10-mm cell at 200–600 nm according to
Snatzke’s method. To form complexes, compound 5 (or 6) was dissolved
in a solution of [Mo2(OAc)4] in DMSO at a 1:1 ratio of the molybdenum
complex to diol.
20.9)
16.4)
5′′
39.2,
CH2
1.63 m
38.6,
CH2
1.54 m
1.70 m
37.9,
CH2
1.57 m
1.67 ddd
(6.9,
10.4,
18.7)
6′′
7′′
78.5, C
144.3,
CH
74.6, C
78.2,
CH
74.7, C
78.5,
CH
Declaration of competing interest
5.80 ddd
(1.3, 11.0,
17.6)
3.50 dd
3.49 dd
(3.2, 7.9)
(3.3, 7.7)
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
8′′
115.8,
CH2
5.21 m
64.0,
CH2
3.55 m
63.9,
CH2
3.55 dd
(7.8,
11.2)
3.80 m
3.75 ddd
(1.8, 3.3,
11.9)
Acknowledgements
9′′
12.4,
CH3
21.9,
CH3
50.4,
CH3
1.83 s
1.28 s
3.17 s
12.5,
CH3
22.1,
CH3
1.87 s
1.16 s
12.5,
CH3
22.8,
CH3
1.87 s
This work was supported by the Basic Science Research Program
through the National Research Foundation of Korea (NRF) grant funded
by the Korean government (MEST) (NRF- 2021R1C1C1011857).
10′′
11′′
1.19 s
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2021.112782.
enzyme.
3.6. Cell Culture B16F10 murine melanoma, HEp-2 laryngeal carci-
noma, and PC3 prostate cancer cell lines were purchased from the
American Type Culture Collection (ATCC). The cells were cultured in
RPMI 1640 and DMEM containing 10% fetal bovine serum and 1%
penicillin/streptomycin under 5% CO2 at 37 ◦C.
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