Structures and Antimutagenic Effects of Onoceranoid-Type Triterpenoids from the Leaves of Lansium domesticum

Article abstract of DOI:10.1021/acs.jnatprod.8b00341

A methanol extract of the dried leaves of Lansium domesticum showed antimutagenic effects against 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-1-methyl-6-phenylimidazo[4,5-bI]pyridine (PhIP) using the Ames assay. Nine new onoceranoid-type triterpenoids, lansium acids I-IX (1-9), and nine known compounds (10-16) were isolated from the extract. The structures of the new compounds were elucidated on the basis of chemical and spectroscopic evidence. The absolute stereostructures of the new compounds were determined via their electronic circular dichroism spectra. Several isolated onoceranoid-type triterpeneoids showed antimutagenic effects in an in vitro Ames assay. Moreover, oral intake of a major constituent, lansionic acid (10), showed antimutagenic effects against PhIP in an in vivo micronucleus test.

Full text of DOI:10.1021/acs.jnatprod.8b00341

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Structures and Antimutagenic Effects of Onoceranoid-Type
Triterpenoids from the Leaves of Lansium domesticum
Takahiro Matsumoto,† Takahiro Kitagawa,† Stephen Teo,‡ Yuuka Anai,† Risa Ikeda,† Daisuke Imahori,†
Haji Sapuan bin Ahmad,‡ and Tetsushi Watanabe*,†
†Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
‡Forest Department Sarawak, Wisma Sumber Alam, Jalan Stadium, Petra Jaya, 93660 Kuching, Sarawak, Malaysia
S
* Supporting Information
ABSTRACT: A methanol extract of the dried leaves of Lansium domesticum showed antimutagenic effects against 3-amino-1,4-
dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-1-methyl-6-phenylimidazo[4,5-bI]pyridine (PhIP) using the Ames
assay. Nine new onoceranoid-type triterpenoids, lansium acids I−IX (1−9), and nine known compounds (10−16) were
isolated from the extract. The structures of the new compounds were elucidated on the basis of chemical and spectroscopic
evidence. The absolute stereostructures of the new compounds were determined via their electronic circular dichroism spectra.
Several isolated onoceranoid-type triterpeneoids showed antimutagenic effects in an in vitro Ames assay. Moreover, oral intake
of a major constituent, lansionic acid (10), showed antimutagenic effects against PhIP in an in vivo micronucleus test.
the isolated onoceranoid-type triterpenoids (10−12, 16, and
17) using in vitro and in vivo bioassays.
Lansium domesticum Corr. (Meliaceae), a fruit-bearing tree,
grows widely in southeastern Asia.1−3 The fruits of this species
are edible and are very popular in desserts. A number of
onoceranoid-type triterpenoids have been reported from L.
domesticum peels.4−6 Previous reports have described the
bioactivities of these triterpenoids, such as toxicity against
brine shrimp,1 inhibition of leukotriene D4-induced contrac-
tion of guinea pig ileum,7 cytotoxic activity,8 and antibacterial
activity against Gram-positive bacteria.9 As part of an ongoing
research program for the discovery of new antimutagenic
agents, our group has reported the structures and antimuta-
genic effects of ent-kaurane diterpenoids,10 coumarins,11 and
oximes.12 As a continuation of these studies, we found that the
MeOH extract of L. domesticum leaves showed antimutagenic
effects against 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole
(Trp-P-1) [inhibition: 80.8% at 125 μg/plate] and 2-amino-
1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [inhibition:
75.7% at 125 μg/plate], using the Ames test. According to
these findings, the isolation of onoceranoid-type triterpenoids
and the investigation of their antimutagenic effects were
pursued. This paper reports the structure elucidation of
lansium acids I−IX (1−9) and the antimutagenic activities of
RESULTS AND DISCUSSION
■
A methanol extract of L. domesticum dried leaves was
partitioned with ethyl acetate−H2O (1:1, v/v), to furnish an
ethyl acetate-soluble fraction (2.1%) and an aqueous layer. The
aqueous layer was further extracted with 1-butanol to give 1-
butanol (1.3%) and H2O (1.6%) soluble fractions. The ethyl
acetate- and 1-butanol-soluble fractions were subjected to
normal- and reversed-phase silica gel column chromatography
and repeated high-performance liquid chromatography
(HPLC) to give nine new compounds, lansium acids I (1,
0.0036%), II (2, 0.0042%), III (3, 0.012%), IV (4, 0.0025%), V
(5, 0.00058%), VI (6, 0.00058%), VII (7, 0.0017%), VIII (8,
0.00073%), and IX (9, 0.00065%) together with nine known
compounds, lansiolic acid (10, 0.06%),7 methyl lansiolate (11,
0.002%),7 ethyl lansiolate (12, 0.0037%),9 lansioside C (13,
0.006%),7 lansioside B (14, 0.0013%),7 8,14-seco-gammacera-
7,14-diene-3,21-dione (15, 0.00047%),5 lansionic acid (16,
Received: April 30, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00341
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Chemical structures of isolated onoceranoid-type triterpenes from Lansium domesticum.
0.11%),1 lansic acid (17, 0.0088%),1 and lamesticumin A (18,
0.00058%).9
4, and C-5; H-25 and C-1, C-5, C-9, and C-10; H-26 and C-7,
C-8, and C-9; H-27 and C-13, C-14, and C-15; H-28 and C-
13, C-17, C-18, and C-19; H-29 and C-17, C-22, and C-30; H-
20 and C-21 (Figure 2). The relative configurations of 1 and 2
were determined via analysis of their nuclear Overhauser effect
spectroscopy (NOESY) spectra (Figure 3). The NOESY cross-
peaks of H-23/H-5, H-24/H-25, H-5/H-9, and H-25/H-11
indicated that the CH2-11, Me-24, and Me-25 moieties are on
one side of each molecule and that H-5 is on the opposite side.
The cross-peaks of H-13/H-15, H-15/H-16β, H-16α/H-30,
H-28/H-30, and H-17/H-19 indicated that H-13, H-15, H-17,
and CH2-19 are all on the same side. The suggested relative
configurations of 1 and 2 were identical to reported
onoceranoid-type triterpenes from L. domesticum.1 Finally,
the absolute configurations were elucidated as being identical
to the known compound lansionic acid (16) via their
electronic circular dichroism (ECD) spectra. ECD maxima of
1 [271.7 nm (Δε +0.6), 317.6 nm (Δε −0.6)] and 2 [274.6
nm (Δε +0.6), 316.7 nm (Δε −0.4)] were identical with those
from the known compounds lansionic acid (16) [274.4 nm
(Δε +0.2), 318.2 nm (Δε −0.2), observed in this study],
lansionic acid methyl ester [275 nm (Δε +0.06), 307 nm (Δε
−0.21), described in the literature],7 and onoceranedione I
[289 nm (Δε ca. +1.6), 302 nm (Δε ca. −0.2), described in
the literature].13 The absolute configurations of these known
compounds have been established via X-ray analysis and
chemical derivatization. On the basis of all this evidence, the
chemical structures of lansium acids I and II (1 and 2) were
characterized as shown in Figure 1.
Lansium acids I and II (1 and 2) were isolated as colorless,
amorphous powders with positive optical rotations (1: [α]25
D
+11.1, 2: [α]25 +11.3 in methanol). Electrospray-ionization
D
mass spectrometry (ESIMS) revealed for each a quasimolec-
ular ion peak (1, m/z 493 [M + Na]+, and 2, 509 [M + Na]+),
which, when combined with data from high-resolution mass
spectrometry (HRESIMS) and 13C NMR, led to the molecular
formulas for 1 (C30H46O4) and 2 (C30H46O5). The 1H and 13
C
NMR (CDCl3) spectra of 1 and 2 (Tables 1 and 2) showed
characteristic signals of onoceranoid-type triterpenoids. They
included five methyl groups [1: δH 0.67 (s, H-28), 0.82 (s, H-
25), 1.01 (s, H-24), 1.09 (s, H-23), and 1.72 (s, H-30), 2: δH
0.63 (s, H-28), 0.83 (s, H-25), 1.02 (s, H-24), 1.09 (s, H-23),
and 1.72 (s, H-30)], three exomethylene groups [1: δC 104.1
(C-27), 107.8 (C-26), 114.4 (C-29), 145.6 (C-22), 147.4 (C-
8), and 149.5 (C-14), 2: δC 105.0 (C-27), 107.8 (C-26), 114.7
(C-29), 145.2 (C-14), 145.4 (C-22), and 147.4 (C-8)], a
ketone carbonyl group [1: δC 217.2 (C-3), 2: δC 217.2 (C-3)],
a carboxylic acid carbonyl [1: δC 177.7 (C-21), 2: δC 178.3 (C-
21)], and a methine group bearing an oxygen function [1: δC
73.2 (C-15), 2: δC 86.2 (C-15)]. The above analysis suggested
that lansium acids I and II (1 and 2) are onoceranoid-type
triterpenoids.7−9
The positions of the functional groups described above were
determined based on COSY double-quantum filter (DQF
COSY) and heteronuclear multiple bond correlation (HMBC)
NMR spectroscopy, as shown Figure 2. Hence, long-range
correlations were observed between the following proton and
carbon pairs: H-9 and C-12; H-13 and C-12; H-23 and C-3, C-
Lansium acids III and IV (3 and 4) were isolated as
colorless, amorphous powders with positive optical rotations
B
DOI: 10.1021/acs.jnatprod.8b00341
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Journal of Natural Products
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Table 1. 13C NMR Data (150 MHz) of Lansium Acids I−IX (1−9) (δ in ppm)
a
a
a
a
b
b
b
b
b
position
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
37.6
34.7
217.2
47.8
55.2
25.1
37.8
147.4
56.4
39.3
22.3
22.5
47.9
149.5
73.2
39.1
48.4
41.1
31.8
27.4
177.7
145.6
26.0
21.6
14.0
107.8
104.1
17.7
114.4
23.1
37.6
34.7
217.2
47.8
55.3
25.1
37.8
147.4
56.3
39.3
22.2
22.3
47.9
145.2
86.2
34.2
48.4
41.4
31.7
27.5
178.3
145.4
25.9
21.6
14.0
107.8
105.0
17.6
114.7
23.1
37.5
34.6
216.6
47.8
55.1
25.1
37.8
147.1
57.9
39.7
23.9
30.5
163.0
133.2
198.3
39.0
46.9
42.1
33.7
29.3
178.2
145.1
26.0
21.6
14.1
107.5
12.2
21.3
115.8
22.6
36.9
27.8
78.5
39.1
54.6
23.6
38.1
147.9
58.7
39.8
24.0
30.6
163.5
133.1
198.4
39.0
47.0
42.1
33.7
29.3
178.5
145.2
28.3
15.4
14.5
106.7
12.2
21.3
115.7
22.5
34.4
30.3
178.6
74.7
53.0
29.2
38.8
152.1
51.4
43.1
21.3
22.3
43.9
150.0
73.7
38.0
44.1
42.4
34.0
28.7
176.8
148.1
35.3
28.3
19.6
107.2
109.1
17.5
114.3
24.4
37.5
27.7
88.7
40.2
55.2
24.4
38.9
152.6
57.7
39.8
22.7
23.0
43.9
150.0
73.4
38.3
44.2
42.4
33.9
29.4
176.8
148.1
28.5
17.1
15.1
107.4
108.5
17.6
114.3
24.6
107.9
75.9
79.0
71.6
67.4
37.3
27.8
88.8
40.2
55.2
24.6
38.9
149.6
57.6
39.8
22.6
23.3
44.6
148.1
86.6
33.9
44.6
42.2
33.8
29.2
176.6
147.4
28.6
17.1
15.1
107.3
112.2
17.5
114.6
24.2
107.9
75.9
79.0
71.6
67.4
37.1
27.4
89.2
39.8
55.1
24.6
38.9
149.6
58.3
39.8
22.7
23.5
44.9
148.1
86.7
33.9
44.6
42.2
33.7
29.3
176.6
147.4
17.1
28.5
15.0
107.3
112.2
17.5
114.6
24.2
105.1
57.9
76.5
72.9
78.6
63.3
170.4
24.1
37.5
27.9
89.0
40.2
55.3
24.7
39.0
149.0
58.9
40.0
25.0
25.1
44.0
85.0
133.6
132.3
49.8
42.3
34.5
29.6
176.6
147.0
28.7
17.2
15.1
107.5
21.9
20.6
116.5
24.0
107.9
75.9
79.0
71.6
67.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
2′
3′
4′
5′
6′
Ac
a
b
Measured in CDCl3. Measured in C5D5N.
(3: [α]25 +10.7, 4: [α]25 +12.6 in methanol). ESIMS
−0.6)] and 4 [252.7 nm (Δε +0.7), 317.2 nm (Δε −0.4)],
similar to the determinations made for 1 and 2. Based on this
evidence, the chemical structures of lansium acids III and IV (3
and 4) were characterized as shown in Figure 1.
D
D
revealed for each a quasimolecular ion peak (m/z 491 [M +
Na]+ for 3, 493 [M + Na]+ for 4), from which the molecular
formulas (3: C30H44O4 and 4: C30H46O4) were determined.
The 1H and 13C NMR (CDCl3) spectra of 3 (Tables 1 and 2)
showed signals due to its onoceranoid-type triterpenoid
structure, which were similar to 1. The main differences
between 3 and 1 were the position of the double bond, the
presence of one more carbonyl group [3: δC 198.3 (C-15)],
and the absence of a hydroxy group. The positions of each
group in 3 were determined based on DQF COSY and HMBC
Lansium acid V (5) was isolated as a colorless, amorphous
powder with a positive optical rotation, [α]25 +4.4, in
D
methanol. ESIMS revealed a sodiated molecular ion peak at m/
z 527 [M + Na]+, from which the molecular formula,
C30H48O6, was determined, in conjunction with the HRESIMS
and 13C NMR data. The 1H and 13C NMR (C5D5N) spectra of
5 (Tables 1 and 2) showed signals for five methyl groups, three
exomethylene groups, a methine bearing an oxygenated
function, a quaternary carbon bearing an oxygenated function,
and two carboxylic acid groups. The above analysis suggested
that lansium acid V (5) is an oxidized analogue of lansic acid
(17). Based on DQF COSY and HMBC spectroscopy, the
positions of two hydroxy groups were elucidated as C-4 and C-
15 (Figure 2). Specifically, long-range correlations were
observed between the following proton and carbon pairs: H-
23 and C-4, -5, H-24 and C-4, -5, and H-27 and C-13, -14, -15
1
NMR spectroscopy, as shown in Figure 2. The H and 13C
NMR (CDCl3) spectra of 4 were very similar to those of 3
except for the absence of the carbonyl group at C-3 and the
presence of one more hydroxy group [4: δH 3.27 (dd, J = 4.1,
12.4), δC 78.5 (C-3)]. From this information and the 2D NMR
experiments, the planar structure of 4 was established. The
relative and the absolute configurations of 3 and 4 were
determined by analysis of their NOESY spectra (Figure 3) and
with the ECD spectra 3 [256.1 nm (Δε +0.9), 316.7 nm (Δε
C
DOI: 10.1021/acs.jnatprod.8b00341
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Journal of Natural Products
Article
Na]+) from which the molecular formulas (6: C35H56O8, 7:
C36H56O9, 8: C38H61NO10) were determined, in conjunction
1
Table 2. H NMR Spectroscopic Data (600 MHz) of
Lansium Acids I−V (1−5) (δ in ppm, J in Hz)
1
with HRESIMS and their 13C NMR data. The H and 13C
a
a
a
a
b
position
1
1
2
3
4
5
NMR (C5D5N) spectra suggested that lansium acids VI and
VIII (6 and 8) are glycosides of lansium acid I (1) and that
lansium acid VII (7) is a glycoside of lansium acid II (2). Acid
hydrolysis of 6, 7, and 8 with 5% aqueous H2SO4−1,4-dioxane
yielded D-xylose (from 6 and 7) and N-acetyl-D-glucosamine
(from 8), which were identified via HPLC of their chiral
tolylthiocarbamoyl thiazolidine derivatives.14,15 The attach-
ment of the glycosyl moiety to C-3 was identified by the
HMBC cross-peak between the aglycone [6: δ C 88.7 (C-3), 7:
δ C 88.8 (C-3), 8: δ C 89.2 (C-3)] and glycosyl moieties [6: δ
H 4.71 (d, J = 8.2 Hz, H-1′), 7: δ H 4.69 (d, J = 7.6 Hz, H-1′),
8: δ H 4.93 (d, J = 8.2, H-1′)]. The relative configurations of 6,
7, and 8 were determined via analysis of their NOESY spectra
(Figure 3). Finally, the absolute configurations were elucidated
from their ECD spectra [6: 268.2 nm (Δε +0.8), 315.8 nm
(Δε −0.8); 7: 273.1 nm (Δε +0.1), 317.6 nm (Δε −0.04); 8:
275.1 nm (Δε +0.4), 317.1 nm (Δε −0.2)], similar to the
procedure used for lansium acid I (1). On the basis of all the
evidence obtained, the structures of lansium acids VI, VII, and
VIII (6, 7, and 8) were characterized as shown in Figure 1.
Lansium acid IX (9) was isolated as a colorless, amorphous
α 2.01 (m) α 2.00 (m) α 2.00 (m) α 1.74 (m) 2.01 (μ)
β 1.44 (m) β 1.47 (m) β 1.57 (m) β 1.20 (m)
2
α 2.37 (m) α 2.36 (dd, α 2.43 (m) α 1.57 (m) 2.75 (m)
3.4, 6.9)
β 2.39 (m) β 2.39 (m) β 2.57 (m) β 1.71 (m) 3.27 (m)
3
5
6
3.27 (dd,
4.1, 12.4)
1.59 (m)
1.59 (m)
1.61 (dd,
2.8, 12.4)
1.09 (m)
1.92 (t-like,
11.7)
α 1.46 (m) α 1.50 (m) α 1.48 (dd, α 1.37 (m) α 1.41 (m)
4.1, 12.4)
β 1.68 (m) β 1.68 (m) β 1.71 (m) β 1.77 (m) β 1.77 (m)
7
9
2.43 (m)
2.44 (m)
α 2.45 (m) α 2.42 (m) α 2.43 (t-
like,
13.7)
β 2.00 (m) β 2.00 (m) β 2.22 (m)
1.61 (m)
1.64 (m)
1.75 (m)
1.65 (m)
2.22 (d-
like,
10.3)
11
12
1.23 (m)
1.64 (m)
1.23 (m)
1.70 (m)
1.56 (m)
1.99 (m)
2.41 (m)
1.74 (m)
2.09 (m)
1.76 (m)
1.77 (m)
13
1.68 (m)
1.70 (m)
3.03 (d-
like,
powder with a negative optical rotation, [α]25 −36.6, in
D
11.0)
methanol. ESIMS revealed a quasimolecular ion peak m/z 643
[M + Na]+, from which the molecular formula, C35H56O9, was
determined in conjunction with the HRESIMS and 13C NMR
15
16
3.97 (dd,
4.8, 11.0)
4.28 (dd,
4.8, 11.6)
4.59 (br s)
α 1.61 (m) α 1.59 (m) α 2.45 (m) α 2.49 (dd, 1.97 (m)
1
data. The H and 13C NMR (CDCl3) spectra of 9 (Tables 1
8.9, 17.4)
β 1.96 (m) β 2.02 (m) β 2.02 (m) β 2.59 (dd,
and 3) showed signals due to the presence of an onoceranoid-
type triterpene glycoside, similar to the data from 7. The main
differences from 7 were the location of the double bond and
the occurrence of a peroxy group. Thus, for 9, the double bond
was located at C-15,16 and the peroxy group located at C-14,
as determined based on DQF COSY, and HMBC spectros-
copy. Long-range correlations were observed between H-27
and C-13, C-14, and C-15. The relative and the absolute
configurations of 9 were determined via analysis of their
NOESY (Figure 3) and ECD spectra [257.2 nm (Δε +0.2),
317.6 nm (Δε −0.2)], similar to the procedure used to
determine 1. On the basis of all this evidence, the structure of
lansium acid IX (9) was characterized as shown in Figure 1.
Antimutagenic activities of selected isolated onoceranoid-
type triterpenes (10, 11, 12, 16, and 17) against Trp-P-1 and
PhIP (Supporting Information, Tables S10 and S11) were
evaluated using the Ames test with S. typhimurium TA98. The
other isolated compounds were not tested, because of their
limited amount available. Trp-P-1 and PhIP are well-known
mutagenic and carcinogenic heterocyclic amines found in
cooked meat. The test results obtained showed that these
onoceranoid-type triterpenes possess antimutagenic effects
against Trp-P-1 and PhIP without antimicrobial activity at
the tested concentrations. Among the compounds tested, the
antimutagenic effect of lansiolic acid (10) [inhibition: 73.8% at
100 nmol/plate] against Trp-P-1 was equivalent to that of the
positive control nobiletin [inhibition: 56% at 80 nmol/plate].
Based on these results, interesting structure−activity relation-
ships could be suggested. Specifically, lansiolic acid (10) with a
carboxylic acid moiety showed more potent antimutagenic
effects than its analogues with either a methyl ester moiety
(11) [inhibition: 29.5% at 100 nmol/plate] or an ethyl ester
moiety (12) [inhibition: 7.9% at 100 nmol/plate]. Among the
esters, the ethyl ester compound showed weaker effects than
8.9, 17.4)
17
19
2.29 (dd,
3.4, 13.0)
2.29 (dd,
3.4, 13.0)
2.64 (m)
1.83 (m)
2.64 (dd,
4.8, 8.9)
3.28 (m)
2.01 (m)
1.59 (d-like, 1.60 (dd-
2.8, 12.0)
1.84 (m)
like, 5.5,
8.3)
1.76 (m)
1.74 (m)
1.91 (m)
1.93 (m)
2.02 (m)
20
2.12 (d-like, 2.13 (m)
2.8, 3.0)
2.13 (dt,
4.8, 11.7)
2.72 (m)
2.86 (m)
2.39 (m)
2.39 (m)
2.33 (dt,
4.8, 11.7)
2.35 (m)
23
24
25
26
1.09 (s)
1.09 (s)
1.01 (s)
1.02 (s)
0.85 (s)
1.00 (s)
0.77 (s)
0.67 (s)
1.37 (s)
1.33 (s)
0.90 (s)
1.01 (s)
1.02 (s)
0.82 (s)
0.83 (s)
4.66 (br s)
4.93 (br s)
4.80 (br s)
5.26 (br s)
0.67 (s)
4.66 (br s)
4.93 (br s)
4.83 (br s)
5.20 (br s)
0.63 (s)
4.66 (br s) 4.59 (br s) 4.78 (br s)
4.97 (br s) 4.90 (br s) 5.04 (br s)
27
1.80 (s)
1.81 (s)
4.86 (br s)
5.20 (br s)
0.76 (s)
28
29
1.14 (s)
1.13 (s)
4.70 (br s)
4.90 (br s)
1.72 (s)
4.70 (br s)
4.91 (br s)
4.76 (br s) 4.76 (br s) 4.84 (br s)
4.91 (br s) 4.91 (br s) 4.91 (br s)
30
1.72 (s)
b
1.67 (s)
1.67 (s)
1.76 (s)
a
Measured in CDCl3. Measured in C5D5N.
(Figure 2). The relative configuration of 5 was determined via
analysis of its NOESY spectrum (Figure 3). The absolute
configuration was assumed to be the same as the other isolated
onoceranoid-type triterpenes. On the basis of all the evidence
obtained, the chemical structure of lansium acid V (5) was
characterized as shown in Figure 1.
Lansium acids VI, VII, and VIII (6, 7, and 8) were isolated
as colorless, amorphous powders with negative and positive
optical rotations (6: [α]25 −17.4, 7: [α]25 −2.0, 8: [α]25
D
D
D
+7.5 in MeOH). ESIMS revealed quasimolecular ion peaks (6:
m/z 627 [M + Na]+, 7: m/z 643 [M + Na]+, 8: m/z 714 [M +
D
DOI: 10.1021/acs.jnatprod.8b00341
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Journal of Natural Products
Article
Figure 2. Important 2D NMR correlations in lansium acids I−IX (1−9).
Figure 3. Important NOESY correlations of lansium acid I−IX (1−9).
the methyl ester. The effect of the compound with a hydroxy
group at the C-3 position (10) was equivalent to the
compound with a carbonyl group (16).
fewer micronucleated reticulocytes (MNRETs) were caused by
PhIP (Figure 4).
EXPERIMENTAL SECTION
To examine the antimutagenic effects of major onoceranoid-
type triterpenes of L. domesticum that showed positive effects in
the Ames test, a micronucleus test was conducted using
peripheral blood of male Institute of Cancer Research (ICR)
mice. The micronucleus test is used to detect chromosomal
damage induced by genotoxic compounds. It has been also
used to evaluate the strength of antimutagenic agents in
vivo.16−18 In this study, the in vivo antimutagenic effects of
lansionic acid (16) were evaluated against PhIP. Normal feed
or sample feed that included lansionic acid (16) was
administrated at either a low or a high dose (0.03% or
0.06%, w/w). Mouse tail vein blood (5 μL) was taken prior to
administration of PhIP and 24, 48, and 72 h after test
compound administration. Samples taken 24 and 48 h after
lansionic acid (16) administration showed that significantly
■
General Experimental Procedures. The following instruments
were used to obtain physical data: specific rotations, a JASCO P-2200
digital polarimeter (l = 5 cm); JASCO FT/IR-4600 Fourier transform
infrared spectrometer; ESIMS and high-resolution ESIMS, JEOL
1
JMS-GCMATE mass spectrometer; H NMR spectra, JEOL ECS400
(400 MHz) and JNM-ECA 600 K (600 MHz) spectrometers; 13C
NMR spectra, JNM-ECA 600 K (150 MHz) spectrometer; 2D NMR
spectra, JEOL JNM-ECA 600 K (600 MHz) spectrometer;
The following experimental materials were used for chromatog-
raphy: normal-phase silica gel column chromatography, 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 chromatography (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 high-
performance TLC, precoated TLC plates with silica gel RP-18 WF254S
(Merck, 0.25 mm); HPLC, a Shimadzu SPD-M10Avp UV−vis
E
DOI: 10.1021/acs.jnatprod.8b00341
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Table 3. 1H NMR Data (600 MHz) of Lansium Acids VI−IX
been deposited in the Forest Herbarium, Forest Department Sarawak,
Kuching, Sarawak, Malaysia.
a
(6−9) (δ in ppm, J in Hz)
Extraction and Isolation. Dried leaves of L. domesticum (550 g)
were extracted three times with methanol under reflux for 3 h.
Evaporation of the solvent provided a methanol extract (27.5 g,
5.0%). The methanol extract was partitioned into an ethyl acetate−
water (1:1, v/v) mixture to furnish an ethyl acetate-soluble fraction
(11.3 g, 2.1%) and an aqueous phase. The aqueous phase was further
extracted with 1-butanol to give a 1-butanol-soluble fraction (7.2 g,
1.3%) and a water-soluble fraction (9.0 g, 1.6%). The ethyl acetate-
soluble fraction was subjected to normal-phase silica gel column
chromatography [250 g, n-hexane−CHCl3 (3:1 → 2:1 → 1:1 → 1:2
→ 0:1, v/v) → CHCl3−MeOH (100:1 → 50:1 → 30:1 → 10:1, v/
v)], to give 10 fractions. Fraction EA5 (0.69 g) was further separated
via reversed-phase silica gel column chromatography, to give eight
fractions. Fraction EA5-4 (100.2 mg) was purified via HPLC [H2O−
CH3CN−CH3COOH (20:80:0.1, v/v/v)] to give 15 (2.6 mg).
Fraction EA6 (1.50 g) was separated via reversed-phase silica gel
column chromatography to give 10 fractions. Fraction EA6-6 (31.1
mg) was purified via HPLC [H2O−CH3CN−CH3COOH (25:75:0.1,
v/v/v)] to give 1 (20.0 mg) and 2 (23.0 mg). Fraction EA6-9 (903.3
mg) was purified via HPLC [H2O−CH3CN−CH3COOH (5:95:0.1,
v/v/v)] to give 16 (603.6 mg). Fraction EA8 (3.11 g) was separated
by reversed-phase silica gel column chromatography to give 12
fractions. Fraction EA8-9 (616.0 mg) was purified via HPLC [H2O−
CH3CN−CH3COOH (10:90:0.1, v/v/v)] to give 10 (328.3 mg) and
18 (3.2 mg). Fraction EA8-10 (383.6 mg) was purified via HPLC
[H2O−CH3CN−CH3COOH (2:98:0.1, v/v/v)] to give 11 (11.2 mg)
and 12 (20.1 mg).
position
1
6
7
8
9
α 1.55 (m)
β 1.22 (m)
α 1.76 (m)
β 1.98 (m)
3.19 (dd, 4.1, 3.23 (dd, 4.1,
11.7)
1.07 (d-like,
13.1)
1.54 (m)
α 1.58 (m)
β 1.31 (m)
α 1.74 (m)
β 2.14 (m)
α 1.41 (m)
β 1.16 (m)
α 1.63 (m)
β 2.17 (m)
3.12 (dd, 1.8, 3.30 (dd, 4.1,
12.4) 11.7)
0.99 (dd, 2.0, 1.11 (dd, 2.8,
10.3)
1.27 (m)
α 1.90 (m)
β 1.43 (m)
α 1.79 (m)
β 2.18 (m)
2
3
5
6
11.7)
1.09 (dd, 1.2,
12.4)
13.1)
α 1.26 (m)
α 1.26 (dd, 4.1,
13.1)
β 1.60 (m)
β 1.60 (d-like,
13.1)
7
2.14 (m)
2.41 (m)
α 2.38 (d-like, α 2.40 (m)
11.7)
β 2.14 (dt, 1.2, β 2.10 (m)
11.7)
2.38 (d, 11.7)
9
1.83 (d-like,
10.3)
1.46 (m)
1.60 (m)
1.85 (m)
3.00 (d-like,
11.7)
4.62 (dr-s)
1.97 (m)
1.82 (m)
4.53 (m)
1.79 (d-like,
10.3)
1.43 (m)
1.79 (m)
11
12
1.49 (m)
1.81 (m)
1.41 (m)
1.52 (m)
13
2.73 (d, 11.0) 2.75 (d, 11.0) 2.67 (d-like,
9.6)
4.76 (m)
15
16
4.77 (br s)
5.91 (d, 10.4)
5.70 (d-like,
10.4)
α 1.89 (dt, 2.8, α 2.19 (m)
6.0)
The BuOH-soluble fraction was subjected to normal-phase silica
gel column chromatography, [150 g, CHCl3−MeOH (100:1 → 50:1
→ 20:1 → 10:1 → 5:1 → 2:1 → 1:1, v/v)], to give nine fractions.
Fraction B3 (1.0 g) was further separated via reversed-phase silica gel
column chromatography, to give eight fractions. Fraction B3-4 (218.8
mg) was purified via HPLC [H2O−CH3CN−CH3COOH (40:60:0.1,
v/v/v)] to give 3 (65.3 mg) and 4 (14.0 mg). Fraction B3-6 (147.1
mg) was purified via HPLC [H2O−CH3CN−CH3COOH (10:90:0.1,
v/v/v)] to give 17 (48.4 mg). Fraction B5 (1.0 g) was further
separated via reversed-phase silica gel column chromatography to give
nine fractions. Fraction B5-4 (184.6 mg) was purified via HPLC
[H2O−CH3CN−CH3COOH (40:60:0.1, v/v/v)] to give 6 (5.0 mg),
7 (9.3 mg), and 9 (3.6 mg). Fraction B5-5 (129.4 mg) was purified via
HPLC [H2O−CH3CN−CH3COOH (20:80:0.1, v/v/v)] to give 13
(32.8 mg). Fraction B7 (0.9 g) was further separated via reversed-
phase silica gel column chromatography, to give nine fractions.
Fraction B7-3 (126.7 mg) was purified via HPLC [H2O−CH3CN−
CH3COOH (60:40:0.1, v/v/v)] to give 5 (3.2 mg). Fraction B7-4
(112.4 mg) was purified via HPLC [H2O−CH3CN−CH3COOH
(60:40:0.1, v/v/v)] to give 8 (4.0 mg). Fraction B7-5 (55.6 mg) was
purified via HPLC [H2O−CH3CN−CH3COOH (60:40:0.1, v/v/v)]
to give 14 (7.2 mg).
β 2.17 (m)
β 1.91 (m)
3.01 (d, 3.4,
9.6)
2.03 (m)
2.60 (m)
2.81 (m)
0.90 (s)
17
3.32 (dd, 6.2, 2.98 (dd, 3.5,
3.26 (br s)
10.3)
17.2)
19
20
1.98 (m)
2.71 (m)
2.95 (m)
1.19 (s)
0.91 (s)
0.61 (s)
4.74 (br s)
4.98 (br s)
4.84 (br s)
5.22 (br s)
0.78 (s)
4.88 (br s)
4.93 (br s)
1.78 (s)
1.98 (m)
2.61 (m)
2.80 (m)
1.18 (s)
0.89 (s)
0.59 (s)
4.77 (br s)
4.96 (br s)
5.03 (br s)
5.35 (br s)
0.72 (s)
4.76 (br s)
4.88 (br s)
1.69 (s)
2.04 (m)
2.69 (m)
2.79 (m)
1.21 (s)
0.90 (s)
0.61 (s)
4.95 (br s)
4.97 (br s)
1.42 (s)
23
24
25
26
1.06 (s)
0.53 (s)
4.75 (br s)
4.97 (br s)
5.05 (br s)
5.37 (br s)
0.74 (s)
4.79 (br s)
4.90 (br s)
1.71 (s)
4.93 (d, 8.2) 4.72 (d, 8.2)
1.75 (m) 3.94 (t, 8.2)
4.36 (t, 10.3) 4.09 (t, 8.2)
4.11 (t, 8.9)
3.88 (m)
27
28
29
1.82 (s)
4.85 (br s)
4.99 (br s)
1.75 (s)
30
1′
2′
3′
4′
5′
4.71 (d, 8.2) 4.69 (d, 7.6)
3.69 (t, 8.2)
4.13 (t, 8.2)
4.19 (m)
3.94 (t, 8.2)
4.09 (t, 8.2)
4.16 (m)
Lansium acid I (1): colorless, amorphous powder; [α]25 +11.3 (c
4.14 (m)
3.64 (t, 11.0)
4.21 (dd, 5.5,
11.0)
D
0.35, MeOH); ECD (MeOH) [271.7 nm (Δε +0.6), 317.6 nm (Δε
−0.6)]; IR (ATR) νmax 2942, 1749, 1705, 1451, 1378, 1218, 1032
cm−1; 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see
Tables 1 and 2; ESIMS m/z 493 [M + Na]+; HRESIMS m/z
493.3290 [M + Na]+ (calcd for C30H46O4, 493.3288).
3.71 (t, 11.7) 3.63 (t, 10.3)
4.29 (dd, 5.5, 4.21 (dd, 5.5,
11.6)
10.3)
6′
4.29 (dd, 5.5,
11.7)
Lansium acid II (2): colorless, amorphous powder; [α]25D +11.1 (c
0.14, MeOH); ECD (MeOH) [274.6 nm (Δε +0.6), 316.7 nm (Δε
−0.4)]; IR (ATR) νmax 2931, 2854, 1701, 1455, 1400, 1377, 1243,
1218, 1065 cm−1; 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150
MHz), see Tables 1 and 2; ESIMS m/z 509 [M + Na]+; HRESIMS
m/z 509.3232 [M + Na]+ (calcd for C30H46O5, 509.3237).
Lansium acid III (3): colorless, amorphous powder; [α]25D +10.7 (c
0.47, MeOH); ECD (MeOH) [256.1 nm (Δε +0.9), 316.7 nm (Δε
−0.6)]; IR (ATR) νmax 2931, 2836, 1705, 1447, 1381 cm−1; 1H
(CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Tables 1
and 2; ESIMS m/z 491 [M + Na]+; HRESIMS m/z 491.3134 (calcd
for C30H44O4 [M + Na]+, 491.3132).
4.49 (d-like,
11.7)
2.10 (s)
Ac
a
Measured in C5D5N.
detector. YMC Triart C18 (250 × 4.6 mm i.d. and 250 × 10 mm i.d.)
columns were used for analytical and preparative purposes.
Plant Material. The leaves of Lansium domesticum were collected
in Ba’kelalan, Sarawak, Malaysia, in November 2015. The plant
identification and the taxonomic authentication were conducted by
one of the authors (S.T.). A voucher specimen (STTM-2015−4) has
F
DOI: 10.1021/acs.jnatprod.8b00341
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Journal of Natural Products
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Figure 4. Frequency of micronucleated reticulocytes (MNRETs) from the peripheral blood of mice treated with a mutagen [PhIP (50 mg/kg body
weight)]. Normal feed or sample feed that included lansionic acid (16) at a low or a high dose (0.03% or 0.06%, w/w) was given ad libitum.
Significant difference: *0.01 < p < 0.05; **p < 0.01 (Student’s t test).
Lansium acid IV (4): colorless, amorphous powder; [α]25D +12.6 (c
0.38, MeOH); ECD (MeOH) [252.7 nm (Δε +0.7), 317.2 nm (Δε
−0.4)]; IR (ATR) νmax 2931, 2872, 1705, 1455, 1414, 1262 cm−1; 1H
(CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Tables 1
and 2; ESIMS m/z 493 [M + Na]+; HRESIMS m/z 493.3291 [M +
Na]+ (calcd for C30H46O4, 493.3288).
method using Salmonella typhimurium TA98 with S9 mix, as described
previously.10,12 PhIP (1 μg) and Trp-P-1 (0.04 μg) were used as
mutagens to examine the antimutagenic activity of the samples. The
experiments were conducted according to the Guidelines for Animal
Experiments at Kyoto Pharmaceutical University (KPU), and the
animal studies were approved by the Experimental Animal Research
Committee (permission number: 17-030).
Lansium acid V (5): colorless, amorphous powder; [α]25 +4.4 (c
D
Evaluation of in Vivo Antimutagenic Effects using Micro-
nucleus Test. The in vivo micronucleus test was carried out
according to a method published previously.11,19 Briefly, for the test
group, sample feed [ansionic acid (16)-containing feed] was ingested
2 days before and throughout the experiment. Feed containing 0.03−
0.06% w/w lansionic acid (16) was prepared by thoroughly mixing
lansionic acid (16) with CE-2 powder diet (Clea Japan, Inc., Tokyo,
Japan). PhIP (50 mg/kg bodyweight) was administered intra-
peritoneally to mice in all groups to induce micronuclei. Mouse tail
vein blood (5 μL) was taken prior to the administration of PhIP and
24, 48, and 72 h after the administration of the test samples.
Observations were made using an excitation filter, and an absorbing
filter (Nikon fluorescence block B-2A, Nikon, Ltd.) epi-equipped with
a fluorescence microscope [power supply HB-10101AF, OPTIPHOT-
2 lens (400 times), Nikon, Ltd.] was used. One thousand reticulocytes
were observed per slide, and those including micronuclei were
counted. Statistics regarding micronucleus frequency were calculated
using Student’s t test. Significance levels were set at 5% and 1%. The
animal studies were approved by the Experimental Animal Research
Committee at KPU (permission number: 17-031).
0.16, MeOH); IR (ATR) νmax 2931, 2887, 1705, 1644, 1385, 1283,
1
1192, 1123 cm−1; H (C5D5N, 600 MHz) and 13C NMR (C5D5N,
150 MHz), see Tables 1 and 2; ESIMS m/z 527 [M + Na]+;
HRESIMS m/z 527.3349 [M + Na]+ (calcd for C30H48O6, 527.3343).
Lansium acid VI (6): colorless, amorphous powder; [α]25 −17.4
D
(c 0.25, MeOH); ECD (MeOH) [268.2 nm (Δε +0.8), 315.8 nm (Δε
−0.8)]; IR (ATR) νmax 2927, 2884, 1705, 1644, 1200, 1163 cm−1; 1H
(C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 1
and 3; ESIMS m/z 627 [M + Na]+; HRESIMS m/z 627.3863 [M +
Na]+ (calcd for C35H56O8, 627.3867).
Lansium acid VII (7): colorless, amorphous powder; [α]25D −2.0 (c
0.47, MeOH); ECD (MeOH) [273.1 nm (Δε +0.1), 317.6 nm (Δε
−0.04)]; IR (ATR) νmax 2931, 2886, 1705, 1644, 1381, 1262, 1090
cm−1; 1H (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see
Tables 1 and 3; ESIMS m/z 643 [M + Na]+; HRESIMS m/z
643.3819 [M + Na]+ (calcd for C35H56O9, 643.3817).
Lansium acid VIII (8): colorless, amorphous powder; [α]25D +7.5 (c
0.2, MeOH); ECD (MeOH) [275.1 nm (Δε +0.4), 317.1 nm (Δε
−0.2)]; IR (ATR) νmax 2890, 2836, 1708, 1644, 1385, 1112 cm−1; 1H
(C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 1
and 3; ESIMS m/z 714 [M + Na]+; HRESIMS m/z 714.4182 [M +
Na]+ (calcd for C38H61NO10, 714.4188).
ASSOCIATED CONTENT
* Supporting Information
■
Lansium acid IX (9): colorless, amorphous powder; [α]25 −36.6
S
D
(c 0.18, MeOH); ECD (MeOH) [257.2 nm (Δε +0.2), 317.6 nm (Δε
−0.2)]; IR (ATR) νmax 2927, 2886, 1708, 1640, 1380, 1163 cm−1; 1H
(C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 1
and 3; ESIMS m/z 643 [M + Na]+; HRESIMS m/z 643.3815 [M +
Na]+ (calcd for C35H56O9, 643.3817).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00341.
1
Experimental details, including ESIMS, HRESIMS, H
and13C NMR spectra, 2D NMR spectra, and the results
of the Ames assay (PDF)
Acid Hydrolysis and Monosaccharide Identification of
Compounds 6−9. Identification of the monosaccharides of 6−9
was performed according to the method reported by Tanaka et al.
with slight modifications.14 After the acid hydrolysis of 6−9, chiral
tolylthiocarbamoyl thiazolidine derivatives were obtained. Each
solution was analyzed via 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); flow rate: 0.8
mL/min; column temperature: 25 °C] to identify the derivatives of
the constituents D-xylose (6, 7, and 9) and N-acetyl-D-glucosamine
(8) by comparison of their retention times with those of authentic
samples (tR: D-xylose, 16.37 min, N-acetyl-D-glucosamine, 35.29 min).
Evaluation of in Vitro Antimutagenic Activity Using the
Ames Test. Antimutagenicity was examined via the preincubation
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +81-75-595-4650. Fax: +81-75-595-4769. E-mail:
watanabe@mb.kyoto-phu.ac.jp.
ORCID
Tetsushi Watanabe: 0000-0002-5190-9244
Notes
The authors declare no competing financial interest.
G
DOI: 10.1021/acs.jnatprod.8b00341
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Journal of Natural Products
Article
ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Grant Number
JP17K15473.
■
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