Lasianosides A–E: New iridoid glucosides from the leaves of lasianthus verticillatus (Lour.) Merr. and their antioxidant activity

Article abstract of DOI:10.3390/molecules24213995

The genus Lasianthus (Rubiaceae) consists of approximately 180 species, of which the greatest species diversity is found in tropical Asia. Some of the Lasianthus species have been used in folk medicine to treat tinnitus, arthritis, fever, and bleeding. Lasianthus verticillatus (Lour.) Merr. (Syn. Lasianthus trichophlebus auct. non Hemsl.) is a shrub, branchlets terete about 1.5–3 m in height. This paper studies the chemical composition of the leaves of L. verticillatus for the first time, which resulted in the isolation of five undescribed iridoid glucosides, lasianosides A–E (1–5), together with three known compounds (6–8). The undescribed structures of isolated compounds (1–5) were characterized by physical and spectroscopic data analyses, including one-dimensional (1D) and two-dimensional (2D) NMR, IR, UV, and high-resolution electrospray ionization mass spectra (HR-ESI-MS). Furthermore, the electronic circular dichroism data determined the absolute configurations of the new compounds. The free radical scavenging properties of isolated compounds was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and their cytotoxicity was assessed toward human lung cancer cell line A549 by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Among the isolated compounds, 3 and 4 displayed potent radical scavenging activities with IC₅₀ values of 30.2 ± 1.8 and 32.0 ± 1.2 μM, which were comparable to that of Trolox (29.2 ± 0.39 μM), respectively, while 5 possessed moderate activity with an IC₅₀ value of 46.4 ± 2.3 μM. None of the isolated compounds exerted cytotoxicity against human cell line A549. As a result, lasianosides C, D, and E have the potential to be non-toxic safe antioxidant agents.

Full text of DOI:10.3390/molecules24213995

molecules
Article
Lasianosides A–E: New Iridoid Glucosides from the
Leaves of Lasianthus verticillatus (Lour.) Merr. and
Their Antioxidant Activity
Gadah Abdulaziz Al-Hamoud 1,2, Raha Saud Orfali 2,*, Shagufta Perveen 2, Kenta Mizuno 3,
Yoshio Takeda 3, Tatsuo Nehira 4, Kazuma Masuda 4, Sachiko Sugimoto 1, Yoshi Yamano 1,
Hideaki Otsuka 1,5 and Katsuyoshi Matsunami 1,
*
1
Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734-8553, Japan; galhamoud@ksu.edu.sa (G.A.A.-H.); ssugimot@hiroshima-u.ac.jp (S.S.);
yamano@hiroshima-u.ac.jp (Y.Y.); otsuka-h@yasuda-u.ac.jp (H.O.)
2
3
4
5
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11495, Saudi Arabia;
Shagufta792000@yahoo.com
Faculty of Integrated Arts and Sciences, The University of Tokushima, 1-1 Minamijosanjima-Cho, Tokushima
770-8502, Japan; tokushis1@yahoo.co.jp (K.M.); takeda@ias.tokushima-u.ac.jp (Y.T.)
Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama,
Higashi-Hiroshima 739-8521, Japan; tnehira@hiroshima-u.ac.jp (T.N.); b163780@hiroshima-u.ac.jp (K.M.)
Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku,
Hiroshima 731-0153, Japan
*
Correspondence: rorfali@ksu.edu.sa (R.S.O.); matunamil@hiroshima-u.ac.jp (K.M.);
Tel.: +966-11-8055014 (R.S.O.); +81-82-257-5335 (K.M.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 7 October 2019; Accepted: 3 November 2019; Published: 5 November 2019
Abstract: The genus Lasianthus (Rubiaceae) consists of approximately 180 species, of which the
greatest species diversity is found in tropical Asia. Some of the Lasianthus species have been
used in folk medicine to treat tinnitus, arthritis, fever, and bleeding. Lasianthus verticillatus (Lour.)
Merr. (Syn. Lasianthus trichophlebus auct. non Hemsl.) is a shrub, branchlets terete about 1.5–3 m
in height. This paper studies the chemical composition of the leaves of L. verticillatus for the
first time, which resulted in the isolation of five undescribed iridoid glucosides, lasianosides
A–E (
1
–
5), together with three known compounds (
6–8). The undescribed structures of isolated
compounds (1–5) were characterized by physical and spectroscopic data analyses, including
one-dimensional (1D) and two-dimensional (2D) NMR, IR, UV, and high-resolution electrospray
ionization mass spectra (HR-ESI-MS). Furthermore, the electronic circular dichroism data determined
the absolute configurations of the new compounds. The free radical scavenging properties of
isolated compounds was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
assay, and their cytotoxicity was assessed toward human lung cancer cell line A549 by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Among the isolated
compounds,
and 32.0 1.2
possessed moderate activity with an IC50 value of 46.4
exerted cytotoxicity against human cell line A549. As a result, lasianosides C, D, and E have the
potential to be non-toxic safe antioxidant agents.
3
and
M, which were comparable to that of Trolox (29.2
2.3 M. None of the isolated compounds
4
displayed potent radical scavenging activities with IC50 values of 30.2
±
1.8
5
±
µ
±
0.39 M), respectively, while
µ
±
µ
Keywords: Lasianthus trichophlebus; Rubiaceae; DPPH; radical scavenging activity; cytotoxicity
Molecules 2019, 24, 3995; doi:10.3390/molecules24213995
www.mdpi.com/journal/molecules

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1. Introduction
Reactive oxygen species (ROS) are derived from exogenous (environmental pollution, cigarette
smoke, UV irradiation, and toxic chemicals) and intracellular (mitochondrial energy generation)
sources. The excessive ROS accumulation leads to oxidative stress through the reaction of ROS with
biomolecules such as cell membrane lipids, proteins, and DNA, which causes a variety of chronic
and degenerative disorders including cataract, rheumatoid arthritis, cancer, cardiovascular diseases,
neurodegenerative disorders, and also aging. Therefore, a supplementation of exogenous antioxidants
as free radical scavengers is thought to be an effective measure for preventing and repairing the
damages caused by ROS [1].
Lung cancer is one of the leading causes of cancerous death for both the male and female in
the world. Treatment options for lung cancer include surgery, chemotherapy, and radiation therapy.
However, the prognosis is still unsatisfactory, probably because of its high invasion and metastatic
activity to various organs (e.g., brain, lymph nodes, bones, and liver), the ability to evade apoptosis,
the resistance to antitumor drugs, and the severe side effects of antitumor drugs and radiotherapy
themselves. Therefore, there is an increased and continued demand for new agents to treat and prevent
this life-threatening disease [2].
A great number of natural bioactive compounds have been isolated from various natural sources
and have contributed to the development of more than 60% of current clinical drugs [3]. Thus, we
still rely on Mother Nature, including plants’ secondary metabolites for the discovery of seed and
lead chemicals in serendipitous way. Plants of the genus Lasianthus (Rubiaceae) are distributed in
Asia, Africa, America, and Australia, with about 180 species in total. Most of the species are found in
tropical Asia, while only one species exists in Australia [
4,
5]. Some of the species have been used in
folk medicine to treat tinnitus, arthritis, fever, and bleeding [6–9]. Previous chemical studies on various
species of genus Lasianthus have resulted in the isolation of iridoids with diverse skeletons, especially
asperuloside and deacetyl asperulosidic acid, in addition to bis-iridoid glucosides, anthraquinones,
megastigmane glucosides, and terpenes [8,10–15]. L. verticillatus (Lour.) Merr. (Syn. L. trichophlebus
auct. non Hemsl.) is a shrub, branchlets terete of about 1.5–3 m in height [16]. L. verticillatus has been
used as a traditional, analeptic, or restorative medicine in Northern Thailand and India [17]. Recently,
Napiroon et al. reported thin-layer chromatography (TLC) and HPLC analyses of the lipophilic fraction
of the leaves of L. verticillatus to reveal the presence of terpenoids, alkaloids, phenolic compounds, and
coumarines by the reaction color of spraying reagents. The seed germination and seedling growth of
weeds and anti-fungal activity against a plant pathogen, Colletotrichum gloeosporioides, were reported.
However, a detailed chemical analysis of the secondary metabolites and their biological activities has
not yet been performed. This study is the first report on the chemical constituents of the polar fraction
(1-BuOH) of the leaves of L. verticillatus, and afforded five new iridoid glucosides, lasianosides A–E
(
1
–
5
), together with three known compounds, an iridoid (
designated hereafter as lasianol by the author, YT), deacetyl daphylloside (
) [18] (Figure 1). This paper deals with the structural elucidation of isolated compounds including
6
) (compound
2
in Takeda et al., 2002 [11],
7), and daphylloside
(8
the absolute configuration of new compounds, and the evaluation of their antioxidant activities by
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, and cytotoxic activity against
human lung cancer cell line (A549) that is frequently used for the study of lung cancer and the initial
drug screening, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

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R1
R2
Glc
H
1
2
6
H
R1
H
Glc
H
7
8
H
Ac
R1
H
R2
H
3
4
5
Ac
H
H
Ac
Figure 1. Isolated compounds from Lasianthus verticillatus (1–8).
2. Results and Discussion
2.1. Isolation and Spectroscopic Analyses of the Compounds
The 1-BuOH fractions of methanolic extract of Lasianthus verticillatus (Lour.) Merr. leaves
were separated by solvent fractionation, various column chromatographies (CC), and HPLC to afford
compounds 1–8 (Figure 1). The chemical structures of the isolated compounds were investigated and
identified through intensive spectroscopic analyses based on UV-visible, one-dimensional (1D) and
two-dimensional (2D) NMR, and high-resolution electrospray ionization mass spectra (HR-ESI-MS) as
follows (Figures S1–S48).
2.1.1. Chemical Structure of Lasianoside A
22
Lasianoside A (
1
), [α
]
D–20.6, was obtained as viscous colorless syrup. Its molecular formula
was determined as C16H24O10 based on a sodium adduct ion at m/z 399.1263 [M + Na]+ (calcd for
C16H24O10Na 399.1262) in HR-ESI-MS, which suggests five degrees of unsaturation. The IR spectrum
exhibited absorption bands at 3390 and 1748 cm−1, which indicated the presence of hydroxy and a
1
five-membered lactone ring, respectively. H-NMR spectrum displayed signals of four methylenes,
including a methylene at δH 2.75 (br d, J = 18.1 Hz) and 2.95 (dd, J = 18.1, 6.3 Hz), and three methylenes
attached to oxygen at δH 3.91 (2H, d, J = 3.9 Hz), 4.20 (br d, J = 13.4 Hz), 4.24 (br d, J = 13.4 Hz), 4.28 (br
d, J = 12.0 Hz), and 4.61 (br d, J = 12.0 Hz) (Table 1). Furthermore, the 1H-NMR data supported the
existence of three methines; two of them showed signals at δH 2.99 (m) and 3.73 (d, J = 6.3 Hz), and an
oxygenated methine proton at δH 5.14 (t, J = 6.3 Hz).
Taking into consideration the above-mentioned molecular formula and degree of unsaturation, the
16 carbon signals in the 13C-NMR spectrum are thus ascribable to the presence of three oxymethylenes
(
δ
c 58.4, 63.3, 64.4), an oxymethine (
134.4, 140.2) and carbonyl carbon ( c 180.9) (Table 2). The COSY (correlation spectroscopy) (Figure 2)
correlations between H2-3/H-4 and H-4/H-5 together with the HMBC (heteronuclear multiple bond
connectivity correlation) (Figure 2) correlations from H-5 to C-11 ( c 180.9), C-3 ( c 63.3), C-8 ( c 140.2),
and C-9 ( c 134.4) and from H-6 to C-11 ( c 180.9) indicated that shared the same core structure as
δc 83.2), as well as two unsaturated quaternary carbons (δc
δ
δ
δ
δ
δ
δ
1

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6
[11]. Moreover, the 1D NMR data of
1
also gave an anomeric proton signal at δH 4.30 (d, J = 7.9 Hz),
in addition to six carbon signals at
δ
c
103.8, 78.0, 78.0, 75.1, 71.6, and 62.8 that belong to a glucose
moiety, which means that
of glucose H-10 (δH 4.30) to C-1 (
on C-1. The presence of a D-glucose moiety was confirmed by the acid hydrolysis of
HPLC analysis with a chiral detector. The coupling constant (J = 7.9 Hz) of H-10 indicated
for glucose moiety. The relative configuration was established by NOESY analysis (Figure 3). The
correlation between H-3/H-5, H-5/H-6, and H-6/H-7 suggested a -orientation, while the absence of
correlation signals between H-4 and H-6 suggested a -orientation. The chemical shift values and the
coupling patterns of aglycone moiety were essentially superimposable to except for the glucosylation
position C-1, which supported the relative stereochemistry of , as shown in Figure 1. The absolute
stereochemistry of was elucidated by comparison of the circular dichroism (CD) spectrum. The
positive cotton effect at 221 nm (∆ε = + 0.854) showed the same absolute stereochemistry with lasianol
) [11], and consequently the structure of was determined to be a (4R, 5R, 6S) configuration, i.e.,
1-O-β-d-glucopyranoside of 6.
1
was the glucoside of
c 64.4) of the aglycone moiety indicated the glucosylation position
followed by
linkage
6. The HMBC correlation from the anomeric proton
δ
1
β
β
α
6
1
1
(6
1
Table 1. 1H NMR data of compounds 1–5 (500 MHz, CD3OD, δ in ppm, J in Hz).
Position
1
2
3
4
5
4.28 br d (12.0)
4.61 br d (12.0)
4.16 br d (13.0)
4.33 br d (13.0)
3.93 dd (9.7, 5.5)
4.23 dd (9.7, 4.0)
3.00 m
3.78 br d (6.3)
5.21 td (6.3, 1.0)
4.16 br d (13.0)
4.27 br d (13.0)
4.02 dd (10.0, 5.0)
4.13 m
4.18 br d (13.4)
4.28 br d (13.4)
4.04 dd (10.0, 5.0)
4.13 dd (10.0, 3.4)
2.99 m
1
4.75 br s
4.04 dd (10.1, 3.5)
4.09 dd (10.1, 4.4)
2.94 m
3.68 br d (6.3)
5.13 t (6.3)
3
3.91 2H d (3.9)
4
5
6
2.99 m
3.73 br d (6.3)
5.14 t (6.3)
2.98 m
3.75 m
5.16 t (6.4)
3.76 m
5.15 t (6.4)
2.75 br d (18.1)
2.95 dd (18.1,
2.73 br d (18.1)
2.94 dd (18.1, 6.3)
2.69 br d (18.0)
2.85 dd (18.0, 6.4)
2.69 br d (18.3)
2.82 dd (18.3, 6.3)
2.62 br d (18.0)
2.82 dd (18.0, 6.4)
7
6.3)
8
9
-
-
-
-
-
-
-
-
-
-
4.20 br d (13.4)
4.24 br d (13.4)
-
4.11 br d (13.7)
4.19 br d (13.7)
10
4.20 2H, br s
-
4.13 2H, br s
-
4.67 2H, br s
11
12
13
10
-
-
-
2.04 3H, s
4.36 d (8.0)
3.24 dd (9.1, 8.0)
3.40 m
-
2.03 3H, s
4.63 d (7.9)
3.23 dd (9.0, 7.9)
3.40 m
4.30 d (7.9)
3.18 dd (9, 7.9)
3.37 t (8.8)
4.33 d (7.9)
3.21 dd (9.1, 7.9)
3.38 t (9.1)
4.37 d (8.0)
3.23 t (8.0)
3.40 m
3.39 m
3.56 m
20
30
40
3.28 m
3.29 m
3.30 m
3.31 m
3.39 m
3.56 m
3.39 m
3.56 m
50
3.68 dd (12, 5.5)
3.90 dd (12, 1.8)
3.72 dd (12, 5.2)
3.89 dd (12, 1.3)
4.34 dd (12.0, 5.7)
4.57 dd (12.0, 1.8)
-
4.43 dd (12.0, 5.7)
4.59 dd (12.0, 2.0)
-
4.35 dd (11.9, 6.0)
4.56 dd (11.9, 2.0)
-
60
1”
2”
3”
4”
5”
6”
7”
8”
9”
7.07 d (1.7)
-
7.05 d (2.0)
-
7.06 d (2.0)
-
-
-
-
6.80 d (8.2)
6.98 dd (8.2, 1.7)
7.57 br d (15.9)
6.30 br d (15.9)
-
6.80 d (8.2)
6.97 dd (8.2, 2.0)
7.56 br d (15.9)
6.28 br d (15.9)
-
6.80 d (8.2)
6.97 dd (8.2, 2.0)
7.56 br d (15.9)
6.30 br d (15.9)
-
m: multiplet or overlapped signals.

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Figure 2. Correlation spectroscopy (COSY) and heteronuclear multiple bond connectivity correlation
(HMBC) correlations of 1–5.

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7
6
7
6
5
5
1
1
4
4
3
3
Aglycone
Aglycone
1
2
7
6
5
4
1
3
4
Aglycone
7
6
5
4
1
3
Aglycone
5
Figure 3. Key NOESY correlations of 1–5.
2.1.2. Chemical Structure of Lasianoside B
22
Lasianoside B (
2), [
α
]
D–26.6, was isolated as viscous colorless syrup and displayed the same
in HR-ESI-MS at m/z 399.1265 [M + Na]+ (calcd for C16H24O10 Na
399.1262). Analysis of 1D and 2D NMR data (Tables 1 and 2) revealed that the data of were very
similar to those of except for apparent differences for the signals of H2-1 [δH 4.16 (br d, J = 13.0 Hz),
4.33 (br d, J = 13.0 Hz)] and H2-3 [δH 3.93 (dd, J = 9.7, 5.5 Hz) and 4.23 (dd, J = 9.7, 4.0 Hz)] in
compared to the corresponding signals of H2-1 and H2-3 in . The downfield shift of H2-3 (δH 3.93 and
4.23) suggested that the glucose moiety in was linked at C-3 instead of C-1 as in . This assumption
was confirmed by HMBC correlation from anomeric proton H-10 (δH 4.33) to C-3 (
c 70.5) (Figure 2).
The relative and absolute configurations of were determined to be the same as those of by NOESY
-d-glucopyranoside
molecular formula C16H24O10 of
1
2
1
2
1
2
1
δ
2
1
(Figure 3) and CD spectral data; thus, the structure of 2 was deduced to be 3-O-β
of 6.
2.1.3. Chemical Structure of Lasianoside C
22
Lasianoside C (
3
), [α
]
D–10.2, was obtained as viscous pale yellow syrup; it had a molecular
formula of C25H30O13 with 11 degrees of unsaturation as determined by HR-ESI-MS (measured at m/z:

Molecules 2019, 24, 3995
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561.1572 [M+Na]+, calcd for C25H30O13Na: 561.1579). The UV spectrum showed absorption maxima
at 329, 300, and 245 nm, suggesting the presence of an aromatic ring. In the same way, the IR spectrum
of
presence of hydroxy, a five-membered lactone ring, conjugated ester carbonyl,
carbons, and an aromatic ring, respectively. The 13C-NMR spectrum closely resembled that of
for the presence of an additional eight sp2 signals (
c 114.8, 115.2, 116.5, 123.1, 127.6, 146.8, 147.3, and
149.7) together with a carboxyl carbon signal ( c 169.1) (Table 2). Thus, compound was expected
to be an acylated derivative of showed three ABX-coupled
. Moreover, the 1H-NMR spectrum of
3
exhibited absorption bands at 3356, 1745, 1712, 1623, 1600, and 1512 cm−1, which suggested the
α
,
β-unsaturated olefinic
2, except
δ
δ
3
2
3
aromatic protons, H-200 at δH 7.07 (d, J = 1.7 Hz), H-500 at δH 6.80 (d, J = 8.2 Hz), and H-600 at δH 6.98
(dd, J = 8.2, 1.7 Hz), and two trans-olefinic protons, H-700 at δH 7.57 (br d, J = 15.9 Hz) and H-800 at δH
6.30 (br d, J = 15.9 Hz) (Table 1). This data implied that the acyl moiety is a trans-caffeoyl group. The
esterification position was indicated to be at C-60 of the glucose moiety, due to the downfield shift of
H2-60 to δH 4.34 (dd, J = 12.0, 5.7 Hz) and 4.57 (dd, J = 12.0, 1.8 Hz). This assumption was confirmed
by HMBC correlation from H2-60 (δH 4.34 and 4.57) to carbonyl carbon C-900
glucosylation position was indicated to be at C-3 due to HMBC correlation from anomeric proton
H-10 (δH 4.37) to C-3 (
c 71.2) (Figure 2). The acid hydrolysis of released d-glucose, while alkaline
hydrolysis afforded caffeic acid. These results were identified by HPLC comparing with authentic
(δc 169.1), while the
δ
3
samples. The configuration of glucopyranose was assigned to be
β according to the coupling constant
of the anomeric proton H-10 at δH 4.37 (d, J = 8.0 Hz), while the caffeoyl configuration was determined
as an E configuration according to the coupling constant of the olefinic protons H-700 and H-800 (J =
15.9 Hz). The relative and absolute configurations of
3
were determined by comparing its NOESY
correlation (Figure 3) and CD spectrum to those of 2. Therefore, the structure of 3 was established to be
60-caffeoyl-3-O-β-d-glucopyranoside of 6, which was designated as lasianoside C.
2.1.4. Chemical Structure of Lasianoside D
22
Lasianoside D (
4
), [
α
]
D–22.9, was also obtained as viscous pale yellow syrup with the molecular
formula C27H32O14 with 12 degrees of unsaturation, which was deduced by the molecular ion peak
1
[M+Na]+ at m/z: 603.1685 (calcd for C27H32O14Na: 603.1684) in HR-ESI-MS. Analysis of H and
13C-NMR (Tables 1 and 2), together with the molecular formula revealed that compound
4
was an
acetyl derivative of
correlation to the acetyl carbonyl carbon at
derivative of . Further analyses of NMR data suggested that the other part of
of . The relative and absolute configurations of
3. In the HMBC spectrum (Figure 2), downfield-shifted H2-1 (δH 4.75) showed
δc 172.6, which indicated that
4
should be a 1-acetyl
3
4 was the same as that
3
4
were also identified to be the same as those of
3
by
comparing the NOESY (Figure 3) and CD spectral data with those of 3; thus, the structure of 4
was
elucidated to be 1-acetyl-60-caffeoyl-3-O-β-d-glucopyranoside of 6, and designated as lasianoside D.
2.1.5. Chemical Structure of Lasianoside E
22
Lasianoside E (
5
), [α
]
D–24.5, has the same molecular formula as that of
4
by HR-ESI-MS (m/z:
603.1682 [M + Na]+, calcd for C27H32O14Na: 603.1684). A further comparison of NMR data (Tables 1
and 2) displayed that compounds and shared the same functional group. In HMBC spectrum
(Figure 2), a significant downfield shift of H2-10 (δH 4.67) showed correlation to acetyl carbonyl carbon
at c 172.6. Detailed analyses of 2D NMR data indicated that the other part of was the same as of
that of . The relative and absolute configurations of were determined by comparison of the NOESY
5
4
δ
5
4
5
experiment (Figure 3) and CD data with those of 4. Therefore, the structure of 5 was elucidated to be
10-acetyl-60-caffeoyl-3-O-β-d-glucopyranoside of 6, and designated as lasianoside E.
2.2. Antioxidant and Cytotoxic Activities of Isolated Compounds
Compounds
scavenging activities. As shown in Table 3, lasianosides C and D (
scavenging activities (IC50: 30.2 1.8 and 32.0 1.2 M, respectively) that were comparable with those
1
–8
from the 1-BuOH fraction of L. verticillatus were evaluated for their DPPH radical
3
and ) displayed potent radical
4
±
±
µ

Molecules 2019, 24, 3995
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of standard Trolox (IC50: 29.2
±
0.39
µ
M), while compound
5
exhibited moderate scavenging activity
(IC50: 46.4
scavenging properties (IC50 >100
may be related to the presence of caffeoyl moieties in their structures [19,
±
2.3
µ
M). Moreover, the remaining compounds did not possess significant DPPH radical
M). These results suggested that the radical scavenging activities of
20]. On other hand, none
µ
3
–5
of the isolated compounds exhibited cytotoxic activity against human lung cancer cell line (A549) as
shown in Table 3, which coincided with our preliminary result for 1-BuOH extract (IC50 > 100 µM).
Table 2. 13C-NMR data of compounds 1–5 (175 MHz, CD3OD, δ in ppm).
Position
1
2
3
4
5
1
3
64.4
63.3
56.8
70.5
58.4
71.2
59.4
71.2
57.1
71.3
4
48.4
46.8
46.9
47.0
46.8
5
53.4
53.0
53.4
53.4
53.3
6
83.2
83.3
83.4
83.3
83.2
7
41.8
41.7
41.6
41.5
41.7
8
9
140.2
134.4
58.4
138.5
137.4
58.4
138.5
137.4
57.0
142.0
132.5
58.5
133.4
140.5
60.9
10
11
12
13
10
20
30
40
50
60
1”
2”
3”
4”
5”
6”
7”
8”
9”
180.9
180.2
180.2
180.0
172.6
20.8
104.8
75.0
77.8
71.6
75.5
64.2
127.6
115.2
146.9
149.8
116.5
123.1
147.3
114.8
169.0
180.0
172.6
20.7
104.9
75.0
77.7
71.6
75.5
64.3
127.6
115.2
146.9
149.7
116.5
123.1
147.3
114.8
169.1
103.8
75.1
78.0
71.6
78.0
62.8
104.6
75.0
77.9
71.5
78.1
62.7
104.8
75.0
77.7
71.6
75.5
64.3
127.6
115.2
146.8
149.7
116.5
123.1
147.3
114.8
169.1
Table 3. Bioactivities of isolated compounds 1–8 (n = 3) *. DPPH: 1,1-diphenyl-2-picrylhydrazyl.
Isolated Compounds
DPPH (IC50 µM)
A549 Cytotoxicity (IC50 µM)
Lasianoside A (1)
Lasianoside B (2)
Lasianoside C (3)
Lasianoside D (4)
Lasianoside E (5)
Lasianol (6)
>100
>100
30.2 ± 1.8
32.0 ± 1.2
46.4 ± 2.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
-
Deacetyl daphylloside (7)
Daphylloside (8)
Trolox
>100
>100
29.2 ± 0.39
Doxorubicin
-
0.90 ± 0.02
* The results were obtained from three independent triplicate experiments, and the IC50 values were calculated by
linear regression on Excel software.

Molecules 2019, 24, 3995
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3. Materials and Methods
3.1. General Method
UV and IR spectra were obtained on a Jasco V-520 UV/Vis spectrophotometer and a Horiba
FT-710 Fourier transform infrared spectrophotometer (Horiba, Kyoto, Japan), respectively. Optical
rotations data were measured on a JASCO P-1030, while CD spectra were recorded on a Jasco J-720
circular dichroism spectrometer (Jasco, Tokyo, Japan). NMR experiments were measured on a Bruker
AVANCE 500-MHz and 700-MHz spectrometers, with tetramethylsilane (TMS) as an internal standard
(Bruker, Billerica, MA, USA). Positive ion HR-ESI-MS spectra were recorded using an LTQ Orbitrap
XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Column chromatography (CC)
was performed on a Diaion HP-20 (Mitsubishi chemical Corp., Japan), silica gel 60 (230–400 mesh,
Merck, Germany), and octadecyl silica (ODS) gel (Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan;
Φ
= 35 mm, L = 350 mm), and TLC was performed on precoated silica gel plates 60 GF254 (0.25 mm
in thickness, Merck). HPLC was performed on ODS gel (Cosmosil 5C18-AR, Nacalai Tesque, Kyoto,
10 mm, 250 mm, flow rate 2.5 mL/min) with a mixture of H2O, acetone, and MeOH, and the elute was
monitored by refractive index and/or a UV detector. An amino column (Shodex Asahipak NH2P-50
4E (4.6 mm
×
250 mm), CH3CN-H2O (3:1) 1mL/min) was used together with a chiral detector (Jasco
OR-2090plus) for the HPLC analysis of sugars obtained after hydrolysis.
3.2. Plant Material
Leaves of Lasianthus verticillatus were collected from Iriomote Island, Okinawa Prefecture, Japan, in
September 2000. The voucher specimen (IR0009-LT) was deposited at the department of pharmacognosy,
faculty of pharmaceutical sciences, Hiroshima university.
3.3. Extraction and Isolation
The air-dried and powdered leaves of L. verticillatus (Lour.) Merr. (7.0 kg) were extracted by
maceration with MeOH (98 L
with 3 L of n-hexane. The remaining solution was evaporated and re-suspended in 1 L of H2O and
×
2) and concentrated to 90% MeOH solution; then, they were defatted
extracted with 3 L of EtOAc and 3 L of 1-BuOH, successively.
Part of the 1-BuOH fraction (124.5 g) was fractionated on a Diaion HP-20 column (
Φ = 10 cm,
L = 350 cm, 2.5 kg), and eluted with H2O (15 L), followed by an MeOH/H2O step gradient solvent
systems (10%, 20%, 30%, 40%, 60%, and 100% MeOH, 15 L each, 1-L fractions being collected), similar
fractions were grouped together to give 20 fractions (Fr. Lt1–Lt20). Fraction Lt8 (18.7 g) was proceeded
on silica gel CC (
Φ = 4.5 cm, L = 50 cm, 400 g), started with CHCl3 (2.5 L), and followed by CHCl3/MeOH
developing solvent systems (7%, 10%, 15%, 20%, 30%, and 100% MeOH, 2.5 L each) to obtain 16
fractions (Fr. Lt8.1–Lt8.16). Each fraction of Lt8.12 (681 mg) and Lt8.13 (240 mg) was subjected to open
reversed phase (ODS) CC with 10% aq. methanol (400 mL) to 100% methanol (400 mL), linear gradient,
to give (Frs. Lt8.12.1–Lt8.12.6 and Frs. Lt8.13.1–Lt8.13.6, respectively). The residue Lt8.12.2 (131 mg)
was purified by HPLC (ODS) with 5% aq. methanol to give
mg) was purified by HPLC (ODS) with 5% aq. methanol to give
(4.3 g) was proceeded on silica gel CC ( = 4 cm, L = 40 cm, 230 g), started with CHCl3 (1.5 L), and
1
(4.0 mg), while residue Lt8.13.2 (174
6
(10.4 mg), (5.0 mg). Fraction Lt13
2
Φ
followed by CHCl3/MeOH developing solvent systems (5%, 7%, 10%, 15%, 20%, 30%, 40%, and 100%
MeOH, 1.5 L each) to give 14 fractions (Frs. Lt13.1–Lt13.14). The residue Lt13.5 (455 mg) was purified
by preparative HPLC (ODS) with 15% aq. acetone to give
Lt13.10 (215 mg) was purified by preparative HPLC (ODS) with 30% aq. methanol to give
Fraction Lt15 (7.2 g) was chromatographed on silica gel CC ( = 5.2 cm, L = 38 cm, 350 g), started with
7
(5.8 mg) and
8
(23.4 mg). The other residue
3
(6.0 mg).
Φ
CHCl3 (2.5 L), and followed by CHCl3/MeOH developing solvent systems (5%, 10%, 12%, 15%, 20%,
30%, and 100% MeOH, 2.5 L each) to obtain 12 fractions (Frs. Lt15.1–Lt15.12). Fraction Lt15.5 (656 mg)
was rechromatographed on silica gel CC (
Φ = 2.5 cm, L = 50 cm, 120 g), started with CHCl3 (500 mL),
and followed by CHCl3/MeOH developing solvent systems (5%, 7%, 10%, 12%, and 100% MeOH,

Molecules 2019, 24, 3995
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500 mL each) to give 11 fractions (Frs. Lt15.5.1–Lt15.5.11). Fraction Lt15.7 (577 mg) was separated by
HPLC (ODS) with 28% aq. acetone to give (3.0 mg). The fraction LtB17 (6.1 g) was further purified by
silica gel column chromatography ( = 5 cm, L = 40 cm, 380 g), eluting with a stepwise CHCl3/MeOH
4
Φ
gradient (100:0 to 70:30, 2.4 L each) to obtain 11 fractions (Frs. LtB17.1–LtB17.11). The residue LtB17.7
(839 mg) was separated by HPLC, 28% aq. acetone to obtain 5 (7.0 mg).
3.4. Spectroscopic Data of Compounds 1–5
22
Lasianoside A (
1
) Viscous colorless syrup [
α
]
–20.6 (c 0.30, MeOH); HR-ESI-MS: m/z: 399.1263
D
[M+Na]+ (calcd for C16H24O10Na, 399.1262); CD λmax (c 2.66
×
10−5 M, MeOH) nm (∆ꢀ): 270 (–0.059),
221 (+0.854); IR (film) νmax: 3390, 2921, 1748, 1194, 1072, 1032 cm−1; 1H and 13C data, see Tables 1 and 2.
22
Lasianoside B (
2
) Viscous colorless syrup [
α
]
D –26.6 (c 0.30, MeOH); HR-ESI-MS: m/z: 399.1265 [M+Na]+
(calcd for C16H24O10Na, 399.1262); CD λmax (c 2.66
×
10−5 M, MeOH) nm (∆ꢀ): 281 (–0.026), 217 (+1.99);
IR (film) νmax: 3372, 2927, 1754, 1171, 1078, 1024 cm−1; 1H and 13C data, see Tables 1 and 2.
22
Lasianoside C (
3
) Viscous pale yellow syrup [
α]
–10.2 (c 0.34, MeOH); HR-ESI-MS: m/z: 561.1572
D
[M+Na]+ (calcd for C25H30O13Na, 561.1579); CD λmax (c 1.85
221 (+6.155); UV (MeOH) λmax nm (log ꢀ): 329 (4.74), 300 (4.63), 245 (4.59); IR (film) νmax: 3356, 2913,
1745, 1712, 1623, 1600, 1512, 1360, 1274, 1165, 1032 cm−1; 1H and 13C data, see Tables 1 and 2.
×
10−5 M, MeOH) nm (∆ꢀ): 283 (–0.357),
22
Lasianoside D (
4
) Viscous pale yellow syrup [
α
]
–22.9 (c 0.27, MeOH); HR-ESI-MS: m/z: 603.1685
D
[M+Na]+ (calcd for C27H32O14Na, 603.1684); CD λmax (c 1.40
208 (+1.010); UV (MeOH) λmax nm (log ꢀ): 329 (4.37), 295 (4.27), 233 (4.42); IR (film) νmax: 3396, 2920,
1746, 1732, 1713, 1634, 1608, 1518, 1355, 1264, 1163, 1032 cm−1; 1H and 13C data, see Tables 1 and 2.
×
10−5 M, MeOH) nm (∆ꢀ): 276 (–0.105),
22
Lasianoside E (
5
) Viscous pale yellow syrup [
α]
–24.5 (c 0.40, MeOH); HR-ESI-MS: m/z: 603.1682
D
[M+Na]+ (calcd for C16H24O10Na, 603.1684); CD λmax (c 1.72
214 (+1.324); UV (MeOH) λmax nm (log ꢀ): 331 (3.98), 292 (3.90), 240 (3.96); IR (film) νmax: 3361, 2928,
1747, 1738, 1714, 1625, 1602, 1509, 1365, 1271, 1179, 1030 cm−1; 1H and 13C data, see Tables 1 and 2.
×
10−5 M, MeOH) nm (∆ꢀ): 281 (–0.297),
3.5. Acid Hydrolysis
The isolated compounds
The reaction mixtures were neutralized with amberlite IRA96SB (OH
1
–
5
(1.0 mg each) were hydrolyzed with 1 M HCl (1.0 mL) at 80 ◦C for 3 h.
form), and the resin was removed
¯
by filtration. Then, the filtrate was extracted with an equal volume of EtOAc. The aqueous layers
were analyzed by HPLC with an amino column [Asahipak NH2P-50 4E, CH3CN-H2O (3:1), 1mL/min]
and a chiral detector (JASCO OR-2090plus) in comparison with d-glucose as authentic standard. The
aqueous layers of hydrolyzed compounds showed a peak at tR 8.15 min, which coincided with that of
d-glucose [21].
3.6. Alkaline Hydrolysis
Compounds 3, 4, and 5 (3.0 mg each) were prepared as solutions in 50% aqueous 1,4-dioxane
(0.5 mL). These solutions were treated with 10% aqueous KOH (0.5 mL) and stirred at 37 ◦C for 1 h.
The reaction mixtures were neutralized with an ion-exchange resin (Amberlite IR-120B (H+-form), and
then filtrated and evaporated. The residues were dissolved in EtOAc and subjected to HPLC analysis
[Cosmosil C18-PAQ, MeOH-H2O (45:50), with 0.1% TFA, 1mL/min)] with UV (245 nm) detector to
identify the caffeoyl moieties at tR 11.42 min [22].
3.7. DPPH Radical Scavenging Activity
Free radical scavenging activity was evaluated by using a quantitative DPPH assay. The absorbance
of various concentrations of test compounds dissolved in 100
was measured at 515 nm as (Ablank). Then, 100 L of DPPH solution (200
well. The plate was incubated in a dark chamber at room temperature for 30 min before measuring the
µL of MeOH in a 96-well microtiter plate
µ
µM) was added to each

Molecules 2019, 24, 3995
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absorbance (Asample) again. The following equation was used to calculate the percentage inhibition of
free radicals:
% Inhibition = [1 − (Asample − Ablank)/(Acontrol − Ablank)] × 100
where Acontrol is the absorbance of control (DMSO and all reagents, except for the test compound).
Trolox was used as the positive control. Their IC50 values were determined based on three independent
experiments [23].
3.8. Cytotoxicity
Human lung cancer cell proliferation assay was carried out by means of colorimetric MTT assay
to evaluate both the potential for antitumor agents and the safety against human cells. The cell
line (A549) was established through explant culture of lung carcinomatous tissue from a 58-year-old
Caucasian male and frequently used for in vitro antitumor drug screening. In this study, we also
aimed to elucidate the safety of our antioxidant compounds using this human cell line because of the
ease of obtaining and culturing indefinitely rather than normal and primary cultures. The cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated
fetal bovine serum, amphotericin B (5.6
various concentrations of test compounds were incubated with A549 cells (5
CO2 incubator at 37 ◦C for 72 h. Then, the cell shape, approximate number, and color of supernatants
were checked by microscope to avoid an over or underestimation of the result obtained by the following
colorimetric evaluation. Then, an MTT solution (0.5 mg/mL) was replaced in each well, and the
plate was further incubated for 1.5 h to generate formazan crystals. Subsequently, the medium was
µ
g/mL), and kanamycin (100
µ
g/mL). In a 96-well plate, the
×
103 cells/well) in a 5%
completely removed; then, 100 µL of DMSO was added to each well to dissolve the crystals. The
optical density (OD) values were measured at 540 nm using a microplate reader. The percentage (%)
inhibition of cell growth was calculated using the following equation:
% Inhibition = [1 − (Asample − Ablank)/(Acontrol − Ablank)] × 100
where Acontrol is the absorbance of control (DMSO and all reagents, except for the test compound).
Their IC50 values were determined based on three independent experiments [24].
4. Conclusions
In this study, the chemical composition of L. verticillatus leaves was studied for the first time and
resulted in the isolation of five undescribed iridoid glucosides, lasianosides A–E (
three known compounds ( ). The chemical structures of the new compounds were elucidated on the
basis of NMR and MS studies. The absolute configuration of the new compounds was determined
by the CD method. The isolated five new compounds ( ) have the same core structure lasianol ( ),
1–5), together with
6–8
1
–5
6
which is an iridane skeleton. It was first isolated from Lasianthus wallichii by our previous research [11].
Lasianol and its derivatives have never been isolated afterward until our present work. The uniqueness
of the structure may be produced through the biosynthetic pathway depicted in Figure 4. The branching
point is the form of intermediate iridotrial, e.g., hemiacetal and keto forms for iridoid and the iridane
skeleton, respectively.

Molecules 2019, 24, 3995
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a)
CHO
H
C
O
H
iridotrial
H
H
H
CHO
H2O
[O]
[O]
H
NADPH
CHO
CHO
NADPH
CHO
iridodial
(keto form)
C
O
H-
HO
geraniol
[O]
H+
10-oxogeranial
(keto form)
-H2
O
HO
HO
-H2O
O
COOH
H
COOH
H
H
H
H
H
Lactonization
OH
NADPH
CHO
H
OH
CHO
HO
OH
HO
CHO
iridane
(monocyclic form)
HO
OH
6
O
HO
iridoid
(hemiacetal form)
UDP-Glc
UDP-Glc
O
O
H
O
O
H
H
H
OH
OGlc
HO
OGlc
HO
OH
1
2
Caffeoyl-CoA
O
O
O
O
O
O
H
H
H
H
H
H
O
O
O
HO
OH
HO
OAc
AcO
OH
O
O
O
O
Acetyl-CoA
O
O
Acetyl-CoA
O
OH
O
OH
O
OH
HO
HO
HO
OH
OH
OH
OH
OH
4
3
5
OH
OH
OH
O
OH
b)
OR
O
O
OR
OH
O
O
OR
O
O
OR
O
ROS
OH
OH
OH
3, 4, 5
ROS
3a, 4a, 5a
3b, 4b, 5b
3c, 4c, 5c
Figure 4. Plausible biosynthetic pathway of compounds
compounds . ( ) Plausible biosynthetic pathway. Iridotrial (keto form) is a common substrate for
the production of iridane and iridoid skeletons. [O]; oxidation. ( ) Possible antioxidant mechanism.
The conjugated para-OH is the expected primary target of reactive oxygen species (ROS) to generate
phenoxy radials 5a. The second ROS attack results in the formation of ortho quinones, 5c, through
the resonance structures 3–5b.
1–5, and possible antioxidant mechanism of
3–5 a
b
3–
3–

Molecules 2019, 24, 3995
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Plant species are potentially good sources for antioxidant secondary metabolites because of their
habitats, being exposed continuously to sunlight, and thus gaining protection mechanisms against
reactive oxygen species produced by UV irradiation [25]. The isolated compounds were tested for
their antioxidant activities by DPPH free radical scavenging assay, and cytotoxicity against human
lung cancer cell line (A549) by MTT assay. Among them, compounds
radical scavenging activity, while compound showed moderate scavenging activity. The free radical
scavenging activity of , and may be related to the presence of caffeoyl moieties in their structures.
3 and 4 exhibited potent
5
3,
4
5
The possible reaction mechanism with reactive oxygen species is shown in Figure 4 according to
the mechanism of catechol [26]. It is noteworthy that none of the isolated compounds exhibited
cytotoxic activity against human lung cancer cell line A549 by the MTT method. Therefore, compounds
3, 4, and 5 have the potential to be safe antioxidant reagents to ameliorate a variety of oxidative
stress-related diseases.
Supplementary Materials: The following are available online, Figures S1–S9: HR-ESI-MS, 1H, 13C NMR, DEPT,
COSY, HSQC, HMBC, UV, and IR spectra of
1, Figures S10–S18: Spectra of 2, Figures S19–S28: Spectra of 3, Figures
S29–S38: Spectra of 4, Figures S39–S48: Spectra of 5.
Author Contributions: K.M. (Katsuyoshi Matsunami) and G.A.A.-H. conceived and designed the experiment;
G.A.A.-H. implement the experiments; G.A.A.-H. analyzed the data and wrote the original draft; K.M. (Kenta
Mizuno), R.S.O., and S.P. analyzed the data; K.M. (Kenta Mizuno), S.S., and Y.Y. helped in experiments and
contributed reagents/material and analysis tools; T.N. and K.M. (Kazuma Masuda) helped in the analysis of
CD; Y.T. and H.O. performed the sample collection; K.M. (Katsuyoshi Matsunami) and R.S.O. supervised, K.M.
(Katsuyoshi Matsunami) wrote, reviewed, and edited the manuscript.
Funding: This work was supported in part by the King Saud University External Joint Supervision Program
(EJSP), Kingdom of Saudi Arabia, and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science
and Technology of Japan, and the Japan Society for the Promotion of Science (Nos., 15H04651 and 17K15465,
17K08336 and 18K06740).
Acknowledgments: The measurements of HR-ESI-MS were performed with an LTQ Orbitrap XL spectrometer at
the Natural Science Center for Basic Research and Development (N-BARD), Hiroshima University. The other
experimental facilities and useful suggestions for publication of this work was kindly supported by a grant
from the “Research Center of the Female Scientific and Medical Colleges”, Deanship of Scientific Research, King
Saud University.
Conflicts of Interest: The authors report no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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