Four new sucrose diesters of substituted truxinic acids from Trigonostemon honbaensis with their anoctamin-1 inhibitory activity

Article abstract of DOI:10.1016/j.bioorg.2020.104058

Truxinic acid sucrose diesters analogs possess interesting chemical structure by the presence of cyclobutane-ring and macrocyclic sucrose diesters moieties which are rarely found from natural sources. This paper describes the isolation and structural elucidation of four new sucrose diesters of substituted truxinic acids, trigohonbanosides A-D (1–4), from the leaves of Trigonostemon honbaensis. Their chemical structures were elucidated by HR-ESI-MS, NMR, and CD spectroscopic methods. At a concentration of 30 μM, compounds 1–4 moderately inhibited ANO-1 activity with inhibitory percentages of 27.7 ± 1.10percent, 35.6 ± 0.92percent, 43.7 ± 1.61percent, and 40.8 ± 1.25percent, respectively.

Full text of DOI:10.1016/j.bioorg.2020.104058

Journal Pre-proofs
Short communication
Four new sucrose diesters of substituted truxinic acids from Trigonostemon
honbaensis with their anoctamin-1 inhibitory activity
Ninh Khac Ban, Luu Hong Truong, Tran Van Tiep, Duong Thi Hai Yen, Vu
Van Doan, Nguyen Xuan Nhiem, Yohan Seo, Wan Namkung, Seung Hyun
Kim, Bui Huu Tai, Phan Van Kiem
PII:
S0045-2068(20)31355-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.104058
YBIOO 104058
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
20 April 2020
8 June 2020
26 June 2020
Please cite this article as: N. Khac Ban, L. Hong Truong, T. Van Tiep, D. Thi Hai Yen, V. Van Doan, N. Xuan
Nhiem, Y. Seo, W. Namkung, S. Hyun Kim, B. Huu Tai, P. Van Kiem, Four new sucrose diesters of substituted
truxinic acids from Trigonostemon honbaensis with their anoctamin-1 inhibitory activity, Bioorganic Chemistry
(
2020), doi: https://doi.org/10.1016/j.bioorg.2020.104058
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Four new sucrose diesters of substituted truxinic acids from Trigonostemon
honbaensis with their anoctamin-1 inhibitory activity
a,b
c
d
a
a
Ninh Khac Ban , Luu Hong Truong , Tran Van Tiep , Duong Thi Hai Yen , Vu Van Doan ,
Nguyen Xuan Nhiem , Yohan Seo , Wan Namkung , Seung Hyun Kim , Bui Huu Taia,b,*, and
a,b
e
e
e
Phan Van Kiema,b,*
a)Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc
Viet, Cau Giay, Hanoi, Vietnam
b)Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
c)Southern Institute of Ecology, VAST, 01 Mac Dinh Chi Street, Ho Chi Minh City, Vietnam
d)Nui Chua National Park, Ninh Thuan Province, Vietnam
eCollege of Pharmacy, Yonsei Institute of Pharmaceutical Science, Yonsei University, Incheon 406-840,
Korea
*
Corresponding author:
Phan Van Kiem, Prof. Ph.D.
Bui Huu Tai, Ph.D.
E-mail: phankiem@yahoo.com
E-mail: bhtaiich@gmail.com
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology
8-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
1
1

Highlights

Four new sucrose diester of substituted truxinic acids (1-4) were isolated from
Trigonostemon honbaensis.


Their structures were established by HR-ESI-MS, NMR, and CD spectra.
Compounds 1-4 (30 µM) inhibited ANO-1 activity at levels of 27.7 ± 1.10%, 35.6 ±
0
.92%, 43.7 ± 1.61%, and 40.8 ± 1.25%, respectively.
2

Graphical abstract
3

Four new sucrose diester of substituted truxinic acids from Trigonostemon honbaensis with
their anoctamin-1 inhibitory activity
Ninh Khac Ban, Luu Hong Truong, Tran Van Tiep, Duong Thi Hai Yen, Vu Van Doan, Nguyen
Xuan Nhiem, Yohan Seo, Wan Namkung, Seung Hyun Kim, Bui Huu Tai, and Phan Van Kiem
All authors declared no conflict of interest
On the behaft of the authors,
Corresponding author:
Phan Van Kiem, Prof. Ph.D.
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18
Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
Phone : Tel.: +84-4-3791-7053 Fax: +84-4-3791-7054 E-mail : phankiem@yahoo.com
4

Abstract
Truxinic acid sucrose diesters analogs possess interesting chemical structure by the presence of
cyclobutane-ring and macrocyclic sucrose diesters moieties which are rarely found from natural
sources. This paper describes the isolation and structural elucidation of four new sucrose diesters
of substituted truxinic acids, trigohonbanosides A-D (1-4), from the leaves of Trigonostemon
honbaensis. Their chemical structures were elucidated by HR-ESI-MS, NMR, and CD
spectroscopic methods. At a concentration of 30 µM, compounds 1-4 moderately inhibited ANO-1
activity with inhibitory percentages of 27.7 ± 1.10 %, 35.6 ± 0.92 %, 43.7 ± 1.61 %, and 40.8 ±
1
.25%, respectively.
Keywords: Trigonostemon honbaensis, Trigohonbanoside, Truxinic acid sucrose diesters,
Anoctamin-1 inhibitor.
Introduction
The genus Trigonostemon Blume (Euphorbiaceae family) consists approximately 85 species which
are mainly distributed in tropical and subtropical regions of the Asia. Of these, Vietnam is
considered to be the center of diversity for this genus with 22 species. Recently, the T. honbaensis
Tagane & Yahara is recorded as a new and endemic Trigonostemon species, growing at Hon Ba
nature reserve of Vietnam [1]. Several parts of the plants from Trigonostemon genus have been
traditionally used as folk medicines for treatment of diarrhea, asthma, and skin diseases. Since
array of interesting diterpenoids, phenolics, steroids, indole and β-carboline alkaloids reviewed
from Trigonostemon genus, the chemical constituents of this plant species were intensively
investigated during the last decade [2, 3]. Over 10 plants in this genus have been phytochemically
5

investigated which resulted in isolation and chemical structural elucidation of over 200 compounds
3-7]. Isolated diterpenes (daphnane diterpenoid orthoesters), indole alkaloids (trigonoliimines)
[
and β-carboline alkaloids (trigonoines and trigonostemines) were performed total synthesis
because of their interesting chemical structure framework and/or valuable biological activities [8-
1
0]. Above mentioned literature prompt us to carry out the phytochemical investigation of the
Trigonostemon honbaensis species and herein, we report the isolation and structural elucidation of
four new sucrose diesters of substituted truxinic acids. Anoctamin-1 (ANO-1) inhibitory activity
of the isolated compounds was also evaluated using yellow fluorescent protein (YFP) reduction
assay. The truxinic acid sucrose diesters analogs are very rare from natural sources. To the best of
our knowledge, around five of those compounds have been isolated so far from Imperata
cylindrica, Coix lachryma-jobi, Bidens parviflora, and oat grains [11-14].
2
. Materials and Methods
2
.1. General experimental procedures
Optical rotations were obtained on a Jasco P2000 polarimeter. CD spectra were recorded on a
Chirascan spectrometer. HR-ESI-MS was performed on an Agilent 6530 Accurate Mass Q-TOF
LC/MS system. NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer.
Preparative HPLC was acquired on an Agilent 1100 HPLC system equipped with quaternary pump
(flow rate 3 mL/min), autosampler, DAD detector, and J'sphere ODS-H80 preparative HPLC
column (20×250 mm, particle size 4 µm). Open column chromatography was performed using
silica gel, reversed phase C18, sephadex LH-20, and diaion HP-20 as stationary phase. Thin layer
chromatography was carried out using pre-coated silica gel 60 F254 and RP-18 F254S plates.
2
.2. Plant material
The leaves of Trigonostemon honbaensis Tagane & Yahara (Euphorbiaceae family) were collected
at Nui Chua national park, Ninh Thuan province, Vietnam in December 2018. Its scientific name
6

was identified by one of the authors, Prof. Ninh Khac Ban. A voucher specimen (No. NCCT-P79)
was kept at the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology.
2
.3. Extraction and isolation
The dried powdered leaves of Trigonostemon honbaensis (4 kg) were extracted three times with
methanol (each 10 L) under sonication (each 60 min) at room temperature. After filtration, filtrated
methanol was evaporated under reduced pressure to yield 450 g dark residue. This methanol
residue was then suspended with distilled water (3.0 L) and partitioned in turn with
dichloromethane and ethyl acetate to give dichloromethane (80.7 g), ethyl acetate (4.1 g), and
water-soluble portions. The water layer was loaded into diaion HP-20 column, washed with water,
and eluted with methanol/water (1/3, 1/1, 3/1, 1/0, v/v, each 1.5 L) to give four fractions (THW1-
THW4). The fraction THW2 was chromatographed on reserved phase C18 (RP-18) column,
eluting with methanol/water (1/1, v/v, 2 L) to yield six fractions (THW2A- THW2F). The fraction
THW2D was then separated by sephadex LH-20 column, eluting with methanol/water (1/1, v/v, 1
L) to four fractions (THW2D1-THW2D4). Compound 1 (6.4 mg, t 36.6 min) was obtained by
R
purifying fraction THW2D4 by preparative-HPLC with isocratic mobile phase of acetonitrile
(20%) in water. The fraction THW2E was separated on sephadex LH-20 column, eluting with
methanol/water (1/1, v/v) to yield four fractions THW2E1-THW2E4. Preparative HPLC
purification of fraction W2E2 with isocratic mobile phase of acetonitrile (20%) in water gave
compound 2 (4.5 mg, t 43.9 min). Compounds 3 (5.0 mg, t 43.4 min), and 4 (4.3 mg, t 40.8
R
R
R
min) were obtained from THW2E4 fraction by preparative HPLC using isocratic mobile phase of
acetonitrile (20%) in water.
2
.3.1. Trigohonbanoside A (1)
7

2
D
5
Light yellow amorphous powder;



: +31.3° (c 0.1, MeOH); CD (MeOH) θ mdeg : −19.1(244);
(λ)
-
1
13
HR-ESI-MS: m/z 729.1824 [M+Cl] (calcd. for C H O Cl, 729.1798); H- and C-NMR data
3
2
38 17
were shown in the Table 1.
.3.2. Trigohonbanoside B (2)
Light yellow amorphous powder;
2
2
D
5



: +48.1° (c 0.1, MeOH); CD (MeOH) mdeg : +21.5(245);
(λ)
-
1
13
HR-ESI-MS: m/z 729.1798 [M+Cl] (calcd. for C H O Cl, 729.1798); H- and C-NMR data
3
2
38 17
were shown in the Table 1.
.3.3. Trigohonbanoside C (3)
Light yellow amorphous powder;
2
2
D
5



: +23.7° (c 0.1, MeOH); CD (MeOH) mdeg : +13.9(243);
(λ)
-
1
13
HR-ESI-MS: m/z 699.1697 [M+Cl] (calcd. for C H O Cl, 699.1692); H- and C-NMR data
3
1
36 16
were shown in the Table 1.
.3.4. Trigohonbanoside D (4)
Light yellow amorphous powder;
2
2
D
5



: +26.5° (c 0.1, MeOH); CD (MeOH) mdeg : +18.1(238);
(λ)
-
1
13
HR-ESI-MS: m/z 699.1710 [M+Cl] (calcd. for C H O Cl, 699.1692); H- and C-NMR data
3
1
36 16
were shown in the Table 1.
.4. Alkaline hydrolysis
2
Compounds 1-4 were each (0.5 mg) dissolved in 1.0 mL solution of KOH (1.0 M) in methanol and
heated at 60°C for 2 h. Reaction was cooled to room temperature and carefully neutralized with
solution of HCl 1.0 M. The solvent was then driven out under nitrogen flow. The residue was re-
dissolved in 1.0 mL of water and extracted twice with equal volume of ethyl acetate. Trace of
sucrose in water layer was confirm by co-TLC analysis in comparison with authentic sucrose
(
CHCl : MeOH : Water = 1.0 : 1.0 : 0.15, R = 0.36).
3
f
2
.5. Yellow fluorescent protein reduction assay
8

YFP reduction analysis was performed using FLUOstar Omega microplate reader and MARS Data
Analysis Software. Experiments were done as previously described [15]. In brief, anoctamin-1
channel and YFP expressing Fisher Rat Thyroid (FRT) cells were plated in black-walled 96-well
4
microplates at a density of 1.5×10 cells per well. Each well was washed twice (each 200 μL) with
phosphate buffered saline (PBS) and then 2 µL of compound solution was added to reach final
concentration of 30 µM. After incubation at 37℃ for 10 min, microplates were transferred to a
plate reader for fluorescence analysis. Each well was individually measured for ANO1-mediated
I− influx by monitoring YFP fluorescence continuously (400 ms per point) for 2 s (baseline). After
−
that, 100 µL of 140 mM I solution containing 200 μM ATP was added at 2 s and then YFP
fluorescence was recorded for 6 s. The initial iodide influx rate was determined from the initial
slope of fluorescence reduction by nonlinear regression after iodide injection with ATP.
3
. Result and Discussion
Dried leaves of T. honbaensis were extracted with methanol and roughly separated into three
polarity fractions. Low-range polarity fraction (dichloromethane extract, notably contained fatty
substances, essential oil, chlorophyll) and middle-range polarity fraction (ethyl acetate extract,
small amount obtained as mentioned in the Experiment) were not subjected for phytochemical
investigation. Water layer were selected for purification using chromatographic methods.
Chemical structures of isolated compounds were elucidated by HR-ESI-MS, NMR, and CD
spectroscopic methods to reveal four new compounds 1-4.
Compound 1 was obtained as light-yellow amorphous powder. The negative mode HR-ESI-MS
-
analysis of 1 exhibited a pseudo-molecular ion peak at m/z 729.1824 [M+Cl] , indicating the
1
molecular formula of C H O (calcd. for C H O Cl, 729.1798). The H-NMR spectrum of 1
3
2
38 17
32 38 17
displayed two set of ABX coupled spin systems [δ 6.34 (1H, d, J = 2.0 Hz); 6.60 (1H, d, J = 8.5
H
Hz), 6.48 (1H, dd, J = 2.0 and 8.5 Hz), 6.43 (1H, d, J = 2.0 Hz), 6.66 (1H, d, J = 8.5 Hz), 6.61 (1H,
9

dd, J = 2.0 and 8.5 Hz)], two methoxy groups [δ 3.61 and 3.63 (each, 3H, s)], and one anomeric
H
1
3
proton [δ 5.37 (1H, d, J = 3.5 Hz)]. The C-NMR spectrum of 1 showed 32 carbon signals, which
H
were assigned thanks to HSQC spectrum into 9 non-protonated carbons, 3 methylenes, 18 methines
and 2 methyl carbons. Twelve olefinic carbons (δ 113.1 ~ 148.7) and two methoxy carbons (δ
C
C
5
6.3 and 56.4) expected a pair of 3-methoxy-4-hydroxylphenyl groups. This deduction was
confirmed by HMBC correlations between H-2 (δ 6.43)/ H-6 (δ 6.61) and C-4 (δ 146.1), H-5
H
H
C
(
δ 6.66)/ 3-OCH (δ 3.63) and C-3 (δ 148.7), H-2′ (δ 6.34)/ H-6′ (δ 6.48) and C-4′ (δ 146.1),
H 3 H C H H C
H-5′ (δ 6.60)/ 3′-OCH (δ 3.61) and C-3′ (δ 148.5). Four aliphatic methines [δ 46.2 (C-7), 44.8
H
3
H
C
C
(
C-8), 45.2 (C-7′), 44.7 (C-8′)] and COSY cross peaks of H-7 (δ 4.20)/ H-8 (δ 3.88)/ H-8′ (δ
H H H
3
.97)/ H-7′ (δ 4.25)/ H-7 were attributed to a cyclobutane ring (Figure 2). Moreover, HMBC
H
correlations between H-7 (δ 4.20) and C-1 (δ 131.8)/ C-2 (δ 113.7)/ C-6 (δ 121.4), H-7′ (δ
H
H
C
C
C
4
.25) and C-1′ (δ 131.7)/ C-2′ (δ 113.1)/ C-6′ (δ 121.6), H-8 (δ 3.88)/ H-8′ (δ 3.97) and
C C C H H
carbonyl carbons C-9 (δ 174.5)/ C-9′ (δ 174.2) proved the presence of truxinic acid moiety.
C
C
Remaining twelve oxygenated carbons (δ 61.9 ~ 106.8) were assigned to a disaccharide moiety.
C
Moreover, resonant signals of an anomeric methine (δ 94.7/ δ 5.37) and non-protonated
C
H
anomeric carbon (δ 106.8) were expected for sucrose disaccharide as previously reported in the
C
literature [16, 17]. The presence of sucrose moiety was also suggested by the small coupling
constant of anomeric proton of glucose unit [δ 5.37 (d, J = 3.5 Hz, Glc H-1″), HMBC correlation
H
of Glc H-1″ (δ 5.37) / Fru C-2‴ (δ 106.8), and later by TLC analysis of alkaline hydrolysis
H
C
product of 1 in comparison with authentic sucrose [16, 18]. Assignments of sucrose backbone were
then elucidated by two set of individual COSY cross peaks Glc H-1″ (δ 5.37)/ Glc H-2″ (δ 4.43)/
H
H
Glc H-3″ (δ 3.74)/ Glc H-4″ (δ 3.13)/ Glc H-5″ (δ 4.05)/ Glc H-6″ (δ 3.83 and 4.85) and Fru
H
H
H
H
H-3‴ (δ 4.23)/ Fru H-4‴ (δ 4.03)/ Fru H-5‴ (δ 4.07)/ Fru H-6‴ (δ 4.22 and 4.33). Connection
H
H
H
H
1
0

between sucrose moiety and truxinic acid derivative was deduced by ester linkages at Glc C-6″
and Fru C-6‴ which supported by HMBC correlations of Glc H-6″ (δ 3.83 and 4.85)/ C-9 (δ
H
C
1
74.5) and Fru H-6‴ (δ 4.22 and 4.33)/ C-9′ (δ 174.2). Finally, stereogenic centers at truxinic
H C
acid backbone were elucidated by NOESY (Figure 3). The NOESY spectrum of 1 exhibited the
correlations between H-2 (δ 6.43)/ H-6 (δ 6.66) and H-8 (δ 3.88), H-2′ (δ 6.34)/ H-6′ (δ 6.48)
H
H
H
H
H
and H-8′ (δ 3.97), indicating trans-orientations between C-7 and C-8, C-7′ and C-8′, respectively.
H
The NOESY correlation between H-2 (δ 6.43) and H-8′ (δ 3.97) also indicated trans-orientation
H
H
between C-7 and C-8′. Thus, relative configurations at truxinic acid backbone were proposed to
be 7α,8β,7′α,8′β orientations. Consequently, structure of compound 1 was established as presented
in Figure 1 and was named as trigohonbanoside A.
The HR-ESI-MS analysis of compound 2 exhibited a pseudo-molecular ion peak at m/z 729.1798
-
[
M+Cl] , suggesting that compound 2 had the same molecular formula, C H O , with compound
3
2
38 17
1
1
. The H- NMR of 2 (Table 1) observed similar pattern with those of 1 such as a pair of ABX
coupled spin system [δ 6.42 (d, J = 1.5 Hz, H-2), 6.68 (d, J = 8.5 Hz, H-5), 6.69 (dd, J = 1.5 and
H
8
.5 Hz, H-6), 6.34 (d, J = 2.0 Hz, H-2′), 6.60 (d, J = 9.0 Hz, H-5′), 6.50 (dd, J = 2.0 and 9.0 Hz,
H-6′)], two methoxy groups [δ 3.62 (s, 3-OCH ) and 3.61 (s, 3′-OCH )], an anomeric proton [δ
H
H
3
3
5
7
.35 (d, J = 3.5 Hz, Glc H-1″ )], and four aliphatic protons [δ 4.29 (dd, J = 4.5 and 10.0 Hz, H-
H
), 3.89 (dd, J = 4.5 and 10.5 Hz, H-8), 4.24 (dd, J = 9.0, 10.0 Hz, H-7′), 4.10 (dd, J = 9.0 and 10.5
Hz, H-8′)]. The COSY cross peaks of H-7/H-8/H-8′/H-7′/H-7 revealed the presence of cyclobutan
ring. The HMBC correlations between H-2 (δ 6.42)/ H-6 (δ 6.69) and C-4 (δ 146.1), H-5 (δ
H
H
C
H
6
.68)/ 3-OCH (δ 3.62) and C-3 (δ 148.6), H-2′ (δ 6.34)/ H-6′ (δ 6.50) and C-4′ (δ 146.1),
3 H C H H C
H-5′ (δ 6.60)/ 3′-OCH (δ 3.61) and C-3′ (δ 148.5), H-7 (δ 4.29) and C-1 (δ 131.7)/ C-2 (δ
C
H
3
H
C
H
C
1
14.1)/ C-6 (δ 121.4), H-7′ (δ 4.24) and C-1′ (δ 131.6)/ C-2′ (δ 113.2)/ C-6′ (δ 121.6), H-8
C H C C C
1
1

(
δ 3.89)/ H-8′ (δ 4.10) and carbonyl carbons C-9/C-9′ (δ 174.2) indicated the presence of 4,4՛-
H
H
C
dihydroxy-3,3՛-dimethoxy-truxinic acid. An anomeric methine (δ 92.8 /δ 5.35), non-protonated
C
H
anomeric carbon (δ 105.6), and HMBC correlation of Glc H-1″ (δ 5.35)/ Fru C-2‴ (δ 105.6)
C
H
C
also suggested that sugar moiety of 2 also to be sucrose. The HMBC correlations between Glc H-
″ (δ 4.58 and 4.02)/C-9 (δ 174.2) and Fru H-6‴ (δ 4.56 and 4.36)/ C-9′ (δ 174.2) confirmed
6
H
C
H
C
sucrose moiety binding with 4,4′-dihydroxy-3,3′-dimethoxy-truxinic acid by ester linkages at Glc
C-6″ and Fru C-6‴. Interestingly, the NOESY spectrum of 2 observed correlations between H-2
(δ 6.42)/ H-6 (δ 6.69) and H-8 (δ 3.89), H-2′ (δ 6.34)/ H-6′ (δ 6.50) and H-8′ (δ 4.10), H-2
H H H H H H
(
δ 6.42) and H-8′ (δ 4.10) which are identical with that of 1. Above mentioned NMR evidence
H
H
indicated that truxinic acid moiety of 1 and 2 are a pair of enantiomers. This deduction was further
confirmed by opposite trend of Cotton effects of 2 (+21.5 at wavelength of 245 nm) compared to
1
(−19.1 at wavelength of 244 nm, Figure 4). Thus, configurations at truxinic acid backbone of
compound 2 were determined to be 7β,8α,7′β,8′α. The structure of 2 was then established and
named as trigohonbanoside B.
The molecular formula of 3 was deduced to be C H O based on the pseudo-molecular ion peak
3
1
36 16
-
1
at m/z 699.1697 [M+Cl] in the HR-ESI-MS (calcd. for C H O Cl, 699.1692). The H-NHR
3
1
36 16
spectrum of 3 exhibited an AA′BB′ spin system [δ 6.93 and 6.61 (each 2H, d, J = 8.5 Hz)] and an
H
ABX spin system [δ 6.31 (1H, d, J = 2.0 Hz), 6.57 (1H, d, J = 8.0 Hz), 6.45 (1H, dd, J = 8.0 and
H
2
.0 Hz)] instead of a pair of ABX spin systems as in the compounds 1 and 2. Careful comparison
1
13
H- and C-NMR spectral data of 3 with those of compounds 1-2 recognized that NMR data of 3
close similarity with compound 2 except signal of A-benzen ring (Table 1 and Figure 1).
Appearance of deshielded carbon signal [δ 156.9 (C-4)] together with HMBC correlations
C
1
2

between H-2/H-6 (δ 6.93) and C-4 indicated that A-benzene ring of 3 to be 4-hydroxyphenyl
H
group which was difference with 3-methoxy-4-hydroxyphenyl group in compound 2. NOESY
correlations between H-2/H-6 (δ 6.93) and H-8 (δ 3.85), H-2′ (δ 6.31)/ H-6′ (δ 6.45) and H-8′
H
H
H
H
(δH 4.11) indicated trans-orientations between C-7 and C-8, C-7′ and C-8′, respectively. The
NOESY correlation between H-2 (δ 6.93) and H-8′ (δ 4.11) also indicated trans-orientation
H
H
between C-7 and C-8′. Furthermore, CD spectrum of compound 3 (+13.9 at wavelength of 243
nm) showed similar Cotton effect with compound 2 (Figure 4), suggesting the identical stereogenic
configuration at truxinic acid moiety between compounds 3 and 2. Consequently, compound 3 was
determined and named as trigohonbanoside C.
The molecular formula of compound 4 was identified as C H O based on the HR-ESI-MS at
3
1
36 16
-
m/z 699.1710 [M+Cl] (calcd. for C H O Cl, 699.1692). The NMR spectral data of 4 also
31
36 16
indicated it to be a sucrose ester of substituted truxinic acid, showing signals of sucrose moiety
twelve oxygenated carbons at δ 63.7 ~ 105.7, one anomeric proton δ 5.35 (1H, d, J = 3.5 Hz)],
[
C
H
signals of cyclobutan ring [four aliphatic carbons (δ 45.9, 45.1, 44.4, 44.6) and their binding
C
protons (δ 4.29, 3.86, 4.23, 4.10)], twelve aromatic carbons (δ 114.0 ~ 157.0), and two carbonyl
H
C
carbons (δ 174.3 and 174.4). Like compound 3, signals of a de-shielded carbon (δ 157.0) and
C
C
AA′BB′ coupled protons [δ 6.78 and 6.56 (each, 2H, d, J = 8.5 Hz)] indicated the presence of 4-
H
hydroxyphenyl group (B-benzen ring). Due to overlapped signals between H-5 and H-6 (δ 6.64),
H
1
the pattern of A-benzene ring is hard to recognize in the H-NMR spectrum. However, the HMBC
correlations between H-2 (δ 6.40)/ H-6 (δ 6.64) and C-4 (δ 146.0), H-5 (δ 6.64)/ 3-OCH (δ
H
H
H
C
H
3
3
.62) and C-3 (δ 148.5), NOESY correlation between H-2 (δ 6.40) and 3-OCH (δ 3.62)
C H 3 H
confirmed the A benzene ring to be 3-methoxy-4-hydroxyphenyl group. NOESY correlations
between H-2 (δ 6.40)/H-6 (δ 6.64) and H-8 (δ 3.86), H-2′/H-6′ (δ 6.78) and H-8′ (δ 4.10)
H
H
H
H
H
1
3

indicated trans-orientations between C-7 and C-8, C-7′ and C-8′, respectively. The NOESY
correlation between H-2 (δ 6.40) and H-8′ (δ 4.10) also indicated trans-orientation between C-
H
H
7
and C-8′. Furthermore, CD spectrum of compound 4 (+18.1 at wavelength of 238 nm) showed
similar Cotton effect with compounds 2-3 (Figure 4), suggesting the identical stereogenic
configurations at truxinic acid moiety between compounds 2-4. Consequently, compound 4 was
determined and named as trigohonbanoside D.
Truxinic acid derivatives are reported naturally arising via head-to-head [2+2]-cyclodimerization
of cinnamic acid derivatives. Cyclodimeric products formed by making C-C bonds between C-7
and C-7′, C-8 and C-8′ [19]. Their chemical structures therefore can be considered as dimeric
derivatives of phenylpropanoid and also called as truxinate lignans by some authors [20].
Compounds 1 and 2 could be generated from cyclodimeric of two caffeic acid moieties meanwhile
compounds 3 and 4 were expected to form by [2+2]-cycloaddition between a caffeic acid moiety
and a coumaric acid moiety. Relative configurations at cyclobutane-ring of compounds 1-4 were
similar. However, CD spectral data indicated that absolute configurations at cyclobutane-ring of
compound 1 were mirror-image with those of compounds 2-4 as described in the Figure 1.
2
+
ANO1 is known as transmembrane protein 16A which is identified to be a Ca -activated chloride
channel expressing in various cell types. It recently considered as an important marker involved in
the regulation of cell proliferation, smooth muscle contraction, epithelial fluid secretion.
Therefore, ANO1 inhibitors would be beneficial to treatments of related diseases such as cancer,
hypertension, asthma, and pain [15, 21, 22]. Recent study indicates natural phenolics could be
good ANO1 inhibitors [15]. Compounds 1-4 were then screened for their effect on ANO1 activity.
At concentration of 30 µM, compounds 1-4 moderately inhibited ANO1 activity with inhibitory
percentages of 27.7 ± 1.10 %, 35.6 ± 0.92 %, 43.7 ± 1.61 %, and 40.8 ± 1.25%, respectively. The
1
4

most potent inhibitor, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-
acetamide (Ani9) was used at 3 µM as a positive control, showing inhibitory rate of 97.5 ± 0.34
%. Significant ANO1 inhibitory activity of compounds 1-4 were consisted with previous reports
that natural phenolics could be good ANO1 inhibitors [15].
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgement
This research is funded by Vietnam National Foundation for Science and Technology
Development (NAFOSTED) under grant number: 104.01-2018.300
Appendix A. Supplementary material
Supplementary data to this article can be found online at https:// doi.org/…
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1
8

Figure 1. Structures of compounds 1-4 isolated from T. honbaensis
HO
HO
5
4
6
''
O
O
6
A
9
5''
O
R1
H CO
3
1
O
3
O
O
2
7
8
OH
OH
4
''
1''
HO
HO
3
'' 2''
2
'
7'
8'
9
'
O
HO
O
R2
HO
H CO
1'
3
O
O
6
'''
3
'
B
O
O
6
'
O
O
4
'
5'
5'''
HO 2'''
HO
HO
HO
1
'''
1
2
4
'''
3'''
2 R = R = OCH
3
OH
OH
1
1
2
3
4
R = H; R = OCH
3
OH
OH
1
2
R = OCH ; R = H
3
1
9

Figure 2. Key HMBC (H → C) and COSY (H ▬ H) correlations of compounds 1-4
HO
HO
HO
O
O
O
H3CO
O
O
H3CO
O
O
O
O
OH
OH
OH
HO
HO
HO
O
HO
O
O
HO
H3CO
H3CO
HO
O
O
O
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
1
and 2
3
4
OH
OH
OH
OH
OH
OH
2
0

Figure 3. Important NOESY correlations in aglycone moieties of compounds 1-4
8
2
2
8
8
'
2
'
8'
2'
1
2-4
2
1

Figure 4. Circular dichroism spectra of compounds 1-4
2
2

1
13
Table 1. H and C-NMR spectral data of compounds 1-4
1
2
3
4
No.
a,bδC
131.8
a,c
δH (mult., J in Hz)
a,bδC
a,c
δH (mult., J in Hz) a,bδC
a,c
δH (mult., J in Hz) a,bδC
a,c
δ (mult., J in Hz)
H
-
-
1
2
3
4
5
6
7
8
9
-
131.7
114.1
148.6
146.1
115.7
121.4
45.9
131.0
130.4
115.9
156.9
115.9
130.4
-
131.7
114.0
148.5
146.0
115.7
121.4
113.7
148.7
146.1
115.7
121.4
46.2
6.43 (d, 2.0)
-
6.42 (d, 1.5)
6.93 (d, 8.5)
6.61 (d, 8.5)
-
6.61 (d, 8.5)
6.93 (d, 8.5)
6.40 (br s)
-
-
-
-
-
*
6.66 (d, 8.5)
6.61 (dd, 2.0, 8.5)
4.20 (dd, 5.0, 10.0)
3.88 (dd, 5.0, 10.5)
6.68 (d, 8.5)
6.69 (dd, 1.5, 8.5)
4.29 (dd, 4.5, 10.0) 45.6
3.89 (dd, 4.5, 10.5) 45.1
6.64
6.64
*
4.28 (dd, 4.5, 10.0) 45.9
3.85 (dd, 4.5, 10.0) 45.1
4.29 (5.0, 9.5)
3.86 (5.0, 10.0)
-
44.8
44.8
174.5
131.7
113.1
148.5
146.1
115.6
121.6
45.2
44.7
174.2
56.4
56.3
94.7
-
174.2
131.6
113.2
148.5
146.1
115.6
121.6
44.9
44.8
174.2
56.4
56.3
92.8
-
-
174.2
131.7
113.0
148.4
146.0
115.5
121.6
-
174.4
131.0
130.1
115.8
157.0
115.8
130.1
-
-
1
2
3
4
5
6
7
8
9
′
-
′
′
′
′
′
′
′
′
6.34 (d, 2.0)
-
6.34 (d, 2.0)
6.31 (d, 2.0)
6.78 (d, 8.5)
6.56 (d, 8.5)
-
6.56 (d, 8.5)
6.78 (d, 8.5)
4.23 (dd, 8.5, 9.5)
4.10 (dd, 8.5, 10.0)
-
-
-
-
-
-
6.60 (d, 8.5)
6.48 (dd, 2.0, 8.5)
4.25 (dd, 8.5, 10.0)
3.97 (dd, 8.5, 10.5)
-
6.60 (d, 9.0)
6.50 (dd, 2.0, 9.0)
4.24 (dd, 9.0, 10.0) 44.7
4.10 (dd, 9.0, 10.5) 45.0
6.57 (d, 8.0)
6.45 (dd, 2.0, 8.0)
4.23 (dd, 8.5, 10.0) 44.4
4.11 (dd, 8.5, 10.0) 44.6
-
174.2
-
-
-
174.3
56.4
-
3
3
-OCH3
′-OCH3
3.63 (s)
3.61 (s)
3.62 (s)
3.61 (s)
3.62 (s)
56.3
92.8
73.0
74.6
72.6
72.4
3.61 (s)
-
1
2
3
4
5
′′
′′
′′
′′
′′
5.37 (d, 3.5)
4.43 (dd, 3.5, 9.5)
3.74 (dd, 9.5, 9.5)
3.13 (dd, 9.5, 9.5)
4.05 (m)
5.35 (d, 3.5)
4.43 (dd, 3.5, 9.5)
3.76 (dd, 9.5, 9.5)
3.12 (dd, 9.5, 9.5)
4.15 (m)
5.35 (d, 3.5)
3.48 (dd, 3.5, 9.5)
3.75 (dd, 9.5, 9.5)
3.12 (dd, 9.0, 9.5)
4.16 (m)
92.9
73.0
74.5
72.6
72.5
5.35 (d, 3.5)
3.47 (dd, 3.5, 9.5)
3.66 (t, 9.5)
3.12 (dd, 9.0, 9.5)
4.16 (m)
73.4
74.1
73.1
73.0
73.0
74.6
72.6
72.5
3
4
.83 (dd, 9.0, 11.5)
.85 (br d, 11.5)
4.02 (dd, 9.0, 11.5)
4.58 (dd, 2.5, 11.5)
3.67 (s)
4.00 (dd, 8.5, 12.0)
4.57 (dd, 1.5, 12.0)
3.66 (s)
4.03 (dd, 9.0, 11.5)
4.57 (dd, 1.5, 11.5)
3.66 (s)
6
′′
67.6
68.6
68.6
68.5
1
2
3
4
5
′′′
′′′
′′′
′′′
′′′
61.9
106.8
79.4
77.7
80.7
3.87 (s)
-
4.23 (d, 7.5)
4.03 (dd, 7.5, 7.5)
4.07 (m)
63.8
105.6
79.2
77.9
79.2
63.8
105.6
79.2
77.9
79.2
63.7
105.7
79.2
77.9
79.2
-
-
-
4.14 (d, 8.0)
4.31 (dd, 8.0, 8.0)
4.16 (m)
4.14 (d, 7.5)
4.32 (dd, 7.5, 7.5)
4.16 (m)
4.15 (d, 7.5)
4.30 (dd, 7.5, 7.5)
4.17 (m)
4
4
.22 (br d, 10.5)
4.36 (dd, 8.0, 10.5)
4.56 (dd, 8.5, 10.5)
4.36 (dd, 8.0, 10.5)
4.56 (dd, 8.5, 10.5)
4.36 (dd, 8.0, 10.5)
4.56 (dd, 8.5, 10.5)
6
′′′
68.4
67.5
67.5
67.5
.33 (dd, 10.5, 10.5)
a)
b)
c)
*)
3
Measured in CD OD, 125 MHz, 500 MHz, Overlapped signals.
2
3

2
4