Inflammation Modulatory Phorbol Esters from the Seeds of Aquilaria malaccensis

Article abstract of DOI:10.1021/acs.jnatprod.6b01096

The tree Aquilaria malaccensis is a valuable source of agarwood, which is used in herbal medicinal preparations. Phytochemical research on A. malaccensis seeds has led to the isolation of four new phorbol esters (1-4), two known phorbol esters (5, isolated from Nature for the first time, and 6), and two known glycerides (7 and 8). The structures of these isolates were elucidated by means of spectroscopic data interpretation. The inflammation-modulatory activities of the isolates on elastase release and superoxide anion generation in human neutrophils were evaluated. Interestingly, phorbol esters 1, 5, and 6 showed potent inhibitory activity on elastase release in human neutrophils, with IC₅₀ values of 2.7, 0.8, and 2.1 μM, respectively. All isolated phorbol esters exerted enhancing activity on superoxide anion generation. The results indicated that phorbol esters may play a bilateral modulatory role in the processes of inflammation. In addition, the compounds were evaluated for their cytotoxic properties against HepG2 (hepatoma), MDA-MB-231 (breast), and A549 (lung) cancer cells, but all compounds were inactive for all cell lines used (IC₅₀ > 10 μM).

Full text of DOI:10.1021/acs.jnatprod.6b01096

Article
pubs.acs.org/jnp
Inflammation Modulatory Phorbol Esters from the Seeds of Aquilaria
malaccensis
Vitthal D. Wagh,†,◇ Michal Korinek,†,‡,◇ I-Wen Lo,† Yu-Ming Hsu,† Shu-Li Chen,† Hsue-Yin Hsu,§
Tsong-Long Hwang,⊥,∥,▽ Yang-Chang Wu,†,# Bing-Hung Chen,*,‡,⬡,δ Yuan-Bin Cheng,*,†,¶,#
and Fang-Rong Chang*,†,¶,□,△
‡
¶
†Graduate Institute of Natural Products, College of Pharmacy, Department of Biotechnology, College of Life Science, Center for
#
Infectious Disease and Cancer Research, and Research Center for Natural Products & Drug Development, Kaohsiung Medical
University, Kaohsiung 807, Taiwan
§Department of Life Sciences, Tzu Chi University, Hualien 970, Taiwan
⊥Graduate Institute of Natural Products, College of Medicine, and Chinese Herbal Medicine Research Team, Healthy Aging Research
Center, Chang Gung University, Taoyuan 333, Taiwan
∥Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic Safety, and Graduate Institute of Health
Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan
▽Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
⬡The Institute of Biomedical Sciences and △Department of Marine Biotechnology and Resources, National Sun Yat-sen University,
Kaohsiung 804, Taiwan
δ
□Cancer Center and Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan
S
* Supporting Information
ABSTRACT: The tree Aquilaria malaccensis is a valuable source of agarwood, which is used in herbal medicinal preparations.
Phytochemical research on A. malaccensis seeds has led to the isolation of four new phorbol esters (1−4), two known phorbol
esters (5, isolated from Nature for the first time, and 6), and two known glycerides (7 and 8). The structures of these isolates
were elucidated by means of spectroscopic data interpretation. The inflammation-modulatory activities of the isolates on elastase
release and superoxide anion generation in human neutrophils were evaluated. Interestingly, phorbol esters 1, 5, and 6 showed
potent inhibitory activity on elastase release in human neutrophils, with IC50 values of 2.7, 0.8, and 2.1 μM, respectively. All
isolated phorbol esters exerted enhancing activity on superoxide anion generation. The results indicated that phorbol esters may
play a bilateral modulatory role in the processes of inflammation. In addition, the compounds were evaluated for their cytotoxic
properties against HepG2 (hepatoma), MDA-MB-231 (breast), and A549 (lung) cancer cells, but all compounds were inactive
for all cell lines used (IC50 > 10 μM).
Aquilaria malaccensis Lam. (family Thymelaeaceae) is a tropical
tree native to Malaysia, known locally as “Karas”. The tree is
distributed also in the rainforests of Cambodia, India, Indonesia,
Laos, the Philippines, Taiwan, and Thailand.1 This species serves
as the source of agarwood, a valuable fragrant resinous wood,
which is used widely in religious ceremonies as well as in
traditional medicinal preparations in Asia.2 Agarwood oil has
been used as antiasthmatic, aphrodisiac, astringent, cardiotonic,
and carminative agents and as a remedy for diarrhea, dysentery,
gout, rheumatism, and paralysis.3 Extracts of A. malaccensis have
been investigated for their cardiotonic,4 cytotoxic,5 antitrypano-
somal,6 antibacterial,7 and antiallergic8,9 activities.
Received: November 25, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b01096
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
In a previously reported study, feruryl glyceride and phorbol
esters were isolated from the bark of A. malaccensis.6 Gas
chromatography−mass spectrometry analysis (GC-MS) of
agarwood (A. malaccensis) revealed the presence of aromatic
terpenes, with sesquiterpenes as the main components, and
chromones, as well as benzenoids, sterols, and fatty acids.10 GC-
MS analysis of A. malaccensis seeds showed high amounts of
unsaturated (oleic acid 68.5%) and saturated fatty acids.11
Currently, there is scarce information on the chemical
composition as well as bioactivities of seeds of A. malaccensis.
Previously, potent antiallergic and anti-inflammatory activities of
a crude extract and chromatographic fractions were observed,
and a promising antiallergic phorbol ester (aquimavitalin) with
nanomolar IC50 values was isolated.9 In the current study, eight
compounds were isolated, namely, four new phorbol esters (1−
4), two known phorbol esters (5 and 6), and two known
diglycerides (7 and 8), from the seeds of A. malaccensis. Their
inflammation-modulatory and cytotoxic activities for cancer cell
lines have been investigated.
(Figure 1) showed correlations between H-10 (δ 3.27)/H-1 (δ
7.59)/Me-19 (δ 1.78), H2-5 (δ 2.49 and 2.55)/H-7 (δ 5.69)/H-8
(δ 3.27), H-14 (δ 1.12), and H-12 (δ 5.51)/H-11 (δ 2.23)/Me-
18 (δ 0.92). The CH3 proton at δH 1.78 (H-19) exhibited HMBC
correlations to the vinylic carbon signal at δC 160.5 (C-1),
quaternary carbon at δC 133.0 (C-2), and ketone at δC 208.7 (C-
3), supporting an α,β-unsaturated carbonyl moiety. The H-7
signal showed a HMBC correlation to the oxygenated methylene
at δC 67.9 (C-20) and the methylene at δC 38.7 (C-5). The
observation of two methyl singlets at δH 1.22 and δH 1.27 (H-16
and H-17) and a methine doublet at δH 1.12 (J = 5.4 Hz, H-14)
together with HMBC correlations from the methyl signal [δH
1.27 (H-17)] to δC 25.9 (C-15) and δC 23.9 (C-16) were used to
establish the presence of a dimethyl cyclopropane moiety.
Furthermore, the connectivity of the H-12 (δ 5.51)/H-11 (δ
2.23)/Me-18 (δ 0.92) moiety with the dimethyl cyclopropane
ring was established by means of HMBC correlations from the
oxymethine proton signal at δH 5.51 (H-12) to δC 43.1 (C-11),
65.5 (C-13), 36.5 (C-14), 25.9 (C-15), and 14.5 (C-18) (Figure
1). Thus, compound 1 was deduced to possess a backbone of a
phorbol-type diterpene.12,13 Moreover, the NMR data of 1 were
similar to those of the known phorbol esters 12-O-(2Z,4E,6E)-
deca-2,4,6-trienoylphorbol-13-acetate5 and aquimavitalin,9 ex-
cept for the fatty acid moiety. There were two substituents
detected in 1, namely, an acetyl group [δC 173.7 (C-21); δH 2.11
(s, H-22), δC 21.1 (C-22)] and a fatty acid moiety possessing four
olefinic protons [δH 6.31 (d, J = 15.6 Hz, H-2′), δC 129.5 (C-2′);
δH 7.40 (dd, J = 15.6 and 11.4 Hz, H-3′), δC 140.8 (C-3′); δH 7.17
(dd, J = 15.6 and 11.4 Hz, H-4′), δC 146.8 (C-4′); δH 6.43 (dd, J =
15.6 and 7.8 Hz, H-5′), δC 137.2 (C-5′)], and also a typical
formyl group proton [δH 9.68 (d, J = 7.8 Hz, H-6′), δC 192.7 (C-
6′)] was evident. The structure of the long-chain fatty acid
moiety was further assigned by COSY correlations between H-2′
(δ 6.31)/H-3′ (δ 7.40)/H-4′ (δ 7.17)/H-5′ (δ 6.43) and HMBC
RESULTS AND DISCUSSION
■
Compound 1 was obtained as a colorless oil. The molecular
formula was calculated as C28H34O9 (12 degrees of unsaturation)
from the analysis of its HRESIMS (m/z 537.20959 [M + Na]+,
calcd 537.20950). Its IR absorbance spectrum showed the
presence of hydroxy (3413 cm−1), carbonyl (1713 cm−1), and
olefinic (1597 cm−1) groups. The 1H, 13C, and HSQC NMR data
of compound 1 (Tables 1 and 2) indicated the occurrence of two
vinylic groups [δH 7.59 (s, H-1) and δC 160.5 (C-1), δC 133.0 (C-
2); δC 140.6 (C-6), δH 5.69, (d, J = 4.2 Hz, H-7), 128.9 (C-7)], an
oxygenated methine [δH 5.51 (d, J = 10.2 Hz, H-12), δC 78.2 (C-
12)], an oxygenated methylene [δH 4.05 (d, J = 12.6 Hz, H-20a),
4.01 (d, J = 12.6 Hz, H-20b), δC 67.9 (C-20)], four methyls, a
methylene, and four methines.13 The H−1H COSY spectrum
1
B
DOI: 10.1021/acs.jnatprod.6b01096
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Article
a
Table 1. 1H NMR Data of Compounds 1−4 in CDCl3
position
1
2
3
4
1
7.59, s
7.60, s
7.60, s
7.60, s
5α
5β
7
2.49, d (19.2)
2.55, d (19.2)
5.69, d (4.2)
3.27, t (5.2)
3.27, brs
2.38, d (18.8)
2.50, d (18.8)
5.70, d (4.4)
2.99, t (5.2)
3.28, brs
2.49, d (19.2)
2.55, d (19.2)
5.69, d (5.4)
3.26, m
2.49, d (19.2)
2.55, d (19.2)
5.69, d (4.8)
3.26, m
8
10
11
12α
12β
14
16
17
18
19
20a
20b
22
2′
3′
4′
5′
6′
7′
8′
3.28, brs
3.28, brs
2.23, m
2.01, m
2.19, m
2.19, m
5.51, d (10.2)
1.57, m
5.48, d (10.2)
5.47, d (10.2)
2.08, m
1.12, d (5.4)
1.22, s
0.83, d (5.2)
1.06, s
1.10, d (5.4)
1.21, s
1.10, d (5.4)
1.21, s
1.27, s
1.19, s
1.26, s
1.26, s
0.92, d (6.6)
1.78, brs
0.88, d (6.0)
1.78, brs
0.91, d (6.6)
1.78, brs
0.90, d (6.6)
1.78, brs
4.05, d (12.6)
4.01, d (12.6)
2.11, s
4.48, d (12.4)
4.43, d (12.4)
2.06, s
4.05, d (12.6)
3.95, d (12.6)
2.10, s
4.05, d (13.2)
4.00, d (13.2)
2.10, s
6.31, d (15.6)
7.40, dd (15.6, 11.4)
7.17, dd (15.6, 11.4)
6.43, dd (15.6, 7.8)
9.68, d (7.8)
2.29, t (7.6)
1.60, m
5.92, d (15.6)
7.27, dd (14.4, 12.0)
6.45, m
5.92, d (15.6)
7.27, dd (14.4, 12.0)
6.48, m
1.25−1.26, m
1.25−1.26, m
1.25−1.26, m
1.25−1.26, m
2.01, m
6.17, dd (15.6, 7.2)
4.24, m
6.12, dd (15.6, 6.0)
4.07, m
3.74, m
3.52, m
1.42, m
1.42, m
9′
5.33, m
1.27−1.29, m
1.27−1.29, m
1.27−1.29, m
1.27−1.29, m
1.27−1.29, m
0.88, t (7.2)
1.26−1.30, m
1.26−1.30, m
1.26−1.30, m
1.26−1.30, m
1.26−1.30, m
0.88, t (7.2)
10′
11′
12′
13′
14′
15′
16′
17′
18′
5.33, m
2.01, m
1.25−1.26, m
1.25−1.26, m
1.25−1.26, m
1.25−1.26, m
1.25−1.26, m
1.25−1.26, m
0.88, t (6.8)
a
1H NMR spectra were measured at 600 MHz (1, 3, 4) and at 400 MHz (2) in CDCl3.
correlations of H-2′/C-4′ (δC 146.8) and H-3′/C-5′ (δC 137.2).
The 1H NMR coupling constants and NOESY correlations were
used to identify the geometry of double bonds. Accordingly, the
NMR data indicated that 1 possesses a (2E,4E)-6-oxohexa-2,4-
dienoic acid moiety. The fatty acid moiety was attached to the
phorbol backbone at C-12 according to the HMBC correlation of
H-12 to C-1′ (δC 165.4); so, therefore, the acetyl moiety was
attached to C-13. The relative configuration of compound 1 was
deduced by comparison of its NOESY correlation data (Figure 2)
with literature values.13 The NOESY correlations between H-11/
H-17 and H-11/H-8 suggested that these protons are β-oriented.
Further correlations between Me-18/H-12α indicated that the
fatty acid moiety is also β-oriented. On the basis of all of the
above information, the structure of 1 was determined as 12-O-
(2′E,4′E)-6-oxohexa-2′,4′-dienoylphorbol-13-acetate.
Compound 2 was also obtained as a colorless oil. It was
assigned with a molecular formula of C40H62O7 (10 degrees of
unsaturation) on the basis of HRESIMS (m/z 677.43884 [M +
Na]+, calcd 677.43878). The IR spectrum indicated the presence
of hydroxy (3409 cm−1), carbonyl (1717 cm−1), and olefinic
(1597 cm−1) groups. A comparison of the 1H and 13C NMR data
(Tables 1 and 2) with 1 suggested that compound 2 possesses a
12-deoxyphorbol type of diterpenoid backbone. Moreover, the
signals typical of a 12-deoxyphorbol derivative were detected [δH
2.99 (t, J = 5.2 Hz, H-8), δC 39.4 (C-8); δH 2.01 (m, H-11), δC
36.3 (C-11); δH 1.57 (m, H-12α), δH 2.08 (m, H-12β), δC 31.9
(C-12)].14,15 Furthermore, signals for acetyl [δC 173.2 (C-21);
δH 2.06 (s, H-22), δC 21.2 (C-22)] and fatty acid moieties were
observed in the NMR spectra. The COSY correlations of
compound 2 (Figure 1) showed connectivities of the H-10 (δ
3.28)/H-1 (δ 7.60)/Me-19 (δ 1.78), H2-5 (δ 2.38 and 2.50)/H-7
(δ 5.70)/H-8 (δ 2.99)/H-14 (δ 0.83), and H2-12 (δ 1.57 and
2.08)/H-11 (δ 2.01)/Me-18 (δ 0.88) substituents for the
skeleton, as well as H-2′ (δ 2.29)/H-3′ (δ 1.60)/H-4′ (δ 1.25),
and H-7′ (δ 1.25)/H-8′ (δ 2.01)/H-9′ (δ 5.33)/H-10′ (δ 5.33)/
H-11′ (δ 2.01)/H-12′ (δ 1.25), for the long-chain fatty acid
moiety.
To confirm the long-chain fatty acid moiety, compound 2 was
subjected to methanolysis using a method reported by Ichihara et
al.16 Compound 2 was reacted with 1.2% HCl in methanol for 1.5
h at 100 °C, and the resulting fatty acid methyl ester was
identified by GC-MS analysis as (9Z)-octadecenoic (oleic) acid
methyl ester (tR 21.30, m/z 296 [M]+). Comparison of the
spectra (Table 1) with the literature revealed that the data of
compound 2 closely matched those of 12-deoxyphorbol-13-
(9′Z)-octadecenoate-20-acetate.15 However, the HMBC corre-
lation from H-20 to C-1′ (δC 173.6) indicated that the fatty acid
moiety is linked to the backbone at C-20. Therefore, the acetyl
C
DOI: 10.1021/acs.jnatprod.6b01096
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Table 2. 13C NMR Data of Compounds 1−4 in CDCl3
a
Compound 3 was isolated as another colorless oil. HRESIMS
analysis indicated this compound to have a molecular formula of
C36H52O10 (m/z 667.34515 [M + Na]+; calcd 667.34527), 10
degrees of unsaturation). The IR absorption bands of 3 showed
hydroxy group (3392 cm−1), carbonyl (1710 cm−1), and olefinic
(1632 cm−1) functionalities. The 1D (Tables 1 and 2) and 2D
(Figure 1) NMR data of the skeleton of compound 3 closely
matched those of compound 1, as a phorbol derivative, with there
being two acyl substituents detected in 3. In addition to an acetyl
group, the 1D NMR data indicated the presence of a long-chain
fatty acid substituent possessing two double bonds along with
position
1
2
3
4
1
160.5, CH
133.0, C
208.7, C
73.7, C
161.3, CH
132.8, C
160.7, CH
132.9, C
160.7, CH
132.9, C
2
3
209.0, C
208.8, C
208.8, C
4
73.6, C
73.7, C
73.7, C
5
38.7, CH2
140.6, C
128.9, CH
39.1, CH
78.2, C
39.0, CH2
135.0, C
38.7, CH2
140.5, C
38.7, CH2
140.5, C
6
7
133.7, CH
39.4, CH
129.2, CH
39.1, CH
78.2, C
129.2, CH
39.1, CH
78.2, C
8
9
75.9, C
1
two hydroxy groups. The H and 13C NMR data showed four
10
11
12
13
14
15
16
17
18
19
20
21
22
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
16′
17′
18′
56.2, CH
43.1, CH
78.2, CH
65.5, C
55.7, CH
56.2, CH
43.1, CH
77.1, CH
65.7, C
56.2, CH
43.1, CH
77.2, CH
65.7, C
vinylic protons at δH 5.92, 7.27, 6.45, and 6.17 with
corresponding carbons at δC 121.7, 143.9, 129.9, and 140.0.
The COSY cross-peaks between the vinylic protons suggested
that the two double bonds are conjugated. The chemical shifts of
H-2′ to H-5′ and C-2′ to C-5′ closely matched literature
chemical shifts for methyl (2E,4E,8E)-6,13-dihydroxytetradeca-
2,4,8-trienoate, indicating a 2′,4′-diene moiety.17 The positions
of two hydroxy groups in the side chain were established
according to 2D NMR data analysis (Figure 1). The COSY
correlations of H-2′ (δ 5.92)/H-3′ (δ 7.27)/H-4′ (δ 6.45)/H-5′
(δ 6.17)/H-6′ (δ 4.24)/H-7′ (δ 3.74)/H-8′ (δ 1.42) and HMBC
correlations of H-2′/C-4′ (δC 129.9), H-4′/C-6′ (δC 74.9), and
H-5′/C-6′ (δC 74.9) revealed the connectivity of conjugated
double bonds with a 6′-hydroxy methine proton [δH 4.24 (H-6′),
δC 74.9, (C-6′)] and a 7′-hydroxy proton [δH 3.74 (H-7′), δC
74.3, (C-7′)]. By comparing the chemical shifts of the 6′,7′-
vicinal diol units of 3 and 4 (see below) with the reference
compounds, methyl 9,10-(erythro)-dihydroxy-11(E)-octadece-
noate and methyl 9,10-(threo)-dihydroxy-11(E)-octadece-
noate,18 two 6′- and 7′-hydroxymethine protons in 3, δH 4.24
(H-6′) and 3.74 (H-7′), were downfield shifted when compared
to those of compound 4, δH 4.07 (H-6′) and 3.52 (H-7′). Thus,
the structure of the fatty acid moiety was assigned in 3 as 12-O-
(2E,4E)-6,7-(erythro)-dihydroxytetradeca-2,4-dienoic acid. The
long-chain fatty acid was attached to the phorbol backbone at C-
12 according to the HMBC correlation of H-12/C-1′ (δC 165.4),
and the acetyl moiety was attached to C-13. The NOESY
correlations of 3 indicated the same relative configuration as in 1
(Figure 2). Therefore, the structure of 3 was proposed as 12-O-
(2′E,4′E)-6′,7′-(erythro)-dihydroxytetradeca-2′,4′-dienoylphor-
bol-13-acetate.
36.3, CH
31.9, CH2
63.5, C
36.5, CH
25.9, C
32.3, CH
36.4, CH
25.8, C
36.4, CH
25.6, C
22.7, C
23.9, CH3
16.9, CH3
14.5, CH3
10.1, CH3
67.9, CH2
173.7, C
21.1, CH3
165.4, C
129.5, CH
140.8, CH
146.8, CH
137.2, CH
192.7, CH
23.2, CH3
15.3, CH3
18.5, CH3
10.1, CH3
69.5, CH2
173.2, C
23.8, CH3
16.7, CH3
14.4, CH3
10.0, CH3
68.0, CH2
173.8, C
23.8, CH3
16.8, CH3
14.4, CH3
10.1, CH3
68.0, CH2
173.8, C
21.1, CH3
173.6, C
21.1, CH3
165.4, C
21.1, CH3
165.4, C
34.2, CH2
24.9, CH2
29.1−29.7, CH2
29.1−29.7, CH2
29.1−29.7, CH2
29.1−29.7, CH2
27.2, CH2
129.8, CH
130.0, CH
27.2, CH
121.7, CH
143.9, CH
129.9, CH
140.0, CH
74.9, CH
74.3, CH
32.2, CH2
29.2, CH2
29.4, CH2
29.7, CH2
31.8, CH2
22.6, CH2
14.0, CH3
121.9, CH
143.8, CH
129.7, CH
141.5, CH
75.0, CH
74.3, CH
33.1, CH2
29.2, CH2
29.5, CH2
29.7, CH2
31.8, CH2
22.6, CH2
14.1, CH3
29.1−29.7, CH2
29.1−29.7, CH2
29.1−29.7, CH2
29.1−29.7, CH2
31.8, CH2
22.7, CH2
14.1, CH3
a
13C NMR spectra were measured at 150 MHz (1, 3, 4) and at 100
MHz (2) in CDCl3.
Compound 4 was isolated as a further colorless oil. Its IR
(3385 cm−1, 1632 cm−1), HRESIMS (m/z 667.34550 [M +
Na]+, calcd 667.34527), and 13C NMR data were identical to
those of compound 3. Therefore, the basic structure was assigned
as a C-12 long-chain fatty acid and C-13 acetyl-disubstituted
group was assigned to C-13. The relative configuration of 2 was
assigned according to the ROESY spectrum (Figure 2). The
correlations between H-11/H-8 and H-17/H-8 indicated that
these protons are β-oriented. On the basis of the interpretation of
the above spectroscopic data, compound 2 was assigned as 12-
deoxy-13-O-acetylphorbol-20-(9′Z)-octadecenoate.
1
phorbol ester. However, in a comparison of the H NMR data
with those of compound 3, the upfield chemical shifts of the 6′,7′-
vicinal diol were evident [δH 4.07 (m, H-6); δH 3.52 (m, H-7)],
Figure 1. COSY (bold bonds) and selected HMBC (arrows) correlations of 1−3.
D
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Journal of Natural Products
Article
Figure 2. Key NOE (left right arrow) correlations of 1−3.
indicating a threo configuration. On the basis of the obtained
data, compounds 3 and 4 were determined as stereoisomers, and
compound 4 was assigned as 12-O-(2′E,4′E)-6′,7′-(threo)-
dihydroxytetradeca-2′,4′-dienoylphorbol-13-acetate.
Table 3. Effects of Pure Compounds on Superoxide Anion
Generation and Elastase Release in FMLF/CB-Induced
Human Neutrophils
superoxide anion
elastase release
Compound 5, 12-O-deoxyphorbol 13-decanoate, was isolated
as a natural product for the first time.19 Compound 6 was
identified as the phorbol ester, mellerin A.14 In addition, known
glycerides were purified, 1,3-dioleoyl glyceride, a major
component of A. malaccensis seeds (7),20 and 1-oleoyl-2-
palmitoyl glyceride (8).21 The NMR spectroscopic data of the
known compounds were in agreement with the literature. To
confirm the fatty acid moiety, compound 7 was subjected to
methanolysis using the method reported by Ichihara et al.16 The
resulting fatty acid methyl ester was determined by GC-MS
analysis as an oleic acid methyl ester. Using the same procedure,
two long-chain substituents of compound 8 were identified by
GC-MS analysis as oleic acid methyl ester and hexadecanoic
(palmitic) acid methyl ester.
All isolated compounds were evaluated for their potential anti-
inflammatory and cytotoxic activities. In the anti-inflammatory
assay, 1 (IC50 2.7 μM), 5 (IC50 0.8 μM), and 6 (IC50 2.1 μM)
showed potent inhibitory activity on N-formyl-L-methionyl-L-
leucyl-L-phenylalanine (fMLF)/cytochalasin B (CB)-induced
elastase release by human neutrophils, comparable to a positive
control, the PI3K inhibitor LY294002 (IC50 3.3 μM) (Table 3).
On the other hand, phorbol 12-myristate 13-acetate (PMA)
showed an enhancing effect on elastase release. The conjugated
aldehydic fatty acid substituent in 1 and saturated fatty acid
substituents in 5 and 6 may play a role in their bioactivity. All
phorbol esters (1−6 and PMA) showed enhancing activities on
superoxide anion generation.
In general, phorbol esters trigger superoxide anion generation
and cause pro-inflammatory effects. However, the extracts of the
seeds of A. malaccensis exhibited an excellent superoxide anion
generation inhibitory effect,9 which could be due to a high
content of free anti-inflammatory fatty acids in the seed oil of
A. malaccensis.11,22 Also, it may be suggested that the fatty acid
substituents, which themselves show significant anti-inflamma-
tory effects,22 play an important role in mediating the effect of
phorbol esters. In addition, for the first time, it was discovered in
the present study that some of the phorbol esters inhibit elastase-
releasing effects. These findings may provide insights for further
research related to the inflammation-modulating activities of
phorbol esters.
a
a
compound
IC50 (μM)
IC50 (μM)
b
1
2
3
4
5
6
7
8
enhancing
2.7 0.6
>10
b
enhancing
b
c
enhancing
enhancing
b
c
enhancing
enhancing
b
enhancing
0.8 0.3
2.1 0.7
>10
b
enhancing
>10
d
enhancing
>10
e
b
c
PMA
enhancing
enhancing
f
LY294002
1.8 0.7
3.3 0.7
a
IC50 values express the concentration of the sample required to
inhibit superoxide anion generation or elastase release by 50%. Results
are presented as means SEM (n = 3−5). The compounds showed
enhancing effects on superoxide generation at 10 μM (1 106.1%, 2
18.1%, 3 106.0%, 4 107.1%, 5 95.3%, 6 109.6%, PMA 102.7%) in the
absence of fMLF/CB. Compared with fMLF/CB (as 100%). The
compounds showed enhancing effects on elastase release at 10 μM (3
10.5%, 4 9.2%, PMA 44.2%) in the absence of fMLF/CB. Compared
with fMLF/CB (as 100%). The compound showed enhancing effects
on superoxide generation at 10 μM (8 74.5%) in the presence of CB.
b
c
d
e
Compared with fMLF/CB (as 100%). Phorbol 12-myristate 13-
acetate (PMA), a PKC activator, was used as a positive control
exerting enhancing effects. LY294002, the PI3K inhibitor, was used as
a positive control exerting suppressive effects.
f
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a JASCO P-2000 digital polarimeter (JASCO Inc.,
Tokyo, Japan). A JASCO V-530 ultraviolet spectrophotometer
(JASCO) was used to run UV spectra. IR spectra were obtained on
an FT-IR-4100 JASCO spectrophotometer (JASCO). NMR spectra
were obtained on a Varian VNMR-600 FT-NMR spectrometer (Varian
Inc., Palo Alto, CA, USA) and on a JEOL JNM-ECS 400 NMR
spectrometer (JEOL Ltd., Tokyo, Japan). Electrospray-ionization mass
spectrometry (ESIMS) data were collected on a Waters micromass ZQ
mass spectrometer (Waters Corporation, Milford, MA, USA). High-
resolution ESIMS data were determined using a Bruker Daltonics APEX
II mass spectrometer (FT-ICR/MS, FTMS) (Bruker Daltonics,
Billerica, MA, USA). GC-MS data were collected using a DSQ II single
quadrupole GC/MS (Thermo Fisher Scientific, Waltham, MA, USA)
E
DOI: 10.1021/acs.jnatprod.6b01096
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Journal of Natural Products
Article
1
equipped with an HP-5MS capillary column (0.25 μm, 30 m × 0.25 mm,
Agilent, Santa Clara, CA, USA). Sephadex LH-20 (GE Healthcare,
Stockholm, Sweden) and silica gel (Kieselgel 60, 0.063−0.200 mm,
Geduran Si 60, 0.040−0.063 mm, Merck KGaA) were used for column
chromatography. Silica gel precoated (Kieselgel 60 F254 and RP-18
F254s, Merck KGaA) TLC plates were used. HPLC analyses were
performed using a Shimadzu LC-10AT VP (Shimadzu Inc., Kyoto,
Japan) pump interface equipped with a Shimadzu SPD-M10A VP diode
array detector using a C18 column (5 μm, 250 × 10 mm, Phenomenex
Inc.). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate
(PMA), LY294002, and doxorubicin hydrochloride were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
2925, 2851, 1710, 1632, 1455, 1375, 1261, 1024, 802, 755 cm−1; H
NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see
Tables 1 and 2; ESIMS m/z 645 [M + H]+; HRESIMS m/z 667.34515
[M + Na]+ (calcd for C36H52O10Na, 667.34527).
12-O-(2′E,4′E)-6′,7′-(threo)-Dihydroxytetradeca-2′,4′-dienoyl-
phorbol-13-acetate (4): colorless oil; [α]2D5 +13.2 (c 0.13, CHCl3); UV
(MeOH) λmax (log ε) 289 (2.79), 249 (2.84) nm; IR (neat) νmax 3385,
2929, 2855, 1632, 1455, 1375, 1261, 1124, 802, 755 cm−1; H NMR
1
(CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Tables 1 and
2; ESIMS m/z 645 [M + H]+; HRESIMS m/z 667.34550 [M + Na]+
(calcd for C36H52O10Na, 667.34527).
1,3-Dioleoyl glyceride (7): C39H72O5; ESIMS m/z 621 [M + H]+;
GC-MS analysis (tR 21.30, m/z 296 [M]+, for oleic acid methyl ester,
fragmentation ions observed at m/z 264, 222, 137, 123, 110, 98, 74, 69,
55).
Plant Material. The seeds of A. malaccensis were collected and
identified by one of the authors (H.-Y.H.) in November 2014. A voucher
specimen (no. KMU-AMS 1) has been deposited in the Graduate
Institute of Natural Products, College of Pharmacy, Kaohsiung Medical
University, Kaohsiung, Taiwan.
1-Oleoyl-2-palmitoyl glyceride (8): C37H70O5; ESIMS m/z 595 [M +
H]+; GC-MS analysis (tR 21.31, m/z 296 [M]+, fragmentation ions
observed at m/z 264, 180, 137, 123, 110, 98, 74, 69, oleic acid methyl
ester; tR 17.71, m/z 270 [M]+, fragmentation ions observed at m/z 227,
185, 143, 87, 74, palmitic acid methyl ester).
Methylation of Samples for GC-MS Analysis. The method of
Ichihara et al. was utilized.16 Lipid sample was placed in a screw-capped
glass test tube and dissolved in 0.20 mL of toluene. To the lipid solution
were added 1.50 mL of methanol (HPLC grade) and 0.30 mL of the
8.0% HCl solution, in order. The final HCl concentration was 1.2%. The
reaction mixture was heated at 100 °C for 1 h. On cooling, the reaction
was quenched by 1 mL of 6% aqueous K2CO3 solution. Then, 1−2 mL
of hexane (HPLC grade) was added, and the mixture was partitioned to
yield a hexane layer (upper layer). The hexane layer was filtered through
a 0.22 μm filter (ChromTech, Apple Valley, MN, USA) into a GC-MS
vial and then subjected to GC-MS analysis.
GC-MS Analysis. The methylated samples were analyzed by gas
chromatography−mass spectrometry (DSQ II single quadrupole GC/
MS, Thermo Fisher Scientific). Derivatized samples were vaporized at
250 °C in standard split mode (1:50) and separated on a 30 m × 0.25
mm HP-5MS capillary column with a 0.25 μm coating (Agilent). The
column oven temperature was set at 70 °C, held for 3 min, then
increased to 180 °C at 20 °C/min, held for 5 min, then increased to 280
°C at 5 °C/min and held for 5 min. A helium gas carrier flow of 1 mL/
min was used. The interface and ion source temperature were set to 250
°C. An electron-impact ionization of 30 eV was utilized, the injection
volume was 1 μL, and the mass range was m/z 38−600. Identification of
the compounds was based on a comparison of mass spectra with the data
from the Wiley/NBS Registry of Mass Spectral Data (version 5.0)/
National Institute of Standards and Technology (NIST) MS Search
(version 2.0).
Superoxide Anion and Elastase Release Assays. Blood was
obtained from healthy human donors (20−30 years old), using a
protocol approved by the Institutional Review Board at Chang Gung
Memorial Hospital (protocol number 102-1595A3). Human neutro-
phils were isolated using a standard method of dextran sedimentation
prior to centrifugation in a Ficoll-Hypaque gradient and hypotonic lysis
of erythrocytes.23 Purified neutrophils containing >98% viable cells were
determined by the trypan-blue exclusion method.24 The neutrophils
were resuspended in a Ca2+-free Hank’s buffered salt solution (HBSS) at
pH 7.4 and were maintained at 4 °C prior to use.
Extraction and Isolation. The dried and powdered seeds of
Aquilaria malaccensis (462 g) were extracted with 90% EtOH (3 × 5 L)
to give an extract (27.7 g), which was suspended in water and further
partitioned between ethyl acetate and H2O (1:1) to obtain an ethyl
acetate layer. The ethyl acetate layer was then partitioned between 90%
aqueous MeOH and n-hexane to afford a 90% MeOH layer (16.2 g) and
an n-hexane partition (7.1 g). The water layer was partitioned with n-
butanol to yield a water partition (1.6 g) and an n-butanol layer (0.4 g).
The MeOH layer (16.2 g) was submitted to column chromatography
(CC) over silica gel 60 (0.063−0.200 mm, Merck), eluted with n-
hexane/CH2Cl2/MeOH (6:3:1 to 6:10:2), to afford six fractions: Fr.1
(2.9 g), Fr.2 (1.3 g), Fr.3 (6.8 g), Fr.4 (3.2 g), Fr.5 (1.7 g), and Fr.6 (97.9
mg). Fraction 4 was fractionated by Sephadex LH-20 CC with CH2Cl2/
MeOH (1:1) as eluant, to yield eight further fractions (Fr.4-1 to Fr.4-8).
Fraction 4-3 (762 mg) was purified using CC on silica gel (30 cm, 1.5
cm, Geduran Si 60, 0.040−0.063 mm, Merck) and eluted with a gradient
of EtOAc/n-hexane (from 1:10 to 4:1) to obtain 15 subfractions (4-3-1
to 4-3-15), with compound 2 (39.5 mg) being purified from subfraction
4-3-6. Subfraction 4-3-3 was further fractionated over a Sephadex LH-20
column (CH2Cl2/MeOH, 8:2) to obtain five subfractions (4-3-3-1 to 4-
3-3-5), and compound 7 (130.5 mg) was purified from subfraction 4-3-
3-3. Subfraction 4-3-3-4 was subjected to silica gel CC and eluted with a
gradient of n-hexane/CH2Cl2/MeOH (10:1:0 to 3:1:0.5), to yield
compound 8 (4.6 mg). Subfraction 4-3-13 (23.5 mg) was separated by
RP-HPLC (C18 column, flow rate = 2.0 mL/min), with MeOH/H2O
(60:40) used for elution, to afford compounds 1 (0.7 mg), 3 (0.9 mg),
and 4 (1.0 mg).
Fraction 4-4 (174 mg) was further purified using column
chromatography on silica gel (30 cm, 1.5 cm, Geduran Si 60, 0.040−
0.063 mm, Merck), eluted with a gradient of EtOAc/n-hexane (from
1:15 to 4:1), to obtain 11 subfractions (4-4-1 to 4-4-11). Subfraction 4-
4-7 (38 mg) was further purified by RP-HPLC (C18 column flow rate =
2.0 mL/min), with MeOH/H2O (85:15), to yield compounds 5 (7.8
mg) and 6 (0.8 mg).
12-O-(2′E,4′E)-6-Oxohexa-2′,4′-dienoylphorbol-13-acetate (1):
colorless oil; [α]D25 +4.2 (c 0.17, CHCl3); UV (MeOH) λmax (log ε)
295 (2.80), 249 (2.84) nm; IR (neat) νmax 3413, 2925, 2855, 2360, 2339,
1713, 1625, 1597, 1261, 1184, 755 cm−1; 1H NMR (CDCl3, 600 MHz)
and 13C NMR (CDCl3, 150 MHz), see Tables 1 and 2; ESIMS found m/
z 515 [M + H]+; HRESIMS m/z 537.20959 [M + Na]+ (calcd for
C28H34O9Na, 537.20950).
Superoxide Anion Generation Assay. Neutrophil superoxide anion
generation was determined using a superoxide dismutase (SOD)-
inhibitory cytochrome reduction assay according to previously
described procedures.25,26 Briefly, after supplementation with 0.5 mg/
mL ferricytochrome c and 1 mM Ca2+, neutrophils (3 × 105 per mL)
were equilibrated at 37 °C for 2 min and incubated with different
concentrations of test compounds or DMSO (as control) for 5 min.
Cells were incubated with cytochalasin B (1 μg/mL) for 3 min prior to
the activation with 100 nM N-formyl-L-methionyl-L-leucyl-L-phenyl-
alanine for 10 min. Changes in absorbance with the reduction of
ferricytochrome c at 550 nm were continuously monitored in a double-
beam, six-cell positioner spectrophotometer with constant stirring
(Hitachi U-3010, Tokyo, Japan). Calculations were based on the
differences in the reactions with and without SOD (100 U/mL) divided
12-Deoxy-13-O-acetylphorbol-20-(9′Z)-octadecenoate (2): color-
less oil; [α]2D5 +2.9 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 285 (2.78),
250 (2.83) nm; IR (neat) νmax 3409, 2922, 2855, 1717, 1375, 1332, 1152,
1021 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100
MHz), see Tables 1 and 2; ESIMS m/z 655 [M + H]+; HRESIMS m/z
677.43884 [M + Na]+ (calcd for C40H62O7Na, 677.43878); GC-MS
analysis (tR 21.30, m/z 296 [M]+, fragmentation ions observed at m/z
264, 222, 137, 123, 110, 98, 83, 74, 69, oleic acid methyl ester).
12-O-(2′E,4′E)-6′,7′-(erythro)-Dihydroxytetradeca-2′,4′-dienoyl-
phorbol-13-acetate (3): colorless oil; [α]2D5 +10.4 (c 0.17, CHCl3); UV
(MeOH) λmax (log ε) 289 (2.79), 249 (2.83) nm; IR (neat) νmax 3392,
F
DOI: 10.1021/acs.jnatprod.6b01096
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Journal of Natural Products
Article
by the extinction coefficient for the reduction of ferricytochrome c (21.1
mM−1 cm−1). All experiments were repeated three times. LY294002 and
PMA were used as positive controls.
Health and Welfare, Taiwan (MOHW106-TDU-B-212-144007,
Health and Welfare Surcharge of Tobacco Products).
Elastase Release Inhibition Assay. Degranulation of azurophilic
granules was determined by measuring the elastase release, as described
previously.26 Experiments were performed using MeOSuc-Ala-Ala-Pro-
Val-p-nitroanilide as the elastase substrate. After supplementation with
MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), neutrophils (3 × 105
per mL) were equilibrated at 37 °C for 2 min and incubated with each
test compound for 5 min. Cells were stimulated with fMLF (100 nM)/
CB (0.5 μg/mL), and changes in the absorbance at 405 nm were
monitored continuously in order to measure the elastase release. The
results were expressed as the percent of elastase release in the fMLF/CB-
activated, drug-free control system. All experiments were repeated three
times. LY294002 and PMA were used as positive controls.
Cytotoxicity Assay. The MTT viability assay was used according to
a previously described method.27 HepG2 (1 × 104 cells), A549 (5 × 103
cells), and MDA-MB-231 (1 × 104 cells) were seeded into 96-well
plates. The cells were treated with the test compounds at a
concentration of 20 μg/mL. After 72 h, the medium was removed and
100 μL of MTT solution (0.5 mg/mL) was added to each well. The
plates were incubated at 37 °C for 1 h to form formazan crystals, which
were then dissolved by DMSO (100 μL). Plates were gently shaken, and
absorbance at 550 nm was measured using a microplate reader. The
degree of cell viability of each sample was calculated as the percentage of
control value (untreated cells). Doxorubicin hydrochloride was used as a
positive control and exhibited IC50 values of 0.5, 0.3, and 1.2 μM against
A549, Hep-G2, and MDA-MB-231 cells, respectively.
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ORCID
Yuan-Bin Cheng: 0000-0001-6581-1320
Author Contributions
◇V. D. Wagh and M. Korinek contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Center for Research Resources and Development
at Kaohsiung Medical University for providing instrumentation
support. This work was supported by grants from Ministry of
Science and Technology, Taiwan (NSC 102-2628-B-037-003-
MY3, MOST 103-2320-B-037-005-MY2, awarded to F.-R.C.,
MOST 103-2628-B-037-001-MY3 awarded to Y.-B.C). This
study was also supported by Kaohsiung Medical University,
Taiwan (Aim for the Top Universities Grant, grant nos. KMU-
TP105E32, KMU-TP104A26, KMU-M106009), and Ministry of
G
DOI: 10.1021/acs.jnatprod.6b01096
J. Nat. Prod. XXXX, XXX, XXX−XXX