A sesquiterpenoid tropolone and 1,2,3,4-tetrahydronaphthalene derivatives from Olax imbricata roots

Article abstract of DOI:10.1016/j.fitote.2018.11.007

The methanol extract of Olax imbricata roots afforded one new sesquiterpenoid tropolone and three new 1,2,3,4-tetrahydronaphthalene derivatives, olaximbrisides A–D (1–4). Their structures were determined by 1D and 2D NMR experiments in combination of HRESIMS. The relative configurations were assigned by the NOESY experiments. The absolute configurations were established by a combination of X-ray diffraction analysis and electronic circular dichroism (ECD) experiments. All isolated compounds were evaluated for their cytotoxic effects against some cancer cell lines. Among them, compound 1 exhibited the cytotoxicities against MCF-7, HepG2 and LU cell lines with IC₅₀ values of 16.3, 34.3 and 8.0 μM, respectively.

Full text of DOI:10.1016/j.fitote.2018.11.007

Fitoterapia132(2019)1–6
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
A sesquiterpenoid tropolone and 1,2,3,4-tetrahydronaphthalene derivatives
from Olax imbricata roots
Huong T.M. Nguyena,1, Nga T. Vob,1, Suong T.M. Huynhc, Lien T.M. Dod, Thammarat Areee,
Santi Tip-pyange, Cam-Tu D. Phanf, Nguyen T. Trungf, Phung K.P. Nguyena,⁎
a University of Science, National University–Ho Chi Minh City, Ho Chi Minh City 748355, Viet Nam
b Ho Chi Minh City University of Technology and Education, Ho Chi Minh City 720214, Viet Nam
c University of Natural Resources and Environment Ho Chi Minh City, Ho Chi Minh City 736400, Viet Nam
d Institute of Environmental-Energy Technology, Sai Gon University, Ho Chi Minh City 748355, Viet Nam
e Center of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok
10330, Thailand
f Department of Chemistry, and Laboratory of Computational Chemistry and Modelling, Quy Nhon University, 55100, Viet Nam
A R T I C L E I N F O
A B S T R A C T
Keywords:
The methanol extract of Olax imbricata roots afforded one new sesquiterpenoid tropolone and three new 1,2,3,4-
tetrahydronaphthalene derivatives, olaximbrisides A–D (1–4). Their structures were determined by 1D and 2D
NMR experiments in combination of HRESIMS. The relative configurations were assigned by the NOESY ex-
periments. The absolute configurations were established by a combination of X-ray diffraction analysis and
electronic circular dichroism (ECD) experiments. All isolated compounds were evaluated for their cytotoxic
effects against some cancer cell lines. Among them, compound 1 exhibited the cytotoxicities against MCF-7,
HepG2 and LU cell lines with IC50 values of 16.3, 34.3 and 8.0 μM, respectively.
Olax imbricata
Sesquiterpenoid
Tropolone
Tetrahydronaphthalene
Olaximbrisides A–D
Cytotoxicity
1. Introduction
mannii leaves.
Up to now, only one report on the phytochemical analysis of O.
Some species of Olax (O.) genus (Olacaceae), growing in the Asia's
rainforest and in Africa, displayed potential bioactivities. The methanol
root extract of O. viridis was proved the ability of protecting the liver
against acetaminophen-induced liver damage [1]. The ethanolic extract
of O. supscorpioidea was sedative and had mild anticonvulsant activity
[2], as well as possessed potent analgesic action [3]. Two triterpenes,
rhoiptelenol and glutinol, isolated from the acetone extract of O.
mannii, were used as folk remedies for the treatment of fever, yellow
fever and snake bite [4]. Although the Olax genus possesses such po-
tential bioactivities, there have been a few reports on their chemical
constituents. From O. andronensis, O. glabriflora and O. psittacorum, a
saponin namely olaxoside was isolated, and this compound had laxative
and anti-inflammatory activities [5]. An amino acid S-ethenylcysteine
was isolated from the ethanolic extract of O. phyllanthi roots [6]. (2E)-3-
Methyl-5-phenyl-2-pentenoic acid was separated from the petroleum
ether extract of O. manni leaves [7]. Olamannoside A-C [8] and ola-
mannoside D-E [9] were separated from the methanol extract of O.
imbricata revealed the presence of polyphenolic compounds, flavonoids,
glycosides, saponins, tannins, alkaloids and some of these compounds
possessed antioxidant, antibacterial activities [10]. In our previous
work, the isolation and structure elucidation of 1-O-(4-hydroxy-2,6-
dimethoxyphenyl)-6-O-(4-hydroxy-3,5-dimethoxybenzoyl)-β-D-gluco-
pyranose and 1-O-(4-hydroxy-2-methoxyphenyl)-6-O-(4-hydroxy-3,5-
dimethoxybenzoyl)-β-D-glucopyranose were reported [11].
In the course of a systematic study for bioactive constituents of this
species, widely distributed in Phu Yen province, south-central of
Vietnam and has been locally used as a traditional remedy for diabetes
and anti-cancer, the methanol extract of O. imbricata roots was ex-
amined. Herein, the isolation and structural elucidation of four new
compounds, olaximbrisides A–D (1–4) (Fig. 1), are described.
Their structures were determined by 1D and 2D NMR experiments
in combination of HRESIMS. The absolute configurations were achieved
by a combination of the single-crystal X-ray crystallographic analyses
and electronic circular dichroism (ECD) experiments. The cytotoxicities
⁎ Corresponding author at: Department of Organic Chemistry, Faculty of Chemistry, University of Science, National University-Ho Chi Minh City, 227 Nguyen van
Cu, District 5th, Ho Chi Minh City, Viet Nam.
E-mail address: kimphiphung@yahoo.fr (P.K.P. Nguyen).
1 Huong T. M. Nguyen and Nga T. Vo contributed equally.
https://doi.org/10.1016/j.fitote.2018.11.007
Received 11 September 2018; Received in revised form 9 November 2018; Accepted 11 November 2018
Availableonline13November2018
0367-326X/©2018ElsevierB.V.Allrightsreserved.

H.T.M. Nguyen et al.
Fitoterapia132(2019)1–6
HO
Table 1
1H NMR spectroscopic data for compounds 1−4.a
HO
4
1
5
5
8
4
1
6
10
9
3
2
3
11
6
7
Pos.
1
2
3
4
7
O
2
12
14
10
8
12
11
9
(DMSO-d6)
(DMSO-d6)
2.57 m
2.12 dd
(C5D5N)
(C5D5N)
(DMSO-d6)
OH
OH
14
13
15
1
2
13
1α
1β
2
2.70 dd
(17.5, 5.0)
2.33 dd
(17.5, 11.5) (16.5, 12.0)
1.50 tt
3.18 dd
(17.0, 4.5)
2.75 m
3.19 dd
(17.0, 4.0)
2.76 m
2.70 dd
(17.0, 4.5)
2.29 m
OGlc
OGlc
12
5
4
1
4
1
5
1.53 tdd
(12.0, 5.0,
2.5)
2.27 tdd
(12.5, 5.0,
2.5)
2.27 tdd
(11.5, 4.5,
2.5)
1.71 tdd
(12.5, 4.5,
2.0)
1.91 d (10.0)
1.16 dd
(12.5. 5.0)
2.36 m
3.08 dd
13
10
3
2
10
3
CH2OH
OH
6
6
7
(11.5, 5.0)
7
9
8
2
9
8
OH
CH2OH
11
3α
3β
1.84 m
1.26 m
1.95 m
2.17 m
2.18 m
11
1.17 dd
(12.5, 5.5)
2.62 m
1.41 dd
(12.5, 5.0)
2.75 m
3.53 dd
(17.0, 3.5)
1.42 dd
(11.5, 5.0)
2.76 m
3.54 dd
(17.0, 3.5)
14
14
3
13
4
12
4α
4β
2.79 dd
(7.5, 5.0)
Fig. 1. Structures of compounds 1–4.
(17.5, 3.1)
5
6
6.98 s
7.18 s
6.29 d (2.5)
7.38 d (8.5)
6.98 d (8.5)
7.39 d (8.5)
6.99 d (8.5)
6.77 d (8.0)
6.88 d (8.0)
against MCF-7 (human breast adenocarcinoma), and HepG2 (human
hepatocellular adenocarcinoma), LU (human lung carcinoma) cell lines
were evaluated on all isolated compounds.
6-OH
7
8
8.79 s
6.38 d (2.5)
11-OH
12
4.14 s
1.12 s
4.04 s
3.99 m
4.00 m
3.30 m,
3.40 m
4.51 m
1.07 s
2. Results and discussion
12-OH
13
14
1.12 s
1.10 s
2.36 s
1.12 s
2.08 s
1.48 s
2.13 s
1.49 s
2.13 s
The ethanol extract of O. imbricata roots was fractionated by liquid -
liquid extraction and the resulting fractions were repeatedly subjected
to different chromatographic methods to yield four compounds, olax-
imbrisides A–D (Fig. 1).
2.11 s
15
1′
2′
2′-OH
3′
3′-OH
4′
5.57 d (7.0)
4.35 m
5.59 d (8.0)
4.37 m
4.67 d (7.5)
3.23 m
5.20 d (5.0)
3.25 m
5.02 d (4.0)
3.17 m
Compound 1 was isolated as a yellow crystal (recrystallized in
4.35 m
4.35 m
4.37 m
4.35 m
chloroform). Its molecular formula was established as
C15H20O3
through the pseudomolecular ion peak at m/z 249.1506 [M + H]+
(calcd 249.1491) in the HRESIMS spectrum. The combination of 13C
NMR and HSQC spectra of 1 showed the presence of 15 carbons in-
cluding three methyl groups [δC 26.6 (two groups), 27.1], three me-
thylene carbons (δC 22.9, 31.0, 36.4), one methine carbon (δC 44.2),
one oxygenated quaternary carbon (δC 70.6), six olefinic carbons, in-
cluding two methines (δC 121.5, 129.6) and four quaternary carbons (δC
137.8, 145.3, 149.1, 162.9) and one conjugated ketone carbon (δC
172.6) (Table 2). This conjugated ketone carbon resonated at a rela-
tively high field, however, such up-field shifted chemical shift value for
this type of carbon had been also observed in the 7-membered ring,
liriosmasides [12] or manicol derivative [13]. The 1H NMR combining
with HSQC spectra displayed three methyl singlet signals at δH 1.10,
1.12 and 2.36 (each 3H), one methine triplet of triplets at δH 1.50, three
methylene signals at δH 1.26, 1.84, 2.33, 2.70 (each 1H) and 2.79 (2H),
and two olefinic singlets at δH 6.98 and 7.18 (Table 1). The NMR data of
1 were highly reminiscent of those of the sesquiterpenoid tropolone
liriosmasides [12]. The 1He1H COSY experiment of 1 revealed the spins
connection between H-1, H-2, H-3 and H-4, shown in bold in Fig. 2. The
HMBC spectrum exhibited correlations of the proton signals at δH 2.70
and 2.33 (H2−1) to the carbon signals at δC 22.9 (C-3), 44.2 (C-2), 70.6
(C-12), 137.8 (C-10), 145.3 (C-11) and 149.1 (C-9), of the signal at δH
2.79 (H2–4) to the carbons C-2, C-3, C-10, C-11 and C-5 (δC 121.5).
These correlations indicated that there was an aliphatic six-membered
ring fused to a conjugated seven-membered ring as in liriosmasides
[12]. The HMBC experiment of 1 (Fig. 2) revealed that the conjugated
seven-membered ring possessing a methyl group at C-9 [the correla-
tions of H3–15 (δH 2.36) to C-8 (δC 129.6), C-9 and C-10], a ketone
group at C-7 and a hydroxy group at C-6 [the correlations of H-5 (δH
6.98) to C-6 (δC 162.9), C-7 (δC 172.6), C-10, C-11, and of H-8 (δH 7.18)
to C-6, C-7, C-9, C-10 and C-15 (δC 26.6)] and the aliphatic six-mem-
bered ring linking to a hydroxyisopropyl group at its C-2 [H3–13 (δH
1.12) and H3–14 (δH 1.10) showing cross-peaks to carbon signals at δC
27.1 (C-13), 26.6 (C-14), 44.2 (C-2) and 70.6 (C-12)]. The relative
configuration of 1 was determined on the basis of NOESY correlations
as well as the 1H NMR coupling constants (Fig. 2). Key correlations of
4′-OH
5′
6′
4.96 d (5.0)
3.28 m
3.68 dd
(12.0, 2.0)
3.46 dd
(12.0, 6.0)
4.51 m
4.10 m
4.56 dd
(12.0, 2.5)
4.40 dd
(12.0, 5.0)
4.37 m
4.56 dd
(12.0, 2.5)
4.42 dd
(12.0, 5.0)
6′-OH
a
Recorded at 500 MHz in DMSO-d6 or pyridine-d5. Chemical shifts (δ) are
expressed in ppm, and J values are presented in Hz.
the signal at δH 1.50 (H-2) with signals at δH 2.70 (Hα-1), 1.84 (Hα-3) as
well as of signals at δH 2.33 (Hβ-1) and 1.26 (Hβ-3) indicated that the
hydrogens H-2, Hα-1, Hα-3 were on the same side of the molecular plan
and Hβ-1, Hβ-3 were at the opposite site of these three-mentioned ones.
To assign the absolute configuration of 1, a single crystal X-ray
crystallographic analysis was successfully performed which both vali-
dated the 2-D structure and determined the configuration of C-2 as R
(Fig. 3). The ECD spectrum of 1 (Fig. 4) showed a positive Cotton effect
at 214 nm (Δε 19.0) implying that the 2R configuration of 1 exhibited
this Cotton effect. Altogether, compound 1 was identified as (2R)-6-
hydroxy-2-(1-hydroxy-1-methylethyl)-9-methylbicyclo[5.4.0]undeca-
5,8,10(11)-triene-7-one and was named olaximbriside A.
Olaximbriside B (2) was isolated as a white amorphous powder with
the molecular formula C14H20O2 as deduced by the HRESIMS spectrum.
The 1H and 13C NMR with HSQC experiment of 2 revealed the signals of
14 carbons: three methyl groups (δC 19.3, 26.6 and 27.1), three me-
thylene carbons (δC 23.9, 27.1 and 30.6), one aliphatic methine carbon
(δC 45.9), one oxygenated quaternary carbon (δC 70.8), six aromatic
carbons including two methines (δC 112.4 and 114.5) and four qua-
ternary carbons with one being oxygenated (δC 125.5, 137.0, 137.3 and
154.3) (Table 2). The comparison of the HRESIMS and 13C NMR of 2
and 1 showed the similarities except that 2 lacked one conjugated ke-
tone group. The 1H NMR spectrum of 2 displayed signals due to three
methyl groups [δH 1.12 (s, 6H) and 2.08 (s, 3H)], three methylenes [δH
1.17 (dd, J = 12.5, 5.5 Hz, 1H), 1.95 (m, 1H), 2.12 (dd, J = 16.5,
2

H.T.M. Nguyen et al.
Fitoterapia132(2019)1–6
Fig. 2. Key COSY, HMBC, NOESY correlations for compounds 1−4.
six-membered ring system like that of 1. As in 1, the six-membered ring
in 2 attached to the benzene ring at its C-1 and C-4 which was proved
by the HMBC cross-peaks of protons H2–1 (δH 2.12, 2.57) to the carbons
C-3 (δC 23.9), C-2 (δC 45.9), C-11 (δC 70.8), C-9 (δC 125.5), C-8 (δC
137.0) and C-10 (δC 137.3), of proton H2–4 (δH 2.62) to the carbons C-
2, C-3, C-5 (δC 112.4), C-9 and C-10, respectively. And as in 1, the
carbon C-2 of this six-membered ring also linked to a hydroxyisopropyl
group with the cross-peak of the two methyl protons, H3–12 and H3–13
(δH 1.12), to carbon signals at C-2 and C-11. The HMBC experiment of 2
(Fig. 2) revealed that the benzene ring also possessed a methyl group at
C-8 and a hydroxy group at C-6. These were proved by cross-peaks of
H3–14 (δH 2.08) to C-8, C-9 and of the two meta-protons [H-5 (δH 6.29,
d, J = 2.5 Hz) and H-7 (δH 6.38, d, J = 2.5 Hz)] to carbon of each other
as well as to C-6 (δC 154.3) and C-9, respectively. The relative config-
uration of 2 was similar to that of 1 which was determined from the
coupling constants of relevant hydrogens (H1-H4) and from the NOESY
experiment (Fig. 2). The absolute configuration at C-2 of 2, an aliphatic
six-membered ring also bearing a hydroxyisopropyl group, was as-
signed as R due to the positive Cotton effect at 214 nm (Δε + 10.0)
Fig. 3. ORTEP drawing of olaximbriside
12.0 Hz, 1H), 2.57 (m, 1H) and 2.62 (m, 2H)], one methine (δH 1.53,
tdd, J = 12.0, 5.0, 2.5 Hz, 1H), two aromatic protons (δH 6.29 and
6.38), and one phenolic hydroxy group [δH 8.79 (s, 1H)] (Table 1). The
1He1H COSY spectrum of 2 (Fig. 2) unequivocally revealed the con-
tiguous arrangement of these three methylenes and one methine of a
Fig. 4. Experimental ECD spectra of compounds 1−4.
3

H.T.M. Nguyen et al.
Fitoterapia132(2019)1–6
Table 2
group at its C-2 supported by correlations of proton signals at δH 3.99
(H2−12) and 1.48 (H3−13) to carbon signals at δC 75.8 (C-11), 69.7
(C-12) 22.3 (C-13) and 42.2 (C-2), and vice-versa, the proton (δH 2.27)
of the latter carbon showed correlations to carbons C-11 and C-12. The
relative configuration of 3 was confirmed by the NOESY experiment
(Fig. 2). The correlations of the signal at δH 2.27 (H-2) and signals at δH
3.18 (Hα-1), 2.17 (Hα-3), 2.75 (Hα-4) and as well as of signal at δH 2.75
(Hβ-1) and 1.41 (Hβ-3) indicated that H-2, Hα-1, Hα-3 and Hα-4 had the
same orientation and Hβ-1 and Hβ-3 were at the opposite site of these
four-mentioned hydrogens. The absolute configuration at C-2 of 3 was
assigned as R as in 1 and 2 due to the similar positive Cotton effect at
214 nm (Δε + 10.0) and the one at C-11 couldn't be determined.
However, the configuration at this stereogenic center would be pro-
posed later due to the comparison of the NOESY spectra of compounds
3 and 4. Therefore, up to this point, 3 could be (2R)-2-(1,2-dihydroxy-
1-methylethyl)-8-methyl-5-O-β-D-glucopyranosyl-1,2,3,4-tetra-
13C NMR spectroscopic data for compounds 1−4.a
Position
1
2
3
4
(DMSO-d6)
(DMSO-d6)
(C5D5N)
(C5D5N)
(DMSO-d6)
1
31.0
27.1
45.9
23.9
30.6
112.4
154.3
114.5
137.0
125.5
137.3
70.8
29.4
42.2
25.1
26.3
155.5
113.1
128.9
131.6
138.5
128.3
75.8
28.9
42.0
24.6
25.8
155.2
112.4
128.1
130.5
138.0
127.6
74.6
27.7
40.5
23.4
24.6
153.8
111.3
127.3
129.5
137.0
126.5
73.9
2
44.2
3
22.9
4
36.4
5
6
7
8
121.5
162.9
172.6
129.6
149.1
137.8
145.3
70.6
9
10
11
12
13
14
15
1’
2’
3’
4’
5’
6’
27.1
26.6
19.3
69.7
22.3
20.3
69.4
22.2
19.6
68.1
22.1
19.2
27.1
26.6
26.6
hydronaphthalene.
Olaximbriside D (4) was isolated as a white amorphous powder. It
well dissolved in both pyridine and DMSO. Its molecular formula was
determined as C20H30O8 (HRESIMS, [M + Na]+ m/z 421.1824, calcd
421.1838). The NMR spectra of 3 and 4 (Tables 1 and 2, DMSO‑d6)
showed the similarities with 20 signals including two methyl groups (δH
1.07, 2.11; δC 19.2 and 22.1), four methylene carbons in which one was
oxygenated (δH 1.16, 1.91, 2.29, 2.36, 2.70, 3.08, 3.30, 3.40, each 1H;
δC 23.4, 24.6, 27.7 and 68.1), one methine (δH 1.71; δC 40.5), one
oxygenated quaternary carbon (δC 73.9), two ortho-aromatic methines
[δH 6.77 (d, J = 8.0 Hz), 6.88 (d, J = 8.0 Hz); δC 111.3 and 127.3], four
quaternary aromatic carbons (δC 126.5, 129.5, 137.0 and 153.8) and six
103.7
75.8
79.2
72.3
79.2
63.3
103.6
75.6
79.3
71.9
79.2
63.0
101.9
73.5
77.4
70.3
77.2
61.3
a
Recorded at 125 MHz in DMSO-d6 or pyridine-d5. Chemical shifts (δ) are
expressed in ppm.
similar to that of 1. Accordingly, the structure of 2 could be proposed as
(2R)-6-hydroxy-2-(1-hydroxy-1-methylethyl)-8-methyl-1,2,3,4-tetra-
hydronaphthalene.
signals belonging to
a β-D-glucopyranose moiety [δH 4.67 (d,
J = 7.5 Hz, H-1′), 3.1–3.7 (H-2’-H-6′); δC 101.9, 77.4, 77.2, 73.5, 70.3
and 61.3]. The sugar unit was expected to be the same as that of 3 as
both were isolated from the same materials and possessing similar
spectral data (Tables 1 and 2).
Olaximbriside C (3) was isolated as a white amorphous solid. It
dissolved well in pyridine but almost did not in DMSO. Its molecular
formula was determined as C20H30O8 by HRESIMS spectrum with a
sodiated molecular ion peak at m/z 421.1840 [M + Na]+ (calcd
421.1838). The 13C NMR and HSQC spectra indicated counts of 20
carbons including two methyl groups (δC 20.3 and 22.3), four methy-
lenes (δC 25.1, 26.3, 29.4 and 69.7), one methine (δC 42.2), one oxy-
genated quaternary carbon (δC 75.8), two aromatic methines (δC 113.1
and 128.9) and four quaternary aromatic carbons (δC 128.3, 131.6,
138.5 and 155.5). Besides, there were six signals belonging to an hex-
opyranose moiety [δC 103.7, 79.2 (2C), 75.8, 72.3 and 63.3] (Table 2).
The combination of 1H NMR with HSQC spectra, 3 displayed singlet
signals for two tertiary methyls (δH 1.48 and 2.13), four methylenes at
δH 1.41, 2.17, 2.75 (2H), 3.18, 3.53 and 3.99 (2H), one methine at δH
2.27, two ortho-aromatic protons at δH 6.98 (d, J = 8.5 Hz) and 7.38 (d,
J = 8.5 Hz). Moreover, the 1H NMR spectrum also showed the presence
of a hexopyranose unit with the anomeric proton at δH 5.57 (d,
J = 7.0 Hz, H-1′) and signals due to sugar protons in the region of
4.1–4.6 ppm. Chemical shifts, multiplicities, coupling constant magni-
tudes in the 1H NMR spectrum along with 13C NMR data of 3 (Tables 1
and 2) indicated that this was a β-D-glucopyranose unit. The nature of
the sugar was further validated upon the anomeric proton analysis of
the sugar obtained after acid hydrolysis [14].
The relative configuration at C-2 of 4 was elucidated by the NOESY
(Fig. 2) as well as the ECD experiment (Fig. 4). The correlations of the
signal at δH 1.71 (H-2) and signals at δH 1.91 (Hα-3), 2.36 (Hα-4) and
2.70 (Hα-1) as well as of signal at δH 2.29 (Hβ-1) and 1.16 (Hβ-3) in-
dicated that H-2, Hα-1, Hα-3 and Hα-4 had the same orientation and Hβ-
1 and Hβ-3 were at the opposite site of the four-mentioned hydrogens.
In the ECD spectrum of 4 (Fig. 4) there were some Cotton effects at
214 nm (Δε + 10.0), 230 nm (Δε − 25.0) and 280 nm (Δε − 3.0) and
among them, the positive Cotton effect at 214 nm (Δε + 10.0) could be
attributed to C-2 because of its similarity with the ones in 1–3. Ac-
cordingly, the absolute configuration at C-2 of 4 was assigned as R as
those in 1–3 and the one at C-11 couldn't be suggested. A closer analysis
of 1D and 2D-NMR spectra of 4 and 3 (Tables 1 and 2, C5D5N) showed
that they would be two diastereoisomers at C-11.
An attempt to assign the configuration at C-11 of 3 and 4 was done
based on a careful examination on their physical properties which
showed some differences. If 3 well dissolved in pyridine, 4 mostly did
not. In their ECD spectra, although having similar Cotton effects in the
region of 214–400 nm, they exhibited opposite Cotton effect in the zone
of 200–207 nm. In this region, 3 showed a strong negative Cotton effect
(Δε from −13.0 to zero) while 4 showed a strong positive one (Δε from
+30.0 to +10.0). Furthermore, if the δH chemical shift values of 3 and
4, measured in the same deuterated solvent, pyridine-d5 (Table 1), were
almost identical, their δC values (Table 2) were relatively different
(Δδc = 0.10–0.70), especially C-11 (Δδc = 1.20).
The multiplicity of the aliphatic protons H2–1, H2–2, H-3 and H2–4
as well as the 1He1H COSY spectrum confirmed their contiguous ar-
rangement. Besides, the HMBC cross-peaks (Fig. 2) of the proton signals
at δH 2.75, 3.18 (H2–1) to the carbon signals at δC 25.1 (C-3), 42.2 (C-
2), 75.8 (C-11), 128.3 (C-10), 131.6 (C-8) and 138.5 (C-9), of signals at
δ
H 2.75, 3.53 (H2–4) to the carbons C-2, C-3, C-5 (δC 155.5), C-9 and C-
The NOESY experiments of 3 and 4 could afford some insights into
the stereochemistry at C-11. In the NOESY spectrum (Fig. 2), of 3 in
pyridine-d5, there were strong correlations of H2–12 to Hα-3 and of
H3–13 to H-2, while in the NOESY spectrum of 4, in DMSO‑d6 as well as
in pyridine-d5 there were cross-peaks of H3–13 to Hα-3 and of H2–12 to
H-2. The CD curves of 3 and 4 (Fig. 4) revealed similar profiles with the
theoretically calculated (2R,11S)- and (2R,11R)-2-(1,2-dihydroxy-1-
methylethyl)-8-methyl-5-O-β-D-glucopyranosyl-1,2,3,4-
10 revealed that compound 3 also possessed an aliphatic six-membered
ring fused with a benzene ring as in 2 as shown. However, the benzene
ring jointed to a methyl group at its C-8 with HMBC cross-peaks of the
signal at δH 2.13 (H3–14) to carbon signals at δC 131.6 (C-8) and 138.5
(C-9) and to a β-D-glucopyranose unit at its C-5 with the correlation of
the anomeric proton to carbon signal at δC 155.5 (C-5). And the ali-
phatic six-membered ring linking to a 1,2-dihydroxy-1-methylethyl
4

H.T.M. Nguyen et al.
Fitoterapia132(2019)1–6
Fig. 5. Caculated CD spectra of 3 (left) and 4 (right) at the level of theory B3LYP/6–311 + G(d,p) using TD-DFT method.
tetrahydronaphthalene, respectively, using Gaussian 09 software
(Fig. 5). These results could lead to assign the absolute configurations as
11S and 11R for 3 and 4, respectively. Therefore, the structures of 3 and
4 could be proposed as shown.
All isolated compounds were evaluated in vitro for their cytotoxic
potential against three cancer cell lines using the modified MTT method
[15] with ellipticine used as a positive control. The in vitro biological
assay of these compounds (Table 3) showed that only olaximbriside A
(1) exhibited cytotoxicities against MCF-7, HepG2 and LU cancer cell
lines with IC50 values of 16.5, 34.3 and 8.0 μM, respectively.
industrial zone, Dong Hoa district, Phu Yen province, Vietnam, in April
2013 and authenticated by Mr. Hoang Xuan Lam, Middle Vietnam
Research and Manufacturing Organic Medicinal Herb Centre in Phu Yen
province. A voucher specimen (No US–C027) was deposited in the
herbarium of the Department of Organic Chemistry, University of
Science, National University–Ho Chi Minh City, Vietnam.
3.3. Extraction and isolation
The roots were cleaned and dried at 60–70 °C to dryness, and then
ground to powder (6.3 kg). This dried powder was exhaustively ex-
tracted with ethanol at room temperature (6 × 15 L) by the maceration
method. The filtrated solution was evaporated under reduced pressure
to afford an ethanolic extract (1.2 kg). This extract was suspended in
ethanol-water (1:9), then partitioned against n-hexane. The obtained
solution was concentrated under reduced pressure to afford the n-
hexane extract (500 g). The procedure was repeated similarly with ethyl
acetate to afford the ethyl acetate extract (70 g). The remaining aqu-
eous solution was evaporated to dryness and was dissolved in methanol
and the methanolic soluble portion was evaporated to dryness to afford
the methanolic extract (430 g). The remaining residue was dried to
obtain the aqueous extract (130 g). The methanolic extract was sub-
jected to silica gel column chromatography using gradient elution of
ethyl acetate–methanol (stepwise 95:5–1:1) to give nine fractions, RM1-
RM9. Fraction RM2 (25 g) was silica gel column chromatographed
eluting with ethyl acetate–methanol (stepwise 98:2–1:1) to give eight
sub-fractions: RM2–1 (2.7 g), RM2–2 (3.5 g), RM2–3 (1.9 g), RM2–4
(2.6 g), RM2–5 (2.9 g), RM2–6 (1.9 g), RM2–7 (4.5 g) and RM2–8
(3.1 g). The sub-fraction RM2–1 (2.7 g) was silica gel chromatographed
eluting with chloroform to give 2 (5 mg). A similar procedure was ap-
plied to the sub-fraction RM2–2 (3.5 g) eluting with chlor-
oform–methanol (95:5) to afford compound 1 (90 mg) and to RM2–7
(4.5 g) eluting with chloroform–methanol (80:20) to give two com-
pounds, 3 (15 mg) and 4 (10 mg).
3. Experimental section
3.1. General experimental procedures
The melting point (uncorrected) was determined by microscope hot
stage, Kofler, Polytherm A. The NMR spectra were recorded on a Bruker
Avance III spectrometer (500 MHz for 1H NMR and 125 MHz for 13C
NMR) using residual solvent signals as internal references: DMSO-d6 δH
2.50, δC 39.51 and pyridine-d5 at δH 8.74, 7.58 and 7.22, δC 123.87,
135.91, 150.35. The HRESIMS were recorded on
a HR–ESI–MS
MicroOTOF–Q mass spectrometer on a LC-Agilent 1100 LC-MSD Trap
spectrometer. The optical rotation was measured on a Kruss digital
polarimeter. The ECD spectra were measured with a JASCO J-815 cir-
cular dichroism spectrometer. Single-crystal X-ray diffraction analysis
was performed on a Bruker X8 PROSPECTOR Kappa CCD area-detector
diffractometer with an IμS X-ray microfocus source (Cu-Kα radiation,
λ = 1.54178 Å) with the help of APEX2 Software Suite. The TLC was
carried out on precoated silica gel 60 F254 and silica gel 60 RP–18 F254
(Merck). Gravity column chromatography was performed with silica gel
60 (0.040–0.063 mm, Silicycle).
S
3.2. Plant material
Olaximbriside A (1): yellow crystal (recrystallized from chloroform);
The roots of Olax imbricata were collected in Hoa Hiep Nam
22
mp 98–100 °C, [a]D
+157.7 (c 0.27, CH3OH). HRESIMS m/z
249.1506 [M + H]+ (calcd for C15H20O3 + H, 249.1491). ECD (Δε)
(0.1 mg/mL, MeOH) 214 (Δε 19.0) nm; 1H and 13C NMR (DMSO–d6)
data, see Tables 1 and 2.
Table 3
Cytotoxicity of compound 1.
Compounds
IC50(μM)a
Olaximbriside
B (2): white amorphous powder. HRESIMS m/z
203.1427 [M–H2O + H]+, (calcd for C14H20O2–H2O + H, 203.1436).
ECD (Δε) (0.1 mg/mL, MeOH) 214 (+10.0), 225 (+4.0), 235 (−4.0)
and 284 (−8.0) nm; 1H and 13C NMR (DMSO–d6) data, see Tables 1 and
2.
MCF-7
HepG2
LU
1
16.32
0.89
0.81
0.12
34.25
1.63
1.21
0.04
7.98
1.42
0.40
0.20
Ellipticineb
a
Olaximbriside C (3): white amorphous powder, well dissolved in
: IC50 values were expressed as the mean values of three experiments SD.
: Ellipticine was tested as a positive control.
pyridine and did not in DMSO. HRESIMS m/z 421.1840 [M + Na]+
b
5

H.T.M. Nguyen et al.
Fitoterapia132(2019)1–6
(calcd for C20H30O8 + Na, 421.1838). ECD (Δε) (0.1 mg/mL, MeOH)
214 (+10.0), 230 (−8.0), 260 (−7.0), 284 (−10.0) nm; 1H and 13C
NMR (C5D5N) data, see Tables 1 and 2.
(human hepatocellular adenocarcinoma) and LU (human lung carci-
noma) cancer cell lines using the modified MTT method [15] with el-
lipticine used as a positive control.
Olaximbriside D (4): white amorphous powder, well dissolved in
pyridine and also in DMSO. HRESIMS m/z 421.1824 [M + Na]+ (calcd
for C20H30O8 + Na, 421.1838). ECD (Δε) (0.1 mg/mL, MeOH) 214
(+10.0), 230 (−25.0), 260 (−3.0) nm; 1H and 13C NMR (DMSO–d6
and in C5D5N) data, see Tables 1 and 2.
Acknowledgments
This work was financially supported by a grant from the Ministry of
Education and Training (No. B2017.SPK.04). Thanks are also extended
for Mr. Hoang Xuan Lam, Middle Vietnam Research and Manufacturing
Organic Medicinal Herb Centre for the gift of the root sample.
3.4. X-ray crystallographic analysis of compound 1
Single crystals of 1 were recrystallized from chloroform in the or-
thorhombic, space group I222 (no. 23) with unit cell parameters:
a = 8.2367(1), b = 17.8310(1), c = 23.1677(2) Å, V = 3402.61(5) Å3,
Z = 8, Dcalc = 1.145 g cm−3. Diffraction data were collected at 296(2)
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2018.11.007.
K
on a Bruker X8 PROSPECTOR Kappa CCD area-detector dif-
fractometer with an IμS X-ray microfocus source (Cu-Kα radiation,
λ = 1.54178 Å) with the help of APEX2 Software Suite.15 Data were
integrated with SAINT+, applied for absorption correction and scaled
by SADABS and then merged by XPREP, implemented in APEX2
Software Suite [16], yielding 3079 unique reflections (Rint = 0.0356).
Structure was solved by intrinsic phasing method with SHELXTL XT
[17] and refined anisotropically by full matrix least-squares on F2 with
SHELXTL XLMP [18]. The disordered solvents in large intermolecular
spaces were removed using PLATON SQUEEZE procedure [19]. The
structure refinement converged to a final R(F2) value of 0.0779 for 2385
data with F2 > 2σ(F2). Data have been deposited with the Cambridge
Crystallographic Data Centre (CCDC no. 1861567). Copies of these data
can be obtained, free of charge, on application to the CCDC via www.
ccdc.cam.ac.uk/conts/retrieving.html (or 12 Union Road, Cambridge
CB2 1EZ, UK, fax: +44 1223 336,033, e-mail: deposit@ccdc.cam.ac.
uk).
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The acid hydrolysis of compound 3 was carried out to obtain the
sugar residue. 3 (5 mg) was treated with HCl 0.2 M (dioxane/H2O, 1/1,
v/v, 200 μL) at 95 °C for 3 h. After cooling, the reaction mixture was
extracted with chloroform (3 × 2 mL) to eliminate the aglycone com-
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the hydrolyzed monosaccharide. The anomeric ratios were obtained by
manual integration with δH 5.26 (d, J = 3.5 Hz, 29.2%) and 4.66 (d,
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3.7.1. Cytotoxic assay
All isolated compounds were evaluated in vitro for their cytotoxic
potential against MCF-7 (human breast adenocarcinoma), HepG2
6