Structures of triterpenoids from the leaves of Lansium domesticum

Article abstract of DOI:10.1007/s11418-019-01319-2

From the methanolic extract of the leaves of Lansium domesticum, three new onoceranoid-type triterpenoids, lansium acids X–XII and a new cycloartane-type triterpene, lansium acid XIII, were isolated. The chemical structures of the isolated new compounds were elucidated on the basis of chemical/physicochemical evidence. For new onoceranoid-type triterpenoids, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. The isolated onoceranoid-type triterpenoids showed antimutagenic effects in the Ames assay against 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1).

Full text of DOI:10.1007/s11418-019-01319-2

Journal of Natural Medicines
https://doi.org/10.1007/s11418-019-01319-2
ORIGINAL PAPER
Structures of triterpenoids from the leaves of Lansium domesticum
Takahiro Matsumoto1 · Takahiro Kitagawa1 · Tomoe Ohta2 · Tatsusada Yoshida2 · Daisuke Imahori1 · Stephen Teo3 ·
Haji Sapuan bin Ahmad3 · Tetsushi Watanabe1
Received: 13 February 2019 / Accepted: 7 May 2019
© The Japanese Society of Pharmacognosy 2019
Abstract
From the methanolic extract of the leaves of Lansium domesticum, three new onoceranoid-type triterpenoids, lansium acids
X–XII and a new cycloartane-type triterpene, lansium acid XIII, were isolated. The chemical structures of the isolated new
compounds were elucidated on the basis of chemical/physicochemical evidence. For new onoceranoid-type triterpenoids,
the absolute confgurations were established by comparison of experimental and predicted electronic circular dichroism
(ECD) data. The isolated onoceranoid-type triterpenoids showed antimutagenic efects in the Ames assay against 3-amino-
1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1).
Keywords Lansium domesticum · Onoceranoid-type triterpenoid · Cycloartane-type triterpene · ECD
Introduction
from the leaves of L. domesticum. The chemical structures of
the new compounds were elucidated on the basis of chemical
and physicochemical evidence such as NMR and HRESIMS.
The absolute confgurations of lansium acids X and XII (1
and 2) were established by comparison of experimental and
predicted electronic circular dichroism (ECD) data. The
antimutagenic efects of lansium acids X–XII (1–3) were
evaluated by the Ames assay.
The plant of Lansium domesticum Corr. (Meliaceae), a fruit-
bearing tree, grows widely in southeastern Asia [1–3]. The
fruits are edible and very popular as a dessert. Previously,
the onoceranoid-type triterpenoids have been reported as the
constituents of the peels [4]. From the seeds, tetranortriter-
penoids have been isolated and showed antimalarial activity
against Plasmodium falciparum [5]. As part of our ongoing
research program for the discovery of new antimutagenic
agents [6–8], we recently reported the chemical structures
and antimutagenic efects of onoceranoid-type triterpenoids
isolated from the MeOH extracts of the leaves of L. domes-
ticum [9]. As a continuing study, we isolated three new
onoceranoid-type triterpenoids, lansium acids X–XII (1–3)
and a new cycloartane-type triterpene, lansium acid XIII (4),
Results and discussion
A methanol extract from the dried leaves of L. domesti-
cum was partitioned with ethyl acetate-H2O, to furnish an
ethyl acetate-soluble fraction and an aqueous layer. The
aqueous layer was further extracted with 1-butanol to give
1-butanol-soluble fraction as described previously [9]. Both
soluble fractions were subjected to normal- and reversed-
phase silica-gel column chromatography and repeated
high-performance liquid chromatography (HPLC) to give
three new onoceranoid-type triterpenoids, lansium acids
X (1, 0.0036%), XI (2, 0.0016%), XII (3, 0.0016%) and
a new cycloartane-type triterpene, lansium acid XIII (4,
0.00032%).
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-019-01319-2) contains
supplementary material, which is available to authorized users.
* Tetsushi Watanabe
watanabe@mb.kyoto-phu.ac.jp
1
Department of Public Health, Kyoto Pharmaceutical
University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Lansium acid X (1) was isolated as colorless amorphous
powder with positive optical rotation ([α]2D5 +34.5 in metha-
nol). Electrospray ionization mass spectrometry (ESIMS)
revealed a quasimolecular ion peak (m/z 493 [M+Na]+).
2
Faculty of Pharmaceutical Sciences, Nagasaki International
University, Nagasaki 859-3298, Japan
3
Forest Department Sarawak, Wisma Sumber Alam, Jalan
Stadium, Petra Jaya, 93660 Kuching, Sarawak, Malaysia
Vol.:(0123456789)
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Journal of Natural Medicines
The high-resolution ESIMS (HRESIMS) and 13C nuclear
magnetic resonance (NMR) led to discerning the molecular
formula as C30H46O4. The 1H and 13C NMR (CDCl3) spectra
of 1 (Table 1) showed signals due to six methyl groups [δH
0.84 (s, H-25), 0.93 (s, H-28), 1.01 (s, H-24), 1.10 (s, H-23),
1.76 (s, H-30), and 1.78 (s, H-27)], two exomethylene groups
[δC 107.5 (C-26), 115.2 (C-29), 146.2 (C-22), and 147.4
(C-8)], a ketone group [δC 217.1 (C-3)], a carboxyl group
[δC 178.6 (C-21)], and a methine group bearing an oxygen
function [δC 69.3 (C-15)]. The above analysis suggested that
lansium acid X (1) is an onoceranoid-type triterpenoid simi-
lar to lansium acid III [9], with the only diferences being the
absence of a carbonyl group and the presence of a hydroxy
group. The positions of the functional groups described
above were determined based on double-quantum filter
correlated spectroscopy (DQF COSY) and heteronuclear
multiple bond correlation (HMBC) spectroscopy (Fig. 2).
Namely, long-range correlations were observed between
the following proton and carbon pairs: H-2 and C-1, 3; H-6
and C-5; H-12 and C-13; H-15 and C-13; H-17 and C-15;
H-19 and C-21; H-20 and C-21; H-23 and C-3, 4, 5; H-24
and C-3, 4, 5; H-25 and C-1, 5, 9, 10; H-26 and C-7, 8, 9;
H-27 and C-13, 14, 15; H-28 and C-13, 17, 18, 19; H-29
and C-17, 22, 30; and H-30 and C-17, 22, 29. The relative
confgurations of 1 were determined via analysis of their
nuclear Overhauser efect spectroscopy (NOESY) spectra
(Fig. 3). The NOESY cross-peaks of H-2β/H-24, H-5/H-9,
H-5/H-23, H-6β/H-24, H-6α/H-23, and H-24/H-25 indicated
that Me-24 and Me-25 moieties were on one side of each
molecule and that H-5 and H-9 were on the opposite side.
The cross-peaks of H-15/H-17 and H-17/H-19 indicated that
H-15, H-17, and CH2-19 moieties were all on the same side.
Finally, the absolute confgurations were elucidated from
the calculated ECD curve (Fig. 4). The calculated ECD
spectra of 1 was identical with that of experimental data,
while the calculated ECD spectra of ent-1 had the oppo-
site sign to the experimental data. These data suggested that
the absolute confgurations were 5R,9S,10R,15S,17S,18S.
Suggested absolute confgurations were identical with pre-
viously reported onoceranoid-type triterpenes [10]. On the
basis of all this evidence, the chemical structure of lansium
acid X (1) was characterized as shown (Fig. 1).
of aglycone of 2 was similar with lansium acid II except for
1H and 13C signals of C-3 [9]. The above analysis suggested
the chemical structure of lansium acid XI (2) is the C-3 gly-
coside of lansium acid II. The HMBC correlation of H-1′
with methine carbon bearing an oxygen function (δC 89.3)
demonstrated that the N-acetyl-d-glucosamine group was
linked to C-3 (Fig. 2). The relative confguration of 2 was
determined via analysis of their NOESY spectrum (Fig. 3).
The absolute confgurations were elucidated from the cal-
culated ECD curve of aglycone of 2 (Fig. 5). The calculated
ECD spectra of aglycone of 2 was identical with that of
experimental data. These data suggested that the absolute
confgurations were 3S,5R,9S,10R,17S,18S.
Lansium acid XII (3) was isolated as colorless amorphous
powder with positive optical rotation ([ꢀ]25 +26.6 in metha-
nol). The molecular formula was deducedDby ESIMS and 13C
NMR as C35H56O8. The 1H and 13C NMR (C5D5N) spectra
of 3 (Table 1) showed signals due to its onoceranoid-type
triterpenoid glycoside structure, similar to 2. The main dif-
ferences between 3 and 2 were the type of sugar, the pres-
ence of one more hydroxy group [3: δC 68.9 (C-15)], and the
absence of a carbonyl group. Acid hydrolysis of 3 yielded
d-xylose, which was identifed by HPLC to be the same as
lansium acid XI (2). The positions of each group in 3 were
determined based on DQF COSY and HMBC NMR spec-
troscopy, as shown in Fig. 2. The relative confguration of
3 was defned by analysis of the NOESY spectrum (Fig. 3).
Finally, the absolute confguration of 3 was assumed to be
the same as other isolated onoceranoid-type triterpens such
as 1 and 2. On the basis of all this evidence, the chemical
structure of lansium acid XII (3) was characterized as shown
in Fig. 1.
Lansium acid XIII (4) was isolated as colorless amor-
phous powder with positive optical rotations ([ꢀ]25 +14.3
in chloroform). ESIMS revealed a quasimolecularDion peak
(m/z 469 [M−H]−). The HRESIMS and 13C NMR led to
discerning the molecular formula as C30H46O4. The 1H and
13C NMR (CDCl3) spectra of 4 (Table 1) showed charac-
teristic signals due to cycloartane-type triterpenoids, such
as fve methyl groups [δH 0.91 (s, H-28), 1.04 (s, H-30),
1.08 (s, H-18), 1.09 (s, H-29), 1.28 (s, H-27), and 1.29 (s,
H-26)], the methylene protons of the cyclopropane ring [δH
0.51 (d, J=4.8, H-19α), 0.78 (d, J=4.8 H-19β)], a ketone
carbonyl group [δC 216.6 (C-3)], a carboxyl group [δC 180.0
(C-21)], a methine group bearing an oxygen function [δC
70.7 (C-25)], and an olefn group [δH 5.55 (dd, J=9.0, 16.2,
Lansium acid XI (2) was isolated as colorless amor-
phous powder with positive optical rotation ([ꢀ]25 +27.6
D
in methanol). ESIMS revealed a quasimolecular ion peak
(m/z 696 [M+Na]+). The HRESIMS and 13C NMR led to
1
1
discerning the molecular formula as C38H59NO9. The H
H-23), 5.62 (d, J = 16.2, H-24)]. The H and 13C NMR
and 13C NMR (C5D5N) spectra of 2 (Table 1) showed char-
acteristic signals due to onoceranoid-type triterpenoid and
N-acetyl-glucosamine. Acid hydrolysis of 2 with 5% aqueous
H2SO4 -1,4-dioxane yielded N-acetyl-d-glucosamine, which
was identifed by HPLC of the chiral tolylthiocarbamoyl
thiazolidine derivative [11]. The 1H and 13C NMR spectra
(CDCl3) spectra of 4 were similar to those of the 25-hydrox-
ycycloart-23-en-3-one except for C-21. The above data and
2D NMR (Fig. 2) suggested the planar structure of lansium
acid XII (4) was C-21 carboxylated 25-hydroxycycloart-
23-en-3-one. The relative and absolute confguration was
deduced to be the same as other cycloartane triterpenes such
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Journal of Natural Medicines
Table 1 13C (150 MHz) and 1H NMR data (600 MHz) of lansium acids X–XIII (1–4, δ in ppm)
Position 1a
2b
3b
4a
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
37.6
α 2.04 (ddd, 2.4, 3.6, 10.4)
β 1.61 (dd-like, 2.4, 12.4)
37.3
27.4
α 1.55 (m)
β 1.06 (m)
37.7
27.9
α 1.86 (m)
β 1.31 (m)
33.4
37.5
α 1.84 (dd-like, 4.2, 13.2)
β 1.49 (dd-like, 2.0, 6.4)
34.7
α 2.42 (m)
α 2.29 (m)
α 1.94 (m)
α 2.28 (m)
β 2.63 (m)
β 1.70 (m)
β 2.33 (m)
β 2.68 (ddd, 6.4, 13.2, 13.2)
3
4
5
6
217.1
47.8
55.1
25.2
89.3
39.9
55.1
24.6
3.33 (dd, 4.1, 11.7)
89.0
40.2
55.3
25.4
3.46 (dd, 4.1, 11.7)
216.6
50.3
48.3
21.4
1.61 (m)
1.06 (m)
1.27 (dd-like, 2.8, 8.8)
1.17 (m)
1.73 (m)
1.69 (dd, 4.8, 12.0)
α 1.49 (m)
1.15 (m)
β 1.70 (m)
0.67 (m)
7
37.9
2.44 (m)
1.68 (m)
38.8
1.95 (m)
2.35 (m)
38.9
2.35 (m)
1.97 (m)
27.0
1.36 (m)
1.15 (m)
1.60 (dd, 4.8, 12.0)
8
9
10
11
147.4
57.8
39.6
28.1
148.9
59.0
40.1
24.1
149.4
59.0
40.3
31.1
47.6
21.0
26.1
26.6
1.60 (m)
1.67 (m)
1.76 (m)
2.15 (m)
1.55 (m)
2.50 (m)
2.81 (t-like, 11.7)
α 1.98 (m)
β 1.17 (m)
12
31.7
1.94 (m)
31.2
34.8
2.00 (m)
2.04 (m)
30.0
1.42 (m)
1.71 (m)
1.60 (m)
13
14
15
141.2
131.2
69.3
163.9
133.3
197.9
140.4
133.3
68.9
48.7
45.3
35.0
3.96 (br-s)
4.31 (br-s)
1.25 (m)
1.36 (m)
16
24.8
1.50 (m)
40.1
2.79 (m)
2.66 (m)
24.6
1.67 (s)
25.8
1.17 (m)
17
18
19
39.9
42.7
33.0
2.54 (d-like, 11.0)
47.7
42.7
35.3
2.77 (m)
41.1
43.4
33.7
3.21 (dd, 2.8, 13.0)
48.3
18.0
29.5
2.37 (ddd, 3.6, 11.4)
1.08 (s)
1.61 (m)
2.16 (m)
2.16 (m)
α 0.51 (d, 4.8)
1.83 (dd-like, 3.6, 13.2)
β 0.78 (d, 4.8)
20
29.7
2.32 (m)
30.6
2.35 (m)
2.58 (m)
28.9
2.04 (br-s)
48.7
2.39 (m)
21
22
178.6
146.2
176.2
146.8
176.9
148.2
180.0
35.1
2.17 (m)
2.30 (m)
23
24
25
26
26.0
21.6
14.1
1.10 (s)
1.01 (s)
0.84 (s)
28.6
17.2
15.0
1.18 (s)
0.95 (s)
0.60 (s)
28.7
17.1
15.2
1.31 (s)
0.96 (s)
0.72 (s)
123.4 5.55 (dd, 9.0, 16.2)
140.7 5.65 (d, 16.2)
70.7
107.5 4.66 (br-s)
107.2 4.70 (br-s)
108.2 4.81 (br-s)
29.5
1.29 (s)
4.93 (br-s)
4.93 (br-s)
4.95 (br-s)
27
28
29
18.4
22.1
1.78 (s)
0.93 (s)
12.8
21.9
1.95 (s)
1.17 (s)
19.4
23.1
2.08 (s)
1.11 (s)
29.8
19.3
22.3
1.28 (s)
0.91 (s)
1.04 (s)
115.2 4.72 (br-s)
115.9 4.85 (br-s)
115.3 4.98 (br-s)
4.97 (br-s)
4.88 (br-s)
5.04 (br-s)
30
1′
2′
3′
4′
5′
22.9
1.76 (s)
23.1
1.66 (s)
23.6
1.81 (s)
20.8
1.09 (s)
105.2 5.10 (d, 8.2)
107.2 4.88 (d, 8.3)
58.4
76.5
72.8
78.7
4.53 (m)
75.9
79.0
71.6
67.5
4.04 (t, 8.3)
4.18 (t, 8.3)
4.23 (m)
4.41 (dd, 8.9, 10.3)
4.19 (d-like, 8.9)
3.95 (m)
3.79 (t, 11.7)
4.38 (dd, 5.5, 11.7)
6′
63.2
4.34 (dd, 4.8, 11.6)
4.50 (dd, 2.8, 11.6)
Ac
170.5
24.3
2.18 (s)
aMeasured in chloroform-d
bMeasured in pyridine-d5
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Journal of Natural Medicines
Fig. 1 Structures of new com-
pounds isolated from leaves of
L. domesticum
Fig. 2 Key 2D NMR correla-
tions of new compounds 1–4
as 25-hydroxycycloart-23-en-3-one [12]. The NOESY spec-
trum suggested the proposed relative confguration. On the
basis of all this evidence, the chemical structure of lansium
acid XIII (4) was characterized as shown in Fig. 1.
X–XII (1–3) on CYP1A2 were examined using the P450-Glo
assay (Promega™ V7700). The lansium acids X–XII (1–3)
showed inhibitory efects on CYP1A2 (Supporting Informa-
tion, S5). Therefore, we assumed that one of the mechanisms
for antimutagenic efects of lansium acids was the inhibitory
efect on CYP1A2.
Antimutagenic activities of onoceranoid-type triterpenoid
lansium acids X–XII (1–3) against Trp-P-1 were evaluated
by the Ames assay using S. typhimurium TA98 strain. As
the results, lansium acids X–XII (1–3) showed antimuta-
genic efects against Trp-P-1 without antimicrobial activity
at tested concentrations (Supporting Information, S4). The
mutagenicity of Trp-P-1 depends on their bioactivation by
CYP1A2. Therefore, the inhibitory efects of lansium acids
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Journal of Natural Medicines
Experimental
General
The following instruments were used to obtain physical
data: specifc rotations, a JASCO P-2200 digital polarim-
eter (l=5 cm); ESIMS, Shimadzu LCMS-8040; HRESIMS,
Shimadzu LCMS-IT-TOF; 1H-NMR spectra, JEOL ECS400
(400 MHz) and JNM-ECA 600 K (600 MHz) spectrom-
eter; 13C-NMR spectra, JNM-ECA 600 K (150 MHz)
spectrometer; 2D-NMR spectra, JEOL JNM-ECA 600 K
(600 MHz) spectrometer; HPLC, a Shimadzu SPD-M10Avp
UV–VIS detector. YMC Triart C18 (250×4.6-mm i.d. and
250×10-mm i.d.) columns were used for analytical and pre-
parative purposes.
The following experimental materials were used for chro-
matography: normal-phase silica gel column chromatogra-
phy, silica gel 60 (Kanto Chemical Co., Inc. 63–210 mesh);
reversed-phase silica gel column chromatography, C18-OPN
(Nakalai Tesque Co., Inc. 140 μm); Thin-layer chromatog-
raphy (TLC), precoated TLC plates with silica gel 60F254
(Merck, 0.25 mm, ordinary phase) and silica gel RP-18 F254S
(Merck, 0.25 mm, reversed phase); reversed-phase HPTLC,
precoated TLC plates with silica gel RP-18 WF254S (Merck,
0.25 mm). Detection was achieved by spraying with 1%
Ce(SO4)2–10% aqueous H2SO4 followed by heating.
Plant material
The leaves of Lansium domesticum were collected in
Ba’kelalan, Sarawak, Malaysia, in November 2015. Iden-
tifcation and taxonomic authentication of the plants were
Fig. 3 Key NOESY correlations of new compounds 1–3
Fig. 4 Comparison of the
experimental ECD spectra with
the calculated curves of lansium
acid X (1)
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Journal of Natural Medicines
Fig. 5 Comparison of the exper-
imental ECD spectra with the
calculated curves of aglycone of
lansium acid XI (2)
conducted by one of the authors (S.T.). A voucher specimen
(STTM-2015-4) has been deposited in the Forest Herbarium,
Forest Department Sarawak, Kuching, Sarawak, Malaysia.
eight fractions. Fraction B8-4 (68.5 mg) was purifed via
HPLC [H2O-CH3CN-CH3COOH (50:50:0.1, v/v/v)] to give
2 (7.8 mg).
Extraction and isolation
Lansium acid X (1)
Dried leaves of L. domesticum (550 g) were extracted
three times with methanol. The EtOAc-, BuOH-, and
H2O-soulble fractions were obtained from the metha-
nol extract described previously [9]. The EtOAc-soluble
fraction was subjected to normal-phase silica gel column
chromatography [2 kg, hexane–CHCl3 (4:1 → 1:1 → 1:4,
v/v) → CHCl3 → CHCl3–MeOH (50:1 → 30:1 → 10:1 → 5
:1→2:1→1:1→1:2, v/v)→MeOH] to give ten fractions
[Fr. EA1–10]. Fraction EA6 (1.50 g) was separated via
reversed-phase silica gel column chromatography to give
ten fractions. Fraction EA6-8 (82.7 mg) was purifed via
HPLC [H2O-CH3CN-CH3COOH (15:85:0.1, v/v/v)] to
give 4 (1.6 mg). Fraction EA9 (1.1 g) was separated via
reversed-phase silica gel column chromatography to give
seven fractions. Fraction EA9-4 (142.3 mg) was purifed
via HPLC [H2O-CH3CN-CH3COOH (30:70:0.1, v/v/v)]
to give 1 (19.7 mg). Fraction EA10 (1.1 g) was separated
via reversed-phase silica gel column chromatography to
give seven fractions. Fraction EA10-3 (249.6 mg) was
purifed via HPLC [H2O-CH3CN-CH3COOH (30:70:0.1,
v/v/v)] to give 3 (7.9 mg). The BuOH-soluble fraction
was subjected to normal-phase silica gel column chroma-
tography [2 kg, CHCl3 → CHCl3–MeOH (50:1 → 20:1 →
10:1 → 5:1 → 2:1 → 1:1, v/v) → MeOH] to give nine frac-
tions [Fr. B1–9]. Fraction B8 (688.7 mg) was separated via
reversed-phase silica gel column chromatography to give
White powder; [α]2D5 +34.5 (c 0.5, MeOH); IR [attenuated
total refectance (ATR)]: νmax 2936, 1705, 1455, 1384, and
1054 cm−1; ECD (MeOH): given in Fig. 4; 1H NMR (chloro-
form-d, 600 MHz) and 13C NMR (chloroform-d, 150 MHz):
given in Table 1; ESIMS: m/z 493 [M+Na]+; HRESIMS: m/z
493.3293 (calcd. for C30H46O4Na [M+Na]+: m/z 493.3288).
Lansium acid XI (2)
White powder; [α]2D5 +27.6 (c 0.5, MeOH); IR (ATR):
νmax 2938, 1658, 1560, and 1375 cm−1; ECD (MeOH):
given in Fig. 5; 1H NMR (pyridine-d5, 600 MHz) and 13
C
NMR (pyridine-d5, 150 MHz): given in Table 1; ESIMS:
m/z 696 [M+Na]+; HRESIMS: m/z 696.4078 (calcd. for
C38H59NO9Na [M+Na]+: m/z 696.4082).
Lansium acid XII (3)
White powder; [α]D25 +26.6 (c 0.5, MeOH); IR (ATR): νmax
2938, 1711, 1455, and 1384 cm−1; ECD (MeOH) [206 nm
(∆ε +6), 230 nm (∆ε −0.06), 248 nm (∆ε +0.05), 280 nm
1
(∆ε − 0.12)]; H NMR (pyridine-d5, 600 MHz) and 13C
NMR (pyridine-d5, 150 MHz): given in Table 1; ESIMS:
m/z 493 [M + Na]+; HRESIMS: m/z 627.3861 (calcd. for
C35H56O8Na [M+Na]+: m/z 627.3867).
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Journal of Natural Medicines
Lansium acid XIII (4)
were converted into Gaussian-type curves with half-bands
(0.26 eV for conformers of 1, and 0.30 eV for conformers of
2) using SpecDis v1.71 [15]. The calculated ECD spectrum
were composed after correction based on the Boltzmann dis-
tribution of the stable conformers. The calculated spectra
of conformers of 1 and 2 were red-shifted by 15 and 13 nm,
respectively.
White powder; [α]D25 +14.3 (c 0.2, CHCl3); IR (ATR): νmax
2941, 1706, 1556, 1443, and 1379 cm−1; 1H NMR (chloro-
form-d, 600 MHz) and 13C NMR (chloroform-d, 150 MHz):
given in Table 1; ESIMS: m/z 469 [M−H]−; HREIMS: m/z
469.3323 (calcd. for C30H45O4 [M−H]−: m/z 469.3326).
Acid hydrolysis of lansium acid XI and XII (2 and 3)
Evaluation of in vitro antimutagenic activity using
the Ames assay
To determine the absolute confgurations of the constitu-
ent monosaccharids of 2 and 3, the reported method by
Tanaka et al. was used with slight modifcations as follows.
Compounds 2 and 3 (0.4 mg) were dissolved in 3 N HCl,
and each solution was heated at 90 °C for 3 h. After extrac-
tion three times with EtOAc, the solutions were neutral-
ized with Amberlite IRA-400 (OH− form). After drying in
vacuo, the residue was dissolved in pyridine (0.1 mL) con-
taining l-cysteine methyl ester hydrochloride (0.5 mg) and
heated at 60 °C for 1 h. A solution of o-tolylisothiocyanate
(0.5 mg) in pyridine (0.1 mL) was added to the mixture and
heated at 60 °C for 1 h. The reaction mixture was analyzed
via reversed-phase HPLC [column: Cosmosil 5C18-AR-II
(Nacalai Tesque), 250×4.6-mm i.d. (5 μm); mobile phase:
25% CH3CN in 50 mM H3PO4; detection: UV (254 nm);
fow rate: 0.8 mL/min; column temperature: 25 °C] to iden-
tify the derivatives of the constituents N-acetyl-d-glucosa-
mine (2) and d-xylose (3) by comparison of their reten-
tion times with those of authentic samples (tR: d-xylose,
16.37 min, N-acetyl-d-glucosamine, 35.29 min).
Antimutagenicity was examined by the preincubation
method using Salmonella typhimurium TA98 with S9 mix
as described previously [9]. The S9 mix (0.5 mL) contained
0.025 mL of S9. The S9 was prepared from the liver of male
Sprague–Dawley rats treated with phenobarbital sodium salt
and β-naphthofavone. Dimethyl sulfoxide (DMSO) was
used as a negative control. Trp-P-1 (0.04 µg) was used as
mutagens to examine the antimutagenic activity of samples.
Antimutagenicity was evaluated by the reduction of revertant
colonies induced by a Trp-P-1 in the presence of samples.
The experiments were conducted according to the “Guide-
lines for Animal Experiments at Kyoto Pharmaceutical Uni-
versity (KPU),” and the animal studies were approved by
the Experimental Animal Research Committee (permission
number: 18-17-030). Values are expressed as mean SEM.
Evaluation of inhibitory efects of onoceranoid‑type
triterpenoids on CYP1A2
Inhibitory efects of isolated onoceranoid-type triterpenoids
on CYP450 enzymatic activities were evaluated using P450-
Glo™ CYP1A2 assay and a screening system (Promega,
V9770). This system provides a luminescent method to
measure CYP1A2 enzyme activity and via the conver-
sion of the inactive luciferin-ME into the active form by
CYP1A2 enzymes to produce luminescence. The amount of
luminescence produced is directly proportional to CYP1A2
activity. Briefy, sample (12.5 µL, test sample was dissolved
into acetone) was added to a 96-well plate, followed by
CYP1A2 reaction mixture (12.5 µL), and incubated at 37 °C
for 10 min. Reaction was initiated by addition of NADPH
regeneration system (25 µL), and the plate was incubated
10 min at 37 °C. Luciferin detection reagent (50 µL) was
added, and the plate was incubated at 20 °C for 20 min.
Luminescence was recorded with a luminometer (Promega,
Glomax®Navigator microplate luminometer, GM2000)
using no flters and an integration time of 1 s/well. Values
are expressed as mean SEM. For statistical analysis, Stu-
dent’s t test was used.
Calculation of theoretical ECD spectra for lansium
acids I and II (1 and 2)
Conformational searches were done with the Spartan’10
program [13]. Geometry optimization and ECD calcula-
tions of conformers were performed using the Gaussian
09 program [14]. The enantiomers (5R,9S,10R,15S,17S,
18S-1, 5S,9R,10S,15R,17R,18-1, 3S,5R,9S,10R,17S,18S-
aglycone of 2, and 3R,5S,9R,10S,17R,18R-aglycone of 2)
were submitted to conformational searches using the Merck
molecular force feld (MMFF) in vacuum. The initial sta-
ble conformers for 5R,9S,10R,15S,17S,18S-1, 5S,9R,10S,
15R,17R,18R-1, 3S,5R,9S,10R,17S,18S-aglycone of 2, and
3R,5S,9R,10S,17R,18R-aglycone of 2 with Boltzmann dis-
tributions over 1% (18, 18, 22, and 22 conformers, respec-
tively) were further optimized by density functional theory
(DFT) calculations at the CAM-B3LYP/6-31G(d,p) level
and subjected to time-dependent DFT (TDDFT) calcula-
tions at the CAM-B3LYP/def2-TZVPP level in the pres-
ence of MeOH with an integral equation formalism polar-
izable continuum model (IEFPCM). The resultant rotatory
strengths of the lowest 30 excited states for each conformer
Supporting information Experimental details; 1H and 13
C
NMR spectra for the new compounds, the DFT-optimized
1 3

Journal of Natural Medicines
9. Matsumoto T, Kitagawa T, Teo S, Anai Y, Ikeda R, Imahori D,
Ahmad HS, Watanabe T (2018) Structures and antimutagenic
efects of onoceranoid-type triterpenoids from the leaves of Lan-
sium domesticum. J Nat Prod 81:2187–2194
chemical structures of conformers of 1 and 2 with their equi-
librium population, and the results of antimutagenic tests are
available in supporting information.
10. Nugroho AE, Inoue D, Wong CP, Hirasawa Y, Kaneda T, Shirota
O, Hadi AHA, Morita H (2018) Reinereins A and B, new onocer-
ane triterpenoids from Reinwardtiodendron cinereum. J Nat Med
72:588–592
Acknowledgements This research was supported by JSPS KAKENHI
grant number JP17K15473.
11. Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I (2007) Facile
discrimination of aldose enantiomers by reversed-phase HPLC.
Chem Pharm Bull 55:899–901
References
12. Gandhe S, Lakavath S, Palatheeya S, Schuehly W, Amancha K,
Kiran Reddy Nallamaddi R, Palepu A, Thakur Y, Rao Adavi Rao
Belvotagi V, Kumar Bobbala R, Narasimha Appa Rao Achanta V,
Kunert O (2018) Cycloartanes from the gum resin of Gardenia
gummifera L.f. Chem Biodivers 10:1613–1622
1. Tanaka T, Ishibashi M, Fujimoto H, Okuyama E, Koyano T,
Kowithayakorn T, Hayashi M, Komiyama K (2002) New onocera-
noid triterpene constituents from Lansium domesticum. J Nat Prod
65:1709–1711
13. Spartan’10. Wavefunction Inc., Irvine CA
14. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,
Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson
GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF,
Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K,
Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao
O, Nakai H, Vreven T, Montgomery JA Jr, Peralta JE, Ogliaro
F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN,
Keith T, Kobayashi R, Normand J, Raghavachari K, Rendell A,
Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM,
Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J,
Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R,
Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski
VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels
AD, Farkas O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ
(2009) Gaussian 09, Revision D.01. Gaussian Inc, Wallingford
15. Bruhn T, Schaumlöffel A, Hemberger Y, Pecitelli G (2013)
SpecDis: quantifying the comparison of calculated and experi-
mental electronic circular dichroism spectra. Chirality 25:243–249
2. Dong SH, Zhang CR, Dong L, Wu Y, Yue JM (2011) Onocer-
anoid-type triterpenoids from Lansium domesticum. J Nat Prod
74:1042–1048
3. Nishizawa M, Nishida H, Kosela S, Hayashi Y (1983) Structure
of lansiosides: biologically active new triterpene glycides from
Lansium domesticum. J Org Chem 48:4462–4466
4. Mayanti T, Tjokronegoro R, Supratman U, Mukhtar MR, Awang
K, Hadi AH (2011) Antifeedant triterpenoids from the seeds and
bark of Lansium domesticum cv Kokossan (Meliaceae). Molecules
16:2785–2795
5. Saewan N, Sutherland JD, Chantrapromma K (2006) Antimalarial
tetranortriterpenoids from the seeds of Lansium domesticum Corr.
Phytochemistry 67:2288–2293
6. Matsumoto T, Nakamura S, Kojima N, Hasei T, Yamashita M,
Watanabe T, Matsuda H (2017) Antimutagenic activity of ent-
kaurane diterpenoids from the aerial parts of Isodon japonicus.
Tetrahedron Lett 58:3574–3578
7. Matsumoto T, Takahashi K, Kanayama S, Nakano Y, Imai H,
Kibi M, Imahori D, Hasei T, Watanabe T (2017) Structures of
antimutagenic constituents in the peels of Citrus limon. J Nat Med
71:735–744
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
8. Matsumoto T, Koike M, Arai C, Kitagawa T, Inoue E, Imahori D,
Watanabe T (2018) Chemical structures and antimutagenic efects
of unusual oximes from the peels of Citrus limon. Phytochem Lett
25:118–121
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